A composition with moisturizing and anti-aging efficacy and application in cosmetics

Abstract

The application discloses a composition with moisturizing and anti-aging effects and application in cosmetics, and relates to a moisturizing and anti-aging composition comprising sodium retinyl hyaluronate and semen phoebe extract. It is verified through experiments that the sodium retinyl hyaluronate and the semen phoebe extract have a synergistic effect, can significantly promote expression of type X collagen gene and generation of AQP3 protein (water channel protein 3), and can be used in cosmetics to improve skin dryness and aging problems, and meanwhile, the composition has good stability and is safe and non-irritating.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to a composition with moisturizing and anti-aging effects and application thereof in cosmetics. BACKGROUND

[0002] In human tissues, collagen accounts for 70-80% of the dry weight, among which type XVII collagen, as a transmembrane glycoprotein, is an important component of epidermal stem cell hemidesmosomes, mainly located in epidermal stem cells at the junction of epidermis and dermis, and its role is to tightly connect epidermis and dermis through the basement membrane zone to promote epidermal replacement and stabilize the basement membrane. Type XVII collagen can effectively prevent the separation of the epidermis and dermis layers, avoid the occurrence of blisters and relaxation of the skin, and maintain the stability and integrity of the overall structure of the skin. With the increase of age, the content of type XVII collagen in the skin gradually decreases, resulting in the decrease of skin elasticity and toughness. Increasing the content of type XVII collagen in the skin can effectively delay the aging process of the skin.

[0003] Sodium hyaluronate retinoate is an esterification product of all-trans retinoic acid and 13-cis retinoic acid and sodium hyaluronate. All-trans retinoic acid, also known as retinoic acid, has the effects of promoting epidermal cell renewal and stimulating collagen synthesis, and sodium hyaluronate has good moisturizing effect. Sodium hyaluronate retinoate has potential moisturizing and anti-aging effects.

[0004] Medicinal plant ingredients are chemical substances with medical efficacy or physiological activity in traditional Chinese medicine, which have been used for thousands of years in China. Medicinal plant ingredients have been widely used in the field of cosmetics and food processing. Cosmetics containing natural Chinese herbal active ingredients are safe to use, widely applicable, and have characteristics that cannot be replaced by chemical cosmetics.

[0005] Most of the skin care products on the market that claim to have anti-aging effects cannot meet the needs of consumers, and some products have a large degree of skin irritation, which can cause dry skin. There is an urgent need to develop anti-aging ingredients with significant anti-aging effects, moisturizing, and mildness without irritation. Sodium hyaluronate retinoate is a potential moisturizing and anti-aging ingredient, and medicinal plant ingredients have good soothing and repairing effects. There is no report on the synergistic moisturizing and anti-aging effects of sodium hyaluronate retinoate and medicinal plant ingredients. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application aims to provide a composition with moisturizing and anti-aging effects and its application in cosmetics, which comprises sodium retinyl hyaluronate and semen phoebe extract, and experiments have verified that the sodium retinyl hyaluronate and semen phoebe extract have a synergistic effect of significantly promoting the expression of collagen type X VII gene and the generation of AQP3 protein (water channel protein 3), and can be used in cosmetics to improve the problems of dry and aging skin, and meanwhile, the composition has good stability, safety and no irritation, thus having good practical promotion and application value.

[0007] In order to achieve the above technical purpose, the technical scheme provided by the present application is as follows:

[0008] In a first aspect, the present application provides a composition with moisturizing and anti-aging effects, which comprises sodium retinyl hyaluronate and semen phoebe extract, wherein the sodium retinyl hyaluronate is 0.01-0.2 parts by mass, and the semen phoebe extract is 4-15 parts by mass.

[0009] Preparation method: mix the sodium retinyl hyaluronate and semen phoebe extract together and dissolve them in purified water (make up to 100 parts).

[0010] Preferably, in the composition with moisturizing and anti-aging effects, the sodium retinyl hyaluronate is 0.08-0.14 parts by mass, and the semen phoebe extract is 8-12 parts by mass.

[0011] The sodium retinyl hyaluronate is an ester prepared by reacting carbonyl diimidazole, retinoic acid and iso-retinoic acid, and hyaluronic acid. The esterification degree (degree of substitution) DS (mol / mol) is 4.0%-7.0%, the retinoic acid / iso-retinoic acid K (mol / mol) combined with HA is 1.6-2.3, and the molecular weight is 5KDa-500KDa. Specifically, a proper amount of carbonyl diimidazole, retinoic acid and iso-retinoic acid are dissolved in dimethyl sulfoxide, and the solution is stirred at 60℃ or below for 10-14h. A proper amount of hyaluronic acid is dissolved in dimethylformamide or dimethyl sulfoxide at 65-75℃, and constant temperature stirring is performed for 20-40min. The molar ratio of carbonyl diimidazole, retinoic acid and iso-retinoic acid, and hyaluronic acid is 28-32:0.4-0.6:1. The solution of carbonyl diimidazole, retinoic acid and iso-retinoic acid is added dropwise to the hyaluronic acid solution, and after reaction at 65-75℃ for 5-8h, saturated sodium chloride solution is added for dilution, ethanol is added for precipitation and washing, the precipitate is collected after filtration, and the precipitate is dried under reduced pressure to obtain a light yellow to yellow granular powder.

[0012] The preparation method of the semen phytosteroli extract is as follows: the semen phytosteroli powder is extracted by heating reflux with 40-60% ethanol solution, the extract is concentrated under reduced pressure, and the concentration is 1.0-1.2 g / mL; then, a proper amount of butanediol is added to the extract to make the concentration of the extract 0.05-0.12 g / mL (calculated by the amount of crude drug), and the semen phytosteroli extract is obtained.

[0013] In a second aspect of the present application, the above-mentioned composition is used in the preparation of a moisturizing and anti-aging skin care cosmetic.

[0014] Therefore, in a third aspect of the present application, a moisturizing and anti-aging skin care cosmetic is provided, which comprises the above-mentioned composition.

[0015] The moisturizing and anti-aging skin care cosmetic can further comprise other raw material components allowed to be added in the cosmetic field, including but not limited to emulsifiers, emollients, humectants and thickening agents, etc.

[0016] Further, the amount of the composition with moisturizing and anti-aging efficacy is 0.1-7.0%, preferably 0.5-3.0%, based on the total mass of the moisturizing and anti-aging skin care cosmetic.

[0017] Meanwhile, the above-mentioned raw material components can also be reasonably added to prepare different cosmetic dosage forms, such as essence water, essence milk, essence cream, etc., and based on the above-mentioned basic cosmetic categories, further derived cosmetic categories obtained are obviously within the protection scope of the present application.

[0018] The above-mentioned one or more technical solutions have the following beneficial effects:

[0019] (1) The moisturizing and anti-aging composition obtained by compounding sodium hyaluronate and semen phytosteroli extract has significant synergistic effect, and good moisturizing and anti-aging efficacy is achieved by promoting the expression of XⅦ collagen gene and the production of AQP3 protein;

[0020] (2) Sodium hyaluronate in the moisturizing and anti-aging composition has slight skin irritation, and the skin irritation of sodium hyaluronate can be obviously reduced by compounding with semen phytosteroli extract;

[0021] (3) It is verified by experiments that semen phytosteroli extract ② (water extraction and alcohol precipitation method) does not have synergistic effect with sodium hyaluronate in moisturizing and anti-aging, and the combination of semen phytosteroli extract ① (reflux extraction method) and sodium hyaluronate can make the product have higher moisturizing and anti-aging effect, which indicates that the extraction method of semen phytosteroli is very critical.

[0022] (4) The moisturizing and anti-aging skin care cosmetic of the present invention is simple and easy to prepare, the raw materials are easy to obtain, and it is suitable for mass production. It has been tested by human efficacy experiments and has good moisturizing and anti-aging effects, so it has good practical application value. Detailed Implementation

[0023] The present invention will be further illustrated below by means of embodiments, but these embodiments are not intended to limit the invention to their scope. Based on the embodiments of the present invention, any modifications to the present invention made by those skilled in the art without inventive effort are within the scope of protection of the present invention. Furthermore, in the embodiments of the present invention, unless otherwise specified, all raw materials used in the preparation are commercially available products well-known to those skilled in the art.

[0024] Examples 1-5 and Comparative Examples 1-18:

[0025] The specific schemes of Examples 1-5 and Comparative Examples 1-18 are shown in Table 1 below, calculated by mass parts. The preparation method is as follows: Sodium retinyl hyaluronic acid and Tiankui seed extract (the comparative examples are replaced with the corresponding components according to Table 1) are mixed together and dissolved in purified water.

[0026] The specific steps for preparing the sodium retinyl hyaluronic acid are as follows:

[0027] Appropriate amounts of carbonyl diimidazole, retinoic acid, and isotretinoin were dissolved in dimethyl sulfoxide, and the solution was stirred at below 60℃ for 12 h. An appropriate amount of hyaluronic acid was dissolved in dimethylformamide at 70℃ and stirred at a constant temperature for 30 min. The molar ratio of carbonyl diimidazole, retinoic acid, isotretinoin, and hyaluronic acid was 30:0.5:1. The solution of carbonyl diimidazole, retinoic acid, and isotretinoin was added dropwise to the hyaluronic acid solution, and the reaction was carried out at 70℃ for 6 h. After dilution, saturated sodium chloride solution was added, ethanol was added for precipitation and washing, the precipitate was collected after filtration, and dried under reduced pressure to obtain a pale yellow to yellow granular powder. Sodium retinyl hyaluronic acid with a degree of esterification (degree of substitution) DS (mol / mol) of 5.0%, a retinoic acid / isoretinoic acid K (mol / mol) ratio with HA of 1.8, and a molecular weight of 8 kDa was obtained.

[0028] The specific steps for preparing the Tiankui seed extract are as follows:

[0029] The extraction method for Tiankui seed extract is as follows:

[0030] (1) Wash the seeds of Tiankui with deionized water, dry them at 40°C, crush them and pass them through an 80-mesh sieve, and place them in a desiccator for later use.

[0031] (2) Accurately weigh an appropriate amount of *Smilax glabra* seed powder, place it in a round-bottom flask, and heat under reflux for extraction. The extraction solvent is 50% ethanol solution. The first material-to-liquid ratio is 1:10, and the second material-to-liquid ratio is 1:6. Combine the two filtrates. Concentrate the filtrate under reduced pressure until the density is 1.1574 g / mL. Add an appropriate amount of butanediol until the extract concentration is 0.1012 g / mL (based on the amount of crude drug), thus obtaining *Smilax glabra* seed extract ①.

[0032] The extraction method for Tiankui seed extract ② is as follows:

[0033] (1) Wash the seeds of Tiankui with deionized water, dry them at 40°C, crush them and pass them through an 80-mesh sieve, and place them in a desiccator for later use.

[0034] (2) Accurately weigh an appropriate amount of Tiankui seeds, add 10 times the amount of water, decoct 3 times, 60 minutes each time, combine the decoctions, concentrate under reduced pressure at 65℃ to a density of 1.1042 g / mL, cool, add alcohol to make the alcohol concentration reach 70%, refrigerate and stand for 12 hours, filter after standing, take the upper liquid and concentrate under reduced pressure to a density of 1.1615 g / mL, add an appropriate amount of butanediol to the extract concentration of 0.1037 g / mL (based on the amount of raw drug), and you will get Tiankui seed extract ②.

[0035] Table 1. List of components for specific embodiments and comparative examples.

[0036]

[0037] Example 6: Essence Cream

[0038] The above-mentioned composition with moisturizing and anti-aging effects is used to prepare an essence cream product containing the composition with moisturizing and anti-aging effects, and the formula is shown in Table 2.

[0039] Table 2. Formula of the Essence Cream

[0040]

[0041] Preparation method of essence cream:

[0042] S1: Preparation of the aqueous phase: Add the raw materials from phase A separately, heat to 85℃ and stir to dissolve;

[0043] S2: Preparation of the oil phase: Add the raw materials from phase B separately and heat to 80℃ to melt;

[0044] S3: Combine phase A and phase B, start homogenization (3000 r / min), and homogenize for 12 min;

[0045] S4: After homogenization is complete, start cooling. When the temperature drops to 70℃, add phase C, stir to dissolve, and continue cooling after dissolution is complete.

[0046] S5: When the temperature drops to 45℃, add phase D components, stir to dissolve and disperse evenly to obtain the essence cream product.

[0047] Experimental Example 1: Stimulus Evaluation Experiment

[0048] The experiment recruited 30 participants (7 males and 23 females) aged 18 to 60.

[0049] Human skin occlusive patch test method: The test samples of Examples 1-5 and Comparative Examples 5, 10, 11, 13, 15, and 17 were diluted with distilled water to a 1% solution as the test substance, and distilled water was used as the control group. A qualified patch applicator was selected, and 0.020–0.025 mL of the test substance was placed in the applicator chamber. The patch applicator containing the test substance was applied to the back of the subject for 24 hours. After 24 hours, the patch applicator was removed, and skin reactions were observed at 0.5 hours, 24 hours, and 48 hours after removal. The results were recorded according to the skin reaction grading standards (Table 3) in the "Cosmetic Safety Technical Specifications 2015". Detailed results are shown in Table 4. The results were interpreted according to the "Cosmetic Hygiene Standards (2007 Edition)". The criteria for interpreting the results of a closed skin patch test are as follows: if more than 5 out of 30 subjects experience a grade 1 adverse skin reaction, or more than 2 out of 30 subjects experience a grade 2 adverse skin reaction, or if any one subject experiences a grade 3 or higher adverse skin reaction, the test substance is considered to have an adverse skin reaction on the human body.

[0050] Table 3. Grading Standards for Skin Reaction

[0051]

[0052] The test results are shown in Table 4. No adverse reactions were observed in Examples 1-5 and Comparative Examples 10, 11, and 15, indicating that they are mild and non-irritating. However, Comparative Examples 5, 13, and 17 all showed adverse reactions of varying degrees, indicating that sodium retinyl hyaluronic acid alone, the combination of sodium retinyl hyaluronic acid and Tiankui seed extract ②, and the combination of 0.4 parts of sodium retinyl hyaluronic acid and Tiankui seed extract ① are skin irritating.

[0053] Table 4 Results of the Stimulus Evaluation Experiment

[0054]

[0055]

[0056] Experimental Example 2: In vitro anti-aging efficacy study

[0057] Human skin fibroblasts (HSF) were used as the model cell line in this study. After cell resuscitation, when the cell plating rate reached approximately 60%, cells were seeded into 6-well plates and incubated overnight in a CO2 incubator (37°C, 5% CO2). Working solutions for the test substances were prepared according to the test protocol (Table 5). When the cell plating rate in the 6-well plates reached 40%–60%, the cells were administered to different groups, with three replicates per group. DMEM culture medium was used. 2 mL of culture medium was added to each well of the blank control group, 2 mL of culture medium containing TGF-β1 was added to each well of the positive control group, and 2 mL of culture medium containing the corresponding concentration of the test sample was added to each well of the sample group. After administration, the 6-well plates were placed in a CO2 incubator (37°C, 5% CO2) for 24 h. After 24 h of incubation, the old medium was discarded, the cells were washed twice with PBS, and 1 mL of RNAiso Plus was added to each well. After cell lysis by pipetting, the samples were collected. RNA was extracted, reverse transcribed into cDNA, and then detected by real-time quantitative PCR. 2... -△△CT The method was used to calculate the Collagen XVII gene upregulation rate. The formula for calculating the Collagen XVII upregulation rate is as follows.

[0058]

[0059] Verify the synergistic effect using the COLBY formula: Where X and Y are the test results of a single component, and E is the result calculated according to the COLBY formula. 测定 For the results of composition determination, E 测定 >E 计算 E represents synergistic effect. 测定 =E 计算 E represents the summation. 测定 <E 计算 This indicates that there is no synergistic effect.

[0060] GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0061] Table 5 Test Plan

[0062]

[0063] Table 6 Summary of Collagen XVII Gene Detection Results

[0064]

[0065]

[0066] Table 7 Summary of Synergistic Effects of In Vitro Anti-Aging

[0067]

[0068]

[0069] As shown in Tables 6 and 7, E in Examples 1-5 测定 >E 计算 This indicates that the combination of sodium retinyl hyaluronic acid and *Smilax china* extract ① in Examples 1-5 has a synergistic effect in promoting the expression of type XVII collagen genes, i.e., it has a synergistic anti-aging effect, with Example 3 showing the most significant synergistic effect. Comparative Examples 11, 13, 15, and 17 showed E... 测定 <E 计算 This indicates that Comparative Example 11, the combination of dimethylsilanol hyaluronic acid ester and *Sedum aizoon* extract ①, did not have a synergistic effect on promoting the expression of type XVII collagen genes; Comparative Example 13, the combination of sodium retinyl hyaluronic acid and *Sedum aizoon* extract ②, did not have a synergistic effect on promoting the expression of type XVII collagen genes; Comparative Example 15, the combination of sodium retinyl hyaluronic acid and 20 parts of *Sedum aizoon* extract ①, did not have a synergistic effect on promoting the expression of type XVII collagen genes; and Comparative Example 17 indicates that the combination of 0.4 parts of sodium retinyl hyaluronic acid and *Sedum aizoon* extract ① did not have a synergistic effect on promoting the expression of type XVII collagen genes.

[0070] Experimental Example 3: Study on the efficacy of in vitro moisturizing (AQP3 enhancement)

[0071] Keratinocytes were used as the model cell line in this experiment. After cell resuscitation, when the cell plating rate reached approximately 60%, cells were seeded into 24-well plates and incubated overnight in a CO2 incubator (37°C, 5% CO2). The working solution of the test substance was prepared according to the test protocol (Table 8). Based on the test groups, when the cell plating rate in the 24-well plates reached 40%–60%, the drugs were administered to the groups, with three replicates per group. The culture medium was KcGrowth medium. 1 mL of culture medium was added to each well of the blank control group, 1 mL of culture medium containing CaCl2 was added to each well of the positive control group, and 1 mL of culture medium containing the corresponding concentration of the test sample was added to each well of the sample group. After drug administration, the 24-well plates were placed in a CO2 incubator (37°C, 5% CO2) for 24 hours.

[0072] The cells were fixed with 4% paraformaldehyde for 30 minutes, followed by immunofluorescence detection, microscopic imaging, and image acquisition and analysis. The AQP3 enhancement rate was calculated using the following formula.

[0073]

[0074] The synergistic effect was verified using the COLBY formula:

[0075] GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0076] Table 8 Test Plan

[0077]

[0078] Table 9: Summary of AQP3 Immunofluorescence Analysis Results

[0079]

[0080]

[0081] Table 10 Summary of Synergistic Effects of In Vitro Moisturizing

[0082]

[0083]

[0084]

[0085] The experimental results are shown in Tables 9 and 10. The E values ​​of Examples 1-5... 测定 >E 计算 This demonstrates that the combination of sodium retinyl hyaluronic acid and *Smilax china* extract ① in Examples 1-5 synergistically promotes the production of AQP3 protein, i.e., it has a synergistic moisturizing effect, with Example 3 showing the most significant synergistic effect. Comparative Examples 11, 13, 15, and 17 showed E... 测定 <E 计算 This indicates that Comparative Example 11, the combination of dimethylsilanol hyaluronic acid ester and *Sedum aizoon* extract ①, did not synergistically promote the production of AQP3 protein; Comparative Example 13, the combination of sodium retinyl hyaluronic acid and *Sedum aizoon* extract ②, did not synergistically promote the production of AQP3 protein; Comparative Example 15, the combination of sodium retinyl hyaluronic acid and 20 parts of *Sedum aizoon* extract ①, did not synergistically promote the production of AQP3 protein; and Comparative Example 17 indicates that the combination of 0.4 parts of sodium retinyl hyaluronic acid and *Sedum aizoon* extract ① did not synergistically promote the production of AQP3 protein.

[0086] Test Example 4: Test Results of the Moisturizing and Anti-aging Composition of the Present Invention on Human Moisturizing and Anti-aging Effects

[0087] Example 6 was tested for its skin moisturizing and anti-aging effects on human skin. A total of 30 people completed the test, including 8 men and 22 women, aged 25 to 55 years, who met the criteria for voluntary inclusion and exclusion of subjects.

[0088] How to use: After using toner, take an appropriate amount of sample and apply it evenly to the face. Pat gently until absorbed, then proceed with your normal skincare routine. Use once in the morning and once in the evening, keeping other skincare habits unchanged, for 28 consecutive days.

[0089] Testing time: before using the sample (D0), 7 days after using the sample (D7), and 28 days after using the sample (D28).

[0090] Test environment: Temperature: 20.0℃-22℃; Relative humidity: 40%-60%.

[0091] Test method:

[0092] 1. The CM825 skin moisture content tester is used to detect the relative moisture content of the skin surface.

[0093] 2. Skin elasticity tester The MPA580 is used to test skin elasticity.

[0094] Experimental parameters:

[0095] 1. Skin moisture content: The higher the skin moisture content value, the higher the skin moisture content and the better the moisturizing effect.

[0096] 2. Skin elasticity: The higher the R2 and R7 values ​​of skin elasticity, the better the skin elasticity and the better the anti-aging effect.

[0097] Test requirements: Perform the test 3 times and take the average value.

[0098] The results are shown in Table 11-13.

[0099] Table 11 Descriptive statistics of skin moisture content (n=30)

[0100]

[0101]

[0102] Statistical analysis results: "***": The difference was statistically significant (P<0.001); "****": The difference was statistically significant (P<0.0001).

[0103] As shown in Table 11, the skin moisture content was significantly increased after 7 days and 28 days of using Example 6, demonstrating good moisturizing effects.

[0104] Table 12 Descriptive statistics of skin elasticity R² values ​​(n=30)

[0105]

[0106] Statistical analysis results: "***": The difference was statistically significant (P<0.001); "****": The difference was statistically significant (P<0.0001).

[0107] Table 13 Descriptive statistics of skin elasticity R7 values ​​(n=30)

[0108]

[0109] Statistical analysis results: "**": The difference is statistically significant (0.001≤P<0.01); "***": The difference is statistically significant (P<0.001).

[0110] As shown in Tables 12 and 13, skin elasticity R2 and R7 were significantly improved after 7 days and 28 days of use in Example 6, demonstrating good anti-aging effects.

[0111] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A composition having moisturizing and anti-aging effects, characterized in that, The composition comprises sodium retinyl hyaluronic acid and *Sedum aizoon* seed extract, wherein, by mass, sodium retinyl hyaluronic acid is 0.08-0.14 parts and *Sedum aizoon* seed extract is 8-12 parts; sodium retinyl hyaluronic acid and *Sedum aizoon* seed extract are mixed together and dissolved in water to make up to 100 parts; The sodium retinyl hyaluronic acid is an esterified product prepared by reacting carbonyl diimidazole, retinoic acid, isotretinoin, and hyaluronic acid. Its degree of esterification is 4.0%~7.0%, the molar ratio of retinoic acid to isotretinoin bound to HA is 1.6~2.3, and the molecular weight is 5KDa~500KDa. The preparation method of the Tiankui seed extract is as follows: Tiankui seed powder is extracted by heating and reflux with 40-60% ethanol solution, the extract is concentrated under reduced pressure to a density of 1.0-1.2 g / mL, and then an appropriate amount of butanediol is added to the extract concentration to 0.05-0.12 g / mL based on the amount of raw drug, thus obtaining the Tiankui seed extract.

2. The use of the composition with moisturizing and anti-aging effects according to claim 1 in the preparation of moisturizing and anti-aging skin care cosmetics.

3. The application as described in claim 2, characterized in that, It achieves moisturizing and anti-aging effects by promoting the expression of type XVII collagen genes and the production of AQP3 protein.

4. A moisturizing and anti-aging skincare cosmetic, characterized in that, The composition comprising the moisturizing and anti-aging effects as described in claim 1.

5. The moisturizing and anti-aging skincare cosmetic as described in claim 4, characterized in that, The composition with moisturizing and anti-aging effects according to claim 1 is added in an amount of 0.1-7.0 wt%.

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