Xian Zhiming,Zheng Chao,Dong Qiang

Tianjin Yumeijing Group Co.,Ltd.,China;

Ma Xue,Pan Yao,Zhao Hua

Beijing Technology and Business University,China

Abstract The moisturizing effect of a fresh-milk cream after single and long-term application was studied by the combination of subjective and instrumental methods.The purpose of this study was to evaluate the moisturizing efficacy of fresh milk as a moisturizer in skin care products.Thirty female subjects aged 18～55 years were enrolled and a randomized,double-blind,placebo-controlled comparison study was applied in this research.The stratum corneum water content and transdermal water loss on the cheek,and VISIA-CR images of the whole face were recorded after using the samples.The subjective evaluation of the subjects were also collected.The results showed that the fresh milk cream played a significant role in increasing the water content of facial stratum corneum,improving the transdermal water loss and skin redness compared with the base formula without fresh milk.It indicated that fresh milk can be added to skin care products as a moisturizing agent,which can replenish moisture and enhance the skin barrier function.

Key words fresh milk; moisturizing; efficacy evaluation

The skin is the largest organ of the human body.It covers the surface of the body and isolates the inside of the body from the external environment.The skin is the first line of defense of the human body,which not only prevent the invasion of external chemical,physical or biological harmful factors,but also prevent the loss of nutrition material and water from the body.Maintaining skin hydration is the basis for keeping the skin in good condition,which can delay skin aging,prevent the occurrence of skin diseases.[1]

Milk is a nutritious substance,contains proteins,fats,amino acids and trace elements,[2]and has the effect of skin care.[3]The casein contained in milk protein can be added to cosmetics as a moisturizer,nutrient and conditioner,while increasing the efficacy of other active ingredients.[4]Colostrum is rich in milk fat and fat-soluble vitamins.[5]Milk fat are a mixture of triglycerides composed of one glycerol molecule and three fatty acid molecules.Conjugated linoleic acid is one of the physiologically active substances of milk fat and has a variety of physiological functions.[6]Conjugated linoleic acid is close to the fatty acids which was secreted by human’s sebum,and has the effect on transdermal absorption.It plays a role in caring the skin through reducing the evaporation of skin moisture,improving the skin’s natural barrier function and protecting the skin from environmental damage.[7]

In this research,we investigated the moisturizing efficacy of a cream containing fresh milk in vivo through analyzing the change of stratum corneum water content and transdermal water loss rate of subjects’ cheek,face image of subjects and the subjective evaluation of subjects.And the results indicated that the fresh milk is effective as a moisturizer in the formula.

1 Experimental section

1.1 Reagents and Instruments

Fresh-milk Cream (the other main ingredients are glycerin,mineral oil,etc.) and control sample (no fresh milk,other ingredients are same with fresh-milk cream)were provided by Tianjin Yumeijing Group Co.,Ltd..

Corneometer CM825 and Tewameter TM300,Courage ＆ Kahazaka,Koln,Germany; VISIA-CR,Canfield Scientific Inc,Fairfield,NJ,USA.

1.2 Subjects

Thirty subjects were enrolled with the following rules:healthy female with dry skin and desquamation,self-reported skin sensitivity,no history of cosmetic allergy,aged 18 to 55 years old.Exclusion criteria:Participate in other clinical trial studies at present or before the beginning of this study; those who are in pregnancy,lactation or within half a year after delivery;taking corticosteroids,antibiotics,tretinoin or other drugs in the past 2 weeks or anti-acne drug treatment;subjects suffering from uncured active skin diseases;the test area has undergone the therapy of laser beauty or chemical peels.This study complies with the provisions of the Declaration of Helsinki revised in 2013.All subjects understood the purpose and process of the research and signed an informed consent form.

1.3 Experiment

1.3.1 Test method

This study was a randomized,double-blind,placebo-controlled study for 30 women.Subjects were randomized to receive twice a day 0.3 g of fresh-milk cream on one randomly selected side of their face and control sample on the other.The subjects must use the corresponding sample on half of the face after daily facial cleaning.The study lasted for 28 days.During the study,it is forbidden to use other similar products.

After cleaning face,the subjects should acclimated for 30 minutes in the testing room.Then,measurement of TEWL,water content in the stratum corneum and the face image at different time were taken (prior to starting samples application and 30min,1h,3h,6h,14d,28d after samples application).During the test,the temperature of environment was maintained at 20.0～22.0 ℃,and relative humidity was maintained at 40.0～60.0%.At the same time,the subjects filled out questionnaires on the improvement of their skin after fresh-milk cream application.Scoring criteria:1-improvement is very insignificant; 2-improvement is not obvious; 3-improvement is relatively obvious; 4-improvement is very obvious.

1.3.2 Test items

Facial image was measured by VISIA-CR facial image analysis system (Canfield Scientific Inc,Fairfield,NJ,USA).Facial images of subjects from different angles (front,left and right sides at 45 ℃ direction) were collected and used for clinical evaluation of skin characteristics and skin color.

Water content in the stratum corneum was measured on the cheeks using a Corneometer? CM 825 stratum corneum water content measurement device (Courage ＆ Khazaka electronic GmbH).Measurements were conducted 5 times in the same location,and the mean was used.

TEWL was measured on the cheeks using a Tewameter TM300? measurement device (Courage＆ Khazaka electronic GmbH).Measurements were conducted 3 times in the same location,and the mean was used.

The questionnaires of the subject’s skin condition and sample’s efficacy were carried out before starting samples application and 6 hours,14 days and 28 days after samples application.

1.3.3 Safety evaluation

The adverse reactions during the 28-days study were investigated.The adverse events occurred during the sample application,such as erythema,infiltration,papules,edema,blisters,bullae and other symptoms on the skin were interrogated,inspected and recorded.

1.4 Statistical analysis

IBM SPSS Statistics (version 21.0) was used for statistical analysis.The Shapiro-Wilk Test is used for testing the normality of the data,pvalue of >0.01 means the normally distributed.Repeated measure ANOVA is used to compare the difference between the data of each group at different time points.The change rate (R) of the experimental group and the control group was compared by paired sample T-test.Apvalue of <0.05 was considered significant.The change rate (R) was calculated using the following equation:

R = (Valuetime-Value0)/Value0×100%

2 Results and Discussion

A total of 30 subjects were enrolled in the study and the average age was 24.06 ± 0.33 years.All subjects completed the study.The study is from October 17 to November 13 in 2019.

2.1 Water content in the stratum corneum

The change of water content in the stratum corneum during the study was shown in Figure 1.Before the samples application,the skin moisture content of experiment group and control group were 55.59±1.82 AU and 56.26±1.75 AU respectively,and there was no significant difference between the two groups (Fig.1A).It can be seen from the data of the short-term test (within 6 h after samples application),the value of both groups were increased significantly compared with the base value (p<0.001).The value of the experiment group were increased to 72.73 ± 2.11 AU,71.90 ± 1.71 AU,66.93 ±2.15 AU,64.72±2.03 AU at 30 min,1h,3h and 6h,and R were 30.83%,29.34%,20.40%,16.42%.Furthermore,the change rate of the experiment group was significantly higher than that of the control group at all the time point (Figure 1B),and the difference was statistically significant (p<0.05).After the long-term application of fresh-milk cream,the skin moisture content increased from 55.59 ± 1.82 to 56.75 ± 1.81 AU at 14 d and 63.10 ± 1.56 AU at 28 d.Compared with the base value,the values are significant differences (p<0.001),and the change rate were 7.47% and 13.51%,respectively.A comparison between the groups showed that the experiment group significantly improved the skin moisture content versus the control sample (p<0.001).In the short-term test,the skin moisture content of the control group was significantly higher than the basic value,but the difference disappeared at 14 d.

Figure 1.Changes of stratum corneum hydration values:A.change trend of stratum corneum hydration (compared with baseline,＊ p < 0.05,＊＊p< 0.01,＊＊＊p < 0.001); B.change rate of stratum corneum hydration (compared with control group,＊ p < 0.05,＊＊p< 0.01,＊＊＊p < 0.001)

The results showed that both samples can significantly increase the moisture content of subjects’cheek no matter in short-term or long-term test,and the effect of fresh-milk cream was better than the control sample.It showed that fresh milk as a moisturizer can increase the moisture content of the skin stratum corneum.

2.2 TEWL

The changes of TEWL were shown in Figure 2.The initial value of TEWL in experiment group is 18.97 ± 0.65 g/(h·m2),18.11±0.62 g/(h·m2) in the control group,and no significant difference was observed between two groups.Compared with the base value,TEWL in experiment group was significantly decreased to 15.02 ± 0.57 g/(h·m2),14.86 ± 0.79 g/(h·m2),15.96 ± 0.52 g/(h·m2),15.27 ± 0.53 g/(h·m2),at 30 min,1 h,3 h and 6 h (p<0.001) in short-term test.The change rate were 20.82%,21.67%,19.35%,19.50%.A comparison between the groups showed that the experiment group significantly improved TEWL compared to the control group (p<0.05).In addition,TEWL in long-term test decreased to 15.25 ± 0.53 g/(h·m2) at 14 d,and 14.16 ± 0.42 g/(h·m2) at 28 d,and there was significantly different compared with base value (p<0.001).The change rate of TEWL in experiment group were 19.61% at 14 d and 25.36% at 28 d.The change rate of TEWL between two groups showed a significant difference (p<0.001) in longterm test.

Figure 2.Results of transepidermal water loss values:A.change trend of transepidermal water loss (compared with baseline,＊＊＊p < 0.001),B.change rate of transepidermal water loss (compared with control group,＊ p < 0.05,＊＊p< 0.01,＊＊＊p < 0.001)

The result indicated that TEWLs of both groups were significant decreased in short-term and longterm test.And the effect of fresh-milk sample is better than the control sample.It indicated that cream containing fresh milk have good effect on barrier function enhancement.

2.3 Facial images

Figure 3 showed that the VISIA-CR images of the two subjects before and after samples application.As shown in Figure 3A and 3B,there was no significant different was observed after using the control sample.The redness of the cheek has not improved.In the contrast,after long-term using cream containing fresh milk.The redness of the cheek significantly reduced,and the area of redness reduced.As a result,the cream containing fresh milk was significantly better than the control sample without fresh milk in improving skin redness.

Figure 3.VISIA-CR image analysis of skin redness before and after samples application

2.4 Subjective evaluation

The result of subjects’ evaluation after freshmilk cream application was shown in Figure 4.The skin dryness,desquamation,moisture retention and the skin barrier function was evaluated.Most of the subjects felt that the skin dryness and desquamation were improved after the application of the freshmilk cream.At the same time,the skin water-locking capacity and barrier function have also been improved.

Figure 4.Results of subjective evaluation

2.5 Safety assessment

During the study,all subjects did not have any adverse reactions,and they had good tolerance to creams containing fresh milk.With reference to the evaluation standards for skin adverse reactions in human trial tests specified “Cosmetics Safety Technical Specifications",the fresh-milk cream have good safety.

3 Conclusion

Moisturizing effect is the basic function of skin care products.The method for evaluating the moisturizing effect of cosmetics requires objectivity,accuracy,and science.In this study,we evaluated the moisturizing effect of fresh milk in formula by instrumental and subjective methods.The results showed that water content in the stratum corneum and TEWL were significant improved after application the fresh-milk cream in short-term and long-term test.The results of the visual image and the subjective evaluation showed that fresh milk has a good effect on improving the skin problems,such as dryness,desquamation,erythema,and poor water retention.In addition,there was no adverse reactions were observed after the fresh-milk application,which indicated that the skin care products containing fresh milk have good safety.In summary,the fresh milk has the effect on moisturizing and enhancing skin barrier function.Skin care products containing fresh milk can be used as moisturizing products in daily skin care.