In vitro and in vivo study of the efficacy of a new sebum control essence

People with oily skin often suffer from skin problems such as oily face, blackheads, acne, and enlarged pores. It is necessary to regulate oily skin with skin care products.

sebaceous glands through the blood circulation. 10 Under the cascade of various metabolic enzymes in sebaceous cells, androgen precursors are converted into active androgen (testosterone, dihydrotestosterone DHT), and 5α-reductase can convert testosterone into DHT, which is 5-10 times more active than testosterone. 11 Through autocrine and paracrine ways, the androgen molecule binding with androgen receptor (AR) in cells increases the phosphorylation level of the target of rapamycin complex 1 (mTORC1), which stimulates sterol regulatory element binding protein-1 (SREBR-1) to increase the number of active sebaceous glands and stimulate the secretion of sebaceous glands. [12][13][14] Besides, sebaceous gland function is affected by a variety of hormones other than androgens: insulin and insulin-like growth factor-1 (IGF-1) increase sebum secretion by stimulating androgen secretion. 15,16 It has been reported that sebum secretion is not directly related to skin water content, and external sebum mixtures cannot correct dry skin problem caused by sebum deficiency. 17 It is investigated that sebum is negatively correlated with the skin's osmotic barrier.
Excessive sebum can disarrange the structure of intercellular lipid, change the cuticle barrier function, and increase the penetration of hydrophilic substances. 18,19 Therefore, people with oily skin also need to pay attention to maintain a healthy cuticle water content in daily care to maintain the stability of skin barrier function.
The common regulation solution of oily skin is to reduce the activity of sebaceous glands by inhibiting 5α-reductase, 20-22 so as to decrease sebum secretion, or to mitigate the excessive keratinization of hair follicles through acids, by dissolving the clogged oil and waste keratin in pores, so as to reduce facial oil secretion rapidly. However, these solutions cannot fully meet the daily care needs of oily skin. It is risky that the skin barrier may be damaged due to overuse of acid. 1 Thus, this study aimed to develop a multi-target essence for the regulation of oily skin, meeting the need for gentle moisturizing and oil control at the same time, and achieving reduction of sebum secretion in both short-and long-term use. According to different sebum control mechanism targets in human skin, three active compounds were selected: saccharomyces/zinc ferment, cleome gynandra leaves extract and epilobium fleischeri extract. By the combination of other auxiliary ingredients, the essence formula was further designed to solve the problems of skin dryness and ingredient compatibility.

| Formula
The stable essence formula was developed in the form of toner in consideration of sensory preference of oily skin. Cleome gynandra leaves extract, saccharomyces/zinc ferment and epilobium fleischeri extract were added to the base formula as the sebum control essence. Ingredients of base and final formula were listed in Table 1.
Both formulas have been stability-tested by centrifugation assay and heat cool cycles.

| Skin hydration
A 3D epidermal skin model (EpiKutis), was used to detect the changes in water content of the epidermal skin before and after applying the essence, so as to evaluate the moisturizing effect. Skin MTT assay was used to obtain the optimal concentration of the essence in cell experiment. SZ95 cells were inoculated into 24-well plates at the inoculation density of 3.5 × 10 4 cells/well, and cultured overnight in an incubator at 37°C and 5% CO 2 . When the cell planking rate in 24-well plates reached 60%-70%, samples was applied in groups, with 1 mL sample added to each well and three wells set in each group. After that, the culture was continued at 37°C and 5% CO 2 for 24 h.
After oil red O staining and washing operation, the cells were photographed and observed with a fluorescence microscope. The absorbance of lipids in the stained sebaceous gland cells was detected at 520 nm by an enzyme marker to obtain OD values.

| In vivo study
The in vivo study was carried out at the SGS Xiamen test center in Xiamen, Fujian Province, China. The study protocol was approved by the ethics committee of SGS clinical study. This study was conducted in accordance with the ethical principles derived from the declaration of Helsinki with Good Clinical Practices (ICH E6) and the law of local regulation clinical studies.

| Long-term sebum control test
Thirty-two eligible subjects applied the essence on their whole face twice a day. After cleansing every day, about 1.5 mL of the essence was evenly smeared on the face and gently patted until completely absorbed. The skin oil content was measured and the subject selfassessment questionnaire were required to fill before using the essence (T 0d) and after 14 days (T 14d), and after 28 days (T 28d).

| Statistical analysis
SPSS was used for data analysis and normal distribution test was conducted on the test data. If the test data were normally distributed, the T test was used for statistical analysis; if the test data was not normally distributed, the rank sum test was used for statistical analysis. p < 0.05 was considered significant.

| In vitro efficacy
The water content of the stratum corneum after applying different samples was shown in Figure 2. Compared with the BC group, the water content of PC group was significantly increased, indicating that the positive control was effective. The essence treatment significantly increased the water content of the skin model, slightly higher than 20% glycerin, indicating excellent moisturizing efficacy.

| Short-term efficacy
According to the close patch test results, the essence showed no skin irritation. After processing the skin oil content change data of 30 subjects within 8 h, the results were obtained as Figure 5. Compared with base formula, there was a significant difference in the changes of skin oil content in the areas where the essence was used, indicating that the essence could effectively control skin sebum secretion within 8 h after use, and the three active ingredients played a key role in achieving the efficacy.
According to the self-assessment results of the subjects (see Table 2), after using the essence for 4 and 8 h, the score of skin sebum secretion and skin dryness decreased significantly (p < 0.001), indicating that the subjects could obviously feel the decrease of skin sebum secretion and the improvement of skin hydration within 8 h in the half face applying sebum control essence.

F I G U R E 4
The OD values under different sample treatments.

F I G U R E 5
The average skin oil content within 8 h (* represents significant difference in oil content after using essence compared with using base formula, i.e., p < 0.05).

| Long-term efficacy
The long-term efficacy of oil control essence is also excellent. The average skin oil content of 32 subjects who used the essence for 28 days changed over time as shown in the Figure 6. Skin oil content decreased by 23.2% on average after 14 days and 30.05% on average after 28 days, compared with before essence treatment.
According to the self-assessment results of the subjects (see Table 3), skin sebum secretion and skin dryness scores decreased significantly (p < 0.001) after 14 and 28 days of using the essence.
Simultaneously, in the self-observation of the subjects, the number and area of pores, the number of blackheads and whiteheads also decreased significantly (p < 0.001). The results prove that long-term use of oil control essence can effectively control oil secretion, improve skin moisture, and reduce a series of problems caused by excessive oil secretion (such as enlarged pores, blackheads, and whiteheads).

| DISCUSS ION
This study developed an essence that can simultaneously achieve moisturizing and oil-control effects, which has been verified in vitro and in vivo experiments, short-and long-term clinical trials. In the long-term clinical trials, pore problems, blackheads and whiteheads problems have been improved to some extent, and the subjects' selfassessment showed a high satisfaction rate (over 90%).  TA B L E 3 Self-assessment results in long-term (28 days) clinical trial.

T 0 T 4 h T 8 h T 4 h T 8 h T 4 h T 8 h
inhibit the activity of 5α reductase, balance the microecology, inhibit the proliferation of harmful bacteria, promote beneficial bacteria, and inhibit the secretion of inflammatory factors at the same time. The combination of these three functional ingredients covers multiple targets for oily skin conditioning. By the other ingredients such as allantoin, glucoside, and tetrasodium glutamate diacetate, the formula was further designed to solve the problems of dryness and compatibility of the ingredients.
The essence is mild and non-irritating, suitable for daily use by oily skin people, and has significant advantages over previous sebum control products that only focus on androgen or exfoliating. It proposes a new design idea for the regulation of oily skin, that is, considering the combination of multiple skin conditioning targets, taking into account the effects of oil control and moisturizing, as well as the immediate and long-term effects while selecting active ingredients, to obtain a comprehensive essence.

| CON CLUS ION
The combination of different active ingredients in this sebum control essence improved excessive sebum secretion in oily skin and achieve rapid and persistent sebum control efficacy at the same time.

AUTH O R CO NTR I B UTI O N S
J.W. and X.M. designed the research study and performed the research, J.W. wrote the paper, X.M. analyzed the data, M.C., S.P. and Y.P. contributed essential reagents and tools.

ACK N OWLED G M ENTS
This work was funded by Hangzhou CNFormulator Technology Co., Ltd.

CO N FLI C T O F I NTE R E S T S TATE M E NT
None of the authors have a conflict of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
I testify on behalf of all co-authors that our article submitted to

Journal of Cosmetic Dermatology:
All authors 1. This material has not been published in whole or in part elsewhere 2. The manuscript is not currently being considered for publication in another journal 3. All authors have been personally and actively involved in substantive work leading to the manuscript, and will hold themselves jointly and individually responsible for its content.