Poly traditional Chinese medicine formulation prepared with skin moisturizing properties

Many traditional Chinese medicine compositions can moisturize the skin and utilize in cosmetics. Using a combination of Chinese Medicine Materials and guided by Traditional Chinese Medicine principles, this study selected Echinacea purpurea to protect the skin barrier, Dendrobium nobile to clear heat and promote fluid production, Sophora flavescens to clear heat for diminished inflammation, and Aloe vera combined Lycium barbarum to nourish yin, to together form a “poly TCM moisturizing formulation.” These poly plant extracts were investigated and optimized for the stability, safety, and moisturizing ability. The combination moisturizing effect was determined by measuring the expression of FLG mRNA, CLDN‐1 mRNA, and AQP3 protein. Toxicological analysis included a red blood cell hemolysis test and a 3T3 phototoxicity test. It has been observed that by using polysaccharide yield as the evaluation criterion showed optimal extraction at a material‐to‐liquid ratio of 1:100, an extraction temperature of 100°C, and an extraction time of 3 hours. Moisturizing effect experiments showed that the expression of FLG mRNA, CLDN‐1 mRNA, and AQP3 protein was significantly increased. Toxicological tests showed that the composition was safe and caused no irritating effects. Based on these results, this poly traditional Chinese medicine moisturizing formulation is safe within moisturizing effects and can be used as a moisturizing raw material in cosmetics.

As the original medical science of the Chinese nation, traditional Chinese medicine (TCM) can reveal the laws of human health and disease occurrence from a macroscopic, systematic and holistic perspective and embody the cognitive style of the Chinese nation, and it has been deeply integrated into the productivity and lives of people, forming a unique health culture and practice. As the core elements of the basic process of TCM diagnosis and problem solving, "symptom, reason, method, prescription, medicine, efficacy" is an approach that is applied consistently throughout the research and application of skin care products. 2 In accordance with the principle of "monarch, minister, assistant and guide" in Chinese medicine, Echinacea purpurea Linn. solid water protects the skin barrier (monarch), Dendrobium nobile Lindl. clear heats and promotes fluid production (minister), Sophora flavescens Ait. clears heat to reduce inflammation (assistant), and Aloe vera L. and Lycium barbarum L. nourish body's essential fluid to together form a "plant moisturizing composition." The plant moisturizing composition uses E purpurea as the monarch and uses it to retain water to protect the skin barrier. Echinacea purpurea is an excellent immune promoter and immunomodulator, and its abundant polysaccharide components (such as 4-methoxyglucaldehyde-arabinose-xylan) exhibit significant enhancement of humoral immune function. 3 It is used as a treatment for trauma, eczema, and so on, and it improves skin barrier function and skin immunity. 4 Dendrobium nobile is commonly known as "millennial nourishment" and is a traditional rehydration herb. Modern studies have shown that D nobile polysaccharide not only has an exogenous moisturizing effect but also promotes the expression of aquaporin 3 in epidermal keratinocytes. 5 Therefore, the plant moisturizing composition takes D nobile as the minister and uses its rehydration and fluid effects.
From the perspective of skin physiology, inflammatory factors and other "hot poisons" speed up water loss, causing endogenous water to be transported to the stratum corneum of the skin surface and thus making the skin dry and dehydrated. Sophora flavescens is a traditional heat-clearing medicine. According to pharmacology, S flavescens has the effect of "heat-treating toxic wind, skin muscle irritating sores". 6 Therefore, the plant moisturizing composition here is based on bitter ginseng, which clears heat and reduces inflammation. 7 A barbadensis has been a skin care product since ancient times.
Some amino acids and metal salts contained in A vera are the same as the natural moisturizing factors contained in human skin, making its moisturizing properties prominent. 8  Materia Medica" states that sweet in flavor and mildly moist in property and is a good product for nourishing yin and health. L barbarum is rich in nutrients such as carotene, vitamin A, calcium, and iron. It can activate skin cells, promote cell metabolism, improve skin's water retention, and help the skin remain full, smooth and delicate. 9 The plant moisturizing composition here includes A vera and L barbarum.
Echinacea purpurea solid water barrier in the prescription can solve the problem of barrier repair in dry skin, reducing skin water loss. 10 D nobile can replenish skin moisture and promote endogenous water production and transport. S flavescens can eliminate inflammatory factors that inhibit endogenous water transport in skin. A vera and L barbarum maintain the skin oil-water balance and nutritional balance by nourishing yin.
This investigation found that the plant composition has a good moisturizing effect. This research will study the extraction process, moisturizing effect and safety of this plant moisturizing composition, aiming to develop new raw materials to increase the moisturizing efficacy of cosmetics.

| Plant material collection and extraction process optimization
In this study, the whole grass of E purpurea (Qianhuyuan, Yunnan,

| Preliminary qualitative phytochemical analysis of plant extracts
The study measured the polysaccharide content of the extract as an index to optimize the extraction process.

| Test for polysaccharide
Take 1 g of sample, add 1 mL 15% TCA solution, add a little 5% TCA solution, pour the supernatant into a 10 mL centrifuge tube, add a little 5% TCA solution to grind, pour the supernatant, and repeat three times. Centrifuge at 3000 rpm for three times. After adding 2 mL of 6 mol/L hydrochloric acid to the colorimetric tube, shake well, and then water-bath in a 96 C water bath for 2 hours. After the water bath, cool with running water and add 2 mL of 6 mol/L sodium hydroxide to shake. Make up to a 25 mL volumetric flask. Pipette 0.2 mL of the sample solution, make up to 2.0 mL by distillation, then add 1.0 mL of 6% phenol and 5.0 mL of concentrated sulfuric acid, shake and cool for 20 minutes at room temperature and measure the optical density at 490 nm. Take two samples for each measurement.
The polysaccharide content was calculated using a standard curve.
The total polysaccharide content was detected. 10-13

| Stability investigation
The cosmetic plant composition was required to be strictly stable. The various conditions for testing the stability of a stock solution included room temperature (placed on the laboratory table top), protected from light (placed in a 28 ± 1 C incubator cassette), illumination (in a 28 ± 1 C constant temperature incubator placed under light), heat (placed in an oven at 45 ± 1 C), alternating hot and cold (ie, a 28 ± 1 C and 4 ± 1 C alternating hot and cold incubator), refrigeration (placed in a 4 ± 1 C refrigerator fresh-keeping layer), and freezing (placed in a − 20 ± 1 C freezer layer) . . 14

| Cell culture of keratinocytes
Keratinocytes were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere with 5% CO 2 at 37 C.
The medium was replaced every 2 days.

| MTT detection of keratinocytes
This test uses the MTT cell activity assay to screen for the maximum safe dose of the sample in keratinocytes. To ensure the validity of the MTT test results, 4% DMSO was used as a positive control (PC). The sample was set up with a gradient of eight concentrations, and three replicate wells were set for each concentration. A solvent control (SC) and a zero well (cell culture) were set up. 15

| Detection of FLG and CLDN-1 cell immunofluorescence
Cells were seeded in six-well plates and incubated overnight at 37 C in a CO 2 incubator. The sample was diluted with DMSO to prepare a plant moisturizing composition at 125, 250, and 500 ppm. When the plating rate of the six-well plate reached 40% to 50%, group administration was performed, and each group was provided in three duplicate wells. A blank control group (1‰ DMSO) was added to the keratinocyte culture medium, and the sample group was added to keratinocyte culture medium containing the corresponding concentration of the test substance. Culture was continued for 24 hours at 37 C in a 5% CO 2 incubator. The culture solution was discarded, 1 mL of TRIzol was added per well, the lysed cells were pipetted, and then a sample was taken. RNA was extracted and reverse transcribed into cDNA, and then quantitative PCR detection was performed.

| Detection of AQP3 cell immunofluorescence
Cells were seeded in 24-well plates and incubated overnight at 37 C in a CO 2 incubator. The sample was diluted with DMSO to prepare a plant moisturizing composition at 125, 250, and 500 ppm. When the cell plating rate in the 24-well plate reached 40% to 50%, group administration was performed, and three duplicate wells were set in groups. The blank control group was supplemented with keratinocyte culture medium containing 1‰ DMSO, the sample group was supplemented with keratinocyte culture medium containing the corresponding concentration of the test substance, and culture was continued for 24 hours at 37 C in a 5% CO 2 incubator. After incubation, the medium in the 24-well plate was discarded, and the slides

| Erythrocyte hemolysis test
Stimulation of the red blood cell membrane broke the red blood cell membrane, causing a certain degree of hemolysis. The absorbance of the red blood cell suspension after the action of chemical substances was measured by spectrophotometry, and the hemolysis rate was calculated. Among the tested chemicals, 0.02% sodium dodecyl sulfate (SDS) is potent as a PC for this test. 16 2.11 | Sample preparation for the 3T3 neutral red test The plant moisturizing composition was formulated into solutions of eight concentrations (7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0, and  1000.0 μg/mL) with Hank's balanced salt solution (HBSS). Chlorpromazine (CPZ) hydrochloride was dissolved in absolute ethanol to prepare the highest-concentration mother liquor (20.00 mg/mL), which was then diluted to different concentrations with 50% absolute ethanol. The concentration was 0.02 mg/mL, and each solution was diluted 100 times with HBSS before use and standby application.

| Cell inoculation for the 3T3 neutral red test
Well-grown Balb/c3 T3 cells were inoculated in the wells of a 96-well culture plate (except for the peripheral wells) according to the guidelines of OECD432 so that the number of cells per well is 1.0 × 10 4 , and then the plate was placed at 37 ± 1 C and 5 ± 1% CO 2 for approximately 24 hours to humidify the culture.

| Dosing and lighting for the 3T3 neutral red test
After culturing of Balb/c 3 T3 cells for 24 hours, the culture solution was removed, and the cells were gently washed twice with 150 μL of prewarmed HBSS. Then, 100 μL of each test substance at different concentrations was added to each well. The control substance was HBSS, and there were six duplicate wells per concentration and two plates for each test substance and the PC, which were labeled "light plate" and "no light plate," respectively. The culture plates were incubated at 37 ± 1 C and 5 ± 1% CO 2 for 1 hour, and the light plates 100 IU penicillin and 100 μg/mL streptomycin, and the cells were cultured at 37 ± 1 C and 5 ± 1% CO 2 for 18 to 22 hours.

| 3 T3 neutral red test
Approximately 3 hours before the end of culture, the medium of each well was discarded, the cells were gently washed once with 150 μL of prewarmed HBSS per well, and then 100 μL of 50.0 μg/mL neutral red was added serum-free per well. DMEM was cultured for 3 hours at 37 ± 1 C and 5 ± 1% CO 2 . After the completion of the culture, the medium-containing neutral red was discarded, and the cells were

| Extraction process optimization
From a range analysis (Table 1), it was concluded that the ratio of material to liquid has the greatest influence on the total polysaccharide content of the extract, the influence of the extraction temperature is second greatest, and the influence of the extraction time is the smallest. In summary, the total polysaccharide content was taken as the parameter to be optimized. The optimum extraction ratio was 1:100, the extraction temperature was 100 C, and the extraction time was 3 hours.

| Results of the keratinocyte MTT assay
The MTT assay results ( Table 2) indicate that the maximum safe concentration of the plant moisturizing composition for keratinocytes was 500 ppm.

| FLG and CLDN-1 cell immunofluorescence
The plant moisturizing composition was set at three concentrations (125, 250, 500 ppm) based on a dose of 500 ppm, and fluorescence quantitative PCR was carried out. The variation trend of the sample at different concentrations is shown in Figures 1 and 2. Figures 1 and 2 show that at the genetic level, the plant moisturizing composition had a significant effect on the expression of FLG and CLDN-1 at doses of 250 ppm and 500 ppm.

| AQP3 cell immunofluorescence
The results showed that the average fluorescence intensity of AQP3 protein expressed by keratinocytes after plant moisturizing composition treatment was significantly higher than that of the blank control group. 18 An immunofluorescence image of the AQP3 protein is shown in Figure 3. The results showed that at the protein level, the plant moisturizing composition significantly improved the expression of AQP3 at a dose of 500 ppm.

| Results of the erythrocyte hemolysis test
The erythrocyte hemolysis test results prove that the plant moisturizing composition has a very low red blood cell hemolysis rate at concentrations below 10% and no stimulating effect (Table 3).

| Results of the 3T3 neutral red test
The judgment criteria are shown in Table 4

| DISCUSSION
The plant moisturizing composition was optimized through a small number of experimental procedures. The optimal conditions were as follows: the material-to-liquid ratio was 1:100, the extraction temperature was 100 C, and the extraction time was 3 hours. According to the production process for this small sample, the scale of a pilot test was enlarged, and trial production proved that the process is feasible and the quality of the produced product meets its requirements, which is basically consistent with the indicators from the small sample.
By  reactions. The plant moisturizing composition can be used as a moisturizing raw material in cosmetics.

| CONCLUSION
Therefore, to solve the problem of dry skin, we must pay attention to repairing the skin barrier and reducing skin inflammation while replenishing the moisture content of the skin. In addition, it is necessary to increase the amount of skin oil and fat and adjust the skin water and oil balance. From the perspective of modern mechanisms, the plant moisturizing composition mainly solves the problem of skin dryness by maintaining skin barrier function, increasing endogenous water production and promoting endogenous water transport. From the perspective of traditional Chinese medicine, there are four ways to solve the problem of dry skin: "solid water screen," "rehydration," "heat-clearing and anti-inflammatory," and "nurturing and nourishing yin" activities.

CONFLICT OF INTEREST
The authors declare that they have no conflicting interests.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are also available from the corresponding author upon reasonable request.