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Skin aging
Humans experience aging in various tissues as they get older, and skin aging is the most sensitive aging phenomenon among the various aging phenomena of the human body because it is most clearly visualized and recognizable by themselves and others. The causes of skin aging can be largely divided into endogenous and exogenous factors. Endogenous factors are mainly genetic factors primarily determining changes in skin cell and tissue structure. For example, tTelomere is a structure located at the end of eukaryotic chromosomes. As the cells age physiologically, the shortening of telomeres occurs, which is known to affect skin aging by affecting the tissue dropout rate.[1]
Extrinsic factors are externally derived factors such as solar ultraviolet rays, smoking, excessive drinking, malnutrition, and stress. Among these exogenous factors, solar UV rays account for more than 80% of skin aging factors, and UV-induced aging is called photoaging.[3] [4] The continuous exposure of the skin to UV promotes the production of reactive oxygen species(ROS) in the skin cell, causing oxidative stress, thus promoting the expression transcription factors of the Activator protein-1, (AP-1) which is involved in cell transformation, growth, differentiation and apoptosis. It is known that promoting them results in inducement of matrix metalloproteinases(MMPs) to destroy collagen, one of the most important component of extracellular matrix(ECM). In conclusion, disassembling collagen leads to changes in skin structure to cause skin aging. [5]~[10]
DNA recovery system for gene preservation.
All organisms have a DNA recovery system to preserve their genes, of which nucleotide excision repair (NER) and base excision repair (BER) to repair photo- damage caused by ultraviolet light. According to how the NER recognizes DNA damage, It is divided into global genome repair (GGR) and transcription coupled repair (TCR). Through these two paths, different proteins are involved in the early stages of DNA damage, and subsequently repair damaged DNA through common stages such as DNA damage identification, double helix release, and damaged strand cutting.[11][12]
Microalgae
Algae, which has a wide range of taxonomically diverse origins, is morphologically divided into single-cell type, colony type, filamentous type, foliated type, and giant multicellular types and its size ranges from microalgae to macroalgae such as seaweed and kelp. Microalgae contain pigments such as chlorophyll, carotenoid, and phycobilins whose cell growth and reproduction is performed through photosynthesis and are known to vary in type or number.[14] It also has explosive propagation power under environmental conditions and is easy to get industrialized because it has relative abundance of useful materials in its body, such as vitamin, carotenoid, polysaccharides, and It can serve as component of bio industries such as hydrogen, hydrocarbons and bio-fuels with low cost and mass production.[15]-[17] Also, Microalgae is also known for its protective instruments through the formation of DNA recovery systems such as NER and ultraviolet absorbers, which enhance resistance and adaptability to ultraviolet rays to avoid damage to cells caused by ultraviolet rays.[18]-[22]
DREAM(novel DNA Repair Regulating Material Discovery) system
Ultraviolet rays are the main cause of skin aging, and these ultraviolet rays are known to increase DNA damage to skin cells, including fibroblasts. By causing genetic mutations in the thymine dimer and cytosine → thymine(C→T), the accumulation of these mutations can develop into skin cell aging and skin malignancy. Therefore, the assessment of the degree of damage to skin cell DNA by ultraviolet rays should be able to quantitatively assess whether thymine dimensions and C→T generate or not. We intend to develop a new assessment method to easily assess it.
To this end, we established modified lentivirus (HPRT) expressing Luciferase and Hypoxanthine phosphoribosyl transferase (HPRT) gene. DREAM-F cells capable of stably expressing luciferase and HPRT were established by infecting human dermal firoblasts WS-1 cells with the modified lentivirus and selecting them with puromycin.[23] As the first step of the DREAM system, as a 96-well-based screening step, about 300 kinds of microalgae extracts provided by the Korea Research Institute of Bioscience and Biotechnology were pre-treated into DREAM-F cells and subjected to UV light B(UVB). After that, cell viability and luciferase activity were analyzed to perform UV protection effect and UVB and then he UVB-induced damage repair effect was confirmed by treating the extract.
In the second step, we evaluated cell cycle analysis and apoptosis of microalgae extracts identified in the first step and DREAM-F cells treated with UVB, and conducted HPRT-DNA sequencing to validate specific extracts identified in the first step (Figure 1).
Anti-wrinkle and anti-aging effects of skin cells induced by UV rays.
To evaluate the irritability of skin cells on selected Scenedesmus Deserticola JD052 extracts through the DREAM system, WST-1-based cell survival tests, inflammatory cytokine(interleukin-1βIL-1β) and interleukin-6IL-6 realtime polymerase chain reaction (qRT-PCR) analysis were performed. As a result, JD052 extracts showed relatively low cytotoxicity and no secretion of inflammatory cytokines IL-1 and IL-6. In conclusion, JD052 extracts had little irritation in the skin cells (Figs. 2A, 2B).
The anti-wrinkle efficacy evaluation for JD052 measured the amount of collagen and collagenase expression through the test method specified in the wrinkle improvement efficacy test guidelines of the Ministry of Food and Drug Safety in Korea.
As a result, JD052 extracts are confirmed to induce high expression of collagen gene COL1A1(Alpha-1 type I collagen gene) and COL3A1's(Alpha-1 Type III Collagen gene) mRNA(Messenger RNA) and suppress expression of Collagen degrading enzyme, MMP-1(Matrix metalloproteinase-1 gene) mRNA. It concludes that JD052 extracts has anti-wrinkle efficacy by enhancing collagen expression with inhibition of collagenase (Figs. 2C, 2D).
Skin aging can be divided into natural aging as you get older and photo-aging due to ultraviolet rays. In particular, photo-aging has the characteristic of inhibiting aging if the effects of skin cells caused by ultraviolet rays are controlled.
Therefore, we checked whether the JD052 extract could inhibit photo-aging by ultraviolet rays. As a result, the JD052 extract inhibits the reduction in cell survival rate caused by UVB(Figure 3A).
In addition, the most representative effect of UVB on skin cells is the increasing the level of active oxygen. Using JD052 extracts, dichlorofluorescin diacetate (DCF-DA) tests were conducted to determine changes in the concentration of active oxygen in skin cells, and JD052 extracts were found to significantly reduce active oxygen level increased by UVB (Figure 3B).
Based on the results above, It is confirmed that the growth and aging of skin cells and DNA damage caused by ultraviolet rays were controlled by JD052 extracts. Experiments have shown that although the growth power of skin cells has decreased significantly by UV rays, growth inhibition by UV rays was controlled at skin cells pre-treated with JD052(Figs. 4A, 4B). Experiments using the aging marker of cells, beta-galactosidase(beta-gal), have shown that Beta-gal positive cells increased by more than 3 times compared to the control group by ultraviolet light, but in the cells pretreated with JD052, it was significantly reduced to the control level and also the expression of the aging molecular indicators in cells, p21 and p16, were reduced by JD052 extracts (Fig. 4C, 4D).
Furthermore, the results of Thymine dimer blotting to measure DNA damage caused by ultraviolet light confirmed that the JD052 extract inhibits the formation of thymine dimer by ultraviolet light (Fig. 4E).
Measuring COL1A1, COL3A1, MMP1, and MMP3 mRNA expressions by qRT-PCR showed that UV rays significantly inhibited the expression of COL-related genes, while enhancing MMP related gene's expression.
However, JD052 treated groups demonstrated significant inhibition of UV-induced effects.(Fig. 5)
JD052 Extracts provide UV-damaged skin irritation calming effects and
It showed excellent effects on skin elasticity, wrinkle improvement, and dermis moisturization through improved dermis density.
Clinical trial evaluation
Using the extract of JD052 whose efficacy ,the ability to protect skin cells from UV rays and the ability to recover cells damaged by UV ray, confirmed through results above, its potential was to be confirmed as a cosmetic raw material for anti-inflammatory UV damage protection.
To this end, a formula (toner, lotion, cream) containing JD052 extracts was applied to subjects of women older than 29 for 4 weeks to verify the effectiveness of skin irritation, dermis density, wrinkles and dermis moisturization improvement by UV rays, and human efficacy assessment was conducted by the Korea Dermatological Research Institute (KISCS-AFH023-RBO).
Using Solar simulator (Multiport Simulator 601-300W, Solar Light Company, Inc., USA) and ANTERA 3D (Miravex, Ireland), Improvement of soothing effect on skin irritation caused by UV rays on left upper arm was analyzed. It showed the change in a* value, a indicator of skin redness. Compared to before applying test substance, a* value showed decrease of 5.30% in the coated area, 1.68% in the uncoated area after 2 weeks and after 4 weeks It showed decrease of 5.54% in the coated area, 3.59% in the uncoated area(Figure 6).
In addition, compared with the Δa* value after 2 weeks and 4 weeks of use of the test substance-free site, the Δa* values after 2 weeks of use and 4 weeks of use at the application site were 0.024 and 0.031, respectively, which were statistically significant (p<0.05) It was confirmed that the test substance helps to soothe skin irritation caused by UV rays.
Using DUB-Skin Scanner (Taberna promedicum, Luneburg, Germany), the improvement in dermal pitch density at the area 3 cm next from the left eye corner were 12.08% after 2 weeks and 21.15% after 4 weeks after applying test substance. In addition, the density value after two weeks of use and after four weeks of use compared to before the test material was applied to be statistically significant (p<0.001) and the test material was found to help improve the dermal pitch density (Figure 7).
An analysis of wrinkle improvement in the right eye tail using ANTERA 3D showed that the wrinkle small value, a marker for shin wrinkles, of the skin was reduced by 5.88% after two weeks of use and 8.35% after four weeks of use compared to before the test material was used. It was also shown statistically significant after two weeks of use and after four weeks of use compared to before the test material was used (p<0.01, p<0.001) and the test substance was found to help improve wrinkles (Figure 8).
Using MoistureMeterD Compact (Delfin Technologies Ltd., Finland), analysis of dermal moisturization improvement on the left cheek showed a 23.54% increase in dermal moisture immediately just after single usage, 4.32% after two weeks of usage , and 6.48% after four weeks of usage. It was also confirmed that the test substance was statistically significant immediately after single usage, two weeks of usage, and four weeks of usage (p<0.001) compared to before using of the test substance the test substance was found to help improve wrinkles (Figure 9).
The survey of the subjects showed no particular skin abnormalities to erythema, edema, phosphorus, itching, pain, burning sensation, stiffness and tingling (Table 1).
These results confirmed through clinical trial that JD052 extract has an excellent effect in soothing skin irritation caused by UV rays and improving skin elasticity, wrinkles and dermal moistureand through improvement of dermal density.
Heterotroph for microalgae mass cultivation
To commercialize microalgae, above all in bulk, It is important to have a cultured system. For mass culture of JD052 selected through this study, subculture using a microbial incubator was performed. To this end, JD052 was cultured under the conditions of phototroph, mixotroph, and heterotroph to produce biomass extracts to compare productivity, ultraviolet protection and recovery activeness for skin cells. As a result, microorganisms in heterotroph and mixotroph conditions has approximately three times more productivy compared to photosynthetic cultures, and for cell survival tests, they did not differ significantly under three incubation conditions (Figs. 10, 11).
A value excluding the control value from the relative cell survival is defined as the relative activity unit, RAU, and RAU productivity using the cell productivity of each culture condition was calculated. As a result, RAU was shown to be phototroph culture 21.46, mixotroph culture 17.20, and heterotroph culture 19.30. Based on those numbers, RAU productivity was calculated showing phototroph culture 6, mixotroph culture 16, and heterotroph culture 16 to conclude that other two conditions recorded approximately 2.7x increase in productivity over photosynthetic culture (Figure 12). Therefore, the cultivation of microalgae by heterotroph cultures is highly productive and easy to produce compared to phototroph cultures, which will greatly benefit the industrialization of microalgae, including JD052.
DESERTICA-Anti-Aging Material
In this study, the world's first system excavating new controling substance of UV-dependent skin cell DNA damage control, the DREAM system, was developed and JD052 microalgae extract, selected through this, to confirm the anti-wrinkle and anti-aging effects of skin cells induced by UV rays. Through clinical trials, it was confirmed that it helps to soothe skin irritation caused by UV rays, improve dermal density, improve wrinkles, and improve dermal moisture. In addition, as a mass production method necessary for the industrialization of JD052, heterotroph culturing technique was established. Based on these results, DESERTICA, a raw material containing JD052 extracts, was developed to protect skin cells from ultraviolet rays and to identified its potential as an anti-photoaging cosmetics material that shows the effectiveness of DNA recovery of skin cells.
Acknowledgements
This research is the result of a study conducted by the Ministry of Health and Welfare under the 'Global Cosmetics New Material, New Technology Research and Development Support' project (task number HN13C0080).