Epigallocatechin gallate (EGCG) belongs to the catechin group of substances and is a polyphenol obtained from green tea (Camellia sinesis L. Ktze (Theaceae). In addition to EGCG, which makes up just under 50% of the catechin content, green tea also contains other catechins such as epicatechin-3-gallate (ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC) and (+)-catechin. The antioxidant and anti-inflammatory effects of EGCG and its effects on proliferation, differentiation and apoptosis have been well researched.
The underlying study investigates the mechanisms by which epigallocatechin gallate (EGCG) improves skin hydration. For this purpose, the gene expression of HAS (hyaluronic acid synthase) and HYAL (hyaluronidase) as well as the anti-oxidative activity and anti-pigmentary properties were analyzed by Western blotting analyses, luciferase assays, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays and reverse transcription polymerase reaction (RT-PCR) analyses. RT-PCR studies showed that EGCG increased the expression of the natural moisturizing factor-related genes filaggrin (FLG), transglutaminase-1, HAS-1 and HAS-2.
Under UVB irradiation, the expression of HYAL in the HaCaT cells was reduced. The study also demonstrated the anti-oxidative activity of EGCG and showed a preventive effect against radical-induced apoptosis by downregulating caspase-8 and -3 in HaCat cells. EGCG reduced the production and secretion of melanin in melanoma cells.
The ability of the human skin to retain moisture is linked to the skin aging process, as it suppresses the formation of wrinkles. Skin ageing occurs through two mechanisms: intrinsic and extrinsic ageing. The process of intrinsic ageing is characterized by reduced proliferative activity of skin cells, which in turn leads to cellular senescence due to reduced synthesis of collagen and elastin. Extrinsic ageing, in turn, is caused by external environmental stimuli such as UV radiation, air pollution and particulate matter. The ageing of the skin causes dehydration of the skin cells. The molecule hyaluronic acid plays a key role in regulating skin moisture. Studies have shown that hyaluronic acid increases skin hydration by regulating the hyaluronic acid synthase genes.
Studies have also shown that epidermal hyaluronic acid promotes the proliferation and differentiation of cells in the wound healing process. Natural moisturizing factors (NMFs) consist of hyaluronic acid and filaggrin (FLG), which have a direct or indirect effect on the skin’s moisture barrier. It is assumed that various components of NMFs are degraded and down-regulated by UV radiation. The mechanism by which UV radiation causes skin damage regulates signaling pathway components such as mitogen-activated protein kinases (MAPKs), NF-κB (nuclear factor “kappa-light-chain-enhancer” of activated B cells) and tumor necrosis factor (TNF)-α. It is also widely documented that hyaluronidase (HYAL) is an enzyme that hydrolyzes hyaluronic acid and that the expression of the hyaluronidase (HYAL) gene is strongly influenced by UV radiation.
The antioxidant system protects our skin from UV radiation, cigarette smoke and oxygen deficiency. Reactive oxygen species (ROS) caused by UV radiation or oxidative stress increase the progression of skin ageing, wrinkling and pigmentation. Excessive production of ROS activates internal programmed cell death, known as apoptosis. This intensifies the ageing process and diseases associated with old age. For this reason, in order to maintain a healthy skin condition or to stop the ageing process, it is recommended to take antioxidants that eliminate free radicals. Melanin is synthesized by transforming L-tyrosine and also protects the skin from external stress. However, excessive melanin production can cause age spots, among other things.
Effects of EGCG on the synthesis activity of NMF
In the underlying study, it was shown that treatment with EGCG increases the expression of genes associated with NMF synthesis, such as filaggrin (FLG), transglutaminase (TGM)-1 and hyaluronic acid synthase (HAS)-1,2 and 3. In particular, the levels of FLG were significantly increased by EGCG compared to treatment with retinol. The levels of TGM-1, HAS-1 and HAS-2 were also increased; the relative band density, based on the data obtained with RT-PCR, was also increased after treatment with EGCG. To determine the upstream proteins regulating the genes related to NMF synthesis, the protein expression of MAPKs and HAS-2 was analyzed. The expression of HAS-2 was increased by 25 µM EGCG. Similarly, EGCG application increased the expression of MAPKs, including c-Jun N-terminal kinases (JNK), extracellular-signal regulated kinases (ERK) and p38. Luciferase activity was also measured with AP-1-Luc plasmid. The application of EGCG increased the luciferase activities influenced by AP-1 in a dose-dependent manner.
To investigate the extent to which EGCG promotes cell proliferation, HaCaT cells were treated with EGCG or retinol for 12 or 24 hours. It was found that EGCG promoted cell proliferation more than retinol. After 12 hours, the percentage increase in proliferation compared to zero hours was 240% for 12.5 µM EGCG and 265% for 25 µM EGCG compared to only 150% for retinol. After 24 hours, the increase in proliferation compared to zero hours was 269% for 12.5 µM EGCG, 310% for 25 µM EGCG and 208% for retinol.
Effects of EGCG on the skin’s ability to retain moisture
To investigate the skin-protective effect against UVB radiation, the cytotoxic effect of EGCG against HaCaT cells was determined by means of MTT testing. After UVB exposure of 30 mJ/cm2, cell viability was reduced by 68.9 % compared to the normal group. In contrast, 12.5 µM EGCG increased cell viability by 72.8 % and 25 µM EGCG significantly increased it by 75.9 %.
This demonstrated that EGCG reduced cell damage caused by UVB radiation. In order to investigate the moisturizing ability of HaCat cells, the expression of the HYAL gene was determined by RT-PCR after UVB-induced cell damage. The expression of HYAL-2, -3 and -4 was reduced in the EGCG group. Especially under UVB damage-induced conditions, EGCG decreased the expression of HYAL-4 in a dose-dependent manner.
Anti-oxidative and anti-apoptopic effects of EGCG
The anti-oxidative effect of EGCG at a target concentration of 0-25 µM was demonstrated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) studies. EGCG significantly reduced DPPH radicals at half maximal inhibitory concentration (IC50) of 13.04 ± 3.95 µM.
In the ABTS study, EGCG clearly captured ABTS; the IC50 value was calculated as 1.57 ± 0.06 µM. Based on these data, the radical scavenging ability of EGCG against nitroprusside (SNP)-induced ROS was investigated. RAW264.7 cells were pretreated with EGCG and then treated with SNP and dihydrorhodamine 123 (DHR123) to determine the extent to which EGCG regulates intercellular ROS levels.
Measurements of intracellular ROS by byflusscytometric analysis showed that EGCG reduced ROS levels in a dose-dependent manner. This confirms that EGCG can regulate not only extracellular but also intracellular radicals. It was also investigated whether the production of nitric oxide caused by SNP is reduced by EGCG. EGCG inhibited the SNP-induced production of nitric oxide in HaCaT cells. The viability of SNP-treated HaCaT cells was simultaneously verified, as free radicals and ROS induced apoptosis.
It is well established in research that antioxidants suppress apoptosis by controlling ROS levels. The expression levels of apoptotic molecules were examined to determine the extent to which EGCG protects against cell death. During apoptosis, caspases are broken down and converted into active forms. The amount of cleaved caspase-3 was greatly reduced by EGCG. Since caspase-3 is involved as a common effector molecule in the extrinsic or intrinsic apoptosis signaling pathway, the study investigated which apoptotic signaling pathway is influenced by EGCG. Immunoblotting of caspase-9 and -8 showed that EGCG only inhibited the formation of cleaved caspase-8. These results demonstrate that EGCG prevents apoptosis by inhibiting the extrinsic apoptosis signaling pathway.
In addition, the extent to which EGCG regulates the formation and secretion of melanin in B16F10 cells was determined. Melanin production was induced by α-melanocyte-stimulating hormone (αMSH) and arbutin was used as a positive control substance. 100 μM EGCG reduced the extracellular secretion of melanin without cytotoxic effects. At the same time, the melanin content in the cells was measured. Although 50 μM EGCG did not affect the secretion of melanin, the production of melanin was reduced at a concentration of 50 μM or higher. The results demonstrate that EGCG regulates melanin pigmentation.
The underlying study confirmed that EGCG improves the moisturizing capacity of keratinocytes. First, the mRNA expression of NMF-related genes (FLG, TGM1, HAS-1, -2 and -3) was analyzed. Subsequently, EGCG was found to increase the expression of all NMF-related genes without cytotoxicity. The study results also suggest that EGCG upregulates FLG, TGM1, HAS-1 and HAS-2 in keratinocytes, thereby moisturizing the epidermis and strengthening the skin barrier.
To determine which proteins regulate NMF, the levels of MAPKs and HAS-2 protein were determined by immunoblotting. EGCG was found to increase the phosphorylation of p38, ERK and JNK to the same extent as retinol (as a positive control substance) and HAS-2 was also upregulated by treatment with 25 µM EGCG. These results indicate that NMF-related genes are regulated by MAPKs. The activity of AP-1, the transcription factor of MAPKs, was investigated via the luciferase system. It was shown that EGCG dose-dependently increased the luciferase activity mediated by AP-1. Thus, the underlying study confirmed that MAPKs regulate the moisturization of keratinocytes.
Under UV irradiation, EGCG reduced cell damage and the expression levels of HYALs. EGCG inhibited the degradation of hyaluronic acid in the epidermis by reducing the levels of HYAL expression and increasing the moisturizing capacity of the skin barrier. EGCG was also observed to increase cell proliferation. Among other things, cell proliferation reduces the formation of wrinkles.
It was also confirmed that EGCG has antioxidant properties and protects keratocytes from radicals mediated by SNP and UV radiation. The anti-apoptotic effects resulting from the anti-oxidative activity of EGCG were also investigated.
Conclusion
The underlying study comes to the conclusion that EGCG is well suited as a cosmetic active ingredient due to its positive effect on skin hydration and its ability to retain moisture, its reducing effect on melanin formation and its effect against wrinkling, as well as its properties as a free radical scavenger.
Underlying study:
Kim, E. et al: Skin Protective Effect of Epigallocatechin Gallate; in: Int. J. Mol. Sci. 2018, 19, 173; doi:10.3390/ijms19010173.