Biological Studies of Kaempferol from Green Tea Seed by Enzymatic Hydrolysis and Their Application to Cosmeceuticals

Abstract

Kaempferol, one of the flavonols in green tea, has many biological activities, but the kaempferol of plant origin is too expensive to be used in commercial products. Recently, it has been confirmed that green tea seed (GTS) contained fairly good amount of kaempferol glycoside. After conducting structural analysis, two forms of kaempferol glycosides i.e. kaempferol-3-O-[2-O-β-D-galactopyranosyl-6-O-α-L-rhamnopyranosyl]-β-D-glucopyranoside (compound 1) and kaempferol-3-O-[2-O-β-D-xylopyranosyl-6-O-α-L-rhamnopyranosyl]-β-D-glucopyranoside (compound 2) were identified. To obtain pure kaempferol in large quantity, their enzymatic hydrolysis was attempted. Through screening of commercially available enzymes, β-galactosidase and hesperidinase were selected and, their mixing ratio was optimized. At the optimized condition, over 95% pure kaempferol was produced.

The purified kaempferol and its glycosides were subjected to various examinations of biological effects. In terms of antioxidant effect on skin anti-aging, Kaempferol was a more efficient scavenger of DPPH radicals and a better inhibitor of xanthin/xanthine oxidase than the compound 1 and compound 2. Kaempferol showed inhibitory effect on lipid peroxidation which was induced by t-butyl hydroperoxide (t-BHP) in keratinocytes. Kaempferol treatment significantly inhibited ultraviolet (UV)-induced matrix metalloproteinase-1 (MMP-1) in mono-culture of normal human fibroblasts or keratinocytes/fibroblasts co-cultured system. However, kaempferol did not change the level of type I pro-collagen synthesis in fibroblasts. Since TNF-α is another inducer of skin-aging and stimulate MMP-1 in fibroblasts, another explanation is that kaempferol inhibits TNF-α production, which in turn repress MMP-1 in keratinocytes/fibroblasts co-culture.

When the effect of kaempferol on the skin wrinkles was examined in vivo, kaempferol showed to decrease wrinkles induced by squalene-hydroperoxide in hairless mice. In addition, topical application of the emulsion containing kaempferol for 8 weeks decreased wrinkles in photo-aged skin area such as crows feet area.

To elucidate its effects on the skin further, the transcriptional profiles of kaempferol-treated HaCaT cells were examined using cDNA microarray analysis. 147 genes exhibited significant changes in expression. Among them, 18 genes were up-regulated and 129 genes were down-regulated. These genes were then classified into 12 categories according to their function: cell adhesion/cytoskeleton, cell cycle, redox homeostasis, immune/defense responses, metabolism, protein biosynthesis/modification, intracellular transport, RNA processing, DNA modification/replication, regulation of transcription, signal transduction and transport. The promoter sequences of the differentially-regulated genes was analyzed, and then over-represented regulatory sites and candidate transcription factors (TFs) for gene regulation by kaempferol were identified as such c-REL, SAP-1, Ahr-ARNT, Nrf-2, Elk-1, SPI-B, NF-κB and p65. In addition, the microarray results and bioinformatic analysis were validated by conventional methods such as real-time PCR and ELISA-based transcriptional factor assay. The inhibitory effect of kaempferol on NF-κB and RelB in HaCaT cells and normal human epidermal keratinocytes (NHEKs) irradiated with UVB was shown by ELISA-based transcription factor assay. Since PPAR activation is one of several mechanisms which could account for the decrease of NF-κB activities, the effect of kaempferol to increase the transcriptional activity of PPARs in HaCaT cells was investigated. Kaempferol stimulated PPAR transcriptional activity in HaCaT cells transiently transfected with the PPRE-tk-Luc reporter gene, suggesting that kaempferol acts as a regulator of epidermal differentiation in human skin. To investigate whether or not kaempferol promotes the differentiation of keratinocytes, BrdU incorporation experiment and a Western blot analysis against transglutamase-1 protein were performed. Kaempferol treatment inhibited BrdU incorporation, and induced transglutamase-1 protein in keratinocytes, suggesting that kaempferol could be an inducer of epidermal keratinocytes differentiation.

In conclusion, kaempferol is demonstrated as a very effective anti-aging reagent that can be used as an anti-aging ingredient, and a possible candidate of cosmeceutical products.