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www.biolandkorea.com Korean Red Ginseng KOREAN RED GINSENG is steamed and dried with the skin unpeeled from the strictly selected fresh ginseng, Panax ginseng C.A. Meyer. It is in light yellowish brown or light reddish brown color. It may be preserved for a long time of 10 years or more. Korean ginseng is well known for its rich saponin content called Ginsenoside that activates the functions of human body's components as well as strengthens immunity. The six-year Korean red ginseng root contains the most saponin among worldwide ginsengs and the composition is so exquisite as to display high efficacy and properties. WHITE GINSENG RED GINSENG www.biolandkorea.com Fermented Korean Red Ginseng It is known that the active components of Ginseng, ginsenosides are hydrolyzed by intestinal bacteria and then the metabolites are absorbed into the body. But the ginsenoside-hydrolyzing ability of intestinal bacteria varies from person to person by person's physical health and the amount and type of intestinal bacteria that exists in the intestines. During fermentation, special enzymes are formed to break down not only the ginsenosides but also other important ingredients of Red Ginseng, thus making it easy for everyone to absorb ingredients of ginseng (It has already been pre-hydrolyzed by fermentation) BIO-FRGE is a fermented Korean Red Ginseng Extract produced by Bioland ‘s high fermentation technology to increase absorption, reduce bitter taste, and also increase anti-aging property of Red Ginseng. Extraction & Fermentation RED GINSENG BIO-FRGE www.biolandkorea.com Metabolism of Ginsenoside Rb1 Ginsenosides Contents of BIO-FRGE 9 Red Ginseng 8 Fermented Red Ginseng Contents (mg/g) 7 6 5 4 3 2 1 Rk1 0 Rg1 Rb1 Rg3 Rk1 Ginsenosides Rd Rg3-S Rg3-R Rk1 Rg5 Total in BIO-FRGE Content (mg/g) 0.11 0.27 0.10 0.15 0.17 3.50 www.biolandkorea.com Cytotoxicity of BIO-FRGE ® Cytotoxicity (MTT method) Cytotoxicity of FRGE by MTT assay. sample MTT treatment treatment Incubation Absorbance measurement (570nm) Incubation Incubation DMSO treatment www.biolandkorea.com Cytotoxicity of BIO-FRGE ® Result (human skin fibroblast cell (ATCC, CRL-2076)) 110 100 90 cell viability (%) 80 70 60 50 40 30 20 10 0 10 25 50 100 250 conc. (ug/ml) Fig 1. Cytotoxicity assay of FRGE. The data were expressed as mean values (± standard deviations) of five experiments. www.biolandkorea.com Cytotoxicity of BIO-FRGE ® Result (human skin keratinocyte (HaCaT)) 110 100 cell viability (%) 90 80 70 60 50 40 30 20 10 0 1 10 25 concentration (ug/ml) 50 100 Fig 2. cell proliferation assay of FRGE. The data were expressed as mean values (± standard deviations) of five experiments. www.biolandkorea.com Anti-aging of BIO-FRGE ® SIRTUIN -It’s looking like these sirtuins serve as guardians of the cell. These enzymes allow cells to survive damage and delay cell death. – David Sinclair / Harvard Medical School researcher Crystallographic structure of yeast sir2 (rainbow colored -Sirtuins are the central regulator of the aging process. – Leonard Guarente / Massachusetts Institute of Technology (MIT) in Cambridge cartoon, N-terminus = blue, C-terminus = red) complexed with ADP (space-filling model, carbon = white, oxygen = red, nitrogen = blue, phosphorous = orange) and a histone H4 peptide (magenta) containing an acylated lysine residue (displayed as spheres) www.biolandkorea.com Anti-aging of BIO-FRGE ® ® Silent Information Regulator 2 (Sir2) proteins (sirtuins) A class of proteins which possess either histone deacetylase or mono-ribosyltransferase activity and are found in organisms ranging from bacteria to humans. Yeast Sir2 and some, but not all, sirtuins are protein deacetylases. Unlike other known protein deacetylases, which simply hydrolyze acetyl-lysine residues, the sirtuin-mediated deacetylation reaction couples lysine deacetylation to NAD hydrolysis. This hydrolysis yields O-acetyl-ADPribose, the deacetylated substrate and nicotinamide, itself an inhibitor of sirtuin activity. The dependence of sirtuins on NAD links their enzymatic activity directly to the energy status of the cell via the cellular NAD:NADH ratio, the absolute levels of NAD, NADH or nicotinamide or a combination of these variables. Sirtuins have been implicated in influencing aging and regulating transcription, apoptosis and stress resistance. Species distribution Whereas bacteria and archaea encode either one or two sirtuins, eukaryotes encode several sirtuins in their genomes. In yeast, roundworms, and fruit flies sir2 is the name of the sirtuin-type protein. This research started in 1991 by Leonard Guarente of MIT. Mammals possess seven sirtuins (SIRT1-7) that occupy different subcellular compartments such as the nucleus (SIRT1, 2, -6, -7), cytoplasm (SIRT1 and SIRT2) and the mitochondria (SIRT3, -4 and -5). www.biolandkorea.com Anti-aging of BIO-FRGE ® Types Sirtuins are classed according to their sequence of amino acids. Prokaryotics are in class U. In yeast (a lower eukaryote), sirtuin was initially found and named sir2. In more complex mammals there are seven known enzymes which act as on cellular regulation as sir2 does in yeast. These genes are designated as belonging to different classes, depending on their amino acid sequence structure Species Yeast Mouse Human Intracellular location Sir2 or Sir2p, Hst1 or Hst1p Sirt1 SIRT1 nucleus deacetylase metabolism inflammation Hst2 or Hst2p Sirt2 SIRT2 cytoplasm deacetylase cell cycle tumorigenesis Sirt3 SIRT3 nucleus and mitrochondira deacetylase metabolism II Sirt4 SIRT4 mitochondria ADP-ribosyl transferase insulin secretion III Sirt5 SIRT5 mitochondria deacetylase unknown a Sirt6 SIRT6 nucleus ADP-ribosyl transferase DNA repair b Sirt7 SIRT7 nucleus unknown rDNA transcription regulation of acetyl-CoA synthetase metabolism Class Subclass Bacteria a I Activity Function b c Hst3 or Hst3p, Hst4 or Hst4p IV U www.biolandkorea.com Anti-aging of BIO-FRGE The yeast sirtuin (Sir2) is a histone deacetylase that modulates yeast replicative life span by suppressing genome instability through chromatin modification. Oberdoerffer et al. (2008) report that SIRT1, the mammalian ortholog of Sir2, is involved in DNA damage-induced chromatin reorganization, which promotes genome stability in mammalian cells. Anti-aging of BIO-FRGE Anti-aging of BIO-FRGE An increasing dose of Sir2 extends yeast replicative life span, whereas its loss reduces natural longevity overexpression of Sir2 orthologs in nematodes and flies also extends life span. In diverse organisms, Sir2 slows the pace of ageing and increases maximum lifespan. Scientists show that the potent activator resveratrol, a polyphenol found in red wine, increases DNA stability and lifespan by 70% by stimulating Sir2. Nature 425, 191 (2003). Hypothesis: A potent activator of Sir2 like resveratrol would be helpful as an anti-aging ingredient in cometics. Anti-aging of BIO-FRGE ® SIRT3 Sirtuin (silent mating type information regulation 2 homolog) 3 (S. cerevisiae), is the third member of the mammalian sirtuin family. The SIRT3 gene encodes the protein SIRT3, which exhibits NAD+-dependent deacetylase activity. SIRT3 is a member of the sirtuin family of proteins, homologs to the yeast Sir2 protein. Members of the sirtuin family are characterized by a sirtuin core domain and grouped into four classes and the protein encoded by this gene is included in class I of the sirtuin family. The human sirtuins have a range of molecular functions and have emerged as important proteins in aging, stress resistance and metabolic regulation. In addition to protein deacetylation, studies suggest that the human sirtuins may function as intracellular regulatory proteins with mono ADP ribosyltransferase activity. Structure While the crystal structure has not yet been solved, SIRT3 is a soluble protein located in the mitochondrial matrix, and contains a mitochondrial processing peptide at the n-terminus. www.biolandkorea.com Anti-aging of BIO-FRGE ® Function - Mitochondrial Three sirtuins, SIRT3, SIRT4 and SIRT5, are located in mitochondria and have been implicated in regulating metabolic processes. Endogenous SIRT3 is a soluble protein located in the mitochondrial matrix. Overexpression of Sirt3 in cultured cells increases respiration and decreases the production of reactive oxygen species. Fasting increases Sirt3 expression in white and brown adipose tissue (WAT and BAT, respectively) and overexpression of SIRT3 in HIB1B brown adipocytes increases the expression of PGC-1α and UCP1, suggesting a role for SIRT3 in adaptive thermogenesis BAT. BAT is different from WAT because it harbors large numbers of mitochondria and is important for thermogenesis in rodents. Thermogenesis in BAT is mediated by the uncoupling protein 1 (UCP1), which induces proton leakage and thereby generates heat instead of ATP. Mechanistic insights into how SIRT3 affects thermogenesis in BAT is lacking and whether SIRT3 affects UCP1 activity directly is not known. In addition to controlling metabolism at the transcriptional level, sirtuins also directly control the activity of metabolic enzymes. In Salmonella enterica, the bacterial sirtuin CobB regulates the activity of the enzyme acetyl-coenzyme A (acetyl-CoA) synthetase. As mentioned above, orthologs of acetyl-CoA synthetase exist in the cytoplasm (AceCS1) and in mitochondria (AceCS2) in mammals. The presence of the sirtuin deacetylase SIRT3 in the mitochondrial matrix suggests the existence of lysine acetylated mitochondrial proteins. Indeed, SIRT3 deacetylates and activates the mammalian mitochondrial acetyl-coA synthetase (AceCS2). Furthermore, SIRT3 and AceCS2 are found complexed with one another, suggesting a critical role for control of AceCS2 activity by SIRT3. Anti-aging of BIO-FRGE ® Nuclear In addition to its reported mitochondrial function, some researchers have proposed a very small pool of active nuclear SIRT3 exists. This pool is reported to consist of the long form of SIRT3 and has been suggested to have histone deacetylase activity. The observation that SIRT3 has nuclear activity came from a report that SIRT3 protected cardiomyocytes from stress mediated cell death and that this effect was due to deacetylation of a nuclear factor,Ku-70. ® Clinical significance The is a strong association between SIRT3 alleles and longevity in males www.biolandkorea.com Anti-aging of BIO-FRGE ® Sirt 3 expression activity (RT-PCR) Measuring Sirt 3 expression level in the human skin fibroblast cell (ATCC, CRL-2076). sample treatment treatment Incubation Incubation Electrophoresis RT-PCR RNA isolation www.biolandkorea.com Anti-aging of BIO-FRGE Effect of BIO-FRGE on the expression of Sirt 3. Control Resveratrol BIO-FRGE 200 % of control 150 100 50 0 Control (FBS 10%) Concentration (µg/ml) Resveratrol 10 uM FRGE 10 ug/ml FRGE 50 ug/ml FRGE 100 ug/ml www.biolandkorea.com Anti-aging of BIO-FRGE - Inflammation and aging - Reference from : Chung HY, Cesari M, Anton S, Marzetti E, Giovannini S, Seo AY, Carter C, Yu BP, Leeuwenburgh C (2009) Molecular inflammation: underpinnings of aging and age-related diseases. Ageing Res Rev, 8, 18-30. Anti-aging of BIO-FRGE RNS inhibition ROS inhibition iNOS expression rate (%) 100 100 80 60 40 28.6 17.79 20 5.76 0.82 C O X - 2 e x p r e s s i o n r a te ( % ) 120 120 100 100 80 60 48.79 43.09 48.21 40 20 3 0 0 10 Control LPS 100 10 250 BIO-FRGE (㎍/㎖) iNOS mRNA Expression inhibition Control LPS 100 250 BIO-FRGE (㎍/㎖) COX-2 mRNA Expression inhibition www.biolandkorea.com