Abstract:
Quercetin, 18 α-glycyrrhetinic and/or hederagenin and/or their derivatives are used as anti-aging and/or whitening agents for the topical treatment of skin. Methods for testing the anti-aging and whitening properties of these compounds are disclosed.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    The present invention relates to the use of natural compounds extracted from plants of the Greek flora with anti-aging properties on human diploid fibroblasts. These anti-ageing properties are mediated through the decrease of cellular oxidative load as well as through the activation and maintenance of the activity of the main proteolytic pathway of the cell and one of the central secondary anti-oxidant system, the proteasome. Furthermore, this invention relates to the use of these compounds as natural whiteners against age spots. 
       SUMMARY OF THE INVENTION 
       [0002]    The aim of the present invention is the identification of natural substances with anti-aging properties, mainly based on the activation and the maintenance of proteasome function and on a secondary base, based on the regulation of cellular oxidative load. Additional goals are the production of anti-aging products that due to their anti-oxidant properties and their rejuvenating effects could be used for as cosmetic products. 
         [0003]    According to the invention, quercetin, 18α-glycyrrhetinic acid and/or hederagenin and/or their derivatives are used as effective compounds in a composition for the topical treatment of skin having anti-aging and/or whitening properties. 
         [0004]    According to the invention, the composition may contain only one of these effective compounds or two, three or more of them. Furthermore, the composition may contain at least one of these effective compounds and at least another compound having anti-aging and/or whitening properties. 
         [0005]    According to the invention, these compounds are effective against senescence of diploid fibroblasts one of the main cell type in skin that play a central rule in the tissue&#39;s aging. 
         [0006]    The invention provides an improvement and maintenance of the young morphology of diploid fibroblasts and whitening of melanocytes, respectively, by topical treatment with these compounds. It has been shown that the increase of the proliferation of diploid fibroblasts during senescence is limited or controllable. The invention also provides preferred methods for testing the anti-aging and whitening properties of a certain compound, preferably in the presence of an oxidizing agent, preferably hydrogen peroxide. 
         [0007]    When the morphology is examined, the ratio of elongated to flattened diploid fibroblast cells can be determined. 
         [0008]    Quercetin has the formula I and its derivatives the formula II. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0009]    Quercetin (formula I) is the aglycon form of a number of other flavonoid (flavonol) glycosides such as rutin (rhamnoglucoside of quecetin-rutinoside), quercitrin (glucoside of quercetin) etc. Main quercetin and many of its derivatives have been found to be among the most active of the flavonoids in studies and many medicinal plants owe much of their activity to their high quercetin and quercetin derivatives content. Quercetin and many of its derivatives have demonstrated significant antiinflammatory and cytotoxic activity, they also exert strong potent antioxidant activity, while extracts rich in quercetin and its derivatives may have positive effects to prevent against allergies-inflammation, heart diseases, etc. 
         [0010]    The following quercetin following derivatives of quercetin have been found to be particularly effective: 
         [0000]    3-:Quercetin caprylate
 
Quercetin 3-β-d-galactoside
 
Quercetin 3-β-d-glucoside
 
Quercetin 3-β-1-rhamnoside
 
Quercetin 3-d-xyloside
 
Quercetin 3-rutinoside
 
Quercetin 3-l-arabinoside
 
Quercetin 3-glucuronide
 
       Hederagenin 
       [0011]    18a-Glycyrrhetinic acid 
         [0012]    The following derivatives of quercetin have been found effective, but to a lesser extent: 
         [0000]    Quercetin 3′-methyl ether
 
Quercetin 4-methylether-7-rutinoside
 
Quercetin 4-methylether
 
Quercetin 7,4′-dimethylether
 
Quercetin 3-methylether
 
Quercetin 3,7-dimethylether
 
Quercetin 7-methyl ether
 
Quercetin 3,3′-dimethylether
 
Quercetin 7,3′-dimethylether
 
Quercetin 7,3′,4′-trimethylether
 
Quercetin 3,4′-dimethylether
 
Quercetin 3,3′,4′-trimethylether
 
Quercetin 3,7,4′-trimathylether
 
Quercetin 4′-methylether-3-O-glucoside
 
Quercetin 3′,4′-dimethylether-3-O-glucoside
 
Quercetin 3,3′-dimethylether-7-O-glucoside
 
       Quercetin 7-O-glucoside 
     Quercetin 5-O-glucoside 
       [0013]    Natural sources rich in quercetin and its derivatives include oak tree ( Quercus  sp.), capers ( Capparis spinosa ), red grapes ( Vitis vinifera ), apples ( Pyrus malus ), onions ( Allium cepa ), tea ( Camellia sinensis ), citrus fruits ( Citrus  sp.), lovage, broccoli, other leafy green vegetables, cherries and many species of berries such as raspberry, bog whortleberry, chokeberry, crowberry etc. Quercetin and its derivatives have been also found in several varieties of honeys. 
         [0014]    The invention comprises the use of quercetin according to formula I as well as of quercetin derivatives according to formula II in which a hydrogen atom of at least one hydroxyl group of quercetin is substituted by another group. The most preferred quercetin derivatives are quercetin caprylate and quercetin glycosides. In quercetin caprylate any hydroxyl group in any position (1, 2, 3, 4, 5) may be substituted with caprylic acid. That means any X in formula II may be an caprylic group. In quercetin glycosides quercetin is substituted in position 3 with sugar. The sugar may be, for instance, a monosaccharide as disaccharide as rutinose in case of rutin. 
         [0015]    18α-glycyrrhetinic acid has the formula III and its derivative the formula IV. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0016]    18α-glycyrrhetinic acid (formula III) is a triterpene mainly occurring in nature from  Glycyrrhiza. Glycyrrhiza  is a genus of about 18 accepted species in the family Fabaceae (Leguminosae), with a subcosmopolitan distribution in Asia, Australia, Europe and America (North and South). The genus is best known for liquirice, which is the product of  Glycyrrhiza glabra , a species native to the Mediterranean region and very common and widespread in Greece. 
         [0017]    The invention also comprises the use of 18α-glycyrrhetinic acid (formula III) as well as of its derivatives according to formula IV in which a hydrogen atom of the hydroxyl group and/or the carboxyl group of 18α-glycyrrhetinic acid is substituted by another group as a sugar. 
         [0018]    Hederagenin has the formula V and its derivative the formula VI. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0019]    Hederagenin is a triterpenic saponin (formula V) which can be found widespread in nature in many plant species but mainly in the leaves of Ivy ( Hedera helix  L., Araliaceae), which is an evergreen medicinal and ornamental and from which it has also taken its name. Such triterpene and similar derivatives can be determined and isolated from the stems of  Humulus lupulus  L. (Hops), from the plant of  Medicago sativa  subsp.  sativa  (Alafalfa), as well from the seeds of  Nigella sativa  L.; (Black Caraway, Black Cumin) and the roots of the  Paeonia  species (such as White Peony  Paeonia lactiflora  PALL.) and  Pulsatilla  sp. (such as  Pulsatilla chinensis ). 
         [0020]    The invention also comprises the use of hederagenin (formula V) as well as its derivatives according to formula VI in which a hydrogen atom of at least one hydroxyl group and/or at least one methyl group and/or the carboxyl group of hederagenin is substituted by another group as a sugar. 
         [0021]    Anti-aging properties of the examined substances in human diploid fibroblasts were evaluated by the following biomarkers: 
         [0000]    (1) Cellular lifespan in the presence of the substances as compared to the lifespan performed by control cultures (diluent-treated) and morphology of the cells following treatment with the substances throughout their lifespan.
 
(2) Detection of β-galactosidase activity.
 
(3) Reversal of senescent phenotype by addition of the substances.
 
(4) Detection of levels of oxidized proteins (Oxyblot analysis) in the presence of the substances.
 
(5) Detection of Reactive Oxygen Species (ROS) in the presence of the substances.
 
(6) Survival cellular ability under oxidative stress conditions in the presence of the substances.
 
(7) Detection of chymotrypsin-like activity of the proteasome following incubation with the substances.
 
(8) Detection of the levels of proteasome subunits following incubation with the substances.
 
         [0022]    Whitening properties of the examined substances on melanocytes were evaluated by: 
         [0000]    (A) Cellular whitening of melanocytes.
 
(B) Detection of tyrosinase expression, the main metalloglycoprotein that catalyzes several steps in the melanin pigment biosynthetic pathway.
 
         [0023]    This invention takes advantage of biological aging markers in order to define the biological action of the examined substances on human diploid fibroblasts. The results indicate that the anti-aging properties of the examined substances are the result of a combined action against cellular oxidation and maintenance of the proteasome activity, the main secondary anti-oxidant mechanism of defense. Oxidation and increase of ROS and, consequently, increase of damaged and oxidized proteins is a hallmark of aging (K. B. Beckman and B. N. Ames (1998) “The free radical theory of aging matures”,  Physiol. Rev.  78: 547-581). The proteasome is the main proteolytic mechanism of the cell responsible for the cellular detoxification and clearance by toxic factors like the oxidized proteins that accumulate with advanced age and accelerate the establishment of senescence (N. Chondrogianni and E. S. Gonos (2005) “Proteasome dysfunction and mammalian aging: involved steps and factors”,  Exp. Gerontol.  40: 931-938). Our previous studies in human diploid fibroblasts have shown that proteasome assembly and function decreases with advanced age in vivo and in vitro and the appearance of senescence can be irreversibly induced upon proteasome inhibition (N. Chondrogianni, I. Petropoulos, C. Franceschi, B. Friguet and E. S. Gonos (2000) “Fibroblast cultures from healthy centenarians have an active proteasome”,  Exp. Gerontol.  35: 721-728; N. Chondrogianni, F. L. L. Stratford, I. P. Trougakos, B. Friguet, A. J. Rivett and E. S. Gonos (2003) “Central role of the proteasome in senescence and survival of human fibroblasts: induction of a senescence-like phenotype upon its inhibition and resistance to stress upon its activation”,  J. Biol. Chem.  278: 28026-28037; N. Chondrogianni and E. S. Gonos (2004) “Proteasome inhibition induces a senescence-like phenotype in primary human fibroblast cultures”,  Biogerontology  5: 55-61). On the other hand, we have also shown that overexpression of the proteasome leads to ameliorated response to oxidative stress whereas, it also confers lifespan extension, thus verifying that the proteasome is a longevity assurance mechanism (N. Chondrogianni, C. Tzavelas, A. J. Pemberton, I. P. Nazis, A. J. Rivett and E. S. Gonos (2005) “Overexpression of proteasome beta 5 subunit increases the amount of assembled proteasome and confers ameliorated response to oxidative stress and higher survival rates”,  J. Biol. Chem.  280: 11840-118500). In addition, our studies have determined that centenarians, the best model of successful aging, possess an active proteasome, regardless their age (N. Chondrogianni, I. Petropoulos, C. Franceschi, B. Friguet and E. S. Gonos (2000) “Fibroblast cultures from healthy centenarians have an active proteasome”,  Exp. Gerontol.  35: 721-728). 
     
    
     
       BRIEF DESCRIPTION OF THE TABLES AND FIGURES 
         [0024]    For a full understanding of the present invention, reference should now be made to the following detailed description of the preferred embodiments of the invention as illustrated in the accompanying tables and figures. 
           [0025]    Table 1: Continuous topical treatment of HFL-1 cells with the substances extends their proliferative lifespan. 
           [0026]    Number of cpds of HFL-1 cells treated with the substances or the relative diluents for 13 consecutive weeks. Continuous topical treatment of primary human fibroblasts with the substances extends their proliferative lifespan as compared to controls. 
           [0027]    Table 2: Amount of β-galactosidase positive cells in the culture. 
           [0028]      FIG. 1 : Continuous topical treatment of HFL-1 cells with the substances delays the appearance of the senescence phenotype. 
           [0029]    Representative photographs of HFL-1 cells treated with the substances or the relative diluents after 8 weeks in culture. Continuous topical treatment of primary human fibroblasts with the substances retains the young morphology and delays the appearance of the senescence phenotype. Arrows indicate cells exhibiting typical senescent morphology. 
           [0030]      FIG. 2 : Continuous topical treatment of HFL-1 cells with the substances results to decreased β-galactosidase activity staining. 
           [0031]    β-galactosidase activity staining (blue color) of HFL-1 cells treated with the substances or the relative diluents after 10 weeks in culture. Cells treated with the substances exhibited less positive cells to β-galactosidase activity staining as compared to the positive cells in the relative control cultures. 
           [0032]      FIG. 3 : Continuous topical treatment of middle aged and terminally senescent HFL-1 cells with the substances results to induction of proliferation. 
           [0033]    Number of (A) middle aged cells and (B) terminally senescent cells that were constantly treated with the substances for 1 week and were then counted. Asterisks reveal statistically significant differences (p&lt;0.05). 
           [0034]      FIG. 4 : Continuous topical treatment of middle aged and terminally senescent HFL-1 cells with the substances results to partial reversal of the senescent phenotype. 
           [0035]    Morphology and β-galactosidase activity staining of (A) middle aged cells and (B) terminally senescent cells treated with the various substances for the 1 week. Arrows indicate cells negative to β-galactosidase activity staining. 
           [0036]      FIG. 5 : Topical treatment of HFL-1 cells with the substances results to decrease of intracellular oxidized proteins. 
           [0037]    Oxyblot® analysis in cells treated with the substances or the relative diluents after 24 h of topical treatment with the substances. 
           [0038]      FIG. 6 : Topical treatment of HFL-1 cells with the substances results to decreased intracellular ROS levels. 
           [0039]    Percentages of ROS levels in cultures treated with the substances for 24 h (Substance), in cultures treated with the substances for 24 h and being subjected to oxidative stress for 2.5 h (Right after) and in the relative control cultures (treated with 0.1% DMSO or 0.1% CAP). ROS levels of control cultures at each time point were arbitrary set to 100%. 
           [0040]      FIG. 7 : Topical treatment of HFL-1 cells with the substances results to increased survival rates. 
           [0041]    Number of cells that survived after treatment with the various substances with or without being subjected to oxidative stress. Asterisks reveal statistically significant differences (p&lt;0.05). 
           [0042]      FIG. 8 : Topical treatment of HFL-1 cells with the substances results to increased proteasome activities. 
           [0043]    Percentage of CT-L activity in cells treated with the substances or the relative diluents after 24 h of treatment with the substances. Use of a proteasome inhibitor (MG132) in control reactions ensured the specificity of the enzymatic reactions. Mean value of activities was set at 100% in control cultures. Each column shows the average of three independent experiments, and error bars denote standard error. 
           [0044]      FIG. 9 : Topical treatment of HFL-1 cells with the substances results to increased protein expression levels of proteasome subunits. 
           [0045]    Immunoblot analysis of representative subunits of 20S complex, β-type subunits (β 5 , 22.9 kDa), and α-type subunits (α 7 , 28.4 kDa) in cells treated with the substances or the relative diluents. Equal protein loading was verified by stripping the membranes and reprobing them with GAPDH (37 kDa) antibody (bottom panels). 
           [0046]      FIG. 10 : Topical treatment of mouse melanocytes with the substances results to cellular whitening. 
           [0047]    Photographs of pellet of melanocytes that were treated with the substances for 72 h. 
           [0048]      FIG. 11 : Topical treatment of mouse melanocytes with the substances results to decreased levels of tyrosinase. 
           [0049]    Immunoblot analysis of tyrosinase (70-80 kDa) in melanocytes treated with the substances or the relative diluents. Equal protein loading was verified by stripping the membrane and reprobing them with GAPDH (37 kDa) antibody (bottom panel). 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0050]    The preferred embodiments of the present invention will now be described with reference to  FIGS. 1-11  of the drawings. Identical elements in the various figures are designated with the same reference numerals. 
       Anti-Aging Properties 
       [0051]    The studies, methods and results that were obtained regarding the anti-aging properties of the examined substances are the following: 
       1) Study of the Lifespan of Human Diploid Fibroblasts in Vitro in the Presence of the Substances. 
       [0052]    1.1. HFL-1 human diploid fibroblasts (HDFs) were used in in vitro conditions (growth at 37° C., 5% CO 2  and 95% humidity). 
         [0053]    1.2. The cells were maintained in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM®; Invitrogen Life Technologies Inc.) supplemented with 10% (v/v) fetal calf serum, 100 Units/ma penicillin, 100 μg/ml strepromycin, 2 mM glutamine and 1% (v/v) non-essential amino acids (complete medium). 
         [0054]    1.3. The cells were constantly cultured in culture medium supplemented with the substances [final concentration for quercetin (QUER), 18α-glycyrrhetinic acid (18α GA), and hederagenin (HED): 2 μg/ml and for quercetin caprylate (QU-CAP) three concentrations were applied: 0.5, 2 and 5 μg/ml]. Quercetin, 18α-glycyrrhetinic acid and hederagenin were diluted in DMSO (dimethyl sulfoxide) whereas quercetin caprylate was diluted in caprilic/capric triglycerides (CAP). Therefore, the control cultures were incubated in medium supplemented with 0.1% DMSO or 0.1% CAP. 
         [0055]    1.4. The cells were replenished with fresh media supplemented with the substances or the diluents every 24 h and their numbers were examined using a Coulter Z2® counter (Coulter Corporation) until they reached senescence (approximately after 13 weeks). The cumulative population doublings performed by each culture were calculated using the following formula: CPD=Σ[PD] where PD=LOG [Nfinal/Ninitial]/LOG [2], where N final represents the number of cells that were measured when each culture reached confluence and N initial represents the number of cells that was initially seeded. 
         [0056]    1.5. Photographs of the cells maintained in cell culture and treated with the various substances for the same time period were taken. 
         [0057]    1.6. Table 1 shows the total number of cumulative population doublings (CPDs) performed by the different HFL-1 cultures that were treated with the various substances. More specifically, cells that were incubated with quercetin caprylate gave up to 2.8% more population doublings as compared to the population doublings performed by the relative control cultures. Cells that were incubated with the other substances gave up to 2.9% more population doublings as compared to their relative control cultures. 
         [0058]    1.7.  FIG. 1  shows the morphology of the cells treated with the various substances for the same time period. Cells that were treated with either of the tested substances maintained the young morphology for longer as compared to the control cultures (DMSO-treated and CAP-treated). Cells treated with the substances are more elongated and they grow in parallel arrays as compared to the control cultures that exhibit a higher number of flattened cells with more irregular shape. It is also worth noting that cells that were treated with the substances exhibited a younger phenotype as compared to their controls although they have performed more population doublings during the time that the photograph was taken. 
       2) Detection of β-Galactosidase Activity. 
       [0059]    2.1. HFL-1 HDFs were cultured as described in 1.1-1.4 for 10 consecutive weeks and were then subjected to β-galactosidase activity staining. 
         [0060]    2.2. Briefly, 10 5  cells from each culture were seeded in 6-well plates. After 24 h cells were washed with PBS (phosphate buffered saline), fixed in 0.2% glutaraldehyde and 2% formaldehyde for 5 min, washed again with PBS, and finally stained for 24 h at 37° C. in the absence of CO 2 , in staining solution (150 mM NaCl, 2 mM MgCl 2 , 5 mM K 3 Fe(CN) 6 , 40 mM citric acid, and 12 mM sodium phosphate, pH 6.0) containing 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactoside. Photographs were then taken. 
         [0061]    2.3.  FIG. 2  shows the β-galactosidase activity positivity of HFL-1 cultures treated with the various substances or the relative diluents for the same time period. Cells treated with the different substances appeared less positive to β-galactosidase activity staining (blue color) as compared to the relative control cultures where almost all cells appeared to be positive to the β-galactosidase activity staining, thus revealing maintenance of the young phenotype for longer. Furthermore, in full accordance with the photograph in  FIG. 1 , the morphology of the cells verifies the above mentioned staining since cultures with less positive cells to β-galactosidase activity exhibited a younger morphology as compared to the rest cultures. 
       3) Reversal of Senescent Phenotype by Addition of the Substances. 
       [0062]    3.1. HFL-1 HDFs that have senesced in vitro were used (growth at 37° C., 5% CO 2  and 95% humidity). More specifically, middle aged HFL-1 HDFs (cpd 59) and terminally senescent HFL-1 HDFs (cpd 65) that had senesced in the absence of any substance were used. 
         [0063]    3.2. The cells were seeded and maintained in complete medium (see 1.2). Culture medium supplemented with the substances [final concentration for quercetin (QUER), 18α-glycyrrhetinic acid (18α GA), and hederagenin (HED): 2 μg/ml and for quercetin caprylate (QU-CAP) three concentrations were applied: 0.5, 2 and 5 μg/ml] or the diluents (0.1% DMSO or 0.1% CAP) were added 24 h later. The cells were replenished with fresh media supplemented with the substances or the diluents every 24 h for 1 week and their numbers were then examined using a Coulter Z2® counter (Coulter Corporation). 
         [0064]    3.3. Detection of β-galactosidase activity was performed in these cells as described above and photographs of the cells following β-galactosidase activity assay were taken. 
         [0065]    3.4.  FIG. 3  shows the number of (A) middle aged cells and (B) terminally senescent cells that were counted following 1 week treatment with the various substances. In middle aged cells, treatment with quercetin, 18α-glycyrrhetinic acid as well as with all three tested concentrations of quercetin caprylate led to higher proliferation rates as compared to the rates exhibited by the control cultures. In terminally senescent cells, treatment with quercetin or quercetin caprylate (2 and 5 μg/ml) induced a limited but significant cell proliferation as compared to the absence of duplication in the control cultures whereas treatment with the rest of the substances did not show any significant differences. 
         [0066]    3.5.  FIG. 4  shows the morphology and the β-galactosidase activity positivity of (A) middle aged cells and (B) terminally senescent cells treated with the various substances for 1 week. In middle aged cells, treatment with quercetin, 18α-glycyrrhetinic acid and all three concentrations of quercetin caprylate, appeared to be less positive to β-galactosidase activity staining but however, since control cultures were not either heavily positive to β-galactosidase activity staining, the main finding was that cells treated with these substances were induced to proliferate and furthermore, acquired a morphology more similar to the one exhibited by young cells (regular size, elongated cells, growth in parallel arrays). In terminally senescent cells, treatment with quercetin and the higher concentrations of quercetin caprylate induced cell proliferation as mentioned above, while many of those cells were negative for β-galactosidase activity. These cells were elongated and they acquired a morphology that exhibits more similarities with the morphology of young fibroblasts as compared to the relative control cultures where almost all cells were positive for β-galactosidase activity with a typical senescent phenotype (enlarged cells with accumulation of autofluorescent material, not growing in parallel arrays). 
       4) Detection of Levels of Oxidized Proteins (Oxyblot Analysis) in the Presence of the Substances. 
       [0067]    4.1. HFL-1 HDFs were cultured as described in 1.1-1.4. Cells treated with the substances for 24 h and were then lysed and proteins were extracted and subjected to SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophorese) followed by OxyBlot analysis that detects the carbonylated groups on proteins. OxyBlot™ is a protein oxidation detection kit. 
         [0068]    4.2.  FIG. 5  shows that cells treated with quercetin caprylate have less oxidized proteins as compared to the relative control cells. Regarding the other substances, cells treated with 18α-glycyrrhetinic acid exhibited slightly lower levels of oxidized carbonylated proteins as compared to the relative control cultures. However it is worth mentioning that OxyBlot analysis detects only carbonylated groups on proteins which is just one oxidative stress-mediated post-translational protein modification. Therefore, this could account for the mild differences that this analysis reveals between treated cells and the relative control cultures. 
       5) Detection of Reactive Oxygen Species (ROS) in the Presence of the Substances. 
       [0069]    5.1. HFL-1 HDFs were seeded and maintained in complete medium (see 1.2). Culture medium supplemented with the substances [final concentration for quercetin (QUER), 18α-glycyrrhetinic acid (18α), and hederagenin (HED): 2 μg/ml and for quercetin caprylate (QU-CAP) three concentrations were applied: 0.5, 2 and 5 μg/ml] or the diluents (0.1% DMSO or 0.1% CAP) were added 24 h later and an additional incubation for 24 h followed. Cells were collected and detection of Reactive Oxygen Species (ROS) was performed in order to reveal ROS levels following the incubation with the substances. In replica cultures, culture medium supplemented with the substances as described above were also supplemented with 300 μM H 2 O 2  for 2.5 h and cells were collected for detection of ROS following oxidative stress in the presence or absence of the substances. 
         [0070]    5.2. 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes, Invitrogen) was used for ROS detection. Cells (10 5 ) were resuspended in prewarmed PBS±the dye at a final concentration of 10 μM (loading buffer) and incubated at 37° C. for 30 min. Cells were then resuspended in prewarmed complete medium and incubated at 37° C. for 5 min. The absorption and the emission of the oxidation product were measured at 493 and 520 nm, respectively. Each sample was measured in quadruplicates. 
         [0071]    5.3.  FIG. 6  shows the percentages of ROS levels in cultures treated with the substances for 24 h (Substance), in cultures treated with the substances for 24 h and being subjected to oxidative stress for 2.5 h (Right after) and in the relative control cultures (treated with 0.1% DMSO or 0.1% CAP). ROS levels of control cultures at each time point were arbitrary set to 100%. Cells treated with quercetin caprylate and quercetin without being subjected to oxidative stress (Substance) exhibited decrease of ROS levels ranging from 11% to 25% as compared to the relative control cultures. Cells treated with the other substances without being subjected to oxidative stress (Substance) exhibited decrease of ROS levels ranging from 30% to 46% as compared to the relative control cultures. When the cells were also challenged with 300 μM H 2 O 2 , treatment with quercetin caprylate or quercetin led to decrease of ROS levels by 12% to 39% while for treatment with the other substances the reduction of ROS levels was ranging from 30% to 75%. 
       6) Survival Cellular Ability Under Oxidative Stress Conditions in the Presence of the Substances. 
       [0072]    6.1. HFL-1 HDFs were cultured as described in 5.1. Following the incubation with the various substances cells were collected and counted using a Coulter Z2® counter (Coulter Corporation). In replica cultures, culture medium supplemented with the substances as described above were also supplemented with 300 μM H 2 O 2  for 2.5 h. The cells were then thoroughly washed with PBS, supplemented with complete medium containing the examined substances and maintained in culture for additional 24 h. They were then collected and counted using a Coulter Z2® counter (Coulter Corporation). 
         [0073]    6.2.  FIG. 7  shows the total number of cells that survived after treatment with the various substances with or without being subjected to oxidative stress. Cells that were just treated with quercetin or quercetin caprylate exhibited higher survival rates ranging from 5.4% to 7.5% as compared to the cells that were treated with the diluents. For the rest of the substances, only treatment with 18α-glycyrrhetinic acid revealed a significantly increased survival rate that reached 3.5%. Additionally, cells that were treated with quercetin or quercetin caprylate and were then subjected to oxidative stress exhibited a higher survival rate reaching to 14.5% as compared to the survival rate of the relative control cultures. Regarding the rest substances, treatment with 18α-glycyrrhetinic acid during oxidative stress had a significant increase of survival that reached 2% as compared to the survival of the control cultures. 
         [0000]    7) Detection of Chymotrypsin-Like Activity of the Proteasome Following Incubation with the Substances. 
         [0074]    7.1. HFL-1 HDFs were cultured as described in 5.1. Following the incubation with the various substances for 24 h, cells were collected, lysed and proteasome activities assays were performed. 
         [0075]    7.2. Detection of the main proteasome activity (chymotrypsin-like; degrading after hydrophobic residues) was performed in crude extracts with the hydrolysis of the fluorogenic peptide LLVY-AMC®, for 30 min at 37° C. Proteasome activity was determined as the difference between the total activity of crude extracts and the remaining activity in the presence of 20 μM MG132 that is a specific inhibitor of the proteasome. Fluorescence was measured using a VersaFluor™ fluorescence spectrophotometer (BioRad Laboratories Inc.). Protein concentrations were determined using the Bradford method with BSA as a standard. 
         [0076]    7.3.  FIG. 8  shows that CT-L activity was induced by ˜1,5-2.5 folds in the presence of quercetin or quercetin caprylate and by ˜2-2.3 folds in the presence of the rest substances as compared to the activity detected in the relative control cultures. 
         [0000]    8) Detection of the Levels of Proteasome Subunits Following Incubation with the Substances. 
         [0077]    8.1. HFL-1 HDFs were cultured as described in 5.1. Following the incubation with the various substances for 24 h, cells were collected, lysed and proteins were extracted and subjected to SDS-PAGE followed by immunoblot analysis against different proteasome subunits. Protein concentrations were determined using the Bradford method with BSA as a standard. 
         [0078]    8.2.  FIG. 9  shows the levels of α- and β-proteasome subunits in cells treated with the substances and their relative control cultures. In the presence of quercetin and quercetin caprylate elevated protein levels of both catalytic (β 5 ) and regulatory (α 7 ) proteasome subunits were revealed. The results were similar for 18α-glycyrrhetinic acid whereas for hederagenin we did not detect any up-regulation regarding proteasome subunits. In total, the increased amount of proteasome subunits expression levels provide an explanation regarding the increased proteasome activities recorded in these cells. 
       Whitening Properties 
       [0079]    The studies, methods and results that were obtained regarding the whitening properties of the examined substances are the following: 
       A) Cellular Whitening of Melanocytes. 
       [0080]    A.1. Mouse melanocytes (B10F16 cell line) were used in in vitro conditions (growth at 37° C., 5% CO 2  and 95% humidity). 
         [0081]    A.2. The cells were maintained in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM®; Invitrogen Life Technologies Inc.) supplemented with 10% (v/v) fetal calf serum, 100 Units/ma penicillin, 100 μg/ml strepromycin, 2 mM glutamine and 1% (v/v) non-essential amino acids (complete medium). 
         [0082]    A.3. 10 5  cells were seeded in petri dishes and the following day they were fed with medium supplemented with the substances [final concentration for all substances: 5 μg/ml and 10 μg/ml]. Quercetin (QUER), 18α-glycyrrhetinic acid (18α GA) and hederagenin (HED) were diluted in DMSO whereas quercetin caprylate was diluted in caprilic/capric triglycerides (CAP). Therefore, the control cultures were incubated in medium supplemented with 0.1% DMSO or 0.1% CAP. The media were replenished every 24 h and the cells were kept under these conditions constantly for 72 h. Cells were then collected and pelleted and the cell pellet was photographed. 
         [0083]    A.4.  FIG. 10  shows the pellet of melanocytes treated either with the various substances or with the diluents. Treatment with both concentrations of quercetin and the lower concentration of hederagenin and 18α-glycyrrhetinic acid revealed whitening potential. However, the effect of quercetin was notably more intensed as compared to the one exhibited by the other substances. The result was positively correlated to the concentration of quercetin used. 
       B) Detection of Tyrosinase Expression. 
       [0084]    B.1. Mouse melanocytes were cultured as described in A.1.-A.3. After 72 h of treatment, the cells were collected, lysed and proteins were extracted and subjected to SDS-PAGE followed by immunoblot analysis against tyrosinase. Protein concentrations were determined using the Bradford method with BSA as a standard. 
         [0085]    B.2.  FIG. 11  shows the protein levels of tyrosinase in cells treated with the substances and their relative control cultures. In the presence of quercetin, quercetin caprylate and a high concentration of hederagenin reduced protein levels of tyrosinase were detected possibly giving an explanation on the whitening effect that was detected in these cells as already described in A.1-A.4. 
       CONCLUSIONS 
     1. Study of the Lifespan of Human Diploid Fibroblasts in Vitro in the Presence of the Substances. 
       [0086]    As described in Sections 1 and 2, cells that were treated with quercetin, quercetin caprylate and 18α-glycyrrhetinic acid exhibited a delay in the appearance of the senescent phenotype. This was accompanied by all the aspects that characterize such a delay, i.e. retention of young morphology, regular shape, lower amount of β-galactosidase positive cells. 
       2. Reversal of the Senescent Phenotype/Rejuvenation Effect 
       [0087]    As described in Section 3, middle aged cells or terminally senescent cells that were treated with quercetin, quercetin caprylate and 18α-glycyrrhetinic acid, regained a younger morphology and were induced to limited but significant proliferation. 
       3. Decrease of the Intracellular Oxidative Load 
       [0088]    As described in Sections 4-6, cells that were treated with quercetin caprylate and 18α-glycyrrhetinic acid possessed lower amounts of oxidized proteins as compared to the relative control cultures. All cultures treated with the tested substances had lower intracellular ROS levels as compared to the control cultures. Furthermore, cells treated with quercetin, quercetin caprylate and 18α-glycyrrhetinic acid survived better to an oxidative stress challenge as compared to the control cells. 
       4. Activation of the Proteasome. 
       [0089]    As described in Sections 7 and 8, treatment with all the tested substances induced an activation of the chymotrypsin-like activity of the proteasome and an increased expression of the proteasome subunits. Consequently, cells treated with these substances possessed enhanced secondary anti-oxidant mechanisms. 
       5. Cell Whitening. 
       [0090]    As described in Sections A and B, treatment of melanocytes with quercetin, hederagenin and 18α-glycyrrhetinic acid revealed whitening potential that was accompanied by a decrease of the protein expression levels of tyrosinase, the main metalloglycoprotein that catalyzes several steps in the melanin pigment biosynthetic pathway that is mainly responsible for the age spots. 
         [0091]    There has thus been shown and described anti-aging and whitening properties of quercetin, 18α-glycyrrhetinic acid and hederagenin and their derivatives which fulfill all the objects and advantages sought therefor. Many changes, modifications, variations and other uses and applications of the subject invention will, however, become apparent to those skilled in the art after considering this specification and the accompanying drawings which disclose the preferred embodiments thereof. All such changes, modifications, variations and other uses and applications which do not depart from the spirit and scope of the invention are deemed to be covered by the invention, which is to be limited only by the claims which follow.