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Pharmaceutical Biology 2007, Vol. 45, No. 6, pp.


Antityrosinase Activity of Some Plant Extracts and Formulations Containing Ellagic Acid
. O zer1, B. Mutlu1, and B. Kvc O ak2 Department of Pharmaceutical Technology; 2Department of Pharmacognosy, Faculty of Pharmacy, Ege University, Bornova, Izmir, Turkey

Ellagic acid (EA) is a naturally occurring polyphenol found in a variety of plants in its free form or in the form of ellagitannin glycosides. In this study, the ellagic acid content of the methanol extracts of Juglans regia L. (Juglandaceae) leaves, Castanea sativa Mill. (Fagaceae) stem bark, and Eucalyptus camaldulensis Dehnh. (Myrtaceae) leaves was determined to develop melanogenesis inhibitors. An improved NaNO2 assay was used for determination of EA. The tyrosinase inhibitory activity of the extracts and synthetic EA was tested in vitro by monitoring the appearance of dopachrome, an intermediate in the melanogenesis process. The results were compared keeping the same total concentration of inhibitor. The efficacy of EA (1%) was compared with arbutin (1%) and hydroquinone monomethyl ether (1%) as reference substances, and it was found to be a more efficient suppressor of pigmentation. The effect of formulation variables on the tyrosinase inhibitory activity was also evaluated. Based on dopachrome tests performed in the formulations, it could be concluded that the combination with plant extracts had a synergistic effect, and gel formulation could be suggested as an effective carrier for treating uneven skin pigmentation. Keywords: Castanea sativa stem bark, ellagic acid, Eucalyptus camaldulensis leaves, Juglans regia leaves, topical formulations, tyrosinase. in melanin synthesis or uneven distribution of melanin can cause local hyperpigmentation or spots. Whitening agents such as hydroquinone, arbutin, kojic acid, or azelaic acid are widely used in cosmetic products as active substances (Prota, 1996; Mora & Baraldi, 2000; rard, 2003). Nakayama et al., 2000; Petit & Pie Plant extracts that have a good inhibitory effect on melanin formation may be a good choice for the cosmetic purposes of whitening facial skin and protection against skin darkening. In addition, they have relatively fewer side effects (Kim & Lee, 1998). In cosmetic preparations, many plant extracts such as Morus alba L. (Moraceae) or Glycyrrhiza glabra Linneva. (Leguminosae) have been used as whitening agents (Lee et al., 1997; Bernard & Berthon, 2000; Baurin et al., 2002). Tyrosinase is the enzyme involved in melanogenesis and catalyzes the oxidation process of tyrosine to dihydroxy-phenylalanine (DOPA) and from DOPA to DOPA quinone. Tyrosinase is known to be a metaloenzyme, containing copper at an active site, catalyzing these reactions through a change in the oxidative site of copper atoms (Lee & Kim, 1995; Prota, 1996; Mora & Baraldi, 2000). Ellagic acid (EA) is a naturally occurring polyphenol found in a variety of plants in its free form or in the form of ellagitannin glycosides. It exists in dicotyledonous woody plants, in the genera of Castanea (Fagaceae), Eucalyptus (Myrtaceae), and Quercus (Fagaceae). It is also found in a variety of fruits and vegetables normally consumed by humans such as grapes, cherry, and walnut (Zee-Cheng & Cheng, 1986; Peng et al., 1991; Bianco et al., 1998; Lei et al., 2001; Amakura et al., 2002). Recently, EA was shown to inhibit skin pigmentation resulting from UV irradiation. Results of in vitro

Hyperpigmentation is the most common facial pigmentary disorder. It has been observed that a local increase
Accepted: January 18, 2007.

zgen O zer, Assoc. Prof. Dr., Department of Pharmaceutical Technology, Faculty of Pharmacy, Ege Address correspondence to: O University, 35100 Bornova, Izmir, Turkey. Fax: 90232 3885258; E-mail: DOI: 10.1080/13880200701446746 # 2007 Informa Healthcare


. O zer et al. O sorbitan oleat, and Synperonic PE= F 127, an ethoxylated propylene oxide copolymer (ICI). Carbopol 954, a synthetic polymer, and Carsoquat CT 429 were obtained from Johnson & Johnson (Turkey). Triethanolamine (TEA) was from Sigma (St. Louis, MO, USA). All other chemicals used for analysis were analytical reagent grade. Quantitative determination of EA from the plant extracts The quantity of free EA existing in natural sources was determined with an improved NaNO2 assay method that was used by Wilson and Hagerman (1990). This assay was first described by Bate-Smith (1972) and was selective for ellagic acid only; gallic acid, gallotannins, ellagitannins, condensed tannins, and flavonoids do not interfere. Different concentrations of EA in dimethyl sulfoxide were added to dimethyl sulfoxide to give a final volume of 9.2 mL in test tubes. Concentrated HCl (0.4 mL) was then added and samples were brought to 30C. The samples were mixed immediately after 0.4 mL 1% (w=v) NaNO2 in H2O was added. The absorbance at 513 nm was recorded, and equation of linear calibration curve was obtained. For determination of EA from plant extracts, 10 mg of extract was dissolved in 10 mL of DMSO. This stock solution (1 mL) was assayed for EA using the described NaNO2 method. Determination of tyrosinase inhibitory effect of synthetic active materials and plant extracts Tyrosinase inhibitory activity is generally determined with a spectrophotometer. The procedure described by Vanni et al. (1990) was followed in our study. Tyrosinase inhibitory activity of EA was determined and compared with the tyrosinase inhibitory activity of arbutin (Nihei & Kubo, 2003) and hydroquinone monomethyl ether (HQMME) (Patrick et al., 1999) as reference substances. Tyrosinase inhibition effect of Cortex Castanea extract, Folium Eucalypti extract, and Folium Juglandis extract was also determined. For evaluation of tyrosinase inhibitory effect, a reaction mixture consisted of 0.9 mL 50% methanol solution of inhibitor and 2 mL L-tyrosine solution (0.244 mM) in aqueous phosphate buffer (pH 6.8; I 0.01 M) was prepared; for the control sample, an equivalent volume of 50% methanol solution was used instead of the inhibitor solution. Oxidation of L-tyrosine was initiated by introducing 0.1 mL of aqueous mushroom tyrosinase solution (0.1 mg=mL). Test mixture and control mixture were incubated for 10 min at 37C. Dopachrome appearance was monitored spectrophotometrically at 475 nm. We determined the effect of test compound on tyrosinase inhibition by IC50, the concentration at which the compound inhibits half the original tyrosinase activity.

experiments have indicated that EA suppresses melanogenesis by inhibiting tyrosinase activity. This inhibition is caused by chelation of the copper atoms on the tyrosinase molecules. It was approved in 1996 for use as an active ingredient in formulations for the prevention of spots and freckles after excessive exposure to sunlight (Shimogaki & Yanagawa, 1996; Shimogaki et al., 2000; Tanaka, 2001). The aim of this study was to determine the EA content and tyrosinase inhibitory effect of Folium Juglandis (Juglans regia leaves), Cortex Castanea (Castanea sativa stem bark), and Folium Eucalypti (Eucalyptus camaldulensis leaves) methanol extracts to develop melanogenesis inhibitors. An improved NaNO2 assay was used for determination of EA (Wilson & Hagerman, 1990). Tyrosinase inhibitory effects of the plant extracts were compared with the synthetic EA by measuring the melanin intermediates (dopachrome) spectrophotometrically (Ferioli et al., 2001). After this study, the tyrosinase inhibitory activity of different formulations containing synthetic EA and plant extract was also studied to determine the effect of formulation variables.

Materials and Methods

Plant materials and preparation of plant extracts Castanea sativa Mill. (Fagaceae) stem bark and Juglans regia L. (Juglandaceae) leaves were collected from Sultanhisar (Malgec emir village) in Aydn in September 2002. Eucalyptus camaldulensis Dehnh. (Myrtaceae) leaves were also collected from the Ege University campus in September 2002 and identified by B. Kivcak. Voucher specimens (no. 1275, no. 1277, no. 1276 in order) were deposited at the herbarium of the Faculty of Pharmacy, Ege University, in Izmir. Plant materials were air-dried and then powdered. Each powdered plant (100 g) was extracted with 80% methanol solution, and after filtration the filtrates were evaporated to dryness under vacuum. These extracts were used for further studies including quantitative determination of ellagic acid and inhibition of tyrosinase.

Drugs and chemicals EA was obtained from Shangai Pudong New Area Li Cheng Group (China). L-Tyrosine and mushroom tyrosinase (EC, activity  127 U=mg of solid) were purchased from Fluka (Sigma-Aldrich, Italy). Methanol was obtained from J. T. Baker (Holland). The oil used was liquid paraffin (Birpa, Turkey). The lipophilic surfactants were Abil EM 90 (Goldschmid, France) and Span 80, a sorbitan oleat (ICI, France), and the hydrophilic surfactants were Tween 80, a polyoxyethylene

Antityrosinase activity of plants containing ellagic acid The percent inhibition of tyrosinase activity was calculated as follows: % inhibition : Acontrol Asample 475 475 Acontrol 475 100


and Asample were the absorbance values in the Acontrol 475 475 presence and absence of inhibitor. Tyrosinase inhibitory activities of plant extracts were evaluated with the same method using the same mixture without plant extract as a control. Preparation of skin-whitening formulations For investigation of the tyrosinase inhibitory effect of formulations, an oil=water (O=W) type emulsion (F1), a W=O=W type multiple emulsion (F2), a gel (F3), a lotion (F4), and a vanishing cream (F5) containing synthetic ellagic acid, plant extracts, and a combination of these were prepared. Determination of tyrosinase inhibitory effect of formulations Blank and spiked cream was weighed accurately into a centrifuge tube (1 g). Ten millilitres of 50% methanol was added and stirred in an ultrasonic bath for 30 min. The remaining suspension was centrifuged at 4000 rpm for 30 min. Aliquots of the resultant solution were further diluted 1:10 with 50% methanol and tested by the dopachrome method for determination of tyrosinase inhibitory effect. Statistical analysis Tests for significant differences between mean values were made by analysis of variance (ANOVA). Reference to significant difference in the following text denotes that the test was carried out at a level of p < 0.05.
Figure 1. Tyrosinase inhibitory activity of ellagic acid (&), arbutin (.), and HQMME (~) at different (0.1 mM, 0.3 mM, 0.5 mM) concentrations (n 6, SD).

sources can be done by acid hydrolysis of the crude tannin extracts. (Zee-Cheng & Cheng, 1986; Peng et al., 1991; Bianco et al., 1998; Amakura et al., 2002). However, the products of acidic hydrolysis process are thought to be a disadvantage for cosmetic products. For this reason, in this study methanol extracts of the plant materials were prepared and the quantity of free ellagic acid was determined spectrophotometrically (Table 1). Folium Juglandis extract was found to have the highest concentration of EA. EA content of Folium Juglandis, Cortex Castanea, and Folium Eucalypti extracts were found to be 16.25%, 2.75%, and 0.28%, respectively. The tyrosinase inhibitory effects of arbutin and HQMME, which are well-known whitening agents, were determined to prove the accuracy of our method. Suppression of tyrosinase could be demonstrated when dose-dependent inhibition was demonstrated using these hydroquinone derivatives as an effective control. The

Results and Discussion

It is known that ellagic acid naturally occurs in Eucalyptus camaldulensis, Castanea sativa, and Juglans regia at high concentrations in its free form or in the form of ellagitannin. Isolation of ellagic acid from natural
Table 1. The quantity of EA in the plant extracts (n 6 SD). Plant sample Folium Eucalypti extract Cortex Castanea extract Folium Juglandis extract EA% 0.28 0.021 2.75 0.335 16.25 0.512 Figure 2. Tyrosinase inhibitory activity of Folium Juglandis extract (&), Cortex Castanea extract (.) and Folium Eucalypti extract (~) at different (100 mg=mL, 250 mg=mL, 500 mg=mL) concentrations (n 6, SD).


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Table 3. Compositions of the skin-lightening formulations. Formulation Mineral oil (Paraffinum liquidum) Span 80 Tween 80 Triethanolamine Abil EM 90 Synperonic PE=F 127 MgSO4 7H2O Propylene glycol Carbopol 954 Methyl paraben Cetyl alcohol Stearic acid Lanoline Glycerine Carsoquat CT 429 Potassium hydroxide Distilled water F1 (g) 20 F2 (g) 24 F3 (g) F4 (g) 10 F5 (g)

Table 2. Concentration (IC50) required for selected tyrosinase inhibitors to reduce mushroom tyrosinase activity by 50%. Plant extracts Folium Juglandis extract Cortex Castanea extract Folium Eucalypti extract IC50 (mg=mL) 505 844 2258

1.75 3.25 0.3 4 0.8 0.7 0.75 49 2 0.15 0.5 3 1 2 2 72.75 70.5 53.85 86.35 1 24 10 1.2 63.8

inhibitory activity was estimated starting from test compound solutions at 0.1 to 0.5 mM. As expected, a concentration-dependent inhibitory effect was observed for each compound tested as shown in Figure 1. The results indicate that the more the concentration of compounds was increased, the more the tyrosinase inhibitory capacity increased. The inhibitory effect of arbutin and HQMME was compared with EA, and except for HQMME at 0.5 mM, significant difference was observed for all concentrations (p < 0.05). The results demonstrated that all of the synthetic active materials showed an inhibitory activity level higher than 50% at the concentration of 0.5 mM, and among these synthetic materials, EA has shown the highest tyrosinase inhibitory effect. IC50 values of the compounds were also compared, and EA was found to be a more potent tyrosinase inhibitor than arbutin and HQMME. IC50 values were calculated as 0.225, 0.311, and 0.267 mM for EA, arbutin, and HQMME, respectively. According to the results of previous studies conducted using the brownish guinea pig, EA was found to be a more efficient skin whitener and suppressor of pigmentation than arbutin or kojic acid at the same dose level (1%). Furthermore, the efficacy of EA was found almost the same to that of HQMME, a well-known depigmentation agent. These results are in good accordance with our results (Patrick, 1999; Shimogaki et al., 2000). The isolation and structural determination of the ellagic acid from plant extracts will provide us new information leading to the development of new skinwhitening products. Current attempts to find new

skin-whitening agents focus on the systematic evaluation of plant extracts. We investigated the inhibitory effect of Folium Eucalypti (Eucalyptus camaldulensis), Cortex Castanea (Castanea sativa), and Folium Juglandis (Juglans regia) extracts on tyrosinase activity for this purpose. These three plant extracts, which demonstrated a significant ability to inhibit mushroom tyrosinase, were described for the first time to possess this biological property. Dopachrome test was performed on these plant extracts at three different concentrations. Folium Juglandis extract exhibited the highest inhibition of tyrosinase at the concentration of 500 mg=mL (Fig. 2). The inhibitory effect of Folium Juglandis was found to be 1.2- and 1.8fold higher than that of Folium Eucalypti and Cortex Castanea extracts, respectively, for all concentrations. The presence of the free form of EA at a high concentration seems to verify the high tyrosinase inhibition of Folium Juglandis extract. When we compared the inhibitory effect of Folium Eucalypti and Cortex Castanea with

Table 4. Combinations that were incorporated into the formulations. Combination C1 C2 C3 C4 C5 C6 C7 C8 Ellagic acid (1%) Folium Eucalypti extract (1%) Folium Juglandis extract (1%) Cortex Castanea extract (1%)

Antityrosinase activity of plants containing ellagic acid

Table 5. Tyrosinase inhibitory effect of formulations (n 6 SD). Combination C1 C2 C3 C4 C5 C6 C7 C8 F1 (%) 43.262 0.512 6.117 0.323 13.475 0.597 19.167 0.466 45.783 0.812 51.950 0.908 58.777 0.840 62.450 0.105 F2 (%) 38.021 0.615 6.337 0.422 15.017 0.718 17.649 0.469 39.843 0.875 47.830 0.831 54.295 0.717 58.631 0.925 F3 (%) 47.553 0.845 6.329 0.557 19.590 0.498 22.779 0.714 51.539 0.918 59.11 0.771 62.450 0.955 66.529 0.720 F4 (%) 39.545 0.986 4.594 0.678 17.235 0.671 21.396 0.791 41.257 0.919 44.257 0.991 51.181 0.928 56.405 0.935 F5 (%)


37.743 0.714 5.115 0.529 18.959 0.564 21.718 0.559 40.740 0.861 51.181 0.964 52.910 0.521 58.082 0.743

Folium Juglandis, a significant difference was observed for all concentrations (p < 0.05), only the difference with Cortex Castanea at 250 mg=mL was found insignificant (p > 0.05). When IC50 values of the compounds were compared, Folium Juglandis extract was found to be more effective, reaching the IC50 at a much lower concentration than Folium Eucalypti and Cortex Castanea extracts (Table 2). Tyrosinase inhibitory effect of formulations Composition of the formulations and combinations that were introduced into the formulations are given in Tables 3 and 4. Ellagic acid (1%), plant extract (1%), and combinations of these were introduced into each formulation. Evaluation of the formulation effects on tyrosinase inhibition demonstrated that the gel formulation containing synthetic EA and three plant extracts together at 1% concentration has the highest inhibitor activity (approximately 66.6%). Methanol extract of blank formulations was used as control sample, and no inhibitory effect caused by the formulation was observed. Tyrosinase inhibitory effects of formulations are given in Table 5. Comparison of the inhibitory activity of the formulations demonstrated that the rank order was approximately the same, F3 > F1 > F2 > F5 > F4, for all combinations. The results of the formulations F2, F5, and F4 were found similar. The inhibitor activity was found higher for gel formulation than for that obtained with the other formulations in all cases. In addition, the combinations C8 and C1 showed the highest inhibitor activity in all formulations, respectively. Comparison of F3-C8 formulation with other formulations containing C8 combination indicated that there were statistically significant differences between gel formulation and the others (p < 0.05). According to the results, it was suggested that gel formulation containing ellagic acid and three plant extracts together will be effective in treating uneven skin pigmentation. In conclusion, the inhibitory effect of Folium Eucalypti (Eucalyptus camaldulensis), Cortex Castanea

(Castanea sativa), and Folium Juglandis (Juglans regia) extracts on tyrosinase activity were described for the first time for this biological property. The highest amount of EA and the strongest inhibitory effect on tyrosinase were observed with Juglans regia extract, followed by Eucalyptus camaldulensis and Castanea sativa extracts. These plant extracts could be suggested as new sources of skin-whitening agents.

We gratefully acknowledge the Ege University Research Foundation for its financial support.

Amakura Y, Umino Y, Tsuji S, Ito H, Hatano T, Yoshida T, Tonogai Y (2002): Constituents and their antioxidative effects in Eucalyptus leaf extract used as natural food additive. Food Chem 77: 4756. Bate-Smith EC (1972): Detection and determination of ellagitannins. Phytochemistry 111: 11531156. Baurin N, Arnoult E, Scior T, Do QT, Bernard P (2002): Preliminary screening of some tropical plants for antityrosinase activity. J Ethnopharmacol 82: 155158. Bernard P, Berthon Y (2000): Resveratrol: An original mechanism on tyrosinase inhibition. Int J Cosmet Sci 22: 219226. Bianco MA, Handaji A, Savolainen H (1998): Quantitative analysis of ellagic acid in hardwood samples. Sci Total Environ 222: 123126. Ferioli V, Rustichelli C, Pavesi G, Gamberini G (2001): New combined treatment of hypermelanosis: Analytical studies on efficacy and stability improvement. Int J Cosmet Sci 23: 333340. Kim J, Lee KT (1998): Inhibitory effects of Ramulus mori extracts on melanogenesis. Cosmetics Toiletries 113(10): 6570. Lee KT, Kim BJ, Kim HJ, Heo MY, Kim HP (1997): Biological screening of 100 plant extracts for cosmetic


. O zer et al. O
Peng S, Scalbaert A, Monties B (1991): Insoluble ellagitannins in Castanea sativa and Quercus petraea woods. Phytochemistry 30: 775778. rard E (2003): Skin-lightening products revisited. Petit L, Pie Int J Cosmet Sci 2: 169181. Prota G (1996): Melanins and melanogenesis. Cosmetics Toiletries 111(55): 4351. Shimogaki H, Tanaka Y, Tamai H, Masuda M (2000): In vitro and in vivo evaluation of ellagic acid on melanogenesis inhibition. Int J Cosmet Sci 22: 291303. Shimogaki H, Yanagawa T (1996): Inhibitory effect of ellagic acid on tyrosinase activity. American Academy of Dermatology 54th Annual Meeting, February 1015, 1996, Washington, DC, p. 280. Tanaka Y (2001): Ellagic acid. Pigment Cell Res 14(6): 495. Vanni A, Gastaldi D (1990): Kinetic investigations on the double enzymic activitiy of the tyrosinase mushroom. Ann Chim 80: 3560. Wilson CT, Hagerman AE (1990): Quantitative determination of ellagic acid. J Agric Food Chem 38: 16781683. Zee-Cheng Y, Cheng CC (1986): Ellagic acid. Drugs Future 11: 10291033.

use (I): Inhibitory activities of tyrosinase and DOPA auto-oxidation. Int J Cosmet Sci 19: 291298. Lee O, Kim E (1995): Skin lightening. Cosmetics Toiletries 110(10): 5156. Lei Z, Jervis J, Helm F (2001): Use of methanolysis for the determination of total ellagic and gallic acid contents of wood and food products. J Agric Food Chem 49: 11651168. Mora PC, Baraldi PG (2000): Dermocosmetic applications of polymeric biomaterials. In: Dumitru S, ed., Polymeric Biomaterials, 2nd ed. New York and Basel, Marcel Dekker, pp. 459490. Nakayama H, Ebihara T, Satoh N, Jinnai T (2000): Depigmenting agents. In: Elsner P, Maibach HI, eds., Cosmeceuticals Drugs vs. Cosmetics. New York and Basel, Marcel Dekker, pp. 123144. Nihei K, Kubo I (2003): Identification of oxidation product of arbutin in mushroom tyrosinase assay system. Bioorg Med Chem Lett 13: 24092412. Patrick E, Juberg R, Odonoghue J, Maibach HI (1999): Depigmentation with tert-butyl hydroquinone using black guinea pigs. Food Chem Toxicol 37: 169175.