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  Inhibitory Effect of an Ellagic Acid-Rich Pomegranate Extracton Tyrosinase Activity and Ultraviolet-Induced Pigmentation Mineka Y OSHIMURA , 1 ; y Yuko W ATANABE , 1 Kouichi K  ASAI , 1 Jun Y AMAKOSHI , 1 and Takuro K  OGA 2 1  Research and Development Division, Kikkoman Corporation, 399 Noda, Noda City, Chiba 278-0037, Japan 2  Biochemical Division, Kikkoman Corporation, 250 Noda, Noda City, Chiba 278-8601, Japan Received July 5, 2005; Accepted September 9, 2005 A pomegranate extract (PE) from the rind containing90% ellagic acid was tested for its skin-whitening effect.PE showed inhibitory activity against mushroom tyro-sinase  in vitro , and the inhibition by the extract wascomparable to that of arbutin, which is a knownwhitening agent. PE, when administered orally, alsoinhibited UV-induced skin pigmentation on the back of brownish guinea pigs. The intensity of the skin-whiten-ing effect was similar between guinea pigs fed with PEand those fed with  L -ascorbic acid. PE reduced thenumber of DOPA-positive melanocytes in the epidermisof UV-irradiated guinea pigs, but  L -ascorbic acid didnot. These results suggest that the skin-whitening effectof PE was probably due to inhibition of the proliferationof melanocytes and melanin synthesis by tyrosinase inmelanocytes. PE, when taken orally, may be used as aneffective whitening agent for the skin.Key words:  pomegranate extract; ellagic acid; tyrosi-nase; pigmentation; whitening The pomegranate ( Punica granatum  L.) has beenextensively used in traditional medicines in manycountries. The Chinese, for example, have used pome-granate as a traditional product in antibacterial, anti-inflammatory, and hemostasis applications. Extractsfrom different parts of this plant such as the juice, 1,2) seed, 3) and peel 4) have been reported to exhibit strongantioxidative activity. Pomegranate juice has a potentantiatherogenic effect in humans and in atheroscleroticmice, which may be attributable to its antioxidativeproperties. 2,5) Dry pomegranate seed contains the ste-roid, estrogen estron, the isoflavone phytoestrogens,genistein and daidzein, the phytoestrogen, coumestrol, 6) and other compounds.Pomegranate is rich in phenolic compounds, and is animportant source of anthocyanins, 3-glucosides, and 3,5-diglucosides of delphinidin, cyanidin, and pelargoni-din. 7) In addition, pomegranate bark is very rich inellagitannins and gallotannins. 8) Ellagic acid (C 14 H 6 O 8 ,Fig. 1) is a naturally occurring phenolic compoundfound in many natural sources, some common onesbeing strawberries, raspberries, blackberries, and pom-egranates. Ellagitannins are also found in various plantsand hydrolyzed to ellagic acid under acidic conditions.Ellagic acid has been found to have anticarcinogenic, 9,10) antifibrosis, 11) and antioxidative 12) properties. It has beenreported that ellagic acid has a high affinity for copper atthe active site of tyrosinase and inhibits its activity. 13) Shimogaki  et al.  have reported that when ellagic acidwas topically applied, it suppressed UV-induced skinpigmentation of brownish guinea pigs. 13) However, relatively little is known about the whiten-ing activity of ellagic acid or of a pomegranate extractrich in ellagic acid when administered orally. In thepresent study, a pomegranate extract rich in ellagic acid(90%) was prepared, and its inhibitory activity towardtyrosinase was investigated  in vitro , together with thewhitening effect on UV-induced pigmentation of brown-ish guinea pig skin by an oral administration. Materials and Methods Pomegranate extract.  Pomegranate ( Punica granatum L.) fruit rind was extracted 3 times with 50% aqueousethyl alcohol at 60  C–70  C for 2 hours. The ethylalcohol was removed under vacuum. The resultingaqueous solution was acidified with hydrochloric acidand then refluxed at 70  C for 6 hours. Upon dilutionwith water, ellagic acid was precipitated. The precipitatewas collected by filtration and dried in a vacuum traydrier. This pomegranate extract (PE) contained 90.16%ellagic acid on a dry basis (confirmed by an HPLCanalysis). This extract was used for the tyrosinaseinhibition assay and the animal study.  Materials.  L -Tyrosine,  L -(+)-ascorbic acid, sodiumdihydrogen phosphate, disodium hydrogen phosphateand dimethyl sulfoxide (DMSO) were purchased fromWako Pure Chemicals (Osaka, Japan). Arbutin waspurchased from Tokyo Kasei Kogyo (Tokyo, Japan).Mushroom tyrosinase was purchased from Calzyme y To whom correspondence should be addressed. Fax: +81-4-7123-5540; E-mail: myoshimura@mail.kikkoman.co.jp  Abbreviation : PE, pomegranate extract  Biosci. Biotechnol. Biochem. ,  69  (12), 2368–2373, 2005  (CA, U.S.A.), and  L -3,4-dihydroxyphenylalanine(DOPA) was purchased from Sigma Chemicals (MO,U.S.A.). Tyrosinase inhibition assay using mushroom tyrosi-nase.  A tyrosinase inhibition assay was performedaccording to the procedure of Lee  et al. 14) with slightmodifications. Fifty  m l of   L -tyrosine (2m M ), 90 m l of a0.1 M  phosphate buffer (pH 6.8) and 10 m l of DMSOwith or without a sample were added to a 96-wellmicroplate. The plate was preincubated at 37  C for 5minutes, before 50 m l of a phosphate buffer with orwithout mushroom tyrosinase (0.125mg/ml) was added.After incubating at 37  C for 15 minutes, the amount of DOPAchrome was determined at 405nm. The percent-age inhibition of tyrosinase activity was calculated asthe inhibition (%) = [(A–B)/A]    100, where A repre-sents the difference in absorbance of the control samplebetween the samples with and without tyrosinase, andB represents the difference in the test sample betweenthe samples with and without tyrosinase.  Animal study on guinea pigs with UV-induced  pigmentation.  The inhibitory effect on UV-inducedpigmentation was investigated by using brownish guineapigs. Female brownish guinea pigs, Kwl:A-1 with abody weight of 246–356g, were purchased from KiwaLaboratory Animals Co. (Wakayama, Japan). All ani-mals had free access to food and water and were keptin an air-conditioned room (20  C–26  C, 40%–70%humidity) under a 12h dark/light cycle. During theexperimental period, the guinea pigs received humanecare consistent with institutional guidelines. Six animalsper group were used. The back of each brownish guineapig was cleanly shaved with electric clippers. A 4-cm 2 area on the shaven skin was irradiated with 64.8J/cm 2 ( 0 : 09mW/cm 2  12min ) from an ultraviolet (UV) Blamp (Toshiba FL20S E, Tokyo, Japan). The guinea pigswere divided into 4 groups and orally administered withwater, PE or  L -ascorbic acid for 35 days as follows:group 1 as the control received only water, group 2received 100mg/kg/day of PE diluted in water at10mg/ml, group 3 received 1,000mg/kg/day of PEdiluted in water at 100mg/ml, and group 4 received600mg/kg/day of   L -ascorbic acid diluted in water at60mg/ml.The guinea pigs were irradiated on days 7, 9, and 11as just described. The whitening effect was determinedby measuring the L  value weekly with a reflectancespectrophotometer (Minolta CR-300, Tokyo, Japan).The blanching effect was quantified by the increase inL  -value: L  ¼ L  (on the measuring day)  L  (on the first day of the test, before UV-irradiation) After the test period, on day 36, the animals weresacrificed according to institutional guidelines. The back skin samples were collected and stored at   80  C.  Measurement of DOPA-positive melanocytes in guin-ea pigs.  The skin tissue samples were taken from UV-irradiated and untreated areas of the tested guinea pigs,and DOPA-staining of the epidermal sheet was per-formed by the method of Staricco  et al. 15) The numberand size of DOPA-positive melanocytes were measuredunder an optical microscope. The size of the melano-cytes was measured across the cytoplasm withoutdendrites. Statistical analysis.  Each data value is expressed asthe mean  þ =   SEM. One-way ANOVA with Dunnett’ssignificant difference test was also used to evaluatedifferences among the groups. Result The tyrosinase inhibitory activity of PE was examinedand compared with that of   L -ascorbic acid and arbutin,which are known tyrosinase inhibitors. PE inhibitedmushroom tyrosinase activity with an IC 50  value of 182.2 m g/ml. This inhibitory activity of PE is compara-ble to that of arbutin, but was about ten times weakerthan that of   L -ascorbic acid (Table 1), indicating that PEis a tyrosinase inhibitor.Further studies on the  in vivo  inhibitory effect of PEon UV-induced pigmentation were performed. Thegeneral condition and behavior of all rats were normal.The body weight and food consumption of animals in allgroups during the course of this study were almost thesame. Figure 2 shows photographs of typical skinsamples after the oral administration of PE,  L -ascorbicacid or water as a control. The UV-induced skinpigmentation was reduced in the PE or  L -ascorbic acidgroup, but not in the control group. A quantitativeevaluation of whitening was done by determining therate of change of L  value after 5 weeks of admin- OOOOHOHOHOHO Fig. 1.  Structure of Ellagic Acid. Table 1.  Tyrosinase Inhibitory ActivityTest sample IC 50  ( m g/ml)Pomegranate Extract 182.2Arbutin 162.2 L -Ascorbic Acid 18.4Inhibition of Tyrosinase and Pigmentation by a Pomegranate Extract 2369  istration of these samples. As shown in Fig. 3, hyper-pigmentation was weakened more effectively by theadministration of both doses of PE or  L -ascorbic acidthan in the control case. PE showed a whitening effect ina dose-dependent manner. The   L  value was notstatistically different among both PE groups and the  L - A: control B: pomegranate extract(100mg/kg)C: pomegranate extract D: L-ascorbic acid (1,000mg/kg) (600mg/kg) Fig. 2.  Lightening Effects of the Samples on UV-Induced Hyperpigmentation.Representative photographs show the lightening effects of the samples (A, control; B, pomegranate extract (100mg/kg); C, pomegranateextract (1,000mg/kg); D,  L -ascorbic acid (600mg/kg)) on UV-induced hyperpigmentation after 35 days of administration ( n ¼ 6 ). Thesephotographs show typical skin from each group. A: control B: pomegranate extract (1000mg/kg)C: L-ascorbic acid (600mg/kg) Fig. 4.  DOPA-Positive Melanocytes in the Epidermis of UV-Irradiated Skin.Photographs are shown of DOPA-positive melanocytes in the epidermal sheet of the UV-irradiated skin of brownish guinea pigs administeredwith each sample for 35 days. A, water (control); B, pomegranate extract (1,000mg/kg); C,  L -ascorbic acid (600mg/kg) ( n ¼ 6 ). Thesephotographs show typical skin samples from each group.2370 M. Y OSHIMURA  et al.  ascorbic acid group.Figure 4 shows photographs of the DOPA-positivemelanocytes in the epidermal sheet of the UV-irradiatedskin of brownish guinea pigs. DOPA-positive melano-cytes with many dendrites were sparse in the untreatedarea of all groups (data not shown). In contrast, in theUV-irradiated areas of the control group, a large numberof melanocytes formed a dense network (Fig. 4A). In thePE group (1000mg/kg), the number of melanocytes waslower than in the control group (Fig. 4B). In the  L -ascorbic acid group, the number of melanocytes wassimilar to the number observed in the control group.However, the intensity of DOPA staining in the guineapigs administered with  L -ascorbic acid was considerablyweaker than that in the control group (Fig. 4C).Figure 5 shows a quantitative measurement of thenumber of DOPA-positive melanocytes. The number of melanocytes in the UV-irradiated skin of the high-dosePE group (1000mg/kg) was significantly lower than inthe control group in a dose-dependent manner (Fig. 5).In contrast, the number of melanocytes in the  L -ascorbicacid group was not reduced (Fig. 5). The size of DOPA-positive melanocytes in the both PE groups wascomparable to that of the control group (Fig. 6). Therewas no difference in the number and size of DOPA-positive melanocytes in the normal skin of all groups(Fig. 5 and 6). Discussion The synthesis and activation of tyrosinase are majorprocesses in the UV-induced pigmentation of mam-mals. 16) Many tyrosinase inhibitors such as proantho-cyanidin, 17) arbutin 18) and ellagic acid 13) have beenisolated from plants. Several of these tyrosinase inhib-itors have been used in cosmetics for topical application,but few are used for oral administration. Oral admin-istration of ellagic acid has been studied in themetabolism of the rat, and its metabolites have beendetected in their urine and feces. 19) We hypothesizedfrom these results that ellagic acid might have awhitening effect if administered orally. To obtain a 0100200300400500600Normal UV-irradiated    N  u  m   b  e  r  o   f   D   O   P   A  -  p  o  s   i   t   i  v  e  m  e   l  a  n  o  c  y   t  e  s   (  c  e   l   l  s   /  m  m    2    )  ControlPomegranate extract(100mg/kg)Pomegranate extract(1000mg/kg)L-ascorbic acid(600mg/kg) * Fig. 5.  Number of DOPA-Positive Melanocytes.The number of DOPA-positive melanocytes in the epidermis of guinea pigs administered with water (control), the pomegranate extract, and L -ascorbic acid for 35 days is shown ( n ¼ 6 ).   ,  p  <  0 : 05  compared with the control. -12-10-8-6-4-200 7 14 21 28 35 days       ∆     L          *    v  a   l  u  e ControlPomegranate extract(100mg/kg)Pomegranate extract(1000mg/kg)L-ascorbic acid (600mg/kg) UV irradiation ********** ***** Fig. 3.  Degree of Pigmentation.The degree of pigmentation change (  L  value) is shown between the start, after UV irradiation and after daily administration of water(control, circle), the pomegranate extract (100mg/kg, unshaded triangle), pomegranate extract (1,000mg/kg, shaded triangle), and  L -ascorbicacid 600mg/kg (square) ( n ¼ 6 ).Inhibition of Tyrosinase and Pigmentation by a Pomegranate Extract 2371
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