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Biol. Pharm. Bull. 33(7) 1242—1245 (2010)
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Vol. 33, No. 7
Melanogenesis Inhibitory Effect of Fatty Acid Alkyl Esters Isolated from Oxalis triangularis Sungran HUH,a, # Young-Soo KIM,a, # Eunsun JUNG,a Jihee LIM,b Kwang Sun JUNG,b Myeong-Ok KIM,b,c Jongsung LEE,a and Deokhoon PARK*, a a Biospectrum Life Science Institute; 101–701 SK Ventium, 522 Dangjung Dong, Gunpo, Gyunggi Do 435–833, Republic of Korea: b SkinCure Life Science Institute, Jeju Bio-industry Development Center; Jeju, Jeju Do 690–121, Republic of Korea: and c Department of Cosmetic & Beauty, Sookmyung Women’s University; Seoul 140–742, Republic of Korea. Received November 23, 2009; accepted March 31, 2010; published online April 16, 2010
Ten fatty acid alkyl esters isolated from Oxalis triangularis, were evaluated for the effects on melanogenesis using mouse B16 melanoma cells. Treatment of methyl linoleate, methyl linolenate, ethyl linoleate and ethyl linolenate significantly blocked forskolin-induced melanogenesis and inhibited tyrosinase activity. In addition, we found that they inhibited cAMP production, suggesting that their anti-melanogenic effect is mediated by the inhibition of cAMP production. We concluded that methyl/ethyl linoleate and linolenate isolated from Oxalis triangularis have pigment inhibition activity. These compounds may be useful as the cosmetic agent to stimulate skin whitening. Key words
Oxalis triangularis; melanogenesis; fatty acid alkyl ester
Oxalis triangularis (purple shamrock or purple clover) in the family Oxalidaceae is an edible perennial plant that is easily cultivated. The leaves of Oxalis triangularis are especially appreciated due to their sour and exotic taste. Oxalis triangularis has intensely purple leaves with a monomeric anthocyanin content of 195 mg/100 g on a malvidin 3,5diglucoside basis, which makes them a potential source of natural colorants.1) Additionally, it is known that treatment with the flavones, C-glycosides, as copigments effects the color and stability of anthocyanins.2—5) To date, no studies have been conducted to investigate the inhibitory effect of Oxalis triangularis on melanogenesis. In this study, the fatty acid alkyl esters methyl linoleate, methyl linolenate, ethyl linoleate and ethyl linolenate from Oxalis triangularis were shown to play an active role in inhibition of the cellular production on melanin. MATERIALS AND METHODS General Methods 1H- and 13C-NMR spectral data were determined using a Varian Unity Inova 500NB NMR instrument (500 MHz) using CDCl3 as the solvent and tetramethylsilane (TMS) as an internal standard. The chemical shifts were reported in d (ppm) units relative to the TMS signal and coupling constants (J) in Hz. Gas chromatography-mass spectrometry (GC-MS) and GC-selected ion monitoring (SIM) were conducted using a 5972 Plus mass spectrometer (electron impact ionization, 70 electron volt, HewlettPackard, Palo Alto, CA, U.S.A.) connected to a 5890 gas chromatograph fitted with a fused silica capillary column (HP-5, 0.25⫻30 m, 0.25 m m film thickness, HewlettPackard). The GC-conditions were as follows: on-column injection mode, He 1 ml/min; oven temperature 60 °C for 2 min; and thermal gradient, 10 °C/min to 260 °C. Medium pressure liquid chromatography (MPLC) was conducted using a Combiflash Companion instrument with an UV/Vis detector (Teledyne ISCO, Inc., U.S.A.). Preparative HPLC (High Pressure Liquid Chromatography) was conducted ∗ To whom correspondence should be addressed. These authors contributed equally to this work.
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using a Prep LC 2000 and 2487 Dual l Absorbance detector (Waters). Silica gel (230—400 mesh, Merck) was used for the column chromatography. All HPLC-grade organic solvents and bulk organic solvents were purchased from J.T. Baker and Duksan Company, South Korea. Plant Material Naturally-grown Oxalis triangularis were collected from Jeju Island, Korea, from June to August 2008. A voucher sample has been deposited at the Jeju Bio Diversity Research Institute of the Jeju Hi-Tech Industry Development Institute. Extraction and Isolation Whole bodies of Oxalis triangularis (1.6 kg dry weight) were homogenized and extracted with 80% ethanol (30 l⫻3). The extracts were then concentrated in vacuo, after which they were re-extracted with chloroform (2 l⫻3). After reducing to dryness in vacuo, the chloroform fraction (138 g) was solvent-partitioned between nhexane and 80% methanol (2 l⫻3). The concentrated nhexane soluble fraction (57.58 g) was then purified by silica gel column chromatography (100 g, Merck) and eluted stepwise with chloroform containing 0, 1, 2, 10 and 100% methanol (1 l each). The fractions eluted in 0% and 1% methanol in chloroform were combined and concentrated in vacuo (17.32 g), after which they were subjected to MPLC (RediSep, silica 40 g, 30⫻140 mm; detection, UV at 254 nm; flow rate, 40 ml/min). The elution was performed stepwise in chloroform–methanol (0%, 2%, 10%, 100% methanol) for 10 min each to give 40 subfractions. Fraction 4 was subjected to reversed phase preparative HPLC (Phenomenex Luna C18(2), 21.2⫻250 mm, 5 m m) and eluted with 40% acetonitrile in water for 10 min, then in a gradient to 100% acetonitrile for 20 min, and finally in 100% acetonitrile for 20 min. The samples were eluted at a flow rate of 17 ml/min and the fractions were collected every minute. HPLC fractions 42— 43 (methyl linolenate), 45 (ethyl linolenate), 47 (methyl linoleate) and 51 (ethyl linoleate) showed biological activity; therefore, these fractions were analyzed by GC-MS and 1Hand 13C-NMR. Methyl Linoleate 1H-NMR (500 MHz, CDCl3) d :
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5.40—5.30 (4H, m, H-9, 10, 12, 13), 3.66 (3H, s, –OCH3), 2.77 (2H, t, J⫽6.5 Hz, H-11), 2.30 (2H, t, J⫽7.5 Hz, H-2), 2.07—2.03 (4H, m, H-8, 14), 1.65—1.59 (2H, m, H-3), 1.38—1.26 (14H, m, H-4, 5, 6, 7, 15, 16, 17), 0.89 (3H, t, J⫽6.5 Hz, H-18); 13C-NMR (500 MHz, CDCl3) d : 174.4 (C1), 130.4 (C-13), 130.3 (C-9), 128.3 (C-12), 128.2 (C-10), 51.7 (–OCH3), 34.3 (C-2), 31.7 (C-16), 29.8 (C-7), 29.6 (C6), 29.4 (C-15), 29.3 (C-5), 29.3 (C-4), 27.4 (C-14), 27.4 (C8), 25.8 (C-11), 25.2 (C-3), 22.8 (C-17), 14.3 (C-18). Methyl Linolenate 1H-NMR (500 MHz, CDCl3) d : 5.42—5.29 (6H, m, H-9, 10, 12, 13, 15, 16), 3.60 (3H, s, –OCH3), 2.82—2.80 (4H, H-11, 14), 2.30 (2H, t, J⫽7.5 Hz, H-2), 2.11—2.03 (4H, m, H-8, 17), 1.65—1.59 (2H, m, H-3), 1.38—1.31 (8H, m, H-4, 5, 6, 7), 0.88 (3H, t, J⫽7.5 Hz, H18); 13C-NMR (500 MHz, CDCl3) d : 174.4 (C-1), 132.1— 127.3 (6C, C-9, 10, 12, 13, 15, 16), 51.6 (–OCH3), 34.3 (C2), 29.7—29.4 (3C, C-5, 6, 7), 29.3, 27.4, 25.8, 25.2 (C-3), 20.8 (C-17), 14.5 (C-18). Ethyl Linoleate 1H-NMR (500 MHz, CDCl3) d : 5.41— 5.30 (4H, m, H-9, 10, 12, 13), 4.13 (2H, m, –OCH2–), 2.77 (2H, t, J⫽7.0 Hz, H-11), 2.28 (2H, t, J⫽7.5 Hz, H-2), 2.08— 2.03 (4H, m, H-8, 14), 1.69—1.60 (2H, m, H-3), 1.38—1.31 (14H, m, H-4, 5, 6, 7, 15, 16, 17), 1.26—1.23 (3H, m, H-20), 0.89 (3H, t, J⫽6.5 Hz, H-18); 13C-NMR (500 MHz, CDCl3) d : 174.1 (C-1), 130.4 (C-13), 130.3 (C-9), 128.3 (C-12), 128.2 (C-10), 60.4 (–OCH2–), 34.6 (C-2), 31.8 (C-16), 29.8 (C-7), 29.6 (C-6), 29.4 (C-15), 29.3 (C-5), 27.4 (C-4), 27.4 (C-14), 27.4 (C-8), 25.8 (C-11), 25.2 (C-3), 22.8 (C-17), 14.4 (C-20), 14.3 (C-18). Ethyl Linolenate 1H-NMR (500 MHz, CDCl3) d : 5.42—5.29 (6H, m, H-9, 10, 12, 13, 15, 16), 4.12 (2H, m, –OCH2–), 2.84—2.78 (4H, H-11, 14), 2.28 (2H, t, J⫽7.0 Hz, H-2), 2.11—2.03 (4H, m, H-8, 17), 1.64—1.59 (2H, m, H-3), 1.38—1.31 (8H, m, H-4, 5, 6, 7), 1.26—1.23 (3H, m, H-20), 0.98 (3H, t, J⫽7.5 Hz, H-18); 13C-NMR (500 MHz, CDCl3) d : 174.0 (C-1), 132.2—127.4 (6C, C-9, 10, 12, 13, 15, 16), 60.3 (–OCH2–), 34.6 (C-2), 29.8—28.3 (3C, C-5, 6, 7), 28.3, 27.4, 25.8, 25.2 (C-3), 20.8 (C-17), 14.5 (2C, C-18, C-20). Cell Culture B16 mouse melanoma cells were purchased from the Korean Cell Bank (Seoul, Korea). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 m g/ml streptomycin at 37 °C in a humidified, 5% CO2 controlled incubator. Melanin Content Assay The melanin content of cultured B16 cells was determined following the method described by Kinoshita et al.6) Briefly, cells were washed with phosphate-buffered saline (PBS) and then dissolved in 1 N NaOH for 1 h at 60 °C. The absorbance of each sample at 475 nm was then measured, after which the melanin content was determined by comparing these values to those of an authentic standard of synthetic melanin. Finally, the protein concentration of each sample was measured using the DC protein assay reagent (Bio-Rad, Hercules, CA, U.S.A.) with bovine serum albumin (BSA) as a standard. MTT Assay The general viability of cultured cells was determined based on the reduction of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan. Briefly, following treatment with fatty acid alkyl esters, cells were incubated for 24 h at 37 °C under 5% CO2. MTT solution (1 mg/ml in PBS) was then added to each well. Next, the
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cells were incubated at 37 °C for 3 h, after which the media was discarded. Dimethyl sulfoxide was then added to dissolve the formazan crystals, after which the absorbance was measured at 570 nm using a spectrophotometer. Tyrosinase Luciferase Reporter Assay To assay for tyrosinase promoter activity, B16 melanoma cells were transfected with tyrosinase reporter along with Renilla luciferase expression vector driven by the thymidine kinase promoter (Promega, Madison, WI, U.S.A.) using SuperfectTM reagent (Qiagen, Germantown, MD, U.S.A.). After incubation for 24 h, B16 melanoma cells were treated for 14 h with fatty acid alkyl esters. The cells were then harvested and lysed, after which the supernatants were assayed for luciferase activity using a Dual Luciferase Assay System (Promega, Madison, WI, U.S.A.). cAMP Immunoassay The cAMP concentration was measured using a cAMP immunoassay kit produced by Cayman. Briefly, B16 cells (7⫻105) were lysed in 0.1 M HCl to inhibit the phosphodiesterase activity. The supernatants were then collected, neutralized, and diluted. Following neutralization and dilution, a fixed amount of cAMP conjugate was added to compete with the cAMP in the cell lysates for sites on a rabbit polyclonal antibody immobilized on a 96 well plate. Next, the samples were washed to remove the excess conjugated and unbound cAMP, after which a substrate solution was added to the wells to determine the activity of the bound enzyme. The color development was then stopped, and the absorbance at 420 nm was then read. The intensity of the color was inversely proportional to the concentration of cAMP in the cell lysates. Statistical Evaluation The means⫾S.E.M. were calculated and compared among groups using a t-test for independent samples. Values of ∗ p⬍0.05 were considered to be significant [ED highlight⫺consider deleting this, e.g., “A p⬍0.05 was considered to be significant.” The asterisk would be useful if there were 2 values (e.g. ∗ p⬍0.05, ∗∗ p⬍0.01) or in a figure legend]. RESULTS AND DISCUSSION Whole bodies of Oxalis triangularis were extracted with 80% aqueous ethanol, followed by chloroform. The extract was then solvent-partitioned between n-hexane and 80% methanol, after which the concentrated n-hexane soluble fraction was purified by silica gel column chromatography and subjected to MPLC. The GC-MS analysis of the MPLC fraction 4 of Oxalis triangularis revealed the presence of five fatty acid methyl esters (FAMEs) and five fatty acid ethyl esters (FAEEs). Based on a comparison with authentic FAMEs and FAEEs, these compounds were identified as methyl palmitate, ethyl palmitate, methyl linoleate, methyl linolenate, methyl oleate, methyl stearate, ethyl linolenate, ethyl oleate, ethyl linoleate, and ethyl stearate (Fig. 1). The endogenous levels of FAMEs and FAEEs in Oxalis triangularis were also determined based on comparison with authentic FAMEs and FAEEs. Specifically, the levels were found to be 248.19 (methyl palmitate), 367.99 (ethyl palmitate), 149.09 (methyl linoleate), 124.10 (methyl linolenate), 186.31 (methyl oleate), 54.36 (methyl stearate), 327.75 (ethyl linolenate), 173.93 (ethyl oleate), 488.55 (ethyl linoleate) and 66.58 (ethyl stearate) m g/g fr. weight. For purification, MPLC
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Fig. 1.
Vol. 33, No. 7
GC-Chromatogram of MPLC Fraction 4 from Oxalis triangularis (A) and Authentic FAMEs and FAEEs (B)
1, Methyl palmitate (17.70 min); 2, ethyl palmitate (18.36 min); 3, methyl linoleate (19.35 min); 4, methyl linolenate (19.40 min); 5, methyl oleate (19.41 min); 6, methyl stearate (19.61 min); 7, ethyl linolenate (19.95 min); 8, ethyl oleate (20.01 min); 9, ethyl linoleate (20.02 min); 10, ethyl stearate (20.21 min).
Fig. 2.
Melanin Content Assay (A) and MTT Assay (B) of FAMEs and FAEEs from Oxalis triangularis
Methyl palmitate (MP), methyl stearate (MS), methyl oleate (MO), methyl linoleate (MLA), methyl linolenate (MLN), ethyl palmitate (EP), ethyl stearate (ES), ethyl oleate (EO), ethyl linoleate (ELA), ethyl linolenate (ELN). Medium only (M), Forskolin 5 m M (F), and arbutin 500 m M (A) were used for control (Cont).
fraction 4 was subjected to reversed phase preparative HPLC. As a result, HPLC fractions 42 and 43 (methyl linolenate), 45 (ethyl linolenate), 47 (methyl linoleate) and 51 (ethyl linoleate), were obtained. To determine the effects of these 5 FAMEs and 5 FAEEs isolated from Oxalis triangularis on melanogenesis, we investigated the levels of melanin content in cultured B16 melanoma cells. We found that methyl linoleate, methyl linolenate, ethyl linoleate and ethyl linolenate reduced the levels of melanin content in a concentration-dependent manner. Specifically, methyl linoleate, methyl linolenate, ethyl linoleate and ethyl linolenate decreased the level of melanin content by 82—54%, 80—25%, 92—47%, and 91—30%, respectively, when compared with forskolin-treated control cells (Fig. 2A). However, there is the possibility that the reduction of melanin content was induced by a cytotoxic effect of these compounds. Therefore, we conducted an MTT assay in B16 melanoma cells. The results revealed that methyl/
ethyl linoleate and linolenate had no cytotoxic effect when administered at 100 m M (Fig. 2B). The melanogenesis-inhibitory activities of the four compounds were further evaluated by determining their half maximal inhibitory concentration (IC50), which is a measure of the effectiveness of a compound at inhibiting biological function. The IC50 of these compounds was determined using a melanin content assay. As shown in Table 1, while the IC50 of methyl and ethyl linoleates was 245 m M and 325 m M, respectively, the IC50 of methyl and ethyl linolenates was 60 m M and 70 m M, respectively. These results indicate that methyl and ethyl linolenates have more potent anti-melanogenic activity than methyl and ethyl linoleates. In a tyrosinase luciferase reporter assay, all of the compounds inhibited tyrosinase promoter activity induced by forskolin, which was consistent with the results of the melanin content assay (Fig. 3). In addition, the compounds reduced forskolin-induced cAMP production (Fig. 4). These findings suggest that these com-
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Table 1. Anti-melanogenesis Effects of FAMEs and FAEEs Isolated from Oxalis triangularis Compounds
Inhibition (IC50 value, m M)
Methyl linoleate Ethyl linoleate Methyl linolenate Ethyl linolenate Linoleate Linolenate
245 325 60 70 35 20
Fig. 3. Effects of Methyl/Ethyl Linoleate and Linolenate on Tyrosinase Promoter Activity Methyl linoleate (MLA), methyl linolenate (MLN), ethyl linoleate (ELA), ethyl linolenate (ELN). Medium only (M) and forskolin 5 m M (F) were used as controls (Cont).
nate and ethyl linolenate. It has been reported that unsaturated fatty acids such as oleic acid, linoleic acid and a linolenic acid decrease melanin synthesis and tyrosinase activity,9) and that these inhibitory effects occur in proportion to the number of unsaturated bonds. Accordingly, melanogenesis is inhibited most effectively by a -linolenic acid, followed by linoleic acid and then oleic acid.9) Consistent with these findings, the results of the present study revealed that methyl and ethyl linolenates exerted higher inhibitory activity against melanin synthesis than methyl and ethyl linoleates. In addition, these results suggest that methyl and ethyl group may act as a negative factor in the antimelanogenic effect. Lotus (Nelumbo nuficera) flower essential oil, comprised of palmitic acid methyl ester (22.6%), linoleic acid methyl ester (11.16%), palmitoleic acid methyl ester (7.55%), and linolenic acid methyl ester (5.16%) was reported to induce melanogenesis through the phosphorylation of extracellular signal-regulated kinase (ERK) and CREB.10) This result is not consistent with our data that linoleic acid methyl ester and linolenic acid methyl ester inhibit melanogenesis through the PKA dependent signaling. However, Choi et al.10) reported that linoleic acid methyl ester inhibits melanogenesis in B16 cells, supporting our data that FAMEs and FAEEs from Oxalis triangularis are involved in the inhibition of melanogenesis.11) Collectively, FAMEs and FAEEs isolated from Oxalis triangularis were characterized and evaluated for their inhibitory effects against melanogenesis in B16 melanoma cells. Among them, methyl/ethyl linoleate and linolenate have a depigmenting activity and may be introduced as a possible therapeutic agent for hyperpigmentation or as a cosmetic lightening agent. Acknowledgements This work was supported by a grant from the Small and Medium Business Administration (S1040155) in Korea. REFERENCES
Fig. 4. Effects of Methyl/Ethyl Linoleate and Linolenate on cAMP Production Methyl linoleate (MLA), methyl linolenate (MLN), ethyl linoleate (ELA), ethyl linolenate (ELN). Medium only (M) and forskolin 5 m M (F) were used as controls (Cont).
pounds inhibit melanogenesis by operating upstream of the cAMP production step. Many investigations have focused on the specific mechanisms involved in melanogenesis to develop new therapeutic agents for skin pigmentation abnormalities.7) Most popular whitening agents, including hydroquinone (HQ), kojic acid and arbutin (HQ b -D-gluconopyranoside), act as tyrosinase inhibitors.8) We found that Oxalis triangularis contains melanin biosynthesis inhibitors, which were isolated and identified as methyl linoleate, ethyl linoleate, methyl linole-
1) Pazmiño-Durán E. A., Giusti M. M., Wrolsta, R. E., Glória M. B. A., Food Chem., 75, 211—216 (2001). 2) Hosino T., Matsumoto U., Goto T., Phytochemistry, 19, 663—667 (1980). 3) Yabuya M., Saito M., Iwashina T., Yamaguchi M., Euphytica, 115, 1— 5 (2000). 4) Bloor S. J., Phytochemistry, 50, 1395—1399 (1999). 5) Fukui Y., Tanaka Y., Kusumi T., Iwashita T., Nomoto K., Phytochemistry, 63, 15—23 (2003). 6) Kinoshita M., Hori N., Aida K., Sugawara T., Ohnishi M., J. Oleo Sci., 56, 645—648 (2007). 7) Kim S. B., Lee J. S., Jung E. S., Lee J. H., Huh S. R., Hwang H. J., Kim Y. S., Park D. H., Arch. Dermatol. Res., 301, 253—258 (2009). 8) Kooyers T. J., Westerhof W., Ned. Tijdschr. Geneeskd., 148, 768—771 (2004). 9) Ando H., Ryu A., Hashimoto A., Oka M., Ichihashi M., Arch. Dermatol. Res., 290, 375—381 (1998). 10) Choi J. Y., Choi E. H., Jung H. W., Oh J. S., Lee W. H., Lee J. G., Son J. K., Kim Y., Lee S. H., Arch. Pharm. Res., 31, 294—299 (2008). 11) Jeon S., Kim N. H., Koo B. S., Kim J. Y., Lee A. Y., Exp. Mol. Med., 41, 517—525 (2009).
