Effect of Oenanthe javanica Ethanolic Extracts on Antioxidant Activity and Melanogenesis in Melanoma Cells

Effect of Oenanthe javanica Ethanolic Extracts on Antioxidant Activity and Melanogenesis in Melanoma Cells

Eun-Jeong Kwon and Moon-Moo Kim*

Department of Chemistry, Dong-Eui University, Busan 614-714, Korea

Received October 25, 2013 /Revised December 10, 2013 /Accepted December 10, 2013

The aim of this study is to investigate the melanogenic effect of Oenanthe javanica ethanolic extracts (OJE) containing quercetin and kaempferol in melanoma cells (B16F1). In order to determine whether OJE inhibits melanin synthesis at the cellular level, the melanoma cells were cultured in the presence of different concentrations of OJE. In the present study, the antioxidant effects of OJE on DPPH radi- cal scavenging, power reduction, lipid peroxidation, and DNA oxidation were evaluated in a cell free system. Furthermore, the effect of OJE on the production of melanin was determined by dopaquinone (DOPA) assay and tyrosinase activity. In addition, the protein expression of tyrosinase, as well as anti- oxidant enzymes such as superoxide dismutase (SOD)-1, SOD-2 and glutathione reductase (GSH), were examined using Western blot analysis. In this study, it was observed that OJE exhibited an inhibitory effect on lipid peroxidation and blocked the DNA oxidation induced by the hydroxyl radical produced by Fenton’s reagent. OJE increased melanin synthesis above 50 μg/ml and tyrosinase activity was de- tected above 50 μg/ml. In Western blot analysis, OJE increased the expression levels of tyrosinase, SOD-1, SOD-2, and GSH in a dose-dependent manner. These findings indicate that OJE with anti- oxidant activity can regulate the tyrosinase activity and melanin production in melanocyte, suggesting that it could promote the development of black hair as well as protect skin from oxidative stress.

Key words : Melanocyte, Oenanthe javanica, superoxide dismutase, tyrosinase

*Corresponding author

*Tel : +82-51-890-1511, Fax : +82-51-890-2620

*E-mail : [email protected]

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Journal of Life Science 2013 Vol. 23. No. 12. 1428~1435 DOI : http://dx.doi.org/10.5352/JLS.2013.23.12.1428

Introduction

Reactive oxygen species (ROS) are generated as by-prod- ucts of cellular metabolism, primarily in the mitochondria [1]. Biomolecules such as lipids, proteins, and DNA are dam- aged by the ROS [11]. In particular, the unpaired electrons of oxygen including superoxide, hydrogen peroxide, hy- droxyl radical, and peroxynitrite react to form partially re- duced ROS [28]. In recent years, some ROS has been known to modulate enzyme activity as well as transcription factors [2]. ROS generated during melanogenesis have been im- plicated as cytotoxic molecules in the immune responses of insects against their internal metazoan parasites [15, 21].

Melanin produced by melanocyte exists in hair, eyes and skin [26]. Melanoma cell stretch swelling and transfer mela- nin to neighboring cells such as keratinocyte [27]. It function as to protect human body that melanin absorb UV [3]. Skin

gets darken than normal one because melanin formation in-

creases in melanocyte [18]. Melanocyte synthesizes melanin

from L-tyrosine through stimulation of such as exposes to

UV [17]. Tyrosinase which is rate-limiting enzyme, change

from L-tyrosine to L-dihydroxy phenylalanine (L-DOPA)

and finally to DOPA quinine [9]. DOPA chrome tautomerase

(DCT) and tyrosinase-related protein-1 (TRP-1) involve syn-

thesis of eumelain, black or brown pigment. Tyrosinase ex-

pression is raised when melanocyte is treated with melano-

cyte-stimulating hormone (α-MSH). The α-MSH augments

cyclic AMP (cAMP) through melanocortin-1-receptor (MC1R)

which increases the expression of microphthalmia-associated

transcription factor (MITF) and activate cAMP responsive el-

ement binding protein (CREB). MITF which is transcription

factor to involve expression of tyrosinase and tyrosinase-re-

lated protein-1 (TRP-1) enhances the gene expression of ty-

rosinase and TRP-1 [16, 31]. Various reports have been pub-

lished on Javan waterdropwort to prevent alcoholic liver dis-

ease over a long period of time. Javan waterdropwort

(Oenanthe javanica) inhibits a wetland in many Asia countries

[14]. It was reported to contain flavonoids, choline, rutamin,

γ -fagarine and coumarine [22, 33]. In addition, it has anti-

oxidant ingredients such as vitamin E, eugenyl beta-D-gluco-

pyranoside, pinoresinol beta-D-glucopyranoside, oenantho-

side A and 2, 3-ethylenedioxy-5-allylphenyl beta-D-gluco- pyranoside [19]. Therefore, in this study it was examined that Oenanthe javanica ethanolic extracts with antioxidant ac- tivity could modulate melanin synthesis

Materials and Methods Materials

Dulbecco’s Modified Eagle’s Medium (DMEM), Trypsin- EDTA, penicillin/ streptomycin/ amphotericin (10,000 U/ml, 10,000 μg/ml, and 2,500 μg/ml, respectively), fetal bovine serum (FBS) reagent were obtained from Gibco BRL, Life Technologies (Paisley, Scotland, UK). B16F1 cells were obtained from American Type of Culture Collection (Manas- sas, VA, USA), MTT reagent, gelatin, agarose, and PMA (phorbol 12-myristate 13-acetate) and other materials were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Extract preparation

For preparing Oenanthe javanica ethanolic extracts (OJE), the air-dried Oenanthe javanica ethanolic leaves and stems were homogenized using a grinder before extraction. The dried powder (1 kg) was extracted with 95% EtOH (1:10 w/v) and evaporated in vacuo. The concentrated OJE were freshly dissolved in DMSO before use.

Cell culture

Cell lines were separately grown as monolayers at 5% CO

and 37℃ humidified atmosphere using appropriate media supplemented with 10% fetal bovine serum, 2 mM gluta- mine and 100 μg/ml penicillin-streptomycin. DMEM was used as the culture medium for B16F1 cells. Cells were pas- saged 3 times a week by treating with trypsin-EDTA and used for experiments after 5 passages.

MTT assay

Cytotoxic levels of OJE on B16F1 cells were measured us- ing MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bro- mide) method as described by Hansen et al [13]. B16F1 cells were grown in 96-well plates at a density of 5×10

cells/well.

After 24 h, cells were washed with fresh medium and were treated with different concentrations of OJE. After 48 h of incubation, cells were rewashed and 20 μl of MTT (5 mg/ml) was added and incubated for 4 h. Finally, DMSO (150 μl) was added to solubilize the formazan salt formed and amount of formazan salt was determined by measuring the

OD at 540 nm using an GENios

microplate reader (Tecan Austria GmbH, Austria). Relative cell viability was de- termined by the amount of MTT converted into formazan salt. Viability of cells was quantified as a percentage com- pared to the blank and dose response curves were developed.

The data were expressed as mean from at least three in- dependent experiments and p<0.05 was considered significant.

DPPH radical scavenging assay

Freshly prepared 500 μl of DPPH (2, 2-diphenyl-1-pic- rylhydrazyl) solution was thoroughly mixed with 500 μl of OJE. The reaction mixture was incubated for 1 h at room temperature. Absorbance of the resultant mixture was re- corded at 532 nm using UV-VIS spectrophotometer. Vitamin C at 10 μg/ml was used as a positive control in this study.

The content of DPPH radical was calculated with absorbance and expressed as a percentage, compared to blank group.

Reducing power

Various concentrations of OJE were mixed with 200 μl of 200 mM sodium phosphate buffer (pH 6.6) and 1% potas- sium ferricynide. The mixture was incubated at 50℃ for 20 min. After added 200 μl of 10% trichloroacetic acid (TCA), the mixture was centrifuged at 3,000 rpm for 10min. The upper layer was mixed with 250 μl distilled water and 50 μ l of 1% ferrichloride and the absorbance was measured at 700 nm using UV-VIS spectrophotometer. Vitamin C at 100 μ g/ml was used as a positive control in this study. The level of reducing power was calculated with absorbance and ex- pressed as a percentage, compared to blank group.

Lipid peroxidation in-vitro using TBARS assay

The inhibitory effect of OJE on lipid peroxidation was in- vestigated using TBARS assay. OJE was added to 9 ml of 0.04 M phosphate buffer and 2.88 ml of 2.51% linolenic acid and then incubated at 40℃. This solution was added to 0.75% TBA and 35% trichloroacetic acid which was then add- ed to the reaction solution, heated for 15 min at 95℃ in a water bath, and cooled immediately. The absorbance at 517 nm was measured using UV-VIS spectrophotometer. The level of lipid peroxidation was calculated with absorbance and expressed as a percentage, compared to blank group.

Genomic DNA isolation

Genomic high molecular weight DNA was extracted from

B16F1 cells using standard phenol/proteinase K procedure

with slight modifications [30]. Briefly, cells culturing in 10 cm dishes were washed twice with PBS and scraped into 1 ml of PBS containing 10 mM EDTA. After centrifugation cells were dissolved in RNase (0.03 mg/ml), sodium acetate (0.175 M), proteinase K (0.25 mg/ml) and SDS (0.6%). The mixture was then incubated for 30 min at 37℃ and 1 h at 55℃. Following incubation, phenol:chloroform:isoamylalcohol was added at 1:1 ratio and mixture was centrifuged at 6,000×

g for 5 min at 4℃. Following centrifugation, supernatant was mixed with 100% ice cold ethanolic at 1:1.5 ratio and kept for 15 min at -20℃. After centrifugation at 14,000× g for 5 min, the pellet was dissolved in TE buffer and purity of DNA was spectrophotometrically determined at 260/

280 nm. Further, the quality of isolated DNA was evaluated with 1% agarose gel electrophoresis in 0.04 M Tris–acetate 0.001 M EDTA buffer.

Determination of radical mediated DNA damage

H2O2 mediated DNA oxidation was determined by a method described previously [25]. For that, 100 μl of DNA reaction mixture was prepared by adding pre-determined concentrations of OJE (or same volume of distilled water as control), 200 μM final concentration of FeSO4, 1 mM final concentration of H

O

and 50 μg/ml final concentration of genomic DNA in the same order. Then the mixture was in- cubated at room temperature for 30 min and reaction was terminated by adding 10 mM final concentration of EDTA.

Aliquot (20 μl) of reaction mixture containing about 1 μg of DNA was electrophorased on a 1% agarose gel for 30 min at 100 V. Gels were then stained with 1 mg/ml ethidium bromide and visualized by UV light using AlphaEase® gel image analysis software (Alpha Innotech, CA, USA). The level of DNA protection was calculated with absorbance and expressed as a percentage, compared to blank group.

Assay of cellular tyrosinase activity

Cellular tyrosinase activity using L-DOPA as the substrate was assayed by the method of Maeda and Fukuda [20].

About 1×10

cells were washed with 10 mM phosphate-buf- fered saline (PBS) and lysed with 45 μl of 1% Triton X-100- PBS. After sonication, 5 μl of 20mML-DOPA were added to each well. The 96 well plates were incubated at 37℃ for 1 h, and the absorbance was measured at 475 nm using an UV-VIS spectrophotometer. The absorbance values were compared with a standard curve obtained with purified mushroom tyrosinase. The standard curve was linear within

the range of experimental values [23].

Melanin synthesis assay

Melanin contents were measured as previously described [29], but with a slight modification. Briefly, the cells were treated with the samples in DMEM containing 10% FBS for 3 d. After concentration by centrifugation, the cell pellets were dissolved in 1 ml of 1 N NaOH at 100℃ for 30 min and centrifuged again for 20 min at 16,000× g. The optical densities of the supernatants were measured at 400 nm using an UV-VIS spectrophotometer [6]. The level of DNA pro- tection was calculated with absorbance and expressed as a percentage, compared to blank group.

Western blot analysis

Western blotting was performed according to standard procedures. Briefly, B16F1 cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.5), 0.4% Nonidet P-40, 120 mM NaCl, 1.5 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride, 80 μ g/ml leupeptin, 3 mM NaF and 1mM DTT at 4℃ for 30 min. 10 μg of cell lysates were resolved on gradient 4-20%

Tris-HCl gels (Novex), electrotransferred onto a nitro- cellulose membrane, and blocked in 5% BSA. The following primary antibodies were used: SOD-1, SOD-2, SOD-3, gluta- thione reductase, tyrosinase and β-actin. Primary antibodies were detected by chemiluminescent ECL kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Image of bands on Western blots was obtained using LAS3000® Luminescent image analyzer (Fujifilm Life Science, Tokyo, Japan).

Results DPPH radical scavenging assay of OJE

DPPH radical scavenging assay is the simplest method to measure the ability of antioxidants to remove free radicals. DPPH is converted from purple into yellow color when exposed to antioxidant. After DPPH was reacted with OJE, the absorbance at 517 nm was measured. As depected in Fig. 1, vitamin C at 10 μg/ml used as a positive control in this experiment clearly showed antioxidant effect on DPPH radical. But OJE is not showed DPPH radical scaveng- ing activity.

Inhibitory effect of OJE on lipid peroxidation

TBARS assay was performed to investigate inhibitory ef-

Fig. 1. Scavenging effect of DPPH radical. Vitamin C (Vit C) at 10 μg/ml was used as a positive control. Data are given as means of values ± S.D. from three independent experiments (*** p <0.001).

Fig. 2. Inhibitory effect of OJE on lipid peroxidation. Vitamin E (Vit E) at 100 μg/ml was used as a positive control.

Lipid peroxidation was determined by TBARS. Data are given as means of values ± S.D. from three independent experiments (* p <0.05, ** p <0.01).

Fig. 3. Reducing power of OJE. Vitamin C (Vit C) at 10 μg/ml was used as a positive control. Data are given as means of values ± S.D. from three independent experiments (** p <0.01, *** p <0.001).

Fig. 4. Protective effect of OJE on DNA oxidative damage in- duced by hydroxyl radical. Genomic DNA purified from B16F1 cells was pre-treated with OJE for 1 h and ex- posed to OH· using fenton reaction. After 30 min, re- action mixture containing 5 μg of DNA was electro- phoresed on a 1% agarose gel for 30 min at 100 V and visualized by UV light after stained with 1 mg/ml ethi- dium bromide. Data are given as means of values ± S.D.

from three independent experiments (** p <0.01, *** p <

0.001).

fect of OJE on lipid peroxidation. Vitamin E at 100 μg/ml was used as a positive control in this experiment. As shown in Fig. 2, OJE significantly inhibited lipid peroxidation at 50 μg/ml compared with blank (p<0.05). It was observed that OJE reduced the lipid peroxidation by 27%.

Reducing Power of OJE

The reducing ability of a compound generally depends on the power of reductants, which exhibit antioxidative po- tential by breaking the free radical chain, donating a hydro- gen atom. Vitamin C at 10 μg/ml was used as a positive control in this experiment. As shown in Fig. 3, OJE at 10 μ g/ ml or higher significantly exerted reducing power com- pared with blank group.

Protective effect of OJE on DNA oxidative damage induced by hydroxyl radical

In a subsequent experiment, genomic DNA was isolated

from B16F1 cells to study the protective effect of OJE against

DNA oxidative damage induced by hydroxyl radical. It was

clearly observed that the genomic DNA of blank group was

almost degraded by hydroxyl radical produced by fenton

reaction, as shown in Fig. 4. Treatment with OJE significantly

inhibited the oxidative damage of DNA at 50 μg/ml or high-

Fig. 5. Viability of B16F1 cells treated with OJE. Data are given as means of values ± S.D. from three independent experiments.

Fig. 6. Effect of OJE on tyrosinase activity in B16F1. Vitamin C (Vit C) at 1,000 μg/ml was used as a positive control.

Data are given as means of values ± S.D. from three independent experiments (* p <0.05 ** p <0.01).

Fig. 7. Effect of OJE on melanin synthesis in B16F1. Vitamin C (Vit C) at 1,000 μg/ml was used as a positive control.

Data are given as means of values ± S.D. from three independent experiments (* p <0.05 , ** p <0.01).

Fig. 8. Effect of OJE on protein expressions of tyrosinase, SOD-1, SOD-2 and GSH in B16F1 cells. The cells were treated with OJE at 1, 5, 10 and 50 μg/ml. Western blot analysis of cell lysates was performed using the antibodies as indicated.

er compared with blank group.

Effect of OJE on cell viability

In order to investigate the cytotoxic effect of OJE on B16F1 cells, MTT assay was carried out following treatment with OJE at the indicated concentration. Fig. 5 showed that OJE at all concentrations did not exert any cytotoxic effect on melanoma cells. On that ground, this result demonstrates that OJE below 100 μg/ml has no cytotoxicity in B16F1 cells used in this study.

Effect of OJE on tyrosinase activity

In order to investigate effect of OJE on tyrosinase activity in B16F1, this experiment was carried out following treat- ment with OJE at the indicated concentration. As shown in Fig. 6. vitamin C at 1,000 μg/ml used as a positive control inhibited tyrosinase activity by 24%. However, OJE did not

inhibit tyrosiase activity. In contrast, it increase tyrosiase ac- tivity by 12% at 1,000 μg/ml in B16F1 cells.

Effect of OJE on melanin generated by L-DOPA

In order to investigate effect of OJE on melanin synthesis B16F1, L-DOPA was used as a substrate following treatment with OJE at the indicated concentration. As shown in Fig.

7. vitamin C at 1,000 μg/ml used as a positive control in- hibited melanin synthesis by 30%. Similar to the result of tyrosinase, OJE did not inhibit melanin synthesis. In contrast, it increase melanin synthesis promoted melanin synthesis B16F1 cells.

Effect of OJE on protein expressions in melanoma cells

In order to clarify how the activity of tyrosinase is in-

creased by OJE, the expression of proteins involved in sig-

naling pathways of melanin synthesis was investigated us-

ing western blot analysis. After B16F1 cells were treated with

OJE at 1, 5, 10 and 50 μg/ml, cell lysates was collected and their protein expressions were evaluated using antibodies including tyrosinase, SOD-1, SOD-2 and glutathione reduc- tase (GSH). As shown in Fig. 8, OJE treatment at 10 μg/ml enhanced the expression level of SOD-1and SOD-2 in B16F1 cells. In addition, the expression level of GSH was increased in the presence of OJE at 10 μg/ml. These results indicate that it could induce the expression of antioxidant enzymes capable of suppressing oxidative stress.

Discussion

Reactive oxygen species (ROS) produced by from mi- tochondria and other cellular organelle have been tradition- ally regarded as toxic by-products of metabolism leading to damage lipids, proteins, and DNA [10, 28]. For this reason, cells possess several antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase to protect themselves from against the potentially damaging effects of ROS. Thus oxidative stress may be broadly defined as an imbalance between oxidant production and the antioxidant capacity of the cell to prevent oxidative injury. These Oxidative stresses have been implicated in a large number of human diseases including atherosclerosis, pulmonary fib- rosis, cancer, neurodegenerative diseases, and aging [8, 12, 28]. Previous studies have reported that these ROS were eliminated by antioxidant like vitamin C, vitamin E, BTH and BTA [4]. In this study we investigated the effect of OJE on melanin production related to antioxidant activity in mel- anocyte, B16F1. OJE exhibited inhibitory effect of lipid per- oxidation and reducing power in in vitro. These results sug- gest that OJE can reacts radical and eliminate free radical.

Next, DNA oxidation assay was carried out using the ge- nomic DNA isolated from melanocyte in order to look into protective effect of OJE on DNA oxidative damage. It was observed that OJE exert the protective effect on DNA dam- age caused by hydroxyl radical. These findings support the assumption that components of OJE strongly reacts phos- phate group with negative charge in nucleic acid, so that they protect DNA [5]. Our results are consistent with pre- vious report that natural compounds with antioxidant activ- ity can inhibit DNA damage induced by hydroxyl radical.

Previous reports suggested that ROS play a key role in mela- nin synthesis. Melanin synthesized by tyrosinase has been widely known to be involved in not only formation of mel- asma and freckle but also generation of skin, hair and pupil

color [21]. Thus, tyrosinase divided from B16F1 was used to investigate effect of OJE on tyrosinase activity. For that, B16F1 cells were exposed to L-DOPA to stimulate melanin synthesis. Our result confirmed that OJE increased ty- rosinase activity. In general tyrosinase oxidizes 3,4-dihy- droxyphenylalanine (DOPA) lead to production of melanin [21, 24]. Unlike our expectation that antioxidant inhibits mel- anin systhesis, even though OJE has antioxidant activity, it could not inhibit oxidation of L-DOPA. In this study it was found that OJE increases the synthesis of melanin in DOPA-induced melanocyte. Therefore, it was suggested that OJE augment melanin synthesis by tyrosinase activation.

These results are in a good agreement with that component of OJE, quercetin, stimulates melanogenesis by increasing ty- rosinase activity [23]. In addition, the effect of OJE on ex- pression of antioxidant enzymes and tyrosinase were exam- ined in B16F1 cells. In this study, OJE increased the ex- pression of antioxidant enzyme such as SOD-1, -2 and GSH.

Previous study demonstrated that quercetin has protective effect in diabetes by decreasing oxidative stress and preser- vation of pancreatic cell integrity [7]. The expression of ty- rosinase was enhanced in the presence of OJE. Therefore, this result reveals that the increase in melanin synthesis is due to the enhanced expression level of tyrosinase [32]. In conclusion, these findings suggest that OJE with antioxidant activity can regulate the tyrosinase activity and melanin pro- duction in melanocyte, suggesting that it could not only pro- tect skin from oxidative stress but also promote develop- ment of black hair.

Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (No. 2013R1A1A1A05005160).

References

1. Adam-Vizi, V. and Chinopoulos, C. 2006. Bioenergetics and the formation of mitochondrial reactive oxygen species.

Trends Pharmacol Sci 27, 639-645.

2. Apel, K. and Hirt, H. 2004. Reactive oxygen species: metabo- lism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55, 373-399.

3. Brenner, M. and Hearing, V. J. 2008. The protective role of

melanin against UV damage in human skin. Photochem

Photobiol 84, 539-549.

4. Chen, X., Touyz, R. M., Park, J. B. and Schiffrin, E. L. 2001.

Antioxidant effects of vitamins C and E are associated with altered activation of vascular NADPH oxidase and super- oxide dismutase in stroke-prone SHR. Hypertension 38, 606-611.

5. Choi, H., You, Y., Hwang, K., Lee, J., Chun, J., Chung, J.

W., Shim, S., Park, C.-S. and Jun, W. 2011. Isolation and identification of compound from dropwort (Oenanthe jav- anica) with protective potential against oxidative stress in HepG2 cells. Food Sci Biotechnol 20, 1743-1746.

6. Chung, S.-Y., Seo, Y.-K., Park, J.-M., Seo, M.-J., Park, J.-K., Kim, J.-W. and Park, C.-S. 2009. Fermented rice bran down- regulates MITF expression and leads to inhibition of α -MSH-induced melanogenesis in B16F1 melanoma. Biosci Biotechnol Biochem 73, 1704-1710.

7. Coskun, O., Kanter, M., Korkmaz, A. and Oter, S. 2005.

Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and β-cell damage in rat pancreas. Pharmacol Res 51, 117-123.

8. Cross, C. E., Halliwell, B., Borish, E. T., Pryor, W. A., Ames, B. N., Saul, R. L., McCord, J. M. and Harman, D. 1987.

Oxygen radicals and human disease. Ann Intern Med 107, 526-545.

9. Fitzpatrick, T. B., Becker, S. W., Lerner, A. B. and Montgomery, H. 1950. Tyrosinase in human skin: demon- stration of its presence and of its role in human melanin formation. Science 112, 223-225.

10. Freeman, B. A. and Crapo, J. D. 1982. Biology of disease:

free radicals and tissue injury. Lab Invest 47, 412-426.

11. Frei, B. 1994. Reactive oxygen species and antioxidant vita- mins: mechanisms of action. Am J Med 97, S5-S13.

12. Halliwell, B., Gutteridge, J. and Cross, C. 1992. Free radicals, antioxidants, and human disease: where are we now?. J Lab Clin Med 119, 598.

13. Hansen, M., Nielsen, S. and Berg, K. 1989. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods 119, 203-210.

14. Jeong, Y.-Y., Lee, Y. J., Lee, K.-M. and Kim, J.-Y. 2009. The effects of Oenanthe javanica extracts on hepatic fat accumu- lation and plasma biochemical profiles in a nonalcoholic fat- ty liver disease model. J Korean Soc Appl Biol Chem 52, 632-637.

15. Jiménez-Cervantes, C., Martínez-Esparza, M., Pérez, C., Daum, N., Solano, F. and García-Borrón, J. C. 2001.

Inhibition of melanogenesis in response to oxidative stress:

transient downregulation of melanocyte differentiation markers and possible involvement of microphthalmia tran- scription factor. J Cell Sci 114, 2335-2344.

16. Kim, S. S., Kim, M.-J., Choi, Y. H., Kim, B. K., Kim, K. S., Park, K. J., Park, S. M., Lee, N. H. and Hyun, C.-G. 2013.

Down-regulation of tyrosinase, TRP-1, TRP-2 and MITF ex- pressions by citrus press-cakes in murine B16 F10 melanoma. Asian Pac J Trop Biomed 3, 617-622.

17. Lerner, A. B. and Fitzpatrick, T. B. 1950. Biochemistry of melanin formation. Physiol Rev 30, 91-126.

18. Lin, J. Y. and Fisher, D. E. 2007. Melanocyte biology and

skin pigmentation. Nature 445, 843-850.

19. Ma, C. J., Lee, K. Y., Jeong, E. J., Kim, S. H., Park, J., Choi, Y. H., Kim, Y. C. and Sung, S. H. 2010. Persicarin from water dropwort (Oenanthe javanica) protects primary cultured rat cortical cells from glutamate-induced neurotoxicity. Phytother Res 24, 913-918.

20. Maeda, K. and Fukuda, M. 1996. Arbutin: mechanism of its depigmenting action in human melanocyte culture. J Pharmacol Exp Ther 276, 765-769.

21. Meyskens Jr, F. L., Farmer, P. and Fruehauf, J. P. 2001.

Redox regulation in human melanocytes and melanoma.

Pigment Cell Res 14, 148-154.

22. Miean, K. H. and Mohamed, S. 2001. Flavonoid (myricetin, quercetin, kaempferol, luteolin, and apigenin) content of edible tropical plants. J Agric Food Chem 49, 3106-3112.

23. Nagata, H., Takekoshi, S., Takeyama, R., Homma, T. and Yoshiyuki Osamura, R. 2004. Quercetin enhances melano- genesis by increasing the activity and synthesis of tyrosinase in human melanoma cells and in normal human melanocytes. Pigment Cell Res 17, 66-73.

24. Napolitano, A., Costantini, C., Crescenzi, O. and Prota, G.

1994. Characterisation of 1, 4-benzothiazine intermediates in the oxidative conversion of 5- S -cysteinyldopa to pheomela- nins. Tetrahedron Lett 35, 6365-6368.

25. Pietraforte, D., Turco, L., Azzini, E. and Minetti, M. 2002.

On-line EPR study of free radicals induced by perox- idase/H

O

in human low-density lipoprotein. Biochim Biophys Acta 1583, 176-184.

26. Rees, J. L. 2003. Genetics of hair and skin color. Annu Rev Genet 37, 67-90.

27. Seiberg, M. 2001. Keratinocyte–melanocyte interactions during melanosome transfer. Pigment Cell Res 14, 236-242.

28. Thannickal, V. J. and Fanburg, B. L. 2000. Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 279, L1005-L1028.

29. Tsuboi, T., Kondoh, H., Hiratsuka, J. and Mishima, Y. 1998.

Enhanced melanogenesis induced by tyrosinase gene-trans- fer increases boron-uptake and killing effect of boron neu- tron capture therapy for amelanotic melanoma. Pigment Cell Res 11, 275-282.

30. Vallyathan, V. and Shi, X. 1997. The role of oxygen free radi- cals in occupational and environmental lung diseases.

Environ Health Perspect 105, 165.

31. Yen, F.-L., Wang, M.-C., Liang, C.-J., Ko, H.-H. and Lee, C.-W. 2012. Melanogenesis Inhibitor (s) from Phyla nodi- flora extract. Evid Based Complement Alternat Med 2012, doi:

10.1155/2012/867494.

32. Zhou, J., Shang, J., Ping, F. and Zhao, G. 2012. Alcohol ex- tract from Vernonia anthelmintica (L.) willd seed enhances melanin synthesis through activation of the p38 MAPK sig- naling pathway in B16F10 cells and primary melanocytes.

J Ethnopharmacol 143, 639-647.

33. Zuo, G.-Y., Zhang, X.-J., Yang, C.-X., Han, J., Wang, G.-C.

and Bian, Z.-Q. 2012. Evaluation of traditional Chinese me-

dicinal plants for anti-MRSA activity with reference to the

treatment record of infectious diseases. Molecules 17,

2955-2967.

초록：항산화 활성과 Melanoma 세포에서 멜라닌조절에 대한 Oenanthe javanica 에탄올 추출액의 효과

권은정․김문무*

(동의대학교 화학과)

본 연구의 목적은 melanocyte (B16F1)에서 quercetin과 kaempferol을 포함하는 미나리 에탄올 추출물(OJE)의

멜라닌 합성효과에 미치는 영향을 조사한 것이다. OJE가 세포수준에서 멜라닌 합성을 억제하는지를 조사하기 위

하여 여러 농도의 OJE 존재 하에서 B16F1세포를 배양하였다. 현재 연구에서 DPPH radical scavenging, reducing

power, lipid peroxidation 및 DNA oxidation에 미치는 항산화 효과는 cell free system에서 평가되었다. 더욱이

멜라닌 생성에 대한 OJE 효과는 dopaquinone (DOPA) assay 및 tyrosinase 활성으로 결정되었다. 뿐만 아니라

superoxide dismutase (SOD)-1, -2, glutathione reductase (GSH)와 같은 항산화 효소 및 tyrosinase의 단백질발현

이 western blot 분석을 이용하여 평가되었다. 본 연구에서 OJE는 지질과산화 억제효과를 나타내었고 fenton 반응

에 의해서 생성되는 hydroxyl radical에 의하여 유발되는 DNA 산화를 보호하였다. OJE는 50 μg/ml 이상에서

멜라닌 합성을 증가시켰고 tyrosinase 활성도 50 μg/ml에서 검출되었다. Western blot 분석에서는 OJE가 농도에

비례하여 tyrosinase SOD-1, -2 및 GSH의 발현 수준을 증가시켰다. 이러한 발견들은 항산화 효과를 가진 OJE가

melanocyte에서 tyrosinase 활성과 melanin 생성을 조절할 수 있어 피부를 산화스트레스로부터 보호할 수 있다는

것을 암시하고 있다.