K. B. W. R. Kim, S. M. Jeong, M. J. Kim1 and D. H. Ahn1*

Institute of Fisheries Sciences, Pukyong National University, Busan 46041, Republic of Korea
1 Department of Food Science and Technology/Institute of Food Science, Pukyong National University, Busan 48513, Republic of Korea

*Corresponding Author:
D. H. Ahn
Department of Food Science and Technology/Institute of Food Science, Pukyong National University, Busan 48513, Republic of Korea
E-mail: dhahn@pknu.ac.kr
Date of Submission 23 August 2019
Date of Revision 12 October 2019
Date of Acceptance 18 December 2019
Indian J Pharm Sci 2020;82(1):171-174  

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DOI: 10.36468/pharmaceutical-sciences.635

Abstract

The aim of this study was to elucidate the effect of Myagropsis myagroides extract on tyrosinase activity. Inhibition of tyrosinase was observed in the presence of an n-hexane fraction of Myagropsis myagroides. Separation of the n-hexane fraction using silica gel column chromatography yielded the chloroform:methanol (50:1) fraction, which showed the highest tyrosinase inhibitory activity. After several separation and purification steps, sargachromanol G, sargachromanol I, and mojabanchromanol b were obtained. These 3 chromene compounds displayed strong tyrosinase inhibitory activity. It could be concluded that Myagropsis myagroides is a potential source of natural antimelagenic compounds.

Keywords

Mojabanchromanol b, Myagropsis myagroides, sargachromanol G, sargachromanol I, Tyrosinase inhibitory effect

Melanogenesis is the biosynthetic pathway for the formation of the pigment melanin in skin melanocyte. Melanocytes produce two types of melanin: pheomelanin and eumelanin formed by conjugation of glutathione or cysteine. Melanogenesis is initiated with the oxidation of l-tyrosine to l-dopaquinone, the precursor of pheomelanin and eumelanin, by tyrosinase[1]; this enzyme is therefore important for mammalian melanin synthesis in melanocytes. Tyrosinase is a multifunctional membrane-bound type 3 copper-containing glycoprotein, and is expressed only in melanocytes. Melanogenesis can be inhibited by inhibition of tyrosinase or by inhibition of melanocyte metabolism[2]. Physiologically, activation of tyrosinase in skin stimulates melanin production to protect skin damaged by UVA and UVB radiation[3,4]; however, the accumulation of melanin can cause hyperpigmentation of skin and dermatological disorders such as freckles and age spots. Tyrosinase inhibition reduces transformation of l-tyrosine to melanin, slowing and decreasing skin hyperpigmentation[5].

Myagropsis myagroides is a brown algae that inhabits the coasts of South Korea, China, and Japan. There are some reports showing that M. myagroides has antibacterial[6], antidiabetic[7], hepatopotective[8] and antiinflammatory[9-12] properties due to the presence of pigments and polyphenols. On the other hand, there is no research on the inhibition of melanogenesis by extract of M. myagroides. In the present study, evidence was provided that tyrosinase activity is inhibited by extracts of M. myagroides, supporting the view that M. myagroides contains antimelanogenic compounds.

M. myagroides was harvested from Song-Jeong, Busan, Korea in March 2011. M. myagroides was washed 3 times with tap water to remove salt, epiphytes, sand and then dried naturally. The dried M. myagroides was lyophilized and pulverized. The powder was stored at -20°. A voucher specimen (MBRB0078) has been deposited at the Marine BrownFigure Algae Resources Bank.

Powdered M. myagroides (1.5 kg) was extracted with methanol (MeOH) for 24 h at room temperature with an agitator (H-8020, Dongwon Science Co., Busan, Korea) and filtered via vacuum suction with a Büchner funnel. The filtrate was evaporated in vacuo to obtain MeOH extract, which were suspended in distilled H2O and then successively partitioned with n-hexane (13 g), CHCl3 (11 g), ethyl acetate (EtOAc, 0.5 g) and n-BuOH (butanol, 4.4 g), respectively. The n-hexane soluble fraction (10 g) was chromatographed on a silica gel column and eluted with CHCl3-MeOH (CM, 100:0~1:1) to yield 6 fractions. The CM (50:1) fraction (1.6 g) was subjected to a Sephadex LH-20 column chromatography and ODS Sepak cartridge (60~90 % methanol) to 7 fractions. Of these fractions, the fraction 4 (242 mg) was further purified by Sephadex LH-20 column chromatography and DOS Sepak cartridge (50~60 % methanol). The fractions were separated using ODS high-performance liquid chromatographyphotodiode array detection (HPLC-PDA; Hitachi, Chatsworth, CA, USA) and found to contain compounds such as sargachromnaol G (conditions- 70 % methanol, 9 ml/min), sargachromanol I (conditions- 70 % methanol, 9 ml/min), and mojabanchromanol b (conditions- 75 % methanol, 3 ml/min), which were isolated with yields of 20, 52 and 2 mg, respectively. The UV spectra of eluted compounds were recorded from 200 to 500 nm. Their structures were elucidated by 1H NMR (600 MHz, CDCl3; JNM-ECA600, Jeol Ltd, Tokyo, Japan) and 13C NMR (150 MHz, CDCl3), 1H-1H COSY, HMQC, HMBC, and EI-MS (JMS-700, Jeol Ltd) spectra.

Tyrosinase inhibition assays were performed using mushroom tyrosinase (Sigma-Aldrich Co., St. Louis, MO, USA) as described previously, with some modification[13]. Briefly, M. myagroides fraction (10 μl) was added to a reaction mixture containing 1500 unit/ml mushroom tyrosinase (10 μl), 0.1 M sodium phosphate buffer (pH 6.5, 110 μl) and 4 mM L-tyrosine (20 μl, Sigma-Aldrich Co.) in a 96-well plate for 20 min at 37°. Tyrosinase activity was measured by monitoring the formation of L-DOPAchrome, indicated by an increase in absorbance at 490 nm using UV/Vis spectrophotometer.

Data was expressed as mean±standard error of the mean. Statistical evaluation was carried out using analysis of variance (ANOVA) in SAS software (SAS Institute, Inc., Cary, NC, USA) and the differences between means were assessed using Duncan’s multiple range test at p<0.05.

Tyrosinase is an enzyme containing copper at the active site and acts on the initial reaction of melanogenesis[1]. To investigate effect of the tyrosinase inhibitory activity, the amount of DOPA chrome was determined. The tyrosinase inhibitory activity of the n-hexane fraction of the methanol extract was 25.64 % at 5 mg/ ml (Table 1), higher than that of the other fractions. Fraction CM (50:1) was obtained by silica gel column chromatography, which showed the highest inhibition activity of 36.46±4.65 % at 5 mg/ml. Seven fractions were obtained from fraction CM (50:1) using a Sephadex LH-20 column chromatography and ODS separk cartridge, among which fractions 3 and 4 inhibited tyrosinase 13.07±1.23 and 38.89±5.70 %, respectively (Table 1). Similar levels of tyrosinase inhibition by seaweed extracts have been reported previously. For example, it was reported that tyrosinase activity is partially inhibited by methanol extracts of Sargassum siliquastrum and Ecklonia cava[14]. In particular, the methanol extract of S. siliquastrum had an inhibitory activity of 50 %. In addition, ethyl acetate extracts of Endarachne binghamiae[15] and Undaria pinnatifida[5] has shown by measurement of activity of intracellular tyrosinase in mouse melanocytes or melanoma cells to exhibit a high inhibition rate of melanin synthesis. However, the melanin synthesis inhibitory compounds in seaweed extracts have not been analyzed.

Fractions Inhibitory activity (%)
n-hexane 25.64±4.64b4)
CM1) 50:12) 36.46±4.65a
33) 13.07±1.23a
4 38.89±5.70a

Table 1: Tyrosinase inhibitory activity of sub-fractions from n-hexane fraction of Myagropsis myagroides

Fraction 4, which exhibited high inhibitory activity, was purified by HPLC and 3 chromene compounds were isolated, which were sargachromanol G (C27H38O4), sargachromanol I (C27H40O4), and mojabanchromanol b (C27H40O4) (Figure 1). Tyrosinase inhibitory activity was detected with all 3 compounds and mojabanchromanol b exhibited the highest inhibitory activity (48.58 ±2.42 %), followed by sargachromanol G (41.27 ±4.70 %) and sargachromanol I (19.92±8.59 %, Table 2). These 3 compounds have already been reported[16,17], but the inhibitory activity of tyrosinase is not reported. Sargachromanol G and I have antiinflammatory and antioxidant effects, which are found in the brown algae family. Sargachromanol G, isolated from S. siliquastrum, was reported to inhibit collagenase activity and exhibited antiinflammatory effect in osteoblasts[18], while sargachromanol I showed antioxidant potency[19] and α-amylase inhibitory activity[7]. Sargachromnenol G was also been isolated from S. horneri and in this case, it was reported to suppress UVA-induced photoaging[20]. Mojabanchromanol b was shown to have an antiinflammatory effect by suppressing the release of cytokines and nitric oxide[9]. Thus, these compounds are known to exert various biological effects. However, further experiments on these 3 compounds, including in vivo experiments, are needed to investigate their mechanisms.

ijpsonline-chromatograms-spectra

Figure 1: HPLC chromatograms and UV spectra of constituents from Myagropsis myagroides methanol extract
HPLC profiles and UV spectra of (A) Sargachromanol G, (B) Sargachromanol I, and (C) Mojabanchromanol b isolated from Myagropsis myagroides methanol extract

Isolated compounds Inhibitory activity (%)
Sargachromanol G 41.27±4.70b1)
Sargachromanol I 19.92±8.59c
Mojabanchromanol b 48.58±2.42b
Arbutin 74.47±1.17a

Table 2: Tyrosinase inhibitory activity of the 3 chrome compounds isolated from fraction 4 of Myagropsis myagroides

In summary, this study demonstrated for the first time that sargachromanol G, sargachromanol I and mojabanchromanol b compounds isolated from M. myagroides exhibited tyrosinase inhibitory activity. In particular, it was found that mojabanchromanol b had greater effect on tyrosinase inhibition than other compounds. This finding suggested that compounds extracted from M. myagroides, especially mojabanchromanol b, could have applications in the food industry, medicine and as a natural skin-whitening agent.

Acknowledgements:

This research was supported by the Basic Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2012R1A6A1028677).

References