EXTRACTION OF FUCOIDAN AND TYROSINASE INHIBITORS FROM BROWN SEAWEED EXTRACTS Work carried out for Gráinne Na Mara as part of Innovation Voucher Number IV-2012-3111 AUTHOR: Dr MARIA HAYES

INTRODUCTION The anti-inflammatory and water retaining characteristics of Fucoidan are well documented. Tyrosinase is a multifunctional, copper containing oxidase that catalyzes three distinct reactions of melanin synthesis. Natural tyrosinase inhibitors found in brown seaweeds could be involved in the control of pigmentation in plants and may act as anti-browning agents in cosmetic products. The aim of this work is to identify Fucoidan in several brown seaweed extracts and to determine the anti-tyrosinsase inhibitory activity of phlorotannin extracts generated from seaweed samples supplied by Gráinne Na Mara. 2

Several brown seaweed extracts examined and these included: Samples to be analysed Several brown seaweed extracts examined and these included: Ascophyllum nodosum Fucus vesiculosus Fucus serratus

Heat treatment for 4 hrs at 100ºC MATERIALS AND METHODS – EXTRACTION ACCORDING TO METHOD OF MARUYAMA AND YAMOMOTO Samples were freeze dried following collection and supply to stablize the fractions Mill (40secs. / 30movs/sec) Heat treatment for 4 hrs at 100ºC 30 grams sample Drying & Concentration Figure 4. Retsch Mixer Mill MM 400 15 parts W/V Ultrapure water Water bath 4 4

MATERIALS AND METHODS – EXTRACTION ACCORDING TO METHOD OF MARUYAMA AND YAMOMOTO 2 g of powder 200 ml 0.09 N HCL, 4ºC, 2 hrs Centrifuged. Water residue Washed and extraction repeated ppt Centrifuged & dissolved in Hot H2O and diluted with Ethanol. This was filtered & left O/N Ethanol added & ppt centrifuged Greyish-white coloured Carbohydrate Fucoidan

Tyrosinase inhibitors Reducing agents causing chemical reduction of dopaquinone such as ascorbic acid, which is used as a melanogenesis inhibitor because of its capacity to reduce back o-dopaquinone to dopa, thus avoiding dopachrome and melanin formations. o-Dopaquinone scavenger such as most thio-containing compounds, which are well-known melanogenesis inhibitors and react with dopaquinone to form colourless products. The melanogenetic process is therefore slowed until all the scavenger is consumed, and then it goes at its original rate. Alternative enzyme substrates such as some phenolic compounds, whose quinoid reaction products absorb in a spectral range different from that of dopachrome. When these phenolics show a good affinity for the enzyme, dopachrome formation is prevented, and they could be mistakenly classified as inhibitors. Nonspecific enzyme inactivators such as acids or bases, which non-specifically denature the enzyme, thus inhibiting its activity. Specific tyrosinase inactivators such as mechanism-based inhibitors, which are also called suicide substrates. These inhibitors can be catalyzed by tyrosinase and form covalent bond with the enzyme, thus irreversibly inactivating the enzyme during catalytic reaction. They inhibit tyrosinase activity by inducing the enzyme catalyzing “suicide reaction.” Specific tyrosinase inhibitors such as most compounds that bind reversibly to tyrosinase and reduce its catalytic activity Reference: Int. J. Mol. Sci. June 2009, 10, 6, 2440-2475

Kojic Acid Kojic acid is the most intensively studied inhibitor of tyrosinase and is a fungal metabolite currently used as a cosmetic skin-whitening agent and as a food additive for preventing enzymatic browning. Kojic acid shows a competitive inhibitory effect on monophenolase activity and a mixed inhibitory effect on the diphenolase activity of mushroom tyrosinase. The ability of kojic acid to chelate copper at the active site of the enzyme may well explain the observed competitive inhibitory effect. In addition, kojic acid is reported to be a slow-binding inhibitor of the diphenolase activity of tyrosinase

Polyphenols Polyphenols represent a diverse group of compounds containing multiple phenolic functionalities and are widely distributed in nature. Polyphenols are also the largest groups in tyrosinase inhibitors until now. Since several polyphenols are accepted as substrates by tyrosinase, it depends on the presence and position of additional subsistent whether a polyphenol may act as an inhibitor. In addition to flavonoids, other polyphenols, which were also identified as tyrosinase inhibitors, contain stilbenes and coumarin derivatives. Many flavonols have been isolated from plants, and some were identified as tyrosinase inhibitors. The inhibitory mode of flavonol inhibitors is usually competitive inhibition for the oxidation of l-dopa by tyrosinase and the 3-hydroxy-4-keto moiety of the flavonol structure acts as the key role in copper chelation.

Polyphenols Polyphenols are a group of chemical substances found in plants characterized by the presence of more than one phenol unit or building block per molecule. Fruits, vegetables, and beverages are the main sources of phenolic compounds in the human diet.

Isolation of phlorotannins from Gráinne na Mara extracts and assessment of bioactivity Fucus vesiculosus Fucus serratus