Journal of Medicinal Plants

A Review on Biosynthesis, Health Benefits and Extraction Methods of Fucoxanthin, Particular Marine Carotenoids in Algae

Irvani N (M.Sc.)1, Hajiaghaee R (Ph.D.)2, Zarekarizi A.R (M.Sc.)2*

1- Faculty of Biological Science and Technology, Shahid Beheshti University, Tehran, Iran 2- Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran * Corresponding author: Institute of Medicinal Plants, ACECR, Karaj, Iran P.O.BOX (Mehr Vila): 31375-369 Tel: +98 - 26 – 34764010-18, Fax: +98 – 26- 34764021 E-mail: [email protected]

Received: 28 June 2017 Accepted: 18 July 2018

Abstract

Fucoxanthin, an allenic carotenoid, is abundant in macro and microalgae as a component of the light-harvesting complex for photosynthesis and photo protection. This carotenoid has shown important pharmaceutical bioactivity, exerting antioxidant, anti-cancer, anti-diabetic, anti- obesity, anti-photoaging, anti-angiogenic, and anti-metastatic effects on a variety of biological models. This carotenoid has been proven to be safe for animal consumption, opening up the opportunity of using this bioactive compound in the treatment of different pathologies. In this paper, an updated account of the research progress in biosynthetic pathway and health benefits of fucoxanthin is presented. Meanwhile, a review on the various methods of extraction of fucoxanthin in macro and microalgae is also revisited. According to these studies providing important background knowledge, fucoxanthin can be utilized into drugs and nutritional products.

Keywords: Fucoxanthin, Carotenoids, Biosynthesis pathway, Extraction methods

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Introduction million species [5]. Microalgae are important Consumer awareness of the importance of a primary producers in marine environments and healthy diet, protection of the environment, play a significant role in supporting aquatic resource sustainability and using all natural animals [6]. Mass production of microalgae resources are continuously increasing and can be carried out outdoors or indoors in fortunately enhance awareness among people bioreactors under optimal conditions [7]. This towards natural products due to their non-toxic makes microalgae a potentially sustainable properties, low pollution and less side effects source of feedstock for fuel, food, chemical, [1]. Microalgae (unicellular organisms) and textile, polymer and even the pharmaceutical macroalgae (multicellular organisms) belong industry [4]. to the large algae group made up of Carotenoids are tetraterpenoids with a photosynthetic organisms [2]. In recent characteristic linear C40 molecular backbone decade, there is a growing interest in using containing up to 11 conjugated double bonds, marine algae as an alternative food source. which are synthesized by all photosynthetic Marine algae are distributing from the polar organisms as well as by many non- region to tropical areas, range in size from photosynthetic bacteria and fungi [8]. There microscopic individual cells of microalgae to are two main classes of naturally occurring huge seaweeds and from nutrient-rich coastal carotenoids: carotenes, which are seas to oligotrophic open oceans [3]. hydrocarbons that are either linear orcyclized Algae have created enormous interest due at one or both ends of the molecule; and to their unconventional growth requirements xanthophylls which are oxygenated derivatives [4]. They provide an opportunity to exploit the of carotenes. In general, a distinction can be underutilized arable lands and oceans without made between primary and secondary reducing the agricultural production surface, carotenoids. Primary xanthophylls are defined which needs to be supplemented in order to as xanthophylls which are structural and feed the growing global population. Seaweeds functional components of the photosynthetic live in the coastal regions of ocean waters, apparatus of the cell and therefore essential for regularly within rocky intertidal or submerged cellular survival. Secondary xanthophylls are reef-like habitats. In this environment, a defined as xanthophylls that algae produce in nutrient struggling for light and space is fierce large quantities only after having been exposed and seaweeds have evolved chemical defense to specific environmental stimuli [9]. mechanisms to ward off predators and increase Fucoxanthin one of the major marine survival. Not surprisingly, many of these xanthophylls, occurs in some macro and chemicals are biologically active and some microalgae and has a unique structure including possess potent pharmacological activity [3]. an allenic bond, a conjugated carbonyl, a 5, 6- Furthermore, Marine microalgae constitute the monoepoxide and acetyl groups [10, 11] (Figure largest group of living organisms in the ocean 1). It serves as an antenna carotenoid in with an estimated range of 2×105 to several

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Figure 1- Structure of Fucoxanthin the light-harvesting complexes where it is identification of biosynthesis pathway and coupled to the thylakoid membrane to transfer improvement of efficient extraction methods excitation energy to the photosynthetic are clearly necessary. Hence, this article electron transport chain via chlorophyll a [12, reviews the current available scientific 13]. Fucoxanthin has attracted considerable literature regarding the Biosynthesis, Health interest because of its potent bioactivities, Benefits and extraction methods of including its antioxidant, anti-inflammatory, fucoxanthin from macro and micro algae. anticancer, anti-obese, antidiabetic, antiangiogenic, and antimalarial activities, and Biosynthesis of fucoxanthin and Enzymatic its protective effects on the liver, blood vessels Reactions of the brain, bones, skin, and eyes [8, 14]. The Some common carotenogenesis genes in effects on adult T-cell leukemia cells, and algae are suggested from homology of the inhibitory effect on the viability of HL-60 cells known genes, but most genes and enzymes for of fucoxanthin are distinctly more potent than algae-specific pathways are still unknown. To that of β-carotene and astaxanthin [15]. This exploit our knowledge regarding this carotenoid has been proven to be safe for carotenoid in the medical fields and industrial animal consumption [16], opening up the applications, resolution of this pathway at the opportunity of using this bioactive compound molecular level is very important [8]. in the treatment of different pathologies [12]. Isopentenyl pyrophosphate (IPP), a C5- Though chemical synthesis of fucoxanthin is compound, is the source of isoprenoids, possible, it is very expensive and hence the terpenes, quinones, sterols, phytol of viability of obtaining directly from brown chlorophylls, and carotenoids. There are two seaweeds should not be overlooked [17]. known independent pathways of IPP synthesis: The promising prospect envisaged in this the classical mevalonate (MVA) pathway and new field of scientific collaboration arose our the alternative, non-mevalonate, 1-deoxy-d- interest to produce fucoxanthin. To perform xylulose-5-phosphate (DOXP) pathway. In the such grave attempt, an intensive work on MVA pathway, acetyl-Coenzyme A is

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converted to IPP through mevalonate. The precursor of fucoxanthin. The neoxanthin pathway is found in plant cytoplasm, animals hypothesis, on the other hand, proposes a and some bacteria [18, 19, 20]. In the DOXP branching of the pathway from neoxanthin to pathway, pyruvate and glycelaldehyde are both diadinoxanthin and fucoxanthin. The converted to IPP and the pathway is found in reasons for these differing hypotheses cyanobacteria, the plastids of algae, land plants regarding the fucoxanthin biosynthetic and some bacteria [9, 14]. pathways are: (1) that no pathway intermediate Farnesyl pyrophosphate (C15) is has been detected by HPLC, and (2) that the synthesized from three IPPs, after which, one genes encoding enzymes involved in the IPP is added to farnesyl pyrophosphate by biosynthesis of fucoxanthin have not been geranylgeranyl pyrophosphate synthase cloned [8]. (GGPS) to yield geranylgeranyl pyrophosphate For conversion of neoxanthin to (C20). In a head-to-head condensation of the fucoxanthin, two sequential reactions are two C20 compounds, the first carotene, necessary: ketolation of neoxanthin and phytoene (C40), is formed by phytoene acetylation of an intermediate [18]. Thus, synthase (Psy) using ATP. Four desaturation biochemical detection of the intermediate steps are needed in the conversion from which is probably fucoxanthinol, and phytoene to lycopene. Phytoene desaturase identification of genes encoding ketolase and (CrtP) catalyzes the first two desaturation acetylase are necessary to support the steps, from phytoene to ζ-carotene through neoxanthin hypothesis. phytofluene, and ζ-carotene desaturase (Zds) catalyzes two additional desaturation steps, Fucoxanthin Extraction from ζ-carotene to lycopene through An important factor determining the market neurosporene (Fig.2.). Lycopene is cyclized for fucoxanthin product development is the into β-carotene through γ-carotene [8, 14]. value of the target compound from the The biosynthetic pathway from organism of choice. The fucoxanthin β-carotenoid to violaxanthin is common to extraction efficiency was highly dependent on both diatoms and brown algae because genes the solvent type. Selecting the proper solvent encoding zeaxanthin epoxidase (ZEP) and to optimize extraction is very important as it violaxanthin de-epoxidase (VDE) are determines the similarities in the chemical conserved in these organisms [18]. According composition of the substances to be extracted to genome analysis, two different pathways are [5]. Different extraction techniques have been proposed which have designated the used to isolate fucoxanthin from the marine diadinoxanthin hypothesis and the neoxanthin algae. However, none of them can be hypothesis (Figure 2). The diadinoxanthin considered as an optimal method for this hypothesis involves a sequential conversion of purpose. violaxanthin to diadinoxanthin which is a

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Figure 2- Biosynthetic Pathway and Health Benefits of Fucoxanthin

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The best solvent to maximize fucoxanthin system of n-hexane/hydroalcoholic phase (0.2, extraction (15.71 mg/g dw) from the v/v). Fortunately, the optimum solvent for microalgae Phaeodactylum tricornutum lipid extraction was the same as that used for among the five solvents tested was Ethanol, fucoxanthin extraction, and a biphasic system whereas water and n-hexane were ineffective can be applied for purifying fucoxanthin. In for extracting fucoxanthin. Acetone, the most order to investigate the possibility of the widely used solvent for marine pigment combined production of lipid and fucoxanthin, extraction, yielded approximately one third of fucoxanthin was dissolved in the same the fucoxanthin extracted with ethanol biphasic system and then, the percentages of (4.60 mg/g dw). In the case of ethyl acetate, fucoxanthin in each phase were analyzed by 2.26 mg/g dw of fucoxanthin was extracted HPLC. In result, most fucoxanthin (over 99%) under the same conditions. Ethanol at high was present in the hydro alcoholic phase, temperatures provides high recoveries of implying lipid and fucoxanthin can be fucoxanthin and other oxygenated carotenoids. separated by this biphasic system after same This process was performed to disrupt the cell extraction process with 100% ethanol solvent. membrane in U. pinnatifida through which, the Another point for consideration in the extraction efficiency of fucoxanthin increases. combined production of lipids and fucoxanthin Due to the long extraction time at the boiling is the different extraction time between lipids point of the ethanol solvent, the possible and fucoxanthin. Longer time (generally over degradation of fucoxanthin due to local 10 h) is required for lipid extraction, while overheating effects might occur [5]. Fucoxanthin fucoxanthin can be extracted within 1 h [5]. concentration from Odontella aurita extracted Liquefied dimethyl ether (DME) was used with methanol (16.18 mg g−1 DW) was the as an extract to enhance extraction of highest among the five tested solvents followed fucoxanthin from U. pinnatifida. DME is the by ethanol (15.83 mg g−1 DW) and acetone simplest form of ether, with the following (13.93 mg g−1 DW). Although ethanol had characteristics: (i) DME has a low normal slightly lower extraction efficiency than boiling point (−24.8 °C) and therefore, it is not methanol, it exerts lower toxicity and thus, was present in the final products at normal selected as the most suitable extraction solvent in temperatures; (ii) Relative permittivity of further research [8]. DME is 1.08 and 5.34 at 30.5 °C, in gaseous Lipids were extracted from Phaeodactylum and liquid states, respectively. Liquefied DME tricornutum with ethanol and a partition has high affinity to oily substances and partial process using a biphasic system. In that report, miscibility with water; (iii) DME has been a water content of 40% (v/v) in the approved as a safe extraction solvent for the hydroalcoholic phase gave the highest lipid production of food ingredients by the recovery and n-hexane was added to European Food Safety Authority (EFSA), by hydroalcoholic phase to extract lipids from the Food Standards of Australia and New hydroalcoholic phase resulting in biphasic Zealand, and by the United States [21].

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Supercritical CO2 is known as an attractive DW when increasing the extraction extraction method for food industries because temperature from 25 °C to 45 °C, attributable the solvent is safe, nontoxic and easily likely to the elevated temperature enhanced removable. The method is fast and extraction solubilization of photosynthetic membranes parameters can be changed in a wide range of and release of fucoxanthin from fucoxanthin- pressure and temperature. However, CO2 is Chla, c-protein complexes. The extracted non-polar fluid and it is the main disadvantage fucoxanthin concentration was also a function in its use for the isolation of antioxidants. of extraction time. At 45 °C, approximately Ethanol was introduced to increase the polarity 80% of fucoxanthin corresponding to 13.53 −1 of CO2 and to improve the fucoxanthin yield. mg g was extracted from algal biomass Yet, respecting to separation process, it is not within the first 10 min, and the maximum only expensive but also difficult [21]. fucoxanthin concentration was obtained at As a type of carotenoids, fucoxanthin is approximately 60 min [21]. highly susceptible to degradation by external Fucoxanthin extracted from agents such as heat, low pH, and light Sargassumbinderiexhibited sensitivity towards exposure promoting changes of color due to different factors such as light, pH and addition several conjugated double bonds. The of antioxidant. Overall, the fucoxanthin degradation process would lead to the pigments were more resistant to degradation in rearrangement or formation of degradation dark condition as compared to pigment compounds such as cis-isomers which are exposed to light which had accelerated rate of thermodynamically less stable and hence, color degradation. Addition of antioxidant resulted in different colors properties and in further protects the fucoxanthin pigments from some cases, volatile compounds [22]. degradation. The fucoxanthin extract Selecting a proper ratio of solvent to dry supplemented with 1.0% w/v of ascorbic acid algal biomass (v/w) is important, as it may displayed greatest pigment retention in both affect the quantity and quality of fucoxanthin. dark and light condition. The most favourable In Odontellaaurita the fucoxanthin extraction pH condition in providing the greatest stability efficiency increased remarkably (11.90–15.74 for the pigment was pH 9, especially in dark mg g−1 DW) with increasing the ethanol to dry condition. Therefore, it could be concluded biomass ratio, up to 20:1 ethanol/dry biomass. that the fucoxanthin pigments were sensitive to When O. auritadry biomass was treated with light exposure, the least stable in acidic pH 30:1 ethanol/dry biomass, the fucoxanthin condition and higher concentration of ascorbic concentration increased slightly (15.90 mg g−1 acid supplementation exerted stabilisation role DW), and further increasing of ethanol did not on fucoxanthin [22]. improve the fucoxanthin extraction efficiency [7]. Health benefits of fucoxanthin The extraction of fucoxanthin was Physicians need to understand the increased from 16.12 mg g−1 to 17.20 mg g−1 biochemical and evidential bases for the use of

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herbs and nutrients to diagnose and treat radical scavenging activities of fucoxanthin patients safely and effectively, to avoid and fucoxanthinol were 13.5 and 1.7 times interactions with standard medications, and to higher than that of α-tocopherol. Interestingly, provide patients with the benefits of alternative fucoxanthin acts as an antioxidant under treatments [23]. Evidence based medicine is a anoxic conditions, whereas other carotenoids, methodological and systematic approach such as β-carotene and lutein, show little or no which is of immense importance to the quenching activities in such chemical treatment of patients. It involves treatment assessment systems [7]. It has been reported processes being clinically tested, not only to that the extracts of H. fusiformis, discover what benefits such treatment has, but C. okamuranus, U. pinnatifida, and also to find what consequences there are for S. fulvellum showed a strong DPPH radical using such treatment. It is also the case that scavenging activity [25, 26]. Recently, evidence based medicine should aim to Airanthi et al. (2011) showed that the discover what treatments are cost-effective, methanol extract of C. hakodatensis was a enabling medical physicians to offer the best good source for antioxidant activity [27]. The treatment available, but at a good price [24]. extract containing fucoxanthin from F. These study reviewsvarious aspects of vesiculosus showed an antioxidant activity ex pharmacological activity and the most reliable vivo through preventing oxidant formation, evidence from clinical studies, scientific scavenging superoxide anion (O2•−) and understanding and medical practice of reducing active intermediates [28]. Sachindra fucoxanthin. et al. (2007) suggested that fucoxanthin and fucoxanthinol exhibited antioxidant activities - Antioxidant activity higher or similar to that of α-tocopherol, and Fucoxanthin has been reported to halocynthiaxanthin showed comparatively effectively scavenge chemically-generated free lower antioxidant activities [29]. Nomura et al. radicals, such as DPPH (1,1-diphenyl-2- (1997) found that, under anoxic conditions, picrylhydrazyl), 12-DS (12-doxyl-stearic fucoxanthinequimolarly reacted with DPPH as acid), NB-L (the radical adduct of radical quencher, whereas β-carotene, β- nitrosobenzene with linolenic acid radical), cryptoxanthin, zeaxanthin, licopene, and lutein AAPH (2,2′-azo-bis- (2-amidinopropane) scarcely reacted with DPPH and had dihydrochloride), ABTS (2,2′-azinobis-3- practically no quenching activities, suggesting ethylbenzothiazoline-6-sulphonate), and that fucoxanthin was more reactive to radicals ABAP (2,2′-azo-bis-2-amidinopropane [7]. than other carotenoids under anoxic conditions Fucoxanthin could effectively inhibit [30]. Sangeetha et al. [31, 32] showed that intracellular reactive oxygen species fucoxanthin and β-carotene protected cell formation, DNA damage, and apoptosis membrane by decreasing Na+ K+-ATPase induced by H2O2, possibly due to the ability of activity and increasing the activities of catalase fucoxanthin to increase catalase. The hydroxyl and glutathione transferase at the tissue and

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Irvani & et al. microsomal level. The results also showed that cells compared to fucoxanthin. During in vivo fucoxanthin had greater potential than studies, fucoxanthin was found to inhibit β-carotene and was somewhat more effective mouse colon carcinogenesis induced by 1,2- than retinol in reducing lipid peroxidation in dimethylhydrazine. In addition, fucoxanthin plasma and liver resulting from retinol has been reported to inhibit duodenal and skin deficiency [32]. Li et al. (2000) indicated that carcinogenesis and liver tumorigenesis in vitamin D2 was oxidized by singlet oxygen in mice. These anti-cancer effects of fucoxanthin the presence of riboflavin, light, and oxygen in are thought to operate by apoptosis induction, a model system, and fucoxanthin and cell cycle arrest and antioxidant activity [8]. β-carotene could minimize the oxidation of The apoptosis-inducing effectof fucoxanthin vitamin D2 by quenching singlet oxygen [33]. on human promyelocytic leukemia HL-60 cell The allenic bond was responsible for the line has been investigated by Hosokawa et al. higher antioxidantactivity of fucoxanthin [30]. (2010), who found that fucoxanthin exhibited In addition, fucoxanthin has six oxygen atoms, strong antiproliferative activity and could and thus might be moresensitive to radicals induce apoptosis of HL-60 cells. In HL-60 especially under anoxic conditions. It is worth cells, fucoxanthin caused cleavages of mentioning that fucoxanthin also contains an procaspase-3 and poly-ADP-ribose α,β-unsaturated carbonyl group, and may polymerase, and apoptosis induction by function as a Michael acceptor which can react fucoxanthin was mediated through with important proteins such as Keap 1 in the mitochondrial membrane permeabilization and Nrf2 system [34]. Liu et al. (2011) recently caspase-9 and caspase-3 activation [38]. Kim showed that fucoxanthin enhanced HO-1 and et al. (2010) showed that fucoxanthin induced NQO1 expression inmurine hepatic BNL CL. reactive oxygen species generation, inactivated 2 cells through activation of the Nrf2/ARE the Bcl-xL signaling pathway, induced system, and suggested that fucoxanthin might caspase-3, -7, and poly-ADP-ribose exert its antioxidant activity, at least partly, polymerase cleavage, and thus triggered the through its pro-oxidant actions [35]. apoptosis of HL-60 cells indicating that the generation of reactive oxygen species was a - Antitumor activity critical target in fucoxanthin-induced apoptosis Fucoxanthin inhibited proliferation of in HL-60 cells [39]. Ganesan et al. (2011) hepatoma HepG2 cells and colon cancer Caco- showed that fucoxanthin, astaxanthin, 2, HT-29 and DLD-1 cells in vitro [36, 37]. siphonaxanthin, neoxanthin, and violaxanthin The induction of apoptosis and suppression of had significant cytotoxic effects against cyclin D levels are proposed mechanisms for cultured HL-60 cells. Both halocynthiaxanthin the observed anti-proliferative effect of and fucoxanthinol showed remarkable fucoxanthin. Furthermore, fucoxanthinolalso induction of apoptosis in HL-60 cells, MCF-7 showed higher apoptosis-inducing activity on breast cancer cells, Caco-2 colon cancer cells, Caco-2 (colon) and MCF-7 (breast) cancer the anti-proliferative and apoptosis-inducing

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effects of halocynthiaxanthin and trigger the terminal differentiation of fucoxanthinol on these cells were significantly cancerous cells in vitro [14]. Kotake Nara et greater than those of fucoxanthin, possibly at al. (2005) showed that fucoxanthin least partly, related to the hydroxyl group in andneoxanthin had nearly the same actions on halocynthiaxanthin and fucoxanthinol [40]. the cultured cells and were more effective in Both fucoxanthinol and amarouciaxanthin A reducing theviability of HCT116 cancer cells also reduced the viability of PC-3 human than that of the other cancer and normal cell prostate cancer cells, and the 50% inhibitory lines, while lycopenewas found to be less concentrations of fucoxanthin, fucoxanthinol, effective [43]. Das et al. (2005) indicated that and amarouciaxanthin A on the proliferation of fucoxanthin inhibited the proliferation of PC-3 cells were 3.0, 2.0, and human colon cancer cell lines WiDr and 4.6 μM, respectively, indicating that the 5,6- HCT116 cells by inducing cell cycle arrest at epoxide in fucoxanthin and fucoxanthinol the G0/G1phase through up-regulating the played important roles in cytotoxicity, and cyclin-dependent kinase inhibitory protein other mechanisms might be also involved in p21WAF1/Cip1 and retinoblastoma protein the antiproliferative effect of epoxycarotenoids (pRb) [44]. Kotake-Nara et al. (2001) showed on PC-3 cells [41]. Adult T-cell leukemia is an that fucoxanthin significantly reduced the incurable malignancy of mature CD4+ T cells viability of threehuman prostate cancer cell caused by human T-cell leukemia virus type 1. lines PC-3, DU 145 and LNCaP to 14.9%, Ishikawa et al. (2008) assayed the 5.0%, and 9.8%, respectively,through antiproliferative effects of some carotenoids apoptosis induction in these cancer cells. The such as fucoxanthin, fucoxanthinol, β-carotene succeeding study demonstrated that and astaxanthin, and found that both fucoxanthin decreased the levels of Bax and fucoxanthin and fucoxanthinol had remarkable Bcl-2 proteins, and induced apoptosis in PC-3 antiproliferative effects on human T-cell cells through caspase-3activation [45]. Satomi leukemia virus type 1-infected T-cell lines and and Nishino (2007) showed that fucoxanthin adult T-cell leukemia cells in vitro, and induced cell cycle arrest at theG1 phase and β-carotene and astaxanthin had mild inhibitory GADD45A expression, and inhibited the effects [42]. growth of DU145 cells [46]. In the sub- The exposure of human non-small-cell sequent study, Satomi and Nishino (2009) Broncho pulmonary carcinoma line NSCLC- showed that several MAPKs modulated the N6 and human lung epithelial cell line A549 to induction of GADD45 andG1 arrest, and fucoxanthin distinctly induced morphological positive regulation by SAPK/JNK was change such as rounding up, reduction of cell involved in GADD45A induction and G1 volume, chromatin condensation, nuclei arrest by fucoxanthin, indicating that fragmentation, and formation of apoptotic GADD45A was closely related with the G1 bodies for the two Broncho pulmonary cells arrest induced by fucoxanthin, and MAPK lines, and suggested that fucoxanthin could pathways were implicated in fucoxanthin-

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Irvani & et al. induced GADD45A expression andG1 cell CyclinB1 and surviving in MGC-803 cells. cycle arrest in tumor cells depending on the The study also showed that fucoxanthin might cell type [47]. reduce Cyclin B1 expression through The molecular mechanisms of fucoxanthin JAK/STAT signal pathway, and thus inhibit against hepatocellular carcinoma using HepG2 proliferation of MGC-803 cells [50]. Bladder cells had been studied by Das et al. (2008), cancer is not only a serious malignancy but who found that the growth-inhibitory effect of also the most expensive cancer to survey and fucoxanthin on the cancer cells was chiefly treat. Zhang et al. (2008) showed that due to an arrest in theG0/G1 phase of the cell fucoxanthin exhibited remarkable cycle, and no apoptosis was observed antiproliferative effects on human urinary indicating that fucoxanthin possessedcytostatic bladder cancer EJ-1 cells and reduced the rather than cytocidal activity in HepG2 cells. viability of EJ-1 cells by inducing apoptosis This study suggested that both the roteolysis which was characterized by morphological and transcriptional suppression might be changes, DNA ladder, and increased responsible for the decreasing levels of cyclin percentage of hypodiploid cells, and activating Ds and the suppression of cyclin D/cdk4 caspase-3 activity with a maximum ratio of activity in fucoxanthin-treated HepG2 cells apoptotic cells of >93% with 20 μM and might be related to the antitumor activity fucoxanthin [51]. Primary effusion lymphoma [48]. Liu et al. (2009) indicated that is a very aggressive type of non-Hodgkin’s fucoxanthin effectively inhibited the lymphoma infected by human herpesvirus 8. It proliferation of human hepatoma SK-Hep-1 was found that fucoxanthin and fucoxanthinol cells but facilitated the growth of murine decreased cell viability in primary effusion embryonic hepatic BNL CL. 2 cells [49]. The lymphoma BCBL-1 and TY-1 cells. study found that fucoxanthin significantly Fucoxanthin and fucoxanthinol induced increased protein and mRNA expressions of cellcycle arrest during G1 phase and caspase- connexin 43 and connexin 32 in SK-Hep-1 dependent apoptosis, inhibited the activation cells, and thus enhanced gap junctional of nuclearfactor-κB, activator protein-1, and intercellularcommunication of SK-Hep-1 cells phosphatidyl inositol 3-kinase/Akt pathways, which might be responsible for the increase of and down-regulated anti-apoptotic proteins the intracellularcalcium level, leading to cell and cell cycle regulators in primary effusion cycle arrest at G0/G1 phase, DNA lymphoma cells. In addition, fucoxanthin fragmentation, and apoptosis of SK-Hep-1 reduced the growth of primary effusion cells. The effects of fucoxanthin on human lymphoma cells in the xenografted mice, gajstric adenocarcinoma MGC-803 cells were suggesting that fucoxanthin could be investigated by Yu et al (2011), who found potentially effective for the treatment of that fucoxanthin induced cell cycle arrest in primary effusion lymphoma [14]. G2/M phaseand apoptosis of MGC-803 cells, An earlier study indicated that fucoxanthin and down-regulated the expressions of inhibited the growth of the human

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neuroblastoma GOTO cells by causing the Although the antitumor effects of arrest in the G0–G1 phase of cell cycle and fucoxanthin are known, the precise mechanism decreasing N-mycgene expression [14]. of action has yet to be elucidated. The Subsequently, Nishino et al. (1992) found that anticancer activity of fucoxanthin was partly halocynthiaxanthin showed a more potent based on the regulative effect of fucoxanthin inhibitory effect on the growth of human on biomolecules related to cell cycle and neuroblastoma GOTO cells, and also inhibited apoptosis. In addition, fucoxanthin was found the growth of other human malignant tumor to be able to selectively inhibit the mammalian cells. The antiproliferative effect of DNA polymerase activities, especially fucoxanthin is dependent on its isomeric replicative DNA polymerases (i.e., pol α, δ, structure [52]. Nakazawa et al. (2009) found and ε), and thus had anti-neoplastic activity that the anti-proliferative activity of 13-cis and [20]. Further investigations are needed to 13′-cis fucoxanthin was significantly higher assess the details of the molecular mechanisms than that of the all-trans or 9′-cis isomeric of fucoxanthin against different types of forms on the growth of cancer cells. In cancer cells with animal models [13]. addition, 13′-cis fucoxanthin had greatest inhibitory effect on the growth of HL-60 cells, - Antiobesity effects followed by 13-cisisomer and all-trans or 9′- Several studies have shown that cis isomers. It was suggested that the stronger fucoxanthin, even with a 0.02% dose, anti proliferative and inhibitory effect of cis- significantly lowered body weight, body fat fucoxanthin might be due to the steric accumulation, visceral fat-pads weights, white hindrances offered by their structure [53]. In adipose tissue weight gain, and the size of animal experiment, fucoxanth in significantly adipocyte in diabetic/obese KK-Ay mice, inhibited the formation and development of a high-fat diet-induced obese C57BL/6N mice berrant crypt foci, a preneoplastic marker for or C57BL/6J mice, and increased brown colon cancer, induced by adipose tissue weight in KK-Ay mice [14, 39]. azoxymethaneand1,2-dimethylhydrazine Moreover, fucoxanthin significantly lowered dihydrochloride in mice. Fucoxanthin had mRNA expression of proliferators activated been proven to suppress spontaneous liver receptor γ and the activity of hepatic tumor genesis in C3H/He male mice and phosphatidatephosphohydrolase, and showed antitumor-promoting activity in a two- significantly increased the mRNA expressions stage carcinogenesis experiment in the skin of of β-oxidation-related acyl-coA oxidase 1, ICR mice, initiated with7,12-dimethylbenz [a] palmitoyl and proliferators activated receptor α anthracite and promoted with 12-O- [54]. In addition, fucoxanthin and teradecanoylphorbol-13-acetate and mezerein. fucoxanthinol inhibited both lymphatic In addition, fucoxanthin was reported to triglyceride absorption and the increase of inhibit duodenal carcinogenesis induced by N- triglyceride concentration in systemic blood, ethyl-N′-nitro-N-nitrosoguanidine in mice [14]. likely due to their inhibitory effects on lipase

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Irvani & et al. activity in the gastrointestinal lumen [55].The 4−7), whereas it enhanced 3T3-L1 adipocyte abdominal white adipose tissue (WAT) differentiation at an early stage (days 0−2) by weights of rats and mice fed fucoxanthin were modulating the expression of key adipogenic significantly lower than those fed a control transcriptional regulators such as peroxisome diet. The daily intake of fucoxanthin in mice proliferator-activated receptor γ, also caused a significant reduction of body CCAAT/enhancer-binding protein α, and weight [15]. sterol regulatory element-binding protein 1c, In experiments in mice testing for anti- and inhibited glucose uptake by suppressing obesity and anti-diabetic effects, an intake of the phosphorylation of insulin receptor more than100 mg fucoxanthin/kg body weight substrate 1 in mature 3T3-L1 adipocytes, (feeding 0.1% fucoxanthin-containing diet) for suggesting that fucoxanthin exerted anti- four weeks was not sufficient to exhibit any obesity effect by inhibiting the expression of benefits [55]. On the other hand, Abidov et al. key transcriptional regulators at intermediate (2010) found that dietary administration of 2.4 and late stages and glucose uptake in mature mg fucoxanthin per day (average body weight adipocytes [60]. of volunteers was 100 kg) increased energy expenditure in the body and resulted in - Antidiabetic activity significant weight loss after 16 weeks [56]. Maeda et al. (2006) found that fucoxanthin The anti-obesity effect of fucoxanthin was markedly decreased the blood glucose and due to oxidation of fatty acids, heat plasma insulin levels, as well as water intake production, and energy dissipation through up- in diabetic/obese KK-Ay mice. It was regulating the expression of uncoupling suggested that fucoxanthin improved insulin protein 1 in the white adipose tissue [15]. resistance and decreased blood glucose level, Shiratori et al. (2005) reported that at least in part, through down regulating fucoxanthin promoted mRNA expression of adipokines such as tumor necrosis factor-α, β3-adrenergic receptor in white adipose tissue monocyte chemo attractant protein-1, of obese mice, which was responsible for interleukin-6, and plasminogen activator lipolysis and thermogenesis [57]. Sugawara et inhibitor-1 via down regulating their mRNA al. (2006) found that fucoxanthin had expression by directly acting on adipocytes significant anti-angiogenic activity which was and macrophages in white adipose tissue and also responsible for its anti-obesity effect [58]. up-regulation of glucose transporter 4 in Maeda et al. (2006) showed that fucoxanthin skeletal muscle in KK-Ay mice [61]. and fucoxanthinol inhibited intercellular lipid Hosokawa et al. (2010) demonstrated that accumulation and decreased glycerol-3- fucoxanthin attenuated hyperglycemia in KK- phosphate dehydrogenase activity [59]. Kang Ay mice, but did not affect blood glucose et al. (2011) found that fucoxanthin inhibited levels in lean C57BL/6J mice [62]. Maeda et 3T3-L1 adipocyte differentiation at al. (2009) and Park et al. (2011) showed that intermediate (days 2−4) and late stages (days fucoxanthin significantly lowered the fasting

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blood glucose concentration, the plasma followed by activation of the degranulating insulin level, and the insulin resistance index signals of mast cells which played important in diet-induced obese mice [63, 64]. Moreover, roles in inflammation and immediate-type Woo allergic reactions [68]. Sakai et al. (2011) et al. (2010) demonstrated that fucoxanthin evaluated the effect of carotenoids on dinitro significantly reduced the blood glucose, fluorobenzene-induced contact hemoglobin A1c, plasma insulin, and resistin hypersensitivity in mice to elucidate their levels, and no change was found in the plasma effect on mast cell degranulation in vivo, and glucagon concentration in high-fat diet fed showed that fucoxanthin significantly inhibited C57BL/6N mice, indicating that the reduction ear swelling and reduced the levels of tumor in the insulin/glucagon ratio could be in part necrosis factor-α and histamine, suggesting responsible to lowering blood glucose that fucoxanthin exerted an anti-inflammatory concentration by fucoxanthin [65]. effect by suppressing mast cell degranulation in vivo [69]. - Antiinflammatory effects Inflammatory response, a self-defensive - Hepatoprotective effect reaction against various pathogenic stimuli, is Woo et al. (2010) found that fucoxanthin characterized by attracting large amounts of significantly lowered the hepatic lipid leukocytes (neutrophiles, monocytes- contents, while feces weight and fecal lipids macrophages, and mast cells) to the inflamed significantly increased by inhibiting lipid area, in which these inflammatory cells are adsorption in high-fat diet fedC57BL/6N mice triggered by inflammation mediators and [70]. Park et al. (2011) also demonstrated that generate superoxide anion and nitric oxide fucoxanthin significantly decreased the hepatic radicals, and may become a harmful self- lipid droplet accumulation in high-fat fed damaging process [14]. Heo et al. (2008) and mice, possibly through reducing the activity of Kim et al. (2010) showed that fucoxanthin hepatic fatty acid synthesis-related enzymes inhibited the inducible nitric oxide synthase [71]. Furthermore, it was shown that there and cyclooxygenase 2 protein expressions, and were no significant dose-dependent effects on reduced the levels of nitric oxide, hepatic lipid changes by 0.05% and 0.2% prostaglandin E2, tumor necrosis factor-α, fucoxanthin and the 0.05% fucoxanthin might interleukin-1β, and interleukin-6 through the be sufficient for improving the hepatic lipid inhibition of nuclear factor-κB activation and content, while no significant change was found the phosphorylation of mitogen-activated in the plasma lipids in Wister rats. In addition, protein kinases [66, 67]. fucoxanthin significantly up-regulated Sakai et al. (2009) indicated that glycolytic enzyme such as glucokinase in the fucoxanthin suppressed the degranulation of liver, and thus increased the ratio of hepatic mast cells by inhibiting antigen-induced glucokinase/glucose-6-phosphatase and aggregation of high affinity IgE receptor glycogen content, indicating that fucoxanthin

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Irvani & et al. normalized the hepatic glycogen content in - Skin protective effect high-fat fed mice [14]. Over exposure to ultraviolet radiation from The reduction of liver lipids might be due sunlight leading to the generation of reactive to the increase of docosahexaenoic acid which oxygen species, inflammatory reaction, and reduces the activity of hepatic enzymes in fatty angiogenesis of the skin is presumed to be the acid synthesis and increases hepatic fatty acid primary causative agent in the damage of β-oxidation, in the liver [14]. Tsukui et al. cellular constituents and some cutaneous (2007) reported that fucoxanthin and disease such as pigmentation, laxity, fucoxanthinol enhanced the amount of wrinkling, erythema, and skin carcer [14]. Heo docosahexaenoic acid in the liver of KK-Ay and Jeon (2009) revealed that fucoxanthin mice, whereas the level of docosahexaenoic significantly decreased intracellular reactive acid in the small intestine remained unaltered. oxygen species generated by exposure to In addition, an increase in arachidonic acid (ω- ultraviolet Bradiation in human fibroblast. 6) was also found in fucoxanthin-fed mice, Fucoxanthin elevated cell survival rate and indicating that fucoxanthin might modify the inhibited cell damage forpre-treated cells, metabolic pathways of ω-3 and ω-6 highly indicating that fucoxanthin could protect skin unsaturated fatty acids [72]. Airanthi et al. against photodamage induced byultraviolet B (2011) showed that the levels of irradiation from sunlight [76]. Shimodaet al. docosahexaenoic acid and arachidonic acid (2010) found that fucoxanthin inhibited inliver lipids of KK-Ay mice given the lipids tyrosinase activity, melanogenesis in from brown seaweeds significantly increased melanoma, and ultraviolet B-induced skin [73]. Furthermore, Liu et al. (2011) showed pigmentation. The results showed that that fucoxanthin significantly recovered cell fucoxanthin significantly suppressed proliferation and increased the levels of expression of cyclooxygenase-2, endothelin glutathione. Moreover, fucoxanthin receptor A, p75neurotrophin receptor, significantly decreased intracellular reactive prostaglandin E receptor 1, melanocortin 1 oxygen species and DNA damage, and receptor and tyrosinase-related protein1, markedly decreased the level of thiobarbituric suggesting that fucoxanthin presented anti- acid-reactive substances and protein carbonyl pigmentary activity by topical or oral contents in BNL CL.2 cells induced by ferric application in ultraviolet B-induced nitrilotriacetate, indicating that fucoxanthin melanogenesis possibly through the effectively protected against ferric suppression of prostaglandin E2 synthesis and nitrilotriacetate-induced hepatotoxicity by melanogenic stimulant receptors [76]. Urikura decreasing intracellular reactive oxygen et al. (2011) showed that fucoxanthin species, thiobarbituric acid-reactive significantly suppressed ultraviolet B-induced substances, and protein carbonyl contents, and epidermal hypertrophy, which may cause increasing glutathione level, associated with wrinkle formation, vascular endothelial growth the antioxidant effects of fucoxanthin [74]. factor, matrix metalloproteinases-13

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expression, and the increase of thiobarbituric scavengingactivity, and suggested that acid reactive substances in the skin of hairless fucoxanthin had a beneficial effect on mice. The results indicated that topical cerebrovascular diseases against ischaemic treatment with fucoxanthin prevented skin neuronal cell death in stroke-prone photoage and wrinkle formation in ultraviolet spontaneously hypertensive rats [79]. B-irradiated hairless mice, possibly through the antioxidant and antiangiogenic effects of - Bone-Protective Effect fucoxanthin. These studies suggest that Using cells from the macrophage cell line fucoxanthin may be an effective ultraviolet RAW264.7 able to differentiate into protect ingredient able to be used in cosmetics osteoclast-like cells when stimulated by and sunscreen in protecting skin from receptor activator of nuclear factor κB ligand, photoaging. Moreover, it would be worthy to Das et al. (2010) showed that fucoxanthin probe into the effect of oral administration of significantly suppressed the differentiation of fucoxanthin on skin photoage [77]. RAW264.7 cells with no toxicity to RAW264.7 cells, induced apoptosis through - Antiangiogenic Effect the activation ofcaspase-3 and sequentially the Sugawara et al. (2006) found that cleavage of poly-ADP-ribose polymerase in fucoxanthin could significantly suppress the osteoclast-like cells, and did not decrease cell differentiation of endothelial progenitor cells viability in the osteoblast-like cell line into endothelial cells and the formation of new MC3T3-E1, indicating that the cytotoxicity of blood vessels and significantly reduced the fucoxanthin against osteoclasts was stronger tube length of endothelial cells. The results than that against osteoblasts. The study showed that fucoxanthin and fucoxanthinol suggested that fucoxanthin suppressed inhibited micro vessel outgrowth in an ex vivo osteoclast genesis through inhibiting osteoclast angiogenesis assay using arat aortic ring, differentiation and inducing apoptosis in suggesting that the antiangiogenic effect of osteoclasts, but did not antagonize bone fucoxanthin might be useful in preventing formation, and fucoxanthinol might play an angiogenesis-related diseases such as cancer, important role in inducing apoptosis in diabetic retinopathy, atherosclerosis and osteoclast-like cells and exert a suppressive psoriasis [78]. effect onosteoclastogenesis. These results indicate that fucoxanthin is helpful for the - Cerebrovascular protective effect prevention of bone diseases such as The preventive effect of fucoxanthin on osteoporosis and rheumatoid arthritis [80]. cultured neuronal cells from hypertensive rats was also investigated by Ikeda et al. (2003). - Ocular protective effect He found that fucoxanthin markedly After-cataract, also known as posterior attenuated neuronal cell injury in hypoxia and capsule opacification, is the main long term re-oxygenation, possibly through its radical- complication of extra capsular cataract

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Irvani & et al. extraction due to the proliferation and weeks no any relevant signs of toxicity migration of lens epithelial cells left in the occurred [82]. Kadekaru et al. (2008) capsular bag after cataract surgery. The growth conducted a toxicity study on the repeated oral of human lens epithelial cell line SRA dosing of fucoxanthin (95% purity) to rats for 01/04was apparently inhibited by fucoxanthin, 28 days, and revealed that fucoxanthin did not indicating that fucoxanthin was an efficient show obvious toxicity [83]. Beppu et al. and safeantiproliferative agent for human lens (2009) conducted a single dose toxicity study epithelial cell line and might be applied to the at doses of 1000 and 2000 mg/kg and a formulation of ocular implant products used in repeated oral dose toxicity study at doses of cataract treatment for the prevention of after- 500 and 1000 mg/kg for 30 days on purified cataract [14]. In addition, Shiratori et al. fucoxanthin (93% purity) in ICR mice. The (2005) studied the anti-ocular inflammatory results showed that no mortality and no effect of fucoxanthin on lipopolysaccharide- abnormalities in gross appearance were found induced uveitis in male Lewis rats, and found in both studies, and no abnormal changes in that fucoxanthin suppressed thedevelopment of liver, kidney, spleen, and gonadal tissues the uveitis [57]. induced by fucoxanthin in the histological observations of the repeated doses study [84]. - Antimalarial effect Subsequently, Beppu et al. (2009) indicated Afolayan et al. (2008) first found that that fucoxanthin had no genotoxic/mutagenic organic extract from brown seaweed effect on the bone marrow cells of mice [85]. S. heterophyllum exhibited promising Iio et al. (2011) investigated the subchronic antiplasmodial activity, and thus separated toxicity of fucoxanthin in rats and genotoxicity sargaquinoic acid, sargahydroquinoic acid, in mice. In the single oral dose study, no sargaquinal, andfucoxanthin from the extract mortality and no change were observed. The for further investigation. The results indicated 50% lethal dose of fucoxanthin was more than that fucoxanthin showedthe highest 2000 mg/kg body weight. In the 13-week oral antiplasmodial activity, while sargaquinal dose study, the no-observed-adverse-effect showed good antiplasmodial activity, level of fucoxanthin was 200 mg/kg body andsargaquinoic acid and sargahydroquinoic weight under the tested subchronic dose acid were only moderately active [81]. condition. These studies suggested that fucoxanthin was a safe compound anddid not Safety of Fucoxanthin exhibit toxicity and mutagenicity under these Fucoxanthin and fucoxanthinol had few experimental conditions. Although adverse effects on normal and uninfected cells fucoxanthin markedly elevated plasma high- both in vitro and in vivo. Zaragozá et al. density lipoprotein cholesterol level, total (2008) studied the toxicity of extracts from cholesterol level in the blood significantly F. vesiculosusin mice and rats, and indicated increased to the same degree while the dose of that even at the dose of 750 mg/kg daily for 4 fucoxanthin was 50 mg/kg daily for 28 days in

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Crl:CD (SD) rat and 1000 mg/kg daily for 30 However, dietary combination of fucoxanthin days in ICR mice. To further ascertain the isolated from brown seaweeds or diatoms and safety of fucoxanthin, the mechanism by edible oil or lipid could increase the absorption which fucoxanthin induces rate of fucoxanthin, and might be developed as hypercholesterolemia and species differences a promising marine drug. More extensive should be elucidated [86]. animal experimentation and well-controlled clinical trials are suggested for further studies. Conclusion Since wild microalgae and macroalgae Fucoxanthin, a carotenoid isolated from harvesting may have a negative environmental marine algae, is considered to be one of major impact due to overexploitation of these natural active compound and has attracted sources, it is expected that extraction from considerable interest because of its potent cultured organisms will have a major role to bioactivities including its antioxidant, anti- play in the coming years. Although both inflammatory, anticancer, anti-obese, microalgae and macroalgae can be grown in antidiabetic, antiangiogenic, and antimalarial controlled environments, carotenoid activities, and its protective effects on the production in macroalgae cultures is still not liver, blood vessels of the brain, bones, skin, commercially feasible on the other hand, and eyes. Particularly, the anti-obese effect, microalgae are organisms that have simpler antiproliferative effects on adult T-cell growth requirements. Moreover, due to their leukemia cells, and inhibitory effect on the simpler structure, energy is directed into viability of HL-60 cells of fucoxanthin are photosynthesis, growth and reproduction distinctly more potent than that of β-carotene processes instead of the maintenance of and astaxanthin. Hence, the results of this differentiated structures, making microalgae study indicate that the fucoxanthin has great organisms of interest for production of potential in the prevention of diseases or biomass and bioactive compounds [11]. management of human health. Orally- The authors think that for a good start, the administered fucoxanthin is metabolized into following should be the directions of fucoxanthinol and amarouciaxanthin A in biotechnological research or cultivation: mice. Therefore, fucoxanthinol, 1. The biosynthetic pathway of fucoxanthin amarouciaxanthin A, or other metabolites of is not yet fully understood. To exploit our fucoxanthin in human should be considered in knowledge regarding this carotenoid in the mechanistic studies of the biological actions of medical and nutraceutical fields, resolution fucoxanthin. of this pathway at the molecular level is very Although some brown seaweed containing important. high levels of fucoxanthin are the most 2. Select the best strains for fucoxanthin common edible delicacies, some studies production from natural or artificial showed that the bioavailability of fucoxanthin populations. in brown seaweeds was low in humans.

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3. Program transplantation of foreign strains 5. Careful collection, identification, for careful investigation. purification and cultivation of currently used 4. Develop research and biotechniques of strains. genetic manipulation of seaweeds and 6. Apply modem tissue/cell culture microalgae. Replace degenerative strains techniques to seaweed preservation. Establish with improved or genetically reformed ones. an efficient system of modem germ preservation and seed stock.

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