Antioxidants from Edible Seaweeds - ACS Symposium Series (ACS

Chapter 19

Antioxidants from Edible Seaweeds Yvonne V . Yuan

Downloaded by UNIV OF PITTSBURGH on May 3, 2015 | http://pubs.acs.org Publication Date: March 12, 2007 | doi: 10.1021/bk-2007-0956.ch019

School of Nutrition, Ryerson University, 350 Victoria Street, Toronto, Ontario M 5 B 2 K 3 , Canada

There is a long history of seaweed consumption including Phaeophyceae, Chlorophyceae and Rhodophyceae taxonomies in Asia and the Pacifics, versus low to zero intakes in Europe and the Americas. This dietary difference between populations coincides with dramatic differences in diet-related chronic disease risks such as breast cancer in these groups. Animal model evidence points to a role for edible algae in antioxidant-mediated effects in vivo. As intertidal organisms, seaweeds require an endogenous antioxidant capacity to protect against oxidative stress from U V light and dessication during tidal fluctuations. The antioxidant capacity of algae include L-ascorbic acid, glutathione, carotenoids, tocopherols, chlorophyll derivatives, polyphenols such as the phlorotannins in brown kelp and mycosporine-like amino acids in red algae. This chapter reviews the evidence underlying the antioxidant mechanisms of these algal constituents and discusses various issues for their analysis.

Interest in naturally sourced antioxidant compounds continues to grow in the food, beverage and cosmetic industries not only as ingredients to take advantage of protective effects against lipid peroxidation in extending product shelf-life, but also as bioactive components of functional foods and nutraceuticals to reduce risk factors of diet-related chronic diseases such as breast, colon and prostate cancer or atherosclerosis. While well known sources of polyphenolic antioxidants in the lay and scientific literature include herbs and spices, various teas, fruits and vegetables, oilseeds and various alcoholic 268

© 2007 American Chemical Society In Antioxidant Measurement and Applications; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.


269 beverages, an underused source of antioxidant molecules in Europe and the Americas are the edible alga or seaweeds, sometimes referred to as sea vegetables. On the other hand, edible seaweeds from the Protista taxonomies Phaeophyceae (brown (B)), Chlorophyceae (green (G)) and Rhodophyceae (red (R)) are common in Pacific and Asian diets. Seaweeds are incorporated into these diets as sushi wrappings such as the red alga known in Japan as 'Nori' or Korea as ' K i m ' (Laver, Porphyra tenera and P. yezoensis); seasonings and condiments such as the brown kelps in Japanese: 'Ararne' (Eisenia bicyclis), 'Hijikï (Hij ikia fusiformis), 'Kombu' (Laminaria digitata), 'Wakame' (Sea Mustard, Undaria pinnatifida) or Chinese cuisines 'Hai dai' (L japonica); green algae in Hawaiian cuisine 'Limu Pâlahalaha' (Ulva fasciata); or snack foods and condiments such as the European/ North American red alga 'Dulse' (Palmaria palmata). Thus, while the bulk of research investigating the antioxidant activity and composition of edible seaweeds has taken place in China (1,2), Japan (5-7) and Korea (8,9), a few researchers are also active in this area in France (10,11) and Canada (12-15). Much of the interest in the antioxidant capacity of algae has arisen from epidemiological evidence linking the habitual consumption of seaweed to reduced risk of particular chronic diseases in the Japanese and Chinese (including Korean, Mongolian and Hong Kong) populations (16). Japanese living in Japan typically consume between 0.4 to 29.2 g seaweed per day (approx. 10-25% of food intake) compared to the low to zero intake levels of migrant Japanese living in Hawaii as well as most Western populations (17,18). These population groups also have strikingly contrasted chronic disease incidences: the one-year prevalence cases per 100,000 for breast cancer in Japan and China were 42.2 and 13.1, respectively versus 125.9 and 106.2 in North America and Europe (16). Similarly, the one-year prevalence cases/100,000 statistics for prostate cancer in Japan and China were 10.4 and 0.7, respectively versus 117.2 and 53.1 in North America and Europe (16). Interestingly, one of the first references to a beneficial effect of seaweed on breast cancer was found in the ancient Egyptian 'Ebers Papyrus' dating back to approximately 1534 B . C . recommending that seaweed be administered as a treatment for breast cancer patients (19). Indeed there is evidence of antioxidant-mediated effects of dietary seaweeds in vivo such as increased antioxidant enzyme activity (GSH peroxidase) and reduced lipid peroxidation in hepatic tissue of rats treated with the mammary cancer carcinogen dimethylbenz[a]anthracene and fed on the brown kelp L. religiosa (20); as well as the inhibition of superoxide anion radical production by peritoneal activated leukocytes incubated with a sulfoglycolipid fraction from the red alga Porphyridum creuntum (21). Therefore, it is not surprising, that similar to terrestrial plants, seaweeds as intertidal organisms require an endogenous antioxidant capacity to protect plant cell membranes and organelle constituents against oxidative stresses associated with U V radiation (22-24) and dessication from tidal fluctuations (25). Indeed, algal antioxidants comprise enzymatic defenses (e.g. superoxide dismutase (SOD), glutathione reductase (GSSG-Red));

In Antioxidant Measurement and Applications; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

270


aqueous and lipophilic molecules including L-ascorbic acid, glutathione (GSH), carotenoids, tocopherols and chlorophyll derivatives (e.g. chlorophyll a, pheophytin a) as well as secondary metabolites such as flavan-3-ols, phenolic acids, lignans, phlorotannins (phloroglucinol polymers) in brown kelp and mycosporine-like amino acids (e.g. mycosporine-glycine and shinorine) in red algae (Table 1). This chapter reviews the evidence and mechanisms underlying the antioxidant activities of edible brown, red and green seaweed constituents. It is noteworthy that antioxidant activity has been reported in both polar as well as lipophilic fractions isolated from these seaweeds; thus, this chapter will also address methodological considerations when analyzing these data where appropriate.

Aqueous low-molecular-weight reducing agents - L-ascorbic acid and glutathione Reducing activity, defined as the donation of electrons to another chemical; the supply of a hydrogen to another chemical; or the removal of oxygen from another chemical is key to maintaining the redox potential of intracellular systems, particularly in the presence of the potential for generation of reactive oxygen species (ROS) such as singlet oxygen CO2), superoxide radical anion (0 "), hydrogen peroxide ( H 0 ) and the highly reactive hydroxyl radical ( Ό Η ) during normal oxidative metabolism. Nonenzymatic aqueous low-molecularweight reducing agents include ascorbic acid and glutathione (GSH, L-γglutamyl-L-cysteinyl-glycine) in vivo in human and animal tissues (26) as well as terrestrial and marine plants (25,27,28). A survey of the G S H contents of a variety of seaweeds harvested from the coast of Mie Prefecture, Japan, was recently carried out by Kakinuma and coworkers (28)\ G S H content was 0.1 200 mg/100 g dry wt in Rhodophyceae, 19-3082 mg/100 g dry wt in Phaeophyceae and 2-113 mg/100 g dry wt in Chlorophyceae. The one extremely low G S H content amongst the red algae was due to Corallina pilulifera, a species unusual for its highly calcified tissue, otherwise the range for red algae was 21-200 mg/100 g dry wt. Other workers investigating the red alga Stictosiphonia arbuscula, harvested in Brighton Beach, Otago, New Zealand, reported that ascorbate and G S H levels in fresh specimens were approx. 2.5 - 2.8 and 0.69 0.71 pmol/g wet wt., respectively (25). Fresh Dulse has been reported to contain 220-520 pg ascorbic acid/g wet wt. harvested from Spitsbergen, Norway and unknown locations in France and elsewhere (24,27). Fresh specimens of Laminaria sp. (B) harvested from Spitsbergen, Norway exhibited lower (170 pg /g wet wt) or only trace amounts of ascorbic acid. However, the air- and sundrying, as well as storage of Dulse is known to reduce the vitamin C content measured in samples (27). The vitamin C content of Dulse harvested from an unknown location in Japan was approximately 2000 pg/g dry wt (29). Similarly, when McDermid and Stuercke (30) surveyed 22 species of algae harvested from various locations in the Hawaiian Islands; the vitamin C content of the oven2

2

2



271 dried samples (60 °C) was minimal with detectable levels reported in only 4 samples: between 1300 and 3000 pg/g dry wt in the Chlorophyceae: Enteromorpha flexuosa, Monostroma oxyspermum, Ulva fasciata and 2000 pg/g dry wt in Eucheuma denticulatum (R). The vitamin C content of the brown alga Ascophyllum nodosum harvested from an unknown location was 550-1650 pg/g dry wt (29). The total content of combined reduced and oxidized ascorbate was not altered in dessicated S. arbuscula, but the concentration of reduced ascorbate did decline at 12 h and remained low at 48 h of dessication (25). Thus, aqueous low-molecular-weight reducing agents are likely most important to the oxidative status of algae prior to harvest, and are found in low concentrations in the dehydrated and processed products available to the consumer purchasing these products. As a reflection of the potential bioactivity of the aqueous low-molecularweight antioxidants discussed above, the reducing activity of water- and alcohol-soluble extracts from a variety of edible algae has been assessed using a variety of in vitro methodologies including the potassium ferricyanide assay of Yen and Chen (37), the hydroxyl radical scavenging deoxyribose assay of Halliwell and coworkers (32) as well as stable free radicals such as 1,1diphenyl-2-picrylhydrazyl (DPPH-; 33) and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical cation ( A B T S ; 34). For example, water extracts of 27 species of edible and non-edible seaweeds harvested in Shandong Province, PRC demonstrated hydroxyl radical scavenging activity in green, brown and red varieties, with the strongest activities in Rhodomela teres (R) and Chorda filum (B; 2). Methanol extracts of these same seaweeds demonstrated relatively weak D P P H radical scavenging activities after 60 and 90 min incubation, with the strongest activities in Phaeophyceae'. Desmarestia viridis, Sargassum kjellmanianum and C. filum. Similarly, a study conducted in Seoul, Korea with crude and fractionated methanol extracts of seaweeds indicated strong DPPH* scavenging activity in U. pinnatifida (B), Enteromorpha sp. (G), P. yezoensis (R) and Dasya pedicellata (R; 8). Matsukawa and coworkers (5) reported that aqueous extracts of seaweeds harvested in Onagawa, Miyagi Prefecture, Japan, generally exhibited lower inhibition of DPPH radical compared to ethanolic extracts after 15 min incubation, regardless of seaweed variety. The DPPH* scavenging activity of the brown kelps was greater than that of the red or green algae studied, with the exception of Porphya sp. which exhibited strong inhibition of DPPH*. More recently, a 1-butanol soluble extract from Dulse (P. palmata) harvested in Grand Manan Island, New Brunswick, Canada, was reported to exhibit moderate Ό Η and DPPH* scavenging activities compared to L-ascorbic acid and butylated hydroxyanisole (BHA) controls (73). It is noteworthy that the DPPH* scavenging activity of the Dulse extract was observed to take place over 105 min compared to 15 and 50 min for L-ascorbic acid and B H A , respectively. Thus, it is important to consider the assay incubation times when comparing DPPH* scavenging activities from different studies, as an insufficient incubation period may influence the results. Moreover, despite being widely used by researchers to evaluate the free radical scavenging $+



Ecklonia kurome

Ecklonia cava

Ascophyllum nodosum

Phaeophyceae

"Kajime'

9

7

Phlorotannins - phloroglucinol, eckol, dieckol, 8,8'-bieckol, phlorofucofuroeckol A, unknown tetramer, other unknowns. Itoshima Peninsula, Japan

11 Phlorotannins - phloroglucinol, eckol, dieckol, 6,6'-bieckol, phlorofucofuroeckol A.

α-, δ-, γ-tocopherol

47

47

53

References

Jeju Island, Korea

Pleubian, France

Epigallocatechin, caffeic acid, Catechol, Hesperidin, Myricetin, Morin.

'Sabotengusa'

Okinawa Prefecture, Japan

Halimeda opuntia

Epigallocatechin, caffeic acid, Catechol, Hesperidin, Myricetin, Morin.

'Hiroha-sabotengusa' Okinawa Prefecture, Japan

Pheophytin a

Antioxidant compounds

Halimeda macroloba

Japan

Harvest location

* Aonori'

Common name

Enteromorpha sp.

Chlorophyceae

Seaweed class

Table 1. Antioxidant profile of edible seaweed varieties.



Pleubian, France

Laminaria digitata

'Kombu'

Pleubian, France

α-, γ-tocopherol, L-ascorbic acid

α-, γ-tocopherol L-ascorbic acid

11, 10

11, 10

12

41

11

11

3

53, 7

Continued on next page.

Mammalian lignans Enterodiol, Enterolactone

Store-bought, Toronto, Canada

Himanthalia elongata

Fucoxanthin, Phlorotannins

Store-bought, Tsukuba, Japan

Hijikia fusiformis


Pleubian, France

Fucus serraîus

Phospholipids Phosphatidyl ethanolamine Phosphatidyl inositol

Jogashima, Kanagawa Prefecture, Japan


Phlorotannins - phloroglucinol, eckol, dieckol, 8,8'-bieckol, phlorofucofuroeckol A , unknown tetramer, other unknowns

Tsuzumi island, Japan; Itoshima Peninsula, Japan

Pleubian, France

'Hijiki'

'Ararne'

Fucus vesiculosus

Eisenia bicyclis



Fucoxanthin

Palmaria palmata

Polysiphonia urceolata

Rhodophyceae

'Dulse'

Lutein

Isle of Wight, UK

40

22, 62

MAAs - Mycosporine-glycine, Shinorine, Porphyra-334, Palythine, Asterina-330, Palythinol, Palythene, Usujirene.

Spitsbergen, Norway; Minami-Kayake, Hokkaido, Japan

12

Bromophenols 56 5-bromo-3,4-dihydroxybenzaldehyde 5-bromo-3,4-dihydroxybenzyl alcohol

Store-bought, Toronto, Canada

'Mekabu'

Undaria pinnatifida

(sporophyll)

Miyagi Prefecture, Japan

Onagawa, Miyagi Prefecture, Japan

'Wakame' 'Sea mustard'

Ondarla pinnatifida

(stipe)

Mammalian lignans Enterodiol, Enterolactone

1,2

High and low MW Phlorotannins

Taipingjiao, Qingdao, PRC

56

61

Chlorophyll a, L-ascorbic acid

Qingdao, PRC

References

'Makonbu' 'Hai dai'

Antioxidant compounds

Harvest location

Common name

Sargassum kjellmanianum

Laminaria japonica

Seaweed class

Table 1. Continued.



Porphyra yezoensis

Porphyra tenera

'Susbinori'

'Non' 'Kim'

'Laver'

Hyogo, Japan

Ota-ku, Tokyo, Japan

Store-bought, Sendai, Japan

M A A s - usujirene, β-carotene, Chlorophyll a, Pheophytin a, A m i n o acids - Alanine, Leucine, Phenylalanine

M A A s - Mycosporine-glycine, Shinorine, P o φ h y r a - 3 3 4

Phospholipids Phosphatidyl choline Phosphatidyl ethanolamine


59

36

276 activity of plant extracts, DPPH» does not solubilize easily, albeit, it does generate strongly colored solutions with methanol and ethanol as solvents (IS). Re and coworkers (34) reported that the decolorization of the ABTS» radical cation reflected the capacity of an antioxidant species to donate electrons or hydrogen atoms to inactivate this stable free radical. The ABTS» radical cation scavenging activity of the Dulse extract and controls was very similar to the DPPH» results, reflecting the reaction stoichiometry of the number of electrons available to inactivate the free radical in the assay (13). For example, slowly reacting free radical scavengers such as butylated hydroxytoluene (BHT), or the closely related B H A , and the Dulse extract, are hypothesized to have a more complex reaction mechanism involving one or more secondary reactions in the quenching of the DPPH» (35) and ABTS» radicals. Essential considerations when determining the reducing activity of seaweeds are the topographic features of the geographic location and water depth of the harvest location of these intertidal species. Seaweeds are exposed to wide variations in photosynthetically available radiation (PAR; 400-700 nm) and more specifically, U V B (280-320 nm) and U V A (320-400 nm) radiation depending upon the season, water depth and turbidity as well as the presence of topographic features such as steep cliff faces (14,22-25). In fact, when Dulse was harvested from two locations known to vary in U V exposure, the sample from a sunny low-lying beach ('Passage' area of Grand Manan Island, N B ) exhibited a 1.68 fold greater reducing activity than the corresponding sample from a shaded high-cliff area (Dark Harbour) on the opposite side of the island (14). Water depth appeared to influence the ability of S. arbuscula (R) to maintain G S H reserves upon dessication/dehydration, in that specimens from deeper water depths exhibited reductions in total G S H at 24 and 48 h of dessication, whereas specimens from shallow waters only experienced a decrease in G S H after 48 h (25). Thus, it is vital to collect water depth and topographic data about the harvest sites of seaweeds in order to compare data from different reports or even the same species from different locations. +


+

+

Lipophilic antioxidants - chlorophyll and its derivatives, carotenoids, tocopherols, and phospholipids The importance of the presence of lipophilic antioxidants to the oxidative status of marine organisms such as algae is implicit to combat cell membrane damage against not only dessication during tidal fluctuations, but also photooxidative stress from U V radiation. This takes on particular importance given that the fatty acid profile of various edible seaweeds are rich in the longchain polyunsaturated ω6 and ω3 fatty acids, including arachidonic acid (C20:4,o)6) and eicosapentaenoic acid (C20:5,œ3; 10,36-38). The fatty acid profiles of Laminaria digitata (Β) and Himenthalia elongata (B) harvested from


277 Pleubian, France were enriched in C20:4, co6 at 24.3 and 33.4%, respectively (10). Similarly, the kelps L. ochroleuca (B) and H. elongata (B) harvested from Bayona and Finisterre, Spain were richest in C20:4, ω6 at 14.20 and 10.69 % total fatty acids (38). Whereas Rhodophyceae such as commercial P. tenera (36), Palmaria sp. harvested from Rinlo, Spain (38) and Porphyridium sp. grown in culture (37) were rich in C20:5, ω3 at 40.8, 24.05 and 11.8 % total fatty acids, respectively. While chlorophylls are the major photosynthetic pigment in algae, carotenoids are also considered to be secondary photosynthetic pigments; thus, it is interesting to note that both classes of pigment also have antioxidant activity. Alpha- and β-carotene are well known as electron donors to free radicals and particularly as 0 quenchers. Similarly, α-, γ- and δtocopherols are well known as electron or hydrogen donors as free radical scavengers as well as 0 quenchers in vitro and in vivo. As photosynthetic pigments, tissue levels of chlorophyll a and carotenoids have been reported to vary seasonally depending upon U V irradiation exposure levels (23,27). When brown, red and green algae were harvested from Spitsbergen, Norway between early June (when the water was still covered by ice) and early August (well after sea-ice break-up), L. saccharina (B), the red alga Dulse and Monostroma sp. (G), as representative species, all exhibited greater levels of chlorophyll a (approx. 2.35 mg/ g wet wt) in early June compared to markedly lower levels (approx. 1.5 mg/g wet wt) in mid to late-July (23). Thus, algal tissues appear to concentrate chlorophyll a during periods of reduced U V radiation (i.e. ice cover, cloudiness) versus lesser pigmentation during periods of enhanced U V radiation (i.e. reduced turbidity, clear skies). l


2

]

2

Carotenoids In contrast to chlorophyll above, tissue levels of carotenoids (a- and βcarotene) in the red alga Dulse appear to peak during the Spring and Summer months (April to September) ranging between 110 and 420 pg/g dry wt in samples harvested from North Berwick, Scotland; Helgoland, Germany and unknown locations in Norway in comparison to the Winter (January) at 37 pg/ g dry wt (27). These data support a role for carotenoids as protective antioxidant molecules in algal tissues; indeed, carotenoids are known to provide photoprotection in plant tissues by preventing the photooxidation of chlorophylls since these pigments exist in close association with each other via weak non-covalent bonds (39). Tissue levels of β-carotene in algae harvested from different locations in the Hawaiian Islands varied widely depending on class, being 27-180, 60-97 and 15-430 IU/g dry wt in the Chlorophyceae, Pheophyceae and Rhodophyceae, respectively (30). Another factor which will impact the carotenoid levels reported in the literature include whether the data reflect total carotenoids, the separate identification of a- and β-carotene, or whether other carotenoids have also been determined, such as lutein, zeaxanthin or fucoxanthin (27,40,41). It is noteworthy that algal a- and β-carotene levels


278 can represent only approximately 1/3 to 1/8 of total carotenoid content in Dulse (27). For example, lutein has been reported to be a significant carotenoid in Dulse at 240 pg/g dry wt in a sample harvested from an unknown location in Norway (27) as well as that from Bembridge, Isle of Wight, U K (40). Moreover, all-/rara-fucoxanthin, an orange colored carotenoid, has been reported to be a major carotenoid in Hijikia fusiformis (B) along with two ds-isomers of this powerful antioxidant (41).


CH3

Fucoxanthin Fucoxanthin exhibits strong antioxidant activity against the D P P H radical. Interestingly, these results differ from a previous report wherein β-carotene, zeaxanthin and lutein did not scavenge the D P P H radical (41). Fucoxanthin has also been identified in the chloroform-soluble fraction of Fucus vesiculus (B) wherein it demonstrated antioxidant activity against lipid peroxidation, but did not exhibit any synergism with vitamin E (77). The structure of fucoxanthin differs from other carotenoids in that there is an unique double allenic carbon at C-7' near one of the two hydroxyl groups in its structure which may confer additional stability and resonance stabilization within the conjugated double bond structure of this molecule. Tocopherols Seaweed tissue levels of the tocopherols vary seasonally with typically low concentrations during Winter and Spring ranging between 22 and 35 p g / g dry wt for the red alga Dulse and trace levels in L. digitata (Β) compared to Summer and Fall levels of approximately 139 pg/g dry wt in Dulse and 2 % dry wt in L. digitata, similar to the phenomenon observed with the carotenoids above (11,27). The brown kelp Ascophyllum nodosum harvested from an unknown location was reported to contain 260-450 pg tocopherols/g dry wt (29). The tocopherol composition of various brown kelps (L digitata, 77. elongata, F. vesiculus, F. serratus and A. nodosum) has also been broken down into the α-, γ- and δisomers, with α-tocopherol making up the majority (77). Certainly, the tocopherol content of the organic solvent extracts of brown kelps was


279 instrumental in the antioxidant effects observed against methyl linoleate oxidation when incubated with extracts of L digitata harvested during the summer, which contained significant amounts of tocopherols, versus the winter when the alga contained only trace amounts of tocopherols (77).


Phospholipids Despite the fact that the phospholipid (PL) fractions of algae are noted to be enriched in the long-chain polyunsaturated fatty acids, there is evidence of the antioxidant activity of these membrane lipids (3,11,36,42). Glycerophospholipids are characterized as lipids containing phosphoric acid as a diester. The phosphoric acid moiety of PL confers the ability to chelate transition metal ions, which may play a part in the antioxidant activity of these molecules. When Kaneda and Ando (36) investigated the antioxidant activity of the chloroform-soluble fraction of P. tenera (R), the P L fraction strongly inhibited the oxidation of the methyl esters of safflower oil fatty acids by extending the induction period on their own and also demonstrated synergistic effects when combined with butylated hydroxytoluene (BHT). Interestingly, the P L fraction of this red alga was enriched with C20:5, ω3 at 54.9 % fatty acids. When the P L fraction of P. tenera was separated into its component molecules, phosphatidylethanolamine and phosphatidylcholine predominated at 30.6 and 23.5 %, respectively, followed by phosphatidylserine (14.0%), lysolecithin (13.2%), sphingomyelin (9.9%) and phosphatidylinositol (4.5%). The chloroform-soluble fraction of Eisenia bicyclis (B) exhibited strong antioxidant activity against the oxidation of safflower oil fatty acid methyl esters at 37 °C (3). Upon chromatographic separation of this extract, the P L fraction was shown to be responsible for the antioxidant activity; phosphatidylethanolamine and phosphatidylinositol were the main PLs. Other workers have reported that the antioxidant activity of PLs is due to synergism with other primary antioxidants such as the tocopherols (11,42). Indeed, crude organic solvent extracts of L. digitata (Β) were not only effective against peroxide production in sunflower oil at 75°C on their own, but also exhibited synergistic effects in combination with vitamin E in extending the induction period during the oxidation of methyl linoleate at 60°C (10). Interestingly, despite the strong inhibition of methyl linoleate oxidation by organic solvent-soluble extracts of F. vesiculus (B), F. serratus (B) and A. nodosum (B) as well as their synergism with vitamin E, the purified phospholipid fractions of these kelps were not observed to be synergistic with vitamin E (77). The phospholipid fractions of these kelps were richest in phosphotidylinositol as opposed to phosphotidylethanolamine as observed in the algae above. Phosphatidylinositol lacks an amino-containing moiety necessary for phospholipid synergism with vitamin E as antioxidants (77). Thus, the synergism of phospholipids is specific to those which contain an


280 amine group in their structure, i.e. phosphatidylcholine, phosphatidylethanol amine and phosphatidylserine. Chlorophyll derivatives It is noteworthy that not only has the photosynthetic pigment chlorophyll a been identified as an antioxidant molecule in various algal extracts, but also that chlorophyll derivatives such as pheophytin a and pyropheophytin a have also been reported to be responsible for the antioxidant activity of organic solvent extracts of seaweeds. Chlorophylls contain a M g within the porphyrin structure (Figure 1) and are thus noted to provide a green-blue pigment to plant tissues. On the other hand, pheophytins are the Mg-free derivatives of chlorophylls which are most often formed during the heating of plant tissues in the presence of acid, leading to the loss of M g from the structure and an olivebrown coloration in tissues. Pyropheophytins are formed through the replacement of the C-10 carbomethoxy group of pheophytins by a hydrogen atom (Figure 1; 39) leading to an olive colouration. Chlorophylls are classified as porphyrins or macrocyclic tetrapyrrole pigments: the four pyrrole rings are linked via methyne bridges forming a closed loop of conjugated double bonds. Thus, the conjugated system of double bonds can provide resonance stabilization to not only chlorophyll a, but also pheophytin and pyropheophytin a as potential antioxidant molecules reacting with free radicals. Moreover, chlorophylls are also noted to undergo a process called 'Allomerization' upon oxidation of the molecule with equimole 0 uptake to the number of chlorophyll molecules present. Oxidation of chlorophylls occurs at the cyclopentanone ring forming either 10-hydroxychlorophylls or 10-methoxylactones (39). Studies evaluating the effect of chlorophyll and pheophytin on the stability of vegetable oil indicated that antioxidant activity was observed in chlorophyll containing oil samples stored at low temperatures in the dark (42). The antioxidant activities of pheophytin a and pyropheophytin a may also include indirect antioxidant activity through the chelation of transition metal ions due to the fact that in pheophytin, and thereby pyropheophytin, the two hydrogen atoms are easily displaced by C u or Zn to form green coloured metallo complexes; moreover, Cu ions appear to be chelated more rapidly than those of Zn (39). Further work to elucidate the mechanism of antioxidant activity of chlorophyll a developed from initial results indicating that hexane-soluble extracts from L. digitata (Β) and 77. elongata (Β) were protective against not only oxidation of methyl linoleate, but also exhibited synergism with vitamin E (77). These workers isolated two unidentified chlorophyll-related compounds from the hexane-soluble extract of H. elongata and chlorophyll a in L. digitata; chlorophyll a was previously reported to have synergism with vitamin E against lipid peroxidation. Le Tutour and coworkers (77) proposed that chlorophyll a can react with peroxyl radicals


2 +

2 +

2


281

:H2CH2C02CH2CH=Ç(CH2CH2CH2ÇH)3CH3 CH3


CH3

H2OCH - ^ ^ r ^ ^ ^ ^ CH

3

H

CH

C H 3

3

C

Pyropheophytin a Figure 1. Chlorophyll a and its derivatives.


H

3

282 (ROO') to form a complex with the π-cation radical of chlorophyll a bound to the negatively charged peroxyl radical: ROO' + chlorophyll a

ROO" + chlorophyll a

+

The complex can then react with another peroxyl radical to form inactive products


ROO" + ROO-chlorophyll a'

+

> inactive products

Moreover, the synergistic antioxidant effect of chlorophyll a in the presence of vitamin E was proposed to involve a non-reactive complex of an a-tocopheryl radical with chlorophyll a: α-tocopheryf + chlorophyll a

>. a-tocopheryl-chlorophyll a

+

which would then block further propagation of lipid peroxidation (11).

Polyphenolic antioxidants-flavonoids, phenolic acids, lignans, phlorotannins and bromophenols As a broad class of compounds, polyphenols comprise the flavonoids including flavones, flavonols, flavanones, flavononols, chalcones and flavan-3ols as well as lignans, lignins, tocopherols (discussed above), tannins and the related marine polyglucinol polymers (i.e. phlorotannins), plus phenolic acids. The flavonoids are synthesized via the Shikimic acid pathway from the aromatic amino acid precursors phenylalanine and tyrosine, giving rise to C - C cinnamic acid derivatives (Figure 2). Cinnamic acid can then be metabolically converted to form flavonoids or various phenolic acids via p-coumaric acid and caffeic acid. Flavonoids are characterized by a C - C - C skeleton as two aromatic rings bridged by a C aliphatic chain often condensed into a pyran or furan ring. As well, flavonoids often occur as glycosides in plant tissues which are deglycosylated to the aglycones during digestion. The diversity of these compounds is also reflected in the antioxidant mechanisms involved such as ROS scavenging, free radical inactivation as well as transition metal ion chelation. It is well known that the position as well as the degree of hydroxylation influences the antioxidant activity of polyphenols. Thus, most monophenols are less effective than polyphenols; addition of a second hydroxyl group ortho- or para- to the first will increase the antioxidant activity (42). Interestinly, methoxy substitutions will also increase the antioxidant activity of a mono-phenol. However, methoxy substitution is noted to be less effective than hydroxylation at increasing antioxidant activity (42). In addition, cinnamic acid and its derivatives with the - C H = C H C O O H side chain are noted to be more 6

6

3

3

6

3


283 efficient antioxidants than benzoic acid and its derivatives. Transition metal ion chelation can occur at the 3-hydroxyl, 4-keto group and/or the 5-hydroxyl, 4keto group when the A ring is hydroxylated at the 5 position of flavonols (43). Generally, structures containing two or more of the following: - O H , - S H , C O O H , - P O 3 H 2 , C=0, - N R , -S- and -O- in a favorable structure-function configuration will exhibit transition metal ion chelation activity (44). Thus, the flavonol quercetin and its glycoside, rutin are noted to chelate transition metal ions (45). Yoshie and coworkers (46) surveyed the flavan-3-ol profiles of Japanese green, brown and red algae, and found that catechin was present in only two Chlorophyceae: Acetabularia ryukyuensis (3330 pg/g dry wt) and Tydemaniz expeditionis (250 pg/g dry wt). It was also present as Phaeophyceae including E. bicyclis (1240 pg/g dry wt), albeit only trace amounts in L. religiosa and none in U. pinnatifida and H fusiformis, the most commonly consumed kelps in Japanese cuisine; it was also present in most Rhodophyceae including P. yezoensis (36 pg/g dry wt). The catechin epimer, epicatechin (EC), was found in only five algae: A. ryukyuensis (500 pg/g dry wt), E. bicyclis (3860 pg/g dry wt), Sargassum muticum (B; 2620 pg/g dry wt), Gelidium elegans (R; 130 pg/g dry wt) and Chondrococcus hornemannii (R; 360 pg/g dry wt). The corresponding gallate esters were not widespread: catechin gallate was found in only G. elegans (70 pg/g dry wt) and epicatechin gallate (ECG) in E. bicyclis (290 pg/g dry wt) and S. muticum (120 pg/g dry wt). Epigallocatechin (EGC) was found in only one Chlorophyceae: T. expeditionis (350 pg/g dry wt); most Phaeophyceae including E. bicyclis (4770 pg/g dry wt), H. fusiformis (3770 pg/g dry wt), but none in U. pinnatifida nor L. religiosa; a few Rhodophyceae including C. hornemannii (16,000 pg/g dry wt), but not P. yezoensis (46). Epigallocatechin gallate (EGCG) was absent from all of the Chlorophyceae investigated and present in only three Phaeophyceae: E. bicyclis (280 pg/g dry wt), Padina arborescens (680 pg/g dry wt) and P. minor (490 pg/g dry wt); and in only three Rhodophyceae: P. yezoensis (32 pg/g dry wt), Gracilaria texorii (24 pg/g dry wt) and Gracukarua asiatica (18 pg/g dry wt). Taken together, these data suggest that the Chlorophyceae are relatively poor sources of the flavan-3-ols; as well, the flavan-3-ol gallate esters are not well represented amongst the Japanese edible algae. Edible algae polyphenols with respect to their hydroxylated cinnamic acid derivatives, flavonols, flavanones and their glycosides have also been investigated (47,48). Analysis of two Chlorophyceae currently not used for food, revealed that Halimeda macroloba and H opuntia were rich in E G C (28000 and 12700 pg/g dry wt, respectively), catechol (1880 and 384 pg/g dry wt), the flavonols myricetin (414 and 147 pg/g dry wt) and morin (429 and 234 pg/g dry wt; 47). In addition, H macroloba also contained caffeic acid (84.9 pg/g dry wt) and the flavanone glycoside hesperidin (hesperitin-7rhamnoglucoside; 144 pg/g dry wt). While most Japanese edible green, brown and red algae were found to be rich in the flavonol morin, ranging between 257 th


2



>2C

I

Phenylalanine

Q-CH*!HCOOH

I

NH2

Tryptophan

Shikimic acid

OH

Cinnamic acid

Isoflavones

Flavonoids

Gallic acid

* G^-X-COOH

Antioxidants from Edible Seaweeds - ACS Symposium Series (ACS

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