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CHAPTER 2 REVIEW OF RELATED LITERATURES AND STUDIES

This chapter presents the literatures relevant to the study. It gives information about the plant material that will be used in the research, its botanical origin, and a background of the properties and other activities that have been previously studied about the Sargassum species. It also includes studies about algal polysaccharides and their uses, and discussions regarding sulfated polysaccharides and its biological activities, specifically its antioxidant activity. Also, presented here are the principles involved in the tests that will be conducted in determining the antioxidant activity of Sargassum siliquosum J. Agardh.

2.1 Plant Material

2.1.1 Botanical Description

Torno and Fortes (as cited in Montano et al., 2005) mentioned that the Genus Sargassums are generally large, tall, and dark brown or yellowish in color. Some Sargassum species grow to several meters forming a bushy habitat while others are found drifting along the currents or near-shores. Sargassum siliquosum J.Agardh is one of the most obvious species of Sargassum along tropical shorelines, but which later break free and drift in the open ocean where they reproduce vegetatively. The 'stems' grow to about 20cm or longer.

UST FACULTY OF PHARMACY


Attached to the stems are leaf-shaped blades and inflated air bladders. The 'leaves' may be narrow, broad or very small (1-5cm long). The small rounds to oval air bladders interspersed among the leaves are often mistaken for fruits.They grow up to 2.3m in height. On the

average, S. siliquosum plants are 1.5 meters in height during its maximum growth season of August to October (Ang, 1985). Like its varied uses, different places in the Philippines have their own local names for this brown seaweed. In the Northern Philippines, it is locally called Aragan, Boto-boto, and Lusay-lusay while In Central Visayas and Northern Mindanao, it is known as Samo (Montano et al., 2005). Figure 1 shows the image of S. siliquosum J. Agardh. http://www.seavegetables.com/handbook/genera/browns/Sargassum/Sargassu m.htm 2.1.2 Taxonomic Classification

Domain: Eukaryota Kingdom: Chromista Phylum: Ochrophyta Class: Phaeophyceae Order: Fucales Family: Sargassaceae Genus: Sargassum Specific descriptor: siliquosum- J. Agardh Scientific name:

UST FACULTY OF PHARMACY Sargassum siliquosum J. Agardh (Bisby FA, Roskov YR et al., 2009).

Figure 2.1 Plant of Sargassum siliquosum J. Agardh

2.1.3

Distribution and Occurence

Sargassum is widely distributed in the intertidal and shallow subtidal rocky substrate of the tropical and subtropical coastal waters including the Philippines (Hurtado & Ragaza, 1999). Sargassum plants are usually found on coralline substratum from low intertidal = 0.5m above mean lower low water level (MLLW) to shallow subtidal of not more than 1.5m below MLLW (Ang, 1985). In a study conducted by Ang (1986) where he studied a Sargassum community located near Balibago, Calatagan, Batangas (13" 56' N, 120" 37' 24" E), he found out that among the eight species reported from that area (his area of study), S. siliquosum

UST FACULTY OF PHARMACY J. Ag is among the dominating Sargassum species constituting 50.4% of the total number of whole Sargassum plants counted.

2.1.4 Constituents and Uses


Sargassum species are the tropical equivalent to Ascophyllum and Fucus in terms of uses and applications. They are used as raw materials for alginate production and are also used as a component of animal feed and a source of liquid plant foods or plant biostimulants. There may also be nutraceutical or

pharmaceutical applications for fucoidan and other bioactive extracts of the genus.

Little is known about the constituents present in Sargassum siliquosum J. Agardh alone. Normally here in the Philippines, S.Siliquosum is studied together with other Sargassum species, and their uses are classified in general. Sargassum, common and abundant brown seaweed (Ang, 1985; Hurtado and Ragaza 1999) in the lower subtidal zones of the Philippines is a potential source of sodium alginate (Ragaza and Hurtado, 1999). Sargassums are used for alginate production in the tropics. Sodium alginate is mainly used as an emulsifier, thickener, stabilizer, or gel-forming agent like agar and carrageenan in the food industry. Harvesting of this seaweed in the central and southern Philippines is mainly for the production of 'kelp' powder which is used as a component of feeds and a source of fertilizer. Several tons of Sargassum are cast to the shoreline after every typhoon during the months of July to October and remain unattended making the resource not utilized (Ragaza & Hurtado, 1999). Edible seaweeds have high nutritional values as sources of vitamins, minerals and non-caloric dietary fibers and as potential sources of biological active ingredients

UST FACULTY OF PHARMACY (Yan, Chuda, Suzuki & Nagata, as cited in Gamal-Eldeen, 2009). Various chemical analyses were performed on Sargassum species and the main components found were sugars, sulfate, amino acids, and small amounts of betaines (Al-Amoudi et al., 2009). Activities of some Sargassum species are hereby summarized in Table 2.1.

Table 2.1 Summary of the Different Activities of Some Sargassum Species Sargassum species S. oligocystum Activities Anticancer Title of Study Anticancer activity of Sargassum oligocystum water extract against human cancer cell lines Author and Year Zandi, Ahmadzadeh, Tajbakhsh, Rastian, Yousefi, Farshadpour, & Sartavi. (2010) Kandhasamy & Arunachalam (2008) Zhu et al. (2004)

S. myricocystum S. tenneerimum S. patens

Antibacterial

Evaluation of in vitro antibacterial property of seaweeds of southeast coast of India Antiviral property and mode of action of a sulphated polysaccharide from Sargassum patens against herpes simplex virus type 2 Sargassum fusiforme fraction is a potent and specific inhibitor of HIV-1 fusion and reverse transcriptase Biological activities of sulfated polysaccharides from tropical seaweeds

Antiviral

S. fusiforme

Antiviral

Paskaleva et al. (2008)

S. filipendula

Antioxidant, Anticoagula nt, Antiproliferat ive

Costa et al. (2010)

UST FACULTY OF PHARMACY S. binderi Antioxidant Antioxidant capacity of sulfated polysaccharides from seaweeds. A kinetic approach Chemical composition and antioxidant activities of Jeddah corniche algae, Saudi Arabia In vitro antioxidant activities of three selected brown seaweeds of India Purification, antitumor and antioxidant activities in vitro of polysaccharides from the brown seaweed Sargassum pallidum Effect of Sargassum thunbergii on ROS mediated oxidative damage and identification of polyunsaturated fatty acid components Antioxidant activities of enzymatic extracts from brown seaweeds Antipyretic, analgesic, and anti-inflammatory activities of the seaweed Sargassum fulvellum and Sargassum thunbergii in mice Barahona et al. (2011)

S. crassifolia

Antioxidant

Al-Amoudi et al. (2009)

S. marginatum S. pallidum

Antioxidant

Antioxidant Antitumor

Kumar Chandini et al. (2008) Ye et al. (2008)

S. thunbergii

Antioxidant

Kim, J., Kong & Kim, S. (2010)

Heo et al. (2005) Kang et al. (2008)

Antipyretic, Analgesic Antiinflammatory S. fulvellum

S. wightii

Antioxidant

Total Flavanoid and in vitro Antioxidant Activity of Two Seaweeds of Rameshwaram Coast

Meenakshi, Gnanambigai, Tamil mozhi, Arumugamnd, & Balasubramani an (2009)

UST FACULTY OF PHARMACY 2.2 Algal Polysaccharides and Their Uses

Normally, brown seaweeds contain four main polysaccharides namely, alginate, polymannurate, laminaran, and fucoidan (Michailovna et al., 2007). Alginate is a natural polysaccharide commonly obtained from brown seaweed. It is composed of mannuronic (M) glucoronic (G) acid with (1,4)-linkages and the

structure varies according to the monomer position on the chain, forming either homopolymeric (MM or GG) or heteropolymeric (MG or GM) segments (Rioux et al., 2007; Yang et al., 2011; Hori et al., 2009). The molecular weight of alginate ranges generally between 500 and 1000kDa. Its solubility is influenced by factors such as pH, concentration, ions in solution, the presence of divealent ions and ionic force (Rioux et al., 2007). Alginate forms a physical hydrogel in the presence of divalent cations such as calcium (Hori et al. 2009). Nowadays, the use of alginate hydrogels in biotechnology and pharmaceutical industry is widespread due to the unique properties alginates possess such as high biocompatibility and biodegradability. Calcium alginate hydrogels are widely used as wound dressings, cell immobilizing materials and scaffolds for tissue engineering. Calciumalginate beads have been used in controlled drug delivery technology for the gastrointestinal administration of protein drug molecules or for ophthalmic drug delivery (Pasparakis, 2006). According to Michailovna et al. (2007), polymannurate is known to posseses anticancer and hypolipidemic activities. Laminarin (also called laminaran) is a -1, 3-D-glucan with (1,6) branching

polysaccharide found in the brown algae containing approximately 25 glucosyl

UST FACULTY OF PHARMACY residues (Rioux et al., 2007). However, in common with many other polysaccharides, laminaran is polydisperse, displaying a degree of structural heterogeneity (Read et al., 1996). Its structure and composition vary according to algae species. The molecular weight of laminaran is approximately 2,600 to 5,000 Da (Friedlaender et al., 1954; Rioux et al., 2007). The solubility of laminarin is influenced by the degree of branching. Highly branched laminaran is soluble in cold water whereas lower levels of ramification induce solubility in warm water (Rioux et al., 2007). However, according to Friedlaender et al. (1954), laminarin occurs in two forms that differ in solubility in cold water. The soluble form is an extract of Laminaria digitate and the insoluble form is an extract of Laminaria cloustoni. Both forms are linear glucosans containing about 20 D-glucopyranose units linked 1:3. In some polysaccharides, it has been reported that -1, 3-glucan composed of -D-glucopyranose linked at the 1 and 3 positions has various biological activities on plant and animal systems (Miyanishi et al., 2004). It has been reported that -1,3-

glucan polymers have a wide variety of biological activities in plant and animal systems. For instance, water-soluble glucans derived from Grifola umbellata or from the fruit body of Sparassis crispa are known to exhibit antitumor activity (Miyanishi et al., 2003). In a study conducted by Cheng, Liang, Li and Jin (2011), they found out that laminarin polysaccharides in a commercially available laminarin powder contained mannose, arabinose, glucose, galactose, and fucose. Furthermore, their

pharmacological tests revealed that laminarin polysaccharides reduced MDA, and increased GSH levels. In addition, they concluded that laminarin polysaccharides are effective in reducing sepsis-induced oxidative stress, lipid peroxidation in rats.

UST FACULTY OF PHARMACY Fucoidans, polysaccharides containing substantial percentages of L-fucose and sulfate ester groups, are constituents of brown seaweed and some marine invertebrates (such as sea urchins and sea cucumbers). The polysaccharide was named fucoidin when it was first isolated from marine brown algae by Kylin in 1913. Now it is named as fucoidan according to IUPAC rules, but some also called it fucan, fucosan or sulfated fucan (Li, Lu, Wei & Zhao, 2008). Fucoidan is a complex sulfated polysaccharide (Yang et al., 2008). The exact structures of it have not yet been discovered unlike the laminarin. And only little regularity in the structure is known at the present time. Fucoidan is composed of fucose, uronic acids, galactose, xylose, and sulfated fucose. Variations are observed between species which have an impact on the determination of the polysaccharide strucuture (Rioux et al., 2007). Studies show a variety of structure sequence. In a study conducted by Chizov et al. (1999) in Chorda filum, a brown seaweed, they stated that the homofucan sulfate from that algae was shown to contain poly-a-(1 3)-fucopyranoside backbone with a high degree of branching, mainly of a-(1 2)-linked single units. In addition, Bilan et al. (2002) concluded that the fucoidan isolated from the Pacific brown alga F. evanescens has a linear backbone of alternating 3- and 4-linked -L-fucopyranose 2-sulfate

residues. It means that the basic structure of the polysaccharide is regular and contains disaccharide-repeating units. Various molecular weights have been reported in the literature for fucoidan. Some authors have established fucoidans weight at approximately 100kDa while others measured a fraction of 1600 kDa and another of 43kDa in the same fucoidan sample. Fucoidan is soluble in water and acid solution (Rioux et al., 2007). Biological

UST FACULTY OF PHARMACY activities of fucoidan will be discussed in the succeeding texts. Table 2.2 provides the summary of the uses of different polysaccharides in brown algae.

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Table 2. 2. Summary of the Uses of Different Polysaccharides in Brown Algae Polysaccharide Alginate Uses/ Activity Wound dressing, cell immobilizong material, scaffolds for tissue engineering, controlled drug delivery technology, ophthalmic drug delivery Polymannurate Anticaner Hypolipidemic activity Laminarin Antitumor Machailovna et al. (2007) Miyanishi et al. (2003) Bruhn et al. (1997) Reduces sepsis-induced oxidative stress, lipid peroxidation Cheng, Liang, Li and Jin (2011) Author and Year Pasparakis et al. (2006)

2.3 Sulfated Polysaccharides (SPs)

In the recent years, many sulfated polysaccharide (SPs) isolated from marine algae have gained much interest in the fields of cosmetics, food, and pharmaceutical industries. SPs consist of an intricate group of macromolecules with a vast array of important biological activities. These polysaccharides are chemically anionic and widespread not only in marine algae but also in animals such as mammals and

UST FACULTY OF PHARMACY invertebrates (Ngo, Wijesekara, Vo, Ta, & Kim, 2011). Marine algae are the most important source of non-animal SPs. They are found in varying amounts in the three major divisions of marine algal groups namely, Rhodophyta, Phaeophyta, and Chlorophyta (Costa et al., 2010). The compounds to be tested for this activity includes ulvan from marine green algae Ulva sp which is mainly composed of rhamnose, xylose, glucuronic acid, iduronic acid, and sulfate, with smaller amounts of glucose, mannose, arabinose, and galactose. The mainly repeating disaccharide units are [ -DGlcpA-(1 4)- -L-Rhap 3s] and [L-IdopA-(1 as cited in Redouan et al., 2011). Sulfated polysaccharides from marine algae have many applications. One 4)- -L-Rhap 3s] (Lahaye & Robic, 2007

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group called carrageenans has been used as a food additive (Shah & Huffman 2003 as cited in Barahona, 2011). Meanwhile, Norziah et al. (as cited in Rocha de Souza et al., 2007), also stated the use of sulfated galactans such as carrageenan for products that require gelling, suspension, thickening or water-holding properties in the food industry. In the studies of Berteau and Mulloy (2003), and Campo et al. (as cited in Barahona et al., 2011), sulfated polysaccharides present significant pharmacological applications such as anticoagulant, antiviral, and antitumor activities. According to Qi et al. and Zhang et al. (as cited in Ngo et al., 2011), the antioxidant activity of SPs depend on their structural features like their degree of sulfation, molecular weight, type of major sugar and glycosidic branching. Sulfated fucans are among the most widely studied of all the sulfated polysaccharides of nonmammalian origin that exhibit biological activities in mammalian systems (Pereira, Mulloy, & Moura, 1999).

UST FACULTY OF PHARMACY In a study performed by Ghosh et al. (2009), they explained that fucan sulfates from marine brown algae usually have complex and heterogenous structures. And these structure complexities account for the varying effect in their biological activities. The bulky acidic sulfate substituents that dominate the physical characteristics of polyanions can equally dominate the effects of such macromolecules in biological systems. In addition, Pereira et al. (1999) pronounced that structures of the sulfated fucans from brown algae also vary from species to species. Fucoidan isolated from Fucus vesiculosus has been reported previously to be an effective anticoagulant in vitro and in vivo (Bruhn et al., 1997). In the study previously conducted by Soeda et al. (1992), it was found that the anticoagulant activity of this sulfated polysaccharide is improved by its further sulfation and that the oversulfated fucoidan (OSF) is effective for the prevention of LPS-induced hepatic vein thrombosis in hyperlipidemic rats. And further studies showed that the fragments of OSF with little or no anticoagulant activity are effective for the prevention of thrombus formation in the above model rats. In addition, the sulfated seaweed extract, fucoidan, has antitumor, antithrombotic, and antiviral activities. (Kim et al., 2010). Fucoidan preparations have been proposed as alternatives to the anticoagulant heparin, which is prepared from mammalian mucosa. Fucoidans, being of vegetable origin are less likely to contain infectious agents, such as viruses or prions. Like heparin, according to the studies of Boisson-Vidal et al. (as cited in Berteau & Molloy, 2003), it has been shown that fucoidans affect many biological activities, such as inflammation, cell proliferation and adhesion, viral infection, and fertilization. According to a study conducted by Zhua et al. (2003), the sulfated polysaccharide isolated from the brown alga Sargassum patens was found to show

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UST FACULTY OF PHARMACY more potent antiviral activity against HSV-2 than that against HSV-1. Table 2.3 presents a summary of activities exhibited by Fucoidan. According to the study conducted by Zhu et al. (2004), the sulfated polysaccharide isolated from the brown alga Sargassum patens was found to show more potent antiviral activity against HSV-2 than that against HSV-1.

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Table 2. 3 Summary of Activities of Fucoidan Uses/Activities Anticoagulant Research Title Preparation of oversulfated fucoidan fragments and evaluation of their antithrombotic activities. Genotoxicity studies onfucoidan from Sporophyll of Undaria pinnaifada. Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide Antiviral property and mode of action of a sulphated polysaccharide from Sargassum patens against herpes simplex virus type 2. Author and Year Soeda et al. (1992)

Antitumor, Antithrombotic, and Antiviral Activities Effects on inflammation, cell proliferation and adhesion, viral infection, and fertilization

Kim et al. 2010

Boisson-Vidal et al. (as cited in Berteau and Molloy, 2003)

Antiviral

Zhu et al (2004)

UST FACULTY OF PHARMACY 2.4 Antioxidants

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There have been numerous studies about antioxidants which possess health benefits because of the currently growing demand from the pharmaceutical industry such as anti-aging and anti-carcinogenic natural bioactive compound (Heo, Park, Lee & Jeon, 2005). Antioxidants are considered key-compounds in the fight against various diseases like cancer, chronic inflammation, atherosclerosis and cardiovascular disorder and aging processes (Zubia, 2009). According to Sun (2009), the oxidative damage of a tissue could be alleviated by antioxidants through increasing the cells natural defenses and directly scavenging the free radical species. By definition, a free radical is a normal by-product of oxidation reactions in metabolism with an unchanged atom or group of atoms (e.g. oxygen) having at least one unpaired electron, which make it highly reactive (The American Heritage Medical Dictionary). Among the major free radical species, those with oxygen called oxygen free radicals (OFRs) are of interest rather than those with ground state molecular oxygen (dioxygen) having two unpaired electrons which is relatively not reactive. OFRs is only a part of reactive oxygen species (ROS) which are strongly oxidizing than molecular oxygen. Due to its highly reactive nature OFRs are very toxic to cells when combined with other molecules, like enzymes, receptors, ion pumps that cause oxidation directly and inactivates the normal functioning of cells. For example when exercising the total oxygen intake has the ability to form free radicals. Hence, these free radicals are generated by metabolic processes by different chain reaction in the

UST FACULTY OF PHARMACY body like electron transport chain, which is found in the inner mitochondrial membrane. Oxygen is utilized to generate energy in the form of ATP and acts as a terminal electron acceptor. And when electron escapes from ETS, it has the ability to form a superoxide radical. The free radicals existence for a long evolutionary process is because of its ability to utilize oxygen as an energy source ( Punchard & Kelly, 1996). H2O2, although not actually a radical, is a weak oxidizing agent that is classified as an ROS because it can generate the hydroxyl radical (OH-). Transition metals, such as Fe2+ or Cu2+ catalyse the formation of the hydroxyl radical from H2O2 in nonezymatic reactions. Because H2O2 is lipid soluble, it can diffuse through membranes and generate OH- at localized Fe2+ or Cu2+-containing sites, such as the components of the electron transport chain within the mitochondria. Hydroxyl radical scavenging capacity of an extract is directly related to its antioxidant activity. This method involves in-vitro generation of hydroxyl radicals using H2O2 system using Fenton reaction. Scavenging of this hydroxyl radical in presence of antioxidant is measured. In one of the methods the hydroxyl radicals formed by the oxidation is made to react with DMSO (dimethyl sulphoxide) to yield formaldehyde. Formaldehyde formed produces intense yellow color with Nash reagent (2M ammonium acetate with 0.05M acetic acid and 0.02M acetyl acetone in distilled water). The intensity of yellow color formed is measured at 412nm spectrophotometrically against reagent blank. The activity is expressed as % hydroxyl radical scavenging. Nitric oxide, because of its unpaired electron, is classified as a free radical and displays important reactivitys with certain types of proteins and other free radicals. In vitro inhibition of nitric oxide radical is also a measure of antioxidant activity. This

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UST FACULTY OF PHARMACY method is based on the inhibition of nitric oxide radical generated from sodium nitroprusside in buffer saline and measured by Griess reagent. In presence of scavengers, the absorbance of the chromophore is evaluated at 546nm. The activity is expressed as % reduction of nitric oxide. (Babu, Shylesh, & Padikkala, 2001) ROS, such as superoxide radical (O2-), hydroxyl radical (OH-), peroxyl radical (ROO), and nitric oxide radical (NO), attack biological molecules, such as lipids, proteins, enzymes, DNA and RNA, leading to cell or tissue injury associated with aging, atherosclerosis, and carcinogenesis (Duan et al., 2006). Hydroxyl radical (OH -), is constantly generated by light, ultraviolet, ionizing radiation, some chemical reactions, metabolic processes and aging. Cytotoxicity and metabolic changes such as chromosome irregularities, protein oxidation, muscle injury, and morphological and central nervous system changes in animals and humans may be a contribution of these reactive species (Chun-hui, Chang-hai, Zhi-liang & Yi, 2007). It is fundamental to develop and extend effective natural antioxidants so that they can protect the human body from free radicals and retard the progress of many chronic diseases (Sun, 2009). Antioxidants are effective in protecting the body against damage by reactive oxygen species (Cho, Lee, Kang, Won & You, 2010). 2.4.1 Antioxidant Properties of Algae

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Algae, as photosynthetic organisms, are exposed to a combination of light and high oxygen concentration at the origin of the formation of free radicals and other oxidative reagents, but they seldom suffer any serious photodynamic damage during metabolism. This fact entails that their cells have some protective antioxidative

UST FACULTY OF PHARMACY mechanisms and compounds (Matsukawa et al. as cited in Heo et al., 2005). The scientific community considers that the protection of algae against oxidation comes from their natural content, or production under stress, in antioxidant substances because of the awareness of the lack of structural damage in their organs (Zubia, 2009). Seaweeds are considered to be a rich source of antioxidants (Heo et. al., 2005). Several researchers have reported the antioxidant properties of both brown and red seaweeds from across the globe (Ganesan, Kumar, & Bhaskar, 2008). Further, there are potential protective effects in seaweeds against oxidative stress in target tissues and lipid oxidation in foods (Ganesan et al., 2007).

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2.4.2 Antioxidant Properties of Sulfated Polysaccharides (SPs)

According to Liu, Dong, Tong, and Zhang (2010), polysaccharides from different resources have strong antioxidant properties and can be explored as a novel potential antioxidant source. Polysaccharides, distributed widely in animals, plants, and microorganisms, have been established to play an important role as dietary free radical scavenger in the prevention of oxidative damage in living organism (Ke et al., 2009). In the study of Wang, Zhang, Q., Zhang, Z., and Li (2008) regarding the antioxidant activities of SPs from brown and red algae, they concluded that among the different polysaccharides extracted, fucoidan and lambda carrageenan exhibit the highest antioxidant activity and free radical scavenging activity. In addition, there is indeed a positive correlation revealed between sulfate content and superoxide radical

UST FACULTY OF PHARMACY scavenging ability of Laminaria japonica. Moreover, they also attest that the ratio of sulfate content/fucose was an indicator to hydroxyl radical scavenger and metal ionchelation, which was applicable in the future experiments understanding the relationship between chemical property of fucoidan and its antioxidant activity. The correlations above are supported by a recent similar study conducted by Redouan et al. (2011), wherein they evaluated the antioxidant capacity of ulvan-like polymer obtained by regioselective oxidation of gellan exopolysaccharide. They found out that the ulvan, a sulfated polysaccharide from green algae, exhibited higher antioxidant activity than oxidized gellan in scavenging activity of DPPH radicals which indicated that uronic acids content and sulfate groups could enhance their antioxidant activity and it was shown that different sulfate content ulvans showed different antioxidant activity. However, these statements are in contrast to a recent study conducted by Barahona et al. (2011). In their study, they concluded that the sulfate content and the antioxidant capacity of the sulfated polysaccharides have no correlation with one another. On the other hand, they also stressed a positive correlation between the monosulfation position in the monosaccharide residues and the antioxidant capacity. Table 2.4 shows a tabulated form of the title of researches undertaken to assess the antioxidant activity of SPs.

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Table 2.4 Studies About the Antioxidant Activity of Sulfated Polysaccharides. Research title Antioxidant activities of sulfated Conclusion There is a positive correlation between Author and Year Rocha de Souza et al. (2007),

UST FACULTY OF PHARMACY polysaccharides from brown and red seaweeds. Antioxidant activity of sulfated polysaccharide fractions extracted from Laminaria japonica There is a positive correlation revealed between sulfate content and superoxide radical scavenging ability. Antioxidant capacity of sulfated polysaccharides from seaweeds. A kinetic approach The sulfate content and the antioxidant capacity of the sulfated polysaccharides have no correlation with one another Also stressed a positive correlation between the monosulfation position in the monosaccharide residues and the antioxidant capacity. Barahona et al. (2011) Wang, Zhang, Q., Zhang, Z., and Li (2008) sulfate content and antioxidant activity.

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The antioxidant capacity of sulfated polysaccharides has been studied by different in vitro methods, including hydrogen peroxide, superoxide anion, and hydroxyl radical scavenging assays. DPPH radical scavenging assay is frequently used for the analysis of food and substances obtained from natural sources (Barahona, Chanda, Encinas, Matsuhiro & Ziga, 2011).

2.5 Determining the polysaccharides

UST FACULTY OF PHARMACY 2.5.1 Reversed Phase High Performance Column Chromatography

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using Refractive Index Detector

High Performance Liquid Chromatography is used to analyze, identify, purify and quantify compounds. It separates compounds in a mixture on the basis of polarity more efficiently and quickly than that of traditional column chromatography. Separation is based on the analytes relative solubility between two liquid phases, the mobile or solvent phase and the stationary or bonded phase. There are two modes of conducting HPLC. First is using the normal phase, polar stationary phase and nonpolar solvent or mobile phase. And the second one is the reverse phase which includes a non-polar stationary phase and a polar solvent or mobile phase. The separation mechanism in reversed phase chromatography depends on the hydrophobic binding interaction between the solute molecule in the mobile phase and the stationary phase. The initial mobile phase binding conditions used in reversed phase chromatography are primarily aqueous which indicates a high degree of organised water structure surrounding both the solute molecule and the stationary phase. As solute binds to the stationary phase, the hydrophobic area exposed to the solvent is minimised. Therefore, the degree of organised water structure is diminished with a corresponding favourable increase in system entropy. In this way, it is advantageous from an energy point of view for the hydrophobic moieties, i.e. solute and solvent, to associate. The mobile phase composition is modified to favour desorption of the solute from the stationary phase back into the mobile phase. Subsequently, the mobile phase composition is modified to favour desorption of the solute from the stationary phase back into the mobile phase. Therefore, reversed

UST FACULTY OF PHARMACY phase chromatography depends on the reversible adsorption/desorption of solute molecules with varying degrees of hydrophobicity to a hydrophobic stationary phase. The mobile phase is continuously pumped at a fixed flow rate through the system and mixed by the pump. The injector is used to introduce a plug of a sample into the mobile phase without having to stop the mobile phase flow and without introducing air into the system. The mixture of components is carried in a narrow band to the top of the column. Some compounds in the sample mixture will have greater preference for stationary phase than the mobile phase and will be retained in the column longer. Those components that are not retained as strongly as are carried by the mobile phase down the column. The detector is then used to respond to a physicochemical property of analyte, this response is digitally amplified and sent to a data system where it is recorded as a 'chromatogram'. The separation is more effective due to greater surface area achieved due to very small particle size of stationary phase in comparison to that used in column chromatography. This decrease in particle size increases has disadvantage that it proportionately enhances the flow time and run time due to increased surface area. To minimize this obstacle the high pressure is applied to the flow of mobile phase through the column by use of pumps. "Like attracts like therefore if the column is non-polar the compound to elute first will be the most polar one. It is used to analyze, identify, purify and quantify compounds. The technique of reversed phase chromatography allows great flexibility in separation conditions so that the researcher can choose to bind the solute of interest, allowing the contaminants to pass unretarded through the column, or to bind the contaminants, allowing the desired solute to pass freely. Generally, it is more appropriate to bind the solute of interest because the desorbed solute elutes from the

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UST FACULTY OF PHARMACY chromatographic medium in a concentrated state. Additionally, since binding under the initial mobile phase conditions is complete, the starting concentration of desired solute in the sample solution is not critical allowing dilute samples to be applied to the column. The refractive index (RI) detector is the only universal detector in HPLC. . It is one of the least sensitive liquid chromatography detectors. It is very sensitive to changes in ambient temperature, pressure changes, flow-rate changes and can not be used for gradient elution. Despite these many disadvantages, this detector is extremely useful for detecting those compounds that are nonionic, do not adsorb in the UV, and do not fluoresce. The detection principle involves measuring of the change in refractive index of the column effluent passing through the flow-cell. The greater the RI difference between sample and mobile phase, the larger the imbalance will become. Thus, the sensitivity will be higher for the higher difference in RI between sample and mobile phase. On the other hand, in complex mixtures, sample components may cover a wide range of refractive index values and some may closely match that of the mobile phase, becoming invisible to the detector. RI detector is a pure differential instrument, and any changes in the eluent composition require the rebalansing of the detector. This factor is severely limiting RI detector application in the analyses requiring the gradient elution, where mobile phase composition is changed during the analysis to effect the separation

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2.5.2

Molisch test

Concentrated sulphuric acid causes dehydration of all carbohydrates to give Furfural compounds, that react wtih -naphthol (Molisch reagent) giving a violet or

UST FACULTY OF PHARMACY purple color complex. Visible result is a violet colored ring at the junction between the two layers. The test is positive for all carbohydrates. Monosaccharides give a rapid positve result while disaccharides and polysaccharides react slower.

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2.5.3

Anthrone test

Carbohydrates exist in plants as free sugars and polysccharides. The basic units of carbohydrates are the monosaccharides which cannot be split by hydrolysis into simpler sugars. This test can measure the carbohydrate content by hydrolysing the polysaccharides into simple sugars by acid hydrolysis and estimating the resultant monosaccharides. Carbohydrates are first hydrolyzed into simple sugars using dilute hydrochloric acid. In hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This compund forms with anthrone a green coloured product with an absorption maximum at 630nm.

2.5.4 FT-IR (Fourier Transform Infrared) Spectroscopy

Fourier Transform Infrared (FT-IR) spectrometry was developed in order to overcome the limitations encountered with dispersive instruments. The main difculty was the slow scanning process. A solution was developed which employed a very simple optical device called an interferometer. The interferometer produces a unique type of signal which has all of the infrared frequencies encoded into it. The signal can be measured very quickly,

UST FACULTY OF PHARMACY usually on the order of one second or so. Thus, the time element per sample is reduced to a matter of a few seconds rather than several minutes. The Interferometer employs a beamsplitter which takes the incoming infrared beam and divides it into two optical beams. One beam reects off of a at mirror which is xed in place. The other beam reects off of a at mirror which allows this mirror to move a very short distance away from the beamsplitter. The two beams reect off of their respective mirrors and are recombined when they meet back at the beamsplitter. Because the path that one beam travels is a xed length and the other is constantly changing as its mirror moves, the signal which exits the interferometer is the result of these two beams interfering with each other. The resulting signal is called an interferogram which has the unique property that every data point (a function of the moving mirror position) which makes up the signal has information about every infrared frequency which comes from the source. This means that as the interferogram is measured; all frequencies are being measured simultaneously. Thus, the use of the interferometer results in extremely fast measurements. Because the analyst requires a frequency spectrum (a plot of the intensity at each individual frequency) in order to make an identication, the measured interferogram signal cannot be interpreted directly. A means of decoding the individual frequencies is required. This can be accomplished via a well-known mathematical technique called the Fourier transformation. This transformation is performed by the computer which then presents the user with the desired spectral information for analysis.

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UST FACULTY OF PHARMACY 2.6 Determining Antioxidant Activity

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2.6.1

DPPH (1,1-Diphenyl-2-picrylhydrazyl) ASSAY

Scavenging of DPPH free radical is the basis of a common antioxidant assay. 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) is a stable free radical which has an unpaired valence electron at one atom of nitrogen bridge. High concentration of DPPH in the reaction mixture gives absorbance beyond the accuracy of spectrophotometric measurements. Light, oxygen, and pH of the reaction mixture also affect the absorbance of DPPH (Sharma & Bhat, 2008).

2.6.2

Fentons Assay

Fenton's assay is used to evaluate oxidation process of various aqueous solutions. The degradation of the sample is strongly dependent on the pH, initial concentration of the sample, H2O2, Fe2+, and [H2O2]o/[Fe2+]o ratio. The optimum conditions are obtained at pH 3 for the H2O2/Fe2+ (Tamimi, Qourzal , Barka, Assabbane, & Ait-Ichou, 2008). Advanced oxidation processes (AOPs) are processes that generate hydroxyl radicals (OH), that are considered to be a promising alternatives to conventional processes due to their efficiency in oxidizing a variety of organic contaminants (Tamimi et al., 2008). Oxidation with Fenton's reagent is based on ferrous ion and hydrogen peroxide, and develops the reactivity of the hydroxyl radicals produced in acidic

UST FACULTY OF PHARMACY solution by catalytic decomposition of H2O2: Fe2+ + H2O2 ---> Fe3+ + OH- + OH-. Hydorxyl radicals may be scavenged by reaction with another Fe2+: OH- + Fe2+ ---> OH- + Fe3+

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The irradiation of Fenton with UV or visible light could increase the degradation rate of the organic substance. The illumination leads to the formation of additional hydroxyl radicals and also to the recycling of ferrous catalyst by reduction of Fe3+. This oxidation using Fenton's reagent has been found to be an effective method on degradation of oxidants. Fenton reagent is a very powerful oxidizing reagent. The process is simple, inexpensive, and it takes place under low temperatures and at atmospheric pressure (Tamimi et al., 2008)

2.6.3 Nitric Oxide Assay

Nitric Oxide (NO), produced endogenously from L-Arginine by nitric oxide synthetases, plays an important role in many physiological processes including vascular regulation, immune responses, and neural communication. It is a diffusible free radical that plays many roles as an effectors molecule in diverse biological systems including neuronal messenger, vasodilatation and antimicrobial and antitumor activities. NO is extremely unstable and undergoes repaid oxidative degradation to nitrite (NO2-) and nitrate (NO3-), which can be spectrophotometrically determined. Oxygen reacts with the excess nitric oxide to generate nitrite and peroxynitrite anions, which act as free radicals. An antioxidant then competes with oxygen to react with nitric oxide and thus inhibits the generation of the anions.

UST FACULTY OF PHARMACY 2.7 Relevance of the Related Literatures to the Present Study

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The prominence of Sargassum siliquosum J.Agardh in the coastal waters of the Philippines is one of the reasons that interest scientists to develop its uses. Among the many uses of Sargassum, it is primarily used as food when broiled with fish and squid and as source of sodium alginate. On the other hand, it has been found out that Sargassum species have other potentials other than providing source for sodium alginates and as a source of food. As shown above, different species of Sargassum have shown anticancer, antibacterial, antiviral, antioxidant, anticoagulant, antiproliferative, antipyretic,

analgesic, and anti-inflammatory activity. It has been associated that most of these activities seen in Sargassum species are related to its sulfated polysaccharide content. Therefore, the present research study is timely relevant to the cited literatures in a way that it can further enhance or prove otherwise the studies that present a correlation between the antioxidant activity and the SPs possessed by Sargassum species. The current undertaking utilizes all the information gathered in Chapter 2 to concretize the knowledge and deepen the understanding available to bring the research into success.

BIBLIO: Amersham Biosciences (1999). Reversed Phase Chromatography. Retreived from http://teachline.ls.huji.ac.il/72682/Booklets/AmershamRPCManual.pdf

UST FACULTY OF PHARMACY

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Refractive Index Detector Retrieved from http://hplc.chem.shu.edu/NEW/HPLC_Book/Detectors/det_ri.html Hedge, J.E. and Hofreiter, B.T. (1962). In: Carbohydrate Chemistry, 17 (Eds. Whistler R.L. and Be Miller, J.N.), Academic Press, New York. <http://www.newagepublishers.com/samplechapter/000091.pdf>

W., Yemm and A. J., Willis. The estimation of carbohydrate in plant extracts by anthrone. Vol 57 (1954), 508-509. 4 January 1954. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1269789/pdf.biochemjo10830159.pdf> http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/molisch/molisch.htm

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