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The search for medicinal compounds has helped to drive the development of natural products chemistry. Natural products have provided challenging synthetic targets and valuable medicines (Hanson, 2003). According to the World Health Organization (WHO), approximately 80% of the world relies on natural sources for primary medicinal while the remaining 20% of the population relies on health care systems which are also incorporate natural sources in their medicinal treatment (Cragg, 2002). Natural product chemistry can be defined as the exploration of nature in the search for novel drugs or drug leads.
Sources of natural products include plants, marine organisms, microbes and fungi. Plants have served as the major source of medicinally useful natural products. This developed from a legacy of folk medicine based on herbal remedies. In the past five decades, many scientists had focused their research on marine natural products due to the diversity of marine organism and habitats, in the belief that the diversity of natural product in marine environment may exceed that of terrestrial habitats because the seas and oceans cover more than seventy percent of the Earth's surfaces.
Marine natural products encompass a wide variety of chemical classes, including terpenes, shikimates, polyketides, acetogenins, peptides, alkaloids of varying structures and a maltitude of compounds of mixed biosynthesis. In the past decade alone, over 5000 structures of marine natural products have been published. To date, two marine-derived natural products have advanced to the pharmaceutical drug market. They are analgesic ziconotides (Figure 1.1) from the venom of fish-hunting cone snails and unusual sponge-derived nucleotides that served as model compounds for antiviral drugs such as Virarabin (Fusetani, 2000). More than ten marine drugs were used for clinical trials in these recent years.
Figure 1.1: Structure of analgesic ziconotides with a molecular formula of C102 H172 N36 O32 S7
Marine natural products are organic compounds produced by microbes sponges, seaweeds and other marine organisms. The host organism synthesizes these compounds as secondary metabolites to protect themselves and to maintain homeostasis (Lipton, 2003). Although marine organisms do not have a long history of medicinal application as the terrestrial plants, some marine organisms have an extensive record of being hazardous to mankind (Cardellina II, 1986). Review of literature reveals that even the seawater has bactericidal properties (Lipton, 2003).
General Introduction on Seaweeds
There are many types of marine flora. Seaweeds or marine algae are the most abundant of these. Seaweeds are among the oldest members of the plant kingdom, extending back many hundreds of millions of years. They have little tissue differentiation, no true vascular tissue, no roots, stems or leaves and flowers. About 30,000 species of seaweeds are found throughout the world. Seaweeds are essential for maintaining the proper balance between chemical and biological environment of the oceans. They supply oxygen to the biosphere, are a source of food for fishes, cattle and man.
The ability of seaweeds to produce secondary metabolites of potential interest has been extensively documented (Faulkner, 1993). There are many reports of compounds derived from macroalgae with a broad range of biological activities, such as antibiotics, antivirals, antitumour, as well as neurotoxins (Ibtissam et al., 2009). These compounds include amino acids from marine algae, guanidine derivatives, phenolic substances, bioluminescence, carotenoids, diterpenoids, biosynthesis of metabolites, indoles, and halogenated compounds. The medicinal importants of marine algae is increasing day by day.
Seaweeds are classified as green algae (Chlorophyta), brown algae (Phaeophyta), red algae (Rhodophyta) and some filamentous blue-green algae (Cyanobacteria). A particular seaweeds is determined first by its photosynthetic pigments, its micro- and macro-morphologies and by its phycopolymers. Most of the seaweeds are red (6000 species) while the rest are either brown (2000 species) or green (1200 species) (Waaland et al., 2000). In this research, antioxidant and antimicrobial activities of Padina antillarum which is a type of brown algae was studied. Approximately 1500 species of brown algae known, almost all are marine. Brown algae is belongs to the phylum Phaeophyta of the kingdom of Prostica. Brown algae contain of chlorophyll a and c, as well as carotenes and xanthophylls, including the brown pigment fucoxanthin which masks the colour of green chlorophyll. Brown algae exist in varieties of forms and sizes, ranging from less than 1mm long to some species that are among the largest photosynthetic organisms on earth. In general, they are not free-floating seaweeds but are attached to rocks, corals and other surfaces (Waaland et al., 1977).
Antioxidants are compounds that prevent cell damage through oxidation. These compounds neutralize free radicals. Free radicals are highly reactive and unstable molecules produced by cigarette smoke, toxic chemicals, excess sunlight and even metabolic processes. Oxidation is the process by which the human body continuously burns calories in order to produce energy. This process which enables life releases free radicals and reactive oxygen species (ROS) (Ramamoorthy & Bono, 2007). ROS is produced in the form of superoxide anion (O2-), hydroxyl radical (Â·OH), hydrogen peroxide (H2O2) and nitric acid (NO). These species may cause mutation in DNA, leading to cancer and other degenerative diseases such as cataracts and heart diseases. Antioxidants obtained from natural sources have been proven to neutralize free radicals and thus prevent their destructive effects (Kumar et al., 2008).
The human body manufactures its own set of antioxidants. These antioxidants are called endogenous antioxidants. Endogenous antioxidants convert free radicals into harmless oxygen particles and water, after which they are changed into less active molecules and excreted. Endogenous antioxidants are function in synergy with immune cells to defend and repair free radical damage (Li et al., 2008). As endogenous antioxidants cannot totally neutralize excess free radicals, nutritionists suggest reinforcing them with a diet rich in antioxidants (Li et al., 2008). Antioxidants obtained through the diet or external sources are called exogenous antioxidants.
In recent years, a relationship between the antioxidant equivalent intake and some chronic diseases has been suggested (Li et al., 2008). There is increasing evidence that antioxidants are active in preventing these disorders. According to World Health Report (World Health Organization, 2003), lung and breast cancer are the most common cancers in men and women respectively. Natural products have been used in the treatment of various chronic human pathologic conditions because they contain high antioxidative ingredients that could reduce the risk of cancers. By neutralizing cell-mutating free radicals, antioxidants may prevent cancer. Some studies have shown that antioxidants are able to prevent heart disease by inhibiting cholesterol deposits on blood vessel walls and by lowering overall cholesterol level. Antioxidants have also been used as anti-inflammatory agents in the treatment of arthritis and bronchitis (Aijith and Jnardhanan, 2007).
Synthetic antioxidants have been used in the stabilization of food products. The most common used synthetic antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylated hydroxyquinone (TBHQ) which are added to fatly and oily foods to prevent oxidative deterioration (Jun & Zhang, 2007). Lipid peroxidation is a cause of food deterioration. It occurs during raw material storage, processing, heat treatment and further storage of final product. BHA and BHT have been found to be anticarcinogenic as well as carcinogenic effects in experimental animals (Cakir et al., 2003). BHA might have tumour-initiating and tumour-promoting action (Jun & Zhang, 2007).
Polyphenols which are effective antioxidants may prevent cardiovascular and inflammatory disease, and cancer (Zhang et al., 2006). Polyphenols are found in terrestrial plants and seaweeds. Phlorotannin is one such special type of polyphenolic compounds widely distributed in marine brown algae (Zhang et al., 2006).
An antimicrobial compound is a substance that kills or slows the growth of microbe like bacteria (antibacterial activity), fungi (antifungal activity), or viruses (antiviral activity). Antimicrobial drugs are used to treat or prevent illness caused by bacteria in humans or animals. However, the effectiveness of antimicrobial drugs has been decreasing due to the increase in bacterial resistance. According to WHO, resistance to antimicrobial agents is a growing problem in many countries. Many researchers have focused on the investigation of natural products for bioactive molecules.
Many substances obtained from marine algae such as alginate, carragenean and agar as phycocolloids have been used for medicinal purposes. Researchers have studied on the algal substances and discussed that some algal substances have bacteriostatic, fungistatic bactericidal and fungicidal properties (Burkholder et al., 1960; Ehresmann et al., 1977; Moreau et al., 1984; Reichelt and Borowitzka, 1984; Hornsey and Hide, 1985; Vlachos et al., 1999; Gonzalez del Val et al., 2001; Nora et al., 2003; Ghosh et al., 2004; Freile-Pelegrin and Morales, 2004; Salvador et al., 2007). These activities are due to a variety of natural products such as fatty acids, terpenes, chlorophylls, brominated phenolic compounds, acrylic acid and polysaccharides (Mtolera and Semesi, 1996). Harder (1917) was the first to study antimicrobial substances in marine algae (Khaleafa et al., 1975). However, it was not until the 1970s that large scale screening antimicrobial activity was done.
In this study, antioxidant and antimicrobial activities of P. antillarum was studied. Previous research has shown that P. antillarum contains of antioxidant and antimicrobial bioactive compounds. However, the structures of the compounds responsible for the antioxidant and antimicrobial activity remain unelucidated. Thus, this study was designed to elucidate their chemical structures.
50% aqueous methanol was chosen to extract antioxidant compounds from P. antillarum because 50% aqueous methanol has the highest extraction efficiency compared to others solvents (Chew et al., 2008). Antioxidant activity of P. antillarum will be evaluated by measuring its total phenolic content (TPC) and antioxidant activity (AOA). TPC was measured using Folin-Ciocalteu's method while 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging was used to study its AOA.
Sequential extraction was performed in order to maximize the extraction of nonpolar and polar antimicrobial compounds from P. antillarum the solvents that were used were hexane followed by diethyl ether, chloroform, ethyl acetate, acetone, methanol and finally water. Colorimetric broth-microdilution method was used to assess the antimicrobial activity. P. iodonitrotetrazolium violet (INT) will be used to indicate the growth of bacteria. The extracts will be tested using 6 types of bacteria. They are Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli (both penicillin-sensitive and penicillin-resistant strains).
The extracts which showed antioxidant and antimicrobial activities were purified by normal phase column chromatography followed by revesred-phase column chromatography. Screening bioassays will be carried out to keep tracking the active compounds. The active fractions were further purified by high performance liquid chromatography. Finally, the pure compounds that obtained were analyzed by 1H-NMR and 13C-NMR spectroscopy analysis in order to elucidate the structure of the compounds.
The objectives of this study are to extract and isolate antioxidant and antimicrobial bioactive compounds from P. antillarum. The antioxidant and antimicrobial active extracts were partially purified by normal phase column chromatography followed by reversed phase column chromatography. The active fractions were further isolate by high performance liquid chromatography until pure compounds are obtained. The structures of the active compounds were elucidated using 1H- nuclear magnetic resonance (NMR) and 13C-NMR.