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Bioactive marine natural products are chemical compounds produced by microbes, sponges, seaweeds, and other marine organisms ( Faulkner, 2001; Jensen and Fenical, 2002). The host organism synthesizes these compounds as non-primary or secondary metabolites. These may exert a biological effect on other organisms. Marine natural products exhibited a wide range of biological activities such as antithelmintic, antibacterial, anticoagulant, antidiabetic, antitumuor, antifungal, anti- inflammatory, antimalarial, antiplatelet, antiprotozoal, antituberculosis, and antiviral activities (Mayer and Hamann, 2005). Considering the potential of marine bioactive natural products, many marine organisms have been extensively studied extensively (Baker et al., 1995).
The seaweeds are the most abundant attached marine plants in the ocean (Bolton et al, 2004). Most of them are green algae (Chlorophyta), brown algae (Phaeophyta), and red algae (Rhodophyta). Each group is characterized by specific combinations of photosynthetic pigments (Hunt, 1978). Most of the seaweeds are grow in intertidal zones and commonly extend to depths of 30 to 40m. In clear tropical seas, they can be found to depths up to 200m. Seaweeds are structurally simpler than terrestrial plants. Being immersed in water, they can absorb nutrients, water, dissolved gasses and sunlight through their entire surface of the plant. Unlike terrestrial plants, seaweeds have no root, leaves nor a complex network to transport food and water around the plants. Some seaweed has root-like holdfasts, but these serve only to anchor the seaweeds on the rocks or in the sand (REF).
Seaweeds greatly influence environmental conditions for other types of marine life by providing food, protection from waves, shade, and a substrate to attach. The seaweeds were one of the first groups of marine organisms whose natural products chemistry was studied extensively because of their abundance in shallow waters. During the past 30 years, a group of marine natural products chemists from several countries has reported a very large number of novel metabolites with useful pharmacological properties (Faulkner, 2000). Majority of the studies on seaweeds occurred after the development of many of the useful mechanism bioassays used today.
2.2.1 Classification of Seaweeds
Three major classes of photosynthetic pigments occur among the algae are chlorophylls, carotenoids (carotenes and xanthophylls), and phycobilins. Generally, seaweeds are classified into 3 major groups which are green, brown and red algae.
The bright green colour characteristic of the green algae (Chlorophyta) is due to the predominance of chlorophyll over other accessory photosynthetic pigments. Although chemical studies of temperate species have not been productive, the tropical green algae are known to produce many interesting biologically active metabolites.
In the brown algae (Phaeophyta), the green hue of chlorophyll is partially masked by the golden xantophylls accessory photosynthetic pigments. Many of the large, more familiar and dominant algae of temperate sea, such as kelps, belong to this division. Natural product studies of this division have been productive, with many reports of biologically active metabolites, predominantly diterpenes.
The red algae (Rhodophyta) contain not only chlorophyll, but the accessory red phycoerythrin and blue phycocyanin pigments. They can display a wide range of colours, form bright green to various shades of red. They are as diverse in structure and habitats as they are in coloration. The chemistry of the red algae has been studied extensively because their propensity to include halogens in their biosynthetic pathways. Indeed, the marine natural products literature was dominated by reports of halogenated metabolites derived from red algae.
2.2.2 Seaweeds as Foods
.Seaweeds have been used as food for quite a long time particularly in Far Eastern countries due to their high nutrition value Seaweeds are rich in vitamins A, vitamin C, vitamin E and Niacin. The concentrations of vitamins B1, B1, panthothetic acid, folic and folinic acids are generally higher in green and red algae than in brown algae (Madlener, 1977). Seaweeds are considered as an ideal food supplement for 21st century, for example as source of protein, lipids, polysaccharides, mineral, vitamins, and enzyme. For example, Porphyra umbilicalis (purple laver) is among the most nutritious seaweeds, with a protein content of 30- 50%, and about 75% of that is digestable. Sugars are low (0.1%) and the vitamin content very high, with significant amounts of Vitamins A, C, niacin and folic acid. But the shield life of vitamin C can be short in the dried product.
Seaweeds for Medicine and Pharmaceuticals
Seaweeds are commonly used in Chinese medicine, for example, the kelps Saccharina japonica and Ecklonia kurome as sources of kunbu. Kunbu is a medicine for scrofula, goiter, tumour, edema, accumulation, testicular pain and swelling. Sargassum is a kind of brown algae which served as the source of haizao. Its applications are same with kunbu. Saccharina and Sargassum have been used in China for the treatment of cancer.
There are reports of algal sulphated polysaccharides exhibiting a variety of activities including antiviral, antithrombic, antitumour, antiulcer, antilipemic and immunomodulating (Renn, 1993). Their inflammatory and immunodulating activities have made them useful in a few pharmacological assays used to discover potential therapeutics (Renn, 1993). More examples are shown in Table 2.1 on medicinal seaweeds.
Table 2.1 Example of seaweeds for medicine and pharmaceuticals
Types of seaweeds
Applications in pharmaceutical filed
Hypotensive and anaesthetic actions
Inhibition of Gram positive and Gram negative bacteria
In vitro antiviral activity
Decrease the level of cholesterol
Treatment for scabies
Medicine to cure scrofula, goitre, tumour, edema, testicular pain and swelling
To dilate the cervix as the stipes swell to several times their original diameter when moistened
Exhibited hypoglycaemic activity
Medicine to cure scrofula, goiter, tumour, edema, testicular pain and swelling
Hypocholesterolaemic and hypoglycaemic agents
Medicine for producing a cooling and blood cleansing effect for the treatment of glandular weakness
Potential anticancer drug
Medicine to cure goitre
Exhibited diuretic activity
Inhibition of marine bacteria
Inhibition of marine bacteria
Medicine for dressing wounds and burns
(Stein & Borden, 1984; Fusatani, 1987; Naqvi et al. 1980; Blunden, 1996; Perveen, 1993; Namboothiry, 2001)
Seaweeds for animal fodders
Historically, seaweeds have been used by Greek as animal fodder from the first century BC (Chapman, 1980). In the coastal areas of Norway, Sweden, Denmark, Iceland, Britain, China, France, Pacific Islands and New Zealand, several seaweeds are used as stock feed for chicken, sheep and cattle. Nowadays the animals are fed in the form of cakes and meals made by grinding the dried seaweeds, which in used as component in the animal feed (Baquar, 2001). Algae usually contain single celled protein, which may supplement animal food (Aaronson, 1986)
Certain species of seaweeds have attracted several farm animals as their source of food such as Ascophyllum nodosum is eaten by cattle, mink, shrimp, pigs etc, while Palmaria palmate is fed to cows and horses. Studies have shown that when fed on seaweeds, cows produce more milk, chicken eggs have better pigmentation (carotene in yolk) and horses and pets are generally healthier. Sargassum species are used as fodder in China (Round, 1973). In India, research conducted on White Leghorn hens indicated that 10% Gracilaria sp. meal can replace yellow maize in feed. When ragi (Eleusine coracana) meal was replaced with 5% level Hypnea musciformis and Gracilaria edulis meal, the chicks recorded increase in weight but the seaweed feed at higher level gave discouraging results. Many commercial feeds from seaweeds are now available in many parts of Europe and Asia.
In the Philippines, Gracilaria verrucosa and Ulva intestinalis are used as supplementary feed for large scale farming of milkfish. Ulva lactuca is used as feed for pigs in Singapore. Fucus serratus and Chirda filum along with the stipes and fronds of Laminaria digitata are given as additional food to cattle in Norway. Alaria is another popular food for cattle, sheep and horses and commonly known as Kutara or cow weed (Kaur, 1997). This was considered as good milk-producing and fattening fodder. Fucus vesiculosus, sometimes called Swine Tang, is supposed to contain vitamin E and is given to pigs (Biswas, 1980). In the past years, some experiments have been conducted on the effects of seaweeds meal on the fertility of sheep improved when using seaweed meal as the only supplement to a basic ration of hay alone, but it was in some cases inferior to herring meal but superior to a conventional admixture of mineral salts (Hallsson, 1961).
Seaweeds for Industries
Many processed foods such as chocolate milk, yogurts, healthy drinks, and even the highest quality German beers contain seaweed polysaccharides such as alginates, agars, and carrageenans.
Alginates are derived from large brown seaweeds generally growing in colder-water areas of the world. The main commercial sources of Phaeophyta are Ascophyllum, Laminaria, and Mycrocystis. Other minor sources include Sargassum, Durvillea, Eklonia, Lessonia, and Turbinaria. They have a wide range of viscosity as the alginates able to absorb a lot more water than their own weight. This can readily form gels and are non-toxic. They have countless uses in the manufactures of pharmaceutical, cosmetic creams, paper and cardboard and processed foods (Chapman, 1970).
Alginate represents the most important seaweed colloid in term of volume. Richards-Radjadurai (1990) described that average production of alginates are 22,000 -25,000 ton per year, and predicts an increase in demand of up to 50,000 ton per year in coming years. Major producers are those countries bordering colder seas, such as the United States, France, Norway, Great Britain and Japan (Huisman, 2000). Alginates are applied in varied industries, but the most important consumers are textiles (50%) and food (30%). As with other phycocolloids various grades of alginate are available for specific applications and associated prices, e.g. Sodium alginate, pharmaceutical grade (US$ 13-15.5 per kg), food grade (US$ 6.5-11.0 per kg). In Japan and Korea, high demands for Laminaria as kombu have resulted in high prices and necessitated the import of supplies for alginate extraction (Critchley, 1997).
The principal seaweed sources of agar are Gracilaria (53%), and Gelidium (44%) with minor amounts (3%) from Gelidiella and Pterocladia (McHugh, 1991). It can be used as a gel in foodstuffs, but mostly use in bacteriology to solidify media. The supply of agar is limited by the relative scarcity of suitable algae, with high prices resulting. Agarose which is a component of agar, is used in pharmaceutical and other industries for the separation and purification of a number of products (Huisman, 2000). Gelidium is the traditionally preferred source of the best quality agar and commands higher prices. Gracilaria tends to give good yields of agar with poor gel strength. However, the discovery that alkali treatment improves the gel strength of agar from Gracilaria and industrialization of the process has increased demand for this genus (Critchley, 1997). Successful cultivation has led to increased availability of Gracilaria as a source of agar and its use now exceeds that of Gelidium.
Carrageenans are also gel-forming or viscous compounds derived from red algae which belong to the genus Eucheuma (Huisman, 2000). The two species originally cultivated in the Philippines were named Eucheuma cottonii and Eucheuma spinosum (McHugh, 2003).
There are three main types of carrageenan are lambda, kappa, and iota. Each of them has their own gel characteristics. Previously the use of carrageenan was restricted by availability of natural resources of Chondrus crispus (commom name: Irish Moss) in Canada, Ireland, Portugal, Spain and France (Trono, 1997). Chondrus contains a mixture of two types (lambda and kappa) that could not be separated during commercial extraction. Limited quantities of wild Chondrus are still used; attempts to cultivate Chondrus in tanks have been successfully biologically, but uneconomic as a raw material for carrageenan (McHugh, 2003). These sources now only contribute 20% of the total processed material (Trono, 1997). Cultivation of seaweeds is expected to increase carrageenan production.
2.3 Oxidative Stress
For decades, oxidative stress has been suspected as a mechanism for some of the processes that lead to aging and diseases. Oxidative stress is defined as an imbalance between oxidants and antioxidants. Oxidative stress has been linked to the pathogenesis of many chronic diseases and has showed links to fatigue, muscle damage and reduced immune function.
Oxidative stress contribute to more than one hundred diseases in humans including atherosclerosis, arthritis, ischemia and reperfusion injury of many tissues, central nervous system injury, gastritis, cancer, AIDS and also degenerative diseases associated with aging. They can damage cells by chain reaction, such as lipid peroxidation or formation of DNA adducts that could cause cancer-promoting mutations or cell death.
Free radicals are generated during metabolism and energy production in the body. To counteract these free radicals, the body produces an armory of antioxidants such as radical scavenging enzymes (i.e. superoxide dismutase) and cellular antioxidant (i.e. vitamin E) to neutralize the free radicals. Antioxidants are able to protect living organisms from damage by inhibiting the initiation or propagation of oxidative chain reactions. However, the body's internal production of antioxidants is not able to neutralize all the free radicals generated. Furthermore, diseases, aging and chemical challenges such as drugs, pesticides, herbicides and various pollutants can disrupt this balance by inhibition of the cellular antioxidant defenses or by stimulation of the formation of free radicals. Therefore, increasing of dietary intake of antioxidants is necessary.
A free radical is defined as any molecule or molecular fragment that contains one or more unpaired electrons. The presence of unpaired electrons makes free radicals more reactive than the corresponding non-radicals because free radicals strive to balance their unpaired electrons with electrons form other molecules. When a radical reacts with a non-radical another free radical is formed, creating a chain reaction. Depending on the free radical and the non-radical molecule involved, a chain reaction can give rise to wide array of free radicals, which potentially could be more or less reactive than the free radical that initiated the chain reaction. There are two major free radical groups which are oxygen- free radicals and nitrogen- free radicals.
Reactive oxygen species (ROS) is used to described oxygen- derived free radicals and other oxygen- derived free radicals. Oxygen generally exists in its diatomic ground state (O2), which by definition is a biradical because it has two unpaired electrons spinning parallel (i.e. they both share the same spin quantum number) to one another in separate orbitals. This means that oxygen is not very reactive towards non-radicals despite its strong oxidizing potential, because non-radical molecules have paired electrons spinning in opposite directions and would not fit the vacant orbital spaces of molecular oxygen, in accordance with Pauli's principle. Consequently, oxygen tends to accept one electron at a time with the potential to form highly reactive oxygen intermediates or ROS, which can potentially cause oxidative stress.
ROS are continuously produced since they are natural byproducts of cell metabolism. Toxic oxygen metabolites may cause tissue damage. Such damage develops whenever the balance between the rate of free radical production and the cell's ability to endogenously eliminate these is disrupted.
Nitric oxide is a free radical species which contains an unpaired electron in the last orbital. It reacts relatively with O2 producing nitrogen dioxide (Â·NO2), a very reactive species. Further reactions of Â·NO with Â·NO2 will eventually produce nitrite (NO2-), which is the major decomposition product of Â·NO. Peroxynitrite is a highly stable and toxic non-radical anion. It is able to oxidize most biological molecules including DNA, RNA, proteins and lipids.
ROS is a collection term that includes both oxygen radicals and certain non-radicals that are oxidizing agents and are easily converted into radicals (HOCl, O3, ONOO-, 1O2, H2O2). Reactive nitrogen species (RNS) is also collective term including nitric oxide and nitrogen dioxide radicals, as well as such nonradicals as HNO2 and N2O4. ONOO- is often included in both categories. Table 2.4 shows the reactive free radicals that exist in the body.
Table 2.4 Reactive Species in the Human Body
Reactive Oxygen Species (ROS)
Hydrogen peroxide, H2O2
Hypochlorous acid, HOCl
Hypobromous acid, HOBr
Reactive Nitrogen Species (RNS)
Nitric oxide (nitrogen monoxide), NOÂ·
Nitrogen dioxide, NO2Â·
Nitrous acid, HNO2
Nitrosyl cation, NO+
Nitroxyl anion, NO-
Dinitrogen tetroxide, N2O4
Dinitrogen trioxide, N2O3
Peroxynitrous acid, ONOOH
Nitronium (nitryl) cayion, NO2+
(e.g. as nitryl chloride, NO2Cl)
Akyl peroxynitrites, ROONO
Antioxidants are chemicals that can react with radicals and prevent the oxidation of other molecules. In order for a chemical to be defined as an antioxidant, it must meet two conditions. When present in low concentrations it will delay or prevent the oxidation of another compound, and the radical formed from the resultant reaction must be relatively stable and must not promote oxidation. A very common way to classify antioxidants is to divide them into mechanistically distinct groups, they are primary and secondary antioxidants. A similar classification of antioxidants is to divide them as (a) chsin breaking (vitamin E, phenolics), (b) preventive (intracellular enzymes, such as catalase, superoxide dismutase and others) and (c) complementary (vitamin C, Î²- carotene, flavanoids).
Primary antioxidants delay or inhibit the initiation step and interrupt the propagation step of the radical chain reaction. Antioxidants act by transferring a hydrogen atom to peroxy radical. The resulting radicals from the oxidized antioxidant are stabilized by resonance and are relatively unreactive and therefore are not capable of initiating or propagating the oxidative reaction (Figure 2.1). Most of the antioxidants used in food protection are primary antioxidants. Basically they are different phenolic compounds with various ring substitutions: phenolics acids, catechins, flavanoids, anthocyanidins, lignins, tannins, coumarins. Synthetic antioxidants, like butylated bydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ), propyl gallate (PG) also have a phenolic structure. Another class of antioxidant is the secondary or preventive antioxidants. They include metal chelating agents, singlet oxygen quenchers, peroxide destructors and some others.
Figure 2.1: Resonance Stabilization of Phenoxy Radical
Theories for antioxidant protective mechanisms in living systems include the donation of electrons or hydrogen atoms to oxidized molecules such as fatty acids, phospholipids, and proteins, which stabilizes them, scavenging radical compounds and atoms such as OHâ€¢, O2-â€¢, and ROOâ€¢, which become oxidized instead of a 'good' molecule, and they coordinate transition metals such as copper and iron, which would prevent the interaction of the metal with an oxidization molecule, therefore, preventing oxidation.
Below are simplified examples of how phenolic antioxidant compounds cam stop lipid oxidation chain reactions, hydroxyl radical and superoxide damage, and metal ion free radical damage by reducing oxidized molecules to less harmful molecules.
Lipid Oxidation : ROOâ€¢ + Antioxidant H ROOH + Antioxidantâ€¢
ROâ€¢ + Antioxidant H ROH + Antioxidantâ€¢
Hydroxyl : OHâ€¢ + Antioxidant H H2O + Antioxidantâ€¢
Superoxide : O2-â€¢ + Antioxidant H O2H + Antioxidantâ€¢
Ferric : Fe3+ + Antioxidant Fe2+ + Antioxidantâ€¢
Cupric : Cu2+ + Antioxidant Cu+ + Antioxidantâ€¢
2.3.2 Natural and Synthetic Antioxidants
Antioxidants can be as natural or synthetic. The best known synthetic antioxidants are BHA, BHT, gallates and TBHQ. The basic action mode of synthetic antioxidants has been described earlier.
The variety of natural antioxidants is much higher. For instance, if one considers the diversity of plant phenolics and the fact that most of them exhibit some radical scavenging properties, the number of possible natural antioxidants become huge.
The biggest advantage of synthetic antioxidants is related to their low cost. Other advantages are their well-studied chemical and technological properties, which in most cases meet the demands of producers. That makes synthetic antioxidants dominating in the world market. Among natural antioxidant, only a small percentage has been thoroughly analyzed and even fewer are actually being used. To date, only tocopherols, carotenoids, ascorbic acid and its derivatives, as well as extracts from rosemary and sage have been industrially applied in foods.
Tocopherols are very important natural antioxidants. They can divide into two groups which are tocols and tocotrienols. Cereals and legumes are rich sources to tocols. The antioxidant mechanism of tocopherol involves reactions with free radicals especially the peroxyl radical, resulting in the formation of a relatively stable phenoxy radical. Another mechanism of tocopherols includes singlet oxygen scavenging and quenching.
Carotenoids also display antioxidant activity. They are yellow, orange, or red pigments. Structurally they are long-chain polyisoprenes, with 40 carbon atoms. The radical trapping ability of carotenoids lies on the delocalization mechanism of unpaired electrons over the conjugated carotene system, making it less likely for the formed radical to take part in chain processes. The reactions of carotenoid with oxidising radicals are anticipated to occur through three distinct pathways. There are electron transfers (prodicing the carotenoid radical cation), hydrogen abstraction (producing the neutral carotenoid radical) and adition (to produce the neutral radical adduct). The equations below showed the three reaction pathways of carotenoid.
Carotenoid + ROOÂ· CarotenoidÂ·+ + ROO- (Electron transfer)
Carotenoid + ROOÂ· Carotenoid(-H) + ROOH (Hydrogen abstraction)
Carotenoid + ROOÂ· (ROO-CarotenoidÂ·) (Addtion)
Ascorbic acid acts as a multifunctional antioxidant and as a synergist for the primary antioxidants. In the presence of higher concentrations of metal ions ascorbic acid and show pro-oxidant properties by reducing back oxidized metal ions after which they can initiate new free radical reactions.
Flavanoids represent a large and diverse group of phenolic compounds which can display a wide range of substitution patterns and oxidation states, and are divided into flavanols, flavanonols, flavones, catechins (flavan-3-ols) and anthicyanins. The antioxidant function and enzyme modifying actions of flavonoids could account for many of their pharmacological activities. The compounds appear to possess variable mechanisms of action, which include radical scavenging and metal ion complexation. Numerous studies have been carried out to determine the necessary structural features for flavonoids to be effective radical scavengers.
Another class of antioxidant are the phenolic acids which act as free radical terminators. Phenolic antioxidants act to inhibit lipid oxidation by trapping the peroxyl radical. This radical abstracts a hydrogen atom (or electron after prior loss of a proton) from the antioxidant to yield a phenoxyl radical.
Other natural antioxidants such as tannins have antioxidant activity due to their ability to scavenge metal ion and free radicals. There are a number of other natural compounds that have been reported to show antioxidant properties such as lignins, curcumin derivatives, hydroquinones, some components of essential oil and diterpenes.
2.4 Classification of medically important bacteria
For more than a century, bacteria have been classified according to "Gram stain reaction" which named after Christian Gram who devised the protocol for his staining process in 1884. On the basis of their reaction to the Gram stain, bacteria can be divided into two major groups which are Gram- positive and Gram- negative. Gram- positive bacteria appear in purple colour when treated with violet dye and iodine due to high amount (90%) of peptidoglycan (thicker layer) in the cell wall (Figure 2.1). In Gram- negative bacteria, the thin peptidoglycan layer in the periplasm does not retain the purple stain and the pink safranin counterstain stains the peptidoglycan layer.
The shape of bacteria is also used to classify them. Three basic shapes of bacteria are round (cocci), rod (bacilli) or spiral (spirilla). 2 types of Gram- positive and 4 types of Gram- negative bacteria were used for this research. The tested Gram- positive bacteria are Staphylococcus aureus (ATCC 6538) and Bacillus cereus (ATCC 11778). Gram- negative bacteria include Klebsiella pneumonia (ATCC 13883), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli penicillin sensitive strain (ATCC 25922) and Escherichia coli penicillin resistant strain (ATCC 35218). These bacteria are explained in depth below.
22.214.171.124 Staphylococcus aureus
S.aureus is a Gram positive cocci which appear in clumps like grapes when viewed under microscope. It is a facultatively anaerobic bacteria and forms large golden yellow colonies on rich medium (Figure 2.4). When S. aureus grows on blood agar plates, Î²-hemolysis often occurs.
S. aureus causes majority of staph infections including skin infections, pneumonia, food poisoning, toxic shock syndrome and blood poisoning (bacteremia). Skin infections are the most common because many healthy people carry S. aureus on their skin and in their noses without getting sick. However, when skin is punctured or broke, S. aureus can enter the wound and cause infections which can lead to other problems as shown in Figure 2.5. They can turn into impetigo, which turns into a crust on the skin.
Figure 2.5 Staphylococcus aureus infections on foot, mouth and cheek.
126.96.36.199 Description of Bacillus cereus
Bacillus cereus is a Gram positive rods and facultative. It is an endemic where the infection is maintained in the population without the need for external inputs. It is also a beta haemolytic bacterium that causes food borne diseases. It is also s and can produce protective endospores. It is known to create heavy nausea, vomiting and abdominal periods.
188.8.131.52 Description of Klebsiella pneumoniae
The genus Klebsiella belongs to the tribe Klebsiellae, a member of the family Enterobacteriaceae. Klebsiellae are nonmotile, rod-shaped, gram-negative bacilli bacteria with a prominent polysaccharide capsule. It has been a recognized pulmonary pathogen since its discovery over 100 years ago. It is also a common hospital- acquired pathogen that can caused urinary tract infections, nosocomial pneumonia and intra-abdominal infections. The extensive use of broad- spectrum antibiotics in hospitalized patients has led to increased carriage of K. pneumoniae and drug resistant. The resistant strain of K. pneumoniae is highly virulent and has a great ability to spread.
184.108.40.206 Description of Escherichia coli
E. coli is a Gram negative bacterium which belongs to the family of Enterobacteriaceae. It is a rod-shaped bacterium, facultatively anaerobic and unable to sporulate. It lives in the intestinal tracts of animals in health and disease. E. coli is one of the most frequent causes of many common bacterial infections, including cholecystitis, bacteremia, cholangtis, urinary tract infections (UTI), and traveler's diarrhea. Two types of E. coli strains were selected for antimicrobial testing in this research. They are E. coli penicillin sensitive strain and E. coli penicillin resistant strain.
As Gram negative bacteria, E. coli are resistant to many antibiotics which are effective against Gram positive organisms. Antibiotic such as amoxicillin, trimethoprim-sulfamethoxazole, ciprofloxacin, nitrofurantoin are used to treat E. coli infection. Antibiotic resistant is a growing problem. The development of Penicillin resistance in bacteria was early found to be a multistep process involving several consecutive mutations and giving rise to clone with gradually increasing Penicillin resistance (Demerec, 1945; Hotchkiss, 1951; Cavalli and Maccacaro, 1952; Banic, 1959). In most bacteria examined, the cause of Penicillin resistance has been attributed to the production of enzymes which inactivate Penicillin. Two enzymes are known to exist in E.coli which is Î²-lactamase and amidase.
220.127.116.11 Description of Pseudomonas aeruginosa
P. aeruginosa is a Gram negative, aerobic rod belonging to the bacterial family Pseudomonadaceae. It is a free- living bacterium, commonly found in soil and water. However, it occurs regularly on the surfaces of plants and occasionally on the surfaces of animals. P. aeruginosa has become increasingly recognized as an emerging opportunistic pathogen of clinical relevance. It causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteraemia, bone and joint infections, gastrointestinal infections and a variety of systemic infections, particularly in patients hospitalized with cancer, cystic fibrosis, and burns.
P.aeruginosa is notorious for its resistance to antibiotics. Therefore, it is a particularly dangerous and dreaded pathogen. It is naturally resistant to many antibiotics due to the permeability barrier afforded by its Gram negative outer membrane. In addition, its tendency to colonize surfaces in a biofilm form makes the cells impervious to therapeutic concentrations antibiotics.
2.4.4 The action of antimicrobial drugs
Antimicrobial drugs are either bacteriacidal (they kill microbes directly) or bacteriostatic (they prevent microbes from growing). In bacteriostasis, the host's own defences, such as phagocytosis and antibody production, usually
18.104.22.168 The inhibition of cell wall synthesis
The cell wall of bacterium consists of a macromolecular network called peptidoglycan. Peptidoglycan is found only in bacterial walls. Penicillin and certain other antibiotics prevent the synthesis of intact peptidoglycan. Consequently, the cell wall is greatly weakened and the cell undergoes lysis. Since penicillin targets the synthesis process, therefore only actively growing cells are affected by these antibiotics. In addition, human cells do not have peptidoglycan cell wall, so, penicillin has very little toxicity for host cells.
22.214.171.124 The inhibition of protein synthesis
Difference between eukaryotes and eukaryotes is the structure of their ribosomes. Eukaryotic cells have 80S ribosomes while prokaryotic cells have 70S ribosomes. The 70S ribosome is made up of a 50S and 30S unit. The S stands for Svedberg unit, which describes the relative rate of sedimentation in a high- speed centrifuge. The difference in ribosomal structure accounts for the selectivity of antibiotics that affect protein synthesis.
Among the antibiotics that interfere with protein synthesis are chloramphenicol, erythromycin, streptomycin, and the tetracylines. Antibiotics targeting the 70S ribosomes can therefore have adverse effects on the cells of the host. Reacting with the 50S portion of the 70S prokaryotic ribosome, chloramphenicol inhibits the formation of peptide bonds in the growing polypeptide chain. Most drugs that inhibit protein synthesis have a broad spectrum of activity because it does not penetrate the Gram- negative cell wall. It affects mostly Gram- positive bacteria.
126.96.36.199 Injury to the plasma membrane
Certain antibiotic, especially polypeptide antibiotics can change the permeability of the plasma membrane which causes the loss of important metabolites from the microbial cell. For example, polymyxin B causes disruption of the plasma membrane by attaching to the phospholipids of the membrane. Some antifungal drugs are effective against a considerable range of fungal diseases. Such drugs combine with sterols on the fungal plasma membrane to disrupt the membrane. These antibiotics do not act on bacteria because bacterial plasma membrane generally lack of sterols.
188.8.131.52 The inhibition of nucleic acid synthesis
A number of antibiotics interfere with the processes of DNA replication and transcription in microorganisms. Some drugs with this mode of action have an extremely limited usefulness because they interfere with mammalian DNA and RNA as well. Others, such as rifampin and the quinolones, are more widely used in chemotherapy because they are more selectivityly toxic.
184.108.40.206 Inhibiting the synthesis of essential metabolites
Some antibiotics act as antimetabolite. They inhibit a particular enzymatic activity of a microorganism by a substance (antimetabolite) that closely resembles the normal substrate for the enzyme.
Previous Research on Padina antillarum
There is very few research had been done on natural product of P. antillarum and no bioactive compound was reported from 1981 to 2008. One of the studies is on carbohydrates of P. antillarumn. It was done in India. They extracted water-soluble and water-insoluble polysaccharides form P. antillarum with hydrochloric acid. The water-soluble polysaccharide was fractionated using centrimethyl ammonium bromide and chromatography on DEAE-cellulose and sephadex G-100. A neutral laminaran like glucan and two new sulphated heterpolysaccharides comprising glucoronic acid, fucose, rhamnose, xylose, araninose, galactose and glucose and half-ester sulphate were obtained (Prasada et al., 1984).
Research on regional and seasonal variations of trace metals in tropical Phaeophyceae form North Queensland was done in Australia. P. antillarum was chosen as indicator organisms to monitor levels of ten metals (Fe, Cu, Co, Cd, Ni, Cr, Ag, Pb, Mn and Zn) in seawater form stations within Halifax Bay, Cleveland Bay and Bowling Green Bay, near Towncille, on the Northest coast of Australia. Determination if regional and seasonal variations of metal concentration were done by collecting samples monthly for one year. It could observed that variability of locations were independent regarding correlations between metal pairs in algae and in those metals which were significantly correlated with temperature and salinity changes.
P. antillarum also selected to study the influence of changes in salinity, pH and temperature on the spores and sporelings. P. antillarum showed tolerance to salinities of 27.0-32.0% only. Besides, P. antillarum had examined on phycochemical. Five saturated fatty acids (isomyristic, palmitic, margaric, stearic and arachidonic) and three unsaturated fatty acids (palmitoleic, oleic and tetradecatrienoic) have been detected and identified by preparation of their methyl esters and gas chromatography- mass spectrophotometry (GC-MS) analysis. Two sterols (24- methyl cholesterol and 24-methylone cholesterol have been isolated and identified by GC-MS electron ionization (EI) and nuclear magnetic resosonance (NMR), which are being reported for the first time from thr genus Padina (Shakh et al., 1991).
Isolation of marine bioactive natural products
Bioactive marine natural products may be obtained from the crushed biological material by extraction with a solvent such as petroleum ether, chloroform, ethyl acetate or methanol. Several solvents of increasing polarity may be used. Thus lipid material (waxes, fatty acids, sterols, carotenoids and simple terpenoids) can be extracted with non-polar solvents such as petroleum ether, but more polar substances such as the alkaloids and glycosides are extracted with methanol, aqueous methanol or even hot water. Table shows that different type of solvents has their own capability on the extraction of different classes of natural products.
Crude extract which exhibit bioactivity will subject to purification in order to obtain the pure bioactive compound. The purification system to be used depends on the polarity of the bioactive compounds. A number of methods can be used to determine the polarity of the natural products in the extracts. One of the most common method is thin layer chromatographic (TLC) with eluents of varying polarity. TLC is performed on precoated TLC plates with silica gel 60 F254 (layer thickness 0.2mm, Merck). It is used to select column chromatography conditions. TLC conditions that give a useful Rf value, i.e., compound separates from the majority of other components without staying at the origin or with the solvent front, can be approximately transferred to column chromatography.
Column chromatography is typically the next step after determination of the polarity of the extract compounds. Flash chromatography is a preparative column chromatography on the basis of optimized prepacked columns and an air pressure driven eluent at high flow rate. It is a simple and quick technique widely used to separate a variety of organic compounds. Normally, the columns are dry silica gel prepacked, vertically clamped and assembled in the system. The stationary phase is saturated with the desired mobile phase just before sample loading. Samples are dissolved in a small volume of the initial solvent used and the resulting mixture is then packed onto the top of the column using a special syringe. The mobile phase (isocratic or gradient elution) is then pumped through the column with the help of air pressure resulting in sample preparation. This technique is considered as a low- to medium-pressure technique and is applied to samples from few milligrams to some grams of sample.
Repeated separation through column chromatography using appropriate stationary phase and mobile phase solvents system previously determined and optimized by TLC. Two most common separation system are normal phase chromatography and reversed phase chromatography. Normal phase chromatography using a polar stationary phase, typically silica gel or Diol, in conjunction with a nonpolar mobile phase with a gradually increasing amount of a polar solvent. Thus, hydrophobic compounds elute quicker than hydrophilic compounds. Reversed phase chromatography using a nonpolar stationary phase and a polar mobile phase. The stationary phase consists of reversed phase material. For instance, C-8 stands for an octanyl chain and C-18 stands for an actodecyl ligand in the matrix. The more hydrophobic the matrix, the greater the tendency of the column to retain hydrophobic compounds. Thus, hydrophilic compounds elute more quickly than do hydrophobic compounds. Elution is performed using water gradually increasing amounts of methanol or acetonitrile.
The semi-purified fractions from flash chromatographic separation will be further purified by analytical high performance liquid chromatography (HPLC). It is also used to identify the distribution of compounds (detected as peaks) from either extracts or fractions, as well as to evaluate the purity of isolated compounds. All peaks are detected by UV-visible photodiode array detector.
Table: Solvents used for active compounds extraction
Extracted active compound