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Bioactive marine natural merchandises are chemical compounds produced by bugs, sponges, seaweeds, and other Marine beings ( Faulkner, 2001 ; Jensen and Fenical, 2002 ) . The host being synthesizes these compounds as non-primary or secondary metabolites. These may exercise a biological consequence on other beings. Marine natural merchandises exhibited a broad scope of biological activities such as antithelmintic, antibacterial, anticoagulant, antidiabetic, antitumuor, fungicidal, anti- inflammatory, antimalarial, antiplatelet, antiprotozoal, antituberculosis, and antiviral activities ( Mayer and Hamann, 2005 ) . Sing the potency of Marine bioactive natural merchandises, many marine beings have been extensively studied extensively ( Baker et al. , 1995 ) .

Seaweeds

The seaweeds are the most abundant affiliated Marine workss in the ocean ( Bolton et al, 2004 ) . Most of them are green algae ( Chlorophyta ) , brown algae ( Phaeophyta ) , and ruddy algae ( Rhodophyta ) . Each group is characterized by specific combinations of photosynthetic pigments ( Hunt, 1978 ) . Most of the seaweeds are grow in intertidal zones and normally extend to deepnesss of 30 to 40m. In clear tropical seas, they can be found to depths up to 200m. Seaweeds are structurally simpler than tellurian workss. Bing immersed in H2O, they can absorb foods, H2O, dissolved gasses and sunlight through their full surface of the works. Unlike tellurian workss, seaweeds have no root, leaves nor a complex web to transport nutrient and H2O around the workss. Some seaweed has root-like fasteners, but these serve merely to ground the seaweeds on the stones or in the sand ( REF ) .

Seaweeds greatly influence environmental conditions for other types of marine life by supplying nutrient, protection from moving ridges, shadiness, and a substrate to attach. The seaweeds were one of the first groups of marine beings whose natural merchandises chemical science was studied extensively because of their copiousness in shallow Waterss. During the past 30 old ages, a group of marine natural merchandises chemists from several states has reported a really big figure of fresh metabolites with utile pharmacological belongingss ( Faulkner, 2000 ) . Majority of the surveies on seaweeds occurred after the development of many of the utile mechanism bio-assaies used today.

Categorization of Seaweeds

Three major categories of photosynthetic pigments occur among the algae are chlorophylls, carotenoids ( provitamin As and luteins ) , and phycobilins. By and large, seaweeds are classified into 3 major groups which are green, brown and ruddy algae.

The bright green coloring material feature of the green algae ( Chlorophyta ) is due to the predomination of chlorophyll over other accessary photosynthetic pigments. Although chemical surveies of temperate species have non been productive, the tropical green algae are known to bring forth many interesting biologically active metabolites.

In the brown algae ( Phaeophyta ) , the green chromaticity of chlorophyll is partly masked by the aureate xantophylls accessary photosynthetic pigments. Many of the big, more familiar and dominant algae of temperate sea, such as kelps, belong to this division. Natural merchandise surveies of this division have been productive, with many studies of biologically active metabolites, preponderantly diterpenes.

The ruddy algae ( Rhodophyta ) contain non merely chlorophyll, but the accoutrement ruddy phycoerythrin and bluish phycocyanin pigments. They can expose a broad scope of colorss, signifier bright green to assorted sunglassess of ruddy. They are as diverse in construction and home grounds as they are in colour. The chemical science of the ruddy algae has been studied extensively because their leaning to include halogens in their biosynthetic tracts. Indeed, the marine natural merchandises literature was dominated by studies of halogenated metabolites derived from ruddy algae.

Seaweeds as Foods

.Seaweeds have been used as nutrient for rather a long clip peculiarly in Far Eastern states 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 by and large higher in green and ruddy algae than in brown algae ( Madlener, 1977 ) . Seaweeds are considered as an ideal nutrient addendum for twenty-first century, for illustration as beginning of protein, lipoids, polyoses, mineral, vitamins, and enzyme. For illustration, Porphyra umbilicalis ( violet Laver ) is among the most alimentary seaweeds, with a protein content of 30- 50 % , and about 75 % of that is digestable. Sugars are low ( 0.1 % ) and the vitamin content really high, with important sums of Vitamins A, C, nicotinic acid and folic acid. But the shield life of vitamin C can be short in the dried merchandise.

Seaweeds for Medicine and Pharmaceuticals

Seaweeds are normally used in Chinese medical specialty, for illustration, the kelps Saccharina japonica and Ecklonia kurome as beginnings of kunbu. Kunbu is a medical specialty for struma, goitre, tumor, hydrops, accretion, testicular hurting and puffiness. Sargassum is a sort of brown algae which served as the beginning of haizao. Its applications are same with kunbu. Saccharina and Sargassum have been used in China for the intervention of malignant neoplastic disease.

There are studies of algal sulphated polyoses exhibiting a assortment of activities including antiviral, antithrombic, anticancer, antiulcer, antilipemic and immunomodulating ( Renn, 1993 ) . Their inflammatory and immunodulating activities have made them utile in a few pharmacological checks used to detect possible therapeutics ( Renn, 1993 ) . More illustrations are shown in Table 2.1 on medicative seaweeds.

Table 2.1 Example of seaweeds for medical specialty and pharmaceuticals

Types of seaweeds

Applications in pharmaceutical filed

Green algae

Caulerpa racemosa

Hypotensive and anesthetic actions

Codium iyengarii

Inhibition of Gram positive and Gram negative bacteriums

Ulva fasciata

In vitro antiviral activity

Ulva prolifera

Decrease the degree of cholesterin

Brown algae

Durvillaea antartica

Treatment for itchs

Ecklonia kurome

Medicine to bring around struma, goiter, tumor, hydrops, testicular hurting and puffiness

Laminaria japonica

To distend the neck as the stipes swell to several times their original diameter when moistened

Padina boryana

Exhibited hypoglycemic activity

Saccharina japonica

Medicine to bring around struma, goitre, tumor, hydrops, testicular hurting and puffiness

Sargassum confusum

Hypocholesterolaemic and hypoglycemic agents

Sargassum fusiformis

Medicine for bring forthing a chilling and blood cleaning consequence for the intervention of glandular failing

Zonaria diesingiana

Potential anticancer drug

Red algae

Asparagopsis taxiformis

Medicine to bring around goiter

Hypnea musciformis

Exhibited diuretic activity

Laurencia pannosa

Inhibition of Marine bacteriums

Laurencia pannosa

Inhibition of Marine bacteriums

Porphyra atropurpurea

Medicine for dressing lesions and Burnss

( Stein & A ; Borden, 1984 ; Fusatani, 1987 ; Naqvi et Al. 1980 ; Blunden, 1996 ; Perveen, 1993 ; Namboothiry, 2001 )

Seaweeds for animate being fresh fishs

Historically, seaweeds have been used by Greek as carnal fresh fish from the first century BC ( Chapman, 1980 ) . In the coastal countries of Norway, Sweden, Denmark, Iceland, Britain, China, France, Pacific Islands and New Zealand, several seaweeds are used as stock provender for poulet, sheep and cowss. Nowadays the animate beings are fed in the signifier of bars and repasts made by crunching the dried seaweeds, which in used as constituent in the carnal provender ( Baquar, 2001 ) . Algae normally contain individual celled protein, which may supplement carnal nutrient ( Aaronson, 1986 )

Certain species of seaweeds have attracted several farm animate beings as their beginning of nutrient such as Ascophyllum nodosum is eaten by cowss, mink, runt, hogs etc, while Palmaria palmate is fed to cattles and Equus caballuss. Surveies have shown that when fed on seaweeds, cattles produce more milk, chicken eggs have better pigmentation ( provitamin A in yolk ) and Equus caballuss and pets are by and large healthier. Sargassum species are used as fresh fish in China ( Round, 1973 ) . In India, research conducted on White Leghorn biddies indicated that 10 % Gracilaria sp. repast can replace xanthous corn in provender. When finger millet ( Eleusine coracana ) repast was replaced with 5 % degree Hypnea musciformis and Gracilaria edulis repast, the biddies recorded addition in weight but the seaweed provender at higher degree gave detering consequences. Many commercial provenders from seaweeds are now available in many parts of Europe and Asia.

In the Philippines, Gracilaria verrucosa and Ulva intestinalis are used as auxiliary provender for big scale agriculture of milkfish. Ulva Lactuca is used as provender for hogs in Singapore. Fucus serratus and Chirda filament along with the stipes and fronds of Laminaria digitata are given as extra nutrient to cattle in Norway. Alaria is another popular nutrient for cowss, sheep and Equus caballuss and normally known as Kutara or overawe weed ( Kaur, 1997 ) . This was considered as good milk-producing and flesh outing fresh fish. Fucus vesiculosus, sometimes called Swine Tang, is supposed to incorporate vitamin E and is given to hogs ( Biswas, 1980 ) . In the past old ages, some experiments have been conducted on the effects of seaweeds meal on the birthrate of sheep improved when utilizing seaweed repast as the lone addendum to a basic ration of hay entirely, but it was in some instances inferior to herring repast but superior to a conventional alloy of mineral salts ( Hallsson, 1961 ) .

Seaweeds for Industries

Many processed nutrients such as cocoa milk, yoghurts, healthy drinks, and even the highest choice German beers contain seaweed polyoses such as alginates, agars, and carrageenins.

Alginate

Alginates are derived from big brown seaweeds by and large turning in colder-water countries of the universe. The chief commercial beginnings of Phaeophyta are Ascophyllum, Laminaria, and Mycrocystis. Other minor beginnings include Sargassum, Durvillea, Eklonia, Lessonia, and Turbinaria. They have a broad scope of viscousness as the alginates able to absorb a batch more H2O than their ain weight. This can readily organize gels and are non-toxic. They have infinite utilizations in the industries of pharmaceutical, decorative picks, paper and composition board and processed nutrients ( Chapman, 1970 ) .

Alginate represents the most of import seaweed colloid in term of volume. Richards-Radjadurai ( 1990 ) described that mean production of alginates are 22,000 -25,000 ton per twelvemonth, and predicts an addition in demand of up to 50,000 ton per twelvemonth in approaching old ages. Major manufacturers are those states surrounding colder seas, such as the United States, France, Norway, Great Britain and Japan ( Huisman, 2000 ) . Alginates are applied in varied industries, but the most of import consumers are fabrics ( 50 % ) and nutrient ( 30 % ) . As with other phycocolloids assorted classs of alginate are available for specific applications and associated monetary values, e.g. Sodium alginate, pharmaceutical class ( US $ 13-15.5 per kilogram ) , nutrient class ( US $ 6.5-11.0 per kilogram ) . In Japan and Korea, high demands for Laminaria as kombu have resulted in high monetary values and necessitated the import of supplies for alginate extraction ( Critchley, 1997 ) .

Agar

The chief seaweed beginnings of agar are Gracilaria ( 53 % ) , and Gelidium ( 44 % ) with minor sums ( 3 % ) from Gelidiella and Pterocladia ( McHugh, 1991 ) . It can be used as a gel in groceries, but largely use in bacteriology to solidify media. The supply of agar is limited by the comparative scarceness of suited algae, with high monetary values ensuing. Agarose which is a constituent of agar, is used in pharmaceutical and other industries for the separation and purification of a figure of merchandises ( Huisman, 2000 ) . Gelidium is the traditionally preferred beginning of the best quality agar and commands higher monetary values. Gracilaria tends to give good outputs of agar with hapless gel strength. However, the find that alkali intervention improves the gel strength of agar from Gracilaria and industrialisation of the procedure has increased demand for this genus ( Critchley, 1997 ) . Successful cultivation has led to increased handiness of Gracilaria as a beginning of agar and its usage now exceeds that of Gelidium.

Carrageenin

Carrageenins are besides gel-forming or syrupy compounds derived from ruddy 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 chief types of carrageenin are lambda, kappa, and shred. Each of them has their ain gel features. Previously the usage of carrageenin was restricted by handiness 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 non be separated during commercial extraction. Limited measures of wild Chondrus are still used ; efforts to cultivate Chondrus in armored combat vehicles have been successfully biologically, but uneconomic as a natural stuff for carrageenin ( McHugh, 2003 ) . These beginnings now merely lend 20 % of the sum processed stuff ( Trono, 1997 ) . Cultivation of seaweeds is expected to increase carrageenan production.

2.3 Oxidative Stress

For decennaries, oxidative emphasis has been suspected as a mechanism for some of the procedures that lead to aging and diseases. Oxidative emphasis is defined as an instability between oxidizers and antioxidants. Oxidative emphasis has been linked to the pathogenesis of many chronic diseases and has showed links to tire, musculus harm and decreased immune map.

Oxidative emphasis contribute to more than one 100 diseases in worlds including coronary artery disease, arthritis, ischaemia and reperfusion hurt of many tissues, cardinal nervous system hurt, gastritis, malignant neoplastic disease, AIDS and besides degenerative diseases associated with aging. They can damage cells by concatenation reaction, such as lipid peroxidation or formation of DNA adducts that could do cancer-promoting mutants or cell decease.

Free groups are generated during metamorphosis and energy production in the organic structure. To antagonize these free groups, the organic structure produces an armoury of antioxidants such as extremist scavenging enzymes ( i.e. superoxide dismutase ) and cellular antioxidant ( i.e. vitamin E ) to neutralize the free groups. Antioxidants are able to protect populating beings from harm by suppressing the induction or extension of oxidative concatenation reactions. However, the organic structure ‘s internal production of antioxidants is non able to neutralize all the free groups generated. Furthermore, diseases, aging and chemical challenges such as drugs, pesticides, weedkillers and assorted pollutants can interrupt this balance by suppression of the cellular antioxidant defences or by stimulation of the formation of free groups. Therefore, increasing of dietetic consumption of antioxidants is necessary.

Free Groups

A free group is defined as any molecule or molecular fragment that contains one or more odd negatrons. The presence of odd negatrons makes free groups more reactive than the corresponding non-radicals because free groups strive to equilibrate their odd negatrons with negatrons form other molecules. When a extremist reacts with a non-radical another free group is formed, making a concatenation reaction. Depending on the free group and the non-radical molecule involved, a concatenation reaction can give rise to broad array of free groups, which potentially could be more or less reactive than the free group that initiated the concatenation reaction. There are two major free group groups which are oxygen- free groups and nitrogen- free groups.

Reactive O species ( ROS ) is used to depict oxygen- derived free groups and other oxygen- derived free groups. Oxygen by and large exists in its diatomic land province ( O2 ) , which by definition is a biradical because it has two odd negatrons whirling analogue ( i.e. they both portion the same spin quantum figure ) to one another in separate orbitals. This means that O is non really reactive towards non-radicals despite its strong oxidizing potency, because non-radical molecules have paired negatrons whirling in opposite waies and would non suit the vacant orbital infinites of molecular O, in conformity with Pauli ‘s rule. Consequently, O tends to accept one negatron at a clip with the possible to organize extremely reactive O intermediates or ROS, which can potentially do oxidative emphasis.

ROS are continuously produced since they are natural by-products of cell metamorphosis. Toxic O metabolites may do tissue harm. Such harm develops whenever the balance between the rate of free extremist production and the cell ‘s ability to endogenously extinguish these is disrupted.

Nitric oxide is a free group species which contains an odd negatron in the last orbital. It reacts comparatively with O2 bring forthing N dioxide ( I‡NO2 ) , a really reactive species. Further reactions of I‡NO with I‡NO2 will finally bring forth nitrite ( NO2- ) , which is the major decomposition merchandise of I‡NO. Peroxynitrite is a extremely stable and toxic non-radical anion. It is able to oxidise most biological molecules including DNA, RNA, proteins and lipoids.

ROS is a aggregation term that includes both O groups and certain non-radicals that are oxidising agents and are easy converted into groups ( HOCl, O3, ONOO- , 1O2, H2O2 ) . Reactive N species ( RNS ) is besides corporate term including azotic oxide and N dioxide groups, every bit good as such nonradicals as HNO2 and N2O4. ONOO- is frequently included in both classs. Table 2.4 shows the reactive free groups that exist in the organic structure.

Table 2.4 Reactive Speciess in the Human Body

Reactive Oxygen Species ( ROS )

Groups

Non-radicals

Superoxide, O2-

Hydroxyl, OHI‡

Peroxyl, RO2I‡

Alkoxyl, ROI‡

Hydroperoxyl, HO2I‡

Hydrogen peroxide, H2O2

Hypochlorous acid, HOCl

Hypobromous acid, HOBr

Ozone, O3

Singlet O

Reactive Nitrogen Species ( RNS )

Groups

Non-radicals

Nitric oxide ( nitrogen monoxide ) , NOI‡

Nitrogen dioxide, NO2I‡

Azotic acid, HNO2

Nitrosyl cation, NO+

Nitroxyl anion, NO-

Dinitrogen tetroxide, N2O4

Dinitrogen trioxide, N2O3

Peroxynitrite, ONOO-

Peroxynitrous acid, ONOOH

Nitronium ( nitryl ) cayion, NO2+

( e.g. as nitryl chloride, NO2Cl )

Akyl peroxynitrites, ROONO

Antioxidant

Antioxidants are chemicals that can respond with groups and forestall the oxidization of other molecules. In order for a chemical to be defined as an antioxidant, it must run into two conditions. When present in low concentrations it will detain or forestall the oxidization of another compound, and the extremist formed from the attendant reaction must be comparatively stable and must non advance oxidization. A really common manner to sort antioxidants is to split them into mechanistically distinguishable groups, they are primary and secondary antioxidants. A similar categorization of antioxidants is to split them as ( a ) chsin breakage ( vitamin E, phenoplasts ) , ( B ) preventative ( intracellular enzymes, such as catalase, superoxide dismutase and others ) and ( degree Celsius ) complementary ( vitamin C, I?- provitamin A, flavanoids ) .

Primary antioxidants hold or suppress the induction measure and disrupt the extension measure of the extremist concatenation reaction. Antioxidants act by reassigning a H atom to peroxy extremist. The ensuing groups from the oxidized antioxidant are stabilized by resonance and are comparatively unreactive and hence are non capable of originating or propagating the oxidative reaction ( Figure 2.1 ) . Most of the antioxidants used in nutrient protection are primary antioxidants. Basically they are different phenolic compounds with assorted pealing permutations: phenoplasts acids, catechins, flavanoids, anthocyanidins, lignins, tannic acids, coumarins. Man-made antioxidants, like butylated bydroxytoluene ( BHT ) , third butylhydroquinone ( TBHQ ) , propyl gallate ( PG ) besides have a phenolic construction. Another category of antioxidant is the secondary or preventative antioxidants. They include metal chelating agents, vest O quenchers, peroxide destructors and some others.

Figure 2.1: Resonance Stabilization of Phenoxy Radical

Theories for antioxidant protective mechanisms in populating systems include the contribution of negatrons or H atoms to oxidise molecules such as fatty acids, phospholipids, and proteins, which stabilizes them, scavenging extremist compounds and atoms such as OHaˆ? , O2-aˆ? , and ROOaˆ? , which become oxidised alternatively of a ‘good ‘ molecule, and they coordinate passage metals such as Cu and Fe, which would forestall the interaction of the metal with an oxidation molecule, hence, forestalling oxidization.

Below are simplified illustrations of how phenolic antioxidant compounds cam stop lipid oxidization concatenation reactions, hydroxyl extremist and superoxide harm, and metal ion free extremist harm by cut downing oxidised molecules to less harmful molecules.

Lipid Oxidation: ROOaˆ? + Antioxidant H ROOH + Antioxidantaˆ?

ROaˆ? + Antioxidant H ROH + Antioxidantaˆ?

Hydroxyl: OHaˆ? + Antioxidant H H2O + Antioxidantaˆ?

Superoxide: O2-aˆ? + Antioxidant H O2H + Antioxidantaˆ?

Ferric: Fe3+ + Antioxidant Fe2+ + Antioxidantaˆ?

Cupric: Cu2+ + Antioxidant Cu+ + Antioxidantaˆ?

2.3.2 Natural and Synthetic Antioxidants

Antioxidants can be as natural or man-made. The best known man-made antioxidants are BHA, BHT, gallates and TBHQ. The basic action manner of man-made antioxidants has been described earlier.

The assortment of natural antioxidants is much higher. For case, if one considers the diverseness of works phenoplasts and the fact that most of them exhibit some extremist scavenging belongingss, the figure of possible natural antioxidants become immense.

The biggest advantage of man-made antioxidants is related to their low cost. Other advantages are their well-studied chemical and technological belongingss, which in most instances meet the demands of manufacturers. That makes man-made antioxidants ruling in the universe market. Among natural antioxidant, merely a little per centum has been exhaustively analyzed and even fewer are really being used. To day of the month, merely tocopherols, carotenoids, ascorbic acid and its derived functions, every bit good as infusions from rosemary and sage have been industrially applied in nutrients.

Vitamin es are really of import natural antioxidants. They can split into two groups which are tocols and tocotrienols. Cereals and leguminous plants are rich beginnings to tocols. The antioxidant mechanism of vitamin E involves reactions with free groups particularly the peroxyl extremist, ensuing in the formation of a comparatively stable phenoxy group. Another mechanism of vitamin Es includes vest O scavenging and extinction.

Carotenoids besides display antioxidant activity. They are xanthous, orange, or ruddy pigments. Structurally they are long-chain polyisoprenes, with 40 C atoms. The extremist caparison ability of carotenoids prevarications on the delocalization mechanism of odd negatrons over the conjugated provitamin A system, doing it less likely for the formed extremist to take portion in concatenation procedures. The reactions of carotenoid with oxidising groups are anticipated to happen through three distinguishable tracts. There are electron transportations ( prodicing the carotenoid extremist cation ) , hydrogen abstraction ( bring forthing the impersonal carotenoid group ) and adition ( to bring forth the impersonal extremist adduct ) . The equations below showed the three reaction tracts of carotenoid.

Carotenoid + ROOA· CarotenoidA·+ + ROO- ( Electron transportation )

Carotenoid + ROOA· Carotenoid ( -H ) + ROOH ( Hydrogen abstraction )

Carotenoid + ROOA· ( ROO-CarotenoidA· ) ( Addtion )

Ascorbic acerb Acts of the Apostless as a multifunctional antioxidant and as a synergist for the primary antioxidants. In the presence of higher concentrations of metal ions ascorbic acid and demo pro-oxidant belongingss by cut downing back oxidized metal ions after which they can originate new free extremist reactions.

Flavanoids represent a big and diverse group of phenolic compounds which can expose a broad scope of permutation forms and oxidization provinces, and are divided into flavanols, flavanonols, flavones, catechins ( flavan-3-ols ) and anthicyanins. The antioxidant map 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 extremist scavenging and metal ion complexation. Numerous surveies have been carried out to find the necessary structural characteristics for flavonoids to be effectual extremist scavengers.

Another category of antioxidant are the phenolic acids which act as free extremist eradicators. Phenolic antioxidants act to suppress lipid oxidization by pin downing the peroxyl extremist. This extremist abstracts a H atom ( or negatron after anterior loss of a proton ) from the antioxidant to give a phenoxyl group.

Other natural antioxidants such as tannic acids have antioxidant activity due to their ability to scavenge metal ion and free groups. There are a figure of other natural compounds that have been reported to demo antioxidant belongingss such as lignins, curcumin derived functions, hydroquinones, some constituents of indispensable oil and diterpenes.

2.4 Categorization of medically of import bacteriums

For more than a century, bacteriums have been classified harmonizing to “ Gram discoloration reaction ” which named after Christian Gram who devised the protocol for his staining procedure in 1884. On the footing of their reaction to the Gram discoloration, bacteriums can be divided into two major groups which are Gram- positive and Gram- negative. Gram- positive bacteriums appear in violet coloring material when treated with violet dye and I due to high sum ( 90 % ) of peptidoglycan ( thicker bed ) in the cell wall ( Figure 2.1 ) . In Gram- negative bacteriums, the thin peptidoglycan bed in the periplasm does non retain the purple discoloration and the pink saffranine counterstain stains the peptidoglycan bed.

The form of bacterium is besides used to sort them. Three basic forms of bacteriums are circular ( coccus ) , rod ( B ) or coiling ( spirillum ) . 2 types of Gram- positive and 4 types of Gram- negative bacteriums were used for this research. The tried Gram- positive bacteriums are Staphylococcus aureus ( ATCC 6538 ) and Bacillus Cereuss ( ATCC 11778 ) . Gram- negative bacteriums include Klebsiella pneumonia ( ATCC 13883 ) , Pseudomonas aeruginosa ( ATCC 27853 ) , Escherichia coli penicillin sensitive strain ( ATCC 25922 ) and Escherichia coli penicillin immune strain ( ATCC 35218 ) . These bacteriums are explained in deepness below.

2.4.1.1 Staphylococcus aureus

S.aureus is a Gram positive coccus which appear in bunchs like grapes when viewed under microscope. It is a facultatively anaerobiotic bacteriums and signifiers big aureate yellow settlements on rich medium ( Figure 2.4 ) . When S. aureus grows on blood agar home bases, I?-hemolysis frequently occurs.

S. aureus causes bulk of staph infections including skin infections, pneumonia, nutrient toxic condition, toxic daze syndrome and blood toxic condition ( bacteriemia ) . Skin infections are the most common because many healthy people carry S. aureus on their tegument and in their olfactory organs without acquiring sick. However, when tegument is punctured or broke, S. aureus can come in the lesion and cause infections which can take to other jobs as shown in Figure 2.5. They can turn into impetigo, which turns into a crust on the tegument.

.

Figure 2.5 Staphylococcus aureus infections on pes, oral cavity and cheek.

2.4.2.1 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 demand for external inputs. It is besides a beta hemolytic bacteria that causes nutrient borne diseases. It is besides s and can bring forth protective endospores. It is known to make heavy sickness, purging and abdominal periods.

2.4.3.1 Description of Klebsiella pneumoniae

The genus Klebsiella belongs to the folk Klebsiellae, a member of the household Enterobacteriaceae. Klebsiellae are immotile, bacillar, Gram-negative B bacteriums with a outstanding polyose capsule. It has been a recognized pulmonary pathogen since its find over 100 old ages ago. It is besides a common hospital- acquired pathogen that can caused urinary piece of land infections, nosocomial pneumonia and intra-abdominal infections. The extended usage of broad- spectrum antibiotics in hospitalized patients has led to increased passenger car of K. pneumoniae and drug resistant. The immune strain of K. pneumoniae is extremely deadly and has a great ability to distribute.

2.4.3.2 Description of Escherichia coli

E. coli is a Gram negative bacteria which belongs to the household of Enterobacteriaceae. It is a bacillar bacteria, facultatively anaerobiotic and unable to sporulate. It lives in the enteric piece of lands of animate beings in wellness and disease. E. coli is one of the most frequent causes of many common bacterial infections, including cholecystitis, bacteriemia, cholangtis, urinary piece of land infections ( UTI ) , and traveller ‘s diarrhoea. Two types of E. coli strains were selected for antimicrobic testing in this research. They are E. coli penicillin sensitive strain and E. coli penicillin immune strain.

As Gram negative bacteriums, E. coli are immune to many antibiotics which are effectual against Gram positive beings. Antibiotic such as Amoxil, trimethoprim-sulfamethoxazole, Cipro, nitrofurantoin are used to handle E. coli infection. Antibiotic resistant is a turning job. The development of Penicillin opposition in bacterium was early found to be a multistep procedure affecting several back-to-back mutants and giving rise to ringer with bit by bit increasing Penicillin opposition ( Demerec, 1945 ; Hotchkiss, 1951 ; Cavalli and Maccacaro, 1952 ; Banic, 1959 ) . In most bacteriums examined, the cause of Penicillin opposition has been attributed to the production of enzymes which inactivate Penicillin. Two enzymes are known to be in E.coli which is I?-lactamase and amidase.

2.4.3.3 Description of Pseudomonas aeruginosa

P. aeruginosa is a Gram negative, aerophilic rod belonging to the bacterial household Pseudomonadaceae. It is a free- life bacteria, normally found in dirt and H2O. However, it occurs on a regular basis on the surfaces of workss and on occasion on the surfaces of animate beings. P. aeruginosa has become progressively recognized as an emerging timeserving pathogen of clinical relevancy. It causes urinary piece of land infections, respiratory system infections, dermatitis, soft tissue infections, bacteraemia, bone and joint infections, GI infections and a assortment of systemic infections, peculiarly in patients hospitalized with malignant neoplastic disease, cystic fibrosis, and Burnss.

P.aeruginosa is ill-famed for its opposition to antibiotics. Therefore, it is a peculiarly unsafe and dreaded pathogen. It is of course immune to many antibiotics due to the permeableness barrier afforded by its Gram negative outer membrane. In add-on, its inclination to colonise surfaces in a biofilm signifier makes the cells imperviable to curative concentrations antibiotics.

2.4.4 The action of antimicrobic drugs

Antimicrobial drugs are either bacteriacidal ( they kill bugs straight ) or bacteriostatic ( they prevent bugs from turning ) . In bacteriostasis, the host ‘s ain defense mechanisms, such as phagocytosis and antibody production, normally

2.4.4.1 The suppression of cell wall synthesis

The cell wall of bacteria consists of a macromolecular web called peptidoglycan. Peptidoglycan is found merely in bacterial walls. Penicillin and certain other antibiotics prevent the synthesis of integral peptidoglycan. Consequently, the cell wall is greatly weakened and the cell undergoes lysis. Since penicillin marks the synthesis procedure, hence merely actively turning cells are affected by these antibiotics. In add-on, human cells do non hold peptidoglycan cell wall, so, penicillin has really small toxicity for host cells.

2.4.4.2 The suppression of protein synthesis

Difference between eucaryotes and eucaryotes is the construction of their ribosomes. Eukaryotic cells have 80S ribosomes while procaryotic 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 comparative rate of deposit in a high- velocity extractor. The difference in ribosomal construction histories for the selectivity of antibiotics that affect protein synthesis.

Among the antibiotics that interfere with protein synthesis are Chloromycetin, Erythrocin, streptomycin, and the tetracylines. Antibiotics aiming the 70S ribosomes can hence hold inauspicious effects on the cells of the host. Reacting with the 50S part of the 70S procaryotic ribosome, Chloromycetin inhibits the formation of peptide bonds in the turning polypeptide concatenation. Most drugs that inhibit protein synthesis have a wide spectrum of activity because it does non perforate the Gram- negative cell wall. It affects largely Gram- positive bacteriums.

2.4.4.3 Injury to the plasma membrane

Certain antibiotic, particularly polypeptide antibiotics can alter the permeableness of the plasma membrane which causes the loss of of import metabolites from the microbic cell. For illustration, polymyxin B causes break of the plasma membrane by attaching to the phospholipids of the membrane. Some fungicidal drugs are effectual against a considerable scope of fungous diseases. Such drugs combine with steroid alcohols on the fungous plasma membrane to interrupt the membrane. These antibiotics do non move on bacteriums because bacterial plasma membrane by and large deficiency of steroid alcohols.

2.4.4.5 The suppression of nucleic acerb synthesis

A figure of antibiotics interfere with the procedures of DNA reproduction and written text in micro-organism. Some drugs with this manner of action have an highly limited utility because they interfere with mammalian DNA and RNA every bit good. Others, such as Rifadin and the quinolones, are more widely used in chemotherapy because they are more selectivityly toxic.

2.4.4.6 Suppressing the synthesis of indispensable metabolites

Some antibiotics act as antimetabolite. They inhibit a peculiar enzymatic activity of a micro-organism by a substance ( antimetabolite ) that closely resembles the normal substrate for the enzyme.

Previous Research on Padina antillarum

There is really few research had been done on natural merchandise of P. antillarum and no bioactive compound was reported from 1981 to 2008. One of the surveies is on saccharides of P. antillarumn. It was done in India. They extracted water-soluble and non-water-soluble polyoses form P. antillarum with hydrochloric acid. The water-soluble polyose was fractionated utilizing centrimethyl ammonium bromide and chromatography on DEAE-cellulose and sephadex G-100. A impersonal laminaran like glucan and two new sulphated heterpolysaccharides consisting glucoronic acid, fucose, rhamnose, xylose, araninose, galactose and glucose and half-ester sulfate were obtained ( Prasada et al. , 1984 ) .

Research on regional and seasonal fluctuations of hint metals in tropical Phaeophyceae signifier North Queensland was done in Australia. P. antillarum was chosen as index organisms to supervise degrees of 10 metals ( Fe, Cu, Co, Cd, Ni, Cr, Ag, Pb, Mn and Zn ) in saltwater signifier Stationss within Halifax Bay, Cleveland Bay and Bowling Green Bay, near Towncille, on the Northest seashore of Australia. Determination if regional and seasonal fluctuations of metal concentration were done by roll uping samples monthly for one twelvemonth. It could detect that variableness of locations were independent sing correlativities between metal braces in algae and in those metals which were significantly correlated with temperature and salt alterations.

P. antillarum besides selected to analyze the influence of alterations in salt, pH and temperature on the spores and sporelings. P. antillarum showed tolerance to salts of 27.0-32.0 % merely. Besides, P. antillarum had examined on phycochemical. Five concentrated fatso acids ( isomyristic, palmitic, margaric, stearic and arachidonic ) and three unsaturated fatty acids ( palmitoleic, oleic and tetradecatrienoic ) have been detected and identified by readying of their methyl esters and gas chromatography- mass spectrophotometry ( GC-MS ) analysis. Two steroid alcohols ( 24- methyl cholesterin and 24-methylone cholesterin have been isolated and identified by GC-MS negatron ionisation ( EI ) and atomic magnetic resosonance ( NMR ) , which are being reported for the first clip from thr genus Padina ( Shakh et al. , 1991 ) .

Isolation of Marine bioactive natural merchandises

Bioactive marine natural merchandises may be obtained from the crushed biological stuff by extraction with a dissolver such as crude oil quintessence, trichloromethane, ethyl ethanoate or methyl alcohol. Several dissolvers of increasing mutual opposition may be used. Thus lipid stuff ( waxes, fatty acids, steroid alcohols, carotenoids and simple terpenoids ) can be extracted with non-polar dissolvers such as crude oil quintessence, but more polar substances such as the alkaloids and glycosides are extracted with methyl alcohol, aqueous methyl alcohol or even hot H2O. Table shows that different type of dissolvers has their ain capableness on the extraction of different categories of natural merchandises.

Crude infusion which exhibit bioactivity will subject to purification in order to obtain the pure bioactive compound. The purification system to be used depends on the mutual opposition of the bioactive compounds. A figure of methods can be used to find the mutual opposition of the natural merchandises in the infusions. One of the most common method is thin bed chromatographic ( TLC ) with eluents of changing mutual opposition. TLC is performed on precoated TLC plates with silicon oxide gel 60 F254 ( layer thickness 0.2mm, Merck ) . It is used to choose column chromatography conditions. TLC conditions that give a utile Rf value, i.e. , compound offprints from the bulk of other constituents without remaining at the beginning or with the dissolver forepart, can be about transferred to column chromatography.

Column chromatography is typically the following measure after finding of the mutual opposition of the extract compounds. Flash chromatography is a preparatory column chromatography on the footing of optimized prepackaged columns and an air force per unit area driven eluent at high flow rate. It is a simple and speedy technique widely used to divide a assortment of organic compounds. Normally, the columns are dry silicon oxide gel prepacked, vertically clamped and assembled in the system. The stationary stage is saturated with the desired nomadic stage merely earlier sample burden. Samples are dissolved in a little volume of the initial dissolver used and the resulting mixture is so packed onto the top of the column utilizing a particular syringe. The nomadic stage ( isocratic or gradient elution ) is so pumped through the column with the aid of air force per unit area ensuing in sample readying. This technique is considered as a low- to medium-pressure technique and is applied to samples from few mgs to some gms of sample.

Repeated separation through column chromatography utilizing appropriate stationary stage and nomadic stage dissolvers system antecedently determined and optimized by TLC. Two most common separation system are normal stage chromatography and reversed stage chromatography. Normal stage chromatography utilizing a polar stationary stage, typically silica gel or Diol, in concurrence with a nonionic nomadic stage with a bit by bit increasing sum of a polar dissolver. Therefore, hydrophobic compounds elute quicker than hydrophilic compounds. Reversed stage chromatography utilizing a nonionic stationary stage and a polar nomadic stage. The stationary stage consists of reversed stage stuff. For case, C-8 stands for an octanyl concatenation and C-18 bases for an actodecyl ligand in the matrix. The more hydrophobic the matrix, the greater the inclination of the column to retain hydrophobic compounds. Therefore, hydrophilic compounds elute more rapidly than make hydrophobic compounds. Elution is performed utilizing H2O bit by bit increasing sums of methyl alcohol or acetonitrile.

The semi-purified fractions from brassy chromatographic separation will be farther purified by analytical high public presentation liquid chromatography ( HPLC ) . It is besides used to place the distribution of compounds ( detected as extremums ) from either infusions or fractions, every bit good as to measure the pureness of stray compounds. All extremums are detected by UV-visible photodiode array sensor.

Table: Solvents used for active compounds extraction

Solvents

Extracted active compound

Quintessence

Alkaloids

Terpenoids

Coumarins

Fatty acids

Dichloromethanol

Terpenoids

Chloroform

Terpenes

Flavanoids

Acetone

Flavanols

Methanol

Anthocyanins

Terpenoids

Saponins

Tannins

Xanthoxyllines

Totarol

Quassinoids

Lactones

Flavones

Phenones

Polyphenols

Ethyl alcohol

Tannins

Polyphenols

Flavanol

Terpenoids

Sterols

Alkaloids

Propolis

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