Mechanism Of Bacterial Resistance Biology Essay


We live in a world which is an ocean of microbes. These microbes are of many types, that which live as autotrophs or phagotrophs or as parasites. The microbes belonging to the third category generally create problems as they lead a life by gaining nutrition from the host (higher forms of life) and in the process, either by its direct activity (creating mechanical injury) or by the metabolites secreted during its life cycle causes disease in the host.

Antibiotics provide the main basis for the therapy of microbial (bacterial and fungal) infections. Since the discovery of these antibiotics and their uses as chemotherapeutic agents there was a belief in the medical society that this would lead to the eventual eradication of infectious diseases. However, overuse of antibiotics has become the major factor for the emergence and dissemination of multi-drug resistant strains of several groups of microorganisms. For example, the worldwide emergence of Escherichia coli, Klebsiella pneumoniae, Haemophilus and many other ß-lactamase producers has become a major therapeutic problem. Multi-drug resistant strains of E. coli and K. pneumoniae are widely distributed in hospitals and are increasingly being isolated from community acquired infections. Candida albicans, also a nosocomial pathogen, has been reported to account for 50-70% of the cases of invasive candidiasis. Alarmingly, the incidence of nosocomial candidemia has risen sharply in the last decade. All this has resulted in severe consequences including increased cost of medicines and mortality of patients.

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Thus, in light of the evidence of rapid global spread of resistant clinical isolates, the need to find new antimicrobial agents is of paramount importance. However, the past record of rapid, widespread emergence of resistance to newly introduced antimicrobial agents indicates that even new families of antimicrobial agents will have a short life expectancy. For this reason, researchers are increasingly turning their attention to herbal products, looking for new leads to develop better drugs against MDR microbe strains.

The use of plants as remedies to treat various forms of an ailment has been a common practice all over the world. According to the WHO report, nearly 70-90% of the world population depends on herbal medicine, which caters for most of African population (Akerele, 1993; Nair and Nathan, 1998). Pharmacological and phytochemical studies done with medicinal plants used traditionally in other countries have led either to isolation of novel structures for the manufacture of new drugs or templates that served for the production of synthetically improved therapeutic agents (Aberra et al., 2005).

Out of 104 new drugs developed over 37 years, 60 originated from plants used in traditional medicine of China. Modern drugs discovered from natural products such as Quinine, Vincristine, Digoxin, Digitoxin, Emetine and Artemisine to mention just a few, exemplifies the huge potential that still exists in plants for the production of many more novel pharmaceuticals (Plotkin, 1988). Pharmacological studies done with essential oils from 15 species of aromatic plants obtained in Brazil have shown activity coherent with the use of the medicinal plants in folk medicine. These studies have dealt with the effects of these oils on muscle contraction, with their anti inflammatory and anti bacterial activity (Holetz et al., 2002).

Laboratories of the world have also literally found thousands of phytochemicals, which have inhibitory effect on all type of microorganisms in vitro. However, their effectiveness in whole organisms, toxicity assays and their possible effects on the beneficial micro biota are not yet conducted for many of these compounds (Cowan, 1999). The prohibitively expensive cost of modern antibiotics to treat various infections and the clinical importance of drug resistant pathogens, particularly in third world countries makes the search of alternative anti-infective agents from natural products more urgent (Shu, 1998). People in developed countries are also shifting back to herbal medicine because of the serious side effects of the modern drugs (Nair and Nathan, 1998).

One more reason to speed up the research in this direction would be due to the fear of extinction of plants. Many feel that it is a driving force for the research to be progressing in this direction for the past 20 years (Lewis, 1995). This factor is causing both the natural-products chemists and microbiologists feel that the multitude of potentially useful phytochemical structures which could be synthesized chemically is at a risk of being lost irretrievably (Borris, 1996.)

1.1 Mechanism of Bacterial Resistance

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The mode of bacterial resistance is either intrinsic (maintained on the bacterial chromosome) or acquired through chromosomal mutations and plasmid transformation, transposition, transductions and conjugation from other species (Dax, 1997). The fundamental mechanisms of resistance generally observable in bacteria include inactivation or degradation of the antibacterial drug by enzymatic action, decreasing or changing of membrane permeability of the bacterial cell wall to antibiotics, the alteration of the bacterial proteins that are antimicrobial targets, and less often, auxotrophic or metabolic by-pass. For example, the resistance to the penicillins and cephalosporins occurs through antibiotic hydrolysis mediated by the bacterial enzyme beta-lactamase (Dax, 1997 ). The resistance to trimethoprim, the sulphonamides, the aminoglycosides, chloramphenicol and the quinoline drugs occurs by the productions of antibiotic- modifying enzymes and the synthesis of antibiotic-insensitive bacterial targets (Dever, 1991).

"It has been found that the degree of resistance may vary based on the level and the capacity of the pathogenic micro-organisms to manipulate, alter and combine all of these biochemical and genetic processes. Several factors, such as inappropriate prescription of drugs (use of doses that are too low), over prescription in trying to meet patients' demand, extravagant prescription due to excessive choices of available drugs, poor infection control practices, the length of therapy (incomplete course taken by patients) and the duration of bacterial exposure to long-acting agents compared to drugs with short half-lives have been found to influence the rate of selection of resistance and play a role in the worsening situation of global resistance" (Claude 2002).

Other determinants such as the state of immunity and the phenotypes of bacterial pathogens also influence the mode of resistance.


The discovery of bacteria in 1683 by van Leuwenhoek helped mankind to understand the infectious pathogens and appropriately develop antiseptic and antibiotic protocol in the following years. By the beginning of the 20th century, Paul Ehrlich proposed the principle of chemotherapy and his work including "structure-activity relationships" significantly contributed to shaping synthetic protocols and helped in the later discoveries of antibacterial drugs (Dax, 1997). Antibiotics were designed either to kill bacteria (bactericidal) or to nullify growth (bacteriostatic) (Walsh, 2000) and three groups of antibacterial agents, which included bacterial cell wall inhibitors, protein synthesis inhibitors (Hanno, 1997) and DNA inhibitors, were developed to fight the bacterial infections. The sulphonamides were the first group of effective antibacterials to be developed following a chance discovery in 1932 by Domagk of antibacterial activity in the synthetic azo dye, prontosil.

Many antibacterial drugs were developed in the late 1940s following the first report of resistance in Staphylococcus aureus (1941) and in Mycobacterium tuberculosis (1940s) (Dax, 1997), a mycobacterium which also developed resistance later to the drugs isoniazid and rifamycin in the 1950s and 1960s. However, by the 1980s most major infectious diseases in the developed world were almost eradicated and half the major pharmaceutical companies in Japan and the USA stopped their antibacterial drug development programs (Williams, 1999). As a result drug-resistant pathogens were on the rise worldwide (Levy, 1998). Streptococci that causes nosocomial infections showed innate resistance to drugs including cephalosporins, clindamycin and aminoglycoside, (Dax, 1997, Williams, 1999). The bacterium (Staphylococcus aureus) has now developed multi-drug resistant strains and threatens to put an end to successful chemotherapy (Mitscher, 1999.). Vancomycin resistance among enterococci became noticeable in 1987 resulting in a true 'super bug'.

For a long period of time, plants have been a valuable source of natural products for maintaining human health, especially in the last decade, with more intensive studies for natural therapies. As of today, approximately 20% of the plants found in the world have been submitted to pharmaceutical or biological test and a substantial number of new antibiotics introduced on the market are obtained from natural or semi synthetic resources. The interest in this area is overwhelming. For example, between the years 1983 and 1994 the increased systematic screening of antibacterial plant extracts emphasizes the continuous efforts that are on the way to find new compounds with the potential to combat these multi-resistant bacteria (Cragg et al., 1999). To top this all, World Health Organization has stated that medicinal plants are the best source to obtain any variety of drug (Santos et al., 1995).

One more reason for present day scientists to show keener interest in these alternative systems is that these phytochemicals are very likely to find their way into the arsenal of antimicrobial drugs prescribed by physicians; and also, the safety of its usage is confirmed as they are already in use (in their crude form) in the alternate forms of medicine. The Phytochemicals that are responsible for this activity are broadly categorized as terpenes (34%), glycosides (32%), alkaloids (16%) and others (18%) (Wilkinson, J. A). The structures and functions of some of the secondary metabolites are listed here.

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Figure Structures of some of the common Antimicrobials (Cowan, 1999)

Table 1 Major Classes of Antimicrobial compounds from plants (Cowan, 1999)

1.2 Antimicrobial compounds of Plant origin

Substances involved in essential metabolic processes within an organism are identified as "primary metabolites". For example lipids, porphyrins, amino acids, polyacids (e.g., citric, tartaric, and the like) and many others are among this class. The term "secondary metabolite" is frequently found in literature that addresses the chemistry of plant-derived natural products. The term has no real scientific basis except that over the past half century it has come to represent those products of plant metabolism that are associated with some readily detectable properties(e.g., taste, color, odor), a biological activity (e.g., toxicity, medicinal or agrochemical use) or simply novel chemical structure (Gibbs,1974).

The secondary metabolites are unique and specific variations of the primary product's metabolism. They are derivatives produced by various enzymatic processes of limited and often specific distribution that generate chemical compounds distinct from the primary metabolic products. Scientists do not have any interest or have a very little interest on the primary metabolites except, for the obvious use as in nutrition, supplements and diet. Other than this, all are interested in the secondary metabolites (Gibbs, 1974). Thus, Secondary metabolites derive a lot of importance and as most of them show antimicrobial property, they find their way easily into the arsenal of antibiotic therapy.

1.2.1 Alkaloids

Alkaloids constitute a major class of chemical group present in plant drugs. Originally, it means "alkali like" which was applied indiscriminately to all the organic bases. Alkaloids may be described as naturally occurring organic substances, having a cyclic nitrogenous nucleus exhibiting basic properties and having a pronounced physiological action (Mukherje,2002). In the plant kingdom, the alkaloids are restricted to certain families and genera. Among the angiosperms, the Apocynaceae, Papaveraceae, Ranunclaceae, Rubiaceae, Solanaceae and Berberidaceae are outstanding as alkaloid yielding families (Lin,2003). Alkaloids are found to have a range of pharmacological activities such as antimalarial, anti-tumor, relaxing heart and respiratory muscle, anesthetic, analgesic etc.

There are significantly many works done on alkaloids as to their antimicrobial activity. Beriberine, a constituent of many plants is a prototype in the group and has been tested against many strains of microorganisms. This alkaloid has a long history of medicinal use in both Ayurvedic and Chinese medicine. It is present in Hydrastis canadensis (goldenseal), Captis chinensis (Coptis or golden thread), Berberis aquifolium, (Oregon grape), Berberis vulgaris (Berbery) and Berberis aristata (Tree turmeric) (Cerankova, 2002). The predominant clinical uses of beriberine include use as a treatment for bacterial diarrhea, intestinal parasite infections and ocular trachoma infections (Cerankova, 2002).

Plant antimicrobials are not used as systemic antibiotics at present as the reported MICs (minimum inhibitory concentration) are often in the range of 100-1000µg/ml and this is higher than those of common broad-spectrum antibiotics from bacteria or fungi. Gram-negative bacteria have an effective permeability barrier; one of the reasons could be the presence of multi-drug resistance (MDR) pumps that extrude toxins across this barrier. In a study to see the permeability barrier of the cell wall, beriberine was taken as a model compound together with other plant antimicrobials against representative human pathogens: Pseudomonas aerogenosa, Escherichia coli, and Salmonella enterica serovar Typhimurium (Tegos,2002).

MDR pumps readily extrude beriberine alkaloids, which are cationic antimicrobials. To inhibit the pumping out of this agent the naturally occurring pump inhibitor synthesized by many plants, 5'-methoxyhydrocarpin, is used. Therefore, direct measurement of the uptake of the model drug confirmed that, disabling of the MDRs strongly increases the level of penetration of beriberine into the cells of gram-negative bacteria. Other compounds that showed comparable potentiation include plumbagin, resveratol, gossypol, and coumestrol. The finding has implication in that plant antimicrobials might be developed into effective and broad-spectrum antibiotics in combination with inhibitors of MDRs (Tegos,(2002). Stermitz,(2000)).

Screening of plant alkaloids for their antimicrobial effect is quite common in the literature but representative samples of alkaloids that are found to have activity are listed in the Table.

Table 2:Alkaloids exhibiting high antimicrobial activity

1.2.2 Tannins

Tannins, commonly referred to as tannic acid are water-soluble polyphenols that are widely distributed in plants and occur in cell sap often in the distinct vacuoles. Tannins have received a great attention in recent years, since they are shown to occur in many plants that are food to humans, which have got many effects on human health. They have been reported to be responsible for decrease in feed intake, growth rate, feed efficiency, net metabolizable energy, and protein digestibility in experimental animals. Therefore foods rich in tannins are considered to be of low nutritional value (Chung,1998).

The antimicrobial activity of tannins is reported here and there in the literature for a number of plants. For example, comparison in the antimicrobial activity of plant extracts from Syzygium jambor (L.) Alston, Hamamalis virginiana, Krametia triandra, Alchemilla vulgaris and Rubus frutilosus have correlated with their tannin content. Elimination of tannins in these plants totally suppressed the antimicrobial activities (Djipa,2000). These compounds were found to form complex with proteins through non-specific forces such as hydrogen bonding and hydrophobic effects, as well as by covalent bond formation. Thus, the mode of antimicrobial action of many types of tannin may be related to their ability to inactivate microbial adhesions, enzymes, and cell envelope transport proteins.

1.2.3 Terpenoids

Terpenoids are widely distributed in nature and are found in abundance in higher plants, fungi, marine organisms and insect pheromones as well as in insect defense secretions. The antimicrobial activity of terpenoids is reported for many plant products. A comparable activity to that of cephatoxim against Gram-positive bacteria was observed from diterpenes obtained fromoleoresin of Copaifera paupera, (MIC <10 µg/ml) (Tincusi ,2002) Similar activities have been found with terpenoids of different plant sources (Ebi,(2001). Scortichin,(1991)).

The mechanism of action of terpenoids is not fully understood but speculated to involve membrane disruption by the lipophillic compounds. Accordingly, increasing the hydrophillicity of kaurene diterpenoids by addition of methyl group drastically reduced the antimicrobial activity.

The volatile oils (essential oils) are very complex mixture of compounds whose constituents of the oils are mainly monoterpenes and sesquiterpenes. Other compounds include phenylpropenes and specific compounds containing sulfur and nitrogen. Basil (Osimum basilicum L.) is a popular culinary herb and its essential oils have been used extensively for many years in food products, perfumery, dental and oral products. Its essential oils and principal constituents were found to exhibit antimicrobial activity against a wide range of Gram-negative and Gram-positive bacteria, yeast and mold. Exhaustive review with regards to its chemical composition, its effect on microorganisms, and possible future use in food preservation or as slow release component of an active package is available elsewhere (Suppakul,2003). Likewise, the antimicrobial activities of many essential oil components of different plant species have been documented (Valero, (2003). Mimica, (2003). Ohno,(2003)).

Generally, the action of essential oils is the result of the combined effect of both their active and inactive compounds. These inactive compounds might influence resorption, rate of reactions and bioavailability of the active compounds. To add to the complexity of volatile oils, there is evidence that the time of harvest influences the oil composition and consequently the potency of their biological effect including its antimicrobial activity (Deans,(1988). Galambosi,(1993). Lis-Balchin, (1992). Marotti, (1994 a), Marotti, (1994 b)). In addition there is ample evidence which shows that Coumarins, Flavonoids and peptides also have a great potential of being antimicrobialcompounds.

With the mainstream medicine becoming increasingly receptive to the use of antimicrobials and as the traditional antibiotics (products of microorganisms or their synthesized derivatives) are becoming ineffective due to Multi-drug Resistance and also due to the emergence of new diseases, particularly viral diseases, which are generally intractable to this type of drug, there is an increased interest in process of developing new antimicrobial compounds. In addition to a downturn in the pace of production of new antibiotics from microorganisms in recent decades, which is about two or three antibiotics per year (Clark, 1996), and due to the increased pace of emergence of these resistant strains, scientists are realizing that the effective life span of any antibiotic is limited.

Thus, there is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action to combat the new and re-emerging infectious diseases (Rojas et al., 2003). Therefore, researchers are increasingly turning their attention to 'alternative medicine', trying to find new leads to develop better drugs against microbial infections (Benkeblia, 2004). Due to this there is a lot of work being undertaken in this area.


The metabolisms often are linked to the production of free radicals; these are very harmful to the machinery if not neutralized. Generally this job is taken up by certain molecules that have the inherent capacity to neutralize these radicals, which are obtained primarily from the food we eat. These are most essential components as radicals can cause damage to the DNA which can cause cancer. They also prevent radicals from causing damage to the blood vessels. It is also found to be effective in preventing Alzheimer's disease. The food we eat determines our health. In this context, anti-oxidant rich compounds, if identified can be useful in adding to the already existing huge array of compounds that can lead to good health.

New types of treatments use the constituents of the diet as the medicine. The food components are graded according to their nutraceutical value. The food a person eats determines the constitution of his body, as is clearly demonstrated by the symptom manifestation of malnutrition. It is also known that the cells are continuously replaced by new ones and the rate of change is variable from very fast in case of skin cells to very rarely in case of neurons. The building blocks obtained from the food are used in constructing the body.

The free radicals (oxygen) are essential for redox signaling, so the activity of antioxidants is actually to keep these radicals in control (Rhee, 2006). So these are referred to as a "necessary evil". The free radicals are formed as part of metabolic reactions and in case of plants, the photo system activity is responsible and in case of animals the major contribution is a result of the lipid break down.

There are theories that state that free radicals are the main cause for aging, (Denham, H.). There are also theories, which state that the free radicals have no effect on the aging process but they just make the consumer healthy (Doonan, 2008). The free radicals are also found to be responsible for causing many neurodegenerative diseases like Alzheimers disease. In addition, reactive oxygen metabolites and free radicals are involved in many pathogenic conditions via DNA damage, inactivation of nitric oxide (NO•) and oxidation of LDL (low density lipoprotein).

Oxidative stress can be defined as the imbalance between biochemical processes, leading to the production of reactive oxygen species (ROS) and species responsible for their removal. In the past few decades, oxidative stress has attracted a great deal of attention because of the increasing evidence supporting its role in the development of a large number of health disorders. Because oxidative stress involves reactions between biological molecules and free radicals, the study of compounds with free-radical-scavenging activity becomes an important area of research aiming to prevent oxidative stress and the consequent molecular damage.

2.1 Antioxidant mechanisms

Ingold (1968) classified all antioxidants into two groups, namely primary or chain breaking antioxidants and secondary or preventive antioxidants. The primary antioxidants can react with lipid radicals to convert them into more stable products, while the secondary antioxidants can reduce the rate of lipid oxidation by a variety of mechanisms (Gordon, 1990). However, it is noteworthy that certain kinds of substances possess more than one mechanism of antioxidant activity (McClements and Decker, 2000).

2.1.1 Primary (chain-breaking) Antioxidants

A primary antioxidant, also known as "chain-breaking" antioxidant, is a substance

that can accept free radicals and further delay the initiation step or interrupt the propagation step of autoxidation (Reische, 1998). Primary antioxidants (AH) can react with lipid and peroxyl radicals and convert them into more stable radicals or nonradical products as shown in the following equations.

R• + AH → RH + A•

RO• + AH → ROH + A•

ROO• + AH → ROOH + A•

The antioxidant radicals (A•) produced by this process are much less reactive than lipid or peroxyl radicals, and therefore do not promote oxidation as lipid or peroxyl radicals do. These antioxidants radicals, in fact, can also terminate the lipid oxidation reaction by reacting with peroxyl radicals, alkoxyl radicals and other antioxidants as shown in the following equations (McClements and Decker, 2000).

RO• + A• → ROA

ROO• + A• → ROOA

A• + A• → AA

2.1.2 Secondary Antioxidants

Secondary antioxidants can retard lipid oxidation through a variety of mechanisms, including chelation of transition metal ions, oxygen scavenging, replenishing hydrogen to primary antioxidants, absorbing UV radiation and deactivation of reactive species (Reische, 1998; Gordon, 1990). The main difference between primary and secondary antioxidants is that secondary antioxidants do not transform or convert free radical species into more stable products. Secondary antioxidants usually only delay the oxidation by interfering with the prooxidant system, such as metals, radiation etc., Many of these show antioxidant activity only if a minor prooxidative component is present in the system. For instance, sequestering agents are only effective in presence of metal ions, and reducing agents such as ascorbic acid are effective in presence of tocopherols or other phenolic antioxidants(Gordon,1990).


2.2.1 Synthetic Antioxidants

Some of the most used synthetic antioxidants are phenolic compounds such as butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ) and propyl gallate (PG). They are used widely in the food industry because of their effectiveness and generally being less expensive than natural antioxidants. Concerns regarding toxicological effects and carcinogenic potential of synthetic antioxidants have prompted the need for natural alternatives in the last few decades (Thompson and Trush, 1988, Thompson and Moldeus, 1988). Since about 1980, natural antioxidants have appeared as a healthier and safer alternative to synthetic antioxidants (Yanishlieva, 2001).

2.2.2 Natural Antioxidants

Due to the increasing concerns regarding safety issues of using synthetic antioxidants, research has focused on the development and utilization of antioxidants from natural sources. The empirical use of natural compounds as antioxidants is very old. The popularity of smoking and spicing in the home for preservation of meat, fish and other fat-rich foods may have be due to the recognition of the rancidity-retarding effect of these treatments (Yanishlieva, 2001). Natural antioxidants are found in almost all plants, microorganisms, fungi, and even in animal tissues (Pokorny, 2001).

Table 2 Different types of antioxidants (Shahidi, F,2000).

The majority of natural antioxidants are phenolic compounds, and the most important groups of natural antioxidants are the tocopherols, flavonoids and phenolic acids. The mechanisms of these natural antioxidants on autoxidation control or rancidity prevention may be different. However, their presence in live plants may be for protecting tissues from injurious damage. Furthermore, the beneficial effects of consuming plant food have been ascribed, at least in part, to the presence of antioxidants in the plant and are associated with lowering the risk of most cardiovascular diseases and cancer, among other degenerative diseases of aging (Cuppett, 1997).

The compounds of plant origin that show this antioxidant activity are: Phenolic compounds

The most active dietary antioxidants belong to the family of phenolic and polyphenolic compounds (Shahidi, 2000). Most phenolic compounds are found in vegetables, fruits, spices and herbs (Vinson, 1998; Kahkonen, 2001; Hu, 2003). Many of these phenolic compounds are effective natural antioxidants. The term "phenolic" or "polyphenol" can be defined chemically as a substance which possesses an aromatic ring bearing one or more hydroxyl substituents, including functional derivatives e.g. esters, methyl ethers, glycosides etc. Most phenolics have two or more hydroxyl groups and are bioactive substances that occur widely in plants. The phenolic compounds which are commonly found in food materials can be classified into three groups including simple phenols and phenolic acids, hydroxycinnamic acid derivatives and flavonoids (Ho, 1992). Phenolic compounds are ubiquitous in plant foods and therefore are an integral part of the human diet. They are closely associated with the sensory and nutritional quality of fresh or processed plant foods. The antioxidant activities of phenolic compounds have been recognized for decades. Recent studies in vitro also confirm that many polyphenols exhibit antioxidant and free radical scavenging properties (Kahkonen, 2001; Cheung, 2003; Shon, 2003). Research on and development of the practice of using natural substances or food ingredients containing phenolic antioxidants will continue to be of great interest to the food industry. In addition, other biological activities besides being antioxidants, of phenolic compounds have also become well known in recent years. The most important biological activity of phenolic compounds is probably their inhibitory effect on mutagenesis and carcinogenesis (Ho, 1992). Flavonoids

Flavonoids have been known as plant pigments for over a century. The first observation regarding their biological activities was published in 1936 by Rusznyak & Szent-Gyorgyi (Rusznyak (1936), Kuo, (1997)). Originally proposed to be required as vitamins, the term "vitamin P" for flavonoids was suggested, although this was later dismissed (Kuo, 1997). Flavonoids belong to a vast group of polyphenolic compounds that are widely distributed in all foods of plant origin. Plant polyphenols have been of interest to scientists for decades, originally owing to their importance in plant physiology, specifically for their roles in plant pigmentation and flavor. Polyphenols are involved in plant growth and reproduction, provide resistance to pathogens and predators, and protect crops from disease and preharvest seed germination (Bravo (1998). Recently, interest in the possible health benefits of polyphenols (particularly flavonoids) has increased owing to their antioxidant and free-radical scavenging abilities observed in vitro.

Flavonoids have been of interest owing to their observed biological effects in vitro such as free-radical scavenging, modulation of enzymatic activity, and inhibition of cellular proliferation, as well as their potential utility as antibiotic, antiallergic, antidiarrheal, antiulcer, and antiinflammatory agents (Bravo, 1998). There is an extensive literature describing each of these biological properties.

Antioxidant activity of flavonoids:

Diets high in fruits and vegetables are protective against a variety of diseases, particularly cardiovascular disease and some types of cancer (WorldCancerRes. Fund in associationwith Am. Inst.Cancer Res. 1997). Antioxidants and dietary fiber are thought to be the principal nutrients responsible for these protective effects. Reactive oxygen species are formed in vivo during normal aerobic metabolism and can cause damage to DNA, proteins, and lipids, despite natural antioxidant defense systems. The accumulation of unrepaired damaged products may be critical to the development of cancer, atherosclerosis, diabetes, and chronic inflammation (Halliwell,1994). Several in vitro studies have shown that the flavonoids, including flavonols, flavones, isoflavones, flavanols, and anthocyanidins, possess antioxidant activity.

Flavonoids, in conjunction with other antioxidants, including vitamins C and E, are thought to inhibit lipid peroxidation in the phospholipid bilayer caused by reactive oxygen species. In contrast to vitamins C and E, which are concentrated in the aqueous phase and phospholipid bilayer, respectively, flavonoids are likely to be localized between the two phases owing to their hydrophilicity. Flavonoids may trap chain-initiating radicals at the interface of the membranes, thus preventing the progression of the radical chain reaction.

Studies have shown many flavonoids to be effective antioxidants in a wide range of chemical oxidation systems, demonstrated by their ability to scavenge peroxyl radicals, alkyl peroxyl radicals, superoxide hydroxyl radicals, and peroxynite in aqueous and organic environments (Duthie, 2000). Recent studies have suggested that dietary flavonoids may protect free-radical-induced damage to DNA by a mechanism other than solely direct free-radical scavenging. Results from pulse radiolysis studies and a plasmid test system have shown that flavonoids can reduce the incidence of single-strand breaks in double-stranded DNA as well as residual base damage through fast chemical repair (Anderson, 2000). In addition to free-radical scavenging properties, some flavonoids can chelate those transition metal ions responsible for the generation of reactive oxygen species and therefore inhibit the initiation of the lipoxygenase reaction. Some evidence has suggested that flavonoids also have antioxidant capacity in nontransition metal-dependent oxidation (McAnlis, 1997).

Flavonoids may also exert antioxidant abilities through protection or enhancement of endogenous antioxidants. Numerous flavonoids have been shown to alleviate oxidative stress by inducing glutathione S-transferase (GST), an enzyme proposed to protect cells against free-radical damage by increasing resistance to oxidative stress caused by hydrogen peroxide (Fiander, 2000). Some flavonoids, including quercetin, myricetin, and fisetin, were shown to cause statistically significant increases in GST-specific activity (Fiander, 2000). GST is thought to play a protective role against cancer by detoxifying xenobiotics with mutagenic potential (Dirven, 1995). Therefore, compounds that upregulate GST may both alleviate oxidative stress and aid in the detoxification of mutagenic xenobiotics.

The antioxidant capacity of phenolic compounds is determined by their structure, in particular the ease with which a hydrogen atom from an aromatic hydroxyl group can be donated to a free radical and the ability of an aromatic compound to support an unpaired electron as the result of delocalization around the M-electron system. Other important structural determinants of the antioxidant capacity of flavonoids appear to be the 4'-OH and 3'-OH groups. The addition of hydroxyl groups to the carbon atoms ortho to the 4-C position appear to further increase antioxidant potential (Lien, 1999). Studies have indicated that the aglycones, including quercetin, luteolin, myricetin, and kaempferol, have greater antioxidant capacity than do the conjugate flavonoids, such as quercetin-3-glucoside, quercitrin, and rutin (Noroozi, 1998). Ioku et al. (36) showed that the antioxidant activity of quercetin glycosides is lower than quercetin aglycone in an artificial membrane system, suggesting that glycosidation weakens the antioxidant activity of flavonoids. This decrease may be caused by increased blocking of the phenolic groups responsible for radical scavenging and metal chelation and possibly to a decrease in accessibility of the membranes owing to the large glycoside group. Reaction rate constants in organic media for several flavonoids exceed that of vitamin E. Suggested reasons include that flavonoids have a more extended conjugated system to support an unpaired electron, two or more reactive OH groups, and less stearic hindrance at the site of abstraction. Noroozi et al. (Noroozi, 1998) demonstrated that, at equimolar concentrations, most flavonoids showed greater antioxidant capacity than did vitamin C. Further, it has been reported that the degree of polymerization of flavonoids may influence antioxidant capabilities, where higher oligomers possess antioxidant capabilities and monomers show little effect (Mao, 1998).

It is important to note that the bioavailability of these compounds determines their activity in vivo. Currently, however, the relevance of in vitro studies to the in vivo situation is unclear. Fremont et al. (Fremont, 1998) demonstrated that in rats fed diets high in both monounsaturated and polyunsaturated fatty acids, supplementation with dietary flavonoids significantly reduced the amounts of dienes produced during copper-induced oxidation, indicating increased resistance of very low density lipoproteins and low density lipoproteins to oxidation. Funabiki et al. (Funabiki, 1999) examined the effects of dietary supplementation of 4-α-glucopyranosylrun (G-rutin), a water-soluble rutin derivative, in rats. Dietary G-rutin significantly inhibited the accumulation of oxidatively damaged DNA and proteins. Terao (Terao, 1999) found that oral administration of (-)-epicatechin and quercetin enhanced the antioxidant capacity of rat plasma, although both flavonoids accumulated mainly as glucuronide and sulfate conjugates in blood plasma. This finding suggests that conjugated metabolites of flavonoids may play a role in the antioxidant defenses of blood plasma.

In humans, Nielsen et al. (Nielsen, 1999) demonstrated apigenin to be absorbed by subjects fed a diet high in parsley and observed an increase in the concentration of the antioxidant enzymes erythrocyte glutathione reductase and superoxide dismutase. Activities of erythrocyte catalase and glutathione peroxidase, however, were unchanged. In a cross-sectional study in Japan, Arai et al. (Arai, 2000) found total intake of flavonoids among women to be inversely correlated with plasma total cholesterol and low density lipoprotein concentrations, after adjustment for age, Body MassIndex, and total energy intake. The study conducted (Julie, 2002) proved that the glycoside moiety actually increased the rate of absorption and it is evident as there is 52% absorption in subjects having fried onions (quercetin glucosides) than in case of quercetin aglycone ( a major component of tea) which was approximately 24%. These were administered orally to subjects who were on quercetin free diet for 12 days.

Further human studies are needed to explore both the bioavailability of the flavonoids along with biomarkers of antioxidant effects. Carotenoids

Carotenoids are a widespread group of naturally occurring fat-soluble pigments. They are especially abundant in yellow-orange fruits and vegetables and in dark green, leafy vegetables. In plant cells, carotenoids are mainly present in lipid membranes or stored in plasma vacuoles (West, (1993). Mangels,(1993)). Literature reports on the various aspects of the biosynthesis of carotenoids and the changes in their accumulation in plants through genetic and environmental factors. Food carotenoids have been compiled in several tables and databases, generally including provitamin A carotenoids such as β-carotene and β-cryptoxanthin, as well as others without that provitamin activity, such as lycopene and lutein, and others less studied in relation to human health such as phytoene or phytofluene.

In human beings, carotenoids can serve several important biological activities. The most widely studied and well- understood nutritional role for carotenoids is their provitamin A activity. Deficiency of vitamin A is a major cause of premature death in developing nations, particularly among children. Vitamin A, which has many vital systemic func- tions in humans, can be produced within the body from certain carotenoids, notably β -carotene (Britton,1995).

Carotenoids also potentially play an important role in human health by acting as biological antioxidants, protecting cells and tissues from the damaging effects of free radicals and singlet oxygen. Lycopene, the hydrocarbon carotenoid that gives tomatoes their red colour, is particularly effective at quenching the destructive potential of singlet oxygen (di Mascio,1989). Lutein and zeaxanthin and xanthophylls found in corn and in leafy greens such as kale and spinach, are believed to function as protective antioxidants in the macular region of the human retina, protection against cataract formation, coronary heart diseases and stroke (Snodderly,(1995). Chrong, (2007). Ribaya,(2004)). Astaxanthin, a xanthophyll found in salmon, shrimp and other seafoods, is another naturally occurring xanthophyll with potent antioxidant properties (di Mascio,1991). Other health benefits of carotenoids that may be related to their antioxidative potential, include enhancement of immune system function (Bendich, 1989), protection from sunburn (Mathews-Roth, 1990) and inhibition of the development of certain types of cancers (Nishino, 1998). Vitamin E (α-Tocopherol)

The association of the tocopherols with lipid peroxidation in biological systems began in the early days of investigations on the chemical nature of these substances (Harris, 1956). Since then a great deal of work has attempted to relate the symptoms of vitamin E deficiency with peroxidative degradation of lipids, primarily those associated with membranous organelles. The signs of vitamin E deficiency in various species of animals are diverse, involving different tissues with different manifestations and different degrees of severity. Thus, it seems clear that the biological function of this vitamin is not specific in the sense of its being a cofactor for an enzymic reaction (as is the case for the B-complex vitamins).

Interest in the chemical nature of vitamin E developed soon after the observation by Evans & Bishop (Evans, 1922) that most foods contained a substance that prevented sterility in rats when the latter were fed a diet formulated to contain only the nutrients that were known to be required at the time. Within a short period following that discovery, Evans and co-workers determined that the factor was, in reality, a family of closely related tocopherols (Emerson,1937), the richest source of which were vegetable oils (Eggitt,(1953). Evans,(1925). Evans,(1928)). The most active of this group of compounds (in terms of preventing sterility in male rats) was determined to be α-tocopherol (Evans, 1922). Since the biological activity of the different tocopherols is proportional to their proficiency as antioxidants (Century,1965), a compelling rationale exists for considering that the biological function of these compounds is to suppress undesirable oxidative processes in the membranous structures of cells. Indeed, one could find a basis for explaining the diverse effects of vitamin E deficiency in various animals if species differences in the molecular organization of the membranous structures in various tissues resulted in differing capacities to take up and retain tocopherol at sites susceptible to oxidative damage. For example, the cerebellum of the chick develops necrotic lesions causing severe ataxia and death after several weeks on a vitamin E-deficient diet (Jungherr, 1956); other species show no such effect on the cerebellum even though they manifest damage in other tissues (McCay,1980). The capacity of certain membranous structures in the cerebellum of the chick to take up and retain tocopherol may be limited compared to the same structures in other species. Burton et al (Burton,1983) provided evidence suggesting that the composition and structure of tissue lipid components may determine tissue levels of α-tocopherol.

As recently as 1980 the question of whether or not α-tocopherol was truly an effective antioxidant compound was still being raised. This doubt persisted to some extent from earlier work reported by Chipault (ChipauJt,1962) that vitamin E was a rather mediocre antioxidant in vitro compared to other phenolic compounds of both natural and synthetic origin. As stated by Burton et al (Burton,1983), the view of Chipault and co-workers had been accepted rather generally even though one would predict from the chemical structure of this antioxidant that it should be an extremely efficient antioxidant, capable of terminating free-radical chain reactions very effectively (Howard,1926). Ingold and co-workers clearly demonstrated that α-tocopherol was a superb chain terminator of free-radical chain reactions by measuring the inhibition rate constant kinh for vitamin E of the radical-mediated chain autoxidation of styrene, in which the abstraction of the phenolic hydrogen is the controlling factor in the reaction. This chemical activity is typical of all phenolic antioxidants according to the following reactions:

Figure .Mechanism of action of Vitamin E (Paul,1985).

Where reaction 1 is the controlling step, and the rate of Reaction 1 is increased by methyl group substituents on the phenolic ring. Furthermore, Ingold and colleagues demonstrated that the special radical-scavenging properties of vitamin E must reside in the fused chroman ring system, and that in vitro at least, the phytyl side chain does not influence the inhibitory properties of this chromanol (Burton,(1980). Howard,(1963)). The chemical nature of the side chain does determine, however, whether or not the particular chromanol possesses vitamin E activity in vivo (Burton,(1980). Simic,(1983)). The highly effective antioxidant properties of the fused ring structure of the chromanols is believed to be due to the positioning of the pair of pi electrons of the ethereal oxygen moiety in the ring 90° from the plane of the ring; this gives stability to the phenoxyl radical formed in Reaction 1 above (Burton,1980). Tocopheroxy radicals produced in chloroform by reaction with diphenyl-picrylhydrazyl are stable for hours at room temperature (Boguth,1971).

The nature of the side chain of the chromanol is important in determining the effectiveness of the antioxidant in vivo, but not necessarily in vitro, a fact that is highly significant. Because vitamin E is associated primarily with membranous organelles (an inevitable consequence of its insolubility in water and its total miscibility with the lipids of biological membranes), the phytyl chain appears to provide the tocopherol molecule with an affinity for the hydrophobic environment of the membrane. Replacement of the phytyl group with a methyl group results in a loss of vitamin E activity in vivo even though the antioxidant activity of the methyl-substituted compound in vitro is essentially the same as that of α-tocopherol in vitro.

In considering the biological function of vitamin E, the requirement for lipid solubility of the compound is interpreted by many as support for prevention of lipid peroxidation being its primary role. However, there is evidence that scavenging of lipid radicals in membranes may not be its only form of activity. Bisby, Ahmed & Cundall (Bisby,1984) recently reported that a vitamin E analogue, 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox C) (III), which has more solubility in water than α-tocopherol, can perform a repair-like function on free radicals of several amino acids that are formed by one-electron oxidations caused by pulse radiolysis of solution containing amino acids.

Radicals of tryptophan, tyrosine, methionine, and histidine are rapidly reduced to their original structure in the presence of this antioxidant (Bisby,1984). The antioxidant is oxidized to the phenoxy radical, which, because of the chroman ring structure discussed above, is relatively stable. The rate constants for the repair reactions were determined by the decay of the amino acid radicals and the formation of Trolox C radicals (Bisby,1984). The enzyme lysozyme was also subjected to the same treatment and formed a radical that was repaired by Trolox C (Bisby,1984).

These findings suggest that hydrophobic proteins that undergo radical attack in biological membranes may also be repaired by vitamin E present in the lipid environment of those proteins. Indeed, it was reported that vitamin E (and other antioxidants) protect cytochrome P-450 from alteration by free radicals produced by the cytochrome's own catalytic activity in the metabolism of carbon tetrachloride (McCay,1983).

A study by Burton, Joyce & Ingold (Burton,1983) showed that vitamin E is the only lipid-soluble antioxidant present in plasma and in erythrocyte membranes. Their findings also indicate that this vitamin is the only radical chain-breaking substance of any significance present in these biological materials. These investigators state that of all the known phenolic antioxidants, the chain­breaking free-radical-scavenging activity of α-tocopherol is the most effective (Burton,1981). Even when an equilibrium existed in the distribution of vitamin E between the plasma and the red cells, there was a 3-to-l difference in favor of the plasma (Burton,1983). As mentioned above, this striking finding suggests that membrane structure is critical in determining how much tocopherol those membranes will be able to absorb when an excess of the vitamin is available. The implications are that, regardless of the amount of vitamin E an animal may consume, the amount that may be taken up in the critical sites in membranes (where protection against initiation of free-radical chain reactions is needed) is inherently limited for a particular type of membranous structure. A further implication is that intake of excess α-tocopherol would not result in deposition at critical sites in membranes in excess of that dictated by the structure of a particular membrane.

Although it has been suggested that the only function of vitamin E is to prevent fortuitous, potentially damaging peroxidation of lipids from occurring (Burton,1983), it is by no means certain that this is its only action. From the standpoint of the nutritional significance of this vitamin in health and disease, this is an extremely important question to resolve. There is a mystique held by the public at large about the enhancement of health by vitamin E supplements. A more pressing question is whether or not the tocopherols are actually beneficial in the treatment of a number of disease states for which they are recommended! In fact, elevation of vitamin E intake in laboratory animals has been shown to increase the number of observable enzyme-generated toxic free-radicals produced in the liver of animals exposed to toxic compounds compared to those formed in similarly exposed animals with normal vitamin E intakes (McCay,1983). Many individuals are consuming considerable quantities not only of vitamin E but also vitamin C on a daily basis.

Thus, the components if administered orally in combinations, there might be more positive response in the form of increased radical scavenging.

The literature that has been collected gives us an understanding that it is a very important area and has a lot of scope to lead to results which would be of benefit to the society as a whole (if fructified).