Aphichart Karnchanatat

*The Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok, Thailand* 

#### **1. Introduction**

144 Antimicrobial Agents

Kone, W. M., Atindehou, K. K., Terreaux, C., Hosetettman, K., Traore, D., and M. Dosso,

Krisharaju A.V. and Rao T. V. N. Sundararaju (2005): Assessment of bioactivity of Indian

Krishna Kishore, G., and Suresh Pande, 2005. Integrated management of the late leaf spot

Lawless, J., 1995. *The Illustrated Encyclopedia of Essential Oils*. Shaftesbury, UK: Element Books

Meena, C., and J. Gopalakrishnan, 2005. Efficacy of plant extracts against bacterial blight.

Mitscher, L. A., Drake, S., Gollapudi, S.R. and S. K. Okwute, 1987. A modern look at folkloric use of anti-infective agents. *Journal of Natural Products,* 50:1025–1040. Mitscher, L. A., R. P. Leu., M. S. Bathala., W. N. Wu., and J. L. Beal, 1972. Antimicrobial

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*International journal of pest management,* 51(4):327-334.

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There are at least three reasons for the need in finding out new alternative antimicrobial substances from natural sources. The first reason is that people nowadays concern about toxic of synthetic substances including daily contact chemicals or even drugs used in medical or healthcare purposes (Hafidh et al., 2009). Any synthetic drugs were avoided in order to keep physiological cleans as belief. Thus, natural substances were used increasingly instead as well as any substances used for antimicrobial purposes. The second reason is that new alternative drugs are human hope for better fighting with existed diseases and pathogens. They may replace currently used drugs in points of more efficiency, more abundant, lower side-effect or safer or even lower production cost. It is fact that most alive organisms should have some mechanisms or substances fight with all time contacting pathogens so that they can be survived in nature. Although a plenty of antibiotics were discovered after first time Fleming's declaration, but they were still relatively low amounts compared with overall real natural antimicrobial substances. This mean the natural sources still flourish with novel antimicrobial substances waiting for discovered. Additional small aspect may be raised here. The natural substances are usually good leading compound sources for mostly synthetic drug from the long past due to their diversities are far from human imagination. New chemical structures are always found in natural resources as higher frequency than artificial deducing structures. The final reason is that the mechanism used to synthesize natural substances are available and they are usually can be imitated in small, medium, and even large scale production with present biotechnological knowledge which looks easier than newly designed plants.

Plants are of primary importance in the global ecosystem. They are, together with a small group of bacteria, the only living organisms which are capable of harvesting and storing solar energy by virtue of their photosynthetic apparatus which converts light energy into chemical energy through the reductive assimilation of carbon dioxide. Marine and terrestrial plants are the first link in the global food chain. Virtually all other life on earth depends on the organic molecules they synthesize. Evidently, the fact that the majority of heterotrophic organisms depends on them makes plants favorite targets of a whole variety of parasites and predators. Therefore, plants must defend themselves against their potential enemies. During the past 15 years, a large number of antimicrobial proteins (AMPs) have been identified in different plants (Broekaert et al., 1997). AMPs constitute a heterogenous class of low molecular mass proteins, which are recognized as important components of defense

Antimicrobial Activity of Lectins from Plants 147

nuclear structures and the extracellular matrix of cells and tissues from throughout the animal and plant kingdoms, down to microorganisms (Brooks and Leathem, 1998). The availability of a large number of lectins with distinct carbohydrate specificities has resulted in the use of these proteins as tools in medical and biological research (Singh et al., 1999), and has attracted great interest because of their remarkable effects in a wide range of biological systems, including the purification and characterization of glycoconjugates and the study of cell-surface architecture. The agglutination activity of these highly specific carbohydrate binding molecules is usually inhibited by a specific simple monosaccharide, but for some lectins di-, tri-, and even poly-saccharides are required. They are classified into a small number of sugar specificity groups, such as mannose, galactose, *N*acetylglucosamine, L-fucose and *N*-acetylneuraminic acid, according to the monosaccharide that is the most effective inhibitor of the lectin-mediated agglutination of erythrocytes (Lis

The lectins represent a large group of plant proteins. Lectins have been found in less than 500 species, which indicates that only a limited number of higher plants, contain detectable levels of lectins (Van Damme et al., 1998). However, the majority of the studies on lectins have been carried out on legume species (Kocourek, 1986; and Lakhtin, 1994) particularly in their seeds where they comprise up to 15% of the total protein. As a result of these studies, many plant lectins have become a very popular class of proteins because of their obvious potential in aiding researchers in other areas of the life sciences. A variety of lectins are presently envisioned to be involved in one or more at least three roles releasing to plant defense. One such defense role for some lectins may be in the recognition of oligosaccharide signals produced by the breakdown of cell wall components of the plant or pathogen upon contact with the plant. A second type of defense role may involve a direct interaction of a lectin with the infectious agent. A third defense type with a considerable support is that some lectins play role in protecting the plant animal predators (Weis and Drickamer, 1996). Legumes and monocots are major sources of plant lectins that have been widely studied (Wood et al., 1999). Plant lectins can be classified into four major families of structurally and evolutionary related proteins: legume lectins, type 2 ribosome inactivating proteins, chitinbinding lectins, and monocot mannose-binding lectins. Three other small lectin families (Cucurbitaceae phloem lectins, amaranthins, and jacalin-related lectins) have also been characterized (Van Damme et al., 1999). Legume lectins represent the largest and most thoroughly studied family of plant lectins. They have been isolated from seeds, stem, and bark of legumes (Imberty et al., 2000). The best known legume lectins are phytohemagglutinin (PHA) from red kidney bean, soybean (SBA), jackbean (Concanavalin A), peanut lectin (PNA), and pea (PSL) (Lis and Sharon, 1998). Type 2 ribosome-inactivating proteins consist of the toxic A subunit and Gal/GalNAc binding subunit of B chain. Whereas the A chain has RNA glycosidase activity, the B chain is responsible for binding to the target cell surface and helping in the internalization of the whole protein into cell membrane (Kaku et al., 1996; and Wood et al., 1999). Ricin from seeds of *Ricinus communis*, the first plant lectin, is an example (Sphyris et al., 1995; and Lisgarten et al., 1999). Chitinbinding lectins containing hevein domains have been prevalently found in cereal. Examples are wheat germ agglutinin, pokeweed mitogen, rice, rye, and barley lectins (Lis and Sharon, 1998; and Wood et al., 1999). Monocot mannose-binding lectins were first reported from the snowdrop (*Galanthus nivalis*) (Van Damme et al., 1997). Later several lectins have been

and Sharon, 1986).

system. They directly interfere with the growth, multiplication and spread of microbial organisms (Lehrer and Ganz, 1999). Different proteins with antibacterial and/or antifungal activity have been isolated from seeds, tubers, and rhizomes, where they accumulate to high levels and may also function as storage proteins. Homologous of the seed proteins have also been identified at very low concentrations in floral and vegetative tissues (Terras et al., 1995; Kheeree et al., 2010; and Charungchitrak et al., 2011). There are several classes of proteins having antimicrobial properties which include thionins, lipid transfer proteins, plant defensins, chitinases, glucanases, 2S albumins, ribosome inactivating proteins and lectin (Ye et al., 2002; and Zhang and Halaweish, 2003).

Lectins are proteins or glycoproteins of a ubiquitous distribution in nature, which have at least one carbohydrate or derivative binding site without catalytic function or immunological characteristics. They have the unique ability to recognize and bind reversibly to specific carbohydrate ligands without any chemical modification; this distinguishes lectins from other carbohydrate binding proteins and enzymes, and makes them invaluable tools in biomedical and glycoconjugate research (Peumans and Van Damme, 1995). Plants were the first discovered source of lectins and, although lectins have since been found to be universally distributed, plants remain the most frequently used source of lectin studies due to both the ease of their extraction and the relatively high yields that can be obtained. Moreover, different families of plants, as well as different tissues within the same plant, can contain different lectins with different bioactivities, including different carbohydratebinding specificities. It has been suggested that plant lectins may have important roles according to their abundance, including in the immune defence, and also that lectins have been co-opted adapted for several functions during evolution (Sharon and Lis, 2001).

The role of lectins in the defense mechanism of plants may have evolved from the ability to lectins to agglutinate and immobilize microorganisms. The supporting evidence for this proposed role in defense against pathogens falls into two main observed categories, namely (a) the presence of lectins at potential sites of invasion by infectious agents, and (b) the binding of lectins to various fungi and their ability to inhibit fungal growth and germination. A number of studies with respect to the potential defense role of plant lectins have been reported. For example, during the imbibition of dry soybean seeds, lectin is released into the water and the presence of this lectin in the vicinity of germinating seeds hints at possible interactions of lectins with potential pathogens. The developmental pattern of the initial accumulation and final disappearance of lectin can be observed during the seed dormancy, germination and maturation, which may implicate the role of lectins in a defense mechanism necessary for plants in the initial stages of growth. Moreover, some lectins may provide some protection to plants against generalist herbivores (Howard et al., 1995). This chapter is intended to provide exposure for recent papers in details of antimicrobial activity of lectins from plants. This omission can be remedied by reading the more detailed reviews listed in the references.

### **2. General properties of plant lectins**

Lectins are proteins or glycoproteins of non-immune origin derived from plants, animals or microorganisms that have specificity for terminal or subterminal carbohydrate residues. The main characteristic of this class of proteins is their ability to interact with carbohydrates and thus combine with glycocomponents of the cell surface, as well as with cytoplasmatic and

system. They directly interfere with the growth, multiplication and spread of microbial organisms (Lehrer and Ganz, 1999). Different proteins with antibacterial and/or antifungal activity have been isolated from seeds, tubers, and rhizomes, where they accumulate to high levels and may also function as storage proteins. Homologous of the seed proteins have also been identified at very low concentrations in floral and vegetative tissues (Terras et al., 1995; Kheeree et al., 2010; and Charungchitrak et al., 2011). There are several classes of proteins having antimicrobial properties which include thionins, lipid transfer proteins, plant defensins, chitinases, glucanases, 2S albumins, ribosome inactivating proteins and lectin (Ye

Lectins are proteins or glycoproteins of a ubiquitous distribution in nature, which have at least one carbohydrate or derivative binding site without catalytic function or immunological characteristics. They have the unique ability to recognize and bind reversibly to specific carbohydrate ligands without any chemical modification; this distinguishes lectins from other carbohydrate binding proteins and enzymes, and makes them invaluable tools in biomedical and glycoconjugate research (Peumans and Van Damme, 1995). Plants were the first discovered source of lectins and, although lectins have since been found to be universally distributed, plants remain the most frequently used source of lectin studies due to both the ease of their extraction and the relatively high yields that can be obtained. Moreover, different families of plants, as well as different tissues within the same plant, can contain different lectins with different bioactivities, including different carbohydratebinding specificities. It has been suggested that plant lectins may have important roles according to their abundance, including in the immune defence, and also that lectins have

been co-opted adapted for several functions during evolution (Sharon and Lis, 2001).

The role of lectins in the defense mechanism of plants may have evolved from the ability to lectins to agglutinate and immobilize microorganisms. The supporting evidence for this proposed role in defense against pathogens falls into two main observed categories, namely (a) the presence of lectins at potential sites of invasion by infectious agents, and (b) the binding of lectins to various fungi and their ability to inhibit fungal growth and germination. A number of studies with respect to the potential defense role of plant lectins have been reported. For example, during the imbibition of dry soybean seeds, lectin is released into the water and the presence of this lectin in the vicinity of germinating seeds hints at possible interactions of lectins with potential pathogens. The developmental pattern of the initial accumulation and final disappearance of lectin can be observed during the seed dormancy, germination and maturation, which may implicate the role of lectins in a defense mechanism necessary for plants in the initial stages of growth. Moreover, some lectins may provide some protection to plants against generalist herbivores (Howard et al., 1995). This chapter is intended to provide exposure for recent papers in details of antimicrobial activity of lectins from plants. This omission can be remedied by reading the more detailed reviews

Lectins are proteins or glycoproteins of non-immune origin derived from plants, animals or microorganisms that have specificity for terminal or subterminal carbohydrate residues. The main characteristic of this class of proteins is their ability to interact with carbohydrates and thus combine with glycocomponents of the cell surface, as well as with cytoplasmatic and

et al., 2002; and Zhang and Halaweish, 2003).

listed in the references.

**2. General properties of plant lectins** 

nuclear structures and the extracellular matrix of cells and tissues from throughout the animal and plant kingdoms, down to microorganisms (Brooks and Leathem, 1998). The availability of a large number of lectins with distinct carbohydrate specificities has resulted in the use of these proteins as tools in medical and biological research (Singh et al., 1999), and has attracted great interest because of their remarkable effects in a wide range of biological systems, including the purification and characterization of glycoconjugates and the study of cell-surface architecture. The agglutination activity of these highly specific carbohydrate binding molecules is usually inhibited by a specific simple monosaccharide, but for some lectins di-, tri-, and even poly-saccharides are required. They are classified into a small number of sugar specificity groups, such as mannose, galactose, *N*acetylglucosamine, L-fucose and *N*-acetylneuraminic acid, according to the monosaccharide that is the most effective inhibitor of the lectin-mediated agglutination of erythrocytes (Lis and Sharon, 1986).

The lectins represent a large group of plant proteins. Lectins have been found in less than 500 species, which indicates that only a limited number of higher plants, contain detectable levels of lectins (Van Damme et al., 1998). However, the majority of the studies on lectins have been carried out on legume species (Kocourek, 1986; and Lakhtin, 1994) particularly in their seeds where they comprise up to 15% of the total protein. As a result of these studies, many plant lectins have become a very popular class of proteins because of their obvious potential in aiding researchers in other areas of the life sciences. A variety of lectins are presently envisioned to be involved in one or more at least three roles releasing to plant defense. One such defense role for some lectins may be in the recognition of oligosaccharide signals produced by the breakdown of cell wall components of the plant or pathogen upon contact with the plant. A second type of defense role may involve a direct interaction of a lectin with the infectious agent. A third defense type with a considerable support is that some lectins play role in protecting the plant animal predators (Weis and Drickamer, 1996).

Legumes and monocots are major sources of plant lectins that have been widely studied (Wood et al., 1999). Plant lectins can be classified into four major families of structurally and evolutionary related proteins: legume lectins, type 2 ribosome inactivating proteins, chitinbinding lectins, and monocot mannose-binding lectins. Three other small lectin families (Cucurbitaceae phloem lectins, amaranthins, and jacalin-related lectins) have also been characterized (Van Damme et al., 1999). Legume lectins represent the largest and most thoroughly studied family of plant lectins. They have been isolated from seeds, stem, and bark of legumes (Imberty et al., 2000). The best known legume lectins are phytohemagglutinin (PHA) from red kidney bean, soybean (SBA), jackbean (Concanavalin A), peanut lectin (PNA), and pea (PSL) (Lis and Sharon, 1998). Type 2 ribosome-inactivating proteins consist of the toxic A subunit and Gal/GalNAc binding subunit of B chain. Whereas the A chain has RNA glycosidase activity, the B chain is responsible for binding to the target cell surface and helping in the internalization of the whole protein into cell membrane (Kaku et al., 1996; and Wood et al., 1999). Ricin from seeds of *Ricinus communis*, the first plant lectin, is an example (Sphyris et al., 1995; and Lisgarten et al., 1999). Chitinbinding lectins containing hevein domains have been prevalently found in cereal. Examples are wheat germ agglutinin, pokeweed mitogen, rice, rye, and barley lectins (Lis and Sharon, 1998; and Wood et al., 1999). Monocot mannose-binding lectins were first reported from the snowdrop (*Galanthus nivalis*) (Van Damme et al., 1997). Later several lectins have been

Antimicrobial Activity of Lectins from Plants 149

Lectins are usually considered as a very large and heterogeneous group of proteins (Goldstein and Poretz, 1986). Although, there is no doubt indeed that numerous plant species of different taxonomic groupings contain lectins. The total number of welldocumented cases is about 400. Assuming that all the close relatives of these plants also contain agglutinins and that some new lectins will be discovered in the future, the expected occurrence of lectins is still limited to a small fraction of the plant kingdom. It can be concluded, therefore, that the occurrence of at least the classical agglutinating lectins in plants is the exception rather than the rule. However, in contrast to the relative scarcity of the agglutinating lectins, chimerolectins belonging to the Class I chitinases seem to be

Lectins are widely distributed throughout the plant kingdom where they have been found in a variety of tissues of a large number of different plants. In plants, lectins are particularly localized in seeds. Howard et al., 1972, reported that seed lectins are particularly seen in cotyledons where they appear during the later stages of maturation of the seeds. In addition to cotyledons, in some cases appreciable amounts of lectins have been reported in the embryos and small amounts in the seed coats (Pueppke et al., 1978). Immunolocalization studies have revealed that lectins are primarily found in the protein bodies of the cotyledon cells (Herman and Shannon, 1984). During the early seedling growth, Weber and Neumann (1980) noticed the decrease in lectin concentration as the cotyledons are resorbed. A short survey of the occurrence and concentration of lectins in seeds as well as in different types of vegetative tissues reveals striking differences in the location and relative abundance of the individual lectins. Usually, seed lectins are confined to cotyledons (e.g. legumes) or endosperm (e.g. castor bean). Normally lectins account for up to 5% of the total seed proteins. Sometimes, they become predominant protein in the seed representing 50% of the total seed protein (e.g. Phaseolus species). The non-seed lectins are found in all kinds of vegetative tissues such as leaves, stem, bark, bulb, tubers, corns, rhizomes, roots, fruits, flowers, ovaries, phloem sap and even in nectar (Peumans and Van Damme, 1995) and are only minor, quantitatively unimportant proteins. Non-seed lectins may occur in different tissues of the same plant. The snowdrop and daffodil lectins, for instance, have been found in all vegetative tissues, although the lectin is most abundant in the bulbs (Van Damme and Peumans, 1990). Similarly, the potato lectin occurs in tubers, stems, leaves and fruits (Kilpatrick, 1980). There are exceptions also. The ground elder berry lectin is confined to the rhizome only (Peumans et al., 1985). In the case of tulip bulbs, lectins are present in large quantities in the bulb but are almost undetectable in stem and leaves (Van Damme and Peumans, 1995). Some legume lectins are found in seeds as well as in bark tissues. A thorough examination of the genes coding for these lectins revealed that the seed and bark lectins are

encoded by different, though highly hommologus, genes (Van Damme et al., 1995).

Lectins are a group of protein that can bind to carbohydrate (which can be in form of sugar, oligosaccharide, or polysaccharide) specifically. Binding of the lectins is differed from those enzymes, anti-lectin antibodies, and other carbohydrate specific binding protein on that they will never change any bound-carbohydrate properties, not convert such carbohydrate to other substances, not come form immune origin, and being reversible binding. In addition to

**5. Hemagglutinating activity by plant lectins** 

**4. Occurrence and distribution** 

present in almost all plant species (Collinge et al., 1993).

extracted and intensively characterized from several monocot families: Alliaceae, Amaryllidaceae, Araceae, Bromeliaceae, Iridaceae, Liliaceae, and Orchidaceae (Wood et al., 1999). For example, *Narcissus pseudonarcissus* (daffodil) and *Scilla campanulata* (bluebell) lectins have been recently reported (Sauerborn et al., 1999; and Wood et al., 1999).
