Preface Organic chemists of the nineteenth century were among the first to recognize and study the diversity of plant secondary metabolites and to analyze them both in terms of structure and function. Nowhere in the plant kingdom do ecology, evolution, and human affairs come together as intricately as phenomena involving secondary metabolites. The importance of these compounds is apparent in some of the earliest annals of human history. Past 60 years have seen the development, testing and refinement of secondary substances. With the functional roles of these compounds broadly acknowledged, several challenges presented themselves to scientists interested in their ecological and evolutionary dynamics. Most obviously, the details of the processes underlying the interactions between each of these compounds and their target pest or pathogen became one of the most vibrant topics in research. Secondary metabolites are compounds biosynthetically derived from primary metabolites but more limited in occurrence in the plant kingdom and may be restricted to a particular taxonomic group. Secondary metabolites are apparently multifunctional in nature. The most disconcerting feature of secondary compounds is their frequent presence in plants as complex mixtures of structures which increases the difficulty of establishing a unique function. In the present account of secondary metabolite functions, substances with growth regulatory properties and those involved in pollination and seed dispersal, as antifungal agents and anti-herbivore agents have been discussed. Beside this, biotechnological aspects for enhancement of secondary metabolites and assay procedures for some important commercially vibrant secondary metabolites have also been covered. We hope that the book would be useful for researchers and teachers associated with vivid biological fields. It is also hoped that the present text will improve and immense value for research workers, planners and above all farmers and individual who are eager to know biochemistry of plant products.

Plants are used for many purposes apart from food such as industrial products like perfumes, pharmaceuticals, agrochemicals, food additives, cosmetics and also for rectifying the side effects of allopathic medicines, etc. Interest was taken in the chemical constituents of plants in the very early days of modern science. Plants were used for very many purposes besides providing food, such as for treating medical ailments and for dyeing in textiles. To the agricultural chemists, any biological organism is ultimately constituted from the elements like C, H, O, N, P, Ca, Mg, S etc. Two or more elements combine to form complex organic compounds such as carbohydrates, fats, proteins, enzymes, alkaloids, glycosides, organic acids and many more complex compounds. These biomolecules can be classified into two main major metabolites. (1) Primary metabolites and (2) Secondary metabolites.

Nowhere in the plant kingdom do ecology, evolution, and human affairs come together as intricately as phenomena involving secondary metabolites. The importance of these compounds is apparent in some of the earliest annals of human history. For example, Papaver somniferum, the original source of morphine and codeine, was known to the Sumerians in 4000 B.C. as hul gil (joy-plant). These two alkaloids, so important in human affairs for many years, probably evolved within the genus Papaver originally as protective compounds against herbivores. As a testament to the importance of secondary metabolites, P. somniferum was one the first plants to be cultivated for a reason other than its caloric or nutritive value. Sadly, its importance has only increased over the time. Organic chemists of the nineteenth century were among the first to recognize and study the diversity of plant secondary metabolites and to analyze them both in terms of structure and function. The German organic chemists developed techniques to acetylate morphine and produce what they claimed at the time to be a non addictive version of the drug. However, acetylation product of morphine trade name Heroin, was vastly more addictive. The economies of several developing nations in Asia still depend on the acetylation of this plant secondary metabolite, and tens of thousands of lives each year are destroyed by its commercial importance. In another striking example of profitable secondary metabolites, acetylation techniques to salicylic acid, derived originally from the bark of willows, Salix, to create aspirin. The modern appreciation of these secondary metabolites from ecological and evolutionary perspectives had its spread outside of the plant-insect ecology community. The overwhelming array of chemicals found in plants was more than simply by-products of primary metabolism that served as waste products for these organisms that lacked excretory systems. Ecologists and evolutionary biologists soon took up the cudgels, and the past 60 years have seen the development, testing, refinement, and, in some cases, rejection of models that describe the ecology and evolution of these “secondary” substances. With the functional roles of these compounds broadly acknowledged, several challenges presented themselves to scientists interested in their ecological and evolutionary dynamics. Most obviously, the details of the processes underlying the interactions between each of these compounds and their target pest or pathogen became one of the most vibrant topics in ecological and physiological research.

Plant cells contain far more compounds than are produced by the basic metabolism. Terpenes, waxes, alkaloids and pigments are just some key words that illustrate what is meant. The term basic metabolism comprises all pathways necessary for the survival of the cells, while secondary plant products are such that occur usually only in special, differentiated cells and are not necessary for the cells themselves but may be useful for the plant as a whole. Examples are flower pigments and scents or stabilizing elements. Both metabolisms are overlapping since it is often not understood why a certain compound is produced. Furthermore, no typical cell exists so that some compounds may be necessary for the survival of some cells but not for that of others. Arguments in support of important functions for plant secondary products are underlined by the organizational complexity and specificity which characterizes many aspects of their biosynthesis. Furthermore – with the very important exception of substances produced in response to stress or pathogen attack – much of secondary metabolism is reproducible from one individual of a species to another and, to a remarkable degree, produces the same principal compounds in broadly the same relative proportions. For example, an Atropa plant extracted for alkaloids can confidently be predicted to contain predominantly atropine, rather than its immediate precursor, tropine, or a more distantly-related alkaloid, nicotine – just as the passenger boarding a train for Mumbai at Anand can be reassured that his train will not terminate prematurely at a “precursor station” (Surat), or even adopt an “alternative pathway” (to Poona). On the other hand, the evolutionary circumstances leading to the development of atropine formation in Atropa, rather than to the formation of other alkaloids with comparable activity (for example, nicotine) remain substantially unknown. A similar question has been discussed regarding two families of plant pigments with apparently similar functions, the anthocyanins and the betalains. With regard to defense (and similarly but perhaps less obviously protection against ionizing radiation), a general hypothesis argues that plants evolved a broad defensive strategy based upon a diversity of antimicrobial and anti feedant substances from which, as a result of chance mutation, more potent substances conferring additional selective advantage from time to time arose. It is conceivable that in some cases enzymes evolved or became adapted to catalyse reactions of benefit which could already occur to some extent (though in a non-stereospecific manner) non-enzymically. Mutation will then have led to the development of modified enzymes able to catalyse reactions with new or different substrates, or to catalyse alternative reactions with existing substrates.

The functional diversity of chemicals within plants is best demonstrated by terpenoids. More than 30,000 terpenoids have been identified, each one composed of 5-carbon (prenyl diphosphate) building blocks added together to make 10-carbon, 15-carbon, and even 2000–500,000-carbon chains, as is the case for rubber. Terpenes are functionally diverse, and many are integral to primary metabolism, e.g., hormones such as gibberellins and abscisic acid, electron carriers such as plastoquinone and ubiquinone, terpene-derived compounds that form structural parts of membranes such as phytosterols, and photosynthetic pigments such as carotenoids. Plant terpenes are also available as essential oil or ethereal oil. Essential oil is defined as the volatile oil obtained by the steam distillation of plants. There is a distinction between fixed or fatty oils and volatile oils. Their volatility (boiling point) and plant origin are characteristic properties of these oils.

The alkaloids represent yet another large group of secondary plant products. Many, but not all, exhibit important pharmacological properties & some of these have been known for hundred of years. For e.g. Cinchona alkaloids, present in bark of Cinchona spp. have quinine as their main constituent, has been known since 1639 as an effective antimalarial agent. An alkaloid contains nitrogen, frequently as a part of a heterocyclic ring, and most are as the general name implies, basic. Alkaloids are the naturally occurring basic, nitrogenous, organic, heterocyclic ring compounds mostly of plant origin with physiological and pharmacological properties. At present, the definition is much more pragmatic and includes all nitrogen containing natural products which are not otherwise classified as peptides, amines, non-protein amino acids, cyanogenic glycosides, glucosinolates, cofactors, phytohormones or primary metabolites (such as purines and pyrimidines bases). Even a number of antibiotics produced by bacteria or fungi are therefore included as alkaloids. Alkaloids evolved early in evolution and were already present (200 million years ago) when the angiosperms began to radiate. Today, more than 12,000 alkaloids are known to occur from more than 4000 species of Angiosperms. Ladenberg defined alkaloid as : “Naturally occurring basic, complex, nitrogenous organic, heterocyclic ring compound of plant origin with physiological and pharmacological properties.”

Glycosides are compounds containing a carbohydrate and a non carbohydrate residue in the same molecule. They are the complex polysaccharides which are chemically heterogeneous collection of substances, which have in common one property that the molecules are linked together and contain at least one sugar unit. The term glycoside represents a group of compound containing glucose and one or more basic or acidic organic compound in chemical combination. When the sugar other than glucose is present, it is called glycoside. Each sugar is linked through a glycosidic bond to either a non sugar component, aglycones, (aglycone or aglucone is the nonsugar molecule in glycosides).

The plant gums are the neutral salts and complex polysaccharide acids which contain methyl pentose, pentose, hexose and uronic acid residues joined glycosidically by linkages involving various carbon atoms within one molecule to form branched chain structure. Uronic acid is generally present in all cases. The sugars include L-fructose, D-xylose, L-arabinose, D-rhamnose, and D-galactose. General Properties of Gums They have high molecular weight. Some are linear while some are branched. They are generally viscous in nature. Some forms a gel also. They have tremendous commercial application in food industry. Gums may be an exudates gum produced by the plant or may be a seed gum, which is accumulated during the seed maturity. Gums of polysaccharide origin are obtained from Acacia spp. (gum Arabic), Cymopsis tetragonolobus (guar gum), Guggle gum, Ficus indica, chia gum etc (Table 7.1).

Plant oleoresins, often referred to as resins, are sticky plant secretions synthesized in large quantities by about 10 % of plant families. Resin is secreted when a plant is disturbed and the sticky substances produced contain many toxic compounds used to defend the plant from herbivores and pathogens. A major component of resin is the terpenoid compounds. Although terpenoid compounds can be quite complex, they are all composed of five-carbon units, with an isoprene unit as the base. The simplest of these terpenes are the monoterpenes that possess 10- carbon atoms derived from two fused isoprene units. Monoterpenes are an important component of the volatile portion of terpenes and often emit very strong odors.

The term rare amino acid is misleading in some respects. It refers to all those amino acids that are not incorporated into proteins. We already got to know some of them like ornithine or citrulline that are rather common intermediates of the basic metabolism. Roughly 220 different structures are known, most of which occur in plant cells in a free state though glutamate-, oxalate- or acetyl- derivatives can also sometimes be found.

Isolation and Purification of Secondary Metabolites Isolation The act of isolating something; setting something apart from others. Purification The process of making pure, free from anything that debases, pollutes, or contaminates, the process of removing impurities.

Role of Biotechnological Approaches In our fore-going chapters, we have studied variety of plant products such as alkaloids, glycosides, volatile or essential oils, gums and mucilage and oleoresins with varied role to play to the plants as well as to human.Knowledge of plants and knowledge of healing have been closely linked. It is well established that industries have many direct and indirect effects on human population. Increased stress is most evident at the same time health awareness goes parallel in the recent times. Most of the natural plant products are compounds biosynthetically derived from primary metabolites such as amino acids, carbohydrate metabolic intermediates and fatty acids. These secondary metabolites are the major sources of pharmaceuticals, food additives, fragrance and pesticides. Application of biotechnology know how helps to synthesize the novel molecules and also could be beneficial to enhance the production of secondary metabolites. Classical definition of biotechnology states that it is “The use of living organisms or their products to enhance our lives and our environment.” The word Pharming is commonly use to describe genetically engineered plants or livestock to produce medically useful products –predominantly protein drugs. Plants or animals are genetically engineered to contain genes capable of producing a biologic or drug compound. Therapeutic proteins are designed to treat illness includes serum proteins, cytokines, growth regulators, anti coagulants, antbiotics, lysosomal enzymes and plant secondary metabolites.

References 1.A rapid method for Determination of Gum in Guar (Cyamopsis Tetoganeloba ( L.) Taub.) . Proccedure of the First of the ICAR Guar Res. Workshop Jan. 11-12,1997, pp. 117-123. 2.Anonymous 1967. Recommended methods for the evaluation of drugs. Analyst, 92, pp. 593-96. 3.Arimura G, R Ozawa, T Shimoda, T Nishioka, W Boland, J Takabyashi Nature 406:512–515. 4.Armbruster WS, JJ Howard, TP Clausen, EM Debevec, JC Loquvam, M Matsuki, B Cerendolo, F Andel. Am Nat 149:461–484. 5.Ashby MN, PA Edwards. J Biol Chem : 265:13157–13164. 6.Ayasse M, FP Schiestl, HF Paulus, C Lofstedt, B Hansson, F Ibarra, W Francke. Evolution 54:1995–2006. 7.Back KW, J Chappell. Proc Natl Acad Sci., 93:6841–6845. 8.Baldwin IT, JC Schultz. Science 221:277–279. 9.Barkman TJ, JH Beaman, DA Gage. Phytochemistry 44:875–882. 10.Barnola, L.F., Hasega Wa, M., and A. Cedeno. Biochem. Syst. Ecol. 22: 437-445. 11.Becerra JX. Science : 276:253–256.