Introduction 1. INTRODUCTION 1. Medicinal plants – Plants have been used as a source of medicine since time immemorial. The earliest documented evidence of the use of medicinal plants in classical Indian texts like the Rigveda, Atharvaveda, Charak Samhita and Sushruta Samhita dates back to about 5000 years along with Unani, Siddha and Amchi. Ayurveda, the traditional Indian medicine and the Chinese traditional medicine remain the most ancient yet living traditions with sound philosophical, experiential and experimental basis (Patwardhan et al., 2005). Thus, the use of herbal or traditional medicines has been derived from the rich traditions of the ancient civilizations and scientific heritage (Kamboj, 2000). This ancient wisdom has now become the basis of modern medicine and an important source for the future medicine and therapeutics. A number of medicinal plants have been and are being studied for their detailed chemical investigations for isolating pure bioactive molecules of interest leading to the discovery of new drugs. Plants produce a diverse array of organic compounds, which are mainly developed for their own defense against microbes, pathogens, insects and predators. These compounds are often referred to as “natural products” or “secondary products” or “secondary metabolites”. Medicinal plants produce secondary products which are the source of fine chemical compounds like plant pigments, alkaloids, isoprenoids, terpenes, waxes, drugs, dyes, flavours, fragrances, insecticides, etc. These secondary products or “secondary metabolites” are either derived from the whole plant or from different organs like the root, stem, bark, leaves, flower, seed etc. or are obtained from the excretory plant product such as gum, resins, latex, etc. The study of such medicinally important plants is carried out for the discovery of novel secondary metabolites which are presently of great interest for their potential use as pharmaceutical drugs, health products, food supplements, nutrients, food additives and cosmetics (Ferrari, 2010; Ahmed and Kim, 2010). Plant secondary metabolites provide an incredible resource for scientific and clinical researches and for the development of new drugs. 2. Secondary Metabolites: Definition, classification and biosynthesis – Plant cells produce a broad spectrum of chemical compounds which are necessary for the basic functions, viz. biochemical pathways for survival and propagation, and are Page | 1 Introduction known as “basic or primary metabolites” which results in respiration, differentiation, assimilation and transport. “Secondary metabolism” produces diverse and relatively less essential or non-essential byproducts which are not directly involved in the growth, development or reproduction of an organism are called as secondary products or “secondary metabolites” (Ahmed and Kim, 2010). A problem of a proper definition has been discussed by many researchers, but the chemical diversity and inadequate knowledge about the role of these secondary metabolites have hindered the attempts of accurately defining this group (Verpoorte, 2000). According to Verpoorte (2000), “Secondary metabolites are compounds with a restricted occurrence in taxonomic groups that are not necessary for a cell (organism) to live, but play a role in the interaction of the cell (organism) with its environment, ensuring the survival of the organism in its ecosystem.” In 1988 the NAPRALERT database contained a record of more than 88,000 secondary metabolites, and the every year there was introduction of approximately 4,000 newly reported secondary metabolites. Taking this estimate into consideration, there should now be more than 1,00,000 secondary metabolites identified (Wink, 2015). Also, many species are studied for a specific compound. Assuming that the existing trend of discovering novel secondary metabolites will continue for the existing plants, at least a million different compounds could be isolated. Each plant has its own unique and complex pattern of secondary metabolites which can exhibit differences at different developmental stages, and/or between individuals and/or between populations as well. Secondary metabolites have been categorized in several ways. Broadly, secondary metabolites from plants are classified into three main classes: terpenoids, alkaloids and phenolics (Kabera et al., 2014). According to Lattanzio (2013), secondary metabolites can be classified into several groups according to their biosynthetic routes and structural features. Secondary metabolites have also been categorized based on chemical characteristics, plant origin or biosynthetic origin. Based on chemical characteristics, the compounds can be divided in a number of groups based on a typical characteristic, example – alkaloids, which are characterized by a basic nitrogen function, or phenolics, which are characterized by aromatic ring systems having a phenolic-hydroxyl group, or presence of a particular type of basic skeleton e.g. anthracene, coumarine, quinone, indole, isoquinoline, etc. Based on plant origin secondary metabolites can be classified, Page | 2 Introduction example – Strychnos alkaloids, Atropa alkaloids, Digitalis cardenolides, etc. this category is mostly connected with pharmaceutical applications. The categorization based on biosynthetic origin has major examples, viz. the terpenoids, phenylpropanoids and polyketides which are derived from just a few building blocks; the isopentenyl diphosphate C5-unit, the phenylalanine/tyrosine-derived C9-unit (phenylpropanoids), the acetate C2-unit (polyketides) and some amino acids (Verpoorte, 1998). The pathways of biosynthesis are responsible for the occurrence of primary and secondary metabolites. Biosynthetic reactions need energy and this energy is obtained from the energy released through the citric acid cycle by glycolysis of carbohydrates. Oxidation of glucose, fatty acids and amino acids leads to the formation of adenosine triphosphate (ATP) which is a high energy molecule formed by catabolism of primary compounds. In fuel anabolic reactions involving intermediate molecules on the pathways ATP is recycled. Catabolism involves oxidation of starting molecules, biosynthesis or anabolism involves reduction reaction. So, a reducing agent or hydrogen donor, usually nicotinamide adenine dinucleotide phosphate (NADP) is needed. These catalysts are called as coenzymes and the most widely occurring is coenzyme A (CoA) made up of adenosine diphosphate (ADP) and phosphopantetheine (Kabera et al., 2014). Thus, generally the most common pathways accepted for biosynthesis are functioned through the pentose for glycosides, polysaccharides; shikimic acid pathway for phenols, aromatic alkaloids, tannins; acetate-malonate pathway for phenols and alkaloids; and mevalonic acid pathway for terpenes, steroids and alkaloids (Dewick, 2002). 3. Phenolic compounds – Phenolics are compounds have one or more hydroxyl groups attached directly to an aromatic ring i.e. a hydroxylated benzene ring. Phenolic compounds are the most widely distributed secondary metabolites in the plant kingdom. This is one of largest group of secondary metabolites occurring abundantly with more than 8,000 phenolic structures currently known, ranging from simple molecules such as phenolic acids to highly polymerized substances such as tannins (Jain et al., 2013). Phenolic compounds are uncommon in bacteria, fungi, and algae, but have been reported from bryophytes, pteridophtyes and gymnosperms and a wide range is present in the vascular plants. It is predicted that approximately 2% of all the carbon which is photosynthesized by the Page | 3 Introduction plants is converted into flavonoids or closely related compounds. Higher plants produce thousands of phenolic compounds and the number of newly characterized and identified ones is increasing rapidly. Common examples of phenolic acids are found in our daily diet which includes citrus fruits, apples, mangoes, onions, tea, coffee, wheat, rice, corn (Lattanzio, 2013; Jain et al., 2013) and many more derivatives are found in nature which are widely distributed and are a part of our nutrition. Apart from food, phenols have most diverse applications in agriculture, pharmaceuticals, cosmetics, health products, food supplements, etc. 3.1 Classification of phenolic compounds – The term “plant phenolics” comprises a highly diverse group of chemical compounds which have been classified in a number of ways. The phenolic compounds have been categorized into groups based on the number of carbons in the molecule (on the basis of their basic skeleton) (Harborne and Simmonds, 1964) (Table 1). Table 1: Classification of phenolic compounds (Harborne and Simmonds, 1964) Structure Class C6 simple phenolics C6 – C1 phenolic acids and related compounds C6 – C2 acetophenones and phenylacetic acids cinnamic acids, cinnamyl aldehydes, cinnamyl C6 – C3 alcohols C6 – C3 coumarins, isocoumarins, and chromones C15 chalcones, aurones, dihydrochalcones C15 Flavans C15 Flavones C15 Flavanones C15 Flavanonols C15 Anthocyanidins C15 Anthocyanins C30 Biflavonyls C6–C1–C6, C6– benzophenones, xanthones, stilbenes C2–C6 Page | 4 Introduction C6, C10, C14 Quinones C18 Betacyanins Lignans, dimers or oligomers neolignans Lignin Polymers Tannins oligomers or polymers Phlobaphenes Polymers Swain and Bate-Smith (1962) used an alternative classification where they grouped the phenols into two categories: “common” and “less common”. Another classification of phenols was given by Ribéreau-Gayon (1972) where they grouped the