LIST OF UNITS
Scheme 1.2: Methods to produce haploids under in vitro conditions
1.6. SECONDARY METABOLITES
Plants and their derivatives form important part of our everyday diet, and as such nutritional value of these have been extensively studied for decades. In addition to essential primary metabolites (e.g. carbohydrates, lipids and amino acids), higher plants also synthesize a wide variety of organic compounds collectively termed as ‘secondary metabolites’. These secondary compounds do not participate in vital metabolic functions of plants (eg. photosynthesis, respiration, protein and lipid biosynthesis, growth and reproduction) but they act primarily as defense molecules that facilitate plant to adapt to new environment. Plant secondary metabolites are biosynthesized from primary metabolites by specific genetically controlled, enzymatically catalyzed reactions that lead to the formation of complex compounds. Higher plants are a major source of natural products, like pharmaceuticals, agrochemicals, flavor and fragrances, food additives, and pesticides (Balandrin and Klocke, 1988). The search for new plant-derived chemicals should, thus, be a priority in current and future efforts toward sustainable conservation and rational utilization of biodiversity (Phillipson, 1990). However, it is becoming increasingly clear that secondary metabolites may play important roles in plant signaling and defense mechanisms (which are shown in Scheme 1.3) (Wink, 2010). In addition, the plants constitute important UV absorbing compounds, thus, preventing serious leaf damages from the light (Li et al. 1993). These secondary metabolites are explained as being antioxidant, antibiotic, antifungal, antiviral and, therefore, are able to protect plants from pathogens (phytoalexins). They are also anti-germinative or toxic to other plants (allelopathy). The plant metabolites act on insects as anti-feeders or even cattle for which forage grasses can express estrogenic properties and interact with fertility (Bourgaud et al. 2001). Due to their diverse biological activities, plant secondary metabolites have been used for centuries in traditional medicine. Based on their biosynthetic pathways
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these compounds are usually classified into three large families: 1. Phenolics, 2. Terpenes and Steroids, and 3. Alkaloids (Harborne 1999).
Among the three classes, phenolics are the largest group of secondary metabolites present in tea. The composition of the tea leaves depends on several factors, such as climate, season, horticulture practices, type, age and physiology of tea plant (Bansal et al., 2012).
The tea leaves contain more than 700 chemical constituents (Mondal et al., 2004). The plucked tea shoots consist of two to three tender leaves and an apical bud which contains 10-35% (dry weight basis) polyphenolic compounds (Balentine et al., 1997; Chaturvedula and Prakash, 2011; Sabhapondit et al., 2011; Bansal et al., 2012). Flavonoids are plant secondary metabolites widely distributed in the plant kingdom, and can be subdivided into six classes: flavones, flavanones, isoflavones, flavonols, flavanols, and anthocyanins, based on the structure and conformation of the heterocyclic oxygen ring (C ring) of the basic molecule (Scheme 1.4). The main classes of flavonoids found in tea are flavanols and flavonols (Balentine et al., 1997; Wang et al., 2000). Flavonoids are basically C15 units consisting of two benzene rings (A and B) connected by three carbon chain. This chain is closed to most flavonoids to form the heterocyclic ring (C). The natural flavonoids are divided into classes based generally on the oxidation state of their C-ring (Stafford, 1990). The major flavonoids present in tea are chatechins. These eight
naturally occurring catechins present in tea are (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (CG), (-)-gallocatechin gallate (GCG), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin
gallate (EGCG) (Scheme 1.4). Maximum percent of catechins in tea are (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate
(ECG), (-)-epicatechin (EC) and (+)-catechin (C) (Balentine et al., 1997; Liang, et al., 2006; Wei et al., 2011). These are colorless, water soluble compounds that contribute the bitterness and astringency to green tea (Balentine et al., 1997). Catechins are considered to be synthesised through phenylpropanoid and flavonoid biosynthetic pathway. The formation of dihydroquercetin and dihydromyricetin, which are the precursors of dihydroxylated catechins (EC and ECG) and trihydroxylated catechins (EGC and EGCG), respectively, is genetically controlled (Gerats & Martin, 1992; Wei et al., 2011).
EGCG is most abundant (50 – 80% of total catechin) of all catechins in green tea (Bansal
et al, 2012). Almost all of the characteristics of manufactured tea, including its taste, color, and aroma, are linked directly or indirectly with modifications to the catechins (Wang et al., 2000). Moreover, it was found that the total catechin of tea leaves increased with exposure to sunlight, suggesting that catechin biosynthesis is also environmentally dependent (Mariya et al., 2003; Wei et al., 2011). It has been published that Assam type cultivars contain higher amount of polyphenols (Bhuyan et al., 2009; Sabhapondit et al., 2012). China variety cultivars generally have quercetin and kaemferol-3-glucosides but these are totally absent or are present only in negligible amount in Assam variety (Hazarika and Mahanta, 1984; Sabhapondit et al., 2012). Flavonols are mainly present as glycosides rather than as their non-glycosylated forms (aglycones). At least 14 glycosides of myricetin, quercetin and kaempferol in fresh tea shoots, green and black teas have been reported (Engelhardt et al., 1992; Wang et al., 2000). Flavonol glycosides are present in tea upto 2 to 3 % of the water soluble extracts (Balentine et al., 1997). Tea leaf contains 2.5 to 4 % of 1,3,7-trimethylxanthine (caffeine) (dry weight basis) and much less quantity of related methylxanthine theobromine (3,7-dimethylxanthine). Theophylline also has been reported as a tea constituent. Theanine is the characteristic and main amino acid in tea (Peng et al., 2008).
In search of alternatives to production of desirable medicinal compounds from plants, biotechnological approaches, specifically, plant tissue culture, is found to have a promising potential as a supplement to traditional agriculture (Rao and Ravishankar, 2002). The explorations of production of secondary metabolites in tea were started from 1821, when caffeine was first prepared in pure form from tea leaves (Spedding and Wilson, 1964). Ogutuga and Northcote (1970a) reported production of caffeine from callus tissue. As high as 30% catechins have been produced from cell culture of tea (Hao et al., 1994). First report on the formation of polyphenols in tea plant as well as in callus tissue was reported by Forrest (1969). He described that the production of simplest catechin and leuco-anthocynin was highly dependent on the original explants and inversely co-related with growth rate of cultured cells of tea. Zaprometov and Zagoskina (1979) reported that callus on medium supplemented with NAA had more soluble phenols, flavans as well as phenolic polymer lignin compared to medium with 2,4-D.
They also reported in 1987, that abscisic acid inhibits the cell growth and production
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Scheme 1.4: Structure of eight catechins and three alkaloids from tea: (+) catechin (C); (-)-epicatechin (EC); (-)-gallocatechin (GC); (-)-epigallocatechin (EGC); (-)-catechin gallate (CG); (-)-epicatechin gallate (ECG); (-)-gallocatechin gallate (GCG); (-)- epigallocatechin gallate (EGCG).
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