Pepper (Capsicum annuum L.) is an inexpensive & one of the essential cultivating crop which belongs to the family Solanaceae. Firstly it was found in American continent mainly in it southern & central region where it was grown after 16th century or in starting of 17th century. Currently it was cultivated as a pungent fruit in all over the India. The genus Capsicum contain 24 wild and 5 domestic species C.annuum L., C.frutescens, C.chinense, C.pubescens and C.baccatum athe.e are generally grown as an spices from past 1000 years (Bosland and Votava, 2000; Wang and Bosland, 2006).
The fruits of chilli are used both as green and ripe (dried form) to impart pungency to food. The pungency, an important ascribe of peppers is due to the presence of six related compounds which constitutes the capsacinoids group capsaicin, dihydrocapsaicin , norcapsacin, nordihydrocapsicin, homocapsaicin and homodihydrocapsaicin. (Perucka and Materska 2001). Capsaicin (CAP) and dihydrocapsaicin (DHCAP) are most abundant which accounts for almost 90% of all capsaicinoids in chilli pepper fruit (Kozukue et al.
2005; Choi et al. 2006). CAP and DHCAP differ only by the saturation of acyl chain moiety (Bernal et al 1993 Walpole et al 1996 Kobata et al 1998).
Capsacinoids are synthesized and accumulated in the epidermal cells of placenta of the fruits (Bosland and Walker, 2010). Capsacinoids are synthesized via convergence of two biosynthetic pathway phenyl propanoid pathway and branched fatty acid pathway. The phenolic structure comes from the phenylpropanoid pathway, in which phenylalanine is the precursor and acyl chain is derived from branched fatty acid pathway, in which valine is the precursor (Ochoa-Alejo and Gomez-Peralta, 1993).
Capsicum has great commercial importance as it is a major component of oleoresin. It has an important role in the production of oleoresin in food processing industry to improve taste and flavor of food products (Mathew and Shakaracharya 1971). Red colour in chilli is mainly due to capsanthin and capsorubin pigment collectively known as oleoresin (Bosland and Votava, 2000). Capsaicinoids have potentially valuable pharmacological and physiological properties including analgesic, anti-cancer, anti-obesity, anti-inflammatory, and anti-oxidant. Hence, they may show potent therapeutic value in cancer prevention, cardiovascular and gastrointestinal systems, pain relief, and weight loss (Luo, X.J.; Peng, J.; Li, Y.J 2011).
Review of literature
Pepper is one of the most important constituent of the cuisines of tropical and subtropical countries and the fourth major crop cultivated globally belongs to Solanaceae family. Genus Capsicum consists of approximately 22 wild species and five domesticated (Bosland, 1994). Capsicum have chromosome number 2n = 24 , Capsicum species may be herb or sub-shrub of height up to 2.5 m with extensively branched stem having hairy growth with purplish spots near the nodes. The tap root is strong with numerous lateral roots. Flowers are generally solitary, terminal, bisexual and pentamerous with campanulate to rotate corolla. Stamens are adnate at the base of the corolla tube with blue to purplish anthers. The ovary is superior having 24 chambers. Filiform style is found with capitate stigma (Bosland, 1992, 1996).
Pertinet literature is reviewed under the following head-
Site of synthesis
Capsaicin biosynthetic pathway
Regulation of capsaisin biosynthetic pathway
Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is a natural vanilloid recognize from various chili peppers. It is found in sufficient amounts in the placental tissue (which holds the seeds) and to a lesser extent in the seeds and pericarp portions of Capsicum (Rios, M.Y.; Olivo, H.F 2014)
This naturally occurring irritant was first isolated in 1816 by Christian Friedrich Bucholz in impure form and named it capsicin (Bucholz, C.F 1816). Later, in 1876, Thresh extracted this compound in almost pure form and renamed it as capsaicin (Thresh, J.C 1876). The pure form of the compound was isolated by Micko in 1898 (Micko, K 1898). Nelson first solved its empirical formula and offered a partial chemical structure in 1919 (Nelson, E.K 1919). The original synthesis of the compound was reported in 1930 by Spath and Darling (Spath, S.; Darling, S.F 1930). Since then, several compounds related to capsaicin grouped as capsaicinoids have been isolated from Capsicum species (Kosuge, S.; Inagaki, Y.; Okumura, H 1961).
Capsaicin is a highly volatile, hydrophobic, odorless, and1colorless alkaloid with a molecular weight of 305.4 kDa and melting point of 6265 ?C. Structurally capsaicin belongs to a group of chemicals known as vanilloids. The structure of capsaicin consists1of a vanillyl (methylcatechol) head group and an aliphatic tail (hydrophobic) linked1by a central amide.
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The pungency of capsaicin is mainly due to the presence of the vanillyl moiety, which is also responsible for its adverse effects in clinical use (Rios, M.Y.; Olivo, H.F 2014). There are more than twenty capsaicinoids are identified from Capsicum species. The most predominant forms present in Capsicum are capsaicin and dihydrocapsaicin that account for 80%90% of the capsaicinoids, whereas others exist in smaller quantities. All capsaicinoids have a similar structure, varying only by the length of aliphatic side chain and degree of saturation (presence or absence of double bonds) in the alkyl side chain region (Luo, X.J.; Peng, J.; Li, Y.J 2011).
The exact percentage of capsaicinoids varies depending on the pepper variety and extraction method. The content of capsaicinoids in naturally occurring spices range from 0.1 mg/g in chili pepper to 2.5 mg/g in red pepper and 60 mg/g in oleoresin red pepper (ONeill, J.; Brock, C.; Olesen, A.E.; Andresen, T.; Nilsson, M.; Dickenson, A.H. 2012) (Al Othman, Z.A.; Ahmed, Y.B.; Habila, M.A.; Ghafar, A.A 2011).
The capsaicin content of different peppers determined by liquid chromatography techniques range from 0.1 to 4.25 mg/g of pepper. Other pepper varieties such as Capsicum frutescens, Capsicum annuum, and Capsicum chinense were found to contain 0.22 to 20 mg of total capsaicinoids/g of pepper (dry weight) (Thomas, B.V.; Schreiber, A.A.; Weisskopf, C.P 1998).
Site of synthesis
Earlier the main site for the synthesis and accumulation of capsaicin was suggested to be placenta in annully grown Capsicun annum by Iwai et al (1979) which was latter on supported by Holden et al (1987) who showed that capsaicin is synthesized only in fruits of perennial Capsicum frutescens and not in any other part of the plant. Recent studies showed that capsaicin is mostly located in vesicles or vacuole like sub cellular organelles of epidermal cells of placenta in the pod (Cheema and Pant 2011). The highest concentration of capsaicin is found in the ovary in the lower flesh (tip) and lower concentration can be found in seeds (Supalkova et al 2007).
Capsaicin Biosynthetic Pathway
Capsaicinoids biosynthetic pathway was studied in terms of organic synthesis and biochemistry using a radiotracer technique at the end of 1960s. It has been propound that capsaicinoids are synthesized in the placenta of chilli fruits by the enzymatic condensation of vanillylamine with C9 to C11 isotype branched-chain fatty acids (Bennett and Kirby, 1968; Iwai et al., 1979; Leete and Louden, 1968; Suzuki et al., 1981). The vanillylamine is procure from the phenylpropanoid pathway and the formation of the methyl-branched, saturated, or trans-monounsaturated fatty acids proceeds from valine and leucine (Suzuki, T.; Iwai 1984).
Capsaicinoids are synthesized through the convergence of two biosynthetic pathway
Phenylpropaoid pathway ii) branched fatty acid biosynthetic pathway (Ochoa-Alejo and Gomez-Peralta, 1993)
Several authors established the participation of phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), coumarate 3-hydroxylase (C3H) and caffeic acid O-methyltransferase (COMT) in phenylpropanoid-mediated capsaicinoid biosynthesis (Fujiwake et al. 1982a, b; Sukrasno and Yeoman 1993). Recently, Stewart et al. (2005) and Mazourek et al. (2009) have propound the participation of some other enzymes, such as 4-coumaroyl-CoA ligase (4CL), hydroxycinnamoyl transferase (HCT), caffeoylCoA O-methyltransferase (CCoAOMT; instead of COMT) and hydroxycinnamoyl-CoA hydratase/lyase (HCHL), in the phenylpropanoid pathway that lead to capsaicinoid formation based on different experimental sources (see for example Gasson et al. 1998; Hoffmann et al. 2003; Merali et al. 2007).
Capasaicin synthase catalysis the formation of capsaicin by condensing vannilylamine and medium chain length fatty acids. The condensing enzyme capsaicinoid synthase (CS) acts specifically on medium chain length fatty acids requiring Mg2+, ATP, and coenzyme A (CoA). CS operates with a 6.5 fold higher speed on the acyl-CoA derivative in comparison to free fatty acids (Perry, L.; Dickau, R.; Zarrillo, S.; Holst, I.; Pearsall, D. M.; Piperno, D. R.; Berman, M. J.; Cooke, R. G.; Rademaker, K.; Ranere, A. J.; Raymond, J. S.; Sandweiss, D. H.; Scaramelli, F.; Tarble, K.; Zeidler, J. A. 2007).
Pepper (Capsicum annuum L.) is an economically and agriculturally important crop that belongs to the Solanaceae family. It was introduced from South and Central America where it was raise around seventeenth century and now it was grown in all parts of India for its pungent fruits. The genus Capsicum comprise of 24 wild and 5 domesticated species Capsicum annuum L., C. frutescens, C. chinense, C. pubescens and C. baccatum which are cultivated basically for use as an spice from thousands of year. (Bosland and Votava, 2000; Wang and Bosland, 2006).
Phenylalanine ammonia lyase (PAL) EC 188.8.131.52 Conversion of phenylalanine to cinnamate
Cinnamate-4-hydroxylase (C4H) EC 184.108.40.206 Conversion of cinnamate to coumarate4 Coumaroyl-CoA ligase( 4CL) EC 220.127.116.11 Conversion of coumarate to 4-coumaroyl- CoA
Hydroxy cinnamoyl transferase (HCT) Converion of 4-coumaroyl-CoA to caffeoyl CoA
Coumaroyl shikimate/quinate3-hydroxylase ( C3H) EC 18.104.22.168 Conversion of
caffeoyl-CoA-3-O-methyltransferase (CCoAOMT) EC 22.214.171.124 Conversion of caffeoyl CoA to feruloyl-CoA
Caffeic acid O-methyltransferase (COMT) EC 126.96.36.199 Conversion of
hydratase/lyase (HCHL) EC 188.8.131.52 Conversion of feruloyl-CoA to vanillin
Putative Aminotransferase pAMT EC Conversion of vanillin to vanillylamineBranched-chain amino acid
transferase (BCAT) EC 184.108.40.206 Conversion of valine to ?ketoisovalerateketoacyl-ACP synthase ( KAS) EC 220.127.116.11 acyl carrier protein (ACL) EC acyl-ACP thioesterase (FAT) EC 18.104.22.168 Conversion of 8-methyl-6-nonenoic acid
Acyl-CoA synthetase (ACS) EC Conversion of 8-methyl-6-nonenoic acid to 8-methyl-6-nonenoyl CoA
Capsaicin synthase (CS) EC Condensation of vanillylamine and 8-methyl-6-nonenoyl CoA
Regulation of Capsaicin biosynthetic pathway
Capsaicinoids (CAP) are nitrogenous metabolite synthesized in the placentas of peppers after 20-30 days of flowering. The ability of pepper to synthesize capsaicinoids is interconnected with the presence of putative acyltransferase encoded by (AT3) gene namely pungent gene 1 (Pun1). Recently, capsaicinoid synthase (CS) activity has been attribute to AT3 (Ogawasa et al. 2015). However both environmental and developmental factors affect CAP accumulation (Sukrasno, N 1993,Contreras-Padilla, M. 1998 ). In this way both genetic factor and plants interaction with the environment attribute to variations in CAP contents.
Non-pungent peppers carry a non-functional allele, that lacks a 2.5 Kb region at the 5 end, spanning the putative promoter and first exon of AT3. (Stewart, C. 2007).
Induction of capsaicinoid accumulation by the addition of SA and MeJa in in vitro cultured placentas require primary nitrogen assimilation, through the activation of glutamine synthetase/glutamine synthase (GS/GOGAT) cycle (Ancona-Escalante, W.R 2013). Being a nitrogenous metabolite nitrogen availability regulates the formation of capsaicinoids (Johnson, C.D.; Decoteau, D.R 1996). An increase in the concentration of nitrate in the placental tissue promoted the acuumulation of capsaicinoids. (Monforte-Gonz?lez, M. 2010) (Aldana-Iuit, J.G.2015).
Capsaicinoids synthesis occur from two amino acids phenylalanine and valine from which phenolic and acyl moieties are derived. Accumulation of capsaicinoids increases in the placenta with the increase of amino acids phenylalanine and valine. Arogenate dehydratase (ADT) and acetolactae synthase (ALS) are the key enzyme involved in the synthesis of phenylalanine and valine. Both of these enzymes play crucial role in the synthesis of each amino acid and were chosen as markers for the functionality of the corresponding pathways (Fray M. Baas-Espinola 2016)
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51054002686051847850211455 Anthranilate Prephenate ?-Acetolactate
1914525339725510540023495017811752349501266825225425 Arogenate ? ketoisovalerate199072532385 ADT
50958752203452371725267970 Tyrosine Phenylalanine Valine231457522478900509587523431500 Vanillylamine 8-Methyl-6-nonenoyl CoA
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