of Nordihydrocapsaicin and Feruloyl-β-D-glucopyranoside."/> 2.20.2 Culture Conditions
Capsicum frutescens cell cultures were capable of producing a maximum of 53 μg capsaicin/g fresh weight exposed to various levels of p-fluorophenylalanine at 100, 400, 1000, and 2000 μM to develop a resistant cell line that over produces capsaicin. Capsaicin accumulation was inhibited in 2000 μM p-fluorophenylalanine-resistant cell line. The profile of phenylalanine ammonia lyase, the key enzyme in phenylpropanoid pathway in resistant cell cultures, was studied and compared with normal cell cultures to understand its role in capsaicin formation. The activity profile of phenylalanine ammonia lyase had no correlation with capsaicin content in both control and p-fluorophenylalanine-resistant cells (Johnson et al. 1998).
Administration of the calcium ionophore A23187 resulted in a 1.43-fold enhancement of the total capsaicin production in the cell suspension cultures. Treatments wherein the calcium channel modulators verapamil and chlorpromazine were administered resulted in lower growth and capsaicin production, suggesting that calcium is involved in the signal transduction of capsaicin pathway in the suspension culture (Lindsey 1985). The activity of Ca2+ ATPase, which is a calmodulin-related enzyme, was also studied under the influence of the calcium channel modulators. Maximum activity of Ca2+ ATPase was observed preceding the capsaicin elicitation. The activity of capsaicin synthase correlated with the increased capsaicin levels under the influence of calcium ionophore (Sudha and Ravishankar 2002). Phycocyanin – a natural blue pigment that is the major light-harvesting biliprotein in the blue–green alga Spirulina platensis – was used as an elicitor to enhance the accumulation of capsaicin and anthocyanin in C. frutescens cell cultures. Phycocyanin at 0.3, 0.6, and 1.2 mg% in Capsicum cell cultures elicited a more than twofold increase in capsaicin content with maximum productivity of 192 μg/g fresh weight (Ramachandra Rao et al. 1996). Capsicum fruits synthesize and accumulate a group of analogues known as capsaicinoids in the placenta tissues. Capsaicinoids are of economic importance because of their use in the food, cosmetic, military, and pharmaceutical industries. In vitro cultures of chili pepper were established, precursors and intermediates were added to the culture medium, and cell lines has been selected as a means to increase the production of capsaicinoids as well as the extraction, separation, and quantification of capsaicinoids from chili pepper cell cultures (Ochoa-Alejo 2006).
Biotransformations of capsaicinoids such as capsaicin and 8-nordihydrocapsaicin and phenylpropanoids such as cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid have been investigated using cultured plant cells (Johnson et al. 1996). Capsain and 8-nordihydrocapsaicin were converted into the corresponding glycosides, which are three glycosides respectively using the cultured cells of Catharanthus roseus. Finally, it was found that the cultured plant cells have the ability to glycosylate the phenolic group of capsaicinoids and phenylpropanoids region selectively (Katsuragi et al. 2011). C. frutescens suspension cultures produced low levels of capsaicin, but in immobilized cells with reticulated polyurethane foam increased the production 100-fold approximately (Lindsey and Yeoman 1984). Further yield was improved by supplying precursors (isocapric acid) in cell cultures of C. frutescens (Lindsey and Yeoman 1984). Lindsey (1985) reported that treatments that suppress cell growth and primary metabolism seem to improve capsaicin synthesis in C. frutescens cells (Lindsey et al. 1983). Elicitation of capsaicin in cell cultures of C. frutescens by spores of Gliocladium deliquescens has been reported (Holden et al. 1988). The effects of nutritional stress on capsaicin production in immobilized cell cultures of C. annuum were thoroughly studied (Ravishankar et al. 1988). Biotransformation of externally fed caffeic acid to capsaicin in freely suspended cell cultures of C. frutescens has also been studied (Ramachandra Rao and Ravishankar 2000).
Capsaicin, an alkaloid, is used mainly as a pungent food additive in formulated foods (Ravishankar et al. 2003). Capsaicin is also used in pharmaceutical preparations as a digestive stimulant and for rheumatic disorders (Sharma et al. 2008). Suspension cultures of C. frutescens produce low levels of capsaicin, but immobilizing the cells in reticulated polyurethane foam can increase production approximately 100-fold (Johnson and Ravishankar 1998).
In callus cultures derived from pericarp and seedling explants of four varieties of C. annuum, viz Punjab Surkh, Punjab Gucchedar, and sweet chili in Punjab Lal, the capsaicin content in seedling-derived callus culture was comparable to that in fruits. Capsaicin content in pericarp-derived callus cultures of all the varieties was much higher than that in seedling-derived callus cultures and in fruits (Varindra et al. 2000). Since the placental callus fresh weight was very high when compared to the stem explant, leaf explant, and pericarp explant, it was further taken for capsaicin estimation. The amount of capsaicin in the placental extract was found to be 1.6 mg/g fresh weight of the callus. The salicylic acid and methyl jasmonate individually enhance capsaicin production, but when administered in combination, there was no further enhancement in capsaicin production in cell suspension cultures of C. frutescence (Sudha and Ravishankar 2003).
Seeds of C. annuum were decontaminated and placed in a culture bottle containing an MS medium, supplemented with BAP and NAA or IAA, and then were incubated in the dark for 10–12 days for germination. Leaf explants excised from four-week-old aseptic seedlings were cultured on an MS medium supplemented with hormones BAP, kinetin (Kin), and the combination of BAP + Kin, BAP with NAA, and BAP with IAA. The BAP with NAA media was observed to be more suitable for callus formation. The highest number of regenerated shoot buds was obtained when shoot explants were cultured on an MS medium supplemented with BAP and IAA (Swamy et al. 2014). Capsaicinoids are acid amides of C9–C11 branched-chain fatty acids and vanillylamine. Moreover, it has been suggested that these compounds play an ecological role in seed dispersal. Recent progress has been made on the biosynthetic pathway, and several of the genes coding for biosynthetic enzymes have been cloned and expression studies performed. With regard to catabolism, cumulative evidence supports that capsaicinoids are oxidized in the pepper by peroxidases. Peroxidases are efficient in catalyzing in vitro oxidation of both capsaicin and dihydrocapsaicin. These enzymes are mainly located in placental and the outermost epidermal cell layers of pepper fruits, as occurs with capsaicinoids and some peroxidases are present in the organelle
