an erect, herbaceous, and perennial habitat, used in the formulation of gaozaban, antibacterial, antifungal, anti-inflammatory, and wound-healing drug (Manjkhola and Dhar 2002). The root contains shikonin, possesses several medicinal properties, and is available in the market with a trade name Ratan Jot (Kirtikar and Basu 1984). The species also possesses stimulant, tonic, diuretic, and expectorant properties. The flowering shoots are used in the treatment of tongue, throat, fever, and cardiac disorders. Arnebin-1 and arnebin-3 isolated from this species possess anticancer activity (Harborne and Baxter 1996).
The Arnebia species is a perennial grass, found in India, Persia, Sudan, Arabia, China, Egypt, Nubia, and Pakistan. Some common species are A. benthamii, A. euchroma, A. guttata, A. nobilis, and A. hispidissima (Anonymous 1985). Roots are recommended for the treatment of ulcers, boils, cuts, heart ailments, headache, and fever. The aqueous extract of flowering shoot is known as remedy for tongue and throat troubles and cardiac complaints, while the whole plant is used as a stimulant, tonic, diuretic, and expectorant. Likewise, the roots of A. euchroma are used in bruises and skin eruptions (Chopra et al. 1956; Kirtikar and Basu 1967; Anonymous 1976). A. hispidissima also possesses anti-inflammatory (Singh and Singh 2003; Singh et al. 2004), antimicrobial (Bhakuni et al. 1969; Shukla et al. 1969; Jain et al. 1999), antitumor (Sankawa et al. 1977; Katti et al. 1979), antiviral (Kashiwada et al. 1995), and inhibition of platelet aggregation activities (Yao et al. 1991).
Most of the naphthaquinones are found in higher plants, distributed among more than 20 families located in leaves, flowers, wood, root bark, and fruits (Thompson 1971). Alkannin, an isomer of shikonin, isolated originally from Alkanna tinctoria (Brockmann 1935) and A. hispidissima (Jain et al. 1999). The petroleum ether extract of roots later yielded a pigment as glittering red solid identified as shikonin (Jain and Mathur 1965). Shikonin has been isolated from A. euchroma (Romanova et al. 1968; Fu et al. 1984). The shikonin content in dried roots of A. euchroma was reported to be 2.47% (Zhang et al. 1989). Hexane fraction of A. nobilis roots yielded the four crystalline naphthaquinones, viz arnebin-1, arnebin-2, arnebin-3, arnebin-4 (Shukla et al. 1969), arnebin-5, arnebin-6, and arnebin-7 (Shukla et al. 1971, 1973). A. euchroma and A. guttata produce acetyl shikonin (Lin et al. 1980; Lu et al. 1983), alkannin-β, β-dimethyl acrylate, β-hydroxyisovalerate (Lin 1981; Khan et al. 1983), β-deoxyshikonin, β-dimethyl acryl shikonin, acetyl shikonin, teracryl shikonin, and β-hydroxyisovaleryl shikonin (Zhu et al. 1984). Arnebia decumbens was found to contain 5,8-dihydroxy-2,1,4-methylpentyl-13-enyl-1,4-naphthaquinones, shikonin isovalerate, and 3,6-dihydroxy-2-isovaleryl-1,2,4-benzoquinones (Afzal and Al-Oriquat 1986a,b; Salim et al. 1996). Teracryl alkannin was found to be present in roots of Arnebia densiflora (Kirimer et al. 1995). Cycloarnebin-7, tiglic acid, and others were isolated from A. hispidissima (Singh and Singh 2003; Singh et al. 2004). Arnebinol and arnebinone were isolated from the roots of A. euchroma and A. hispidissima (Eisai Co. Ltd 1983; Yao et al. 1983a,b). The A. hispidissima and A. nobilis roots' ethanolic extract provided β-sitosterol (Nigam and Mitra 1964), lupeol, betulin, and β-amyrin acetate (Sharma et al. 1972). Two triterpenic acids identified as tormentic acid and 2-α-hydroxyursolic acid have been isolated from A. euchroma (Yang et al. 1992). Several flavonoids were separated from fresh flowers of A. hispidissima (Hamdard et al. 1988). A. hispidissima gave echimidine, monocrotaline (Gamila et al. 1987), O9-angeloyl retronecine, and minor amounts of O7-angeloyl retronecine were isolated from A. euchroma (Roeder and Rengel-Meyer 1993; Srivastava et al. 1999). The presence of 7- and 9-tigloyl retronecine, supinine, heliotrine, lycopsamine, and europine was confirmed by gas-liquid chromatography (GLC) and gas chromatography–mass spectrometry (GC–MS) in A. decumbens (El-Dahmy et al. 1995).
2.12.2 Culture Conditions
Cell suspension culture studies of A. nobilis were established from friable callus Murashige and Skoog (MS) culture medium supplemented with 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA). Growth kinetic studies were conducted by adopting settled cell volume and fresh/dry cell weight method. Two culture systems were used as growth medium (MS) and production medium (modified M9 medium) for production of cell biomass and naphthaquinones. The experimental results revealed that incubation under dark period enhanced the production of cell biomass and naphthaquinones. In case of production medium, absence of Na2SO4 was not effective in increasing the production naphthaquinones but increased the level of naphthaquinones production in air-lift bioreactor (Malik et al. 2008; Gupta et al. 2014). Similarly, a Box–Behnken statistical design was established as a bioreactor, which increased the production of acetyl shikonin in A. nobilis (Subramaniam et al. 2015). Increased naphthaquinones production was reported in culture media supplemented with KH2PO4 and K2HPO4 rather than NaH2PO4. In situ extraction resulted in significantly higher yield of naphthoquinone derivatives than control, along with higher volumetric and specific productivities. High-performance liquid chromatography (HPLC) analysis revealed that acetyl shikonin synthesized as the major naphthoquinone derivative than other compounds (Kumar et al. 2011; Malik et al. 2016). The nutritional factors like as phosphate supplementation in medium was observed as one of the most important nutritional factor affecting yield of both cell biomass and naphthaquinones (Saito 1993). The M9 medium containing KH2PO4 as the phosphate source increased on yield of naphthaquinones (Fujita et al. 1981a,b).
The hairy root cultures provide an efficient method for increasing the production of shikonin in A. hispidissima. The nodal of plant material were infected with Agrobacterium rhizogenes followed for the development of hairy roots. The composition of medium was optimized for the induction of shikonin and found that RC medium showed rapid growth of cell biomass and induction of accumulation of shikonin. The production of shikonin was eightfold higher than control (Chaudhury and Pal 2010). The media combination was found to be the most suitable factor for induction of root cultures as well as shikonin production. Higher levels of IBA induced callusing from the roots. Some surprising results were also observed wherein roots were not submerged in the culture medium first, increased in length, and then started alkannin production. The synthesis alkannin occurred only in the upper surface of the roots tissue while synthesis was not reported in the inner surface in case of A. hispidissima (Bozan et al. 1999; Shekhawat 2012).
Deoxyalkannin, alkannin, acetylalkannin, isobutyryl alkannin, β-hydroxyisovalerylalkannin, 2″-(S)-α-methylbutyryl alkannin, propionyl alkannin, teracrylalkannin, and acetyl shikonin were isolated from A. euchroma in vitro cultures. Similarly, methyl jasmonate and 1-monoglyceryl oleate, palmitate and stearate were also isolated from this genus (Damianakos et al. 2012). Arnebacene and arnebidin, along with arnebin-7 and vanillic acid, were isolated from the A. hispidissima roots (Ahmed et al. 2014). The cell culture medium of A. euchroma was elicited with phenylalanine to increase the production of shikonin, acetyl shikonin, and isobutyryl shikonin. Phenylalanine is considered as a key factor of phenylpropanoid pathway and found that it induced cell proliferation to promote the production of compounds. Phenylalanine was induced the cell biomass production up to 12-fold and production of compounds up to
