Tikkam Singh

Industrial Crops & Products 151 - Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop Isolation and optimization of plumbagin production in root callus of Plumbago zeylanica L. augmented with chitosan and yeast extract T Tikkam Singh, Upasana Sharma, Veena Agrawal* Department of Botany, University of Delhi, Delhi, 110007, India ARTICLE INFO ABSTRACT Keywords: Plumbago zeylanica Roots Isolation Elicitation Plumbagin Chitrak (Plumbago zeylanica L.), a renowned traditional medicinal plant, is being exploited extensively for its roots which are employed in the preparations of many important herbal products (e.g. Dashmularisht, Chitrakadivati) possessing anticancer, anti-atherogenic, cardiotonic, hepatoprotective and neuroprotective properties. P. zeylanica roots, being the major source of plumbagin, were used for its isolation. Plumbagin isolation from in vivo roots was done employing column chromatography and thin layer chromatography (TLC) using a hexane: ethyl acetate (70:30) mobile phase. A total of 65 fractions were obtained which pooled down to 8 (F1–F8) based on their similar Rf values. Of the 8 fractions, F2 gave a single spot corresponding to that of the standard plumbagin which subsequently was validated and confirmed through 1H-NMR and FT-IR. For elicitation to increase plumbagin, in vitro root callus initially raised on Murashige and Skoog medium +5 μM TDZ was augmented with different concentrations of precursors (sodium acetate, L-tyrosine), biotic (chitosan, proline, lysine, salicylic acid, yeast extract) and abiotic [Pb(NO3)2, CdCl2] elicitors either alone or in combinations. The optimum increase of up to 2.07 and 2.64–folds in plumbagin was seen when callus cultures were fed individually with sodium acetate (1 mg/L) and L-tyrosine (25 mg/L), respectively. Similarly, chitosan and yeast extract when used alone, the plumbagin content was enhanced 4.58–fold at 50 mg/L and 6.50–fold at 100 mg/L, respectively. However, an enormous increase of 12.08–fold plumbagin content occurred when a combination of 50 mg/L chitosan + 100 mg/L yeast extract was used. A combination of salicylic acid (25 μM) and yeast extract (100 mg/ L) also enhanced plumbagin content up to 8.23–fold. Thus, this is our first report of scaling up to 12.08–fold plumbagin content using simple and cost effective elicitors. 1. Introduction Plumbago zeylanica L., commonly known as Chitrak, is a medicinal plant extensively utilized in complementary and alternative medicine around the world including India (Jaiswal et al., 2018). The aerial parts of plant have been used to cure various ailments such as rheumatic pain, scabies, sprains, skin diseases and wounds. Also, P. zeylanica is being exploited extensively for its roots employed in the preparations of many important herbal products (e.g., Dashmularisht, Chitrakadivati) possessing anticancer, anti-atherogenic, cardiotonic, hepatoprotective and neuroprotective properties (Kumar et al., 2009; Son et al., 2010; Sundari et al., 2017). The main bioactive compounds occurring in P. zeylanica are plumbagin, plumbagic acid, zeylanone, chitranone, betasitosterol and elliptinone (Kishore et al., 2012). These bioactive compounds including plumbagin are synthesized and accumulated mainly in the roots. Plumbagin, a 5-hydroxy-2-methyl-1, 4-naphthoquinone, is a promising drug with a diverse pharmacological properties including ⁎ strong anticancer activity. There are some studies pertaining to anticancer activity of plumbagin against various human cancer cell lines including esophagus (Cao et al., 2018), prostrate (Chrastina et al., 2018), leukemia (Fu et al., 2016), cervical (Jaiswal et al., 2018), brain (Khaw et al., 2015), liver (Li et al., 2019), lung (Xu et al., 2013) and breast (Dandawate et al., 2012). Plumbagin also exhibited activity against cancer stem cells such as breast cancer stem cells and prostate cancer stem-like cells (PC-3 and DU145 cells) (Somasundaram et al., 2016; Reshma et al., 2016). These studies showed that plumbagin inhibited malignant activity of human cancer cells via numerous mechanisms such as growth inhibition, caused apoptosis, cell cycle arrest, antiangiogenesis, etc. In addition, there are also a few reports regarding antibacterial activity of plumbagin and root extract of Plumbago sp. against different bacterial strains (Jeyachandran et al., 2009; Kaewbumrung and Panichayupakaranant, 2014; Nair et al., 2016). Such reports put this important therapeutic biomolecule in high demand, and in order to reach the needs of the pharmaceutical, Corresponding author at: Medicinal Plant Biotechnology and Applied Research Laboratory, Department of Botany, University of Delhi, Delhi 110007, India. E-mail address:-(V. Agrawal). https://doi.org/10.1016/j.indcrop- Received 10 December 2019; Received in revised form 3 April 2020; Accepted 5 April-/ © 2020 Elsevier B.V. All rights reserved. Industrial Crops & Products 151 - T. Singh, et al. nutraceutical industries (Sinlikhitkul et al., 2019) and folk medicine (Poosarla et al., 2011), the production of this valuable bioactive compound needs to be increased using in vitro elicitation and precursors feeding techniques. Medicinal plants produce a large number of bioactive compounds in response to biotic or abiotic stress not only involved in the normal growth, development or reproduction but also have a crucial role in plant defense mechanism. In recent times, these bioactive compounds acquired utmost interest to pharmaceutical industries for drug designing and many other therapeutic uses (Chetri et al., 2016). However, the synthesis of these therapeutic bioactive molecules is site-specific for a particular plant, depends on growth and developmental stages, environmental conditions and availability of micro- and macronutrients (Ramirez-Estrada et al., 2016). Such conditions may also contribute towards poor quality and quantity of the bioactive compounds and may also change the medicinal property of the active constituent. Therefore, an alternative, sustainable and eco-friendly technique must be employed for the production of plumbagin which is not affected by environmental conditions. Plant cell and organ cultures are being currently employed as an alternative system for the production of valuable bioactive compounds (Ramirez-Estrada et al., 2016). Plant organ induced callus tissues are considered as one of the best source materials, for recurrent and rapid proliferation of cells for the production and enhancement of bioactive compounds (Mulabagal and Tsay, 2004; Ramirez-Estrada et al., 2016). Although different approaches are available for the enhancement of bioactive compounds from cells, organs and plant systems, elicitation and precursors feeding are two viable strategies presently being used (Qu et al., 2011; Naik and AlKhayri, 2016). Precursor feeding is one of the techniques employed to improve the production of secondary metabolites in plant cell and organ cultures. In precursor feeding, precursors of the target bioactive compounds are added to the growth medium which is absorbed by the plant cell and organ cultures. However, these precursors may also induce stress in plant cell and organ cultures while they enhance the quantity of secondary metabolites (Qu et al., 2011). Similarly, elicitors may be biotic, abiotic or signaling molecules that trigger the production of bioactive compounds in in vivo or in vitro raised cultures (Baenas et al., 2014). Elicitors act as ligand/ secondary messengers which bind to the receptors on the plasma membrane and consequently trigger the signal cascade to influence the physiological and biochemical changes in a plant. The activation of the intracellular transduction system, NADPH cascade, production of reactive oxygen species, expression of defense related genes, GTP binding proteins, high intracellular cAMP and Ca2+, and other secondary messengers with mitogen-activated protein kinases, are the responses of elicitors which may induce secondary metabolites production (Vasconsuelo and Boland, 2007; Ferrari, 2010; Goel et al., 2011; Zhang et al., 2012). The present study focused on isolation, characterization and elicitation of potential therapeutic agents from root callus of P. zeylanica. The fractions were collected through column chromatography, identified and characterized through 1H-NMR and FT-IR. In addition, in vitro elicitation of plumbagin has been achieved using precursors feeding (Ltyrosine and sodium acetate) and supplementing exogenous elicitors such as chitosan, yeast extract, salicylic acid, amino acids (proline and lysine) and heavy metals [Pb(NO3)2 and CdCl2] employed either alone or in combinations in root callus cultures of P. zeylanica. cut into small pieces, washed vigorously under running tap water and then dried at room temperature (25 ± 2 °C) for 7 days. Further, these roots were ground into fine powder and then extracted with methanol (99.0 %, Methanol LR, SDFCL, Mumbai, India) (Fig. 1C, D). The mixture of solvent and root powder was kept on an incubator shaker with continuous stirring for 48 h at 20 ± 2 °C in the dark and then filtered with Whatman No.1 filter paper (Whatman™, GE Healthcare UK Limited, Amersham Place, UK). Finally, the filtrate was evaporated to concentrate and stored in an amber coated bottle at 4 °C. 2.2. Isolation of fractions from root extract 2.2.1. Thin layer chromatography Selection of suitable solvent system and separation of bioactive compounds present in methanolic root extract was done using thin layer chromatography (TLC). The saturation chambers (Borosil®, Mumbai, India), were filled with different solvents such as chloroform: methanol (80: 20; v: v), hexane: chloroform (75: 25; v: v), chloroform: toluene (60: 40; v: v), toluene: ethyl acetate (70: 30; v: v), hexane: ethyl acetate (70: 30; v: v), and chloroform: ethyl acetate: methanol (65: 25: 10; v: v: v). The saturation chambers were covered and kept undisturbed for 10 min to get fully saturated with solvent vapours. Silica Gel 60 F254 precoated aluminum TLC plates (Merck, Mumbai, India) were used as a stationary phase. The crude root extract was loaded on the TLC plates (10 × 2 cm) and kept in the jars containing solvents. To visualize resolution, the plates were placed into a closed glass chamber containing iodine vapours and the Rf values calculated using the formula given by Touchstone (1992). 2.2.2. Column chromatography Isolation of bioactive compounds from the in vivo root extract was done through column chromatography using the mobile phase selected on the basis of resolution of compounds on TLC plates. For isolation of compounds, 5 g of root crude extract was mixed with 8 g of dry silica gel (100–200 mesh) to prepare the slurry. An amber coated glass column (100 × 3 cm) was filled carefully with silica gel powder and was then loaded with the root crude extract using 100 % hexane. Subsequently, the compounds of the root extract were isolated by using a gradient of 0, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 % ethyl acetate in hexane as mobile phase. The elution rate of the fractions through the column was kept at 0.20 mL/s. Afterward, these isolated fractions were analyzed through TLC, using hexane: ethyl acetate (70: 30; v: v) as the mobile phase. The isolated fractions containing compounds having similar Rf values were pooled together. However, the fractions having a single spot on TLC plates showing similar Rf values were also pooled together and subjected to further characterization and identification. 2.2.3. Characterization of isolated compounds 2.2.3.1. Fourier transform infrared spectroscopy (FT-IR) analysis. For Infrared (IR) spectra of isolated compound, 1 mg crystals were dissolved in 1 mL of methanol and a drop was made on the KBr plate. Absorption peaks of functional groups were recorded using Fourier transform spectrometry instrument (Perkin Elmer, Spectrum RXI, resolution 400 cm−1, detector LiTaO3). 2.2.3.2. Nuclear magnetic resonance spectroscopy (1H-NMR). 1H-nuclear magnetic resonance (NMR) spectrum of the isolated and purified compound was observed in methanol-d4 using NMR (Bruker Spectrospec), DPX-300 MHz, where tetramethylsilane was the internal standard. 2. Material and methods 2.1. Preparation of root extract of Plumbago zeylanica For the preparation of in vivo root extract, roots of P. zeylanica (Fig.1 A, B) were collected from the Hamirpur, Himachal Pradesh, India (76.52 °E, 31.68 °N; 746 m) and a herbarium specimen submitted to Herbarium of Delhi University, Delhi, India (DUH14245). Roots were 2.3. Elicitation of plumbagin in root callus cultures 2.3.1. Production of root callus Nodal explants were excised from plants collected from the 2 Industrial Crops & Products 151 - T. Singh, et al. Fig. 1. A-G. Isolation of bioactive compounds from methanolic extract of roots of Plumbago zeylanica. (A) A twig of P. zeylanica bearing flowers; (B) uprooted plant with roots (Inset: dried roots); (C) dried root powder; (D) methanolic root extract; (E) column chromatography; (F) isolated fractions (F1–F8) obtained through column chromatography; and (G) TLC of standard compound plumbagin (s) and isolated fractions (F1-F8); (H) multiple spots of root extract on TLC plate using hexane: ethyl acetate (70: 30; v: v) as mobile phase. Red arrows indicate single spot of F2 corresponding to the standard compound plumbagin (s). Hamirpur, Himachal Pradesh, India and sterilized and cultured using the established protocol (Sharma and Agrawal, 2018). Roots induced on the nodal explants were used for callus induction (Fig. 3A). The root callus was achieved on MS (Murashige and Skoog, 1962) medium supplemented with 5 μM thidiazuron (TDZ) for 45-d (Fig. 3B) and was maintained by sub-culturing at 30-d intervals using the same medium 3 Industrial Crops & Products 151 - T. Singh, et al. as that used for callus induction (Fig. 3C) prior to conducting experiments. chemicals used were of HPLC grade. 2.3.2. Preparation of precursors and elicitors Precursors of plumbagin such as L-tyrosine (HiMedia Laboratories Pvt. Ltd., Mumbai, India) and sodium acetate (Merck Specialities Private Limited, Mumbai, India) and elicitors: chitosan (Medium molecular weight; 90 % Deacetylation degree; Sisco Research Laboratories Pvt. Ltd., Mumbai, India), yeast extract (HiMedia Laboratories Pvt. Ltd., Mumbai, India), proline (Sigma Chemical Co., USA), lysine (TM Media, India), salicylic acid (Sisco Research Laboratories Pvt. Ltd., Mumbai, India), lead nitrate [Pb(NO3)2] (Ranbaxy Laboratories Limited, India) and cadmium chloride (CdCl2) (Qualigens Fine Chemicals, Mumbai, India) were employed in the current investigation for increased production of in vitro plumbagin. Chitosan was initially dissolved in a minimum volume of 1 N HCl then made up to final concentration with sterile distilled water. In contrast, salicylic acid was initially dissolved in a minimum volume of 1 N NaOH and then sterile distilled water was added to make up final stock solution. Salicylic acid and L-tyrosine were filter sterilized using 0.2–μm microfilter (Axiva Sichem Pvt. Ltd., India) and then added to culture medium prior to autoclaving. However, yeast extract, proline, lysine, sodium acetate, lead nitrate and cadmium chloride were dissolved in sterile distilled water only. 2.4. Data analysis and statistics Data was represented as mean ± SE and each experiment consisted of twelve replicates was performed in triplicates. Statistical analysis comparing the means of control and different precursors and elicitor treatments was done by one way ANOVA using Duncan’s Multiple Range Test at P ≤ 0.05 employing SPSS software. Pearson coefficient (r) was calculated to determine correlation between the callus biomass (mg d.w.) and plumbagin production (μg/g d.w.). 3. Results and discussion 3.1. Isolation of fractions from root extract Root extract (5 g) obtained after extraction was subjected to isolation of bioactive compounds through the TLC and column chromatography using different solvent systems. Of all the tried solvent systems, hexane: ethyl acetate (70: 30; v: v) proved to be the best mobile phase for the separation of compounds from the methanolic root extract on TLC plate. A maximum of 6 spots were resolved on the TLC plate (Fig. 1H) having Rf values of 0.93, 0.85, 0.75, 0.59, 0.45, 0.35 (Table 1). Hence, the aforesaid solvent system was selected for further elution of compounds in the root extract through column chromatography (Fig. 1E). At the end, a total of 65 fractions each were of 50 mL obtained. The isolated fractions having compounds with similar Rf values were pooled into 8 fractions (Fig. 1F). Of the 8 fractions (F1–F8), only F2 gave a single, yellow coloured spot (Rf value: 0.74) corresponding to the standard compound plumbagin, whereas the other fractions gave multiple spots (Fig. 1G). Based on the above data, F2 was chosen for further characterization and validation using 1H-NMR and FT-IR (Fig. 2). 2.3.3. Treatment of precursors and elicitors The root calluses (90–95 mg) were cultured on MS + 5 μM TDZ supplemented with different concentrations (1, 5, 25, 50, 100 and 200 mg/L) of abovementioned freshly prepared precursors and elicitors. These cultures were then maintained under light intensity of- μmol m−2 s-1 provided by cool white fluorescent tubes (Philips India Ltd., Kolkata, India) with a 16-h photoperiod in growth room at 25 ± 2 °C for 30-d. The root calluses of each treatment were harvested and dried at 25 ± 2 °C in the dark for 7-d and their fresh weights (f.w.) and dry weights (d.w.) were recorded. These calluses were further used for the quantification of plumbagin employing High Performance Liquid Chromatography (HPLC). 3.2. Identification and characterization of purified compound through 1HNMR and FT-IR 2.3.4. HPLC analysis of plumbagin After treatment with precursors of plumbagin and different elicitors, the root calluses of P. zeylanica were crushed separately in liquid nitrogen and extracted in methanol (Merck, Kenilworth, NJ) for 48 h with continuous shaking in the dark at 20 ± 2 °C. The methanolic extracts were filtered using Whatman No.1 filter paper and allowed to concentrate in the dark at room temperature. Amber glass bottles were used to store dried extracts. Further, the HPLC analysis was carried out with photo diode array (PDA; Waters, Milford, MA; Model; Waters, 2998) detector of the HPLC unit (Waters, Pump model: Waters 515 HLPL pump; Pump control module: Model code PC2 serial # K08PC2 519G) while using a Supelcosil C18 column (L × I.D. 250 × 4.6 mm; Sigma-Aldrich®). Plumbagin content was determined using methanol: water (80: 20) as mobile phase (Beigmohamadi et al., 2019) and monitored at 266 nm employing Empower2 software, Waters. Peak identification was done by comparing the retention time of standard plumbagin (Sigma-Aldrich, USA) with that of the samples. All the The 1H-NMR spectrum of the isolated compound F2 showed peaks at δ 2.13 (3H, s), δ 6.83 (1H, s), δ 7.21 (1H, s), and δ 7.55−7.65 (2H, m). NMR peaks at δ 4.81 (s) and δ 3.28 (s) regions belonged to the methanol-d4 solvent. Therefore, the isolated compound F2 was confirmed as 5-hydroxy-2-methyl-1, 4-naphthoquinone; plumbagin (Fig. 2A, Table 2). The FT-IR spectra of isolated F2 corresponding to the standard plumbagin revealed the characteristic absorption peaks at 3315.21 (NeH stretch), 2942.81 (C–H stretch), 2830.76 (C–H stretch), 1449.80 (CeC stretch), 1114.67 (CeO stretch), 1021.53 (CeN stretch), and 632.75 (-C≡C–H: C–H bend) representing the presence of functional groups such as 1°, 2° amines, amides, alkanes, aldehydes, aromatics, alcohols, carboxylic acids, esters, ethers, aliphatic amines and alkynes (Fig. 2B, Table 3). Table 1 Resolution of compounds from root extract of P. zeylanica on TLC plates using different inorganic solvents. Mobile solvents Volume (v:v; mL:mL) Number of spots on TLC plate Rf values Chloroform:Methanol Hexane:Chloroform Chloroform:Toluene Toluene:Ethyl acetate Hexane:Ethyl acetate Chloroform:Ethyl acetate:Methanol 80:20 75:25 60:40 70:30 70:30 65:25:10 5 1 5 2 6 4 0.92, 0.84, 0.80, 0.69,-, 0.59, 0.51, 0.42, 0.16 0.96, 0.81 0.93, 0.85, 0.75, 0.59, 0.45, 0.35 0.87, 0.72, 0.56, 0.36 Bold values show the maximum number of spots and their Rf values in respective mobile solvents. 4 Industrial Crops & Products 151 - T. Singh, et al. Fig. 2. A-B. Characterization of isolated compound (F2). (A) 1H-NMR spectrum of the isolated fraction 2 confirming the structure of plumbagin; (B) FT-IR spectrum of isolated fraction 2. 3.3. Elicitation of plumbagin in root callus cultures 200 mg/L), proline (1, 5, 25, 50, 100 and 200 mg/L), lysine (1, 5, 25, 50, 100 and 200 mg/L), lead nitrate [Pb(NO3)2] (1, 5, 25, 50, 100 and 200 mg/L), cadmium chloride (CdCl2) (1, 5, 25, 50, 100 and 200 mg/L), or in combinations such as yeast extract (100 mg/L) + chitosan (1, 5, 25 and 50 mg/L) and yeast extract (100 mg/L) + salicylic acid (1, 5, 25 and 50 μM) to evaluate their impact on plumbagin content in root callus Root calluses raised on MS + 5 μM TDZ were augmented with precursors of plumbagin such as sodium acetate (1, 5, 25, 50, 100 and 200 mg/L) and L-tyrosine (1, 5, 25, 50, 100 and 200 mg/L) and different elicitors used either alone such as chitosan (1, 5, 25, 50, 100 and 5 Industrial Crops & Products 151 - T. Singh, et al. d.w.) (Fig. 4B, Table 4). However, at 50 mg/L and beyond, plumbagin content reduced even below the control. When root callus cultures of P. zeylanica treated with different concentrations of L-tyrosine, a positive correlation (r = 0.676, P < 0.05) was observed between the callus biomass (mg d.w.) and plumbagin (μg/g d.w.) (Fig. 5A). L-tyrosine is an aromatic amino acid which acts as a precursor molecule in the homogentisic ring cleavage pathway, where tyrosine is catabolized to give acetate moieties. After subsequent enzyme guided reactions, these acetate moieties are incorporated into plumbagin. Durand and Zenk (1974b), showed that feeding Drosophyllum lusitanicum cell suspension cultures with tyrosine-[β14-C] yielded plumbagin and resulted in 20 % of the total plumbagin extracted from cultures that had incorporated labeled carbon. Supplementing the culture medium with L-tyrosine led to enhancement of total bio-accessible phenolic content by 30 % in Fagopyrum esculentum (Świeca, 2016). In the present study, increase in plumbagin yield with increasing tyrosine up to a certain concentration could be attributed to the fact that precursor molecules may enhance the production of bioactive compounds. Table 2 1 H-NMR data of fraction 2 from root extract of P. zeylanica in solvent Methanold4. S. No. δH (Hz) Multiplicity No. of protons H-1 H-2 H-3 H-4 -−7.65 s s s m 3 1 1 2 cultures after 30-d of treatment were quantified by using HPLC. 3.3.1. Impact of precursors feeding Two precursors of plumbagin biosynthetic pathway, sodium acetate and L-tyrosine were used to improve the production of plumbagin in root callus cultures of P. zeylanica. 3.3.1.1. Sodium acetate. Of the different concentrations (1, 5, 25, 50, 100 and 200 mg/L) of sodium acetate used, maximum root callus biomass of 932.93 ± 4.86l mg f.w. (143.52 ± 2.12h mg d.w.) was observed on media with the lowest concentration at 1 mg/L sodium acetate which was statistically different to the control of 835.95 ± 7.04hi mg f.w. (128.67 ± 4.39f mg d.w.) (Fig. 3D, E, Table 4). The largest increase of 2.07–fold (218.15 ± 6.32g μg/g d.w.) in plumbagin content was also recorded at this concentration, when compared to the control (71.0 ± 1.35d μg/g d.w.) (Fig. 4B, Table 4). Thereafter, a steep decline in plumbagin accumulation was seen as the sodium acetate concentration was raised beyond 1 mg/L. The contents at 100 and 200 mg/L were the lowest at 0.82 ± 0.12a and 0.32 ± 0.002a μg/g d.w, respectively. A significant positive correlation (r = 0.906, P < 0.05) was observed between the callus biomass (mg d.w.) and plumbagin (μg/g d.w.) in sodium acetate treatment (Fig. 5A). Prior to this, an increase in intracellular plumbagin content was reported when root callus culture medium of Plumbago indica was supplemented with 5 mM sodium acetate (Jaisi and Panichayupakaranant, 2016). The probable reason for decline in plumbagin content in the presence of increasing concentrations of sodium acetate could be due to the Na+ ions interfering with absorption and mobilization of K+ and Ca2+ and may also affect the efficient stomatal regulation which eventually leads to reduced photosynthetic activity (Tavakkoli et al., 2010). Higher Na+ in cell apoplast leads to cellular membrane damage (Flowers et al., 2014), which might result in leaching of secondary metabolites into the extracellular environment. 3.3.2. Impact of elicitors 3.3.2.1. Impact of chitosan. A significant increase in plumbagin content and callus biomass proliferation was observed when root callus medium was supplemented with increasing concentrations (1, 5, 25, 50, 100, 200 mg/L) of chitosan. Maximum callus growth of 1193.60 ± 5.98d mg f.w. (171.92 ± 2.98f mg d.w.) was found with 50 mg/L chitosan over control of 835.95 ± 7.04b mg f.w. (128.67 ± 4.39b mg d.w.) (Fig. 3D, G, Table 5). Interestingly, the highest plumbagin synthesis of 408.50 ± 6.12d μg/g d.w. (4.58–fold) was also recorded with 50 mg/L chitosan compared with the control (73.17 ± 1.24a μg/g d.w.) (Fig. 4C, Table 5). A decline in plumbagin content and callus growth was observed beyond 50 mg/L. Positive correlation (r = 0.562, P < 0.05) was observed between the callus biomass (mg d.w.) and plumbagin (μg/g d.w.) in chitosan treatment (Fig. 5B). Chitosan, a deacetylated chitin derivative mimics the effects of numerous pathogenic fungi to trigger the defense responses and production of secondary metabolites in plants (Muxika et al., 2017). Osman et al. (2018), reported that a 1.3–fold enhancement in gallic acid content was obtained when 150 mg/L chitosan was added to cell suspension cultures of Barringtonia racemosa. In another publication (Jiao et al., 2018), it was observed that hairy root cultures of Isatis tinctoria exposed to 150 mg/L chitosan showed a 7.08–fold increment in flavonoid content and up-regulation of the chalcone synthase and overexpression of the flavonoid 3′-hydroxylase genes of the flavonoid biosynthetic pathway. More recently, Ahmad et al. (2019), reported that a 1.4–fold enhanced quantity of 2-hydroxy-4-methoxybenzaldehyde at 200 μM chitosan over control in suspension cultures of root tuber derived callus of Decalepis salicifolia. Also, a 3–fold enhancement in oleanolic acid content was seen in hairy root cultures of Calendula officinalis when treated with 100 mg/L chitosan compared with the control (Alsoufi et al., 2019). More recently, enhancement in daidzein and psoralen contents up to 11.2–fold and 7.2–fold, respectively, at 50 mg/L chitosan and 6.21–fold genistein with supplements of 100 mg/L chitosan in cotyledon callus cultures of Cullen corylifolium has been reported (Singh et al., 2020). Besides, cell cultures of Scrophularia striata treated 3.3.1.2. L-Tyrosine. When root callus medium of P. zeylanica was supplemented with various concentrations (1, 5, 25, 50, 100 and 200 mg/L) of L-tyrosine, optimum callus biomass of 971.21 ± 4.04m mg f.w. (134.55 ± 1.81fg mg d.w.) was obtained at 25 mg/L compared with the control of 835.95 ± 7.04hi mg f.w. (128.67 ± 4.39f mg d.w.) (Fig. 3D, F, Table 4). At the same time, a significant enhancement in plumbagin content was observed in the root callus, such that the plumbagin accumulation of 258.85 ± 3.9h μg/g d.w. (2.64–fold) was also observed at 25 mg/L compared to the control (71.0 ± 1.35d μg/g Table 3 FT-IR analysis of isolated fraction 2 from P. zeylanica root extract. S.N. Absorption Peak (cm−1) Wavelength (cm−1) Functional group - - NeH stretch -) C–H stretch -) C–H stretch (2850−2695) CeC stretch (1500−1400) CeO stretch (1320−1000) CeN stretch (1250−1020) eC^C–H: C–H bend (700−610) 1•, 2• amines, amides Alkanes Aldehydes Aromatics Alcohols, carboxylic acids, esters, ethers Aliphatic amines Alkynes 6 Industrial Crops & Products 151 - T. Singh, et al. Fig. 3. A-K. Induction and proliferation of callus from in vitro roots of P. zeylanica and effect of different elicitors on plumbagin content in root callus. (A) in vitro raised plantlet with numerous roots; (B) in vitro roots inducing callus on MS +5 μM TDZ medium after 14-d of culture (inset: roots at the time of inoculation); (C) proliferation of root callus on MS +5 μM TDZ medium; (D) root callus raised on MS +5 μM TDZ without elicitors (control); (E) optimum root callus growth at 1 mg/L sodium acetate; (F) optimum proliferation of callus at 25 mg/L L-tyrosine; (G) optimum growth of root callus at 50 mg/ L chitosan; (H) optimum proliferation of root callus at 50 mg/L proline; (I) optimum growth of root callus at 100 mg/L lysine; (J) root callus showing decline in growth at 25 mg/L Pb(NO3)2; (K) root callus showing optimum growth at 5 mg/L CdCl2; (L) optimum proliferation of root callus at 100 mg/L yeast extract and 25 μM salicylic acid; and (M) optimum growth of root callus at 100 mg/L yeast extract and 50 mg/L chitosan. Red bars = 1 cm. with chitosan showed increased activities of scavenging enzymes and phenylalanine ammonia lyase indicating an increased antioxidant response (Kamalipourazad et al., 2016). The possible mechanism of action of chitosan in the elicitation of secondary metabolites (Fig. 6) could be due to induction of plant defense responses via the activation of NADPH oxidase, G-protein activation, MAPKs phosphorylation, production of reactive oxygen species (ROS), cytoplasmic acidification through influx of H+ and efflux of K+/Cl−, and also increased cAMP concentration resulting in elevation of Ca2+ in the cytosol (Iriti and Faoro, 2009; Ramirez-Estrada et al., 2016). Moreover, chitosan is also involves in the signal transduction pathways and binds with transcription factors which regulate over-expression of defense genes and biosynthesis of secondary metabolites. 3.3.2.2. Impact of amino acids. A slight increment in callus growth and plumbagin content was seen when root calluses were exposed to increasing concentrations (1, 5, 25, 50, 100, 200 mg/L) of proline and lysine. Maximum callus growth of 1556.00 ± 6.50k mg f.w. (177.81 ± 8.17g mg d.w.) and 1124.96 ± 0.90f mg f.w. (153.06 ± 4.56f mg d.w.) was observed at 50 mg/L proline and 100 mg/L lysine, respectively (Fig. 3H, I, Table 5). Maximum plumbagin accumulation of 177.00 ± 5.37g μg/g d.w. (1.41–fold) was also achieved with 50 mg/L proline (Fig. 4D, Table 5). Subsequently, there was a sharp decline in callus growth and plumbagin content beyond 50 mg/L proline. A significant positive correlation was observed between the callus biomass (mg d.w.) and plumbagin (μg/g d.w.) in proline treatment (r = 0.869, P < 0.05) 7 Industrial Crops & Products 151 - T. Singh, et al. Table 4 Effect of precursors on plumbagin content and callus biomass in root callus cultures of P. zeylanica. Precursors of plumbagin Plumbagin1 (μg/g d.w.) Callus biomass1 (mg f.w.) Callus biomass1 (mg d.w.) Types Control Sodium acetate Concentration – 1 mg/L 5 mg/L 25 mg/L 50 mg/L 100 mg/L 200 mg/L 71.0 ± 1.35d 218.95 ± 6.33g 198.88 ± 4.23f 160.86 ± 4.23e 18.90 ± 1.20b 0.82 ± 0.12a 0.32 ± 0.002a 835.95 ± 7.04hi 932.93 ± 4.86l 888.48 ± 5.74k 869.44 ± 1.88j 239.69 ± 4.53e 134.38 ± 2.89b 112.72 ± 4.19a 128.67 ± 4.39f 143.52 ± 2.12h 138.91 ± 0.76gh 133.78 ± 0.48fg 32.19 ± 1.24c 23.88 ± 0.94b 15.16 ± 1.98a L-Tyrosine 1 mg/L 5 mg/L 25 mg/L 50 mg/L 100 mg/L 200 mg/L 68.22 ± 2.9d 82.29 ± 6.03d 258.86 ± 3.92h 41.72 ± 4.24c 14.23 ± 0.25ab 3.00 ± 0.21a 823.93 ± 4.99g 845.64 ± 2.93i 971.21 ± 4.04m 459.89 ± 6.27f 226.24 ± 5.92d 183.75 ± 4.06c 121.39 ± 2.18e 130.14 ± 5.35f 134.55 ± 1.81fg 80.11 ± 1.63d 31.93 ± 1.06c 22.60 ± 2.02b d.w.; dry weight, f.w.; fresh weight. Sodium acetate (r = 0.906, P ≤ 0.05); L-Tyrosine (r = 0.676, P ≤ 0.05). Each treatment consisted of 12 replicates and was repeated thrice. Bold values show maximum plumbagin and callus biomass in respective treatments. 1 Different superscript letters after mean values represent significant differences P ≤ 0.05 within the groups using one-way ANOVA. (Fig. 5C). Similarly, maximum plumbagin synthesis of 160.72 ± 5.92fg μg/g d.w. (1.19–fold) was observed at 100 mg/L lysine over control (73.17 ± 1.24d μg/g d.w.) with maximum callus growth (Fig. 4D, Table 5). Also, at 200 mg/L lysine a sharp decrease was seen in callus growth and plumbagin content. Weak correlation (r = 0.507, P < 0.05) was observed between the callus biomass (mg d.w.) and plumbagin (μg/g d.w.) in lysine treatment (Fig. 5C). Amino acids are considered as a source of nitrogen and they trigger the production of secondary metabolites. Aljibouri et al. (2012), reported a 58.03 % increase in hyoscyamine content with 50 mg/L proline over control in callus cultures of Hyoscyamus niger. In another study, comarin and eugenol content enhanced up to 2752 % and 290 % respectively when exposed to proline (150 mg/L and 50 mg/L) in callus cultures of Verbascus thapsus (Al-Jibouri et al., 2016). Connessine content improved to 3.24–fold when bark derived callus cultures of Hollarrhena antidysentrica were exposed to 100 mg/L proline (Kumar et al., 2018). A recent study reported, increases in psoralen and genistein contents up to 3.9–fold and 1.74–fold, respectively with 50 mg/L proline and 1.55–fold daidzein with supplements of 100 mg/ L proline in cotyledon callus cultures of Cullen corylifolium (Singh et al., 2020). There are no previous reports pertaining to lysine as an elicitor in plants. To our best knowledge, this is our first report of lysine as an elicitor in P. zeylanica. However, Yan et al. (2014), reported a 40 % higher huperzine A with 200 mg/L lysine over control in fungus Shiraia sp. Slf14 cultures through fermentation. significant correlation was observed between the callus biomass (mg d.w.) and plumbagin (μg/g d.w.) in CdCl2 treatment (r = 0.786, P < 0.05) (Fig. 5D). As found for the other elicitors, higher concentrations of heavy metals (> 25 mg/L) were inhibitory for accumulation of plumbagin content and biomass growth in root callus cultures. Heavy metals are considered one of the main abiotic elicitors which may affect the production of secondary metabolites (Nasim and Dhir, 2010). Heavy metals may alter the metabolic activity of plants which would affect the production of bioactive compounds, photosynthetic pigments, sugars, and proteins (Gorelick and Bernstein, 2014). Reactive oxygen species (ROS) produced in plants upon heavy metals stress interfere with secondary metabolism of plants (Sytar et al., 2013; Kumar and Prasad, 2018). Cadmium (Cd) and Lead (Pb) in plants may increase the level of cytoplasmic Ca2+, activate MAPKs cascades and signaling pathways which lead to the induction of stress responsive genes (Cetin et al., 2014; Kumar and Majeti, 2014). Phosphorylation through MAPK, and activation of downstream transcription factors generally lead to the transcriptional reprogramming of secondary metabolism in plants (Fig. 6) (Vasconsuelo and Boland, 2007; Schluttenhofer and Yuan, 2015; Phukan et al., 2016). Pitta–Alvarez et al. (2000), have reported that CdCl2 (1, 2 mM) stimulated the quantity of scopolamine (3 to 8–fold), and hyoscyamine (4 to 24–fold) in hairy root cultures of Brugmansia candida. Cai et al. (2013), also observed that a low concentration (5 μM) of heavy metals (Co2+, Ag+, Cd2+) was effective for the enhancement of resveratrol of up to 1.6–fold in cell suspension cultures of Vitis vinifera. The highest accumulation of gymnemic acid content of up to 6.8–fold was observed with 2 mM CdCl2 supplements in cell suspension cultures of Gymnema sylvestre (Ch et al., 2012). There is another report in which diosgenin production was enhanced by 40 and 41–fold with CdCl2 and CoCl2, respectively, in seedlings of Trigonella foenum-graecum soaked with ½ strength MS medium on double layered filter paper. A recent study of Kumar et al. (2018), also showed 5.41 and 3.54–fold increases in conessine content with 100 mg/L Pb(NO3)2 and 50 mg/L CdCl2, respectively, in green bark callus of Holarrhena antidysenterica. 3.3.2.3. Impact of heavy metals. An increase in callus biomass and plumbagin content was observed when root callus medium was supplemented with different concentrations (1, 5, 25, 50, 100, 200 mg/L) of heavy metals such as Pb(NO3)2 and CdCl2. Optimum callus biomass of 966.95 ± 10.30i mg f.w. (153.06 ± 7.10h mg d.w.) and 876.11 ± 4.56g mg f.w. (135.77 ± 0.94fg mg d.w.) was recorded with 25 mg/L Pb(NO3)2 and 5 mg/L CdCl2, respectively (Fig. 3J, K, Table 6). Optimum amount of plumbagin 151.85 ± 5.95h μg/g d.w. (1.07–fold) was achieved with 25 mg/L Pb(NO3)2 compared to the control (73.17 ± 1.24d μg/g d.w.) (Fig. 4E, Table 6). A gradual decline in plumbagin content was observed beyond 25 mg/L Pb(NO3)2. Positive correlation (r = 0.604, P < 0.05) was observed between the callus biomass (mg d.w.) and plumbagin (μg/g d.w.) Pb(NO3)2 treatment (Fig. 5D). Similarly, Maximum amount of plumbagin 156.76 ± 7.88h μg/g d.w. (1.14–fold) was observed with 5 mg/L CdCl2 compared to the control (73.17 ± 1.24d μg/g d.w.) (Fig. 4E, Table 6). Subsequently, there was a steep decline in plumbagin content beyond 5 mg/L CdCl2. A 3.3.2.4. Combined impact of elicitors. A tremendous increase in root callus biomass and plumbagin production was seen in root calluses when 100 mg/L yeast extract was added in combination with increasing concentrations (1, 5, 25, 50 mg/L) of chitosan. Maximum root callus biomass of 1825.81 ± 9.59f mg f.w. (191.63 ± 1.47d mg d.w.) and 2254.51 ± 18.84i mg f.w. (207.15 ± 0.60f mg d.w.) was achieved 8 Industrial Crops & Products 151 - T. Singh, et al. Fig. 4. A-G. HPLC analysis representing chromatogram of standard compound plumbagin and impact of different elicitors on plumbagin accumulation in root callus cultures of P. zeylanica after 30-d treatment. (A) Chromatogram of standard compound plumbagin showing peak at retention time of 6.688 min; (B) impact of different concentrations (0, 1, 5, 25, 50, 100 and 200 mg/L) of precursors such as sodium acetate and L-tyrosine; (C) impact of chitosan (0, 1, 5, 25, 50,100 and 200 mg/L); (D) impact of different concentrations (0, 1, 5, 25, 50,100 and 200 mg/L) of amino acids such as proline and lysine; (E) impact of heavy metals such as Pb (NO3)2 and CdCl2 (0, 1, 5, 25, 50,100 and 200 mg/L); (F) impact of combination of yeast extract (100 mg/L) and salicylic acid (0, 1, 5, 25 and 50 μM); (G) impact of combination of yeast extract (100 mg/L) and chitosan (0, 1, 5, 25 and 50 mg/L). Different superscript letters on bars represents significant differences P ≤ 0.05, d.w. dry weight, d; day, PL; plumbagin. with medium supplements of 100 mg/L yeast extract + 25 μM salicylic acid and 100 mg/L yeast extract +50 mg/L chitosan, respectively, compared to the control of 835.95 ± 7.04a mg f.w. (123.67 ± 4.39b mg d.w.) (Fig. 3L, M, Table 5). An increase in plumbagin synthesis was observed when the calluses were augmented with 100 mg/L yeast extract in combinations with increasing concentrations (1, 5, 25, 9 Industrial Crops & Products 151 - T. Singh, et al. Fig. 5. Correlation of callus biomass (mg d.w.) and plumbagin (μg/g d.w.) in root callus cultures of P. zeylanica treated with different precursors or elicitors. (A) Precursors (sodium acetate and L-tyrosine); callus biomass-SoA, biomass of root callus cultures treated with sodium acetate; callus biomass-LTy, biomass of root callus cultures treated with L-tyrosine; plumbagin-SoA, plumbagin in sodium acetate treatment; plumbagin-LTy, plumbagin in L-tyrosine treatment; (B) chitosan; (C) amino acids (proline and lysine); callus biomass-Pro, biomass of root callus cultures treated with proline; callus biomass-Lys, biomass of root callus cultures treated with lysine; plumbagin-Pro, plumbagin in proline treatment; plumbagin-Lys, plumbagin in lysine treatment; (D) heavy metals (Pb(NO3)2 and CdCl2); callus biomass-Pb, biomass of root callus cultures treated with (Pb(NO3)2; callus biomass-Cd, biomass of root callus cultures treated with CdCl2; plumbagin-Pb, plumbagin in (Pb(NO3)2 treatment; plumbagin-Cd, plumbagin in CdCl2 treatment; (E) yeast extract and salicylic acid; (F) yeast extract and chitosan. Different superscript letters on bars represents significant differences P ≤ 0.05, d.w. dry weight, d; day, PL; plumbagin. 50 μM) of salicylic acid. The optimum quantity of plumbagin was 675.91 ± 6.91d μg/g d.w. (8.23–fold) at a combination of 100 mg/L yeast extract and 25 μM salicylic acid (Fig. 4F, Table 5). As found before, a decline in plumbagin accumulation was observed beyond 25 μM. A significant correlation was observed between the callus biomass (mg d.w.) and plumbagin (μg/g d.w.) in combination of yeast extract and salicylic acid treatment (r = 0.715, P < 0.05) (Fig. 5E). As seen before with the other tested precursors and elicitors, the highest plumbagin content of 957.52 ± 4.73e μg/g d.w. (12.08–fold) was achieved on the MS medium that gave maximum callus growth, supplemented with a combination of 100 mg/L yeast extract and 50 mg/L chitosan (Fig. 4G, Table 5). A strong significant positive correlation was observed between the callus biomass (mg d.w.) and plumbagin (μg/g d.w.) production in combination of yeast extract and chitosan (r = 0.986, P < 0.05) (Fig. 5F). To our best knowledge, this is the first report of enhancement of plumbagin up to 12.08–fold using a combination of yeast extract and chitosan in P. zeylanica root callus cultures. Earlier Sharma and Agrawal (2018), used yeast extract and salicylic acid alone and observed that plumbagin content of 544.60 ± 10.11 μg/g d.w. (6.5–fold) and 319.16 ± 0.76 μg/g d.w. (3.4–fold) was achieved with 100 mg/L yeast extract and 25 μM salicylic acid supplements, respectively, when compared with the control (71.0 ± 1.35 μg/g d.w.). Zhao et al. (2010), reported enhanced production of tanshinone with combinations of either two elicitors (Ag+ + yeast extract/ Cd2+ + yeast extract) or three elicitors (Ag+ + Cd2+ + yeast extract) of up to 20 % and 40 %, respectively, compared to a single elicitor (Ag+/ Cd2+/ yeast extract) in cell suspension culture of Salvia miltiorrhiza. It was also reported that the level of anthocyanin biosynthesis increased by 4.6–fold over control with a combination of 10 Industrial Crops & Products 151 - T. Singh, et al. Table 5 Effect of different elicitors on plumbagin content and callus biomass in root callus of P. zeylanica. Elicitors Plumbagin1 (μg/g d.w.) Callus biomass1 (mg f.w.) Callus biomass1 (mg d.w.) Types Chitosan Concentration Control 1 mg/L 5 mg/L 25 mg/L 50 mg/L 100 mg/L 200 mg/L 73.17 ± 1.24a 196.51 ± 4.66b 199.88 ± 7.57b 213.40 ± 6.65b 408.50 ± 6.12d 264.79 ± 4.36c 248.58 ± 6.44c 835.95 ± 7.04b 838.59 ± 9.43b 853.72 ± 7.89b 935.17 ± 8.76c 1193.60 ± 5.98d 837.13 ± 7.48b 721.43 ± 7.66a 128.67 ± 4.39b 141.44 ± 2.32cd 149.78 ± 3.25de 157.22 ± 2.83e 171.92 ± 2.98f 138.48 ± 5.45bc 114.78 ± 2.24a Proline Control 1 mg/L 5 mg/L 25 mg/L 50 mg/L 100 mg/L 200 mg/L 73.17 ± 1.24ab 78.07 ± 0.93ab 107.86 ± 8.13c 119.66 ± 2.60cd 177.00 ± 5.37g 155.23 ± 6.38f 70.03 ± 8.81a 835.95 ± 7.04b 1254.33 ± 16.22g 1428.36 ± 4.94i 1507.00 ± 8.02j 1556.00 ± 6.50k 1315.22 ± 7.82h 797.46 ± 4.29a 128.67 ± 4.39bc 135.73 ± 2.90bcd 144.34 ± 4.78def 144.78 ± 8.97def 177.81 ± 8.17g 149.70 ± 6.42ef 99.25 ± 5.66a Lysine Control 1 mg/L 5 mg/L 25 mg/L 50 mg/L 100 mg/L 200 mg/L 73.17 ± 1.24ab 77.72 ± 5.99ab 90.55 ± 5.83b 110.33 ± 8.38c 135.33 ± 5.48de 160.72 ± 5.92fg 142.90 ± 4.91ef 835.95 ± 7.04b 908.38 ± 2.88c 934.50 ± 0.82d 943.82 ± 10.99d 952.50 ± 3.27d 1124.96 ± 0.90f 1077.33 ± 0.41e 128.67 ± 4.39bc 133.10 ± 0.19bcd 136.83 ± 1.68bcde 142.43 ± 7.16def 140.91 ± 6.58cdef 153.06 ± 4.56f 125.46 ± 3.08b Yeast extract + salicylic acid Control 100 mg/L +0 μM 100 mg/L +1 μM 100 mg/L +5 μM 100 mg/L +25 μM 100 mg/L +50 μM 73.17 ± 1.24a 534.31 ± 5.75b 645.63 ± 7.05c 668.78 ± 6.81d 675.91 ± 6.91d 551.47 ± 6.45b 835.95 ± 7.04a 1638.59 ± 8.64c 1687.82 ± 8.56d 1762.65 ± 6.92e 1825.81 ± 9.59f 1413.39 ± 14.41b 128.67 ± 4.39b 172.92 ± 1.91c 176.23 ± 3.91c 186.07 ± 3.37d 191.63 ± 1.47d 125.30 ± 1.47a Yeast extract + chitosan Control 100 mg/L +0 mg/L 100 mg/L +1 mg/L 100 mg/L +5 mg/L 100 mg/L +25 mg/L 100 mg/L +50 mg/L 73.17 ± 1.24a 538.42 ± 6.10b 784.79 ± 6.99c 801.29 ± 5.17c 856.30 ± 6.12d 957.52 ± 4.73e 835.95 ± 7.04a 1647.11 ± 7.46c 1743.03 ± 7.06e 1879.88 ± 12.70g 1979.51 ± 12.24h 2254.51 ± 18.84i 128.67 ± 4.39b 176.89 ± 1.12c 184.85 ± 1.01d 190.07 ± 0.81d 200.41 ± 0.51e 207.15 ± 0.60f d.w.; dry weight, f.w.; fresh weight. Chitosan (r = 0.562, P ≤ 0.05); Proline (r = 0.869, P ≤ 0.05); Lysine (r = 0507, P ≤ 0.05); Yeast extract and salicylic acid (r = 0.715, P ≤ 0.05); Yeast extract and chitosan (r = 0.986, P ≤ 0.05). Each treatment consisted of 12 replicates and was repeated thrice. Bold values show maximum plumbagin and callus biomass in respective treatments. 1 Different superscript letters after mean values represent significant differences P ≤ 0.05 within the groups using one-way ANOVA. 5 mg/L phenylalanine and 50 mg/L methyl jasmonate in cell suspension culture of Vitis vinifera (Qu et al., 2011). In contrast, Piątczak et al. (2016) showed enhanced combined effect of methyl jasmonate and salicylic acid on catalpol, harpagide, verbascoside and isoverbascoside contents, but enhancement was less when methyl jasmonate alone was added to hairy root cultures of Rehmannia glutinosa. In a recent study, a combination of 2.5 mg/L yeast extract and 100 μM methyl jasmonate stimulated the accumulation of parthenolide content up to 0.05 % and up-regulated the expression of parthenolide synthase gene in hairy root culture of Tanacetum parthenium (Pourianezhad et al., 2019). Cell suspension cultures of Cannabis sativa supplemented with combination of 100 μM methyl jasmonate and 1 mM tyrosine showed an 82 % increase in phenolic content over the control (Gabotti et al., 2019). In the current study, yeast extract showed direct relation with the plumbagin content accumulation and biomass production. Yeast extract is a mixture of fungal cell wall polysaccharides (chitin and β-glucan), amino acid residues, minerals, and vitamins (Pitta-Alvarez et al., 2000). Amino acid residues and minerals have stimulated the callus biomass production (El-Sharabasy et al., 2012). Chitin and β-glucan serve as microbe-associated molecular patterns (MAMP) that are recognized by pattern recognition receptors (PRRs) present in plants, and thereby induce the expression of certain stress-related genes, leading to increased secondary metabolite production (Jumali et al., 2011; Fesel and zuccaro, 2016). On the other hand, salicylic acid is a signaling molecule that modulates the activities of major antioxidant enzymes participating in certain metabolic pathways that lead to increased secondary metabolite production (Rahimi et al., 2014; Fesel and Zuccaro, 2016) (Fig. 6). The complete biosynthetic pathway of plumbagin has not yet been worked out (Durand and Zenk, 1974a, b), therefore the exact enzyme target for salicylic acid, causing the increased plumbagin production, remains to be elucidated. Similar to our study, a dose dependent increase in plumbagin content (1.5–fold) at 50 μM salicylic acid was observed in P. indica root cultures (Jaisi and Panichayupakaranant, 2016). Based on the above reports, it can also be postulated that when different elicitors supplemented in combination to growth medium may exert synergistic or additive effect on plumbagin synthesis/production. It is therefore evidently interpreted that an appropriate combination of chitosan and yeast extract has proved beneficial over the single elicitor for the scaling up of plumbagin in the root callus cultures of P. zeylanica. 4. Conclusion The current investigation reports the isolation of plumbagin from methanolic root extract of P. zeylanica. Roots are the principal site for the synthesis and accumulation of plumbagin. Therefore, in vitro raised 11 Industrial Crops & Products 151 - T. Singh, et al. Fig. 6. Possible mechanism of action of elicitors on enhancement of plumbagin: various elicitors recognized by plasma membrane bound receptors result in ROS production, activation of NADPH oxidase, G-protein activation, MAPKs phosphorylation, increase in Ca2+ and cAMP concentration in cytoplasm, cytoplasmic acidification through influx of H+ and efflux of K+/Cl– and ion fluxes through ion channels. Cytosolic receptors also bind with metal ions or other signaling molecules may also contribute towards the formation of ROS, activation of MAPKs, etc. which in return activates downstream transduction pathway leads to the activation/ phosphorylation of transcription factors (TFs). These TFs may regulate the expression of homogentisate oxygenase/defense genes which leads to the production of bioactive compounds such as plumbagin. PLC; Phospholipase C, cAMP; Cyclic adenosine monophosphate, IP3; Inositol trisphosphate, DAG; Diacylglycerol, MAPKs; Mitogen-activated protein kinases, P; Phosphorylation, ROS; Reactive oxygen species, SA; Salicylic acid, JA; Jasmonic acid, CAT; Catalase, SOD; Superoxide dismutase, APX; Ascorbate peroxidase, GPX; Glutathione peroxidase, TFs; Transcription factors. (Source: Durand and Zenk, 1974a; Iriti and Faoro, 2009; Ramirez-Estrada et al., 2016). roots were selected as an alternate source in lieu of in vivo roots for elicitation of plumbagin bioactive compound. It has been established that root callus has the potential to synthesize enormous quantity of plumbagin if exposed to suitable elicitors. Optimum increases in plumbagin accumulation of up to 2.07 and 2.64–fold was achieved with precursors sodium acetate (1 mg/L) and L-tyrosine (25 mg/L), Table 6 Effect of heavy metals on plumbagin content and callus biomass in root callus of P. zeylanica. Elicitors Plumbagin1 (μg/g d.w.) Callus biomass1 (mg f.w.) Callus biomass1 (mg d.w.) Types Control Concentration – 73.17 ± 1.24d 835.95 ± 7.04b 128.67 ± 4.39b Lead Nitrate 1 mg/L 5 mg/L 25 mg/L 50 mg/L 100 mg/L 200 mg/L 94.63 ± 2.60e 116.80 ± 3.05f 151.85 ± 5.95h 131.54 ± 6.99g 105.15 ± 4.25e 35.71 ± 2.26b 843.40 ± 3.53f 933.85 ± 7.25h 966.95 ± 10.30i 627.18 ± 3.73f 460.51 ± 5.53d 354.02 ± 5.62c 129.13 ± 0.49f 138.67 ± 2.90g 153.25 ± 7.10h 89.87 ± 0.97e 68.86 ± 2.65d 40.62 ± 0.64b Cadmium Chloride 1 mg/L 5 mg/L 25 mg/L 50 mg/L 100 mg/L 200 mg/L 98.80 ± 3.08e 156.76 ± 7.88h 130.01 ± 4.28g 51.95 ± 3.58c 16.16 ± 1.76a 11.56 ± 1.20a 836.96 ± 5.96f 876.11 ± 4.56g 477.56 ± 6.22e 146.45 ± 6.28b 94.00 ± 5.35a 95.69 ± 2.81a 130.41 ± 1.64fg 135.77 ± 0.94fg 83.01 ± 2.28e 54.70 ± 1.89c 38.59 ± 0.91ab 30.62 ± 0.64a d.w.; dry weight, f.w.; fresh weight. Lead nitrate (r = 0.604, P ≤ 0.05); Cadmium chloride (r = 0.786, P ≤ 0.05). Each treatment consisted of 12 replicates and was repeated thrice. Bold values show maximum plumbagin and callus biomass in respective treatments. 1 Different superscript letters after mean values represent significant differences P ≤ 0.05 within the groups using one-way ANOVA. 12 Industrial Crops & Products 151 - T. Singh, et al. respectively. Of all elicitors tested individually, maximum production of plumbagin of 4.58–fold was observed with supplements of 50 mg/L chitosan in root callus culture. Plumbagin content increased only up to 8.23–fold when a combination of 100 mg/L yeast extract and 25 μM salicylic acid was tried. However, a tremendous enhancement in plumbagin content of up to 12.08–fold was seen in root callus grown on MS + 5 μM TDZ augmented with a combination of 100 mg/L yeast extract and 50 mg/L chitosan. Therefore, it is concluded that elicitors when used in certain combinations could tremendously enhance production of plumbagin in root callus cultures of P. zeylanica at higher levels than previously reported. 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