Tikkam Singh

Industrial Crops & Products 143 - Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop Effective protocol for isolation and marked enhancement of psoralen, daidzein and genistein in the cotyledon callus cultures of Cullen corylifolium (L.) Medik T Tikkam Singh, Renuka Yadav, Veena Agrawal* Department of Botany, University of Delhi, Delhi, 110007, India A R T I C LE I N FO A B S T R A C T Keywords: Cullen corylifolium Isolation Elicitation Daidzein Psoralen Genistein Cullen corylifolium (L.) Medik. (Babachi), a traditional herb is being utilized to cure several serious ailments such as psoriasis, leprosy, leucoderma, osteoporosis including leukemia due to the occurrence of many important furanocoumarins and flavonoids especially in the green seeds. Since these compounds occur in extremely low amounts, in vitro elicitation has been proved to be an effective, sustainable, and eco-friendly tool for enhanced production of such compounds. The current study aimed at the isolation of some marker bioactive compounds such as psoralen, daidzein and genistein from the green seed extract, their elicitation and callus biomass production employing various organic elicitors such as chitosan, proline and salicylic acid. The isolation of bioactive anticancerous compounds were done using the gradient of Toluene: Ethyl acetate (v:v) as a solvent system through column chromatography and thin layer chromatography (TLC). The results showed that a total of 101 fractions were collected through column chromatography, which were pooled down to 10 fractions (F1-F10) on the basis of their similar Rf values on TLC plates. The isolated fractions F2, F3 and F5 were characterized and identified as daidzein, psoralen and genistein, respectively, through NMR and HR-MS. For elicitation of such compounds the callus was induced from cotyledons of green seeds on Gamborg B5 medium +5 μM BA +10 μM IBA. Maximum cotyledon callus biomass was observed at 25 mg/L proline followed by chitosan at 50 mg/L and salicylic acid at 5 μM. Maximum psoralen content (14.8–fold) was achieved with 5 μM salicylic acid over control. Whereas, in case of daidzein 11.2–fold enhancement was seen at 50 mg/L chitosan over control. Incidentally, optimum genistein content (6.21–fold) was achieved with 100 mg/L chitosan over control. Thus, the organic elicitors especially the salicylic acid proved to enhance psoralen content, whereas, chitosan was found to be effective for the increment of daidzein and genistein content in the cotyledon callus cultures of Cullen corylifolium. This is a cost effective and industrially viable protocol for the production of important bioactive compounds psoralen, daidzein and genistein. 1. Introduction Cullen corylifolium (L.) Medik., commonly known as babachi, is a popular traditional medicinal plant of Fabaceae, being extensively used against several skin diseases such as psoriasis, leprosy and leucoderma (Sah et al., 2006; Qiao et al., 2006; Khushboo et al., 2010). Besides, it also has properties like antioxidant, anticancer, anti-inflammatory, hepatoprotective, antidiabetic, antimycobacterial, and antihelminthic, etc. due to the occurrence of a number of important furanocoumarins and isoflavonoids (Newton et al., 2002; Jiangning et al., 2005; Wang et al., 2011; Chanda et al., 2011; Kamboj et al., 2011; Chopra et al., 2013). Furanocoumarins and isoflavonoids are synthesized in the different parts of C. corylifolium and widely employed as anticancerous ⁎ agents. The main bioactive compounds occurred in C. corylifolium are psoralen, daidzein and genistein synthesized mainly in green seeds via phenylpropanoid pathway (Yu et al., 2003; Parast et al., 2011). There are some reports regarding the anticancerous activity of psoralen against different human cancer cell lines such as leukemia, breast, lung, central nervous system, oral, erythroleukemia and liver (Viola et al., 2004; Oliveira et al., 2006; Wang et al., 2011; Jiang and Xiong, 2014; Panno and Giordano, 2014; Hsieh et al., 2014; Jiang et al., 2016). Similarly, daidzein and genistein were also seen to inhibit cell proliferation, caused cell cycle arrest and induced apoptosis through mitochondrial pathway or increased caspase-3 activity in breast, colon, and ovarian cancer cells (Choi and Kim, 2008; Jin et al., 2009; Guo et al., 2004; Shafiee et al., 2016; Chan et al., 2018). Such reports cause Corresponding author at: Medicinal Plant Biotechnology and Applied Research Laboratory, Department of Botany, University of Delhi, Delhi, 110007, India. E-mail addresses:-,-(V. Agrawal). https://doi.org/10.1016/j.indcrop- Received 19 July 2019; Received in revised form 17 September 2019; Accepted 23 October-/ © 2019 Elsevier B.V. All rights reserved. Industrial Crops & Products 143 - T. Singh, et al. placed into a closed glass chamber containing iodine vapours. The Rf values were then calculated using the formulae (Touchstone, 1992): these bioactive compounds in high demand, and in order to meet the needs of the pharmaceutical industry, the production of these valuable compounds need to be enhanced using in vitro elicitation technique. Medicinal plants are known to synthesize plethora of important bioactive compounds in response to biotic or abiotic stress for their biological functions. At present, they are acquiring substantial interest for the production of these valuable compounds having pharmaceutical importance (Chetri et al., 2016). However, the production of these valuable secondary metabolites is specific to the particular plant part, depends on growth and developmental stages, season, stress and availability of nutrients and incidentally, they occur in very low amount. (Ramirez-Estrada et al., 2016). Moreover, the extraction of these bioactive molecules is quite an expensive process with low yields. Plant cell and organ cultures are being employed as an alternative, sustainable, controllable and eco-friendly tool for the production of bioactive secondary metabolites (Ochoa-Villarreal et al., 2016). Callus tissue is considered as best material for recurrent and rapid multiplication of cells for the synthesis and elicitation of bioactive compounds (Mulabagal and Tsay, 2004). Although various strategies are available for the amelioration of bioactive secondary metabolites from cells, organs and plant systems, yet elicitation is one of the feasible strategies currently being used (Naik and Al-Khayri, 2016). Elicitors may be biotic, abiotic or phytohormones, which stimulate the synthesis of secondary metabolites (Baenas et al., 2014). The possible mechanisms involved in the elicitation are intracellular transduction systems including elicitor receptors, GTP binding proteins, Ca2+ and other secondary messengers with mitogen-activated protein kinases (MAPKs) pathway for the biosynthesis of secondary metabolites (Vasconsuelo and Boland, 2007; Goel et al., 2011). The present study focused on isolation, characterization and elicitation of potent cancer inhibitors from green seeds derived cotyledon callus of C. corylifolium. The isolated fractions collected through column chromatography and characterized through NMR and HR-MS. Besides, in vitro elicitation has been achieved employing exogenous elicitors such as chitosan, proline, and salicylic acid on psoralen, daidzein and genistein accumulation in cotyledon callus cultures of C. corylifolium. Rf = Distance travelled by the solute Distance travelled by the solvent 2.2.2. Column chromatography Mobile phase was carefully chosen on the basis of resolution of crude extract on TLC plates and proceeded for the isolation of bioactive compounds through column chromatography. Fifteen grams of crude extract was mixed with 8 g of dry silica gel (100–200 mesh) to prepare the slurry. An amber glass column (120 × 3 cm) was then packed carefully with silica gel powder and loaded the slurry of the crude extract using hexane. Once the column was packed, the compounds of the crude extract were isolated by using the Toluene: Ethyl acetate as mobile phase of increasing polarity (0, 0.5, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 %). The flow rate of the column was kept at 0.16 mL/s. The eluted fractions from column chromatography were again analyzed through TLC, using Toluene: Ethyl acetate (75: 25 v/v) as mobile phase. The Rf values of each spot on TLC plates of every fraction were recorded. Afterward, the fractions having compounds with similar Rf values were pooled together. Subsequently, the weight (in milligrams) of each respective fraction was measured. 2.2.3. Characterization of isolated compounds 1 H & C13-nuclear magnetic resonance (NMR) spectra of the isolated and purified compounds were observed in solvents CDCl3 and DMSO-d6 using NMR (Bruker Spectrospec), DPX-300 MHz, where internal standard was tetramethylsilane. High Resolution Mass Spectrometry (HRMS) spectrum was recorded using a Shimadzu UV-2501PC instrument (Agilent Technology). 2.3. Elicitation of psoralen, daidzein and genistein in cotyledon callus cultures 2.3.1. Induction of cotyledon callus Green seeds were collected from previously mentioned place and washed thoroughly under running tap water for 15 min and treated with 3 % (v/v) Teepol detergent (Rickett and Column India Ltd., Kolkata, India) with vigorously shaking for 10 min, followed by thorough rinsing with distilled water. These seeds were treated with 1 % (w/v) bavistin fungicide (Bavistin® Carbendazim 50 % WP, BASF India Ltd., Mumbai, India) + 150 mg/L cefotaxime (Alkem, Sikkim, India), for 15 min with constant shaking and then further rinsed with distilled water to eliminate any traces of bavistin. These seeds were surface sterilized with 0.1% (w/v) aqueous Mercuric Chloride (Merck, Mumbai, India) solution for 1 min under laminar airflow cabinet and finally rinsed 3 times with autoclaved distilled water. Cotyledons were excised from the green seeds for callus induction. Gamborg B5 (Gamborg et al., 1968) medium containing growth regulators N6- benzyladenine (BA) and Indole-3-butyric acid (IBA) (Sigma Aldrich, USA) in different concentrations (1, 5 and 10 μM) were used either alone or in combination for cotyledon callus induction. The pH was adjusted to 5.8. Cotyledon callus cultures were maintained in the culture room at 25 ± 2 °C temperature and 45 ± 10 % relative humidity with photoperiod of 16/ 8 h (Light/Dark) under the 30–40 μmol/m2 s cool white fluorescent tubes (Philips India Ltd., Kolkata, India). 2. Materials and methods 2.1. Preparation of crude extract of green seeds For the preparation of crude extract, the green seeds (100 g) of C. corylifolium were collected from the plants grown in the fields of Botanical Garden, Department of Botany, University of Delhi, India (77.21 °E, 28.68 °N) and washed thoroughly under running tap water. The green seeds were then coarsely ground and then extracted with 100 % methanol. The solvent-tissue slurry was then kept on incubator shaker for 48 h at 25 ± 2 °C with continuous stirring, and further filtered with Whatman No.1 filter paper (Whatman™, GE Healthcare UK Limited, Amersham Place, UK). Subsequently, the filtrate was air-dried in dark and stored at 4 °C for further experiments. 2.2. Isolation of bioactive compounds from the crude extract of green seeds 2.2.1. Thin layer chromatography Thin layer chromatography (TLC) was carried out for the selection of appropriate solvent system and separation of bioactive compounds available in crude extract of green seeds. The glass jars (Borosil®, Mumbai, India), were filled with different solvents such as Chloroform (100 %), Ethyl acetate: Methanol (30:70), Ethyl acetate: Acetone (40:60), Toluene: Ethyl acetate (75:25), and Toluene: Ethyl acetate (93:7). The jars were then covered and kept untouched for 10 min to get fully saturated with solvent vapour. Silica Gel 60 F254 precoated aluminum TLC plates (Merck, Mumbai, India) were used as a stationary phase. The extract was loaded on the TLC plates (15 × 2 cm) and kept in the jars containing solvents. To visualize resolution, the plates were 2.3.2. Elicitor preparation Elicitors such as chitosan (Medium molecular weight; 90 % Deacetylation degree; Sisco Research Laboratories Pvt. Ltd., Mumbai, India), proline (Sigma Chemical Co., USA) and salicylic acid (Sisco Research Laboratories Pvt. Ltd., Mumbai, India) were used in the current study for elicitation of anticancerous compounds such as psoralen, daidzein and genistein. Chitosan was initially dissolved in a minimum volume of 1 N HCl then made up to final concentration with sterile 2 Industrial Crops & Products 143 - T. Singh, et al. as F1 (fractions 1–2), F2 (fractions 3–4), F3 (fractions 5–10), F4 (fractions 11–12), F5 (fractions 13–14), F6 (fractions 15–18), F7 (fractions 19–22), F8 (fractions 23–35), F9 (fractions 36–65) and F10 (fractions 66–101), respectively. The TLC of fractions F2, F3 and F5 revealed the presence of single spot each. These spots were then labelled as compounds A, B, and C with Rf values of 0.19, 0.31 and 0.24, respectively. The Rf values (0.19, 0.31 and 0.24) of compounds A, B and C matched with the Rf values of standard compounds daidzein, psoralen and genistein, respectively. Further the fractions F2, F3, and F5 were identified and characterized through HReMS & NMR (Fig. 1). The final amount of the isolated compounds A, B and C were 18, 14 and 12 mg, respectively. 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. Prepared salicylic acid was then filter sterilized using 0.2–μm microfilter (Axiva Sichem Pvt. Ltd., India) before adding to the culture medium. However, proline was dissolved in sterile distilled water only. 2.3.3. Elicitation treatments The cotyledon calluses (87–96 mg FW) were cultured on B5 (Gamborg et al., 1968) medium containing optimized growth regulators and supplemented with different concentrations of chitosan (0, 1, 5, 25, 50, 100, 200 mg/L), proline (0, 1, 5, 25, 50, 100, 200 mg/L) and salicylic acid (0, 1, 5, 25, 50, 100, 200 μM). Culture medium without elicitors considered as control. The calluses of each treatment were harvested and fresh weight was measured after 30 d of incubation period. These calluses were further used for quantification of bioactive compounds such as psoralen, daidzein and genistein. 3.2. Characterization of isolated compounds The 1H-NMR spectrum of the compound F3 showed peaks at δ 6.35 (s), 7.80 (s), 7.69 (s), 7.67 (s), 6.69 (s), and 7.46 (s), which corresponds to the protons H-1, H-2, H-3, H-4, H-5 and H-6, respectively. NMR peak at δ 7.25 (s) region belonged to the deuterated chloroform (CDCl3) solvent (Fig. 1A, Table 2). In correlation to this, the C13-NMR spectrum of the compound F3 showed peaks at δ 115.22 (CH), 152.18 (C), 120.0 (C), 106.44 (CH), 156.58 (C), 144.51 (C), 99.85 (CH), 125.10 (C), 114.52 (CH), 147.06 (CH) and 161.33 (C) which corresponds to the carbon C1, C2, C3, C4, C5, C6, C7, C8, C9, C10 and C11, respectively. Whereas, three peaks at δ 77.73, 77.20 and 76.66 region belongs to the deuterated chloroform (CDCl3) solvent (Fig. 1B, Table 2). Similarly, 1H-NMR spectrum of the compound F2 showed peaks at δ 6.80 (s), 7.90 (s), 7.33 (d, J = 8.7 Hz), 6.76 (d, J = 8.7 Hz), 8.24 (s) and 6.87 (s), which has been attributed to protons H-1, H-2, H-3, 6, H-4, 5, H-7 and H-8, respectively (Fig. 1C, Table 2). The 1H-NMR spectrum of the compound F5 showed peaks at δ 6.34 (s), 7.33 (d, 17.7 Hz), 6.78 (d, 8.7 Hz), 8.29 (s) and 6.20 (s), which has been attributed to protons H-1, H-2, 5, H-3,4, H-6, and H-7, respectively (Fig. 1D, Table 2). Besides, three broad singlet peaks at δ 12.94, 9.56 and 10.84 represented the presence of hydroxyl group. According to the HR-MS analysis, the molecular formulae of the extracted compounds A, B and C from their positive ESI-TOF-MS data (m/z-,- and- [M+H]+) was calculated as C15H10O4, C11H6O3, and C15H10O5, respectively (Fig. 1A). However, the HR-MS data also revealed the presence of active compounds such as bavachin, daidzein, psoralen, bakuchiol, genistein, corylifol E and bavachalcone or bavachinin in the respective isolated fractions F1, F2, F3, F4, F5, F6, and F7. The TLC of these fractions also revealed the presence of many more compounds among which few compounds were identified. On the basis of the proton NMR spectrum, molecular mass was obtained through HR-MS and their respective Rf values, the fractions F2, F3, and F5 were identified as purified compounds; Compound A (daidzein): 7-hydroxy-3-(4-hydroxyphenyl) chromen-4-one, compound B (psoralen): Furo (3, 2-g) chromen-7-one, and compound C (genistein): 5, 7-dihydroxy-3-(4-hydroxyphenyl) chromen-4-one. This is our first report of isolation of genistein from green seed crude extract of C. corylifolium. 2.3.4. HPLC analysis and quantification of psoralen, daidzein and genistein After treatments with different organic elicitors such as chitosan, proline and salicylic acid, fresh samples of green seeds and cotyledon callus of C. corylifolium were crushed in liquid N2 and extracted in methanol (Merck, Kenilworth, NJ) for 48 h with continuous shaking in dark at 25 ± 2 °C. The methanolic extract was filtered using Whatman No.1 filter paper and allowed to concentrate in dark at room temperature. The amber glass bottles were used to store dried extract. 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 519 G). Bioactive compounds were separated by using Supelcosil C18 column (L × I.D. 250 × 4.6 mm; Sigma-Aldrich®). Psoralen content was determined using methanol (100 %) as mobile phase and monitored at 243 nm. In contrast, daidzein and genistein content were quantified using freshly prepared acetonitrile: water (40: 60 v/v) and monitored at 250 nm. Peak identification was done by comparing the retention time of standards psoralen, daidzein and genistein (Sigma-Aldrich, USA) with that of the samples. All the chemicals used were of HPLC grade. 2.4. Data analysis and statistics Each experiment was carried out thrice and the data were represented as Mean ± SE. Significant difference between the means of control and different elicitor treatments were analysed by one way ANOVA using Duncan’s Multiple Range Test at P ≤ 0.05 through SPSS software. 3. Results and discussion 3.1. Isolation of bioactive compounds from crude extract of green seeds In order to isolate the bioactive compounds the methanolic green seed crude extract of C. corylifolium was subjected to fractionation using column chromatography. Of all the different solvent system combinations [chloroform (100 %), Ethyl acetate: Methanol (30:70), Ethyl acetate: Acetone (40:60), Toluene: Ethyl acetate (75:25) and Toluene: Ethyl acetate (93:7)] evaluated, Toluene: Ethyl acetate (75:25 v/v) showed best resolution of maximum 10 spots on TLC plates, with respective Rf values being 0.75, 0.72, 0.60, 0.48, 0.43, 0.40, 0.30, 0.19, 0.16, 0.09 (Table 1). Hence, the solvent combination of Toluene: Ethyl acetate (75:25 v/v) was chosen for the elution of compounds in the crude extract. While performing the column chromatography, it was observed that the green seed extract resolved into various colored bands under the influence of Toluene: Ethyl acetate gradient. At the end, a total of 101 fractions were obtained. The isolated fractions having compounds with similar Rf values were pooled into ten fractions 3.3. Elicitation of psoralen, daidzein and genistein in cotyledon callus cultures 3.3.1. Induction of cotyledon callus Cotyledon excised from green seeds when cultured on Gamborg B5 medium augmented with 5 μM BA + 10 μM IBA, a significant amount of callus was obtained within 30-d (Fig. 2A–C). The callus was used both for elicitation and further proliferation to increase biomass production. 3.3.2. Elicitation treatments A significant enhancement in callus biomass was recorded when 3 Industrial Crops & Products 143 - T. Singh, et al. Table 1 Resolution of the C. corylifolium green seed extract loaded on the TLC plate using different solvents. Solvents Volume (mL : mL) Number of spots Rf values Chloroform Ethyl acetate: Methanol Ethyl acetate: Acetone Toluene: Ethyl acetate 100 30 : 40 : 75 : 93 : 6 3 2 10 4 0.66, 0.71, 0.71, 0.75, 0.47, - 0.51, 0.65, 0.67 0.72, 0.33, 0.35, 0.22, 0.12,-, 0.48, 0.43, 0.40, 0.30, 0.19, 0.16, 0.09 0.18, 0.12 Fig. 1. A–B. 1H-NMR and C13-NMR spectra of the isolated fraction 3 (F3 inset), confirming the structure of psoralen; (C) 1H-NMR spectrum of the isolated fraction 2 (F2 inset), confirming the structure of daidzein; (D) 1H-NMR spectrum of the isolated fraction 5 (F5 inset), confirming the structure of genistein. (760.61 ± 34.35 mg FW) after 30-d (Figs. 2D and E, 3A, and Table 3). However, higher concentrations such as 100 mg/L and 200 mg/L of chitosan significantly inhibited the growth and callus biomass production (Fig. 2F). Similar report has appeared recently, Khan et al. (2019), who found that 65.5 μM chitosan increased the production of callus biomass in callus cultures of Fagonia indica. The maximum biomass production was also observed at 100 μM chitosan in micropropagated Stevia rebaudiana (Bayraktar et al., 2016). Apart from this, chitosan stimulates plant growth and development (Brasili et al., 2016), improves seed germination, quality of fruits with enhanced storage life (Jiang and Li, 2001) and alters the metabolic profile of plants (Sathiyabama et al., 2016). Similar to chitosan, cotyledon callus biomass production increased when exposed to increasing concentrations upto 25 mg/L of proline where maximum 3184.16 ± 112.29 mg FW (150 %) callus biomass was observed over control (1271.20 ± 109.38 mg FW) after 30-d (Figs. 2G, 3B, and Table 3). However, biomass started declining when concentration of proline increased from 50 mg/L to 200 mg/L (Fig. 2H). Earlier, Gupta et al. (2015a), reported increased biomass production in Table 2 1 H-NMR data of compounds A, B & C in solvents CDCl3 and DMSO-d6 . No. Compound A Compound B Compound C H-1 H-2 H-3 H-4 H-5 H-6 H-7 H-8 OH-a OH-b OH-c - ― ― ― ― ― - ― ― ― 6.34 (s) 7.33 (d) [17.7 Hz] 6.78 (d) [8.7 Hz] 6.78 (d) [8.7 Hz] 7.33 (d) [17.7 Hz] 8.29 (s) 6.20 (s) ― 12.94 (s) 9.56 (s) 10.84 (s) (s) (s) (s) (s) (s) (s) (s) (s) (d) (d) (d) (d) (s) (s) [8.7 Hz] [8.7 Hz] [8.7 Hz] [8.7 Hz] cotyledon calluses were treated with increasing concentrations (1, 5, 25, 50, 100, 200 mg/L) of chitosan. Maximum cotyledon callus biomass production was achieved with 50 mg/L of chitosan 1895.04 ± 86.16 mg fresh weight (FW) (149 %) over control 4 Industrial Crops & Products 143 - T. Singh, et al. Fig. 2. A–J. Induction and proliferation of callus biomass of C. corylifolium through green seed cotyledon using elicitors. (A) A twig of C. corylifolium bearing flowers and seeds; (B) green seeds of C. corylifolium; (C) cotyledons from green seeds inducing callus on B5 + 5 μM BA + 10 μM IBA medium after 30-d of culture; (D) callus raised on B5 + 5 μM BA + 10 μM IBA, without elicitors (control); (E) optimum callus proliferation at 50 mg/L chitosan; (F) callus showing declined growth at 200 mg/L chitosan; (G) optimum growth of callus at 25 mg/L proline; (H) callus showing declined growth at 200 mg/L proline; (I) optimum growth of callus at 5 μM salicylic acid; (J) callus showing extremely reduced and inhibited growth at 200 μM salicylic acid. (Red scale bars = 1 cm). with increasing concentrations (1, 5, 25, 50, 100, 200 mg/L) of chitosan (Fig. 4C and Table 4). In contrast, a steep increase in daidzein content was recorded with increasing concentrations of chitosan. Maximum psoralen and daidzein synthesis of 4728.27 ± 7.10 μg/g FW (7.2–fold) and- ± 8.81 μg/g FW (11.2–fold), respectively, were achieved with 50 mg/L chitosan over their respective controls (573.96 ± 6.12 μg/g FW and 1395.18 ± 2.02 μg/g FW) after 30-d treatment. However, highest genistein accumulation of 1596.47 ± 5.86 μg/g FW (6.21–fold) was observed at 100 mg/L chitosan over control of 244.41 ± 2.8 μg/g FW. A decrease in psoralen content was seen beyond 50 mg/L. However, at 200 mg/L chitosan a sharp decline in psoralen content was observed (Fig. 4C). Chitosan, a deacetylated chitin derivative has been reported to enhance the accumulation of bioactive compounds such as; Ahmed and Baig (2014), have reported increased psoralen quantity upto 7.4–fold at 125 mg/L chitosan in cell suspension cultures of Psoralea corylifolia. Chitosan treatment also increased the production of phenolic compounds especially brasilixanthone B in root cultures of Hypericum perforatum (Brasili et al., 2016). Cell suspension cultures of Barringtonia racemosa treated with 150 mg/L chitosan showed 1.3–fold increment in gallic acid content (Osman et al., 2018). Similarly, accumulation of plumbagin content increased upto 6.6-fold at 150 mg/L chitosan in root cultures of Plumbago indica (Jaisi and Panichayupakaranant, 2017). A recent study of Jiao et al. (2018), showed 7.08–fold enhancement in flavonoid content employing 150 mg/L chitosan in hairy root cultures of Isatis tinctoria and also reported the up-regulation of flavonoid biosynthesis precursor genes such as chalcone synthase and flavonoid 3′-hydroxylase. Kamalipourazad et al. (2016), reported that application of chitosan in cell cultures of Scrophularia striata induced antioxidant responses through enhanced activity of scavenging enzymes and phenylpropanoid compounds through induction of phenylalanine ammonia lyase and increase in amino acid content. The probable role of chitosan in the elicitation of secondary metabolites may be due to the induction of plant defense response through activation of MAP-kinases, elevation of cytosolic H+ and Ca2+, oxidative burst and hypersensitive response (Iriti and Faoro, 2009). Moreover, chitosan also interferes with plant signal transduction pathways and directly interacts with callus and cell suspension cultures of Stevia rebaudiana using 5 mM proline as an elicitor. Similarly, they also observed that higher concentrations of proline were inhibitory to the growth and production of callus biomass. In another study, Aljibouri et al. (2012) have shown highest callus biomass accumulation with 100 mg/L proline in Hyoscyamus niger. In contrast, Al-Jibouri et al. (2016), found proline not significantly enhanced the biomass production in callus cultures of Verbascus thapsus. Contrarily, in case of salicylic acid where cotyledon callus biomass production increased initially then started to decline beyond 5 μM concentration (Fig. 2I and J). It was observed that salicylic acid at 5 μM produced 1447.33 ± 20.66 mg FW (31 %) callus biomass over control (1104.66 ± 10.65 mg FW) after 30-d (Figs. 2I, 3 C, and Table 3). A negative effect of salicylic acid on the growth and biomass production have already been reported by many workers in different plant systems which included Stemona sp. (Chaichana and Dheeranupattana, 2012), Rosa hybrida (Ram et al., 2013), Rhinacanthus nasutus (Cheruvathur and Thomas, 2014). Besides this, salicylic acid also plays a critical role in the regulation of physiological and biochemical processes such as respiration (Rhoads and McIntosh, 1992), senescence (Morris et al., 2000), photosynthesis (Khodary, 2004), flowering (Martínez et al., 2004), and seed germination (Rajjou et al., 2006). Salicylic acid also plays an important role in the plant defense and induces defense related genes against pathogens (Loake and Grant, 2007). 3.3.3. Elicitation of psoralen, daidzein and genistein in cotyledon callus cultures Psoralen, daidzein and genistein content in cotyledon callus cultures were evaluated and quantified by HPLC (Fig. 4). Cotyledon derived calluses were exposed to different concentrations of elicitors such as chitosan, proline (1, 5, 25, 50, 100, 200 mg/L) and salicylic acid (1, 5, 25, 50, 100, 200 μM) to evaluate their effect on enhancement of psoralen, daidzein and genistein content. The cultures were analyzed after every 30-d. 3.3.3.1. Effect of chitosan. A gradual increase in psoralen and genistein synthesis was observed when cotyledon derived calluses were treated 5 Industrial Crops & Products 143 - T. Singh, et al. Fig. 3. A–C. Effect of different concentrations of elicitors on cotyledon callus biomass production in C. corylifolium. (A) chitosan (0, 1, 5, 25, 50, 100 and 200 mg/L); (B) proline (0, 1, 5, 25, 50, 100 and 200 mg/L), and; (C) salicylic acid (0, 1, 5, 25, 50, 100 and 200 μM). Different lowercase alphabets on bars represents significant differences P ≤ 0.05, FW; fresh weight. Table 3 Biomass production of C. corylifolium cotyledon callus cultures treated with different concentrations of organic elicitors. Elicitor Concentration* Biomass of cotyledon callus treated with elicitors (mg FW) Chitosan Control- Proline a Salicylic acid a 1271.20 ±- ± 25.62bc 3093.56 ± 133.77cd 3184.16 ± 112.29d 2792.40 ± 37.51b 2782.00 ± 21.07b 2689.00 ± 153.44b 760.61 ±- ± 11.79a 1157.33 ± 140.45bc 1216.66 ± 117.42c 1895.04 ± 86.16d 1015.50 ± 24.95ab 802.80 ± 14.40a 1104.66 ± 10.65e 1320.53 ± 19.44f 1447.33 ± 20.66g 738.43 ± 37.18d 523.00 ± 38.37c 357.10 ± 13.30b 257.66 ± 16.33a FW; fresh weight. * Concentration: mg/L (chitosan and proline); μM (salicylic acid). daidzein 2448.57 ± 5.04 μg/g FW (1.55–fold) was achieved with 100 mg/L over control 958.77 ± 13.06 μg/g FW. Regarding amino acids, they are considered as a good source of nitrogen and trigger the secondary metabolite production (Parast et al., 2011; Kumar et al., 2018). Similarly, hyoscyamine content increased to 58.03 % with 50 mg/L proline compared with the control in callus cultures of Hyoscyamus niger (Al-Jibouri et al., 2012). Steviol glycosides content increased upto 1.09 and 5.03 % using 7.5 mM proline in callus and cell suspension cultures of Stevia rebaudiana, respectively. Al-Jibouri et al. (2016) have also reported enhanced production of comarin (2752 %) and eugenol (290 %) at 150 mg/L and 50 mg/L proline, respectively, in callus cultures of Verbascus thapsus. In another recent study, Kumar chromatin which regulates gene expression. 3.3.3.2. Effect of proline. In contrast to the above two elicitors tried, a slight increase in psoralen, daidzein and genistein synthesis was observed when cotyledon calluses were treated with increasing concentrations (1, 5, 25, 50, 100, 200 mg/L) of proline (Fig. 4D and Table 4). Maximum accumulation of psoralen and genistein 1809.67 ± 13.38 μg/g FW (3.9–fold) and 1982.79 ± 3.57 μg/g FW (1.74–fold) was achieved with 50 mg/L over their respective controls (368.26 ± 7.31 μg/g FW and 721.64 ± 5.09 μg/g FW) after 30-d treatment. Beyond 50 mg/L gradual decline in quantity of psoralen and genistein was seen (Fig. 4D). Besides, maximum amount of 6 Industrial Crops & Products 143 - T. Singh, et al. Fig. 4. A–E. HPLC analysis showing chromatograms of standard compounds and effect of different elicitors on psoralen, daidzein and genistein synthesis in C. corylifolium callus cultures (after 30-d treatment). (A) Chromatogram of standard compound psoralen revealing peak at retention time of 2.996 min; (B) chromatogram of standard compounds daidzein and genistein revealing peaks at retention time of 10.55 and 16.94 min respectively; (C) effect of chitosan (0, 1, 5, 25, 50, 100 and 200 mg/L); (D) effect of proline (0, 1, 5, 25, 50, 100 and 200 mg/L), and; (E) effect of salicylic acid (0, 1, 5, 25, 50, 100 and 200 μM). Different lowercase alphabets on bars represents significant differences P ≤ 0.05, FW; fresh weight, d; day, pso; psoralen, D; daidzein, G; genistein. calluses of C. corylifolium were treated with different concentrations (0, 1, 5, 25, 50, 100 and 200 μM) of salicylic acid for 30-d (Fig. 4E and Table 4). It was observed that a tremendous enhancement in psoralen content was achieved at 5 μM of salicylic acid where the quantity increased to 5337.84 ± 6.33 μg/g FW (14.8–fold) over control (336.90 ± 1.15 μg/g FW). A sharp decrease in psoralen, content was seen beyond 5 μM to 200 μM (Fig. 4E). Prior to this, Hari et al. (2018), though have also reported 24.9–fold enhancement in psoralen in the leaf callus derived cell suspension culture but they had used a combination of very high amount of cinnamic acid (16.8 mM) and methyl jasmonate (100 μM). However, when they used combination of high amount of cinnamic acid (16.8 mM) and salicylic acid (100 μM), 1.57–fold lesser psoralen content was seen compared with former combination. While methyl jasmonate (100 μM) and salicylic acid et al. (2018) showed elicitation of connessine content upto 3.24–fold using 100 mg/L proline in green bark callus cultures of Hollarrhena antidysentrica. Apart from this, exogenous application of proline increased different antioxidative enzymes activities to protect cell membrane (Yan et al., 2000; Sobahan, 2018) and promoted seedling growth and germination in Oryza sativa (Singh et al., 2018) under salt stress. Proline acts like a precursor as well as inductor, an osmolyte and protective agent of the plasma membrane integrity. It has therefore been proved that proline is an effective elicitor whose treatment increases the level of amino acids in the free pool to get metabolized into secondary metabolites (Chetri et al., 2016). 3.3.3.3. Effect of salicylic acid. Dose dependent enhancement in psoralen, daidzein and genistein content was seen when cotyledon 7 Industrial Crops & Products 143 - T. Singh, et al. Table 4 Effect of organic elicitors on psoralen, daidzein and genistein content in cotyledon callus cultures of C. corylifolium. Organic Elicitors Type Concentration Chitosan Control 1 mg/L 5 mg/L 25 mg/L 50 mg/L 100 mg/L 200 mg/L Control 1 mg/L 5 mg/L 25 mg/L 50 mg/L 100 mg/L 200 mg/L Control 1 μM 5 μM 25 μM 50 μM 100 μM 200 μM Proline Salicylic acid Psoralen content (μg/g FW) Daidzein content (μg/g FW) Genistein content (μg/g FW) 573.96 ± 6.12b 801.77 ± 3.73c 1212.34 ± 6.62d 1381.82 ± 6.71de 4728.27 ± 7.10j 3625.44 ± 3.80h 984.70 ± 4.10c 368.26 ± 7.31ab 473.53 ± 11.20d 588.73 ± 6.88e 1120.93 ± 34.35i 1809.66 ± 13.38l 1717.86 ± 11.20k 603.60 ± 22.00e 336.90 ± 1.15d 1505.53 ± 2.33k 5337.83 ± 6.33n 723.10 ± 8.18h 450.90 ± 3.21g 411.76 ± 3.48f 384.93 ± 33.57ef 1395.18 ± 2.02de 2729.51 ± 5.48g 3785.26 ± 2.84h 4109.73 ± 6.02i- ± 8.81l 7491.9 ± 5.77k 114.23 ± 0.88a 958.77 ± 13.06h 1098.34 ± 4.41i 1145.69 ± 4.60i 1289.56 ± 6.22j 2078.56 ± 10.46n 2448.57 ± 5.04° 450.98 ± 4.58cd 909.09 ± 9.00j 1753.79 ± 10.32l 5331.73 ± 17.64n 230.43 ± 8.16c 108.46 ± 4.24b 53.74 ± 4.66a 37.49 ± 6.71a 244.41 ± 2.80a 535.17 ± 2.64b 571.04 ± 2.33b 644.25 ± 2.13c 1596.47 ± 5.86ef 1762.6 ± 9.17f 95.92 ± 3.17a 721.64 ± 5.09f 811.14 ± 3.80g 934.09 ± 2.36h 1739.36 ± 2.92l 1982.79 ± 3.57m 402.77 ± 3.55bc 315.24 ± 2.37a 374.88 ± 5.32e 791.53 ± 2.65i 2438.74 ± 3.93m 107.12 ± 4.05b 96.86 ± 0.74b 64.91 ± 2.65a 50.22 ± 2.57a FW, fresh weight. (100 μM) when used alone showed 13.6 and 10.2–fold increment in psoralen content, respectively. Contrary to this, by using very less amount of salicylic acid (5 μM) alone an enormous enhancement, i.e. 14.8–fold was achieved in psoralen in present investigation. Interestingly, maximum content of daidzein and genistein was also achieved at 5 μM of salicylic acid where their quantities increased to 5331.73 ± 17.64 μg/g FW (4.8–fold) and 2438.74 ± 3.93 μg/g FW (5.5–fold), respectively, when compared with their respective controls (909.09 ± 9.00 μg/g FW and 374.88 ± 5.32 μg/g FW). A sharp decrease in daidzein and genistein contents were also seen beyond 5 μM to 200 μM. Daidzein and genistein synthesis were also reported previously in Psoralea corylifolia cell suspension and hairy root cultures, but enhancement was very low. Shinde et al. (2009b) reported 2.25–fold increased production of daidzein (3.4 % dry wt) and genistein (0.41 % dry wt) using 1 mM salicylic acid as elicitor in root callus derived cell suspension cultures of Psoralea corylifolia. Similarly, in hairy root cultures of Psoralea corylifolia enhanced daidzein (2.2 % dry wt) and genistein (0.228 % dry wt) content was observed using 1 mM salicylic acid after 2 days of elicitation (Shinde et al., 2009a). Contrarily, Gueven and Knorr (2011) found that salicylic acid had negative effect on biosynthesis of isoflavones in cell suspension cultures of Glycine max. Incidentally, during current investigation, daidzein and genistein synthesis enhanced upto 4.8–fold and 5.5–fold, respectively, using very low concentration of salicylic acid (5 μM) in cotyledon callus cultures of C. corylifolium. The probable reason for an enhancement using salicylic acid could be due to the activation of key intermediate enzymes of biosynthetic pathways of secondary metabolites, generation of ROS, ion fluxes and cytoplasm acidification (Vasconsuelo and Boland, 2007; Rahimi et al., 2014; Fesel and Zuccaro, 2016). Similar to this, some reports are also on record where salicylic acid enhanced the accumulation of bioactive compounds. Yu et al. (2006) have reported 2.5 and 2.7–folds increase in jaceosidin and syringin content, respectively, using 20 μM salicylic acid in cell cultures of Saussurea medusa. Yousefzadi et al. (2010) also reported improved podophyllotoxin content three times more than control in Linum album using 10 μM salicylic acid. Hypericin and pseudohypericin content increased to 7.98 and 13.58–folds, respectively, with 50 μM salicylic acid in shoot cultures of Hypericum hirsutum (Coste et al., 2011). Similarly, Sharma and Agrawal (2018) also found 25 μM salicylic acid effective for enhancing the plumbagin content upto 3.4–fold in root callus cultures of Plumbago zeylanica. It is also proved that lower concentrations of salicylic acid (10 and 20 μM) were effective for improving the production of secondary metabolites in callus cultures of Fagonia indica (Khan et al., 2019). Contrarily, higher concentrations of salicylic acid also seen to enhanced the bioactive compounds production. Taxol production enhanced using 20 mg/L (Wang et al., 2007) and 200 mg/L (Khosroushahi et al., 2006) salicylic acid in cell suspension cultures of Taxus chinensis var. mairei and T. baccata, respectively. Coste et al. (2011) proved 200 μM salicylic acid effective for enhancing the accumulation of hypericin and pseudohypericin upto 2.2 and 3.94–folds, respectively, in shoot cultures of Hypericum maculatum. Similarly, Chodisetti et al. (2015) showed 4.9–fold enhancement in gymnemic acid using 200 μM salicylic acid in cell suspension cultures of Gymnema sylvestre. 3.4. Comparative analysis of bioactive compounds Green seeds of C. corylifolium possess maximum amount of psoralen content (3772.78 ± 8.1 μg/g FW) followed by daidzein (1183.45 ± 8.85 μg/g FW) and genistein (362.78 ± 5.05 μg/g FW). However, cotyledon callus being the young and meristematic tissue significantly contains less amount of psoralen (526.37 ± 44.76 μg/g FW) and genistein FW), daidzein (922.72 ± 18.19 μg/g (310.86 ± 37.68 μg/g FW) as shown in Fig. 5. However, by exposing callus tissue to different elicitors such as chitosan, proline and salicylic acid tremendous enhancement in all the compounds psoralen (14.8–fold), daidzein (11.2–fold) and genistein (6.21–fold) was observed. Seeds are considered as main site for the synthesis and storage of essential compounds including secondary metabolites required for plant growth and development, defense etc. Parast et al. (2012) showed that seeds of Psoralea corylifolia contain highest psoralen content compared to other plant parts. 4. Conclusion The current investigation for the first time reports the isolation of genistein, besides, two already reported compounds psoralen and daidzein from crude extract of green seeds of C. corylifolium. Green seeds possess higher quantities of psoralen, daidzein and genistein compared with cotyledon callus of C. corylifolium. However, callus has 8 Industrial Crops & Products 143 - T. Singh, et al. V., Tocci, N., Valletta, A., Pasqua, G., 2016. Metabolic profile and root development of Hypericum perforatum L. in vitro roots under stress conditions due to chitosan treatment and culture time. Front. Plant Sci. 507, 1–12. Chaichana, N., Dheeranupattana, S., 2012. 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Khosroushahi, A.Y., Valizadeh, M., Ghasempour, A., Khosrowshahli, M., Naghdibadi, H., Fig. 5. Graph representing comparison of psoralen, daidzein and genistein contents in between green seeds and cotyledon callus of C. corylifolium. Different lowercase alphabets on bars represents significant differences P ≤ 0.05, FW; fresh weight. strongly proved its potential to proliferate and synthesize large amount of compounds if exposed to different elicitors. The cotyledon callus cultures of C. corylifolium showed maximum production of biomass using 25 mg/L proline and 50 mg/L chitosan. Contrarily, salicylic acid beyond 5 μM showed negative effect on biomass production. The current investigation have also showed a remarkable enhancement in psoralen, daidzein, and genistein content upto 14.8–fold, 11.2–fold, and 6.21–fold, respectively, in the cotyledon callus cultures of C. corylifolium using low concentrations of salicylic acid and chitosan. Thus, the results of the current study support that elicitation is an effective strategy for enhancing the accumulation of bioactive compounds in in vitro culture system. This is a cost effective and industrially viable protocol for the production of important bioactive compounds psoralen, daidzein and genistein. Author’s contributions VA designed experiments, monitored, assisted in writing and edited the manuscript. TS and RY performed the experiments and also wrote the manuscript. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgements The authors are grateful to the Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India, New Delhi for the sanction of a Major Research Project Grant No. (EMR/2016/001673) to VA. TS is also thankful to SERB for Junior Research Fellowship. RY is thankful to DU-UGC for UGC Non-NET Fellowship. References Ahmed, S.A., Baig, M.M.V., 2014. 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