http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2015; 53(5): 735–738 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.942786
ORIGINAL ARTICLE
In vitro enhancement of psoralen as an important anticancer compound in Psoralea corylifolia through precursor feeding Behrooz Mohammadparast1, Ali Reza Rustaiee2, Mousa Rasouli3, Sannaz Zardari4, and Veena Agrawal5 Department of Biology, Faculty of Science, University of Malayer, Malayer, Iran, 2Department of Horticultural Sciences, Agriculture Research Center, University of Zabol, Zabol, Iran, 3Department of Landscape Engineering, Faculty of Agriculture, University of Malayer, Malayer, Iran, 4Faculty of Agriculture, University of Tabriz, Tabriz, Iran, and 5Department of Botany, North Campus, University of Delhi, Delhi, India
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1
Abstract
Keywords
Context: Psoralea corylifolia L. (Fabacese) is rich source of bioactive compounds, which endows the plant with immense value for its use in pharmaceuticals, health, and body-care products. Objective: The current study was designed (i) for the determination of psoralen from callus derived from different plant parts, and (ii) for the enhancement of psoralen in in vitro condition with the treatment of various psoralen pathway precursors. Materials and methods: B5 media were employed for raising the cultures from different plant parts such as leaf, node, root, and green seeds. Cotyledons’ calluses were derived from cotyledon of green seeds that were elicited on MS + 10 mM BA + 5 mM IBA medium supplemented at 0.1, 1, 2.5, 5, 25, and 50 mg/L of various psoralen pathway precursors such as umbelliferone, cinnamic acid, and NADPH. The method for extraction of psoralen was modified from the Singh method and the content of psoralen was measured using HPLC. Results: HPLC analysis of callus derived from different parts of P. corylifolia revealed that a maximum of psoralen (2601.8 mg/g fresh wt.) was recorded in cotyledons’ callus. Cotyledonary callus was chosen for the enhancement of psoralens because of higher amount of psoralen in it. In vitro evaluation showed that all the precursors enhanced the psoralen amount dramatically so that the optimum amount of psoralen (2518.8 mg/g fresh wt.) was obtained at 2.5 mg/L cinnamic acid. Discussion and conclusion: The results obtained indicate that cinnamic acid is one of the important precursors of psoralen pathway that induced a maximum amount of psoralen with in vitro conditions.
Anticancerous, biosynthesis pathway, cinnamic acid, medicinal plants, umbelliferone
Introduction Medicinal plants are nearly unlimited sources of secondary metabolites. The presence of comparatively higher content of secondary metabolites in medicinal plants has endowed them as important sources of phytomedicines. Therefore, it is imperative to develop approaches for enhancing the bioactive compounds, isolating the phytochemical constituents, developing methods for large-scale in vitro productions (Pandey & Agrawal, 2009). Some strategies for the production of the metabolites in culture have been developed to improve the yield of plant secondary metabolites. These include treatment with various elicitors, signal compounds, and abiotic stresses (Zhao et al., 2005). Many such treatments indeed effectively promote the production of a wide range of plant secondary metabolites, both in vivo and in vitro. Psoralea corylifolia L. (Fabaceae) is an important medicinal plant that has been included in the list of threatened
Correspondence: Behrooz Mohammadparast, Department of Biology, Faculty of Science, University of Malayer, Malayer 65719-95863, Iran. Tel: +98 9188724625. E-mail: [email protected]
History Received 23 June 2013 Revised 13 April 2014 Accepted 23 June 2014 Published online 21 October 2014
plants (Anonymous, 1989; Bhattacharjee, 1998; Jain, 1994). Psoralea corylifolia is a rich source of bioactive compounds, which endows the plant with an immense value for its use in pharmaceuticals, health, and body-care products. It is being used for various biomedical applications. The seeds of this plant are anthelminthic, antibacterial, aphrodisiac, astringent, cardiac, cytotoxic, deobstruent, diaphoretic, diuretic, stimulant, stomachic, and tonic (Anonymous, 1989; Joshi, 2000). It is also externally used to treat various skin ailments including leprosy, leucoderma, hair loss, and treating vitiligo as well as psoriasis (Anonymous, 1989; Joshi, 2000; Kornhauser et al., 1982). Triplex-formation oligonucleotides, attached with a photoreactive psoralen molecule, can be used to induce site-specific DNA damage and control gene expression (Ping & Pana, 2005). Seed extract of P. corylifolia inhibits tumor growth and stimulates natural killer activity (Latha et al., 2000). Psoralen has been evaluated to inhibit the in vitro growth of three human tumor cell lines, representing different tumor types, MCF-7 (breast cancer), NCI-H460 (non-small cell lung cancer), and SF-268 (CNS cancer). The results showed the efficiency of the psoralen in inhibition of cancer (Oliveira et al., 2006). Due to the complex bioactivity
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Pharm Biol, 2015; 53(5): 735–738
of psoralen, its biosynthesis pathway may enable us to influence its formation in a direct way, for example, by metabolic pathway engineering. The biosynthetic pathways to the linear furanocoumarin (psoralen) involved precursors and enzyme cofactors (Buchanan et al., 2000). The basis of linear furanocoumarin formation was mostly established by the end of the 1980s by a combination of precursor-feeding experiments and the biochemical characterization of major enzymes of the pathway (Larbat et al., 2007). The present study highlights (i) the evaluation of the psoralen content in callus derived from different plant parts of P. corylifolia, and (ii) in vitro elicitation of psoralenemploying precursors of the psoralen biosynthetic pathway.
Sample preparation and estimation of psoralen
Material and methods
HPLC determination of psoralen from callus derived from different plant parts
Plant materials The seeds of P. corylifolia were procured from Homeopathic Pharmacopoeia Laboratory, Gaziabad (Uttar Pradesh, India) and were sown in the seed beds of Botanical Garden, Department of Botany, University of Delhi, in the month of March. Voucher specimen was deposited in the Herbarium section (Accession no. DUH-13553) of the Department of Botany, University of Delhi, Delhi, India. The leaf, node, root, and green seeds of P. corylifolia were taken from field-grown mature plants. These explants were washed thoroughly under running tap water for 20 min and treated with 1% bavistin (w/v), for 10 min with constant vigorous shaking on the rotary table top shaker at 150 rpm, to provide better surface content with the fungicide. These were rewashed under running tap water to remove any traces of bavistin. After pouring out excess of water, the explants were surface sterilized with 0.1% (w/v) aqueous mercuric chloride solution for 2 min and finally washed 4–5 times with sterilized distilled water. Culture media B5 media (Gamborg et al., 1968) was employed to raise the cultures of P. corylifolia. Analytical grade (AR) salts (Qualigens or Glaxo Fine Chemicals, Mumbai, India) were used to prepare the stock solutions. The basal medium was supplemented with various growth regulators such as N6-benzyladenin (BA), indole-3-butyric acid (IBA) (Sigma Aldrich, St. Louis, MO), and precursors of psoralen (cinnamic acid, NADPH, and umbelliferone: 0.1, 1, 2.5, 5, 25, and 50 mg/L). The media were gelled with 0.8 % agar (Qualigens, Mumbai, India) and the pH of media was adjusted to 5.8 using 0.1 N NaOH or HCl before autoclaving. Approximately, 20 mL media was dispensed in each 2.15 15 cm test tubes (Brosil, Qualigens, Mumbai, India) plugged with nonabsorbent cotton wrapped in muslin cloth and was autoclaved at 1.06 kgcm 2 at 121 C for 15 min. Raising and incubation of cultures Cultures were incubated in continuous light of 400–500 mw/ cm2 by cool day light fluorescent incandescent tubes (40 W, Philips, Kolkata, India). The cultures were maintained in a culture room at the temperature of 25 ± 2 C and 55 ± 10% relative humidity. Observations were recorded at an interval of 7 d. The final data were recorded after 30 d of inoculation.
The method for extraction of psoralen was modified from the Singh (2003) method. The fresh samples (1 g, each) of plant tissue were crushed with liquid nitrogen and were soaked in ethanol for 24 h, darkness and then homogenized using pestle and mortar. After evaporation of ethanol, the semisolid form of extract was mixed in methanol (HPLC grade). This mixture was transferred to a centrifuge tube and centrifuged for 15 min at 12 000 rpm at room temperature. The pellet was discarded and the supernatant was filtered using a 0.22 mm milipore filter.
Results
In this study, psoralen as an important anticancer compound has been reported from P. corylifolia cotyledon callus for the first time. Prior to this, psoralen has been extracted from seeds of P. corylifolia (Qiao et al., 2006; Rajput et al., 2008; Ruan et al., 2007). HPLC chromatogram of psoralen standard showed a retention time at 5.4 min. Quantitative analysis of psoralen from the callus derived from different plant parts showed that a maximum of 2601.8 mg/g fresh wt. of psoralen was recorded in callus derived from cotyledons (Table 1). The quantity of psoralen in node-, leaf-, and root-derived callus was 1876.6, 1468.0, and 1062.0 mg/g fresh wt., respectively. It may be possible that cotyledons of seed contain a specific gene responsible for the synthesis of psoralen. In the absence of cotyledons, this particular gene is not fully expressed or less expressed in plant-derived callus. It can also be due to the development of secondary metabolism mechanism in callus derived from cotyledons. Contrary to some results (Innocenti et al., 1997; Rakhmankulov & Korotkova, 1975), the amount of psoralen in root was in fewer amounts when compared with other parts of P. corylifolia. In vitro evaluation of secondary metabolites from the cultures grown along with various precursors of psoralen through HPLC Precursors of biosynthetic pathways have been used in various plant cell and tissue cultures to increase the yield of secondary metabolites. Different precursors, namely, cinnamic acid, umbelliferone, and NADPH with the aim of increasing the psoralen content, were used in the present study. A marked variation in the psoralen content in cotyledonary callus cultures has been analyzed when they were elicited on MS + 10 mM BA + 5 mM IBA medium adjuvanted with different concentrations of umbelliferone, cinnamic acid, and NADPH. Although every concentration of umbelliferone enhanced the psoralen content compared with control, higher concentrations (25–50 mg/L) of umbelliferone proved to be better (Table 2). Umbelliferone at 25 mg/L induced the maximum amount of psoralen (2371.7 mg/g fresh wt.). Thus, it can be deduced that psoralen production in P. corylifolia in vitro cultures is explant and the precursor is dosedependent. In case of cinnamic acid, every concentration of it improved the psoralen content, but lower concentrations (0.1–5 mg/L) proved to be beneficial over higher level. The
Psoralea corylifolia and psoralen production
DOI: 10.3109/13880209.2014.942786
Table 1. Psoralen estimates obtained from callus derived from different parts P. corylifolia. Callus derived from different plant parts Plant parts
Area (mAU)a
Psoralen quantity (mg/g fresh wt.)
139 008 100 262 78 429 56 729
2601.8 1876.6 1468.0 1062.0
Cotyledon callus Node callus Leaf callus Root callus a
Milliabsorbance units.
Table 2. Effect of some precursors on psoralen accumulation (mg/g fresh wt.) in cotyledonary callus of P. corylifolia, after 30d of incubation.
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Cotyledonary callus
Control 0 Umbelliferone (mg/l) 0.1 1 2.5 5 25 50 Cinnamic acid (mg/l) 0.1 1 2.5 5 25 50 NADPH (mg/l) 0.1 1 2.5 5 25
Area (mAU)a
Psoralen quantity (mg/g fresh wt.)
83 193.0
1932.8
107 494.1 108 910.0 120 175.0 123 138.0 126 718.0 125 731.0
2011.9 2038.4 2249.3 2304.8 2371.7 2353.3
130 108.0 130 782.0 134 577.0 133 519.0 121 398.0 112 563.0
2435.2 2447.8 2518.8 2499.4 2272.2 2106.8
109 059.0 113 921.6 126 814.2 124 153.0 123 789.0
2041.2 2132.2 2373.7 2333.7 2315.4
a
Milliabsorbance units.
maximum (2518.8 mg/g fresh wt.) of psoralen content was found at 2.5 mg/L of cinnamic acid that was the highest amount of psoralen which detected among all the precursors tried. The optimum psoralen content (2373.7 mg/g fresh wt.) was found at 2.5 mg/L of NADPH. Thereafter, a gradual decline in the response has been observed and 5 and 25 mg/L NADPH could induce only 2333.7 and 2315.4 mg/g fresh wt. of psoralen, respectively, which was significantly higher than that obtained on control (1932.8 mg/g fresh wt.) (Table 2).
737
to function as a precursor in other compounds such as taxoid, shikonin, phenylethanoid glycosides, melatonin, and serotonin synthesis as well as in the biosynthesis of other compounds in various plants (Liu et al., 2007; Mei et al., 2001). Additionally, psoralen synthesis could be activated with high concentrations of L-phenylalanine. It is possible that high concentration of L-phenylalanine promotes PAL (phenylalanine ammonia-lyase) pathway activity to catalyze cinnamic acid. The effectiveness of psoralen accumulation in callus cultures of P. corylifolia could be due to the limiting factor of the flux whereby an exogenous supply of a biosynthetic precursor to the culture medium might improve alkaloid accumulation if the endogenous level of these precursors is a limiting factor of the flux. All these observations suggest that exogenous application of organic elicitors could have induced a subset of secondary metabolite biosynthetic genes, which may modulate expression of genes and accumulation of compounds induced by elicitors. The biotic and abiotic elicitors can result in an enhancement of the secondary metabolite production. The stimuli are perceived by receptors which then result in the activation of the secondary messengers. All these messengers compose paralleling or crosslinking pathways to integrate these signals to regulation of transcription factors (TFs), which subsequently activate gene expression by transcription machinery. Most of the genes for secondary metabolites synthesis are late response genes (Zhao et al., 2005).
Conclusion In summary, short based on all the above points, the maximum quantity of psoralen was recorded in callus derived from cotyledons. The HPLC analysis of the derived cotyledon callus supplemented with different precursors of psoralen pathway such as umbelliferone, cinnamic acid, and NADPH indicated that cinnamic acid induced maximum amount of psoralen.
Declaration of interest The authors explicitly declare that there is no financial or personal relationship with any third parties or organizations whose interests could be positively or negatively influenced by the content of the article. The data presented in this article are from a Ph.D. Thesis (Behrooz Mohammadprast) performed in the Botany Department of Delhi University. Financial support of this study by Prof. Veena Agrawal is acknowledged.
Discussion Psoralen, an important furanocoumarin, is seen abundantly in the seeds of P. corylifolia. Due to the complex bioactivity of furanocoumarins, its biosynthesis has received continuous attention. The biosynthetic pathways to the linear furanocoumarin (psoralen) involved enzymes (and their cofactors) and precursors which are as follows: cinnamic acid, p-coumaric acid, umbelliferone, demethylsuberison, and marmasin (Buchanan et al., 2000). Precursors, namely cinnamic acid, umbelliferone, and NADPH, have substantially improved the production of psoralen. Incidentally, optimum enhancement was seen with cinnamic acid. Cinnamic acid was also shown
References Anonymous. (1989). Psoralea spp. In: The Wealth of India: A Dictionary of Indian Raw Materials and Industrial Products. New Delhi, India: PID, CSIR, 8:295–8. Bhattacharjee S. (1998). Membrane lipid peroxidation, free radical scavengers and ethylene evolution in Amaranthus as affected by lead and cadmium. Biol Plan 40:131–5. Buchanan BB, Gruissem W, Jones RL. (2000). Biochemistry and Molecular Biology of Plants. New Delhi, India: IK International Publishing House Private Limited. Gamborg OL, Miller RA, Ojima K. (1968). Plant cell cultures. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–8.
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Innocenti G, Dall-Acqua F, Guiotto A, Caporale G. (1977). Investigation of skin-photosensitizing activity of various kinds of Psoralea. Planta Med 31:151–5. Jain SK. (1994). Ethnobotany and Research in Medicinal Plants in India. New Delhi, India: National Book Trust, 185:153–68. Joshi SG. (2000). Medicinal Plants. New Delhi, India: Oxford & IBH Publishing Company Private Limited. Kornhauser A, Wamer WG, Giles AL. (1982). Psoralen phototoxicity: Correlation with serum and epidermal 8-methoxypsoralen and 5-methoxypsoralen in Guinea pig. Science 217:733–5. Larbat R, Kellner S, Specker S, et al. (2007). Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis. J Biol Chem 282: 542–54. Latha PG, Evans DA, Panikkar KR, Jayavardhanan KK. (2000). Immunomodulatory and antitumour properties of Psoralea corylifolia seeds. Fitoterapia 71:223–31. Liu JY, Guo ZG, Zeng ZL. (2007). Improved accumulation of phenylethanoid glycosides by precursor feeding to suspension culture of Cistanche salsa. Biochem Eng J 33:88–93. Mei X, Wu D, Cheng Q, et al. (2001). Effects of metabolic regulators on the biosynthesis of taxol and its analogue. Nat Prod Res Dev 13: 17–20. Oliveira AM, Raposo MM, Oliveira Campos AM, et al. (2006). Psoralen analogues: Synthesis, inhibitory activity of growth of
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human tumor cell lines and computational studies. Eur J Med Chem 41:367–72. Pandey V, Agrawal V. (2009). Bioprospecting of Spilanthes species – Micropropagation and bioassay guided isolation of larvicidal compounds against malaria and filarial vectors [Ph.D. thesis]. Delhi: Department of Botany, University of Delhi. Ping KH, Pana TM. (2005). Mechanisms of site specific psoralen photoadducts formation in triplex DNA directly by psoralenconjugated oligonocleotides. Biochemistry 44:2301–9. Qiao CF, Han QB, Mo SF, et al. (2006). Psoralenoside and isopsoralenoside, two new benzofuran glycosides from Psoralea corylifolia. Chem Pharm Bull 54:14–716. Rajput SJ, Zade V, Rai P. (2008). Studies on extraction, isolation and estimation of psoralen from the fruits of Psoralea corylifolia. Pharmacogn Mag 4:52–6. Rakhmankulov U, Korotkova EE. (1975). Dynamics of furocoumarine content in different parts of Psoralea drupacea. Rastitel Nye’Resursy 11:98–104. Ruan B, Kong LY, Takaya Y, Niwa M. (2007). Studies on the chemical constituents of Psoralea corylifolia L. J Asian Nat Prod Res 9:41–4. Zhao J, Davis LC, Verpoorte R. (2005). Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23:283–333.
ORIGINAL ARTICLE
In vitro enhancement of psoralen as an important anticancer compound in Psoralea corylifolia through precursor feeding Behrooz Mohammadparast1, Ali Reza Rustaiee2, Mousa Rasouli3, Sannaz Zardari4, and Veena Agrawal5 Department of Biology, Faculty of Science, University of Malayer, Malayer, Iran, 2Department of Horticultural Sciences, Agriculture Research Center, University of Zabol, Zabol, Iran, 3Department of Landscape Engineering, Faculty of Agriculture, University of Malayer, Malayer, Iran, 4Faculty of Agriculture, University of Tabriz, Tabriz, Iran, and 5Department of Botany, North Campus, University of Delhi, Delhi, India
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1
Abstract
Keywords
Context: Psoralea corylifolia L. (Fabacese) is rich source of bioactive compounds, which endows the plant with immense value for its use in pharmaceuticals, health, and body-care products. Objective: The current study was designed (i) for the determination of psoralen from callus derived from different plant parts, and (ii) for the enhancement of psoralen in in vitro condition with the treatment of various psoralen pathway precursors. Materials and methods: B5 media were employed for raising the cultures from different plant parts such as leaf, node, root, and green seeds. Cotyledons’ calluses were derived from cotyledon of green seeds that were elicited on MS + 10 mM BA + 5 mM IBA medium supplemented at 0.1, 1, 2.5, 5, 25, and 50 mg/L of various psoralen pathway precursors such as umbelliferone, cinnamic acid, and NADPH. The method for extraction of psoralen was modified from the Singh method and the content of psoralen was measured using HPLC. Results: HPLC analysis of callus derived from different parts of P. corylifolia revealed that a maximum of psoralen (2601.8 mg/g fresh wt.) was recorded in cotyledons’ callus. Cotyledonary callus was chosen for the enhancement of psoralens because of higher amount of psoralen in it. In vitro evaluation showed that all the precursors enhanced the psoralen amount dramatically so that the optimum amount of psoralen (2518.8 mg/g fresh wt.) was obtained at 2.5 mg/L cinnamic acid. Discussion and conclusion: The results obtained indicate that cinnamic acid is one of the important precursors of psoralen pathway that induced a maximum amount of psoralen with in vitro conditions.
Anticancerous, biosynthesis pathway, cinnamic acid, medicinal plants, umbelliferone
Introduction Medicinal plants are nearly unlimited sources of secondary metabolites. The presence of comparatively higher content of secondary metabolites in medicinal plants has endowed them as important sources of phytomedicines. Therefore, it is imperative to develop approaches for enhancing the bioactive compounds, isolating the phytochemical constituents, developing methods for large-scale in vitro productions (Pandey & Agrawal, 2009). Some strategies for the production of the metabolites in culture have been developed to improve the yield of plant secondary metabolites. These include treatment with various elicitors, signal compounds, and abiotic stresses (Zhao et al., 2005). Many such treatments indeed effectively promote the production of a wide range of plant secondary metabolites, both in vivo and in vitro. Psoralea corylifolia L. (Fabaceae) is an important medicinal plant that has been included in the list of threatened
Correspondence: Behrooz Mohammadparast, Department of Biology, Faculty of Science, University of Malayer, Malayer 65719-95863, Iran. Tel: +98 9188724625. E-mail: [email protected]
History Received 23 June 2013 Revised 13 April 2014 Accepted 23 June 2014 Published online 21 October 2014
plants (Anonymous, 1989; Bhattacharjee, 1998; Jain, 1994). Psoralea corylifolia is a rich source of bioactive compounds, which endows the plant with an immense value for its use in pharmaceuticals, health, and body-care products. It is being used for various biomedical applications. The seeds of this plant are anthelminthic, antibacterial, aphrodisiac, astringent, cardiac, cytotoxic, deobstruent, diaphoretic, diuretic, stimulant, stomachic, and tonic (Anonymous, 1989; Joshi, 2000). It is also externally used to treat various skin ailments including leprosy, leucoderma, hair loss, and treating vitiligo as well as psoriasis (Anonymous, 1989; Joshi, 2000; Kornhauser et al., 1982). Triplex-formation oligonucleotides, attached with a photoreactive psoralen molecule, can be used to induce site-specific DNA damage and control gene expression (Ping & Pana, 2005). Seed extract of P. corylifolia inhibits tumor growth and stimulates natural killer activity (Latha et al., 2000). Psoralen has been evaluated to inhibit the in vitro growth of three human tumor cell lines, representing different tumor types, MCF-7 (breast cancer), NCI-H460 (non-small cell lung cancer), and SF-268 (CNS cancer). The results showed the efficiency of the psoralen in inhibition of cancer (Oliveira et al., 2006). Due to the complex bioactivity
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of psoralen, its biosynthesis pathway may enable us to influence its formation in a direct way, for example, by metabolic pathway engineering. The biosynthetic pathways to the linear furanocoumarin (psoralen) involved precursors and enzyme cofactors (Buchanan et al., 2000). The basis of linear furanocoumarin formation was mostly established by the end of the 1980s by a combination of precursor-feeding experiments and the biochemical characterization of major enzymes of the pathway (Larbat et al., 2007). The present study highlights (i) the evaluation of the psoralen content in callus derived from different plant parts of P. corylifolia, and (ii) in vitro elicitation of psoralenemploying precursors of the psoralen biosynthetic pathway.
Sample preparation and estimation of psoralen
Material and methods
HPLC determination of psoralen from callus derived from different plant parts
Plant materials The seeds of P. corylifolia were procured from Homeopathic Pharmacopoeia Laboratory, Gaziabad (Uttar Pradesh, India) and were sown in the seed beds of Botanical Garden, Department of Botany, University of Delhi, in the month of March. Voucher specimen was deposited in the Herbarium section (Accession no. DUH-13553) of the Department of Botany, University of Delhi, Delhi, India. The leaf, node, root, and green seeds of P. corylifolia were taken from field-grown mature plants. These explants were washed thoroughly under running tap water for 20 min and treated with 1% bavistin (w/v), for 10 min with constant vigorous shaking on the rotary table top shaker at 150 rpm, to provide better surface content with the fungicide. These were rewashed under running tap water to remove any traces of bavistin. After pouring out excess of water, the explants were surface sterilized with 0.1% (w/v) aqueous mercuric chloride solution for 2 min and finally washed 4–5 times with sterilized distilled water. Culture media B5 media (Gamborg et al., 1968) was employed to raise the cultures of P. corylifolia. Analytical grade (AR) salts (Qualigens or Glaxo Fine Chemicals, Mumbai, India) were used to prepare the stock solutions. The basal medium was supplemented with various growth regulators such as N6-benzyladenin (BA), indole-3-butyric acid (IBA) (Sigma Aldrich, St. Louis, MO), and precursors of psoralen (cinnamic acid, NADPH, and umbelliferone: 0.1, 1, 2.5, 5, 25, and 50 mg/L). The media were gelled with 0.8 % agar (Qualigens, Mumbai, India) and the pH of media was adjusted to 5.8 using 0.1 N NaOH or HCl before autoclaving. Approximately, 20 mL media was dispensed in each 2.15 15 cm test tubes (Brosil, Qualigens, Mumbai, India) plugged with nonabsorbent cotton wrapped in muslin cloth and was autoclaved at 1.06 kgcm 2 at 121 C for 15 min. Raising and incubation of cultures Cultures were incubated in continuous light of 400–500 mw/ cm2 by cool day light fluorescent incandescent tubes (40 W, Philips, Kolkata, India). The cultures were maintained in a culture room at the temperature of 25 ± 2 C and 55 ± 10% relative humidity. Observations were recorded at an interval of 7 d. The final data were recorded after 30 d of inoculation.
The method for extraction of psoralen was modified from the Singh (2003) method. The fresh samples (1 g, each) of plant tissue were crushed with liquid nitrogen and were soaked in ethanol for 24 h, darkness and then homogenized using pestle and mortar. After evaporation of ethanol, the semisolid form of extract was mixed in methanol (HPLC grade). This mixture was transferred to a centrifuge tube and centrifuged for 15 min at 12 000 rpm at room temperature. The pellet was discarded and the supernatant was filtered using a 0.22 mm milipore filter.
Results
In this study, psoralen as an important anticancer compound has been reported from P. corylifolia cotyledon callus for the first time. Prior to this, psoralen has been extracted from seeds of P. corylifolia (Qiao et al., 2006; Rajput et al., 2008; Ruan et al., 2007). HPLC chromatogram of psoralen standard showed a retention time at 5.4 min. Quantitative analysis of psoralen from the callus derived from different plant parts showed that a maximum of 2601.8 mg/g fresh wt. of psoralen was recorded in callus derived from cotyledons (Table 1). The quantity of psoralen in node-, leaf-, and root-derived callus was 1876.6, 1468.0, and 1062.0 mg/g fresh wt., respectively. It may be possible that cotyledons of seed contain a specific gene responsible for the synthesis of psoralen. In the absence of cotyledons, this particular gene is not fully expressed or less expressed in plant-derived callus. It can also be due to the development of secondary metabolism mechanism in callus derived from cotyledons. Contrary to some results (Innocenti et al., 1997; Rakhmankulov & Korotkova, 1975), the amount of psoralen in root was in fewer amounts when compared with other parts of P. corylifolia. In vitro evaluation of secondary metabolites from the cultures grown along with various precursors of psoralen through HPLC Precursors of biosynthetic pathways have been used in various plant cell and tissue cultures to increase the yield of secondary metabolites. Different precursors, namely, cinnamic acid, umbelliferone, and NADPH with the aim of increasing the psoralen content, were used in the present study. A marked variation in the psoralen content in cotyledonary callus cultures has been analyzed when they were elicited on MS + 10 mM BA + 5 mM IBA medium adjuvanted with different concentrations of umbelliferone, cinnamic acid, and NADPH. Although every concentration of umbelliferone enhanced the psoralen content compared with control, higher concentrations (25–50 mg/L) of umbelliferone proved to be better (Table 2). Umbelliferone at 25 mg/L induced the maximum amount of psoralen (2371.7 mg/g fresh wt.). Thus, it can be deduced that psoralen production in P. corylifolia in vitro cultures is explant and the precursor is dosedependent. In case of cinnamic acid, every concentration of it improved the psoralen content, but lower concentrations (0.1–5 mg/L) proved to be beneficial over higher level. The
Psoralea corylifolia and psoralen production
DOI: 10.3109/13880209.2014.942786
Table 1. Psoralen estimates obtained from callus derived from different parts P. corylifolia. Callus derived from different plant parts Plant parts
Area (mAU)a
Psoralen quantity (mg/g fresh wt.)
139 008 100 262 78 429 56 729
2601.8 1876.6 1468.0 1062.0
Cotyledon callus Node callus Leaf callus Root callus a
Milliabsorbance units.
Table 2. Effect of some precursors on psoralen accumulation (mg/g fresh wt.) in cotyledonary callus of P. corylifolia, after 30d of incubation.
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Cotyledonary callus
Control 0 Umbelliferone (mg/l) 0.1 1 2.5 5 25 50 Cinnamic acid (mg/l) 0.1 1 2.5 5 25 50 NADPH (mg/l) 0.1 1 2.5 5 25
Area (mAU)a
Psoralen quantity (mg/g fresh wt.)
83 193.0
1932.8
107 494.1 108 910.0 120 175.0 123 138.0 126 718.0 125 731.0
2011.9 2038.4 2249.3 2304.8 2371.7 2353.3
130 108.0 130 782.0 134 577.0 133 519.0 121 398.0 112 563.0
2435.2 2447.8 2518.8 2499.4 2272.2 2106.8
109 059.0 113 921.6 126 814.2 124 153.0 123 789.0
2041.2 2132.2 2373.7 2333.7 2315.4
a
Milliabsorbance units.
maximum (2518.8 mg/g fresh wt.) of psoralen content was found at 2.5 mg/L of cinnamic acid that was the highest amount of psoralen which detected among all the precursors tried. The optimum psoralen content (2373.7 mg/g fresh wt.) was found at 2.5 mg/L of NADPH. Thereafter, a gradual decline in the response has been observed and 5 and 25 mg/L NADPH could induce only 2333.7 and 2315.4 mg/g fresh wt. of psoralen, respectively, which was significantly higher than that obtained on control (1932.8 mg/g fresh wt.) (Table 2).
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to function as a precursor in other compounds such as taxoid, shikonin, phenylethanoid glycosides, melatonin, and serotonin synthesis as well as in the biosynthesis of other compounds in various plants (Liu et al., 2007; Mei et al., 2001). Additionally, psoralen synthesis could be activated with high concentrations of L-phenylalanine. It is possible that high concentration of L-phenylalanine promotes PAL (phenylalanine ammonia-lyase) pathway activity to catalyze cinnamic acid. The effectiveness of psoralen accumulation in callus cultures of P. corylifolia could be due to the limiting factor of the flux whereby an exogenous supply of a biosynthetic precursor to the culture medium might improve alkaloid accumulation if the endogenous level of these precursors is a limiting factor of the flux. All these observations suggest that exogenous application of organic elicitors could have induced a subset of secondary metabolite biosynthetic genes, which may modulate expression of genes and accumulation of compounds induced by elicitors. The biotic and abiotic elicitors can result in an enhancement of the secondary metabolite production. The stimuli are perceived by receptors which then result in the activation of the secondary messengers. All these messengers compose paralleling or crosslinking pathways to integrate these signals to regulation of transcription factors (TFs), which subsequently activate gene expression by transcription machinery. Most of the genes for secondary metabolites synthesis are late response genes (Zhao et al., 2005).
Conclusion In summary, short based on all the above points, the maximum quantity of psoralen was recorded in callus derived from cotyledons. The HPLC analysis of the derived cotyledon callus supplemented with different precursors of psoralen pathway such as umbelliferone, cinnamic acid, and NADPH indicated that cinnamic acid induced maximum amount of psoralen.
Declaration of interest The authors explicitly declare that there is no financial or personal relationship with any third parties or organizations whose interests could be positively or negatively influenced by the content of the article. The data presented in this article are from a Ph.D. Thesis (Behrooz Mohammadprast) performed in the Botany Department of Delhi University. Financial support of this study by Prof. Veena Agrawal is acknowledged.
Discussion Psoralen, an important furanocoumarin, is seen abundantly in the seeds of P. corylifolia. Due to the complex bioactivity of furanocoumarins, its biosynthesis has received continuous attention. The biosynthetic pathways to the linear furanocoumarin (psoralen) involved enzymes (and their cofactors) and precursors which are as follows: cinnamic acid, p-coumaric acid, umbelliferone, demethylsuberison, and marmasin (Buchanan et al., 2000). Precursors, namely cinnamic acid, umbelliferone, and NADPH, have substantially improved the production of psoralen. Incidentally, optimum enhancement was seen with cinnamic acid. Cinnamic acid was also shown
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