In vitro isolation, elicitation of psoralen in callus cultures of Psoralea corylifolia and cloning of psoralen synthase gene

Plant Physiology and Biochemistry 49 (2011) 1138e1146

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Research article

In vitro isolation, elicitation of psoralen in callus cultures of Psoralea corylifolia and cloning of psoralen synthase gene Behrooz M. Parast a, Siva K. Chetri b, Kuldeep Sharma b, Veena Agrawal b, * a b

University of Malayer, 4Km Malayer-Arak Road, Malayer 65719-95863, Iran Department of Botany, University of Delhi, Delhi 110007, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 January 2011 Accepted 30 March 2011 Available online 9 April 2011

Psoralen, an important furanocoumarin occurring abundantly in seeds of Psoralea corylifolia is used as an anticancerous compound against leukemia and other cancer cell lines. Evaluation and isolation of psoralen from the calluses derived from different plant parts, viz. cotyledons, nodes, leaves and roots have been done in the present case for the first time. Amongst all, a maximum of 1934.75 mg/g f.w. of psoralen was recorded in callus derived from cotyledons, followed by 1875.50 and 1465.75 mg/g f.w. of psoralen in node and leaf derived calluses, respectively. Amount of psoralen enhanced further when cotyledonary calluses were exposed to different concentrations of organic elicitors (yeast extract, proline, inositol, casein hydrolyzate (CH), glycine, glutamine and sucrose) and precursors of psoralen (umbelliferone, cinnamic acid and NADPH). Isolation of psoralen was done using methanol as solvent through column chromatography and TLC. FT-IR and NMR further characterized and confirmed the structure of psoralen. In addition, the putative gene, psoralen synthase involved in psoralen synthesis pathway has been isolated, cloned and sequenced which comprised 1237 bp length. BLAST analysis of the gene sequence of psoralen synthase revealed that its nucleotide sequence showed 93% homology with psoralen synthase isolated from Ammi majus. This is the first report of isolation, cloning and characterization of psoralen synthase from Psoralea corylifolia. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Anticancerous Biosynthesis Cotyledonary callus Psoralen Psoralen synthase

1. Introduction Psoralea corylifolia is one of the promising medicinal plants, possessing immense biomedical applications against several diseases such as leucoderma, leprosy, psoriasis, vitiligo due to the presence of several isoflavonoids and furanocoumarins. Of the several bioactive compounds, psoralen, is one of the important furanocoumarin abundantly available in this plant which is also widely employed as an anticancerous agent against leukemia and other cancer lines [16,27,46,48]. Furanocoumarins are natural plant metabolites characterized by a furane moiety fused to benzopyran-2-one. Furanocoumarins intercalate in double-stranded DNA, and psoralens are known to cross-link pyrimidine bases under irradiation by [2 þ 2] cycloaddition via their 3, 4- and 2, 3-double bonds [12]. The position of the

Abbreviations: CDCl3, deuterated chloroform; DMAPP, dimethylallyl pyrophosphate; FT-IR, Fourier transform infra red; F.W., fresh weight; IPTG, isopropyl-b-thio galactopyranoside; LiTaO3, lithium tantalate; NMR, nuclear magnetic resonance; TMS, tetramethylsilane; X-gal, 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside. * Corresponding author. E-mail address: [email protected] (V. Agrawal). 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.03.017

furane substitution distinguishes two large groups of compounds, the linear (psoralens) and the angular furanocoumarins (angelicin and derivatives) [24]. Psoralen, an important furanocoumarin in particular is known for its photosensitizing and phototoxic effects and has been used in photochemotherapy of skin disorders (psoriasis, vitiligo, and mycosis). Due to the complex bioactivity of furanocoumarins, its biosynthesis has received continuous attention. Knowledge of the biosynthetic pathway of psoralen 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 enzymes (and their cofactors) which are as follows 1. DMAPP-umbelliferone dimethylallyl transferase 2. marmesin synthase (O2, cytochrome P450, NADPH), 3. psoralen synthase (O2, Cyt, P450, NADPH) [5] (Fig. 1). 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 [15]. Nevertheless, until recently, the key enzyme genes in the pathway of psoralen synthesis in P. corylifolia had not yet been cloned and sequenced. The present study highlights the evaluation of the psoralen content in (i) calluses derived from different plant parts of

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Fig. 1. Schematic representation of the biosynthetic pathway of psoralen. (Source: Croteau et al., 2000 [5]).

P. corylifolia, viz. cotyledons, nodes, leaves and roots (ii) in vitro elicitation of psoralen employing organic elicitors and precursors of the psoralen biosynthetic pathway (iii) isolation and characterization of psoralen from callus and (iv) isolation and characterization of putative gene, psoralen synthase involved in psoralen biosynthetic pathway.

The media were gelled with 0.8% agar (Qualigens Mumbai) 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 (Borosil) plugged with non-absorbent cotton wrapped in muslin cloth and was autoclaved at 1.06 kg cm2 at 121  C for 15 min.

2. Materials & methods

2.3. Raising and incubation of cultures

2.1. Plant materials

Cultures were incubated in continuous light (28e35 mmol m2 s1) by cool day light fluorescent incandescent tubes (40 W, Philips, Kolkata). The cultures were maintained in a culture room at the temperature of 252  C and 55  10% relative humidity. The explants were sub-cultured after every 30e32 d interval on the same but fresh medium. Observations were recorded at an interval of seven days. The final data were recorded after 30 d of inoculation.

The seeds of P. corylifolia were procured from Homeopathic Pharmacopoeia Laboratory, Ghaziabad (Uttar Pradesh, India) and were sown in the seed beds of Botanical Garden, Department of Botany, University of Delhi, in the month of March. 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 rotary table top shaker at 150 rpm, to provide better surface contact 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 or 5 times with sterilized distilled water.

2.4. Statistical analysis The evaluation of psoralen from various callus samples of P. corylifolia was performed with four replicates each and the data obtained were analyzed statistically. The statistical analyses were performed by ANOVA using SPSS. The differences between means were tested for significance by Duncan’s multiple range test at p ¼ 0.05.

2.2. Culture media 2.5. Phytochemical analysis MS (Murashige and Skoog, 1962) [23] and B5 (Gamborg et al., 1968) [8] media were employed for raising the cultures of P. corylifolia. Analytical grade (AR) salts (Qualigens or Glaxo Fine Chemicals, Mumbai) were used to prepare the stock solutions. The basal medium was supplemented with various growth regulators such as N6-benzyladenine (BA), a-Naphthaleneacetic acid (NAA), Indole-3-butyric acid (IBA) (SigmaeAldrich, USA), organic compounds (Casein hydrolyzate, Glycine, Myo-Inositol, Glutamine, Proline, Yeast extract in the range of 0, 1, 5, 25, 50, 100, 200 & 300 mg/l and Sucrose; 0, 1.5, 3, 4.5, 6, 7.5 & 9%) and precursors of psoralen (Cinnamic acid, NADPH and Umbelliferone; 0, 0.1, 1, 2.5, 5, 25 & 50) (Sigma Aldrich, USA). As a source of carbon, 3% (w/v) sucrose (Daurala, DCM, Meerut) was used in all experiments unless mentioned specifically.

The fresh samples (1 g, each) of different plant tissue were crushed with liquid nitrogen and soaked in ethanol for 24 h, under dark 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 centrifuge tube and centrifuged for 15 min at 12,000 rpm at room temperature. The pellet was discarded and the supernatant was filtered using 0.22 mm millipore filter for further analysis. The HPLC unit of Shimadzu-4A type, equipped with UV detector and printer plotters was operated under the following parameters: Column: C18; Column packing: Zorbex ODS (Octadecyl silane); Solvent: Methanol (HPLC grade); Injection volume: 20 ml; Flow rate: 0.5 ml/min; Detection: UV 244 nm for psoralen.

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The dried cotyledonary callus (500 g) of P. corylifolia L. was extracted with methanol by soxhlate apparatus for 48 h. The extract was suspended in H2O (1 L) and extracted with petroleum ether. The silica gel column chromatography of cotyledonary callus of P. corylifolia was done with modified protocol of Li et al. (2007) [17]. Separation of different components of the crude extract was done by running the mobile phase of petroleum ether: ethyl acetate, through the column in order of increasing polarity (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12%). The column was run at the flow rate of 0.16 ml/s at room temp. A total of 60 fractions, of 100 ml each were collected and subsequently concentrated to dryness in a rotary vacuum evaporator. The concentrated fractions were stored in glass vials at room temperature. The separated fractions were further analyzed through TLC, following the aforesaid mentioned protocol using hexaneeethyl acetate mobile phase in all cases. For visualization of the resolved compounds, the TLC plates were exposed to fluorescent indicator UV rays (l ¼ 254 nm) and the visible spots were marked. The respective Rf values of different components of each fraction were calculated. The fraction having compounds with similar Rf values were mixed together. FT-IR spectra of the crude extract of callus of P. corylifolia was obtained with Fourier Transform Spectrometry instrument (Spectrum RXI), resolution: 400 cm1, detector: LiTaO3. The dried cotyledonary callus of P. corylifolia was extracted with methanol using soxhlate apparatus for 48 h. A small drop of the crude extract was placed on one of the KBr plates. Further, the second plate was placed on top and a quarter turn was done to obtain a nice even film. The plates were placed into the sample holder and the spectrum was run. 1 mg of pure psoralen powder (SigmaeAldrich, USA), was dissolved in few drops of ethanol and the final volume was made to 1 ml with methanol. 1H-NMR spectra of the isolated and purified compounds from cotyledonary callus of P. corylifolia were obtained with NMR instrument (Brucker Spectrospec), DPX-300 MHz using tetramethylsilane (TMS) as internal standard. 2.6. Total RNA isolation and cDNA synthesis Leaves of P. corylifolia were used for isolation of total RNA using the modified RNA isolation method of Salzman et al. (1999) [37]. First strand cDNA synthesis was done using the manufacturer’s (Revertad H Minuts, First strand cDNA synthesis kit, Fermentas) protocol. Based on the known nucleotide sequence of psoralen synthase of Ammi majus [15], forward and reverse primers were synthesized for the synthesis of cDNA. The gene specific primers were designed with primer3 software and purchased from Sigma Aldrich, USA. The sequence of the forward primers used were RBF (50 -GCAGAGTGCAGAGCAATAGAAATGAAGATGC-30 ), RBL1 (50 -GAG GCTTTGAAAACCCATGA-30 ) and RBL2 (50 -CGCAAGCATCACTCAGAG AC-30 ) and the reverse primers used included RBR (50 -GAGCTGG GAATGAGTATTGACATGG-30 ) RBR1 (50 -CAAACATGTGGTCTGGCAAC30 ) and RBR2 (50 -CAAACATGTGGTCTGGCAAC). The PCR was performed using 2 ml Template, 2 ml of dNTPs (2.5 mM each), 0.5 ml Forward primer (10 mM), 0.5 ml of Reverse primer (10 mM), 2 ml of 10 X Taq buffer, 0.3 ml of Taq polymerase and 12.7 ml water. Amplification was performed in a thermal cycler (Mycycler, Bio-Rad) as follows: 95  C for 3 min (1 cycle); 95  C of 1 min (25 cycles) and 72  C for 10 min (25 cycles). The PCR products were loaded in agarose gel and samples were run at 80 V. The amplified fragments were eluted from agarose gels using Mini Elute Gel Extraction Kit from (Qiagen). 2.7. Cloning of psoralen synthase from P. corylifolia The cDNA prepared from leaf of P. corylifolia was used to amplify the psoralen synthase with gene specific primers. The amplified

PCR product was purified and cloned into pGEM-T Easy vector (Promega) followed by transformation into Escherichia coli (strain DH5a) competent cells. The competent cells were thawed on ice. The ligation mixture (10 ml) was taken and spun shortly to mix the contents. The ligation mixture was added to the competent cells and mixed gently with pipette and given an incubation of 30 min on ice. A heat shock, exactly for 90 s was given at 42  C. 900 ml of LB was added, mixed properly and incubated on shaker at 37  C for 45 min with slow shaking (150 rpm). The LB-Amp plates were plated with 7 ml of IPTG (20%) and 40 ml of X-gal (2%). After the incubation of 45 min at 37  C, 200 ml of culture was taken and spread on the plates containing IPTG and X-gal. The remaining culture was then centrifuged at 4000 rpm for 1 min at room temperature. The supernatant was discarded by inversion and the pellet was resuspended in the LB remaining in it, and spread on the IPTG and X-gal containing indicator plate. The plates were kept at 37  C overnight and observed next morning for blue/white colonies. The transformants were screened for the presence of inserts. The screening strategies used were blue/white screening and the positive clones were further confirmed by colony PCR. The plasmid was isolated from transformants using alkaline lysis method [39] and the transformants were screened for the presence of insert by colony PCR using T7 (50 -TAATACGACTCACTATAGGG-30 ) and SP6 (50 -ATTTAGGTGAACACTATAG-30 ) primers which flank the multiple cloning site of p-GEM-T Easy vector. The plasmid DNA was sequenced using automated DNA sequencer (ABI Prism) which is a modified version of Sanger et al. (1977) [40] method for the determination of precise sequence of nucleotides in the sample of DNA. Sequence similarity searches were performed using the basic local alignment search tool (BLAST, National Center for Biotechnology Information, Bethesda, MD) [1] and Alignment software. 3. Results 3.1. Quantitative analysis of psoralen in callus derived from different plant sources Calluses derived from different sources were analyzed which showed different yields of psoralen (Fig. 2). Quantitative analysis of psoralen carried out from calluses derived from different plant parts showed that a maximum of 1934.75  0.85 mg/g f.w. of psoralen was recorded in callus derived from green cotyledons of P. corylifolia seeds, followed by node derived callus i.e. 1875.50  1.19 mg/g f.w. The quantities of psoralen in the leaf and root derived calluses were 1465.75  1.65 and 1062.75  0.85 mg/g f.w., respectively. Since callus derived from cotyledons proved optimum in terms of psoralen content, it was further used for enhancement of psoralen content using various elicitors such as organic elicitors and precursors of psoralen biosynthetic pathway. 3.2. Effect of various elicitors on psoralen synthesis The callus cultures reared on MS þ 10 mM BA þ 5 mM IBA were feeded with different concentrations (1, 5, 25, 50, 100, 200 and 300 mg/l) of various elicitors such as adjuvants like yeast extract, myo-inositol, casein hydrolyzate and sucrose (1.5, 3, 4.5, 6, 7.5, 9%) and amino acids, i.e. proline, glycine and glutamine to improve the psoralen content. However, the psoralen yield was elicitor specific and concentration dependant. A significant enhancement in psoralen quantity was observed in cotyledon calluses reared on MS medium supplemented with different concentrations of yeast extract. Its lower concentrations (1, 5 and 25 mg/l) failed to elevate the psoralen content over control however, the quantities increased gradually, the

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Fig. 2. Estimation of psoralen through HPLC from calli derived from different plant parts of Psoralea corylifolia. Results for the Psoralen content through HPLC for different calli source are the mean of four replicates. Letters on the top of the columns indicate significant differences according to Duncan’s multiple range test at p ¼ 0.05.

respective quantities were 1965.50  0.64, 2043.50  0.64, 2146.50  0.64 and 2226.00  0.91 mg/g f.w. at 25, 50, 100 & 200 mg/l (Fig. 3A). A maximum of 2884.25  0.85 mg/g f.w. of psoralen was obtained at 300 mg/l (Fig. 3A). Lower levels of myo-inositol proved effective for enhancement of psoralen over control but higher levels (200e300 mg/l) failed to elevate the psoralen content in the callus cultures. The optimum quantity of psoralen (2292.50  1.04 mg/g f.w.) was at 25 mg/l followed by 2272.25  0.85 mg/g f.w. at 5 mg/l whereas, 1964.75  0.85 and 1956.50  0.64 mg/g f.w. was estimated on 200 and 300 mg/l, respectively (Fig. 3A). Similar to myo-inositol, psoralen content decreased gradually with the increasing concentrations of casein hydrolyzate being optimum (2263.25  0.85 mg/g f.w.) on 50 mg/l (Fig. 3A). A sudden decline was observed at 300 mg/l where 1543.00  0.70 mg/g f.w. of psoralen was achieved (Fig. 3A). Addition of different concentrations (1.5%, 3%, 4.5%, 6%, 7.5% and 9%) of sucrose in the medium enhanced the quantity of psoralen significantly. With the gradual increase in concentration of sucrose from 1.5% to 6%, there was a gradual increase in the psoralen content. At 1.5, 3 and 4.5% sucrose, psoralen content was 2031.50  0.64, 2035.50  0.64 and 2075.75  0.85 mg/g f.w., respectively (Fig. 3B). Interestingly, a sudden increase in the psoralen content was analyzed at 6% sucrose, which elevated the content upto 2812.75  0.85 mg/g f.w. (Fig. 3B). However, 7.5% too enhanced it upto 2335.25  0.85 mg/g f.w. of psoralen. Subsequently, low level was seen at 9% sucrose (Fig. 3B). Thus, 6% sucrose proved optimum among all the aforesaid elicitors tried. Psoralen content enhanced markedly when callus was exposed to 1, 5, 25, 50, 100, 200 and 300 mg/l of proline and of all the levels tried, 50 mg/l proline elicited the maximum content (2437.25  0.85 mg/g f.w.) followed by 25 mg/l (2303.25  1.10 mg/g f.w.) (Fig. 3C). Contrary to proline, glycine did not prove effective for elicitation of psoralen in callus cultures as the optimum quantity of psoralen (2084.50  0.64 mg/g f.w.) was found at 200 mg/l (Fig. 3C). At 1, 5, 25 and 50 mg/l of glycine, the quantities were 1816.75  0.85, 1894.50  0.64, 1682.25  0.85 and 1884.50  0.64 mg/g f.w., respectively (Fig. 3C). However, 100 and 300 mg/l glycine could induce, 1976.00  0.91 and 1987.50  0.64 mg/g f.w. psoralen content, respectively (Fig. 3C). In addition, glutamine proved effective in elevation of the psoralen content, the maximum quantity being 2520.50  0.64 mg/g f.w. at 25 mg/l glutamine followed by 2097.50  0.64 mg/g fresh wt at 50 mg/l (Fig. 3C). However, a significant amount of 2272.50  0.64 mg/g f.w. at 100 mg/l, 2107.50  0.64 mg/g f.w. at 200 mg/l and 2097.50  0.64 mg/g f.w. at 300 mg/l of glutamine was recorded (Fig. 3C). Three precursors namely umbelliferone, cinnamic acid and NADH at 0.1, 1, 2.5, 5 & 50 mg/l were tried to study their impact on

psoralen content in callus cultures reared on MS þ 10 mM BA þ 5 mM IBA. Higher concentrations (25e50 mg/1) of umbelliferone proved better. Maximum amount of psoralen, i.e. 2371.50  0.64 mg/g f.w. was achieved in callus reared on 25 mg/l of umbelliferone (Fig. 3D). Comparatively, lesser amounts i.e. 2013.75  0.85, 2038.25  0.47, 2248.50  0.64, 2305.50  0.64 and 2353.50  0.64 mg/g f.w. of psoralen was detected on 0.1, 1, 2.5, 5 and 50 mg/l of umbelliferone, respectively (Fig. 3D). Though, every concentration of cinnamic acid significantly improved the psoralen content but lower concentrations (0.1e5 mg/1) proved beneficial over higher levels (25e50 mg/1). A maximum of 2517.50  0.64 mg/g f.w. psoralen was found at 2.5 mg/l of cinnamic acid (Fig. 3D). It is noteworthy here that this was the maximum quantity of psoralen detected among all the precursors tried (Fig. 3D). Akin to this, a marked elevation in the psoralen content was observed when callus was grown on medium feeded with different concentrations of NADH. Nearly, 2042.50  0.64 and 2133.00  0.70 mg/g f.w. psoralen was estimated on 0.1 and 1 mg/l NADPH, respectively (Fig. 3D). The maximum yield (2374.50  0.64 mg/g f.w.) was at 2.5 mg/l of NADPH (Fig. 3D). Thereafter, a gradual decline in the response occurred and 5 and 25 mg/l NADPH could induce only 2335.50  0.64 and 2316.50  0.64 mg/g f.w. of psoralen, respectively which was significantly higher than that obtained on control (1934.75  0.85 mg/g f.w.) (Fig. 3D).

3.3. Phytochemical analysis of crude extract of cotyledon derived callus of P. corylifolia Fourier Transform-Infra Red (FT-IR) spectroscopy at resolution of 4.00 cm1 with detector of LiTaO3 provided evidence about the presence of different compounds in the standard and crude extract. The IR spectrum of the standard and sample revealed that the existing peak at 3436.06 cm1 and 3509.1 cm1 were basically for the OH absorbance which could appear because of the moisture or ethanol content used to separate out the psoralen. The next characteristic absorbance in sample and standard at 1612.77 cm1 and 1635.38 cm1 was due to banding of the OH functional group (Fig. 4A and B). The other characteristic peak in standard and sample at 1723.87 cm1 and 1715.60 cm1 were due to the carbonyl peak (C]O) (Fig. 4A and B). The patterns of the standard and crude extract were quite similar. The petroleum ether soluble fraction was subjected to silica gel chromatography and increasing polarity with ethyl acetate (1%). Finally, 60 fractions were collected. Thin layer chromatography with mobile phase of ethyl acetate/cyclohexane (90:10) for development of psoralen spot was achieved. The Rf value of the spots has been calculated. In fraction No. 35e38 brown compounds at 91:9

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revealed one spot on the TLC plates with the respective Rf value being 0.72. Incidentally, two different spots were visible in fraction 22e29. Their respective Rf values were 0.72 and 0.68, respectively. Fraction 30e33 revealed three spots (Rf ¼ 0.68, 0.54 and 0.57). However, fraction 34 contained two spots, with Rf values being 0.57 and 0.4. Interestingly, fraction 35e38 revealed only a single spot (Rf ¼ 0.4). It was characterized as psoralen. Further fractions (39e46) contained compounds with different Rf values, still two or three spots with Rf values similar to those recorded in previous fractions were also found. No spot was visible in fractions 47 and 48. Rest of the fractions (49e60) revealed either single spot or moved along the TLC plate without any separation or spot. Finally, the fractions containing psoralen compound were pooled together. 3.4.

1

H NMR spectrum of isolated fractions of P. corylifolia

The 1H NMR spectrum of the psoralen compound revealed signals at d 7.79 (d), 7.69 (d), 7.68 (s), 7.48 (s), 6.82 (d) and 6.38 (d) which has been attributed to the protons Ha, Hb, Hc, Hd, He and Hf, respectively (Table 1; Fig. 5A and B). There was one signal at 7.26 which belong to the CDCl3 solvent. This information in 1H NMR indicates the presence of psoralen (Fig. 5B) in cotyledonary callus of P. corylifolia. 3.5. Molecular analysis of putative gene encoding psoralen synthase from P. corylifolia To obtain the nucleotide sequence of psoralen synthase, cDNA prepared from leaf of P. corylifolia was used to amplify with three sets of gene specific forward (RBF, RBL1 and RBL2) and reverse primers (RBR, RBR1 and RBR2). Of the forward and reverse primers used, RBL2 (50 -CGCAAGCATCACTCAGAGAC-30 ) and RBR1 (50 -CAAAC ATGTGGTCTGGCAAC-30 ), respectively proved positive for the synthesis of the nucleotide sequence of psoralen synthase (Fig. 6). Twenty-five white colonies were selected among the blue/white colonies and colony PCR with gene specific primers was done. Among the 25 colonies selected, colony 1 showed the amplification of gene which denotes that this colony was having the insert of corresponding size. Sequencing was done using dideoxy chain termination method [40]. Analyses of the sequences were done using bioinformatics databases http://www.ncbi.nlm.nih.gov. The nucleotide sequence of psoralen synthase comprises 1237 bp length (Fig. 7). The clone showed 93% homology with psoralen synthase of A. majus (Gene Bank ACC. No. : AY532370) [15] (Fig. 8). 4. Discussion

Fig. 3. Impact of different adjuvants (1, 5, 25, 50, 100, 200 & 300 mg/l) (A), sucrose (1.5, 3, 4.5, 6, 7.5 & 9%) (B), amino acids (1, 5, 25, 50, 100, 200 & 300 mg/l) (C) and precursors of psoralen (1, 5, 25, 50, 100, 200 & 300 mg/l) (D) on biosynthesis of psoralen in cotyledonary callus cultures. Results for the Psoralen content through HPLC for different elicitors used are the mean of four replicates (SE). Letters on the top of the columns indicate significant differences according to Duncan’s multiple range test at p ¼ 0.05.

(petroleum ether/ethyl acetate) with Rf value of 0.4 was characterized as psoralen. The TLC analysis of the separated fractions revealed that the first ten (1e10) fractions contained more impurities as nothing was visible on the TLC plates. Fractions 11e15 revealed single spot with the respective Rf value being 0.85. The next three fractions (16e21)

Psoralen, an important anticancerous compound has been reported for the first time from P. corylifolia callus. Prior to this, Rajput et al. (2008) [34], Ruan et al. (2007) [36] and Qiao et al. (2006) [32] had extracted psoralen from seeds of P. corylifolia. Girard et al. (2004) [9] extracted psoralen from dried powdered twig and leaves of Phebalium brachycalyx. Lakshmi and Reddy (2007) [14] isolated psoralen from leaf callus of Cullen corylifolium. Enhancement of bioactive compound through different elicitors such as organic elicitors and precursors has been achieved at all levels. Earlier reports on biochemical analysis of psoralen too has revealed the presence and distribution patterns of psoralen [6,7,10,22,25,26,30,33] in Psoralea genus but none of them reported psoralen from callus which could be used as a source of psoralen production at commercial level. Not only this, Bouque et al. (1998) [4] gave negative report of the presence of psoralen from the callus lines they generated. Furthermore, the cotyledonary callus derived from green cotyledons contained highest quantity

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Fig. 4. FT-IR spectrum of standard of psoralen (A) and crude extract of cotyledonary callus cultures of P. corylifolia (B).

(1934.75  0.85 mg/g f.w.) of psoralen while node, leaf and root derived calluses revealed lesser amount of psoralen. It is, therefore proved that elicitation was concentration dependant and elicitor specific [2,47]. Among all the adjuvants, a significant enhancement of psoralen bioactive compound occurred under the influence of yeast extract (Fig. 3A). Cotyledonary callus cultures produced a maximum of 2884.25  0.85 mg/g of psoralen at 300 mg/l of yeast extract amongst all the organic elicitors tried. The yeast extract significantly increased the intracellular content of psoralen in comparison to control. The possible reason could be attributed to the content of some cations like Zn, Ca, and Co, present in yeast extract which could act as abiotic elicitors [44]. As yeast extract is composed of a variety of compounds, apart from amino acids, vitamins, and minerals, it is also possible that the elicitation effects might be due to other components still not identified. Similar to our results, an enhancement in metabolite content using yeast extract was also reported in Orthosiphon aristatus, Salvia miltiorrhiza [43,50]. The effect of yeast extract might be due to its role in increasing the phenylalanine ammonia lyase activity which is a key enzyme of phenylpropanoid pathway that catalyzes L-phenylalanine deamination and trans cinnamic acid production which links primary metabolism to the secondary one, and formation of vast secondary metabolites with phenylpropanoid skeleton [41]. Akin to this, amino acid also enhanced the psoralen content in P. corylifolia callus cultures and this may be due to production of some plant hormones under the influence of amino Table 1 1 H-NMR Spectral Data for psoralen compound S.No. 1 2 3 4 5 6

D 7.79 7.69 7.68 7.48 6.82 6.38

Protons

Multiplicity

J value

Ha Hb Hc Hd He Hf

d d s s d d

9.98 Hz 2.38 Hz e e 2.38 Hz 9.99 Hz

(d values, at 400 MHz, in CDCl3, TMS as internal standard).

acids. Many authors have described the biochemical basis of synthesis of other secondary metabolites in different plants [31]. Bonner (1972) [3] suggested that some amino acids are incorporated into intermediate compounds of glycolysis and TCA cycle after deamination. Acetyl CoA is a precursor of secondary metabolites and is converted into isopentyl pyrophosphate which is a precursor of terpenoids. Precursors, namely cinnamic acid, umbelliferone and NADPH have also substantially improved the production of psoralen. Incidentally, optimum enhancement was seen with Cinnamic acid. Cinnamic acid was also shown to function as 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 [19e21]. Additionally, psoralen synthesis could be activated with high concentrations of L-phenylalanine. It is possible that high concentrations of L-phenylalanine promote PAL (phenylalanine ammonia-lyase) pathway activity to catalyze cinnamic acid synthesis. 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 [51]. All these messengers compose paralleling or cross-linking pathways to integrate these signals on to regulation of transcription factors (Tfs) which subsequently activate gene expression by transcription machinery. Most genes for secondary metabolites synthesis are late response genes [51]. Identification of appropriate solvents to include in pharmaceutical study design is the key to extraction of meaningful and efficient studies [49]. Psoralen is a tricyclic furanocoumarin and

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Fig. 5. 1H NMR spectrum of psoralen compound in CDCl3 solvent (A) and structure of psoralen revealed through NMR analysis (B) from cotyledonary callus cultures of Psoralea corylifolia.

slightly polar with potent photosensitizing properties. Different solvents such as ethyl acetate, cyclohexane, hexane, methanol, petroleum ether, chloroform, benzene were used for isolation of psoralen and in ethyl acetate/cyclohexane (90:10) psoralen spot has been achieved. During present study methanolic extract of cotyledonary callus of P. corylifolia was subjected to liquideliquid solutions, using ethyl acetate/cyclohexane mobile phase to recover the different fractions eluted through column chromatography. The fractions were subsequently used for carrying out TLC for developing the psoralen spot in ethylacetate/cyclohexane. Chromatography coupled with spectroscopy is a powerful analytical tool for fast structural elucidation of complex mixtures [29]. The FT-IR spectra and 1H NMR of the standard (psoralen) and crude extract of cotyledonary callus of P. corylifolia indicated the presence of psoralen in cotyledonary callus of P. corylifolia. Despite the considerable interest in furanocoumarins as therapeutics or plants’ herbivore defense factors, the biosynthesis

and genetics of these compounds, particularly the linear furanocoumarins, are still poorly understood [18,42]. Psoralen synthase is a key enzyme and catalyzes the oxidation of (þ)-marmesin to psoralen through a unique carbon chain cleavage reaction concomitantly releasing acetone carbon by syn-elimination [5]. Several earlier workers [11,13,15,28,35,38,45] isolated the key enzymes from different species in different family. Larbat et al. (2007) [15] reported the molecular cloning and functional characterization of psoralen synthase from A. majus L. Comparison of the nucleotide sequence of putative gene, psoralen synthase from P. corylifolia with those of psoralen synthase of A. majus revealed a significant degree of similarity. The sequence showed highest similarity of 93% to the putative sequence of psoralen synthase from A. majus L. (Gene Bank ACC. No. AY532370) [15]. Contrary to this, this sequence showed significantly less (77%) homology with angelicin synthase (ACC. No. EF191021; an isomer of psoralen synthase). It is thus amply clear that our sequence pertains to psoralen synthase. In conclusion, the current study concentrates on the in vitro elicitation of psoralen in callus cultures of P. corylifolia, its extraction, isolation, cloning and characterization of putative gene, psoralen synthase, involved in psoralen biosynthetic pathway. The characterization of the psoralen synthase gene will constitute GAG GCT TTG AAA ACC CAT GAT CTT GTT TTC GCA CCC CGG CCT TAT TCA AGT GTG GCC AAT AAA ATC TTC TAC AAT GGG AAC GAA ATG GTG TTT GCT TAT TGG AGA CAC AGA GTA AAG AGT ATC TGC GTT ACT CAG CTC CTA AGT AAC AAA AGG GTT AAT TGT TTT CAC TAT GTC AGA GAA GAA GAA GTT GAT CTT TTT GTC CAA AAT CTC GAA AAT TCT CAA TCG TCG AAA GTA GCA AAT TTA ACT GAA CTG TTA ATC GAA GTA ACT GGC AAT GTA GTC GAA GTA AC T GGC AAT GTA GTC TGC AGG GTT TCA GTA GGA AGT GGT GAC AAA GTG GAT TCA TAC AAG ATT TTT ATC CTG GAA AAA TAA TGG ATA TGT TAG GCT ATT CCT GGA GCA TAG AGG ATT TTT TAC CAT TGC TCG GTT GGG TTG AAT GGC TTA CTG GAT TGA GGG GAA AGG TTG CGG AAG CAG CCA AAG GGG TTG ATA CTT TTC TGG AAG GAG TTC TTA AAG AAC ATC TAA GTA CTA CTG GAT CCA AAT ACA ATG ACT TTG TAT GCA TTT TGC TCG AGA TTC AGG AGG CAG ATG CTG GCT CTT CTA TGG AAA ATG AAT GTA TCA AAT CTC GTA TCT GGG ATA TGT TGG GTG CCG GAA CTG AAA AAA TAT CGA CAG CTT TGG AAT GGA CAC TAG CAG GGC TAA TAA AAA ATC CGG CGT ACC CCA TGT TCA AAT TGC AAA ATG AGG TCA GAG AAA TTG GCA AAG GCA AAT CAA AGA TAT CAG AGG GTG ATC TAG TCA AAA TGA AGT TCC TGC AAG CAG TAA TGA AAG AGA GGA GCA TGC GAT TGT ATT TTT CAG CCC CAC TAC TAG TTC CTA GAG AAG CGA GGC AGG ACA TAA AAT TTA TGG GGT ATG ACA TAA GCT CAG GAA CAC AAG TAC TGA TAA ATG CAT GGG CTG CAA TTG CAA GAG ACC CTT TAT TGT GGG ACA AAA GAG AGG AGT TTC CGG CCT GAG AGG TTC TTG AAA AGT CCA ATA GAT TCC CAA GGC TTT CAC TAT GAG TTT CTT CCC TTT GGA GCC GGT CGG AGG GGT TGT CCT GGT ATC CAC TTT GCA ATG TGT ATT AAA GAG CTA GTA GTG GCA AAT CTT GTG CAC ATG TTT AAT TTC GAA TTG CCT GAT GGG AAA AGA TTG GAA GAT TTG GAT ATG ACT GCT GCC AGT GGC ATT ACT CTT CGT AAA AAA TCT CCT CTC TTG GGG GTT GCC AGA CCA CAT GTT T

Fig. 6. PCR amplification of cDNA of P. corylifolia with different primers. Lane M: 2.5 kb DNA ladder Lane 1: RBF, RBR primers; Lane 2: RBL1, RBR1 primers; Lane 3: RBL2, RBR1 primers.

Fig. 7. The nucleotide sequence of psoralen synthase.

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psoralen from callus cultures and cloning of putative psoralen synthase gene from P. corylifolia. Acknowledgments V. Agrawal is grateful to University Grants Commission for providing the financial assistance in the form of a UGC’s Major Research Project and to the University of Delhi, Delhi for sanctioning funds for Research and Development. Authors are also thankful to Dr. A.K. Sinha, NIPGR, New Delhi for helping in Molecular Biology work. B.M. Parast is debted to the Iran Embassy (India) for providing funds. The authors also acknowledge the work of Larbat et al. [15] which helped in designing the primers of psoralen synthase for the present work. References

Fig. 8. Sequence analysis of psoralen synthase employing bioinformatic databases, http://www.ncbi.nlm.nih.gov.

a major step forward to our understanding of the furanocoumarin pathways in higher plants. Further work related to the expression and over expression of psoralen synthase gene are in progress. To the best of our knowledge this is the first report on detection of

[1] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403e410. [2] M.A. Bernards, L.M. Susag, D.L. Bedgar, A.M. Anterola, N.G. Lewis, Induced phenylpropanoid metabolism during suberization and lignification: a comparative analysis, J. Plant Physiol. 157 (2000) 601e607. [3] J. Bonner, The isoprenoids. in: James Bonner, J.E. Varner (Eds.), Plant Biochemistry. Academic Press, New York, 1972, pp. 665e690. [4] V. Bouque, F. Bourgaud, C. Nguyen, A. Guckert, Production of daidzein by callus cultures of Psoralea species and comparison with plants, Plant Cell Tiss. Org. Cult. 53 (1998) 39e40. [5] R. Croteau, T.M. Kutchan, N.G. Lewis, Natural products (secondary metabolites). in: B. Buchanan, W. Gruissem, R. Jones (Eds.), Biochemistry & Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, Maryland, USA, 2000, pp. 1250e1318. [6] J. Ding, Q. Zhang, L. Zhang, Determination of psoralen and isopsoralen in Psoralea corylifolia L. from different habitats, J. Chin. Med. Mat. 27 (2004) 817e818. [7] T.N. Dong, K. Bae, Y.H. Kim, G.S. Hwang, O.S. Heo, S.E. Kim, J.S. Kang, Quantitative determination of psoralen and angelicin from some medicinal herbs by high performance liquid chromatography, Arch. Pharmacol. Res. 26 (2003) 516e520. [8] O.L. Gamborg, R.A. Miller, K. Ojima, Plant cell cultures. Nutrient requirements of suspension cultures of soybean root cells, Exp. Cell Res. 50 (1968) 151e158. [9] C. Girard, F. Muyard, M. Columbian, F. Tillequin, P.L.G. Waterman, F. Bevalot, Coumarin from Phelabium brachycalyx, Biochem. Syst. Ecol. 33 (2004) 1193e1201. [10] G. Innocenti, A. Piovan, R. Fillippini, R. Caniato, E.M. Cappelletti, Quantitative recovery of furanocoumarins from Psoralea bituminosa, Phytochem. Anal. 8 (1997) 84e86. [11] W. Jung, O. Yu, S.M. Lau, D.P. Okeefe, J. Odell, G. Fader, B. Mcgonigle, Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes, Nat. Biotechnol. 18 (2000) 208e212. [12] D. Kanne, K. Straub, H. Rapoport, J.E. Hearst, Psoralenedeoxyribonucleic acid photoreaction. Characterization of the monoaddition products from 8methoxypsoralen and 4,5’,8-trimethylpsoralen, Biochem 21 (1982) 861e871. [13] I. Kasajima, T. Fujiwar, Identification of novel Arabidopsis thaliana genes which are induced by high levels of boron, Plant Biotechnol. 24 (2007) 355e360. [14] L.S. Lakshmi, K.J. Reddy, Influence of growth medium source of explants and micronutrients on daidzein and psoralen contents in cell cultures of Cullen corylifolium (L.), J. Plant Biol. 34 (2007) 33e41. [15] R. Larbat, S. Kellner, S. Specker, A. Hehn, E. Gontier, J. Hans, F. Bourgaud, U. Matern, Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis, J. Biol. Chem. 282 (2007) 242e254. [16] P.G. Latha, D.A. Evans, K.R. Panikkar, K.K. Jayavardhanan, Immunomodulatory and antitumour properties of Psoralea corylifolia seeds, Fitoterapia 71 (2000) 223e231. [17] H.L. Li, W.D. Zhang, T. Han, C. Zhang, R.H. Liu, H.S. Chen, Tetrahydroprotoberberine alkaloids from Corydalis saxicola, Chem. Nat. Comp. 43 (2007) 173e175. [18] W. Li, M.A. Schuler, M.R. Berenbaum, Diversification of furanocoumarinmetabolizing cytochrome P450 monooxygenases in two papilionids: specificity and substrate encounter rate, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 14593e14598. [19] J.Y. Liu, Z.G. Guo, Z.L. Zeng, Improved accumulation of phenylethanoid glycosides by precursor feeding to suspension culture of Cistanche salsa, Biochem. Eng. J. 33 (2007) 88e93. [20] D.P. Lu, D.X. Zhao, Y. Huang, Q. Zhao, The effects of precursor feeding on flavonoids biosynthesis in cell suspension cultures of Saussurea medusa, Acta Bot. Yunnanica 23 (2001) 497e503. [21] X. Mei, D. Wu, Q. Cheng, X. Shu, W. Huang, Effects of metabolic regulators on the biosynthesis of taxol and its analogue, Nat. Prod. Res. Dev. 13 (2001) 17e20. [22] B. Murali, A. Amit, M.S. Anand, B.J. Venkataraman, An HPLC method for simultaneous estimation of psoralen bakuchicin and bakuchiol in Psoralea corylifolia, J. Nat. Remedies 2 (2002) 76e79.

1146

B.M. Parast et al. / Plant Physiology and Biochemistry 49 (2011) 1138e1146

[23] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant 15 (1962) 473e497. [24] R.D.H. Murray, The naturally occurring coumarins, Prog. Chem. Org. Nat. Prod. 83 (2002) 1e19. [25] C. Nguyen, F. Bourgaud, P. Forlot, A. Guckert, Establishment of hairy root cultures of Psoralea species, Plant Cell Rep. 11 (1992) 424e427. [26] C. Nguyen, V. Bouque, F. Bourgaud, A. Guckert, Quantification of daidzein and furanocoumarin conjugates of P. cinerea L. (Leguminosae), Phytochem. Anal. 8 (1997) 27e31. [27] A.M.A.G. Oliveira, M.M.M. Raposo, A.M.F. Oliveira-Campos, A.E.H. Machado, P. Puapairoj, M. Pedro, Psoralen analogues: synthesis, inhibitory activity of growth of human tumor cell lines and computational studies, Eur. J. Medi. Chem. 41 (2006) 367e372. [28] U. Oster, R. Tanaka, A. Tanaka, W. Rüdiger, Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana, Plant J. 21 (2001) 305e310. [29] V. Pandey, M. Chopra, V. Agrawal, In vitro isolation and characterization of biolarvicidal compounds from micropropagated plants of Spilanthes acmella L, Parasitol. Res. 108 (2011) 297e304. [30] J.T. Paturde, S.G. Wankhade, P.P. Khode, D.D. Deo, P.U. Ghotol, Evaluation of improved babchi (Psoralea corylifolia) genotypes with high psoralen content, Agric. Sci. Dig. 20 (2000) 104e105. [31] M. Perassolo, C. Quevedo, V. Busto, F. Ianone, A.M. Giulietti, J. Rodriguez Talou, Enhancement of anthraquinone production by effect of proline and aminoindan-2-phosphonic acid in Rubia tinctorum suspension cultures, Enzym. Microbial. Technol. 41 (2007) 181e185. [32] C.F. Qiao, Q.B. Han, S.F. Mo, J.Z. Song, L.J. Xu, S.L. Chen, D.J. Yang, L.D. Kong, H.F. Kung, H.X. Xu, Psoralenoside and isopsoralenoside, two new benzofuran glycosides from Psoralea corylifolia, Chem. Pharm. Bull. 54 (2006) 714e716. [33] C.F. Qiao, Q.B. Han, J.Z. Kong, S.F. Mo, L.D. Kong, H.F. Kung, H. Xu, Chemical fingerprint and quantitative analysis of Fructus psoraleae by high-performance liquid chromatography, J. Sep. Sci. 30 (2007) 813e818. [34] S.J. Rajput, V. Zade, P. Rai, Studies on extraction, isolation and estimation of psoralen from the fruits of Psoralea corylifolia, Pharmacogn Mag. 4 (2008) 52e56. [35] A. Raychaudhuri, P.A. Tipton, Cloning and expression of the gene for soybean hydroxyisourate hydrolase localization and implications for function and mechanism, Plant Physiol. 130 (2002) 2061e2068. [36] B. Ruan, L.Y. Kong, Y. Takaya, M. Niwa, Studies on the chemical constituents of Psoralea corylifolia L, J. Asian Nat. Prod. Res. 9 (2007) 41e44.

[37] R.A. Salzman, T. Fujita, Z. Salzman, P.M. Hasegawa, R.A. Bressan, An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates, Plant Mol. Biol. Rep. 17 (1999) 11e17. [38] N. Samanani, S.U. Park, P.J. Facchini, Cell type specific localization of transcripts encoding nine consecutive enzymes involved in protoberberine alkaloid biosynthesis, Plant Cell. 17 (2005) 915e926. [39] J. Sambrook, E.F. Fritsch, T. Mianiatis, Molecular Cloning: a Laboratory Manual, second ed. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York, 1989. [40] F. Sanger, S. Nickles, A.R. Coulson, DNA sequencing with chain-terminating inhibitors, Proc. Natl. Acad. Sci. U. S. A. 74 (1977) 5463e5467. [41] V. Seidel, J. Windhövel, G. Eaton, A.W. Alfermann, R.R.J. Arroo, M. Medarde, M. Petersen, J.G. Wolley, Biosynthesis of podophyllotoxin in Linum album cell cultures, Planta 215 (2002) 1013e1039. [42] R.S. Stern, Psoralen and ultraviolet A light therapy for psoriasis, New Eng. J. Med. 357 (2007) 682e690. [43] W. Sumaryono, P. Proksch, T. Hartmann, M. Nimtz, V. Wray, Induction of rosmarinic acid accumulation in cell suspension cultures of Orthosiphon aristatus after treatment with yeast extract, Phytochem 30 (1991) 3267e3271. [44] T. Suzuki, H. Mori, T. Yamame, S. Shimuzu, Automatich supplementation of minerals in fed-batch culture to high cell mass concentration, Biotech. Bioeng. 27 (1985) 192e201. [45] M. Takizawa, K. Hori, K. Inai, H. Takase, T. Hashimoto, Y. Watanabe, A virusinduced gene silencing approach for the suppression of nicotine content in, Nicotiana benthamiana. Plant Biotechnol. 24 (2007) 295e300. [46] Y. Wang, C. Hong, C. Zhou, D. Xu, H. Qu, Y. Cheng, Screening antitumor compounds psoralen and isopsoralen from Psoralea corylifolia L, Seeds (2009). doi:10.1093/ecam/nen087. [47] B. Weisshaar, G.I. Jenkins, Phenylpropanoid biosynthesis and its regulation, Curr. Opin. Plant Biol. 1 (1998) 251e257. [48] D. Xin, H. Wang, J. Yang, Y.F. Su, G.W. Fan, Y.F. Wang, Y. Zhu, X.M. Gao, Phytoestrogens from Psoralea corylifolia reveal estrogen receptor-subtype selectivity, Phytomed 17 (2010) 126e131. [49] D. Xu, N. Redman-Furey, Statistical cluster analysis of pharmaceutical solvent, Int. J. Pharm. 339 (2007) 175e188. [50] Q. Yan, Z.D. Hu, J.Y. Wu, Synergistic effects of biotic and abiotic elicitors on the production of tanshinones in Salvia miltiorrhiza hairy root culture, China J. Chinese Material Media 31 (2006) 188e191. [51] J. Zhao, L.C. Davis, R. Verpoorte, Elicitor signal transduction leading to production of plant secondary metabolites, Biotechnol. Adv. 23 (2005) 283e333.