Enhanced daidzin production from jasmonic and acetyl salicylic acid elicited hairy root cultures of Psoralea corylifolia L. (Fabaceae)

Daidzin (7-O-glucoside of daidzein) has several pharmacological benefits in herbal remedy, as antioxidant and shown antidipsotropic activity. Hairy root culture of Psoralea corylifolia L. was developed for biomass and enhanced daidzin production using signalling compounds such as jasmonic acid (JA) and acetyl salicylic acid (ASA). Best response of 2.8-fold daidzin (5.09% DW) with 1 μM JA treatment after second week and 7.3-fold (3.43% DW) with 10 μM JA elicitation after 10th week was obtained from hairy roots compared to untreated control. ASA at 10 μM promoted 1.7-fold increase in daidzin (1.49% DW) content after seventh week compared to control (0.83% DW). Addition of 25 μM ASA resulted in 1.44% DW daidzin (1.5-fold increase) with 0.91% DW in control after fifth week and 1.44% DW daidzin (2.3-fold increase) after eighth week when compared to untreated control (0.62% DW). Reduced biomass with increased daidzin content was facilitated by elicited hairy root cultures. Graphical Abstract


Introduction
Psoralea corylifolia L. (Fabaceae) produces many isoflavonoids (daidzein, genistein, daidzin, genistin, etc.) which are derived from the malonate-shikimate biosynthetic pathway (Gholami et al. 2014). P. corylifolia L. is widely used in Indian and Chinese medicine for antibacterial, anti-inflammatory, antitumor, hepatoprotective, antidiabetic, antioxidant, antihelminthic GRAPHICAL ABSTRACT activities and it acts against diseases such as hypertension, osteoporosis and dermatitis (Chopra et al. 2013). Daidzin is one of the major isoflavone glycosides. The pharmacological importance of daidzin can be traced back to the report of Xie et al. (1994) and the finding described daidzin as an antioxidant isoflavonoid. Daidzin intake was found beneficial for the treatment of alcohol toxicity with the reduction in blood ethanol level in animal models. Antidipsotropic activity and herbal remedy uses of daidzin for addiction to alcohol have also been reported (Rooke et al. 2000;Lowe et al. 2008).
Plant tissue and organ cultures are considered as alternative for the supply of bioactive pharmaceutical secondary metabolites (Begum et al. 2009;Murthy et al. 2014). The hairy roots induced by Agrobacterium rhizogenes infection are genetically stable and grow rapidly in hormone-free media (Giri et al. 2003;Chandra & Chandra 2011, Sharma et al. 2013Murthy et al. 2014). Hairy root culture of P. corylifolia was developed in our laboratory for the production of valuable bioactive compounds. The metabolic profiling of P. corylifolia hairy root clones through LC-MS and AFLP analysis was studied (Abhyankar et al. 2005). The only report available on this plant species pertain to molecular analysis of phenylpropanoid pathway is the isolation and characterisation of isoflavone synthase gene (Misra et al. 2010). Recently, study on the isolation of gene and detailed expressed sequence tag analysis was performed on methyl jasmonate treated Centella asiatica (Kim et al. 2014). The elicitation of P. corylifolia hairy roots with signalling molecules may open up possibilities for enhanced production of isoflavonoids. Several findings on elicitation of hairy root cultures and improvement in biosynthetic competence in vitro were reported (Goel et al. 2011;Ramakrishna & Ravishankar 2011;Kastell et al. 2013;Qin et al. 2014;Thakore et al. 2015).
Earlier study on P. corylifolia hairy roots revealed higher accumulation of isoflavonoids (daidzein and genistein) following elicitation with biotic and abiotic elicitors (Shinde et al. 2009). However, the present communication reports for first time the influence of chemical elicitors (jasmonate and acetyl salicylic acid (ASA)) on biomass and enhanced daidzin (7-O-glucoside of daidzein) production from hairy root cultures of P. corylifolia.

Influence of chemical elicitors on biomass and enhanced daidzin production
Jasmonic acid (JA) and its derivatives are stress signalling molecules involved in plant defence and regulate various metabolic pathways along with other plant hormones (Ramakrishna & Ravishankar 2011;Pirbalouti et al. 2014). The influence of JA and ASA on biomass and daidzin production using hairy root cultures of P. corylifolia was investigated in this study which revealed variable responses.

Effect of JA on biomass and daidzin production
Growth kinetics study of P. corylifolia untreated control hairy roots revealed that there was an overall increase up to sixth week and decline in growth thereafter. The fresh weight (FW) and dry weight (DW) of untreated control and JA-treated hairy roots were similar in first week with a marginal decrease after second and third week, compared to control. The growth of hairy roots was less with 1 μM JA and it was further less in 5 and 10 μM treated roots compared to control after fourth and fifth week (Figures S1 and S2). In Sinapis alba, all the concentrations of JA treatment failed to increase biomass of hairy roots compared to control (Kastell et al. 2013). More biomass of hairy roots was obtained after seventh week of treatment with 1, 5 and 10 μM JA compared to control roots. FW and DW of hairy roots were more with 1 and 5 μM JA and insignificant increase in growth was obtained with 10 μM JA compared to control after eighth and ninth weeks. Marginal increase in FW and DW was observed only with 1 μM JA compared to control hairy roots after 10th week. High concentrations of JA (25, 50 and 100 μM) had an inhibitory effect on the growth of hairy roots as evidenced by blackening of roots from first to 10th weeks. In an earlier finding with Withania somnifera also similar response with suppressed growth of hairy roots was observed at higher JA concentrations (Doma et al. 2012).
Treatment of hairy roots with 1, 5 and 10 μM JA resulted in daidzin production in the range of 2.70, 2.95 and 2.02% DW, respectively; after first week when compared to control (1.34% DW) hairy roots. Highest and maximum daidzin production of 5.09% DW (2.8-fold increase) was observed after second week when 1 μM JA was used in comparison with untreated control (1.79% DW) hairy root cultures. Lower JA concentrations (1, 5 and 10 μM) resulted in 2.88, 4.01 and 2.97% DW daidzin production, respectively; compared to control (2.28% DW) after seventh week. Hairy roots treatment with JA at 1, 5 and 10 concentrations resulted in daidzin production and was in the range of 3.29, 3.59 and 3.61% DW, respectively; after eighth week. Addition of 1, 5 and 10 μM JA resulted in 2.73, 3.22 and 3.50% DW, respectively; when compared to control (1.83% DW) after ninth week ( Figure S3).
High concentrations of JA (25, 50 and 100 μM) resulted in decreased daidzin content in hairy roots. Baicalin production in hairy root cultures of Scutellaria baicalensis using methyl jasmonate revealed analogous trend (Hwang 2006). After 10th week, the production of daidzin was 2.55%, 3.43% (maximum 7.3-fold increase), 2.07%, 1.19%, 1.13% DW when 5, 10, 25, 50 and 100 μM JA were used, respectively; but inhibited root growth after 10th week ( Figure S3) compared to untreated control (0.47% DW). A study on the production of alkamide from hairy root cultures of all the three Echinaceae species adds credence to the present finding (Romero et al. 2009). However, an increase was evident in higher concentration only after 10th week. Previously, glucosinolates levels increased in hairy root cultures of S. alba and Brassica rapa in response to JA, but root growth was inhibited (Kastell et al. 2013). Low concentrations of JA (1, 5 and 10 μM) promoted growth and content in hairy root cultures. These results imply that the daidzin accumulation could be enhanced with low concentrations of JA in the culture medium. Recently, the action of signal compound in the presence of perfluorodecalin was found favourable to increase taxane production in taxus hairy root cultures (Sykłowska-Baranek et al. 2015).

Effect of ASA on biomass and daidzin production
ASA a chemical derivative (analogue) of salicylic acid was found to enhance the yield of secondary metabolites using in vitro cultures (Doma et al. 2012;Qin et al. 2014). The FW and DW of untreated control and all concentrations of ASA (1, 10 and 25 μM) treated hairy roots showed similar pattern of reduced growth from first to fourth week. Wielanek and Urbanek (2006) showed that ASA had a negative influence on growth of Tropaeolum majus hairy roots. Marginal increase in FW and DW was observed only with 1 μM ASA compared to control roots without ASA treatment after fifth and sixth week. Increase in FW and DW was observed with treatment of 1 and 10 μM ASA compared to control roots after seventh week. Meagre increase in FW and DW of roots was obtained only with 1 μM ASA, but there was negligible increase with 10 μM ASA compared to control after eighth week. ASA at 25 μM has a drastic effect on growth of hairy roots from first to 10th week when compared to other concentrations of ASA and untreated control roots (Figures S4 and S5). Further, 50 and 100 μM ASA did not promote higher biomass generation. Hence, higher ASA concentrations were excluded from the evaluation of elicitors for biomass and daidzin production. This is the first report on ASA usage for the daidzin production in hairy root cultures of P. corylifolia. Additionally, various concentrations of ASA improved daidzin production compared to control. Addition of 1, 10 and 25 μM ASA enhanced the daidzin content up to 0.67, 0.83 and 0.73% DW, respectively; after second week when compared to untreated control (0.48% DW) cultures. All the concentrations of ASA evaluated showed decrease in daidzin production after third week compared to control ( Figure S6). ASA at 25 μM produced 1.24%, 1.44% DW after fourth and fifth week, respectively; when compared to other concentrations of ASA and untreated control cultures. After sixth week, a little increase in daidzin content was observed with all the concentrations of ASA when compared to control. Maximum daidzin production of about 1.45 and 1.49% DW (1.7-fold increase) was obtained with 1 and 10 μM ASA, respectively; after seventh week when compared to control cultures (0.83% DW). Daidzin content was 1.03% DW with 10 μM ASA and 1.44% DW (2.3-fold increase) with 25 μM ASA when compared to 1 μM ASA and control cultures after eight week. ASA at 1 μM produced 1.22% DW daidzin which is more than the roots treated with 25 μM ASA (0.88% DW) after ninth week when compared to 10 μM ASA (0.45% DW) and control (0.52% DW). All the concentrations of ASA were proved to be effective after 10th week when compared to untreated control hairy roots Figure S6. ASA was proved to be the best chemical elicitor among all other abiotic elicitors used for the production of glucotropaeolin in hairy roots of T. majus (Wielanek & Urbanek 2006). ASA also enhanced the production of withanolide in W. somnifera and tropane alkaloids in Atropa baetica (el Jaber-Vazdekis et al. 2008;Doma et al. 2012).
In this study, high concentration of ASA (25 μM) enhanced the production of daidzin after fourth, fifth, eighth and 10th weeks. High concentration of ASA increased the biosynthesis of tropane alkaloid in Anisodus luridus hairy root cultures (Qin et al. 2014). All the concentrations of ASA promoted increased daidzin production except first and third week compared to control. These results showed that elicitation by ASA did not depend on concentration and duration of treatment for enhanced production of secondary metabolites. Further experiments using ASA with the involvement of other plant species may strengthen the present claim. Similar reports on isoflavones in hairy root cultures of P. corylifolia (Shinde et al. 2009) and Pueraria candollei (Udomsuk et al. 2011) lend credence to our work. Higher concentrations of ASA suppressed the growth of hairy roots but stimulated accumulation of daidzin. Even though the daidzin content was increased with ASA treatment, highest production of daidzin was obtained with JA-treated hairy root cultures. This proves that comparatively JA is a more effective elicitor to work with P. corylifolia when compared to ASA.

Conclusions
JA and ASA driven elicitation of hairy root cultures facilitated enhanced daidzin production. This finding may be helpful for bioprocess development for large-scale production of pharmaceutically important daidzin using P. corylifolia hairy root cultures.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Authors would like to thank sponsor OU-DST-PURSE project, Department of Science and Technology (DST), New Delhi for financial support.

Supplemental data and research materials
Supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419 .2015.1054823.