Enhanced production of bioactive compound in the callus culture of Glycyrrhiza glabra L. with the improved biomass accumulation

Licorice ( Glycyrrhiza glabra ) of Fabaceae family is known to have wide range of medicinal properties due to the metabolites found in the plant’s tissue. Callus cultures from Glycyrrhiza glabra were previously initiated in vitro using leaf as an explant. The current study was designed in a way to examine the possible role of different elicitors concentrations on the stimulation of biomass and their effects on different metabolite content such as total carbohydrates, protein, proline, phenol, alkaloid, avonoid and glycyrrhizin. Elicitation with different concentration of elicitors increased the biomass and metabolite content in callus culture of G. glabra at different rates. The optimum concentration of adenine sulphate for maximum biomass accumulation (16.79 g/ask) was found to be at 50 mg/l on the incubation of 20 th days. Adenine sulphate as well as putrescine was also found to stimulate the different metabolite content to 3-4 folds in the callus cultures as compared to that of control. Results showed that the metabolite content and antioxidant enzyme of Glycyrrhiza glabra can be enhanced by appropriate forms of elicitation.


Introduction
Glycyrrhiza glabra (licorice), a perennial herb of Fabaceae family, is abundantly rich in ameliorating bioactive secondary metabolites used for the treatment of a wide range of human ailments (Wang et al. 2013). The major constituents of licorice are triterpenoids and avonoids. A variety of additional constituents such as triterpene, saponins, avonoids, polysaccharides, pectins, simple sugars, amino acids, mineral salts, starch, gums, mucilage, essential oil, fat, tannins, glycosides, protein, resins, sterols, volatile oils and various other compounds have also been derived from licorice (Blumenthal et al. 2000).
Glycyrrhizin (potassium-calcium-magnesium salts of glycyrrhizic acid), a triterpenoid, is the most active constituent which re ects the sweet taste of licorice root. It is exclusively obtained from the dried roots and stolons of licorice and used in the treatment of liver and allergic diseases. Glycyrrhizin is 50 times or more sweeter than sugar and demands high prices globally as a non-nutritive sweetener (Jaiswal et al. 2017). It is manufactured in the form of injections (such as Stronger Neo-Minophagen®C) and tablets (such as Glycyron®) which are available in India and many other countries (Hayashi and Sudo 2009). Gels containing glycyrrhizin are used for the treatment of oral diseases and genital lesions caused due to Herpes simplex virus (Varsha et al. 2009).
Attempts were made to understand the biochemistry of licorice and its derivatives. Glycyrrhizin are synthesized through their speci c metabolite pathways and possess speci c structural and functional characteristics. Biochemical synthesis of the metabolite for industrial use is often not feasible due to complex metabolic pathways, complicated structures and chirality exhibited by these compounds (Namdeo 2007). The commercial demand can only be met by obtaining them directly from eld grown plants. However, G. glabra accumulates glycyrrhizin in small amounts in specialized tissues probably after attaining a certain stage in their life cycle. Apart from this, the yields from eld grown plants are in uenced by many factors like climate, pests and diseases which are di cult to control and in turn affect their consistent production, due to which the commercial exploitation becomes a challenging task (Oksman-Caldenteyl and Inze 2004).
Recent developments in plant tissue culture techniques alongwith their processing have shown promising results to improve the productivity to many folds and have made it possible to gradually replace the whole plant cultivation as a source of useful secondary metabolites. Today, various tissue culture techniques are being used to enhance yield of secondary metabolites by invigorating plant defense and triggering stress response in plant cells with the help of elicitors (Chattopadhyay et al. 2002). Elicitors are being used as an enhancement strategy in plant secondary metabolite synthesis as they play an important role in stimulating the biosynthetic pathways leading to enhanced production of commercially important compounds. This provides an opportunity for intensive research in the eld of plant sciences not only to exploit plant cells for increased yield of secondary metabolites, but also for the investigation of plant defense mechanism and regulation of secondary metabolism (Radman et al. 2003).

Plant material
Cuttings of Glycyrrhiza glabra were procured from the nursery of Central Institute of Medicinal Aromatic Plants (CIMAP), Lucknow, and maintained in the greenhouse (relative humidity 70-90%; temperature 25-30 o C). The stem and leaves were excised and washed under running tap water for 20-30 minutes to remove the contaminants. Explants were then surface sterilized with 2-3 drops of Tween 20 and 0.1% mercuric chloride for 5-10 minutes followed by repeated rinses with sterile distilled water prior to in vitro culture.
Establishment of callus culture and culture conditions Callus induction was initiated from the stem segments and young leaves of one-month old plants of licorice, sterilized as above and then gently cut or scraped using a scalpel. The explants were then inoculated on MS medium (Murashige and Skoog 1962) supplemented with the optimized combinations of phytohormones BAP (2.0 mg/l) and 2,4-D (0.5 mg/l) along with 3% sucrose and 0.8% agar, as reported by Jaiswal et al. (2017). The medium was adjusted to pH 5.8 and autoclaved at 15 psi for 15 minutes. After inoculation the cultures were incubated at 25+2°C under at photoperiod of 12 hours with a light intensity of 40 μmolm -2 s -1 provided from cool white uorescent tubes. Callus thus formed was sub-cultured every 30 days on the same, fresh medium.

Elicitation
The effect on plant growth and biochemical metabolites of various abiotic elicitors including adenine sulphate, biotin, salicylic acid and polyamines (putrescine, spermine and spermidine) at three different concentrations (30, 60 and 90 mg/l) was studied. All elicitors above were prepared as a stock solution and then added to the callusing media.

Estimation of primary and secondary metabolites
The Anthrone method was used for total carbohydrates determination (Hedge and Hofreiter 1962) and the results were expressed as glucose in milligram per gram of sample. Total protein was determined according to the method given by Lowry et al. (1951) and recorded as bovine albumin serum in milligram per gram of sample. Total phenol was assessed according to the method by Bray and Thorpe (1954) with results expressed as catechol in milligram per gram of sample. Proline was determined according to the method given by Bates et al. (1973). Total alkaloid, expressed in milligram per gram of sample, was measured according to Fazel et al. (2008) using atropine as a standard. Total avonoid was evaluated according to the method of Chang et al. (2002) using quercertin as a standard and expressed in milligram per gram of sample.
Glycyrrhizin estimation HPLC analysis of glycyrrhizin was carried out using the method by Hurst et al. (1983). The callus as well as the leaves and roots of eld grown plants were weighed, crushed and extracted using 50% methanol under ultra-sonication (Elma, Germany), for 30 mins at 45 MHz. The extract was ltered (Millipore lter 0.45 μm) and concentrated on a rotary-evaporator (Buchi, Switzerland) and 2 ml of solvent was added to the sample before analysis. The resultant extracts were then used for subsequent HPLC analysis. A 10μl aliquot of extract was analysed by HPLC at 30°C. The HPLC system consisted of Waters HPLC 510 pump, a Nova-pak C18 column (3.9 × 150 mm, Waters, United States), a Waters 2478 detector, and a Millennium chromatography data system (Waters). The separation was performed with an isocratic elution using methanol-water-acetic acid (60: 34: 6) at a ow rate of 1 ml/min with UV absorption detection at 254 nm. Routine sample calculations were made by comparison of the peak area with that of the standard.

Assay of antioxidant enzyme activity
Approximately 0.5 g fresh samples were homogenized in 8 ml of 50 mM PBS (pH 7.6) including 0.1 mM Na-EDTA, and then centrifuged for 15 mins at 20,000 rpm and 4°C. The superoxide dismutase (SOD) activity was estimated by recording the decrease in optical density of formazone made by superoxide radicals and nitroblue tetrazolium chloride (NBT) dye by the enzyme (Cakmak and Marschner 1992). One unit of SOD activity was de ned as the amount of enzyme required to cause 50% inhibition of the rate of NBT reduction measured at 560 nm. Peroxidase (POX) activity was measured as described by Castillo et al. (1984) and the enzyme activity was calculated as per extinction coe cient of its oxidation product, tetra-guaiacol. The activity of ascorbate peroxidase (APX) was measured according to Cakmak (1994) and measured by monitoring the rate of H 2 O 2 -dependent oxidation of ascorbic acid.

Effect of various elicitor concentrations on biomass accumulation
After 15 days callus were sub-cultured on to the best callusing media (MS + 2mg/l BAP + 0.5mg/l 2,4-D) forti ed with the various elicitors (biotin, adenine sulphate, salicylic acid, putrescine, spermine or spermidine) at different concentrations to optimize biomass accumulation. Biomass accumulation (25, 50, 75 and 100 mg/l) was recorded for different time intervals (10, 20 and 30 days) as shown in Table 1.
Biomass accumulation in control was 3.15, 5.66 and 8.21 g/ ask after 10, 20 and 30 days of incubation respectively. Elicitation with different elicitors increased the biomass in callus culture of G. glabra at different, signi cantly different rates ( Table 1). The optimum concentration of biotin (B), salicylic acid (SA) and spermidine (SD) was 75 mg/l, whereas that of adenine sulphate (AdS), putrescine (P) and spermine (SP) was 50 mg/l after incubation for 20 days.
Maximum biomass production was found with adenine sulphate (16.79 g/ ask) elicitation followed by putrescine (14.23 g/ ask) at 50 mg/l. Elicitation fostered, a 2-6 fold increase in biomass as compared to the control culture. Biomass production (callus weight) varies during incubation i.e., rst it increases and reaches its peak on the 20 th day and then it abruptly begins to decline.
The change in biomass production induced by various elicitors was found to be dependent on their concentration as well as on the incubation time. The biomass accumulation was affected by high concentration of elicitors (75-100 mg/l) in the medium as well as by prolonged incubation period (beyond 20 days) resulting into cell browning with decreased viability and cell death. Thus, increased callus growth in G. glabra is triggered when an adequate concentration of the appropriate elicitor is incorporated in the medium and callus are incubated for a speci ed period.

Effect of various elicitors on the production of different biochemical metabolites
The effect of the exogenous elicitors on the variation of various phytoconstituents present in licorice was evaluated and is summarized in Table 2 and Table 3. The concentration at which elicitors (biotin, adenine sulphate, salicylic acid, putrescine, spermine and spermidine) were showing highest biomass accumulation was selected for further biochemical analysis. The time of incubation of callus with the elicitors was of 20 days.
Elicitation seems to enhance the production of different metabolites in G. glabra at different rates (Table 2 and Table 3). The elicitors applied chemical stress to the callus culture triggering the production of different primary and secondary metabolites at rates that are normally not produced. Seven different biochemical parameters viz. carbohydrates, proteins, proline, phenols, alkaloids, avonoids and glycyrrhizin were noted to be higher in the callus culture as compared to the leaves and roots of in vivo plants. Callus treated with the elicitors also showed higher metabolite content than the untreated callus.
Among the different elicitors, adenine sulphate and putrescine proved to be most effective in enhancing primary metabolite content in callus culture of licorice. The callus treated with 50 mg/l putrescine showed the highest carbohydrates content (54.82 mg/g) followed by those treated with 50 mg/l adenine sulphate (49.71 mg/g) whereas the highest content of protein (28.41 mg/g), phenol (36.46 mg/g) and proline (0.082 µmol/g) were found in callus treated with 50 mg/l adenine sulphate. The lowest metabolite content was found in callus treated with 75 mg/l spermidine. (Fig. 1) Similarly, among the different elicitors tried, putrescine (75 mg/l) gave the highest alkaloid content (13.71 mg/g) whereas adenine sulphate (75 mg/l) showed the highest avonoid content (16.29 mg/g). The least alkaloid and avonoid content (4.69 and 7.12 mg/g) were found in spermidine treated callus. Glycyrrhizin was absent in the leaves of the eld grown plant while glycyrrhizin content was highest in adenine sulphate treated callus (35.44µg/g) and lowest (8.02 µg/g) in untreated callus. (Fig.2) It may thus be assumed that the in vitro raised tissues have higher content of metabolites than the in vivo plants and that these are enhanced by all the elicitors although at different concentrations and varying rates. Also, the callus treated with elicitors had higher metabolite content than the untreated callus (control).
Effect of various elicitors on the activity of antioxidant enzyme The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POD) have been analyzed to gain some insights of the cellular antioxidant responses triggered by elicitation. The antioxidant enzyme activities of callus increased rapidly after elicitation (Table 4). The enzyme activities of calluses treated with elicitors were found to be higher than that of leaves, roots and control callus. Superoxide dismutase (1.382 unit/mg protein) and ascorbate peroxidase (0.531 unit/mg protein) activity was maximum in callus treated with adenine sulphate while peroxidase activity (0.733 unit/mg protein) was highest in putrescine treated callus.

Discussion
Elicitation studies in callus culture using a wide range of elicitors have been documented to be successful in boosting the accumulation of biomass and metabolite production (Ram et al. 2013). During elicitation the optimal age of the culture varies between different plant cell system (Ahmed et al. 2014). The reaction of the cells to elicitor exposure depends on the growth stage of the culture thereby in uencing the production of biomass and secondary metabolite (Deepthi and Satheeskumar 2016).
The e ciency of elicitors on cell growth and product yield varied according to their concentration, genotype, incubation period and culture characteristic, which may be due to the variations between different species, different cell lines and different cell physiology  (Ram et al.2015) and D. hamiltonii (Zang et al. 2016). Adenine sulphate as nitrogen source can be drawn more rapidly by the cell than the inorganic nitrogen (Singh and Patel 2014). Nitrogen is essential for plants and can in uence various physiological processes that regulate growth and morphogenesis according to its availability, source and concentration (Ramage and Williams 2002). Chandler and Dodds (1983) noticed that nitrogen in uenced in vitro production of secondary products of S. laciniatum. Adenine sulphate (a nucleoside base) assist sulphate assimilation and amino acid biosynthesis necessary for the synthesis of metabolites (Sharma et al. 2014). In a variety of cultures, it serves as a cytokine synergist, a substrate for cytokinin synthesis and may delay cytokinin degradation by feed-back inhibition, or by competing for cytokinins metabolism enzymes (Van Staden et al. 2008). Adenine sulphate was found to enhance the different metabolite contents in several other plant species including C. asiatica L. (Sharma et al. 2014), T. grandis L. (Akram and Aftab 2015), W. somnifera L. (Sivanandhan et al. 2015) In conjugation with other media components, the exogenous provision of vitamins has been documented to have a direct and indirect impact on different processes, such as callus growth, embryonic development, rooting and somatic growth (Abrahamian and Kantharajab 2011), acting as a cofactor of several enzymes considered essential for the metabolism of nutrients (Hildebrand et al. 2005). Biotin (water soluble B complex vitamin) is a source of nitrogen, and it manages and transport cytokinins needed for plant growth, development and metabolism. Being a heterocyclic compound, biotin serves as a cofactor for enzymes involved in several reactions (carboxylation, decarboxylation, transcarboxylation and transamination) concerned with the fatty acid and carbohydrates metabolism and protein synthesis (Alban et al. 2000). Biotin promoted callus induction and proliferation in several other species such as Capsicum annuum (Kintzios et al. 2001), Curcuma mangga (Tamil et al. 2012) and Phoenix dactylifera (Diab 2015). However, no reports are available on metabolite production from callus culture treated with biotin.
Salicylic acid is extensively used to regulate the physiological, metabolic and biochemical activities of plants thereby affecting their growth (Orenes et al. 2013;Khan et al. 2015). SA stimulatory effects on callus and plant development have been reported in C. o cinalis L. (Bayat et al. 2012), Ziziphus spina christii (Galal 2012) and Vigna mungo (Lingakumar et al. 2014). SA enhances both the primary as well as secondary metabolite in plants (Babel et al. 2014). It induces pathogen resistance protein and was successful in providing systemic acquired resistance to pathogens . SA potentially alters the metabolic pathways leading to the accumulation of phytoconstituents during in vitro culture (Ram et al. 2013), may be because SA directly or indirectly affects the synthesis and signalling pathways of auxins, ethylene and jasmonic acid (Vlot et al. 2009). Salicylic acid elicits different metabolites content in several plant species such as Andrographis paniculata L. (Zaheer and Giri 2015), Bacopa monnieri (Largia et al. 2015) and Achillea millefolium L. (Gorni and Pacheco 2016).
Polyamines (PAs) are small aliphatic amines (particularly, putrescine, spermine and spermidine) found in almost all plant cells (Sawhney et al. 2003). They engage in several stress reactions triggering cell division and differentiation (Liu et al. 2015), and could be an endogenous regulator for plant growth or hormonal messengers (Davis 2004). Several ndings showed a strong association between polyamine level and several fundamental processes (cell division, tissue differentiation, organogenesis and embryogenesis alongwith macromolecular biosynthesis) (Aydin et al. 2016). Polyamines interact with phytohormones enhancing the production of callus in M. charantia (Thiruvengadam et al. 2012), P. gerardiana (Ravindra and Nataraja 2013) and Phoenix dactylifera (Ibrahim et al. 2014). PAs in uence the membrane transport by regulating the proton pump which plays an important role in secondary metabolism (Janicka-Russak et al. 2010). PAs incorporation with phytohormones enhanced the secondary metabolite production in other species such as P. corylifolia L. (Shinde et al. 2009), N. cataria L. (Yang et al. 2010) and P. ginseng (Marsik et al. 2014).
Elicitors trigger the plant defense signals by an immediate cellular response which increases the accumulation of reactive oxygen species (ROS) including hydroxyl free radicals (OH -), hydrogen peroxide (H 2 O 2 ) and superoxide anions (O 2 -). In order to reduce these excessive ROS, different cell compartments use antioxidant enzymes like ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), peroxidase (POD) and superoxide dismutase (SOD) to activate the cell defensive systems (Martinez et al. 2016). Under light, chloroplast and peroxisomes are the main source of ROS production while in dark it is mitochondria (Foyer and Noctor 2005). The severe discrepancy between ROS production and antioxidant defenses leads to oxidative stress which causes cellular damages thereby increasing the production of secondary metabolites (Zhao et al. 2005). The application of elicitors induces ROS burst in the cultured cells of Parsley, Taxus chinensis and as well as in protoplasts of Arabidopsis (Wang and Wu 2005;Sasaki-Sekimoto et al. 2005). H 2 O 2 enhanced the production of phytoalexin by activating the signaling defense genes and coordinating with antioxidant response (Ramos-Valdivia et al. 2012).
During the ROS detoxi cation process SOD catalyzed the primary reaction by providing rst line of defense against the toxic effects of ROS (Ali et al. 2006), and abiotic elicitors have been shown to increase SOD, CAT, APX and POX activities (Martinez et al. 2016).
Under unfavorable conditions SOD is the rst to scavenge off toxic O 2 − level, and other antioxidant enzymes (APX, CAT, GR and POX) convert H 2 O 2 into H 2 O and molecular oxygen, preventing cellular damage (Scandalios 2005). In several plant genera, elicitation was found to increase stress which in turn resulted in an increased SOD activity (Samar et al. 2011). During elicitation, a similar pattern was observed for CAT, POD and APX activity, except for tissue and dose speci c variation (Elkahoui et al. 2005).
SA induces the production of ROS thereby developing systemic acquired resistance (SAR) (Kawano and Muto 2000). Depending on the H 2 O 2 concentration SA increases and reduces the activities of catalase (CAT) and peroxidase (POX) (Guan and Scandalios 2006). Defensive teams such as CAT and POX targeted protective cells from oxidative damage (Mittler 2002). SA enhanced the antioxidant activities in many other plant species (Rehman et al. 2017). Adenine sulphate was also reported to elicit different antioxidant enzyme activities in other plant species (Ahmad et al. 2017). Polyamines have effectively improved both enzymatic and non-enzymatic antioxidant activities (Asthir et al. 2012). Abundant studies have emphasized that the plants under stress showed an interaction between polyamines and reactive oxygen species (Pottosin et al. 2014). Tables   Table 1. Effect of different elicitors on biomass accumulation of in vitro grown callus of G. glabra on MS media supplemented with 2 mg/l BAP, 0.5 mg/l 2,4-D and 40 mg/l Ascorbic acid conc.