The Effect of Elicitors on the Production of Vasicine from Justicia adhatoda L. Cell Suspension Cultures

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1 Chapter 4 The Effect of Elicitors on the Production of Vasicine from Justicia adhatoda L. Cell Suspension Cultures 4.1. ABSTRACT In this study, α-naphthyl acetic acid and mannitol were used as elicitors to improve the productivity of useful metabolite, vasicine for archiving high concentration in Justicia adhatoda L. cell suspension cultures. When the cells were treated with 3 mg 100 ml -1 suspension of α- naphthyl acetic acid and 0.32M 100ml -1 of mannitol, the vasicine concentration was increased in the cell cultures. After 15 days of the cell culture, it was found that the vasicine concentration was significantly higher when compared to untreated control cell cultures. Key Words: vasicine, elicitor, α-naphthol acetic acid, mannitol, Justicia adhatoda L.

2 80 Chapter INTRODUCTION Plant cell culture is often an effective system to study the biological significance of bioactive metabolites under in vitro conditions, as well as for producing natural products for bioprocessing applications (Walker et al., 2002). It has been industrially used for the synthesis of secondary metabolites (Riedel et al., 2012). The regular increase in demand in world market for natural, renewable products has refocused attention on in vitro plant materials as potential factories for secondary phytochemical products, and has paved the way for new research exploring secondary product expression in vitro (Anand, 2010). The synthesis of plant secondary metabolites can be stimulated by acting on different parameters: environmental factors, use of precursors of the targeted molecules, use of elicitors and, genetic transformation of the plant (Vu et al., 2006). Secondary metabolites production in entire plants depends on endogenous (Hashimoto et al., 1991) and exogenous factors (Boitel-Conti et al., 2000). Plants express a number of morphological and physiological responses to a range of physical and chemical factors from the environment, known as elicitors, which activate chemical defenses in plants (Bota and Deliu, 2011). Enhancement of secondary metabolites by elicitor application is one of the few strategies which have recently found commercial application. Elicitors can be abiotic or biotic compounds of mainly microbial origin or non-biological origin, which upon contact with higher plant cells; trigger the increased production of pigments, flavones, phytoalexins and other defense related compounds (Pirian and Piri, 2012).

3 The Effect of Elicitors on the Production of Vasicine 81 The recent development of elicitation has opened a new avenue for the production of secondary compounds. Secondary metabolite synthesis and accumulation in cell cultures can be triggered by the application of elicitors to the culture medium (Subathra and Srinivasan, 2011). Vasicine is found to be effective for the treatment of various diseases and disorders, mostly for respiratory tract ailments. This work reports the successful approach for the elevated production of vasicine by cell suspension cultures after using α- naphthyl acetic acid (NAA) and mannitol as elicitors REVIEW OF LITERATURE Plant based secondary compounds have been incorporated into a wide range of commercial and industrial applications, and fortuitously, in many cases, rigorously controlled plant in vitro cultures can generate valuable natural products. There is a series of distinct advantages for producing a valuable secondary product in plant cell culture, rather than in vivo in the whole crop plant. Several strategies are being followed to improve yields of secondary metabolites in plant cell cultures (Anand, 2010). Plant cells are biosynthetically totiopotent (Rao and Ravishankar, 2002). Secondary products however are generally not produced in significant quantities until a culture reaches stationary phase. During the last decades many approaches have been used to improve the usually low content of secondary metabolites in cell cultures. The optimization of culture conditions has resulted in fold increase in metabolites production. Plant cell culture technology and its optimization provide a continuous and constant year round supply of natural plant products. Growth regulator concentration is often a crucial factor in secondary product accumulation. Mantell and

4 82 Chapter 4 Smith (1984) reported that the type and concentration of auxins and cytokinins or the auxin/cytokinin ratio, dramatically alters both the growth and the product formation in culture plant cells. Under some stress-inducing conditions, the regulation of amino acids pathways may be dominated by the need for secondary metabolites derived from the pathway rather than protein synthesis (Zia et al., 2007). However, the industrial application of plant cell suspension cultures has, to date, been limited. One of the difficulties of industrial production by plant cell culture is the low productivity caused by low concentration of the secondary metabolites. However, the low yield of secondary metabolites in plant cell culture has been a bottleneck for commercialization. Consequently there have been numerous attempts to improve the productivity of secondary metabolites in plant cell culture such as medium optimization, cell line selection, cell immobilization, precursor addition, elicitation, and metabolic engineering. Among these manipulation techniques, elicitation is a very attractive strategy for increasing the metabolite production in cell culture system, which can lead to increased yields and shortened culture times. Elicitation is the induction of secondary metabolite production by either biotic or abiotic treatments (Li et al., 2011). The production of secondary metabolites can be enhanced by treating the undifferentiated cells with elicitors (Raskin et al., 2002). The molecules which are able to stimulate the production of secondary metabolites are called Elicitors, and the phenomenon is known as Elicitation (Aijaz et al., 2011). Wu et al. (2003) and Riedel et al. (2012) stated that in nature, the process of elicitation induces the synthesis of plant secondary metabolites to ensure their survival, persistence and competitiveness. Elicitors produced

5 The Effect of Elicitors on the Production of Vasicine 83 within plant cells are termed as endogenous elicitors, while supplied from outside are called exogenous elicitors. Elicitors are classified as physical or chemical, biotic or abiotic and complex or defined depending on their origin and molecular structure. Elicitors induce different types of effects in plant cells which are reflected by the influenced cell metabolism. Secondary metabolite synthesis in the plant cell tissue cultures can be targeted by the application of physical, chemical and biological elicitors. The elicitors mimic the effects of stresses and thereby activate the plant biochemical system, which results in increased contents of secondary metabolites in plant tissues (Saw et al., 2010). Elicitors of plant origin are polysaccharides derived from cell walls, e.g., pectin, pectic acid, cellulose, while those of microorganism origin are cell wall components like chitin, chitosan or glucans. These elicitors are of biological origin; hence they are called biotic elicitors. Low concentrations ( mg L -1 ) of elicitors added to the culture medium dramatically enhance secondary metabolite production by plant cells. Biotic elicitors bind to a receptor protein in plasma membrane, which ultimately leads to the accumulation of secondary metabolites. The production of these compounds is a dynamic defense response exhibited by plant cells when challenged by an elicitor. The most frequently used elicitors in previous studies were fungal carbohydrates, yeast extract, methyl jasmonate (MJ), pectin and chitosan. Fungal homogenates from families such as Phytophthora, Aspergillus, and Alternaria, and abiotic elicitors, e.g., inorganic salts of cadmium, copper, and vanadyl, Jasmonates (plant hormones that have a dual effect on plant growth and development) etc. were also used as elicitors. From early studies carbohydrates have been implicated in the over production of secondary metabolites in plant cell cultures (Aijaz et al., 2011). Anand (2010) stated

6 84 Chapter 4 that elicitor- induced production of secondary metabolites is important for optimizing their commercial production. Different elicitors, jasmonic acid (JA) and salicylic acid (SA) were independently used to enhance the synthesis of phenolics in suspension cultures of Vitis vinifera c.v. Muscat de Frontignan followed by chemical analysis with high-performance liquid chromatography (Riedel et al., 2012). A combination of inappropriate medium and elicitor as well as unsuitable concentration of the latter can result in ineffective elicitation. For example, phenyl propanoid pathway was not induced in all cultures of Vanilla planifolia by yeast extract, indicating that a successful elicitation is a very challenging process requiring intense screening procedures (Pirian and Piri, 2012). In plant cell cultures, UV light acts as an abiotic factor which stimulates the biosynthesis of secondary metabolites (Broeckling et al., 2005). Thus, it has been shown that UV-B light induces both the formation of dimeric terpenoid indole alkaloids and tryptophan decarboxylase and strictosidine synthase mrna accumulation in Catharanthus roseus (Almagro et al., 2011) Abiotic elicitor molecules such as salicylic acid (SA) and methyl jasmonate (MJ) are known to induce the production of secondary metabolites when added to culture medium and promote the production of terpenes (Penuelas et al., 2007) including taxol (Yuan et al., 2002; Wang et al., 2004). According to Holopainen et al. (2009) elicitors are compounds, which activate chemical defenses in plants. Various biosynthetic pathways are activated in treated plants depending on the compound used. The most

7 The Effect of Elicitors on the Production of Vasicine 85 intensively studied elicitor for manipulating defense pathways in plants is methyl jasmonate, which modifies e.g. the production of terpenoids, the main constituents of conifer oleoresin. Other commonly tested chemical elicitors are salicylic acid, methyl salicylate and benzothiadiazole, which affect production of phenolic compounds in plants. Both jasmonate-based and salicylate-based elicitors have been shown to have suppressive effects on fungal diseases and insect pests of plants. Zia et al. (2007) were studied the effect of amino acids and growth regulators on the production of artemisinin, an antimalarial compound, in the callus of Artemisia absinthium. The presence of valine and cystine enhanced the production of artemisinin. Bhambhani et al. (2012) used the addition of selected elicitors like methyl jasmonate (MJ), chitosan, yeast extract, ascorbic acid, and sodium salicylate at optimized concentrations for the enhanced production of vasicine in J. adhatoda cell culture. Bhojwani and Razdan (1966) stated that plant growth regulators affect cell growth and differentiation and therefore affect secondary metabolite production by cultured cells. According to them the alkaloid yield was remarkably increased by the combined presence of an auxin and a cytokinin. Of the various auxins tested NAA was most effective. Coste et al. (2011) proved that cultivation of Hypericum maculatum on modified M S medium with NAA resulted in approximately two fold increased production of hypericins compared to controls. By the studies on Dioscorea bulbifera Srivastava et al. (2003) also proved the effect of NAA to trigger the secondary metabolite production.

8 86 Chapter 4 The yield of the vasicine from different samples in India ranged from to 1.105% on dry basis. Yield as high as 2.18% on dry basis has been reported from a foreign sample of which more than half was the l -form and the remainder the dl-form of the alkaloid (Arambewela et al., 1988). 4.4 SPECIFIC OBJECTIVES The study was designed with the following objectives 1. To study the effect of α-naphthyl acetic acid, as elicitor on the production of vasicine from cell suspension cultures of J. adhatoda. 2. To study the effect of mannitol, as elicitor on the production of vasicine from cell suspension cultures of J. adhatoda MATERIALS AND METHODS SUSPENSION CULTURE In order to initiate suspension culture of J. adhatoda, 50 gm of friable calli from culture tubes were transferred to 250 ml Erlenmeyer flasks containing 100 ml of MS medium with1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mg of NAA (Central Drug House (P) LTD, India). And another set of suspension culture with mannitol of 0.08 M, 0.16 M, 0.32 M and 0.64 M (E. Merck India) were also prepared. 250 ml Erlenmeyer flasks containing 100 ml of MS medium with 50gm of friable calli, without elicitors, was kept as control. The flasks were incubated on a rotary shaker at 120 rpm using the same culture conditions used for callus culture.

9 The Effect of Elicitors on the Production of Vasicine EXTRACTION OF SECONDARY METABOLITE FROM SUSPENSION CULTURE From the suspension culture of friable calli, the supernatant was collected and extracted with methanol for 30 minutes by using a separating funnel. The extracts collected and concentrated to half the volume QUANTIFICATION OF VASICINE Quantification of total alkaloids was done by spectrophotometric method with tropaeolin OO (Sigma Aldrich, India). Coloured complex developed was measured at 545 nm against blank. The amount of total alkaloids in the samples was calculated using standard curve of vasicine (SPIC India Ltd, Chennai). The content of the total alkaloids was expressed as vasicine (Soni et al., 2008). All the procedures were done in three replicates 4.6 RESULT EFFECT OF NAA AND MANNITOL IN VASICINE PRODUCTION Vasicine content was found to be 6.73 mg ml -1 and 6.56 mg ml -1 in suspension cultures (Table 4.1 and 4.2) with 3 mg 100 ml -1 NAA and 0.32 M 100 ml -1 Mannitol respectively which were higher than that of field grown plants (Soni et al., 2008) and control culture (5.08 mg ml -1 ). All the observations strongly support the use of NAA and Mannitol as elicitors for vasicine production from J. adhatoda. But after the 20 th day of inoculation, a slight reduction in the concentration of vasicine was noticed (Figure 4.1 and 4.2) and in the culture without elicitors, the reduction was detected after the 15 th day of inoculation.

10 88 Chapter 4 Table.4.1. Influence of NAA as elicitor for enhancing the production of vasicine in suspension cultures of J. adhatoda Concentration of NAA in mg 100ml -1 On 5 th Production of vasicine in mg ml -1 On 10 th On 15 th On 20 th On 25 th ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.10 Control 1.85± ± ± ± ±0.12

11 The Effect of Elicitors on the Production of Vasicine 89 Figure 4.1. Effect of NAA as Elicitor for vasicine production from J. adhatoda suspension culture with Control Table Influence of mannitol as elicitor for enhancing the production of vasicine in suspension cultures of J. adhatoda Concentration of mannitol in moll -1 On 5 th Production of vasicine in mg ml -1 On 10 th On 15 th On 20 th On 25 th ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.10 Control (Noelicitor) 1.85± ± ± ± ±0.12

12 90 Chapter 4 Figure 4.2. Effect of Mannitol as Elicitor for vasicine production from J. adhatoda suspension culture with Control 4.7. DISCUSSION Present study was conducted to examine the effect of elicitors, NAA and mannitol, on the production of vasicine in the callus suspension cultures of J. adhatoda. Mannitol is a polyol normally synthesized in numerous species of vascular plants, which serves as an alternate metabolic reserve as well as an osmoprotectant. Mannitol accumulation increases when plants are exposed to a low water potential and the most obvious function of this compound is in osmotic adjustment. Therefore, mannitol has been proposed to enhance tolerance to water deficit stress primarily through osmotic adjustment. It is already described the role of mannitol as hydroxy radical scavenger. The mannitol added to cultures causes a mild osmotic stress to cell cultures and, in time, this treatment may activate an adaptive mechanism that can render the cells tolerant to more severe treatments. During the culture on medium supplemented mannitol, the cells take up mannitol that

13 The Effect of Elicitors on the Production of Vasicine 91 can act as an osmoprotectant. However, accumulation of mannitol may have adverse effects, even though mannitol is a compatible solute, the target plant may not tolerate high levels of mannitol and can mimic a water stress environment (Mitoi et al., 2009). According to Bagchi et al. (2003) and Arambewela et al. (1988) J. adhatoda showed seasonal variation and the vasicine content was high in dry climate. The plant shows wide seasonal variation in vasicine content in its leaves. It exhibited higher levels of vasicine twice in a year i.e. 3.0% in March and 1.4% in September. Interestingly, it coincided with the flowering of the plant. In March, it is in full bloom condition and in September, it is in partial flowering. During the vegetative stage, the plant contained very low concentration of vasicine (Bagchi et al., 2003). So mannitol induced water stress might have been a reason for observed enhancement in vasicine accumulation in comparison to control. The effect of NAA to trigger the secondary metabolite production was supported by the findings of Bhojwani and Razdan (1966) and the studies of Srivastava et al. (2003) on Dioscorea bulbifera. Work of Coste et al. (2011) with Hypericum maculatum also gave appreciable result with NAA. In the present work the cell cultures showed great sensitivity towards elicitors by producing vasicine when supplemented with NAA and mannitol at concentration as 3 mg 100 ml -1 and 0.32M 100ml -1 respectively (Table 4.1 and 4.2). The increased production, through elicitation, of the secondary metabolites from plant cell culture has opened up a new area of research, which could have important economical benefits for bio industry. Elicitation studies have shown promise in increasing yields and cutting production costs.