Carbohydrate-induced biomass accumulation and elicitation of secondary metabolites in callus cultures of Fagonia indica

Industrial Crops & Products 126 (2018) 168–176

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Carbohydrate-induced biomass accumulation and elicitation of secondary metabolites in callus cultures of Fagonia indica ⁎

T

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Tariq Khana,b,c, , Bilal Haider Abbasia, , Alam Zebb, Gul Shad Alic a

Department of Biotechnology, Quaid-i-Azam University Islamabad, 45320, Pakistan Department of Biotechnology, University of Malakand, Chakdara, KP, Pakistan c Mid-Florida Research and Education Center and Department of Plant Pathology, University of Florida/Institute of Food and Agricultural Sciences, 2725 Binion Rd., Apopka, FL, 32703, United States b

A R T I C LE I N FO

A B S T R A C T

Keywords: Carbohydrates F. indica Callus Biomass Antioxidant Phenolic compounds

Elicitation is a very feasible strategy to enhance important secondary metabolites, such as phenolic compounds, flavonoids, and terpenoids in plant cell cultures. In this report, we studied the effects of different carbohydrate sources on biomass and secondary metabolism of Fagonia indica callus cultures. The results showed that disaccharides, especially sucrose, are favorable for biomass accumulation when applied in higher concentration. Maximum biomass (Fresh weight = 52.05 g/100 mL and dry weight = 2.72 g/100 mL) was recorded in callus cultures raised in vitro in 5% sucrose. Maltose-supplemented callus cultures (5%) also responded with higher biomass (fresh weight = 43.75 g/100 mL and Dry weight 2.715 g/100 mL). Considering the production of secondary metabolites, 3% glucose produced the highest total phenolic content (TPC) in callus cultures (1.677 mg GAE/g DW) followed by fructose (1.625 mg GAE/g DW). The high-performance liquid chromatography data showed that a higher concentration of carbohydrates in the media elicited higher quantities of important phenolic compounds. It is concluded that the antioxidant potential of callus cultures is directly related to the secretion of phenolic compounds because 3% glucose-treated callus cultures gave the highest 82.11% antioxidant activity. The total chlorophyll content was found to decrease with the increasing concentration of carbohydrates. In conclusion, maltose, as a source of carbohydrate in F. indica callus cultures elicits the production of secondary metabolites including Gallic acid, Caffeic acid, Salicylic Acid, Quercetin, Myricetin, and Ellagic acid.

1. Introduction

exploitation for the preparation of pharmaceuticals can endanger the plant. Research programs must be initiated and implemented for sustainable utilization and conservation of this important medicinal plant (Khan et al., 2015). The plant is being investigated in vitro through different culture systems for increased production of medicinally important secondary metabolites (Ebrahimi and Payan, 2013; Khan et al., 2017, 2016). Cell cultures, especially callus cultures, can produce a handful of secondary metabolites (Nikolaeva et al., 2009). Callus cultures can be exploited further to provide inoculum to establish and develop cell suspension (Mustafa et al., 2011), as a source of explant for plantlet regeneration (Ikeuchi et al., 2013), for induction of somatic embryos (Abbasi et al., 2016), and adventitious root cultures (Sivakumar et al., 2005). Callus cultures are usually affected by various in vitro conditions including explant type, plant growth regulators, nutrient supply, carbohydrate source and other environmental conditions (Khan et al., 2016; Kumar et al., 2015; Lee et al., 2011; Yan et al., 2009). In vitro cultures depend on the growth-room light for their

The medicinal plant, Fagonia indica is well known for its anticancer potential (Waheed et al., 2012). It is rich in medicinally important secondary metabolites, such as phenolic compounds, flavonoids, and terpenoids. F. indica products have been employed as tea or decoction for the treatment of various disorders (El-hadidi et al., 1988; Hope, 2012; Shaker et al., 2000; Shehab et al., 2011). The importance of F. indica has been highlighted in numerous studies for being active as the antimicrobial, antihemorrhagic, hepatoprotective, antidiabetic, anthelminthic, thrombolytic plant (Bagban et al., 2012; Rasool et al., 2014; Shehab et al., 2011; Soomro and Jafarey, 2003). The most prominent activity of F. indica has been its anticancer potential. Lam et al. (2012) have demonstrated the potent role of F. indica extract against breast cancer cell lines, MCF-7 and MDA-MB-231 via FOXO3a and p53 expression. Owing to its medicinal importance, a drastic increase in market demand of F. indica has been observed in recent years. Over-

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Corresponding author. E-mail addresses: [email protected] (T. Khan), [email protected] (B.H. Abbasi).

https://doi.org/10.1016/j.indcrop.2018.10.023 Received 10 June 2018; Received in revised form 5 October 2018; Accepted 7 October 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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in triplicate from culture flasks in the form of (i) callus initiation day, (ii) callus induction frequency (%), (iii) callus dimension (width and height in cm), and (iv) callus biomass (FW g/100 mL and DW g/ 100 mL). Callus cultures grown in response to different carbohydrate concentrations were sub-cultured by putting 400 mg of fresh callus in MS media supplemented with TDZ 1.0 mg/L and various concentrations (1, 3, and 5%) of the different carbohydrates. For each set of cultures, data was recorded as FW and DW with an interval of 7 days for a total of the 49-day culture period. Samples were oven dried for 24 h at 40 °C to determine DW. 3% sucrose, which is usually applied to cultures, was treated as a control treatment. The general growth conditions were maintained as described in the previous section.

photosynthesis (Tariq et al., 2014). However, the growth-room light is not enough for photosynthetic activity and thus the plant cells become heterotrophic and start relying upon the exogenously supplied carbohydrate source. Monosaccharides and disaccharides have been employed in vitro in various concentrations for their stimulating effects on biomass accumulation and production of useful compounds in various commercially important plant species (Kretzschmar et al., 2007; Kumar et al., 2015). Amorim et al. (1977) evaluated the effects of exogenous glucose on phenol synthesis in Paul's Scarlet Rose cells grown in liquid suspension or solid culture and concluded that higher concentrations of glucose in the culture medium resulted in an increased synthesis of phenols. In another study, Pourtau et al. (2006) showed that increased intake of external sugars promotes symptoms of senescence such as the leaf yellowing in Arabidopsis thaliana. This has been linked with the production of phenolic compounds and thus subsequent oxidation, yellowing, and deterioration of leaves of the plant. Similarly, Lux-Endrich et al. (2000) concluded that the increased sucrose content resulted in an enhanced amount of phenolic compounds. Conventionally, 3% sucrose is defined as a standard carbohydrate amount and type in plant culture medium for feasible in vitro growth (Murashige and Skoog, 1962). Sucrose is preferred for growth because of its rapid intake, long-distance intercellular transport and long half-life inside cells (Tognetti et al., 2013). Other types of carbohydrates are also used for growth and development in plant in vitro cultures. For instance, Maltose has been shown to produce an optimum biomass accumulation in callus cultures of Gossypium hirsutum (Kumar et al., 2015). Similarly, the exogenous application of fructose and glucose have also been reported to influence the growth and development of plant cells, organs or tissues (Ma et al., 2017). Apart from being crucial components of the general metabolism, carbohydrates are important signaling molecules in cellular and metabolic processes during biotic and abiotic stress situations (Lastdrager et al., 2014; Rolland et al., 2006). Sugars also act as a signal for regulating the plant defense system (Morkunas and Ratajczak, 2014). Sugars stimulate the biosynthesis of secondary metabolites such as phenolic and flavonoids to confer anti-oxidative properties to the plant system. Sugar optimization was found to affect the production of flavonoid in callus and cell suspension cultures of Coleus blumei (Qian et al., 2009). This suggests that changes in sugar supplementation to the callus culture medium can be used as a strategy to stimulate the production of medicinally important phenolic compounds. The alteration in carbohydrate source and concentration during cell cultures, especially callus cultures, is a relatively novel approach for elicitation of secondary metabolites (Kumar et al., 2015). This study, for the first time, reports the impacts of different carbohydrate types and concentrations on the biomass accumulation, antioxidant activity, and the secretion of phenolic and flavonoid compounds in callus cultures of F. indica.

2.2. Analytical scheme 2.2.1. Total phenolic, antioxidant and chlorophyll content analysis For determination of total phenolic content (TPC), extraction from samples was performed according to the method used in our previous study (Khan et al., 2016). For confirmation of yield enhancement in terms of TPC, the assays were performed on the samples obtained from the cell suspension, harvested at an interval of seven days. The reaction mixture was incubated for 30 min at room temperature. The optical density (OD) of the samples was read at 630 nm with a microplate reader (Synergy H1 Hybrid Multi-Mode Reader, Biotek Inc; USA). The results are expressed as mg gallic acid equivalent (GAE) per gram of DW. Free radical scavenging activity (FRSA) in the samples was determined according to Amarowicz et al. (2004). Furthermore, Chlorophyll “a’’ and “b’’ were investigated following the method of (Tariq et al., 2014). The extract color intensity was measured using spectrophotometer (Shimadzu, UV-120-01) at 645 nm and 663 nm to estimate chlorophyll “a’’ and chlorophyll “b’’. The chlorophyll content (mg/g fresh weight) was calculated using the following formulas. Chla = (mg/ g FW of leaf) = [0.0127(OD663)-0.00269(OD645)]x 100 Chlb = (mg/g FW of leaf) = [0.0229(OD645)-0.00648(OD663)]x 100 Here, O.D. = Optical Density. 2.2.2. HPLC based quantification of phenolic compounds For the quantification of phenolic compounds, the protocol of Zeb (2015) was followed. Briefly, samples in duplicates were mixed with water-methanol in 1:9, v/v and were kept in shaker overnight at 30 °C. The extraction was performed three times. Afterward, 90 mm Whatman® general purpose filter paper from Sigma-Aldrich, Germany was used to filter the samples which were then centrifuged for 30 min at 4000 rpm. Furthermore, for the concentration of the filtrate, a vacuum concentrator was used, and the concentrate was filtered again through the PFTE filter paper (0.45 μm; Agilent Technologies, Germany) and transferred directly into HPLC vials (2 mL). The Agilent 1260 infinity HPLC-DAD system of the Agilent Technologies, Germany was used for determination of phenolic compounds in callus cultures. A 3.5 μm C18 Agilent rapid resolution column with dimensions of 4.6 × 100 mm was used for the separation (Agilent Technologies, Germany). A gradient elution system was employed for the separation which comprised of solvent A in the composition of 10-288, v/v (methanol-acetic acid-deionized water) and solvent B in the composition of 90-2-8, v/v (methanol-acetic acid-deionized water). The gradient elution was such that it started with 100% solvent A for 5 min, 85% A after 5 min, 50% A after 20 min, 30% A after 25 min, and ultimately reaching 100% B after 30 to 40 min. The spectra ranging from 200 to 600 nm were noted for detecting phenolic compounds. Further, available standards, UV spectra and retention times or comparison with the literature were used for the identification of phenolic compounds. The peak area against the standard calibration curves was used to quantify the compounds identified in this method. The calibration curve was prepared from caffeic acid, ellagic acid, gallic acid, phloroglucinol, quercetin, quercetin-3-glucoside, and salicylic acid. The quantity of phenolic compounds was drawn through the formula given

2. Materials and methods 2.1. Effect of varying levels of different carbohydrates on callus growth and biomass accumulation in F. indica Callus cultures were established by following our previously published report (Khan et al. (2016). Briefly, stem explants (∼1.0 cm) from 50 days old lab-grown plantlet of F. indica were cultured in Murashige and Skoog basal medium (MS0, 1962) containing 0.8% (w/v) agar, 3% sucrose and Thidiazuron (TDZ = 1.0 mg/L). In subsequent experiments, the callus cultures were grown in varying concentrations (1% = 10 g/L, 3% = 30 g/L 5% = 50 g/L) of sucrose, glucose, fructose, and maltose supplied in the MS medium. Before autoclaving (121 °C for 20 min) the pH of the growth media was adjusted to 5.8. Further, culture flasks were maintained under controlled growth conditions i.e. 25 ± 2 °C and a 16 h light & 8 h dark. Data on callus growth parameters were collected 169

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cultures of Gossypium hirsutum (Kumar et al., 2015). Glucose and fructose supplementation resulted in a comparatively lesser biomass (Table 1). Both glucose and fructose are monosaccharides and are degraded readily in the cell, which makes them less feasible for biomass accumulation (Tognetti et al., 2013). These hexoses responded differently to fresh and dry biomass accumulation. For instance, higher FW (37.8 g/100 mL) was recorded in response to lower concentrations (1%) of glucose while the highest DW (2.015 g/100 mL) in response to glucose was recorded at higher concentrations (5%) (Table 1). In addition, carbohydrates at lower concentrations, produced granular and light or yellowish green callus while increasing the concentration turned the callus into brownish green and compact mass of cells (Fig. 1).

below.

mg mg ⎞ ⎛ ⎞ × V (mL) × D⎞/ Wt Ax ⎛ Phenolic compound ⎛⎜ ⎟ = mL g ⎝ ⎠ ⎝ ⎠ ⎝ ⎠ Ax represents the concentration of each phenolic compound (in mg/ mL) derived from the standard calibration curve, V means volume of the extraction (mL), D gives the dilution factor, and Wt represents the weight (grams) of the dry sample. 2.3. Statistical analysis All the experiments were repeated at least three times and were performed in a completely randomized fashion. Statistical analysis was carried out using SPSS 22.0 and GraphPad Prism 7.0. Two-way ANOVA and Tukey’s multiple range tests (HSD) for post-hoc analysis was used to check the significant mean difference using p < 0.05. All the figures were made using Origin 8.5 pro.

3.2. Effects of carbohydrate source and concentration on growth kinetics Carbohydrates are the main drivers of primary metabolism, growth, and development of plants (Lloyd and Zakhleniuk, 2004). The results of the present study showed that the carbohydrate types and concentrations have varying effects on the growth kinetics of F. indica callus cultures. The callus cultures supplemented with all types and concentrations of carbohydrates remained in a shorter lag phase of 7 days. In sucrose-supplemented callus cultures, the biomass increased after the lag phase and the cultures did not enter the stationary phase even after 49 days (Fig. 2). For glucose, however, the callus growth achieved a stationary phase on the 42nd day of culture. Optimal concentration (3%) of fructose resulted in biomass accumulation even after 49 days. However, callus cultures supplemented with both lower (1%) and higher (5%) concentrations of fructose remained in the log phase for about 42 days and then entered the stationary phase followed by decline phase (Fig. 2). Statistical analysis (two-way ANOVA) revealed that there was a significant difference (p < 0.05) between DW of callus grown under the effect of different types of carbohydrates. Interestingly, the effect of concentration of these carbohydrates was also significant (p < 0.0001). When applied in optimal concentrations (3%), the difference in response of carbohydrate type was significant (p < 0.0001). However, at higher concentration, the effect of the carbohydrate type became less significant and most of the variation was attributed to concentration rather than type. The favorable response of sucrose-treated callus cultures in terms of biomass accumulation might be due to the stability of sucrose which can remain longer in cells compared with glucose and fructose. Glucose and fructose are readily taken, rapidly metabolized and are not transported or accumulated between cells. Sucrose signals the initiation of biosynthesis of starch through up-regulation of many different genes involved in starch biosynthetic pathways, a response glucose and fructose are unable to elicit (Tognetti et al., 2013). Starch is a recognized energy reservoir which

3. Results and discussion 3.1. Effects of different carbohydrate types on callus induction and biomass accumulation Sugars play vital regulatory and physiological roles in the growth and development of plants (Stokes et al., 2013). In the present study, 3% sucrose resulted in the highest callus induction frequency (90%) in explants than all other concentrations and types of sugars used. At this treatment, callus formation was observed on the third day of culture cultivation (Table. 1). Moreover, sucrose at higher concentrations (5%) produced the highest FW (52.05 g/100 mL) and the highest DW (2.72 g/100 mL) in 42 days old callus cultures. Sucrose is an important product of photosynthesis which is hydrolyzed through the cell’s metabolism into hexoses such as glucose and fructose by either of the invertases in the cell wall, cytoplasm and/or vacuole. The intracellular hexoses are then employed in glycolysis for the synthesis of sugar polymers such as starch, cellulose, and fructan (Rolland et al., 2006). Sucrose promotes growth through cell division as shown in different plant parts such as the roots of pisum (Tognetti et al., 2013). Moreover, studies have reported the removal of sucrose from cell cultures of A. thaliana caused cell cycle cessation. In a study, Riou-Khamlichi et al. (2000) reported that sucrose causes the expression of cyclin B and D involved in cell cycle progression. Maltose produced more biomass (FW = 43.75 and DW 2.715 g/ 100 mL) at higher concentration (5%) (Table 1). Maltose is also a disaccharide and has been shown to positively affect the growth of callus

Table 1 The effects of different carbohydrate sources and concentrations on different growth parameters and secondary metabolites accumulation in 42 days old callus cultures of Fagonia indica. The letters represent significant difference between the values. Carbohydrate Source

Type

Concentration (%)

Sucrose

1 3 5 1 3 5 1 3 5 1 3 5

Glucose

Fructose

Maltose

Initiation day

5rd 3th 4th 4th 6th 7th 5th 7th 8th 6th 4th 8th

Percent indu ction

70%b 90%a 65%bc 60%c 68%bc 55%cd 62%bc 65%bc 72%b 55%cd 57%cd 48%d

Callus dimensions

Maximum biomass (g/100 mL)

Max. Width (cm)

Max. Height (cm)

FW

DW

5.26 ± 0.19b 5.94 ± 0.54b 7.04 ± 0.68a 4.62 ± 0.32bc 4.80 ± 0.53bc 6.00 ± 0.60ab 5.42 ± 0.56b 4.51 ± 0.66bc 5.03 ± 0.14b 5.37 ± 0.60b 4.61 ± 0.42bc 5.61 ± 0.34b

4.13 ± 0.25bc 5.62 ± 0.29a 4.33 ± 0.48b 4.18 ± 0.14b 4.70 ± 0.65ab 3.37 ± 0.27c 3.51 ± 0.17c 4.05 ± 0.16b 4.54 ± 0.13ab 4.33 ± 0.17b 4.07 ± 0.50bc 3.77 ± 0.22c

34.5 ± 0.71b 49.35 ± 0.50a 52.05 ± 0.16a 37.8 ± 1.05b 32.8 ± 0.43b 31.7 ± 0.54b 22.8 ± 0.18c 38.7 ± 0.55b 30.65 ± 0.17b 36.4 ± 0.70b 32.1 ± 0.21b 43.75 ± 0.35ab

0.75 ± 0.09c 2.035 ± 0.02ab 2.965 ± 0.04a 0.782 ± 0.08c 1.335 ± 0.09b 2.015 ± 0.07ab 0.3 ± 0.01cd 1.792 ± 0.07b 1.535 ± 0.08b 0.655 ± 0.01c 1.33 ± 0.01b 2.715 ± 0.03a

170

Optimum TPC (mg GAE/g DW)

Total Phenolic Production (TPCxDW)

Morphology/ Characteristics

1.292 ± 0.034c 1.371 ± 0.026bc 1.297 ± 0.005c 1.247 ± 0.023c 1.677 ± 0.010a 1.645 ± 0.008a 0.908 ± 0.003cd 1.625 ± 0.007a 1.542 ± 0.013ab 0.994 ± 0.032cd 1.605 ± 0.030a 1.423 ± 0.021b

0.969 2.789 3.845 0.975 2.238 3.314 0.272 2.912 2.366 0.651 2.134 3.863

LG, granular LG, granular BG, compact LG, granular BG, granular BG, granular YG, granular YG, compact BG, compact LG, compact BG, compact BG, compact

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Fig. 1. Callus cultures as affected by various concentrations of Sucrose (S), Glucose (G), Fructose (F) and Maltose (M) 42 days after culture initiation (The numbers 1, 3 and 5 represents the percent concentrations of each carbohydrate used).

3.3. Effects of carbohydrate-type and source on the accumulation of useful secondary metabolites

provides glucose for glycolysis in case of carbohydrate deprivation. Like sucrose, maltose supplemented callus cultures were observed to grow even after 49 days of culture for all the concentrations (Fig. 2). The highest biomass was recorded in callus cultures supplemented with 5% maltose. This can be linked with the concept that maltose, a disaccharide, is metabolized in a cell at a slower rate compared with sucrose and it remains in the cell un-metabolized for a longer time (Kumar et al., 2015). In contrast, glucose- and fructose-treated callus cultures observed a limited log phase and entered the stationary phase and then a decline phase around the 5th and 6th week of culture initiation (Fig. 2). This might be due to the fact that cells sense hexoses very rapidly through hexokinases and fructokinases, which phosphorylate glucose and fructose, respectively (Smeekens and Hellmann, 2014). Another factor might be the lesser mobility of hexoses intracellularly as compared with sucrose which has a properly controlled long-distance transport and subcellular distribution (Tiessen and Padilla-Chacon, 2012). Furthermore, Glucose is also thought to regulate senescence and lifespan in plants by activating TOR kinases and that is why the increased concentration of the hexose might result in a rapid decline of plant cells (Sheen, 2014) (Fig. 1).

In addition to their most prominent role in growth, development and gene expression, carbohydrates have been found to play an active role in secondary metabolism (Wang and Weathers, 2007). It was observed that with increasing the time of culture the amount of phenolic compounds was increasing for all types of carbohydrates (Fig. 3). Our results indicate that the callus cultures accumulated higher phenolic content at moderate to higher concentrations of all the carbohydrate types. In sucrose-supplemented callus cultures, which were kept under control, the most TPC recorded was 1.375 mg GAE/g in response to sucrose 3% after 49 days of culture (Fig. 3). Increase in sucrose concentration has been reported as a trigger for secretion of important phenolic compounds in apple shoot cultures by Lux-Endrich et al. (2000). In our study, the highest TPC was observed in glucose supplemented callus cultures (1.677 mg GAE/g DW) in response to 3% glucose after 42 days of culture. Fructose supplementation in 3% concentration gave comparative TPC (1.625 mg GAE/g DW) after 42 days of culture. Similarly, maltose-supplemented callus cultures responded with the most TPC (1.605 mg GAE/g DW) after 49 days of culture (Fig. 3). Statistical analysis (ANOVA) showed that both concentration and type of 171

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Fig. 2. Effects of different concentrations of sucrose, glucose, fructose, and maltose on dry matter accumulation over time in callus cultures of Fagonia indica. The results were analyzed through two-way ANOVA and Tukey’s multiple comparison tests (p < 0.05).

Fig. 3. Effect of different carbohydrate sources and concentrations on the accumulation over time of phenolic compounds in callus cultures of Fagonia indica. The results were analyzed through two-way ANOVA and Tukey’s multiple comparison tests (p < 0.05). 172

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higher since they produce a higher callus DW as compared to Glucose. This was confirmed by multiplying TPC obtained in callus cultures grown under the effect of different carbohydrate type and concentrations with the DW obtained at the respective concentration (Table 1). Maltose is shown to increase the secretion of phenolic compounds in the wheat plant (Ibrahim and Abdellatif, 2016). This may be linked to the stress produced by excess maltose as indigenous maltose is synthesized by the plant cells in dark condition as a product of transitory starch conversion (Grennan, 2006). In our previous study, we reported the production of important phenolic compounds such as gallic acid, myricetin, caffeic acid, catechin, and apigenin in sucrose (3%) supplemented callus cultures (Khan et al., 2016). This study showed that increasing the concentration of sucrose (5%) increased the production of phenolic compounds in callus cultures of F. indica.

carbohydrates had a significant effect on the accumulation of TPC in callus cultures of F. indica. Point by point ANOVA results showed that all the concentrations tested had a significant effect (p < 0.0001) on the temporal accumulation of TPC. However, at lower concentrations (1%), the effect of carbohydrate type was not significant (p < 0.06). While, at higher concentrations, the carbohydrate type was a significant factor (p < 0.0001) in inducing temporal TPC accumulation. Therefore, it can be assumed that both concentration and type of carbohydrates were drivers of TPC accumulation in callus cultures of F. indica. Sugars act as signal molecules which interact with hormones and regulate plant defense system. This means that sugars can stimulate the synthesis of antioxidant flavonoids in case of any external stress stimulus (Morkunas and Ratajczak, 2014). The higher response in terms of TPC in glucose-treated callus cultures of F. indica is linked directly with the fact that glucose generates precursors of the phenylpropanoid pathway. Glucose is metabolized readily in glycolysis generating phosphoenolpyruvate (PEP), which ultimately enter the shikimate pathway through chorismate. Chorismate may then be degraded into the aromatic amino acids (tryptophan, tyrosine, and phenylalanine) and other related compounds. Phenylalanine ammonia-lyase (PAL) further transform phenylalanine or tyrosine into trans-cinnamic acid or p-coumaric acid. This generates a diverse class of aromatic secondary metabolites (Seigler, 2012). Therefore, glucose input may speed up the pathway and thus generate a handful of these molecules. This is in accordance with the study by Wang and Weathers (2007), where they reported an increased production of Artemisinin in the 3% glucosesupplemented cultures of Artemisia annua. Compared with glucose, sucrose is involved in primary metabolism and growth/cell division signaling, therefore, having a lesser impact on secondary metabolism (Tognetti et al., 2013). In accordance with our data, Kumar et al. (2015) also reported comparatively higher levels of phenolic compounds secreted in glucose- and fructose-treated callus cultures of Gossypium hirsutum. However, unlike our study, they reported the lowest concentration of phenolic compounds in maltosetreated callus cultures. TPC accumulated in a linear fashion in response to different concentrations of all the carbohydrates kept on increasing even after 49 days of culture (Fig. 3). There was no prominent lag phase in terms of TPC production as the subculture callus started to accumulate TPC in the first week which remained in the log phase until the end of the cultures and it might be suggested that the inoculum (initial callus transferred), when introduced to new media, started adapting to the stress situation by different carbohydrate types and concentration thus activating its secondary metabolism. It was observed that all the carbohydrates at lower concentrations (1%) accumulated the least TPC (Fig. 3).

3.5. Effects of carbohydrate source and concentration on the antioxidant activity There is a strong relationship between carbohydrate signaling and antioxidant defense system of plants (Bolouri-Moghaddam et al., 2010). In this study, varied antioxidant activity in response to different carbohydrate sources and concentrations was observed in 42-day old callus culture samples. Among the carbohydrate sources tested, 3% glucosetreated, 42-day old callus cultures displayed the highest FRSA (82.11%) (Fig. 4). While 1% maltose supplementation resulted in lowest antioxidant activity (35.17%), 5% fructose- and sucrose-treated cultures also showed higher antioxidant activity (75.02% and 67.64%), respectively (Fig. 4). It was, however, interesting to note that the callus cultures displayed maximum levels of antioxidant activities when given in higher concentrations except glucose-treated cultures which showed maximum activity when provided in 3% concentration. This was shown through two-way ANOVA of the DPPH results which confirmed that there was a significant difference among the different concentrations applied (p < 0.05). The Tukey’s multiple comparison tests also revealed a significant difference between the concentrations tested (p < 0.05). Similarly, statistical analysis revealed that 85.74% of the variation lies in the comparisons among concentrations of carbohydrates. On the other hand, ANOVA of DPPH results indicated that the difference between FRSA of different carbohydrates types was not significant (p > 0.05). However, Fisher’s LSD tests showed that Glucose showed significant effects on FRSA compared to Fructose and Maltose when applied in optimal concentrations (3%). Higher concentrations of glucose resulted in the quicker decline of callus cultures and thus lesser antioxidant activity. Furthermore, when correlated with secondary metabolites, the antioxidant activity in glucose-induced cultures was found dependent on total phenolic content. These results are in accordance with Ali et al. (2016), where they found a positive correlation between antioxidant activity and secondary metabolites. For other carbohydrates, at higher concentrations, the higher antioxidant and comparatively lesser phenolic content indicate the involvement of antioxidant metabolites other than phenolics (Fig. 4). The disaccharides such as sucrose have been shown to be more effective in plant growth. This attribute has been linked to the findings that sucrose is a strong antioxidant for plants and readily scavenge free radicals generated from internal oxidation (Bolouri-Moghaddam et al., 2010; Couee et al., 2006). This means that sucrose itself acts strongly as antioxidant metabolite and thus allows feasible growth of cultures. Therefore, sucrosesupplemented callus cultures of F. indica produces lower quantities of phenolic compounds (Fig. 4). However, there is a differential effect of glucose and sucrose in oxidative stress regulation. Lower concentrations of soluble monosaccharides are involved in oxidative stress reduction through reactive oxygen species (ROS) signaling (Couee et al., 2006). For instance, low levels of glucose and fructose enhance the production of glutathione which in turn contribute to FRSA. However, higher concentrations of glucose increase the photosynthetic activity of cells resulting in excessive metabolism and thus the generation of excessive

3.4. Identification and quantification of phenolic compounds in callus cultures grown in different carbohydrate sources The quantitative analysis of phenolic compounds from callus culture extracts of samples was studied using HPLC (Table 2). Based on the comparison of the retention time, and UV spectra of authentic standards, 16 phenolic compounds were identified in the different samples. Among the different callus extracts, maltose (5%) supplemented extracts produced the highest quantities of different metabolites such as Kaempferol hexoside-malonate (6.91 mg/g), Quercetin-3,7-di-O-glucoside (5.14 mg/g), Myricetin-3-hexoside (3.23 mg/g), and Ellagic acid (4.5 mg/g). Sucrose (5%) supplemented callus extract also showed higher quantities of the phenolic compounds as compared with control (3% sucrose supplemented callus). Overall, the HPLC results showed that disaccharide maltose induced higher quantities of reference phenolic compounds compared with glucose. This can be interpreted through the results that the abundance of different phenolic compounds can be attributed to maltose producing higher quantities in terms of dry weight, which indicate that the total TPC production of maltose may be 173

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Table 2 Identification and quantification of phenolic compounds (Mn: mean, SD: standard deviation) in 42-day old callus cultures grown under the effect of different types of carbohydrates at 3% concentration. The alphabet letters represent significant difference between the values. Peak

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Rt (min)

1.1 3.1 8.2 9.3 15.1 15.6 16.9 17.4 18.3 20.8 22.6 23.1 24.1 24.4 27 29.1

Phenolic Compound

Plurglucinol Gallic acid Caffeic acid Salicylic Acid Quercetin-3-O-rhamnoside Luteolin-7-O-glucuronide Kaempferol hexoside-malonate Quercetin-3-O-glucoside Quercetin pentosylhexoside Quercetin-3,7-di-O-glucoside Myricetin-3-hexoside Ellagic acid hexoside Ellagic acid deoxyhexoside Galloyl-HHDP-hexose Ellagic acid Quercetin

Maximum Absorption (nm)

267 271 323, 302, 348, 348, 348, 352, 352, 350, 360, 362, 362, 365, 370, 372,

298, 238 236 266 254 264 255 266 256 271 270 256 266, 230 256 255, 232

Quantity detected (mg/g) Control

Fruct-Callus

Glu-Callus

Malt-Callus

Suc-Callus

144 ± 3.2a 1.0 ± 0.01a 0.50 ± 0.01c 1.43 ± 0.1cd 1.42 ± 0.1cd 0.73 ± 0.1bc 4.55 ± 0.3b 2.97 ± 0.2cd 1.26 ± 0.1d 1.60 ± 0.1d 1.40 ± 0.01cd 0.9 ± 0.01a 1.42 ± 0.1ab 0.8 ± 0.02b 0.58 ± 0.02d 0.33 ± 0.01c

60.6 ± 2.1b 0.61 ± 0.01ab 0.91 ± 0.1ab 1.54 ± 0.1c 2.71 ± 0.1c 0.97 ± 0.01a 4.73 ± 0.2b 3.29 ± 0.1b 3.52 ± 0.1c 2.32 ± 0.2c 2.06 ± 0.01c 0.49 ± 0.01c 1.18 ± 0.02b 0.4 ± 0.01c 2.31 ± 0.1b 1.06 ± 0.01b

40.9 ± 0.5c 0.56 ± 0.01b 0.35 ± 0.01b 0.37 ± 0.01e 0.93 ± 0.03d 0.35 ± 0.02c 1.2 ± 0.03d 0.73 ± 0.01d 3.26 ± 0.1cd 2.06 ± 0.1cd 0.38 ± 0.03d 0.48 ± 0.01c 0.9 ± 0.01bc 0.89 ± 0.02b 0.86 ± 0.01c 0.88 ± 0.02bc

52.3 ± 2.1bc 0.57 ± 0.01b 0.97 ± 0.01ab 1.64 ± 0.1b 3.08 ± 0.2b 1.0 ± 0.01a 6.91 ± 0.2a 4.25 ± 0.1a 4.54 ± 0.2b 5.14 ± 0.1a 3.23 ± 0.2a 0.95 ± 0.01a 6.47 ± 0.1a 1.57 ± 0.01a 4.5 ± 0.01a 1.0 ± 0.02b

38.4 0.36 1.59 1.81 3.67 0.76 4.34 2.57 5.04 3.14 2.89 0.63 0.86 0.39 0.86 1.34

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.2cd 0.01bc 0.01a 0.01a 0.1a 0.01b 0.2c 0.01c 0.2a 0.01b 0.01b 0.01b 0.01c 0.02c 0.01c 0.01a

HHDP: hexahydroxydiphenoyl. Table 3 Effect of different carbohydrate concentrations and sources on the total chlorophyll content in callus cultures of Fagonia indica. The results were analyzed through two-way ANOVA and Tukey’s multiple comparison tests (p < 0.05). The alphabet letters represent significant difference between the values. Carbohydrate Source

Chlorophyll content (mg/g)

Type

Chl a

Sucrose

Glucose

Fructose

Maltose

Fig. 4. The effects of different carbohydrate sources and concentrations on antioxidant activity of callus cultures of Fagonia indica. The results were analyzed through two-way ANOVA and Tukey’s multiple comparison tests (p < 0.05).

Concentration (%) 1 3 5 1 3 5 1 3 5 1 3 5

Chl b ab

2.60593 ± 0.1065 1.95733 ± 0.0337bc 1.24177 ± 0.0585d 2.94933 ± 0.0149a 1.62724 ± 0.0276c 0.76375 ± 0.0364de 2.46165 ± 0.0936b 1.52073 ± 0.0504cd 0.83267 ± 0.0376de 2.50438 ± 0.0558b 0.7224 ± 0.0493de 1.19978 ± 0.0567c

1.3058 ± 0.04811ab 0.93502 ± 0.0042bc 0.71014 ± 0.0115c 1.4407 ± 0.01355a 0.81785 ± 0.0156bc 0.4525 ± 0.09586cd 1.13769 ± 0.0914cd 0.59712 ± 0.0846b 0.47603 ± 0.0358cd 1.16357 ± 0.0187cd 0.39763 ± 0.0146d 0.62629 ± 0.0336cd

concentration of carbohydrates (Table 3). 3% maltose- and 5% glucosetreated calli produced the least chlorophyll content. A two-way ANOVA of the results from chlorophyll content analysis confirmed that there was a significant difference among the different concentrations applied (p < 0.05). The Tukey’s multiple comparison tests also revealed a significant difference between the different concentrations tested (p < 0.05) for both types of chlorophyll. Just as that for antioxidant activity, the effect of carbohydrate type on chlorophyll content was found non-significant (p > 0.05). This concludes that the chlorophyll content is more affected by the concentration of carbohydrates than the type. Plant cells synthesize endogenous carbohydrates through photosynthetic carbohydrate fixation by photosynthetic machinery (Ramon et al., 2008). The entire process of making and using carbohydrates are performed through shuffling from source (where they are synthesized) to a sink (where they are utilized). At higher concentrations of carbohydrates, plant cell switches off genes responsible for photosynthesis in a process termed sugar sensing and start relying on the available sugars for metabolism (Avonce et al., 2005). This might be one of the reasons for lesser photosynthetic pigments (chlorophyll content) at higher carbohydrate concentrations and subsequent brownish appearance of cells (Fig. 1). In our study, at higher concentrations of sugars, glucose has shown the lowest chlorophyll content (Chl a = 0.7637 mg/g FW

ROS. This leads to the activation of antioxidant defense mechanisms generating excess phenolic compounds for ROS scavenging (Keunen et al., 2013). This might be one of the reasons of strong antioxidant activity of glucose-supplemented callus cultures in the present study. 3.6. Effects of carbohydrate source and concentration on total chlorophyll content Carbohydrate signaling plays an important role in the regulation of the photosynthetic apparatus of plants (Eckstein et al., 2011). In this study, the chlorophyll a and b content showed a decreasing pattern with an increase in carbohydrate concentration (Table 3). The highest chlorophyll a and b content (2.94 and 1.44 mg/g FW, respectively) were detected in 1% glucose-treated callus cultures. Similarly, comparatively higher chlorophyll content was detected in 1% sucrose treated callus cultures. Overall, chlorophyll a was present in abundance compared with chlorophyll b. It is worth mentioning that the more the browning of callus, the more the total chlorophyll content decreased in calli with the least value observed in callus cultures treated with higher 174

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and Chl b = 0.4525 mg/g FW). Excess glucose is readily sensed through hexokinase1, the most important glucose sensor (Ramon et al., 2008) which is then metabolized readily coupled with oxidation in the process. This may be the reason for lesser chlorophyll content in 5% glucose-treated callus cultures of F. indica. (Table 3).

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