Enhanced production of anthraquinones by gamma-irradiated cell cultures of Rubia cordifolia in a bioreactor

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Enhanced production of anthraquinones by gamma-irradiated cell cultures of Rubia cordifolia in a bioreactor Mariadoss Aa, Ramesh Satdiveb, Devanand P. Fulzeleb, Siva Ramamoorthya,*, George Priya Doss Ca, Hatem Zayedc, Salma Younesc, Rajasekaran Ca a

School of Bio-Sciences and Technology, Vellore Institute of Technology, Vellore, 632 014, India Plant Biotechnology and Secondary Metabolites Section, NA & BTD, Bhabha Atomic Research Centre, Mumbai, 400085, India c Hatem Zayed, College of Health Sciences, Department of Biomedical Sciences, Qatar University, Doha, Qatar b

A R T I C LE I N FO

A B S T R A C T

Keywords: Rubia cordifolia Gamma irradiation Anthraquinones Bioreactor Rushton disc turbine Helical ribbon impeller

The aim of this study was to obtain high-yielding cell cultures of Rubia cordifolia by applying gamma irradiation and subsequently scaling up for anthraquinone production in a bioreactor. Calli cultured on MS medium was irradiated at variable doses between 2 Gy and 30 Gy. The callus cultures that were irradiated at 8 Gy accumulated a maximum alizarin level of 26.86 mg g-1 DW and a purpurin level of 44.85 mg g-1 DW during the M1T4 (fourth sub-cultures after gamma irradiation treatment) subculture, which was 6-fold and 11-fold higher than those of the nonirradiated callus cultures, respectively. Suspension cultures that originated from high-yielding callus cultures irradiated at 8 Gy were scaled up in an 8-L stirred bioreactor equipped with different impellers. The callus cultures in a bioreactor using a helical ribbon for homogenous mixing synthesized 63.58 % more anthraquinones than those in the bioreactor equipped with a Rushton disc turbine. This is the first report on the production of alizarin and purpurin in a bioreactor.

1. Introduction Larger quantities of synthetic dyes are manufactured by the textile industry and are used for the production of different items in various market areas compared to the amount of natural dyes that are used. In fact, synthetic dyes have superior colorfastness; they are more economical and have a wider color variety. However, the use of synthetic dyes has become a cause of concern due to the mutagenic, carcinogenic, and toxicological properties they possess, which can adversely affect the natural ecosystem (Amin et al., 2010; Gita et al., 2017; Mathur et al., 2012). In addition, contact dermatitis has been widely reported in the textile industry (Malinauskiene et al., 2012; Siva, 2010; Shahid and Mohammad, 2013). Recently, natural dyes of plant origin have gradually been taken into consideration because of their biodegradability, low toxicity, eco-friendliness, low association with allergic reactions, and high compatibility with the environment (Siva, 2014; Bechtold et al., 2009; Sinha et al., 2012; Rungruangkitkrai et al., 2013). Various industries, including the cosmetic, textile and food industries, have been implementing approaches to end the use of toxic raw materials and achieve cleaner production (CP) (Bai et al., 2015; Ortolano et al., 2014). Consequently, the growing interest in natural dyes has come to the forefront, encouraging their exploration and evaluation for ⁎

potential use as a substitute for synthetic dyes. Furthermore, this research has indicated that natural dyes obtained from plants are substitutes for synthetic dyes in the pharmaceutical, textile, and food industries. In the past, natural pigments and dyes have been isolated from different biological sources, including flowers, leaves, seeds, stems, bark, roots, and wood (Siva, 2007). In the textile industry, natural dyes from plants have created a significant resurgence through innovative means of production, which could be a potential alternative to synthetic dyes. Natural dyes have been extracted from different plant species, such as Eucalyptus grandis, Amaranthus tricolor, Tamarindus indica, Butea monosperma, Thespesia populnea, and Rubia cordifolia, are used for the natural textile dyeing of multiple fabrics (Prabhu and Teli, 2014; Sinha et al., 2012; Ramanarayanan et al., 2017; Yusuf et al., 2017; Vedaraman et al., 2017; Rossi et al., 2017; Kuchekar et al., 2018). Furthermore, there are a constant increase in interest regarding the use of natural dyes to dye hair (Boonsong et al., 2012), leather (Selvi et al., 2013), plastics (van den Oever et al., 2004) and textiles (Yusuf et al., 2017). Experimental findings demonstrate the uses of natural dyes as food additives, functional foods, cosmetic products, and in pharmaceuticals, which signifies an opportunity to use natural dyes in several industries (Carocho et al., 2015; Fernández-García et al., 2012; Kapoor, 2005; Kostick et al., 2006; Chigurupati et al., 2002).

Corresponding author. E-mail addresses: [email protected], [email protected] (S. Ramamoorthy).

https://doi.org/10.1016/j.indcrop.2019.111987 Received 13 August 2019; Received in revised form 25 October 2019; Accepted 12 November 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Mariadoss A, et al., Industrial Crops & Products, https://doi.org/10.1016/j.indcrop.2019.111987

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Fig. 1. Chemical structures of alizarin and purpurin.

the impeller and mass transfer causes improved cell growth and productivity. Cell suspension cultures of Digitalis lanata were cultured in a 40 l bioreactor equipped with a helix impeller to produce deacetyl lantoside C (Fulzele et al., 1992). Critical shear stress was caused by a flat-blade turbine impeller in a bioreactor, which reduced L-DOPA production by Stizolobium hassjoo (Chen and Huang, 2000). The present investigation focuses on the influence of low gamma irradiation dosages and the successive selection of high-yielding cell lines following M1T4 subculture. Eventually, suspension cultures were established from gamma-irradiated callus cultures and successfully cultivated in the bioreactor. To date, the effects of applying low-dose gamma irradiation on alizarin and purpurin production and R. cordifolia cell cultures have not been reported. In this study, cell suspension cultures of R. cordifolia were cultivated using two different 8 l bioreactors containing a Rushton turbine and a helical ribbon impeller. An ideal impeller imparting low shear stress was identified, and it enhanced cell growth and boosted the yield of the natural dyes, alizarin, and purpurin.

Rubia cordifolia Lin. belongs to the Rubiaceae family and is commonly referred to as “Indian Madder.” It serves multiple purposes, including its use for the production of essential dyes on the Indian subcontinent, and this species is widely dispersed throughout the lower hills of the Indian Himalayas (Shekhar et al., 2010). Different plant parts of R. cordifolia are enriched with red dye, which is used by villagers for dyeing hair, clothes, handmade ornaments, and handmade bamboo articles. Akhtar et al. (2006) identified the various chemical constituents of R. cordifolia, such as anthraquinones. The roots of this plant contain substantial amounts of purpurin and alizarin (Fig. 1) in the free form or bound as glucosides that contain an aglycone group, which is a derivative of anthraquinone (Mouri and Laursen, 2012; Son et al., 2008). A great deal of research has been conducted on the dyeing performance of R. cordifolia on textile substrates due to its outstanding dyeing performance and beautiful red color (Sarkar, 2004; Gupta et al., 2004; Parvinzadeh, 2007). In addition, the roots have been used as a traditional medicine for the treatment of different illnesses for many years, and its extract has multiple pharmacological properties, including hepatoprotective (Babita et al., 2007), gastroprotective (Deoda et al., 2011), immunomodulating (Kannan et al., 2009), antidiabetic (Baskar et al., 2006), potential radioprotective (Tripathi and Singh, 2007), antimicrobial (Mariselvam et al., 2013), antiviral (Ho et al., 1996), and anticancer activities (Son et al., 2008; Patel et al., 2013). Plant cell culture has been considered a promising approach for improving plant secondary metabolites production. Cell culture techniques might offer higher yields and better selectivity of the desired bioactive products since the cells can be exposed to gamma irradiation for higher compound production than in any other parts of the plant (Hoshyar et al., 2017). Low doses of gamma irradiation have been used to improve the yield of secondary plant metabolites by cell cultures. In the past, a remarkable increase in shikonin production of 400 % was observed in a suspension culture of Lithospermum erythrorhizon when using low doses of gamma irradiation (Chung et al., 2006). In addition, leaf-derived callus cultures of Stevia rebaudiana that were exposed to gamma rays showed significantly enhanced growth and stevioside production (Khalil et al. (2015). Fulzele et al. (2015) reported the beneficial effects of using low-dose gamma irradiation on the production of camptothecin from Nothapodytes foetida callus cultures. Furthermore, low-dose gamma irradiation was shown to produce a 13-fold higher yield of 20-hydroxyecdysone in the in vitro shoots of Sesuvium portulacastrum during M1T4 subculture (Kapare et al., 2017). To boost the production of secondary metabolites, different types of impellers have been used in bioreactors. In the past, secondary metabolites were produced through cell suspension cultures in bioreactors (Fulzele and Heble, 1994; Fulzele et al., 1992; Georgiev et al., 2009; Nasim et al., 2010; Wilson and Roberts, 2012; Alam, 2013). The suspension cultures of Lithospermum erythrorhizon produced 830-fold more shikonin in a stirred tank bioreactor as compared to the production from field-cultivated plants (Tabata and Fujita, 1985). In addition, a low shear stress impeller ensures cell viability and the appropriate mixing of suspension cultures in the bioreactor. The uniform mixing by

2. Materials and methods 2.1. Establishment of callus cultures Leaves from Rubia cordifolia were used as an explant material collected from the Yercaud hills, Salem, and Shevaroys range of hills in the Eastern Ghats of India. They were rinsed for 30 min under tap water, washed for five min with Dettol, and subsequently washed with distilled water to remove traces of the germicidal agent. The explant material was then collected under aseptic conditions in a laminar flow hood and treated with ethyl alcohol (70 %) for 3 min. After that, the material was treated for 2 min with mercuric chloride (0.1 % w/v) solution and rinsed thoroughly under aseptic conditions five to six times with sterile distilled water. Last, the disinfected explants were incised into small pieces (10−12 mm) and placed in Murashige and Skoog (1962) medium supplemented with N6-benzyl amino purine (BA) (1 mg l-1) and indole-3-acetic acid (IAA) (0.2 mg l-1) to initiate multiple shoots. Subsequently, the cultures were transferred to MS medium supplemented with identical concentrations (1 mg l-1) of indole-3-acetic acid (IAA), N6-benzyl amino purine (BA), and 1-naphthaleneacetic acid (NAA) for the callus cultures. The pH was adjusted to 5.8 ± 0.02 with 0.1 N HCl or 0.1 N NaOH before the addition of agar (8.5 g/L). The medium (20 mL) was dispensed into test tubes and autoclaved at 103.42 kPa for 20 min. Surface-sterilized explants were transferred into test tubes aseptically then incubated at 25 ± 1°C and maintained at a photoperiod of 16 h with cooling white fluorescent tubes at a 40 μmol m-2 s-1 light intensity. The cultures were regularly subcultured on the same medium every four weeks and maintained under the same conditions.

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subculture (M1T1, M1T2, M1T3, and M1T4) and analyzed separately for their purpurin and alizarin contents. The collected biomass materials were placed into a drying oven at a temperature of 60 ± 2 °C for 24 h and were then ground into a powder. Then, 100 mg of finely powdered calli were transferred to Eppendorf tubes and dissolved in 1 ml of methanol. The mixture was then sonicated at 33 kHz for 15 min. Finally, the samples were centrifuged at 14,000×g for 10 min. The supernatant was collected and transferred to glass vials and analyzed using highperformance liquid chromatography (HPLC). Similarly, the suspension cultures were harvested from the bioreactor, filtered and dried in an oven at a temperature of 55 ± 2 °C for 24 h, and then ground and extracted as described above. The spent medium was extracted with chloroform, which was then concentrated by separation and evaporation. Finally it was dissolved in methanol for further analysis.

2.2. Radiation sensitivity test Gamma Chamber 2000 (BRIT, Mumbai) was used for gamma irradiation experiment, which emits 1 Gy minute-1 from 60Cobalt source. The R. cordifolia callus cultures were irradiated with a 5, 10, 15, 20, 25, and 30 Gy to identify the lethal dose that would reduce 50 % of the fresh weight compared to that of the nonirradiated callus (LD50) (Kapare et al., 2017). The survival percentage was calculated after the M1T1 subcultures, and LD50 values were determined using the regression equation. 2.3. Gamma irradiation sensitivity test Callus cultures were irradiated at different low doses ranging from 2 to 30 Gy. For the sensitivity test, the callus cultures were irradiated at 5, 10, 15, 20, 25, and 30 Gy. After the sensitivity test, the cultures were exposed to 2, 4, 6, 8, 10, 12, 14, and 16 Gy for the kinetic study of cell growth and anthraquinone contents. The irradiation was performed at room temperature (25 °C ± 1 °C) with a stable source of 60Cobalt (Gamma chamber, supplied by Bhabha Atomic Research Centre, Trombay, Mumbai, India). Nonirradiated callus cultures were used as controls. After exposure to gamma radiation at different doses, the cultures were transferred to MS basal medium fortified with a 1 mg l1 concentration of IAA, NAA, and BA and sustained at room temperature (25 °C ± 1 °C) and a photoperiod of 16 h. To investigate gamma irradiation effects, every four weeks, gamma-irradiated cell cultures were subcultured, and cell growth was evaluated; the alizarin and purpurin contents were quantified for four successive subcultures denoted M1T1, M1T2, M1T3, and M1T4.

2.7. Quantification of anthraquinones Isocratic analytical HPLC was conducted using an HPLC system consisting of a PU-2080 plus pump (Jasco Corporation, Japan), a UV/ VIS detector (Model No. UV-2075 plus, Japan), and an autoinjector (AS2055 plus Japan) set at a 20 μl capacity per injection. The C18 HPLC column was purchased from Thermo Scientific (150 mm × 4.6 mm, 5 μm particle size). The mobile phase for purpurin and alizarin elution consisted of acetonitrile:1 % formic acid (65:35, v/v) with a 1 mL/min flow rate, and the detection wavelength was set at 254 nm. Data were collected using Jasco Chrompass software. A stock solution of 1 mg/ 10 ml of standard purpurin and alizarin was prepared in methanol. For the calibration curve, 0.1 μg/mL to 1 μg/mL dilutions of the standard purpurin and alizarin solution were injected in triplicate into the HPLC. The calibration curves of purpurin and alizarin were generated by plotting the peak areas against the corresponding concentrations. The linearity was addressed using a linear least-squares regression of the peak areas plotted against the corresponding concentrations of the standard purpurin and alizarin solution over the range of 0.1 μg/mL to 1 μg/mL. There was no endogenous interference observed when acetonitrile:formic acid (1 %) was used as the mobile phase for alizarin and purpurin. This method was characterized by high sensitivity, accuracy, and reproducibility. The method validation was performed five times with samples. The results showed similar retention times for the five injections of the same samples at five concentrations from 0.1 μg/ml to 0.5 μg/mL, which were determined to be 5.8 min for alizarin and 7.4 min for purpurin. Chromatographic peaks were identified by comparison with those of the purpurin and alizarin standards (Sigma, USA).

2.4. Establishment of suspension cultures To initiate the cell suspension cultures, the callus cultures that were irradiated at 8 Gy to produce high concentrations of anthraquinones were transferred to MS liquid medium supplemented with IAA (1 mg l1 ), NAA (1 mg l-1) and BA (1 mg l-1) with 30 g mg l-1 sucrose. 5 g of inoculum was added to Erlenmeyer flasks (500 ml) containing 200 ml of liquid medium, and the cultures were agitated on a gyratory shaker at 60 rpm and 25 ± 2 °C under a 16 h photoperiod. Subcultures were performed at four-week intervals. 2.5. Bioreactor configuration Suspension cultures were scaled up in the 8 l bioreactors. The first bioreactor was equipped with a helical ribbon impeller (shaft height: 360 mm, impeller length: 180 mm, width: 90 mm). A total of 5 blade pitches were used, and the distance between each blade pitch is 45 mm. The second bioreactor was equipped with a standard 6- vertical bladed Rushton turbine impeller, with an impeller length of 200 mm and a width of 50 mm. A schematic diagram of the helical ribbon and Rushton turbine impellers is shown in Fig. 2. These bioreactors were installed with a top-driven Remi Rq-40 agitator to measure the agitation speed. The agitation speed of the impeller was 60 rpm in both reactors. Suspension cultures were aerated by sterile air, and aeration was supplied at 5.5 h-1 by a stainless steel orifice at the bottom of the impeller. The temperature was maintained at 25 ± 0.1 °C. Three-week-old suspension cultures were cultivated in the bioreactor, which was operated at 60 rpm. The bioreactor was stopped on the 40th day, and samples were withdrawn every tenth day to determine the growth and anthraquinone contents. All experimental data were collected in triplicate from each impeller, and their averages with standard deviations are given in the results.

2.8. Growth measurement To determine the fresh weight (FW), the harvested biomass was dried by gentle pressing on filter papers (Whatman No. 1) and was subsequently weighed. The biomass was then placed in a drying oven at 55 °C for 24 h, after which the dry weight (DW) was determined. 2.9. Statistical analysis The differences in the mean cell growth and the alizarin and purpurin yield values were determined using ANOVA. All experiments were repeated in triplicate, and the data are expressed as the mean ± S.E. of three different measurements. 3. Results and discussion 3.1. Establishment of callus cultures Leaf segments of R. cordifolia were cultured on MS medium fortified with BA (1 mg l-1) and a reduced concentration of IAA (0.2 mg l-1) to induce multiple shoots over four weeks. In vitro shoots and buds were transferred to MS basal medium containing NAA (1 mg l-1) and BA

2.6. Extraction of anthraquinones In vitro irradiated callus cultures were harvested from every 3

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Fig. 2. Lab-scale schematic diagram of a 8-L stirrer tank bioreactor equipped with a Rushton turbine impeller (on the left) and a helical ribbon impeller (on the right). Fig. 3. Establishment of in vitro cell cultures of R. cordifolia: a - multiple shoot cultures of R. cordifolia grown on MS + BAP (mg l-1) NAA 0.5 (mg l-1); c- compact and green colour callus culture on MS + BAP (mg l-1); c &d. callus cultures on 1st and 2nd subcultured respectively on MS + BAP (mg l-1), NAA (mg l-1) and IAA (mg l-1) ; e- friable callus on MS + (mg l-1), NAA (mg l-1) and IAA (mg l-1); f-Suspension cultures established in shake flasks MS + BAP (mg l-1), NAA (mg l-1) and IAA (mg l-1).

(1 mg l-1) and proliferated into green and compact calli within four weeks. After three successive subcultures, each green compact callus became a friable and reddish-brown color to orange callus when cultured on MS medium containing identical concentrations (1 mg l-1) of BA, NAA and IAA (Fig. 3).

3.2. Callus culture radiation sensitivity test, LD50 In this study, lethal concentrations (LD50) were calculated according to the overall fresh weight reduction of the friable and orange callus cultures of R. cordifolia obtained on MS medium supplemented with

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cultures were exposed to 16 Gy in the M1T1 subcultures. However, cell growth was improved, and no variation was observed during the M1T2, M1T3, and M1T4 subcultures (Fig. 6). In addition, these results were supported by Ling et al. (2010), whereby the gamma-irradiated callus cultures of O. stamineus did not affect the growth rate in successive subcultures. Our results are consistent with those of Tshilenge-Lukanda et al. (2013), who reported that the cultures were irradiated at higher doses to inhibit the accumulation of biomass. Variable gamma doses significantly reduced the growth compared to the nonirradiated callus cultures (M1T1 subculture), which can be observed in Fig. 6. These findings are consistent with those of Hasbullah et al. (2012), who reported that higher radiation doses significantly inhibited the growth of Gerbera jamesonii in comparison to the nonirradiated cultures. Additionally, gamma irradiation is a type of ionization radiation that generates free radicals, and high doses are known to cause morphological, anatomical, biochemical, and physiological alterations in plants. Bajaj (1970) documented that higher doses of gamma irradiation inhibit biomass accumulation and could be caused by the failure of RNA and protein synthesis that directly inhibited cell growth. The possible reason for the gradual reduction in cell growth rate may be due to the adverse effect of higher gamma irradiation doses on endogenous growth regulators (Khalil et al., 2015). In this study, the irradiated callus cultures were stable and did not show significant changes in biomass during the successive M1T2, M1T3, and M1T4 subcultures. The findings of the present study demonstrate that callus cultures of R. cordifolia have the ability to withstand gamma irradiation at high doses, and they accumulated higher concentrations of anthraquinones than those not subjected to gamma irradiation.

Fig. 4. LD50 regression equations of different gamma irradiation doses on callus cultures of R. cordifolia.

identical concentrations (1 mg l-1) of BA, NAA and IAA following exposure to different low doses of gamma radiation and compared to the fresh weight of the nonirradiated control samples. The radiation sensitivity results for the LD50 were determined according to the percentage of fresh weight reduction with respect to the increasing gamma radiation dose of irradiated and nonirradiated in vitro cultured cells. Callus cultures were treated with gamma irradiation at various doses ranging from 5 Gy to 30 Gy, and the survival rate was observed to be 100 % in the first in vitro M1T1 subcultures. The LD 50 value was calculated by plotting new weight reduction in response to increase in gamma dose. The LD 50 value was found to be 16 Gy (Fig. 4), which is consistent with the radiation sensitivity test conducted by Kapare et al. (2017), who reported that exposure to increased doses of gamma irradiation (30 Gy) caused a significant reduction in the percentage of survival of Sesuvium portulacastrum shoot cultures. In addition, a similar observation was studied in Orthosiphon stamineus cell cultures exposed to low-dose (30 Gy) gamma irradiation (Kiong et al., 2008). Applying an optimum irradiation dose leads to minimize damage to the in vitro cultures while promoting maximum mutation. In accordance with our results, a radio-sensitivity analysis by Sianipar et al. (2015) was applied, in which the lethal dose determined the reduction in the percentage of survival of Typhonium flagelliforme callus cultures.

3.4. Effect of gamma irradiation on the production of anthraquinones by callus cultures from M1T1 to M1T4 subcultures Alizarin and purpurin were detected in all callus cultures exposed to gamma irradiation. However, the concentrations of anthraquinones showed considerable dose-dependent variability. These variations are exhibited in Fig. 6, which shows the distribution of alizarin and purpurin contents within the M1T1, M1T2, M1T3, and M1T4 subcultures. The callus cultures that originated following exposure to low doses of gamma irradiation exhibited higher anthraquinone concentrations compared to those observed in the nonirradiated callus cultures. The alizarin and purpurin concentrations were variable in the successive, M1T2, M1T3, and M1T4 subcultures. In M1T1, the 8 Gy and 10 Gy doses enhanced the percentages of alizarin and purpurin by 18.41 % and 49.97 %, respectively, relative to that of nonirradiated callus cultures. Overall, low doses of irradiation improved the production of anthraquinones more than the higher doses. A similar variation was observed in the M1T2, M1T3, and M1T4 subcultures. Higher accumulations of anthraquinones were obtained in cultures irradiated with the 8 Gy dose in the M1T4 subculture, which produced 11-fold and 6-fold more alizarin and purpurin, respectively, than those in the nonirradiated callus cultures. It appears that low-dose gamma irradiation stimulated the metabolic activity of the callus cultures, which continuously maintained anthraquinone synthesis throughout successive subcultures. From the findings of this study, it can also be observed that the gamma irradiation stimulation effect on the production of alizarin and purpurin was immediate, which might have been because low-dose gamma radiation induced the production of reactive oxygen species (ROS) (Al-safadi and Simon, 1990; Hong et al., 2018; Moghaddam et al., 2011). HPLC analysis revealed that the anthraquinone level in M1T1 was significantly lower than in the successive M1T2, M1T3, and M1T4 subcultures. However, the alizarin and purpurin contents gradually increased, and the maximum content was obtained from the M1T4 subcultures (Fig. 6). These phenomenal changes in content are possibly due to the oxidation of polyphenols, which then act as “free radical scavengers” as generated by gamma irradiation (Urbain, 1996). In this study, the alizarin

3.3. Effect of gamma irradiation on callus morphology and biomass Gamma irradiation has not shown any significant inhibitory effects on the morphology or color of callus in M1T1, M1T2, M1T3, and M1T4 subcultures. In addition, no significant morphological alteration appeared to occur in the phenotype of the irradiated callus cultures. All the irradiated callus cultures were found to be nonembryogenic and friable with a reddish-brown color and an unorganized appearance. In the past, irradiated callus cultures of Stevia rebaudiana showed significantly changed colors and morphology (Khalil et al., 2015). Irradiated callus cultures were not differentiated into shoots and buds, as can be observed in Fig. 5. Similarly, no apparent changes were observed in terms of the color and morphology of gamma-irradiated callus cultures of Orthosiphon stamineus (Ling et al., 2010). Our results were contrary to those reported by Hoshyar et al. (2017), who stated that irradiated callus cultures of Hypericum triquetrifolium were differentiated into vegetative buds and shoots after successive subcultures. The cell growth of irradiated and nonirradiated callus cultures was recorded for M1T1, M1T2, M1T3, and M1T4 subcultures. A steady decline was found in the dry weights of callus cultures, which were exposed to higher radiation doses during the M1T1 subculture. Higher dry weights were observed in nonirradiated callus cultures than in the irradiated cultures, whereas a 41 % reduced dry weight was found when the 5

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Fig. 5. Callus cultures of R. cordifolia grown on MS medium supplemented with BA (1 mg l-1), NAA (1 mg l-1) and IAA (1 mg l-1) and their exposure to different doses of gamma irradiation.

Fig. 6. Effects of low doses of gamma irradiation on the growth and accumulation of alizarin and purpurin in callus cultures of R. cordifolia on MS medium supplemented with BA (1 mg l-1), NAA (1 mg l-1) and IAA (1 mg l-1) during the successive M1T1, M1T2, M1T3, and M1T4 subcultures.

level was higher than that of nontreated callus cultures. Therefore, our study has confirmed that gamma radiation plays a significant role in enhancing alizarin and purpurin production. The differentiation in callus cultures obtained from irradiated calli is useful for optimizing cell suspension cultures in bioreactors to increase the yields of medicinally valuable secondary metabolites (Khalil et al., 2015). In this study, a friable appearance, reddish-brown color, and high-yielding callus culture proliferated in response to 8 Gy, which was useful for the initiation of suspension cultures and for scaling up the culture in the bioreactor.

(26.61 mg/g) and purpurin (45.98 mg/g) production at 8 Gy was almost 11-fold and 6-fold more than that of nonirradiated cultures (2.50 mg/g and 7.75 mg/g), respectively. This result shows that low-dose gamma irradiation enhanced alizarin and purpurin accumulation. The ability of gamma irradiation to enhance plumbagin by 4-fold has also been observed in the root cultures of Plumbago indica (Jaisi et al., 2013). Similarly, Chung et al. (2006) reported that low doses of gamma irradiation increased shikonin production in Lithospermum erythrorhizon callus cultures, but doses higher than 32 Gy did not affect the yield significantly. Furthermore, Fulzele et al. (2015) reported that high-dose gamma irradiation inhibited camptothecin production in Nothapodytes foetida, while lower doses significantly influenced the secondary metabolites and exhibited dry weight percentages of 0.098 % and 0.0043 % for camptothecin and 9-methoxy camptothecin, respectively, which were almost 20-fold higher than those of the nonirradiated cultures. The impact of different radiation doses activated stress conditions in the cell cultures after the first subculture, and, consequently, these conditions remained during the successive subculture and led to a continuously enhanced level of secondary metabolites (Kapare et al., 2017). From the experimental data, cultures exposed to higher doses of gamma radiation produced higher concentrations of anthraquinones during successive subculture, and at the same time, the anthraquinone

3.5. Suspension cultures in shake flasks and anthraquinone contents Friable callus with a reddish-brown color and a high yield of anthraquinones in cultures exposed to 8 Gy were transferred to MS liquid medium containing similar media constituents to achieve an initial concentration of biomass ∼0.045 to 0.050 g ml-1 FW. The cell growth in the shake flasks, in terms of dry weight (DW), as illustrated in Fig. 7. The biomass was calculated based on the increase in biomass from the initial inoculum (Chung et al., 2006). A typical growth curve was obtained with an exponential growth phase that started from day 10 and was sustained until day 40, after which a declining phase was finally 6

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Fig. 7. Time course of cell growth and accumulation for alizarin and purpurin in suspension cultures of R. cordifolia in MS medium supplemented with BA (1 mg l-1), NAA (1 mg l-1) and IAA (1 mg l-1) in shake flasks.

observed. During the total culture period in the shake flasks of 50 days, a maximum dry weight of 17.09 g l-1 was found after 40 days. The average absolute cell growth rate of the suspension culture in the shake flasks reached 0.427 dry weight per day. The pH of the medium fluctuated between 5.4 and 4.1 during the culture period. A time course of accumulated alizarin and purpurin production by cultured R. cordifolia cells in the shake flasks, as illustrated in Fig. 7. The suspension cultures showed remarkable changes in the accumulation of alizarin and purpurin during different growth phases. Alizarin and purpurin production by cultured cells in the shake flasks was started and then increased consistently. The maximum production of alizarin was 25.02 mg/g, and that of purpurin was 56.19 mg/g at 40 and 30 days, respectively. After that, the production capacity of the cells slowly declined. However, the purpurin production was higher on day 30 than that of alizarin, which produced higher concentrations on day 40. HPLC analysis revealed that alizarin and purpurin were released into the medium in trace amounts. As observed in the present study, alizarin and purpurin production was associated with the accumulation of biomass. Our results are consistent with those of Mathur and Shekhawat (2013), who reported that suspension cultures of Stevia rebaudiana increased stevioside production during the exponential phase. Similar effects on other plant cell cultures showed that increased biomass production was interconnected with anolide-A accumulation in Withania somnifera suspension cultures (Nagella and Murthy, 2010). Similar observations were found in phytoestrogens produced by suspension cultures of Psoralea corylifolia (Shinde et al., 2009).

Fig. 8. Scale-up of suspension cultures containing R. cordifolia originating from the gamma-irradiated callus cultures in an 8-L stirrer bioreactor.

3.6. Production of anthraquinones in the bioreactor The suspension cultures of R. cordifolia were scaled up to an 8 l stirred tank bioreactor for the production of anthraquinones (Fig. 8). Three-week-old suspension cultures (1 L) were transferred to a bioreactor that contained 5 l of fortified MS medium with mg l-1 of IAA, NAA, and BA. We observed the effect of different impellers in the bioreactor on the cell growth and accumulation of alizarin and purpurin in R. cordifolia suspension cultures. A helical ribbon agitation speed of 60 rpm generated the homogeneous bulk flow of the suspension cultures in the bioreactor without shear damage to the cells and resulted in maximum biomass of 32.53 g/l DW on day 30 (Fig. 9). The bioreactor equipped with a Rushton turbine showed reduced cell growth and maximum biomass of 21.65 g/l DW on the 30th day (Fig. 10). The present study revealed that the helical ribbon yielded an improved homogeneous mixing as compared to that of the Rushton turbine with maximum biomass.

Fig. 9. Cell growth and accumulation of alizarin and purpurin by suspension cultures of R. cordifolia in an 8-L bioreactor equipped with a helical ribbon impeller.

To the use of impellers in a bioreactor involves dead zone formation, with volumes reaching almost 50 % of the total volume (Bridgeman, 2012). This study suggested that the Rushton turbine impeller may not be the best choice for homogenous mixing of suspension cultures. Additionally, Patel et al. (2014) demonstrated an efficient method of visualizing nonideal flows, such as dead zones, in stirred tank bioreactors containing Rushton turbine impellers. The present results showed that the helical ribbon impeller improved the mixing performance and efficiency in the bioreactor. In addition, the liquid-gas mass transfer 7

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Fig. 10. Cell growth and accumulation of alizarin and purpurin by suspension cultures of R. cordifolia in an 8-L bioreactor equipped with a Rushton Turbine impeller.

Fig. 11. Comparison of the effects of the helical ribbon and Rushton turbine impellers on the accumulation of total anthraquinone (alizarin and purpurin) by suspension cultures of R. cordifolia.

coefficient was improved, yielding high biomass. The decrease in biomass could be related to the accrual of nonideal flows by the Rushton turbine impeller. A significant change was noted in pH variation in relation to the bioreactor equipped with different impellers (data not shown). These findings could be due to the uniform flow and non-ideal flow of suspension cultures in the bioreactor. The bioreactor equipped with a helical ribbon was more progressive, and the increase in pH was noticed during the height of anthraquinone production, whereas the pH did not increase in the presence of the Rushton turbine, which favors non-ideal flows in suspension cultures. These findings indicated that the cell growth associated with uniform mixing in the bioreactor was achieved by the implementation of the appropriate impeller type, which depends on the characteristics of the cells suspended in the bioreactor. Both the impeller design and configuration should be considered when mixing the suspension culture entirely and increasing the oxygen transfer. However, the type of impeller has a remarkable effect on the accumulation and production of secondary plant metabolites. In this study, suspension cultures grown in a bioreactor equipped with the helical ribbon impeller produced 37.96 mg/g DW alizarin and 78.93 mg/g DW purpurin (Fig. 9), while at the same agitation rate of 60 rpm, there was a lower accumulation of alizarin (21.04 mg/g DW) and purpurin (51.28 mg/g DW) when the Rushton turbine impeller was used as an agitator in the bioreactor (Fig. 10). Using these values, it can be inferred that the maximum volume of the suspension cultures in the bioreactor equipped with a Rushton turbine led to the formation of a dead zone, while this value dropped dramatically in the bioreactor containing a helical ribbon impeller and accumulated more biomass. In addition, the effects of impellers on the productivity of the natural dyes were observed. Higher productivity was found with suspension cultures cultivated in the bioreactor using a helical ribbon and yielded 63.6 % more total anthraquinones than those yielded using the Rushton turbine. The total anthroquinone signifies the combined yield of alizarin and purpurin (Fig. 11). It could be possible to install an appropriate impeller for the oxygen transfer and homogeneous mixture of suspension cultures in the bioreactor. During bioprocessing, a bioreactor equipped with a Rushton turbine conveys a radial flow to the suspension cultures. Although the proper transfer of oxygen is provided, this setup generates nonuniform mixtures with high shear stress near the blade tips and mild shear at the bioreactor periphery (Badino et al., 2001; Patel et al., 2009). These findings suggested that an appropriate impeller in the bioreactor favored biomass accumulation and secondary metabolite production. It was observed that homogeneous mixing was

improved in the bioreactor fitted with the helical ribbon, which imparts an axial flow and yielded the maximum biomass accumulation and productivity. In addition, a bioreactor equipped with a helical ribbon impeller operating at 60 rpm exhibited a higher production of anthraquinones and probably better homogenization performance. Paul et al. (2004) reported the benefits of using a helical impeller in the bioreactor in comparison with using the Rushton turbine. Our results are in line with those of Lebranchu et al. (2017), who reported that a helical impeller was found to be more beneficial for suitable oxygen transfer and an appropriate homogenization in the bioreactor. These findings demonstrate the enormous potential of helical ribbons in bioreactors for producing alizarin and purpurin. 4. Conclusion Our study indicates that it is possible to attain high-yielding cell lines by applying low doses of gamma irradiation to callus cultures. Callus cultures irradiated at 8 Gy proliferated the friable calluses and enriched them with anthraquinones, which was ultimately useful for the suspension cultures and did not allow the cultures to form cell aggregates. Here, we report that the maximum alizarin and purpurin productivity was achieved with 8 Gy, and when the same suspension cultures were scaled up in the bioreactor equipped with a helical ribbon impeller at 60 rpm, they produced 13-fold more alizarin and 9.4-fold more purpurin than those produced by the nonirradiated cultures. These results confirmed that using a helical ribbon in a bioreactor increased the anthraquinone production rate by 63 % compared with that produced by cultures in the bioreactor equipped with a Rushton turbine. This study encourages researchers to screen cell lines, followed by scaling up cultures in a bioreactor, to enhance plant secondary metabolite production. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments The research reported in this manuscript was supported by grant no. 2013/35/14/BRNS from the Board of Research in Nuclear Sciences of Mumbai, India. We sincerely thank VIT management for their generous support and encouragement. 8

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References

characteristics and production of bioactive compounds during callogenesis in Stevia rebaudiana (Bert.). New Negatives Plant Sci 11–15. Kiong, A.L.P., Lai, A.G., Hussein, S., Harun, A.R., 2008. Physiological responses of Orthosiphon stamineus plantlets to gamma irradiation. Am.-Eurasian J. Sustain. Agric. 2, 135–149. Kostick, R., Wang, S., Wang, J., 2006. Color Cosmetics-Coloring/Staining the Skin from Fruit and Plant Color Pigments. Patent US20060280762. Kuchekar, M., Landge, T., Navghare, V., 2018. Dataset on analysis of dyeing property of natural dye from Thespesia populnea bark on different fabrics. Data Brief 16, 401–410. Lebranchu, A., Delaunay, S., Marchal, P., Blanchard, F., Pacaud, S., Fick, M., Olmos, E., 2017. Impact of shear stress and impeller design on the production of biogas in anaerobic digesters. Bioresour. Technol. 245, 1139–1147. Ling, A.P.K., Ong, J.X.C., Hussein, S., Harun, A.R., 2010. Morphological and physiological responses of Orthosiphon stamineus callus to gamma irradiation at different doses. World J. Agri. Sci. 6, 58–66. Malinauskiene, L., Zimerson, E., Bruze, M., Ryberg, K., Isaksson, M., 2012. Are allergenic disperse dyes used for dyeing textiles? Contact Derm. 67, 141–148. Mariselvam, R., Ranjitsingh, A.J.A., Nanthini, A.U.R., 2013. Preparation and characterization of silver nanoparticles using Rubia cordifolia plant root extract and their microbial properties. Int. J. Adv. Res. 1, 56–61. Mathur, N., Bhatnagar, P., Sharma, P., 2012. Review of the mutagenicity of textile dye products. Universal J. Environ. Res. Technol. 2 (2). Mouri, C., Laursen, R., 2012. Identification of anthraquinone markers for distinguishing Rubia species in madder-dyed textiles by HPLC. Microchim. Acta 179, 105–113. Moghaddam, S.S., Jaafar, H., Ibrahim, R., Rahmat, A., Aziz, M.A., Philip, E., 2011. Effects of acute gamma irradiation on physiological traits and flavonoid accumulation of Centella asiatica. Molecules 16 (6), 4994–5007. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15 (3), 473–497. Mathur, S., Shekhawat, G.S., 2013. Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside. Acta Physiol. Plant. 35 (3), 931–939. Patel, N., Choy, V., Malouf, P., Thibault, J., 2009. Growth of Trichoderma reesei RUT C30in stirred tank and reciprocating plate bioreactors. Process Biochem. 44, 1164–1171. Nagella, P., Murthy, H.N., 2010. Establishment of cell suspension cultures of Withania somnifera for the production of withanolide A. Bioresour. Technol. 101, 6735–6739. Nasim, S.A., Aslam, J., Kapoor, R., Khan, S.A., 2010. Secondary metabolites production through biotechnological intervention: a Review. Emirates J. Food Agric. 22 (3), 147. Ortolano, L., Sanchez-Triana, E., Afzal, J., Ali, C.L., Rebellon, S.A., 2014. Cleaner production in Pakistan’s leather and textile sectors. J. Clean. Prod. 68, 121–129. Parvinzadeh, M., 2007. Effect of proteolytic enzyme on dyeing of wool with madder. Enzyme Microb. Technol. 40, 1719–1722. Patel, D., Ein-Mozaffari, F., Mehrvar, M., 2014. Using tomography to visualize the continuous-flow mixing of biopolymer solutions inside a stirred tank reactor. Chem. Eng. J. 239, 257–273. Patel, P.R., Patel, N.N., Suthar, M.P., Rajesh, K.S., Patel, L.D., 2013. In vitro anticancer activity of Rubia cordifolia against HepG 32 cell line. Pharmagene 1, 1–3. Paul, E.L., Atiemo-Obeng, V.A., Kresta, S.M., 2004. Handbook of Industrial Mixing: Science and Practice. John Wiley & Sons, Hoboken, New Jersey. Prabhu, K.H., Teli, M.D., 2014. Eco-dyeing using Tamarindus indica L. seed coat tannin as a natural mordant for textiles with antibacterial activity. J. Saudi Chem. Soc. 18, 864–872. Ramanarayanan, R., Nijisha, P., Niveditha, C.V., Sindhu, S., 2017. Natural dyes from red amaranth leaves as light-harvesting pigments for dye-sensitized solar cells. Mat. Res. Bull. 90, 156–161. Rungruangkitkrai, N., Mongkholrattanasit, R., Wongphakdee, W., Studnickova, J., 2013. Eco-friendly dyeing and UV protection properties of wool fabric using natural dye from eucalyptus. Res. J. Text. Appar. 17, 29–37. Sarkar, A.K., 2004. An evaluation of UV protection imparted by cotton fabrics dyed with natural colorants. BMC Dermatol. 4, 15. Selvi, A.T., Aravindhan, R., Madhan, B., Rao, J.R., 2013. Studies on the application of natural dye extract from Bixa orellana seeds for dyeing and finishing of leather. J. Clean. Prod. 43, 84–86. Shahid, M., Mohammad, F., 2013. Recent advancements in natural dye applications: a review. J. Clean. Prod. 53, 310–331. Shekhar, T.C., Bahuguna, Y.M., Vijender, S., 2010. Anti-Inflammatory activity of ethanolic stem extracts of Rubia cordifolia Linn. In Rats. Int. J. Res. Ayur. Pharm. 1, 126–130. Shinde, A.N., Malpathak, N., Fulzele, D.P., 2009. Enhanced production of phytoestrogenic isoflavones from hairy root cultures of Psoralea corylifolia L. using elicitation and precursor feeding. Biotechnol. Bioprocess Eng. 14, 288–294. Sianipar, N.F., Ariandana, Maarisit, W., 2015. Detection of gamma-irradiated mutant of rodent tuber (Typhonium flagelliforme lodd.) in vitro culture by RAPD molecular marker. Procedia Chem. 14, 285–294. Sinha, K., Saha, P.D., Datta, S., 2012. Extraction of natural dye from petals of Flame of forest (Butea monosperma) flower: process optimization using response surface methodology (RSM). Dye. Pigment. 94 (2), 212–216. Siva, R., 2007. Status of natural dyes and dye yielding plants in India – an overview. Curr. Sci. 92, 916–926. Siva, R., 2010. Plant dyes. In: Singh, B. (Ed.), Industrial Crop and Uses. CAB publication, UK, pp. 349–357. Siva, R., 2014. Food colorants and health issues: are we aware? Curr. Sci. 106, 143–144. Son, J.K., Jung, S.J., Jung, J.H., Fang, Z., Lee, C.S., Seo, C.S., Moon, D.C., Min, B.S., Kim, M.R., Woo, M.H., 2008. Anticancer constituents from the roots of Rubia cordifolia L. Chem. Pharm. Bull. 56 (2), 213–216.

Alam, E.A., 2013. In vitro cultures for the production of some anticancer agents. Life Sci. J. 10, 297–310. Al-Safadi, B., Simon, P.W., 1990. The effects of gamma irradiation on the growth and cytology of carrot (Daucus carota L.) tissue culture. Environ. Exp. Bot. 30, 361–371. Akhtar, M.S., Ali, M., Madhurima, Mir, S.R., Onkar, S., 2006. New anthraquinones from Rubia cordifolia roots. Indian J. Chem. 45 (B), 1945–1950. Amin, K.A., Hameid II, H.A., Elsttar, A.A., 2010. Effect of food azo dyes tartrazine and carmoisine on biochemical parameters related to renal, hepatic function and oxidative stress biomarkers in young male rats. Food Chem. Toxicol. 48 (10), 2994–2999. Babita, M.H., Chhaya, G., Goldee, P., 2007. Hepatoprotective activity of Rubia cordifolia. Pharmacologyonline. 3, 73–79. Badino, A.C., Facciotti, M.C.R., Schmidell, W., 2001. Volumetric oxygen transfer coefficients (kLa) in batch cultivations involving non-Newtonian broths. Biochem. Eng. J. 8, 111–119. Bai, Y., Yin, J., Yuan, Y., Guo, Y., Song, D., 2015. An innovative system for promoting cleaner production: mandatory cleaner production audits in China. J. Clean. Prod. 108, 883–890. Bajaj, Y.P.S., 1970. Effect of gamma-irradiation on growth, RNA, protein, and nitrogen contents of bean callus cultures. Ann. Bot. 34, 1089–1096. Baskar, R., Bhakshu, L.M., Bharathi, G.V., Reddy, S.S., Karuna, R., Reddy, G.K., Saralakumari, D., 2006. Antihyperglycemic activity of aqueous root extract of Rubia cordifolia in Streptozotocin-induced diabetic rats. Pharm. Biol. 44, 478–479. Bechtold, T., Mahmud-Ali, A., Komboonchoo, S., 2009. Sustainable dyes from agrifood chain co-products. In: Walden, K.W., Moates, G.K., Faulds, C.B. (Eds.), Total Food. RSC Publishing, Cambridge, UK, pp. 211–218. Boonsong, P., Laohakunjit, N., Kerdchoechuen, O., 2012. Natural pigments from six species of Thai plants extracted by water for hair dyeing product application. J. Clean. Prod. 37, 93–106. Bridgeman, J., 2012. Computational fluid dynamics modelling of sewage sludge mixing in an anaerobic digester. Adv. Eng. Softw. 44 (1), 54–62. Carocho, M., Morales, P., Ferreira, I.C.F.R., 2015. Natural food additives: quo vadis? Trends Food Sci. Technol. 45, 284–295. Chen, S.Y., Huang, S.Y., 2000. Shear stress effects on cell growth and L-DOPA production by suspension culture of Stizolobium hassjoo cells in an agitated bioreactor. Bioprocess Eng. 22 (1), 5–12. Chigurupati, N., Saiki, L., Gayser Jr, C., Dash, A.K., 2002. Evaluation of red cabbage dye as a potential natural color for pharmaceutical use. Int. J. Pharm. 241, 293–299. Chung, B.Y., Lee, Y.B., Baek, M.H., Kim, J.H., Wi, S.G., Kim, J.S., 2006. Effects of low-dose gamma-irradiation on production of shikonin derivatives in callus cultures of Lithospermum erythrorhizon. S. Radiat. Phys. Chem. 75, 1018–1023. Deoda, R.S., Kumar, D., Bhujbal, S.S., 2011. Gastroprotective effect of Rubiacordifolia Linn. on aspirin plus pylorus-ligated ulcer. Evidence-Based Complement. Altern. Med. 2011, 1–5. Fernández-García, E., Carvajal-Lérida, I., Jarén-Galán, M., Garrido-Fernández, J., PérezGálvez, A., Hornero-Méndez, D., 2012. Carotenoids bioavailability from foods: from plant pigments to efficient biological activities. Food Res. Int. 46, 438–450. Fulzele, D., Kreis, W., Reinhard, E., 1992. Cardenolide biotransformation by cultured Digitalis lanata cells: semi-continuous cell growth and production of deacetyl lanatoside C in a 40-l stirred-tank bioreactor. Planta Med. 58 (S1), 601–602. Fulzele, D., Satdive, R., Kamble, S., 2015. Improvement of anticancer drug camptothecin production by gamma irradiation on callus cultures of Nothapodytes foetida. Int. J Pharma Res. Allied Sci. 4 (1), 19–27. Fulzele, D.P., Heble, M.R., 1994. Large-scale cultivation of Catharanthus roseus cells: production of ajmalicine in a 20-l airlift bioreactor. J. Biotechnol. 35 (1), 1–7. Georgiev, M.I., Weber, J., Maciuk, A., 2009. Bioprocessing of plant cell cultures for mass production of targeted compounds. Appl. Microbiol. Biotechnol. 83 (5), 809–823. Gita, S., Hussan, A., Choudhury, T.G., 2017. Impact of textile dyes waste on aquatic environments and its treatment. Environ. Ecol. 35 (3C), 2349–2353. Gupta, D., Gulrajani, M.L., Kumari, S., 2004. Light fastness of naturally occurring anthraquinone dyes on nylon. Color. Technol. 120, 205–212. Hasbullah, N.A., Taha, R.M., Saleh, A., Mahmad, N., 2012. Irradiation effect on in vitro organogenesis, callus growth and plantlet development of Gerbera jamesonii. Hortic. Bras. 30, 252–257. Ho, L.K., Don, M.J., Chen, F.C., Yeh, S.F., Chen, J.M., 1996. Inhibition of hepatitis B surface antigen secretion on human hepatoma cells components from Rubia cordifolia. J. Nat. Prod. 59, 330–333. Hong, M.J., Kim, D.Y., Ahn, J.W., Kang, S.Y., Seo, Y.W., Kim, J.B., 2018. Comparison of radiosensitivity response to acute and chronic gamma irradiation in colored wheat. Genet. Mol. Biol. 41 (3), 611–623. Hoshyar, A., Ibrahim, K., Pop, R., Pamfil, D., Hârţa, M., Bobiș, O., 2017. Changes induced by gamma ray irradiation on biomass production and secondary metabolites accumulation in Hypericum triquetrifolium Turra callus cultures. Ind. Crops Prod. 108, 183–189. Jaisi, A., Sakunphueak, A., Panichayupakaranant, P., 2013. Increased production of plumbagin in Plumbago indica root cultures by gamma ray irradiation. Pharm. Biol. 51, 1047–1051. Kannan, M., Singh, R., Narayanan, 2009. Phytochemistry and immunopharmacological investigation of Rubia cordifolia Linn. (Rubiaceae). Pharmacologyonline 3, 653–662. Kapare, V., Satdive, R., Fulzele, D.P., Malpathak, N., 2017. Impact of gamma irradiation induced variation in cell growth and phytoecdysteroid production in Sesuvium portulacastrum. J. Plant Growth Regul. 37, 919–930. Kapoor, V.P., 2005. Herbal cosmetics for skin and hair care. Nat. Prod. Rad. 4, 306–314. Khalil, S.A., Ahmad, N., Zamir, R., 2015. Gamma radiation induced variation in growth

9

Industrial Crops & Products xxx (xxxx) xxxx

M. A, et al.

on renewable resources and food-grade colorants for application in thermoplastics. J. Appl. Polym. Sci. 92, 2961–2969. Vedaraman, V., Sandhya, K.V., Charukesh, N.R.B., Venkatakrishnan, B., Haribabu, K., Sridharan, M.R., Nagarajan, R., 2017. Ultrasonic extraction of natural dye from Rubia Cordifolia, optimisation using response surface methodology (RSM) & comparison with artificial neural network (ANN) model and its dyeing properties on different substrates. Chem. Eng. Processing: Process Intens. 114, 46–54. Wilson, S.A., Roberts, S.C., 2012. Recent advances towards development and commercialization of plant cell culture processes for the synthesis of biomolecules. Plant Biotechnol. J. 10 (3), 249–268. Yusuf, M., Mohammad, F., Shabbir, M., 2017. Eco-friendly and effective dyeing of wool with anthraquinone colorants extracted from Rubia cordifolia roots: optimization, colorimetric and fastness assay. J. King Saud Univ. Sci. 29, 137–144.

Rossi, T., Silva, P.M.S., De Moura, L.F., Araújo, M.C., Brito, J.O., Freeman, H.S., 2017. Waste from eucalyptus wood steaming as a natural dye source for textile fibers. J Cleaner Prod. 143, 303–310. Tabata, M., Fujita, Y., 1985. Production of shikonin by plant cell cultures. In: Zaitlin, M., Day, P., Hollaender, M.A. (Eds.), Biotechnology in Plant Science. Academic Press, Orlando, pp. 207–218. Tripathi, Y.B., Singh, A.V., 2007. Role of Rubia cordifolia Linn in radiation protection. Ind. J. Exp. Biol 45 (7), 620–625. Tshilenge-Lukanda, L., Kalonji-Mbuyi, A.C., Nkongolo, K.K., Kizungu, R.V., 2013. Effect of gamma irradiation on morpho-agronomic characteristics of groundnut. Am. J. Plant Sci. 4, 2186–2192. Urbain, M.W., 1996. Fruits vegetables and nuts. In: Schweigert, B.S. (Ed.), Food Irradiation. Academic Press Inc, Orlando, pp. 170–216. van den Oever, M.J.A., Boeriu, C.G., Blaauw, R., van Haveren, J., 2004. Colorants based

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