Article pubs.acs.org/JAFC

Evaluation of Total Phenolic Compounds and Insecticidal and Antioxidant Activities of Tomato Hairy Root Extract Harpal Singh, Sameer Dixit, Praveen Chandra Verma,* and Pradhyumna Kumar Singh National Botanical Research Institute (NBRI), Council of Scientific and Industrial Research (CSIR), Rana Pratap Marg, Lucknow, 226001 Uttar Pradesh, India ABSTRACT: Tomatoes are one of the most consumed crops in the whole world because of their versatile importance in dietary food as well as many industrial applications. They are also a rich source of secondary metabolites, such as phenolics and flavonoids. In the present study, we described a method to produce these compounds from hairy roots of tomato (THRs). Agrobacterium rhizogenes strain A4 was used to induce hairy roots in the tomato explants. The Ri T-DNA was confirmed by polymerase chain reaction amplification of the rolC gene. Biomass accumulation of hairy root lines was 1.7−3.7-fold higher compared to in vitro grown roots. Moreover, THRs efficiently produced several phenolic compounds, such as rutin, quercetin, kaempferol, gallic acid, protocatechuic acid, ferulic acid, colorogenic acid, and caffeic acid. Gallic acid [34.02 μg/g of dry weight (DW)] and rutin (20.26 μg/g of DW) were the major phenolic acid and flavonoid produced by THRs, respectively. The activities of reactive oxygen species enzymes (catalase, ascorbate peroxidase, and superoxide dismutase) were quantified. The activity of catalase in THRs was 0.97 ± 0.03 mM H2O2 min−1 g−1, which was 1.22-fold (0.79 ± 0.09 mM H2O2 min−1 g−1) and 1.59-fold (0.61 ± 0.06 mM H2O2 min−1 g−1) higher than field grown and in vitro grown roots, respectively. At 100 μL/g concentration, the phenolic compound extract caused 53.34 and 40.00% mortality against Helicoverpa armigera and Spodoptera litura, respectively, after 6 days. Surviving larvae of H. armigera and S. litura on the phenolic compound extract after 6 days showed 85.43 and 86.90% growth retardation, respectively. KEYWORDS: flavonoid, hairy roots, Helicoverpa armigera, phenolic acid, ROS, Spodoptera litura



INTRODUCTION Plant roots release a range of compounds that are not directly involved in the growth and development of the plant but are very much important for plants during stress conditions (biotic/abiotic). These compounds include aliphatic acids, aromatic acids, fatty acids, sterols, phenolics, enzymes, and other secondary metabolites, including flavonoids.1,2 Flavonoids are phenylpropanoid metabolites, most of which are synthesized from p-coumaroyl-CoA and malonyl-CoA, and share their precursors with the biosynthetic pathway for lignin biosynthesis.3 Flavonoids are low-molecular-weight compounds having approximately 15 atoms of carbon, which are organized in a C6−C3−C6 configuration.4 More than 9000 flavonoids have thus far been identified in plants.5 Phenolic acids are considered as simple phenolics. They are categorized into two groups, i.e., the hydroxybenzoic and hydroxycinnamic acids. Hydroxybenzoic acids have C6−C1 arrangement, such as protocatechuic, vanillic, gallic, syringic, and p-hydroxybenzoic acids, while hydroxycinnamic acids have C6−C3 arrangement, such as p-coumaric, sinapic, caffeic, and ferulic acids.4 Flavonoids and phenolic acids are considered as potentially health-promoting substances and have a number of physiological properties, such as antithrombotic, antiallergenic, antimicrobial, cardioprotective, anti-inflammatory, antioxidant, artherogenic, and vasodilatory effects.6−10 Tomatoes (Solanum lycopersicum L. or Lycopersicon lycopersicum L.) are among the most consumed vegetables worldwide and play an important role in the human diet. The occurrence of flavonoids and phenolics in fruits of tomatoes is almost exclusively restricted to their skin, leaving only negligible © 2014 American Chemical Society

quantities in the remaining parts of the fruit. The main flavonoids in fruits of tomatoes identified and reported in previous literature are rutin, naringenin, and chalconaringenin.11,12 Moreover, some minor flavonoids have also been identified from tomato fruits, such as kaempferol 3-rutinoside and naringenin 7-glucoside.13 The pharmaceutical importance of flavonoids would yield a valuable commercial prospect for these plants. Traditionally, flavonoids are extracted as byproducts of the food and oil industry.4,14 Recovery of flavonoids from tomato fruit skin has also been reported;15 however, fruit peel is difficult to harvest and is not normally available as a raw material under normal agronomic practice. Several other flavonoids are also isolated and identified from 24 species of the genus Solanum.16 Present study demonstrated a protocol for the development of the Agrobacterium rhizogenes-mediated hairy root system in the tomato plant to produce such (rutin, quercetin, kaempferol, gallic acid, and many more) medicinally important compounds. The study was extended to qualitative and quantitative characterizations of the major flavonoids isolated from tomato hairy roots (THRs), followed by quantification of the major antioxidant enzyme and the insecticidal activity of the THR extract. Received: Revised: Accepted: Published: 2588

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water (25:75, v/v) at room temperature on a rocking platform, and filtered. The filtrate was collected, and the residue was extracted twice more at 4 h intervals with the same volume of extractant. The filtrate were pooled and extracted with n-hexane (3 × 60 mL). The n-hexane fraction was discarded, and the methanol−water fraction was further extracted with chloroform (3 × 60 mL). The chloroform fractions were pooled and concentrated on a rotary evaporator. Further, any residues were removed by freeze-drying. Dry powder (5 mg) was dissolved in high-performance liquid chromatography (HPLC)-grade methanol (1 mL), filtered through a Millipore Sample Clarification Kit (Millx GV, 13 mm, 0.22 N μm), and subjected to HPLC analysis. Qualitative and Quantitative Analyses of Phenolics. Separation for qualitative and quantitative analyses of phenolics was performed by HPLC−photodiode array (PDA) with a LC-10 (Shimadzu, Japan). Phenolics were separated on a 250 × 4.6 mm (inner diameter), 5 mm pore size RP-C18 column (Merck) protected by a guard column packed with the same matrix. The gradient was prepared from 0.5% (v/v) phosphoric acid in HPLC-grade water (component A) and methanol (component B) in mobile phase. Nylon filters (0.45 μm) were used for filtration of all components and deaerated in an ultrasonic bath. The gradient from 25 to 50% B in 0−3 min, from 50 to 80% B in 3−18 min, from 80 to 25% B in 25 min, and 25% B in 30 min was used for conditioning of the column with a flow rate of 0.8 mL/min. Shimadzu class VP series software was used for data integration, and quantification was carried out by comparison to standard curves, which was constructed according to a previously reported method.19 All samples and solutions were filtered through 0.45 μm nylon filters (Millipore, Billerica, MA) before analysis by HPLC. A simple mobile phase was used as a control for identification of blank peaks. Extraction and Quantification of Antioxidant Enzymes. Extraction of Enzymes from THRs. Fresh tissue (200 mg) was weighed and ground to a fine powder in liquid nitrogen, allowed to thaw, and then homogenized in 1.5 mL of 0.2 M potassium phosphate buffer (pH 7.8) with 0.1 mM ethylenediaminetetraacetic acid (EDTA). Samples were centrifuged at 15000g at 4 °C. The supernatant was collected in a fresh tube and stored at 4 °C for further experiments. Quantification of Enzymatic Activity. Enzymatic activity of three enzymes, i.e., ascorbate peroxidase (APX), catalase (CAT), and superoxide dismutase (SOD), which play a vital role in the reactive oxygen species (ROS) detoxification pathway, was determined spectrophotometrically, as previously described by Elavarthi and Martin.20 Antifeeding Activity of Hairy Root Extract against Chewing Insects. The phenolic compound extract from THRs was tested against two polyphagous insects Helicoverpa armigera and Spodoptera litura. The insect bioassay was performed as described earlier.21 The extract of the phenolic compound was mixed with an artificial diet at different concentrations in Petri dishes. The ethanol-mixed diet and only artificial diet were taken as the control and blank, respectively. Petri dishes were kept open overnight at room temperature to let the solvent evaporate. Mortality data were recorded after 6 days. Experiment was performed in triplicate, and data were analyzed by one-way analysis of variation (ANOVA) (p < 0.05). Means were compared using Duncan’s multiple range test (DMRT) using SPSS software.

MATERIALS AND METHODS

Chemicals. All of the flavonoid standards were obtained from Sigma-Aldrich. The purities of the reference substances were determined by gas chromatography (GC) and gas chromatography− mass spectrometry (GC−MS). Bacterial Strain. Agrobacterium rhizogenes A4 strain was taken from the Central Institute of Medicinal and Aromatic Plants, Lucknow, India. The bacterial culture was maintained at 25 ± 2 °C on yeast mannitol broth (YMB) medium (Himedia). Plant Material and in Vitro Culture Establishment. Tomato (Lycopersicon esculentum, variety Pusa Early Dwarf) seeds were purchased from the Indian Agricultural Research Institute (IARI), New Delhi, India. Seeds were washed thoroughly with Teepol (10%, w/v), followed by several washings with distilled water. Seeds were kept for 3 h in water prior to being inoculated onto solid Murashige and Skoog (MS) medium with Gamborg’s B5 vitamins containing 100 mg/L myoinositol, 30.0 g/L sucrose, and 8.0 g/L agar−agar, which was used for the induction and maintenance of plant cultures through the seeds. All of the cultures were incubated under continuous light of 300 lux at 25 ± 2 °C. The young leaves of 8-week-old plants were used for genetic transformation. Agrobacterium-Mediated Genetic Transformation. A. rhizogenes A4 strain was cultivated at 25 ± 2 °C on YMB. The second and third leaves of in vitro grown tomato plants were used to transform, as described previously by Verma et al.17 Transgenic hairy roots were excised from each wound site and maintained further at 24 ± 2 °C. Transgenic hairy roots were micropropagated on MS medium,18 supplemented with 100 mg/L Augmentin and 250 mg/L Cefotaxime (Figure 1).

Figure 1. Flowchart of hairy root development in tomato. Screening of Putative Transformed THRs. DNA was isolated from hairy roots using DNeasy Mini Kit (Qiagen, Valencia, CA). Polymerase chain reaction (PCR) was performed to detect Ri plasmid genes (rolB) in putative hairy root lines. Further, any possibility of Agrobacterium contamination in THR was ruled out by the amplification of the virC gene. Growth Kinetics of THR. Growth of THR was regularly monitored. The fresh weight of THR was determined every 7 days until 35 days of culture to calculate the growth index (GI) at different time points. At each time interval, samples were harvested, dried on blotting paper to remove unwanted liquid medium, and dried further at 35 ± 2 °C for 7 days, followed by vacuum drying in desiccators until a constant dry weight was obtained. Samples were weighed and stored at −80 °C for further studies. Extraction of Phenolics. Phenolics were extracted from THRs, and their level was compared to the roots of field grown and in vitro grown plants. To extract the phenolics, hairy roots (4 g) were finely powdered in liquid nitrogen, kept overnight in 20 mL of methanol/



RESULTS AND DISCUSSION Genetic Transformation and Screening of Putative THRs. The second and third leaves of in vitro grown tomato plants were infected with A. rhizogenes A4 strain, and hairy roots were propagated on MS medium. Hairy roots that did not show necrosis and proliferated well in media were taken for further study. Seven different THRs that showed maximum elongation and branching potentials were selected and screened using PCR. Genomic DNA was isolated from all selected THRs individually, and PCR was performed with a gene-specific primer of the rolB gene. An amplicon of ∼650 bp was obtained 2589

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in THRs that were absent in field grown and in vitro grown roots. This confirmed the transformation of THR (Figure 2). A.

root clones have been studied in medicinal plants, and variations in the growth level during different growth phases have been noted among hairy root clones of the same origin.28 Growth of all THRs was regularly monitored during different growth phases, i.e., 7, 14, 21, and 35 days (Figure 3). All THRs showed healthy appearance and lateral branching in liquid media. A gradual increase in the fresh weight up to 28 days of culture could be observed with all of the hairy root clones, which further declined in all lines, except THR-4 (slightly increased after 35 days) and THR-5 (unchanged after 35 days; Figure 4). After 28 days, maximum elongation and branching

Figure 2. PCR amplification of rolB and virC genes for confirmation of transformants: (gel A) lane M, 100 bp molecular weight marker; lanes 1−7, different clones of THRs; and lane +ve, positive control and (gel B) lane M, 500 bp molecular weight marker; lanes 1−7, different clones of THRs; and lane +ve, positive control.

rhizogenes-mediated induction of hairy roots has already been reported in a large number of dicotyledonous plants, such as Atropa, Beta, Brugmansia, Catharanthus, Glycyrrhiza, Hyoscamus, Solanum, Trachelium, and many others.22,23 A variety of A. rhizogenes strains are available for induction of hairy roots. Different groups have used strain LBA 9402 to initiate hairy roots from tomato to see the response of salt on endogenous levels of jasmonate and octadecanoid,24 effect of salt on peroxidase activity25 and for bioremediation of phenol using THRs26 but we selected A4, which is very commonly used (approximately 22% used worldwide) for biotransformation.27 Agrobacterium contamination in THRs was ruled out by the amplification of the virC gene because it was not present between the left and right borders of the Ri plasmid. PCR of THRs with the virC primer did not amplify any PCR product. All further studies were restricted to these seven selected THR lines. Growth Kinetics of Selected THRs. The fact that different root clones differ distinctly, owing to each and every transformation event, has made it mandatory to screen and select the best performing hairy root line to obtain a maximum biomass in a minimum time period. Various independent hairy

Figure 4. Graph showing growth kinetics of different THR clones.

Figure 3. Different stages of development of the THR culture: (A) induction of hairy root by A. rhizogenes A4 and (B−E) growth of THR at different time intervals, i.e., (B) 14 days, (C) 21 days, (D) 28 days, and (E) 35 days. 2590

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Figure 5. Graph representing the level of different phenolic compounds in THR culture.

Figure 6. HPLC chromatograms of extract of different root samples: (A) standard peaks, (B) in vitro grown roots, and (C) hairy roots. HPLC chromatogram of standards: (1) gallic acid, (2) protocatechuic acid, (3) chlorogenic acid, (4) caffeic acid, (5) rutin, (6) ferulic acid, (7) quercetin, and (8) kaempferol.

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Table 1. Activity of ROS Detoxifying Enzymes in THRs enzyme

field grown roots

in vitro grown roots

hairy root

ascorbate peroxidase (mM ascorbate min−1 g−1) catalase (mM H2O2 min−1 g−1) superoxide dismutase (units mg−1)

5.01 ± 0.24 0.79 ± 0.09 0.87 ± 0.05

4.50 ± 0.19 0.61 ± 0.06 0.71 ± 0.03

7.41 ± 0.30 0.97 ± 0.03 1.45 ± 0.05

Table 2. Insect Bioassay on an Artificial Diet with H. armigera and S. litura at Different Concentrations of THR Extract insect bioassay on an artificial diet with different concentrations of THR extract after 6 days H. armigera extract concentration (μL/g of diet)

percent mortality (%)

100 μL/g 50 μL/g 10 μL/g control blank

53.34 30.00 16.67 3.34 0.00

S. litura

percent weight reduction (%) (average weight of larvae, mg) 85.43 68.93 23.30 0.97 NA

(0.50 (1.06 (2.63 (3.40 (3.43

± ± ± ± ±

0.17) 0.15) 0.15) 0.20) 0.30)

percent mortality (%) 40.00 26.67 6.67 0.00 0.00

percent weight reduction (%) (average weight of larvae, mg) 86.90 52.38 28.57 1.19 NA

(0.36 (1.34 (2.00 (2.76 (2.80

± ± ± ± ±

0.15) 0.15) 0.26) 0.15) 0.3)

2.94-fold (9.94 μg/g), and 6.22-fold (4.32 μg/g) higher when compared to in vitro grown roots (Figures 5 and 6). Hairy root development has been reported in various plant species. They have a very high growth rate, low maintenance cost, capablity of producing a number of products in a single step, easy recovery and purification of these compounds in biologically active form, and many other benefits. Therefore, it always offers a very good system for producing biological compounds for commercial purposes. Enzymatic Activity. It was well-established that, when plants were exposed to stress, either biotic or abiotic, the production of ROS increases and can cause significant damage to the cells; therefore, the plant cell produces a number of enzymes, i.e., catalase and ascorbate peroxidase, which can maintain the ROS level.35,36 Thus, the activities of enzymes involved in the ROS detoxification pathway were analyzed in THR and control samples. The activities of catalase, ascorbate peroxidase, and superoxide dismutase were quantified. The activity of catalase in THRs was 0.97 ± 0.03 mM H2O2 min−1 g−1, which was 1.22-fold (0.79 ± 0.09 mM H2O2 min−1 g−1) and 1.59-fold (0.61 ± 0.06 mM H2O2 min−1 g−1) higher than field grown and in vitro grown roots, respectively (Table 1). The activities of ascorbate peroxidase and superoxide dismutase in THRs were 7.41 ± 0.30 mM ascorbate min−1 g−1 and 1.45 ± 0.05 units mg−1, respectively. These were also 1.47-fold (5.01 ± 0.24 mM ascorbate min−1 g−1) and 1.66-fold (0.87 ± 0.05 units mg−1) higher than field grown roots, while 1.64-fold (4.50 ± 0.19 mM ascorbate min−1 g−1) and 2.04-fold (0.71 ± 0.03 units mg−1) higher when compared to in vitro grown roots (Table 1). The Ri-plasmid-transformed calli of Rubia cordifolia showed enhanced expression of catalase, ascorbate peroxidase, and superoxide dismutase genes encoding ROS-detoxifying enzymes.37 The reason for enhanced activity of catalase, ascorbate peroxidase, and superoxide dismutase was due to bacterial (A. rhizogenes) infection. Many hairy root systems, i.e., Brassica napus, Daucus carota, Ipomoea batatas, and Solanum aviculare, were available for peroxidase production.38,39 Peroxidase production was further increased by adding elicitors, such as yeast extract, V2O5, and Hg2Cl2, which increase phenylalanine ammonia lyase synthesis.40 Antifeeding Activity of Hairy Root Extract against Chewing Insects. Many reports suggested that flavonoids in their biological form were toxic to insects. The extract of Nothofagus dombeyi and Nothofagus pumilio leaves consisted of

was observed in THR-7, followed by THR-3 and THR-2. Fresh weight of THR-7 (4.75 ± 0.05 g), THR-3 (4.22 ± 0.09 g), and THR-2 (4.14 ± 0.11 g) was 4.2-, 3.76-, and 3.69-fold higher than non-transformed root (1.12 ± 0.13 g), respectively (Figure 4). The clonal nature of the individually selected hairy root lines resulting from site-specific insertion of Ri TDNA, and the difference in the incorporated copy numbers might finally be responsible for the interclonal variation in terms of growth.29 Profuse adventitious root growth was attained after 21 days of growth in various medicinal plants and reached its maximum up to 35 days of culture.30 Similar observations of decline in growth were observed in Plumbago zeylanica, Plumbago kurroa, and Gossypium hirsutum.17,30,31 The decline in growth is attributed to the secretion of secondary metabolites and other phenolics in the medium, which hamper the adventitious growth of root hairs. Qualitative and Quantitative Analyses of Phenolics. Potential of flavonoid production in hairy root cultures of tomato is not yet reported. Most of the studies are on either flavonoid production through tomato fruits or peels.32 However, some other systems have been explored for flavonoid production through hairy roots. Hairy root cultures of Psoralea sp. in different stages displayed more concentrations of daidzein and coumestrol compared to callus cultures from the same plant.33 The hairy root line THR-7 showed distinct superiority over other lines in terms of growth indices (after 21 days) and was used to quantify the level of different phenolics. HPLC analysis confirmed the presence of several flavonoid and phenolic acids in hairy roots after 21 days of culture (Figures 5 and 6). Many flavonoids, such as rutin, quercetin, and kaempferol, were present in considerable quantity, along with the number of phenolic acids, such as gallic, protocatechuic, ferulic, colorogenic, and caffeic acids. AtMYB12 transformed hairy roots of Fagopyrum esculentumproduced rutin up to 0.9 mg/g of dry weight, but in the present study, THRs produce up to 20.26 μg/g of rutin.34 The content of gallic, protocatechuic, chlorogenic, caffeic, and ferulic acids in THR-7 was 34.02 μg/g, 6.11 μg/g, 11.35 μg/g, 29.24 μg/g, and 26.89 μg/g after 21 days. It was 1.4-fold (24.3 μg/g), 1.4-fold (4.32 μg/g), 1.7-fold (6.37 μg/g), 1.3-fold (22.5 μg/g), and 2.49-fold (10.78 μg/g) higher than field grown roots, while 2.5fold (13.34 μg/g), 3.28-fold (1.86 μg/g), 4.15-fold (2.73 μg/g), 2592

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triterpenes and flavonoids and showed antifeeding activity against the fifth instar larvae of Ctenopsteustis obliquana.41 The insect-resistant genotype of soybean (Glycine max PI 227687) accumulated a high amount of rutin.42 On the basis of previous findings, we tested the phenolic compounds extracted (PCE) from THR against H. armigera and S. litura. PCE showed significant mortality against both of the insects, while the surviving larvae showed significant growth retardation and have a very low average weight. At 100 μL/g concentration, PCE caused 53.34 and 40.00% mortality against H. armigera and S. litura, respectively, after 6 days, whereas no toxicity was observed in a blank diet (Table 2). Surviving larvae of H. armigera and S. litura on PCE after 6 days showed 85.43 and 86.90% growth retardation, respectively (Table 2). Our HPLC data clearly showed the presence of numerous flavonoids and phenolic acids in PCE; this might be a reason for insect toxicity. Insect mortality also revealed that extracted phenolic compounds were in their biologically active form.



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AUTHOR INFORMATION

Corresponding Author

*Telephone: +91-0522-2297922. Fax: +91-0522-2205836 and +91-0522-2205839. E-mail: [email protected] Funding

Harpal Singh is thankful to CSIR, India, for a Senior Research Fellowship. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS The authors are thankful to Dr. Abhishek Niranjan, CIF CSIR− NBRI, for the HPLC experiments. REFERENCES

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dx.doi.org/10.1021/jf405695y | J. Agric. Food Chem. 2014, 62, 2588−2594

Evaluation of total phenolic compounds and insecticidal and antioxidant activities of tomato hairy root extract.

Tomatoes are one of the most consumed crops in the whole world because of their versatile importance in dietary food as well as many industrial applic...
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