Transformation of Lactuca sativa L. with rol C gene results in increased antioxidant potential and enhanced analgesic, anti-inflammatory and antidepressant activities in vivo

Lettuce is an important edible crop which possesses various medicinal properties. In this study Lactuca sativa L. (cv Grand Rapids) was transformed by Agrobacterium-mediated transformation with rol C gene. Transgene integration and expression was confirmed through PCR and semiquantitative RT-PCR. The transformed extracts were evaluated for their in vitro antioxidant and in vivo analgesic, anti-inflammatory and antidepressant activities in rats. The transformed plants showed 53–98 % increase in total phenolic and 45–58 % increase in total flavonoid contents compared with untransformed plants. Results of total reducing power and total antioxidant capacity exhibited 90–118 and 61–75 % increase in transformed plants, respectively. In contrast to control, DPPH, lipid peroxidation and DNA protection assay showed up to 37, 20 and 50 % enhancement in transformed plants, respectively. The extracts showed similar but significant enhancement behavior in hot plate analgesic and carrageenan-induced hind paw edema test. The transformed extracts showed 72.1 and 78.5 % increase for analgesic and anti-inflammatory activities, respectively. The transformants of rol C gene exhibited prominent antidepressant activity with 64–73 % increase compared with untransformed plants. In conclusion, the present work suggests that transformation with rol C gene can be used to generate lettuce with enhanced medicinally important properties, such as antioxidant, analgesic, anti-inflammatory and antidepressant potential.


Introduction
Plant secondary metabolites are exclusive sources for food additives, flavors, pharmaceuticals and economically significant substances (Hussain et al. 2012). Plant cell cultures for extraction and multiplication of secondary metabolites can be produced characteristically under sterile conditions from explants, for example plant stems, meristems, leaves and roots (Hussain et al. 2012). Genetic transformation can manipulate biochemical pathways and produce transgenic crops in which nature, scope and range of existing natural products are modified to offer valuable agronomic and commercial post-harvest processing features (Karuppusamy 2009). Various methods are currently available for the genetic transformation of lettuce. The commonly used approach is Agrobacterium-mediated transformation. Previously, lettuce has been transformed with the gene-containing GDP-mannose pyrophosphorylase gene with 2.5fold increase in vitamin C content (Wang et al. 2011). In another study, lettuce was transformed with synthetic folE and Atpsy gene, and a threefold increase in b-carotene content and 1.8-fold increase folic acid was measured (Li et al. 2012). These reports placed the basis for altering the metabolic pathways by transformation and earning the new lettuce varieties rich in pharmacological properties.
A number of phytochemicals including secondary metabolites and vitamins have antioxidant properties, which help in fending off the injury caused by stress-induced reactive oxygen species (El-beltagi and Mohamed 2013). Thus, the accumulation of antioxidants has a distinct adaptive advantage in plants. Typical phytochemicals with antioxidant properties are vitamins, such as a-tocopherol, ascorbic acid, carotenoids, phenolic and flavonoid compounds (Blokhina et al. 2003). These phytochemicals also have been reported to exert anticarcinogenic, anxiolytic and anti-inflammatory properties (Seeram et al. 2008;. Lettuce has been used in the treatment of stomach problems to enhance appetite, to stimulate digestion and relieve inflammation (Sayyah et al. 2004). In addition, seed oil of lettuce has been reported for hypnotic, sedative, anticonvulsant and analgesic properties (Yakoot et al. 2011). Inflammation is normally characterized by pain, heat, swelling and redness while depression is common in chronic pain patients and it has been recommended that depression and pain share similar neurochemical mechanisms (Micó et al. 2006).
Antidepressants have been used as analgesic agent for several pain associated disorders like neuropathic and nonneuropathic pain as they possess intrinsic antinociceptive activity (McQuay et al. 1998). They inhibit the uptake of monoamines which leads to increase the amount of serotonin and noradrenaline in the synaptic cleft at both supraspinal and spinal levels that reinforce reducing pain inhibitory pathways (McQuay et al. 1998). Several reports proposed about the analgesic and anti-nociceptive activity of antidepressant drugs (reviewed by McCleane 2008), and they are generally identified for controlling chronic pain (Sindrup et al. 2005). Additionally, anti-inflammatory activity of clomipramine, fluoxetine and imipramine has been reported by using carrageenan-induced model (Abdel-Salam et al. 2003). In another investigation (Kast 2003) the potential for bupropion to decrease inflammation was evaluated. Although the exact mechanism is unknown but it can be suggested from the above reports that there is a link between all these activities. Considering the various activities reported in Lactuca sativa, this study was carried out to investigate the enhancement in its phytochemical, antioxidant, analgesic, anti-inflammatory and antidepressant potential by transformation with rol C gene.

Seed germination
The commercially available seeds of Lactuca sativa L. (cv. Grand Rapids) were surface sterilized with 70 % ethanol for 1 minute and sodium hypochlorite (10 %) for 30 seconds. Germination of sterilized seeds was carried out on MS medium (Murashige and Skoog 1962).

Bacterial strains and plasmids
Agrobacterium tumefaciens strain GV3101 containing plasmid pPCV002-CaMVC kindly provided by Dr. A. Spena, Max-Planck-Institute fur Zuchtungsforschung, 5000 Koin 30, FRG (Spena et al. 1987) were used for transformation. The T-DNA region of the plasmid pPCV002-CaMVC contained the coding sequence of rol C genes under the control of CaMV35S promoter. T-DNA of pPCV002-CaMVC also contained the neomycin phosphotransferase (NPTII) gene with nopaline synthase (NOS) promoter and NOS terminator sequences. Agrobacterium tumefaciens containing the plasmid pPCV002-CaMVC was grown overnight in Luria broth medium at 28°C and 120 rpm in a shaking incubator. This bacterial culture was used for transformation purpose.

Transformation and regeneration
One month old nodes and internode explants were used for transformation purpose. Explants were pre-cultured for 2 days on shooting media (0.1 mg/L NAA and 0.5 mg/L BAP), which were then given infection with bacterial strains containing the desired construct for 5 minutes. Explants were co-cultivated with bacteria for 2 days on shooting media supplemented with 200 lM acetosyringone. After 2 days of incubation in dark at 28°C, explants were washed with antibiotics and placed on selection medium (0.1 mg/L NAA, 0.5 mg/L BAP, 50 mg/L kanamycin, 300 mg/L cefotaxime). Subculturing was done after every 14 days on selection media. Regeneration occurred within 1 month; three to four cycles of selection were given until complete transformed plants were regenerated on selection medium.

Semiquantitative RT-PCR
Expression of rol C gene was analyzed by semiquantitative reverse transcriptase PCR. Total RNA was extracted from transformed and untransformed (WT) plants according to the reported procedure (Shirzadegan et al. 1991). DNAse treatment was given to the extracted RNA to confirm complete elimination of DNA. The purity and quantity of extracted RNA was checked by taking absorbance at 260 nm and 280 nm. Then 1-2 lg of RNA was reverse transcribed at 42°C in a 20 ll reaction mixture containing 200 units of RevertAid M-MuLV (Moloney murine leukemia virus) reverse transcriptase (Thermo Scientific #K1622), according to the manufacturer's instructions in the presence of RNAse inhibitor. PCR was carried out with rol C gene primers by using 1-2 lL of cDNA reaction mixture as template. The PCR products were evaluated on 1.5 % agarose gel containing ethidium bromide and photographed under UV. The band on agarose gel indicated the presence of mRNA and intensity of the bands indicated the quantity of mRNA levels.

Preparation of the plant extracts
Biomass extraction was carried out from untransformed and transformed aerial parts of L. sativa to carry out antioxidant assays. 1 g of dried powdered plant material was extracted with 20 mL methanol at room temperature in a sonication bath (Kerry Ultrasonic, UK) for 1 hour. The extracts were filtered using Whatman #1 filter paper and concentrated in vaco to generate the crude extracts. For experiments, extracts were prepared as 50 mg/mL in distilled water.

Total phenolic contents (TPC)
TPC measurement was carried out according to the reported procedure (Moein et al. 2008) with some modifications. Briefly, 4 lL of each extract (50 mg/mL in distilled water) was transferred into each well of 96-well plate and 98 lL of Folin-Ciocalteu reagent diluted with distilled water (tenfold) was added. After mixing well, it was kept at 25°C for 5 minute and then 98 lL 6 % Na 2 CO 3 was added. Incubation of the resulting reaction mixture was carried out at 25°C for 90 minutes and thereafter absorbance was measured at 725 nm. Here, distilled water was used as negative control and TPC were expressed as gallic acid equivalents.

Total flavonoid contents (TFC)
Aluminum chloride colorimetric method was used for the total flavonoid content determination (Moein et al. 2008). 4 lL of each plant extract (50 mg/mL in distilled water) was mixed with 10 lL of 10 % AlCl 3 , 10 lL of 1 M potassium acetate and 176 lL of distilled water in each well of 96-well plate. Incubation of reaction mixture was carried out at 25°C for 30 minutes. Absorbance was measured at 405 nm. Distilled water was used as negative control and TFC were expressed as quercetin equivalents.

Total antioxidant capacity (TAC)
TAC was determined according to the previously reported method (Phatak and Hendre 2014). 4 lL (50 mg/mL in distilled water) of each plant extract was mixed with 196 lL of mixture containing 28 mM sodium phosphate, 4 mM ammonium molybdate and 0.6 M sulfuric acid. Then mixture was kept at 95°C for 90 minute. After cooling reaction mixture at room temperature, absorbance was measured at 630 nm. Distilled water was used as negative control. TAC was expressed as ascorbic acid equivalent for each extract.
Total reducing power (TRP) TRP was measured according to the reported method (Moein et al. 2008). The TRP of the extracts was determined using 20 lL (50 mg/mL in distilled water) of extract mixed with 490 lL of 0.2 M of phosphate buffer and 490 lL of 1 % potassium ferricyanide. The reaction mixture was incubated for 20 minutes at 50°C. Then 500 lL of 10 % trichloroacetic acid was added to the reaction mixture; the mixture was then centrifuged for 10 minutes at 3000 rpm. After taking 500 lL of the upper layer of the mixture in new eppendorf, 100 lL of 0.1 % ferric cyanide was added. Absorbance was measured at 630 nm. 100 lL of distilled water was used to blank the instrument. TRP of each sample was expressed as ascorbic acid equivalent.
DPPH free radical scavenging assay DPPH free radical scavenging activity of the plant extracts was determined according to the method reported by Ismail et al. (2015). DPPH (316 lM) was prepared in methanol. 2 lL of test sample was mixed with 98 lL of DPPH solution and then added in each well of 96-well plate. Incubation was carried out for 1 hour at 37°C. Afterwards absorbance was measured at 515 nm. Assay was carried out at three different concentrations (1000, 500, 250 and 125 lg/mL) of each plant extract. Ascorbic acid was used as positive while distilled water was used as negative control. Percentage scavenging was calculated by the given formula and IC 50 values were determined Percentage scavenging % ð Þ ¼ 1 À absorbance of extract absorbance of control Â 100: DNA protection assay DNA protection activity of the extracts was measured in vitro (Ismail et al. 2015). The reaction mixture was prepared in PCR tube with total volume of 15 lL, having 3 lL pBR322 plasmid DNA (0.5 lg), 4 lL of 30 % H 2 O 2 , 3 lL of 2 mM FeSO 4 and 5 lL of plant extracts at final concentration of 10, 100 and 1000 lg/mL. A positive control was used which contained pBR322 DNA treated with 2 mM FeSO 4 ? 30 % H 2 O 2 , untreated pBR322 DNA was used as negative control (P). Extract and pBR322 (C ? P) was also used as a control to check the natural damaging or protective effect on DNA. Then the mixture was incubated for 1 hour at 37°C. All reaction mixtures were subjected to 1 % agarose gel electrophoresis in 19 TBE buffer with 1 kb ladder (L). Gels were analyzed by scanning with Gel-Doc (BioRad) computer program and intensities of the bands were determined.

Lipid peroxidation assay
The lipid peroxidation activity was determined according to the previously reported method (Gülen et al. 2008 where ''Ac'' means absorbance of control and ''As'' means absorbance of test sample.

Animals and treatment groups
Albino rats between 150 and 200 g of weight were used for the experiments. Rats were kept in standard aluminum cages and bred with water ad libitum and standard diet in the Primate facility of Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan. The study design was approved by the Institutional Animal Ethics Committee and all provisions were carried to minimize animal suffering. Extracts and standard drugs were administered orally with the concentration of 500 and 10 mg/kg of the rat body weight, respectively to each group having five rats per treatment.

Hot plate analgesic assay
Analgesic activity was determined by hot plate method first reported by Eddy and Leimbach (Eddy and Leimbach 1952), which is based on stimulation of pain by heat. Saline and aspirin (10 mg/kg) were used as negative and positive controls, respectively. Prior to oral dosage, rats were placed on the hot plate at 55 ± 2°C to determine the jumping and paw licking response which was noted down as initial reaction time (IRT). After 30 minutes of dosage, each rat was placed on hot plate (55 ± 2°C) and the basal response time was recorded by observing paw licking and jumping reaction (whichever seems first) was taken as final reaction time (FRT). The reaction time in seconds was recorded at different time intervals of 0.5, 1 and 2 hours after dosage with a cut-off period of 30 s. Percentage analgesic activity was calculated by the formula: Percentage analgesic activity = FRT À IRT IRT Â 100 :

Carrageenan-induced hind paw edema test
Anti-inflammatory activity was determined by using carrageenan-induced hind paw edema test (Winter et al. 1962). Diclofenac potassium was used as positive control, whereas saline was used as negative control. After 1 hour of oral dosage of the plant extract, edema was induced by injecting 100 lL of carrageenan prepared in 1 % saline into the subplanter region of left hind paw. The paw volume was measured quickly before and after the carrageenan injection by using Plethysmometer (UGO Basile 7140), which served as the control readings of paw. Paw volume was measured after regular interval of 1 hour; readings were taken up to 4 hours. The percentage edema inhibition was determined by the formula: where ''EDC'' is edema of control rats and ''EDT'' is edema of treatment rats.

Forced swimming test
The anti-depressant activity of L. sativa was determined by forced swim test as described before (Slattery and Cryan 2012). One day before the experiment, rats were engaged in water containing vertical cylinder (18 cm diameter, 40 cm height and 15 cm; retained at 25°C) separately and were forced to swim. In 5-6 minutes, the animal became steady and stayed motionless for almost 80 % of the time.
After 15 minutes rats were evacuated, dried and placed back to their cages. This whole procedure was called preswimming. Fluoxetine HCl and saline were used as positive and negative controls, respectively. On the experiment day, after 30 minutes of dosage, rats were again placed in the water filled vertical cylinder and the camera was positioned to the side of the cylinder. Video recording was started and then the rats were placed in the cylinder. After 6 min, recording was stopped; the rats were removed from the cylinder, dried and placed back to their cages. When the experiment was completed, all videos were observed carefully (last 4 min of total recorded video) to estimate the total immobility time.

Statistical analysis
The data obtained were statistically analyzed by one-way analysis of variance (ANOVA) and Turkey multiple comparison test. Results are represented as mean ± SD and p \ 0.05 was considered to be significant. Percentage change of transformed lines (TransL) in comparison of untransformed plant (WT) was calculated by the following formula: Percentage change ¼ TransL À WT WT Â 100:

Transformation and regeneration of lettuce plants
Lettuce was transformed with Agrobacterium tumefaciens GV3101 harbouring rol C gene. About 300 explants were transformed with the construct and the transformation efficiency was calculated as 65-75 %; but only three transgenic lines survived till maturity. In contrast to control, phenotypic alterations were observed in all transformed plants exhibiting reduction in inflorescence, stem heights, internodal lengths and leaf area (Fig. 1).

Confirmation of integration and expression of rol C gene
The integration of rol C gene in the genome of Lactuca sativa was confirmed by performing PCR which showed the amplified products of 540 bp of rol C and 780 bp of nptII genes as shown in Fig. 2a, b. Plasmid DNA of GV3101-rol C was used as positive control which showed the respective amplified products for rol C and nptII genes. Wild type untransformed plants were used as negative control (WT) which showed no amplification. Semiquantitative reverse transcriptase PCR confirmed that the rol C gene was expressed in all transformed plants (Fig. 2c). For this expression analysis, RNA sample without reverse transcription was used as negative control (NC) and GADPH (provided with the kit) was used as positive control. The result showed that the expression of rol C gene in each line was not uniform. The transgenic line rol C1 exhibited highest expression while rol C2 showed moderate and rol C3 showed lowest expression (Fig. 2c).

Quantification of antioxidants
Total phenolic contents (TPC), total flavonoid contents (TFC), total antioxidant capacity (TAC) and total reducing power (TRP) of rol C gene transformed and untransformed plants of Lactuca sativa were determined and their comparative analysis was performed to find the correlation between them. TPC were expressed as equivalent of gallic acid per gram of plant dry weight (Table 1)

Measurement of antioxidant activities
DPPH free radical scavenging assay is a spectrophotometric method for the screening of antioxidant potential of plant extracts. In this study, comparative estimation of antioxidant activity was carried out for rol C transgenic plants and untransformed wild type plants. Ascorbic acid (IC 50 0.01 mg/mL) and distilled water served as positive control and blank, respectively. Results showed significant enhancement in antioxidant activity for all transgenic lines than that of untransformed plants (  (Table 1). Overall, about 20 % increase in activity was observed in transformed plants as compared to untransformed plants.

Analgesic assay
The extracts of Lactuca sativa transformed with rol C gene showed significant increase in analgesic activity in rats. Latency period of each rat was determined and percentage inhibition was calculated which showed gradual increase in activity for 0.5 and 1 hour after oral dosage followed by decrease in activity at 2 hours (Fig. 4). Aspirin was used as positive control (95 % inhibition) and saline was used as negative control (22 % inhibition). The results were compared with untransformed wild type extract (WT) which showed 45.9 % analgesic activity. In contrast to control, the highest activity was calculated by rol C1 with 79.1 % reduction in analgesia which is about 72.1 % higher than that of untransformed plants. In case of rol C2 and rol C3 transgenic lines, 65.1 and 60.7 % increase in analgesic activities were recorded, respectively.
Each data represent mean ± SD (n = 3) of extract with * p \ 0.05, ** p \ 0.01 statistically significant. The values are expressed as milligram equivalents of gallic acid (TPC), quercetin (TFC) and ascorbic acid (TAC and TRP) per gram of plant dry weight, respectively. DPPH and LPA assays are expressed as IC 50 (lg/ml). Ascorbic acid (IC 50 9.52 lg/mL) and vitamin E (IC 50 8.16 lg/mL) were used as positive controls for DPPH and LPA assays, respectively. In DNA protection assay ''?'' represent the slight, ''??'' moderate and ''???'' good protection as positive control (95 % inhibition) and saline served as negative control (0 % inhibition). The extracts of rol C1 line showed maximum activity with 80.8 % edema inhibition which is about 78.5 % higher activity than that of untransformed control plants (45.3 % inhibition). The extracts of transgenic lines rol C2 and rol C3 showed 64.1 and 48.1 % increase in anti-inflammatory activities, respectively.

Antidepressant assay
Antidepressant potential of transformed and untransformed lettuce extracts were investigated in rats by forced swimming test. The transgenic line rol C1 exhibited highest antidepressant activity with the immobility time of 36 ± 3 s as compared with untransformed extracts (131 ± 5 s; Fig. 6). Fluoxetine-HCl and saline were used as positive and negative controls with the immobility time of 11 ± 1 and 180 ± 3 s, respectively. Overall, the rol C1, rol C2 and rol C3 transgenic lines displayed antidepressant activities with 72.5, 67.2 and 64.1 % enhancement, respectively, as compared to untransformed control plants.

Discussion
The use of the medicinal plants for curing disease has been documented in history of almost all civilizations (Gousia et al. 2013). One such plant Lactuca sativa is famous for its medicinal properties, such as antioxidant, anti-  Each group (n = 5) is expressed in mean ± SD. *p \ 0.05, **p \ 0.01 statistically significant inflammatory, analgesic and sedative-hypnotic effects (Fu et al. 2012). Complete profile of pharmacological activities of lettuce as well as molecular modes of actions can further elucidate the full potential of this important crop. The aim of this study was to enhance the production of secondary metabolites in Lactuca sativa (cv. Grand Rapids). To achieve this, lettuce was transformed with rol C gene by using Agrobacterium tumefaciens. PCR analysis confirmed the presence of transgene and semiquantitative reverse transcriptase PCR was performed to study the expression which measures the mRNA level specific to DNA level. It has been seen that the rol genes of Agrobacterium rhizogenes played an essential role in the activation of secondary metabolites in transformed plants. We also report some morphological differences that existed among transgenic and non-transgenic plants. Such features were similar in all transformed plants which show that these changes are associated with transformation and not due to somaclonal variation. Previously, similar type of growth reduction was observed in Lactuca sativa L. (cv. Lake Nyah) when transformed with Agrobacterium rhizogenes rol AB genes (Curtis et al. 1996).
It is well known that consumption of fruits and vegetables is linked with reduced risk of diseases. This is due to their high levels of phytochemicals that prevent diseases related to oxidative stress in the human body. Understanding the distribution of phytochemical profile in vegetables and fruits is of primary importance. So, all the transformed and untransformed plants were evaluated for their TPF, TFC, TRP, TAC and antioxidant activity by using DPPH, DNA protection and lipid peroxidation assays. A significant positive correlation was found among all enhanced phytochemicals and antioxidant activities in transformed plants. The phytochemical studies on lettuce have already reported that it contains a number of flavonoids (Caldwell 2003). Synergistically, these dietary antioxidants provide bioactive mechanisms to reduce free radical induced oxidative stress. The DNA damage and oxidative stress conditions are the results of free radical production initiated by imbalance state of antioxidants and oxidants. In our results, transformed plants showed enhanced DNA protection response due to the effect of rol C gene as compared with previous report . The protective effect of extracts on DNA may be These results are supported by the previous findings (Bulgakov 2008) which states that suppressed levels of reactive oxygen species (ROS) is as a consequence of higher antioxidant activity in rol C transformed cells. Likewise, light stimulated elevated levels of ROS were decreased in the rol C transformed cells as compared to the controls (Bulgakov 2008). Enhanced antioxidant activity by transformation with rol genes may improve the plant defence response by suppressing the oxidative damages. Transgenic lines of rol C gene were investigated for their analgesic, anti-inflammatory and anti-depressant activities in rats. Hot plate assay is one of the most suitable and easy methods for the investigation of centrally acting analgesic involving spinal reflexes (Sharma et al. 2013) while inflammation process is biphasic regulating process induced by carrageenan. The initial phase (1-2 h) of the inflammation is caused by the release of serotonin ad histamine while the final phase (3-4 h) is considered by the peak volume of hind paw (Khan et al. 2009). Analgesic and anti-inflammatory studies showed significant enhancement with a strong positive correlation representing that they might share a similar mechanism at some level. Recently, the presence of flavonoids has been studied in Lactuca species and these flavonoids have been reported to halt prostaglandin synthetase (Hugar et al. 2010). Since prostaglandins are involved in pain perception as well as released in the inflammation response, it could be proposed that limited accessibility of prostaglandins by flavonoids might be involved in its analgesic effect and reduced inflammation. We also investigated the anti-depressant effect of L. sativa via forced swimming test that represents the pharmacological model and produces a state similar to human depression (Glory et al. 2014). In literature, it has been reported that treatment with antidepressants reduces the oxidative stress related to depressive disorder and flavonoids contain antioxidant property which is demonstrated experimentally by the rise of the plasma antioxidant status (Pietta 2000). Although, it has been identified that plants containing flavonoids exhibit antidepressant activity (Moallem et al. 2007), antidepressant effect of various flavonoids and phenolics contained in Lactuca sativa needs to be further explored.

Conclusion
The results of current study on Lactuca sativa (CV. Grand Rapid) transformed with rol C gene showed significant enhancement in secondary metabolites compared with untransformed wild type plants. It is interesting to note that three transgenic lines showed a fixed pattern in all the activities. All three transgenic lines showed increase in phytochemicals and antioxidants and enhanced analgesic, anti-inflammatory and anti-depressant activities. In short, it can be concluded that rol genes can be potentially used to enhance antioxidant and medicinal properties.