Introduction

One of the indispensable components of our life is oxygen. Although oxygen may exist in oxidized, reduced or intermediate form, these intermediates are highly reactive and unstable owing to the presence of unpaired electrons in their orbitals. Hence, they are all regarded as Reactive Oxygen Species (ROS). Usually, for energy derived processes within the cells by mitochondrial respiration or by certain oxidases, ROS are produced as a by- product, consisting mainly of hydroxyl (HO·) radicals, superoxide anion (O2·), non-free radical species such as H2O2 and singlet oxygen (1O2) (Lobo et al. 2010). It has been reported that, under non-stressed conditions, production as well as removal of ROS is in equilibrium within the body. Stress factors including temperature, drought, pollution, excessive light intensities, and nutritional limitation increases the production of ROS (Ehling-Schulz and Scherer 1999). Cellular as well as extracellular Antioxidant Defense Systems (AODS) including ROS quencher enzymes such as Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), Gluthathione Reductase (GRed), Gluthathione-S-Transferase, Monamine Oxidase, Xanthine Oxidase, vitamin C, vitamin E, uric acid etc. act against ROS production (Halliwell and Gutteridge 1989; Sies 1993). But an imbalance between the generation and neutralization of ROS due to altered redox homoeostasis within the cell mediates Oxidative Stress (OS) namely peroxidation of the membrane lipids, aggression to tissue proteins and membranes, on damage to DNA and enzymes, degenerative or pathological processes, such as aging (Husain et al. 1987) cancer, coronary heart disease, Alzheimer’s disease (Diaz et al. 1997), neurodegenerative disorders, atherosclerosis, diabetes, and inflammation.

Epidemiological reports suggest that the dietary phytochemicals can lead to the risk of developing malignancies when administered in high doses due to a prooxidant activity of antioxidant rich phytochemicals present in high concentrations in vivo. Every antioxidant is in fact a redox agent and might become a pro-oxidant to accelerate lipid peroxidation and induce DNA damage under special conditions and concentrations (Lastra and Villegas 2007). When being not able to step in to the chain of electron transport the antioxidant compounds will be saturated and become a free radical source. Probably this is the explanation of the failure of the ‘Natural’ Cancer Prevention Trial (Peterson 1999). Studies have revealed pro-oxidant effects of antioxidant vitamins and several classes of plant-derived polyphenols such as flavonoids, tannins and curcumin (Rahman et al. 1990; Singh et al. 2001; Ahsan and Hadi 1998). Experimental results also proved that high doses of ß-carotene and selenium produce superoxide radicals. Excess of inorganic selenium seem to react with thiols functional group (Glutathione) present in tissues and form selenium trisulphides. These selenium trisulphides in turn react with more thiols group to generate ROS by redox catalysis (Mézes and Balogh 2009). Presence of oxygen in tissues, helps the transition metals (Cu and Fe) for catalysis of redox cycling of antioxidant compounds such as phenolic components and leading to the generation of ROS (Yasuko et al. 2002).

Nature has always been the best source of medicine for humans since ancient times. There are many plants available abundantly that have medicinal importance. In this regard, Ruellia tuberosa Linn of Acanthaceae family is no exceptional. Ruellia or wild Petunias, is a flowering plant and is known by many names such as cracker plant, Minnie root, Fever root, Snapdragon root and Sheep potato. It grows often as a weed in the rainy season. It is native of Central America but soon it has spread in many countries of tropical South and Southeast Asia. This herb possesses strong antioxidant and antimicrobial properties (Chen et al. 2006; Arirudran et al. 2011; Lin et al. 2006). It has many important uses in folk and Ayurvedic medicine. It also owns strong in vitro anti-cancerous and anti-inflammatory property (Alam et al. 2009). Here, in this study, we report both antioxidant and prooxidant behaviour of aqueous extract of Ruellia tuberosa Linn.

Due to its reported therapeutic properties, the weed is reflected to be one of the potential sources for the development of many important drugs including chemopreventive drugs in future. Therefore, this study was carried out to investigate the antioxidant and pro-oxidant activity of Ruellia tuberosa.

Materials and methods

Plant material and sample preparation

The green leafy herb Ruellia tuberosa Linn (cracker plant) sourced from field of Mednipur district of West Bengal (Fig. 1), and characterized and identified by experts of Botany Department, University of Calcutta, India. All chemicals used were of analytical grade purchased from Sigma, Merck and Himedia.

Fig. 1
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A twig of Ruellia tuberosa in its native state

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The aqueous extract of leaves of cracker plant was prepared according to Dutta and Singh (2011). Firstly, fresh leaves of cracker plant were washed thoroughly in running tap water, followed by rinsing twice in double distilled water. The thoroughly washed leaves were subjected to sun dry. The sun dried leaves were powdered in a mechanical grinder. Ten grams of powered sample was added to 100 ml of double distilled water and concentrated by boiling to one-tenth of its value. Following boiling, the sample was filtered using Whatman no. 1 filter paper. The crude extract was concentrated by freeze dryer, designated as AE and was stored at −20 °C for analytical evaluation.

Determination of total phenolic content (TPC)

Total phenol content of the extract was determined by Folin-Ciocalteau method (McDonald et al. 2001) with minor modification. 100 μl of AE was added to 900 μl of double distilled water. Then 500 μl of 1 N Folin Ciocalteu reagent was added to each set. The mixture was strongly shaken and thereafter 1 ml of 20 % Na2CO3 was added. The mixture was boiled for 1 min in an incubator and then cooled to room temperature. The absorbance of the developed colour was recorded at 765 nm using an UV-VIS spectrophotometer and was expressed as Gallic acid equivalents (GAE) per gm of plant weight.

Determination of flavonoids content (TPC)

Ebrahimzadeh and co-worker (2010) procedure was followed for determination of flavonoids content of the plant extracts with minor modification. In brief, AE was mixed with solution of hydrated Aluminium chloride (2 %). Subsequently the reactant solution was incubated at room temperature for 10 min. Finally, the absorbance of the solution was recorded at 440 nm.

Determination of total antioxidant capacity (TAC)

For determination of Total antioxidant activity (TAC), the reduction of molybdenum (VI) to molybdenum (V) by the phytochemicals of the plant extract was followed (Prieto et al. 1999). In brief, 100 μl AE was mixed with 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) in screw capped tubes. The capped tubes were incubated at 95 °C for 90 min. After completion of the incubation period, the samples were cooled to room temperature and the absorbance was measured at 695 nm against blank. The water soluble antioxidant capacities present in AW were expressed as equivalents of ascorbic acid (Ascorbic acid equivalents mg/g).

Determination of DPPH radical scavenging capacity

Scavenging of the stable DPPH• free radical with the extract was assayed following the methods of Blois (1958) with slight modification. In brief, aqueous extract (5–50 μg/ml) was taken in total volume of 1 ml and mixed with 4 ml of 0.1 mM methanolic DPPH• solution. The reaction mixture was mixed well and allowed to stand for 20 min at room temperature. The change in absorbance of the samples was measured at 517 nm. Radical scavenging activity was expressed as the inhibition percentage calculated using the following formula,

$$ \mathrm{Percentage}\ \mathrm{inhibition}\ \mathrm{activity}=\left[\left(\mathrm{A}-\mathrm{B}\right)/\mathrm{A}\right)]\times 100 $$

where, A = absorbance of the blank solution and B = absorbance of the test solution.

Determination of anti-lipid peroxidation capacity

Thiobarbituric acid-reactive species (TBARS) assay (Dutta and Singh 2011) with minor modification was followed to quantify anti-lipid effect of the extract against goat liver cells. The supernatant (0.5 ml) of 10 % goat liver homogenates in 0.1 M phosphate buffer (pH 7.0) was taken in screw capped tubes containing varying concentrations of extract (5–100 μg/ml). Total volume was adjusted to 1 ml by double distilled water. FeSO4 (0.07 M) was added to each test tube for initiation of peroxidation and incubated for 30 min at 37 °C. Lastly, 20 % acetic acid (1.5 ml), 0.8 % TBA in 1.1 % SDS (1.5 ml) and ice chilled 20 % TCA (50 μl) were added to all the test tubes and were gently vortexes. The reaction mixtures were further kept at 95oC for 1 h. After completion of incubation period, samples were cooled to room temperature; 5 ml of butan-1-ol were added to each tube and centrifuged at 300 rpm for 10 min. Organic layer was pipetted out and absorbance at 532 nm was recorded. Similar experiment was performed devoid of extract as control. Anti-lipid peroxidation (ALPO) was calculated by using the formula,

$$ \mathrm{ALPO}\%=1-\left(\mathrm{E}/\mathrm{C}\right)\times 100 $$

where, E was absorbance of sample and C was absorbance of blank.

Determination of metal chelating capacity

UV-Visible spectra were analysed to detect metal chelating activity of the extract following the procedure described by Dutta and Singh (2011). Briefly, 100 μg/ml of extract was added to 10 mM PBS (pH 7.4), and the absorption spectra was recorded between 200 and 600 nm. This spectrum was designated as native spectra. A number of scans with increasing concentration of CuSO4 (25, 50, 75 or 100 μM) were taken after 10 min of incubation and compared with native spectra of the extract. Then the effect of EDTA (100 μM) was tested for the extract supplemented with Cu2+ complex.

Effect on high concentration of water extract on plasmid (pUC18) DNA

Briefly, 5 μg of plasmid DNA (pUC18) and with increasing concentration of AE (0–100 μg/ml) was added in a total volume of 20 μl and then incubated at room temperature for 30 min. After completion of treatment period, the DNA was immediately subjected to electrophoresis in 0.9 % Agarose gel, in 1× TAE buffer (40 mM-Trise-acatate, 1 mM EDTA), followed by ethidium bromide staining (50 μg/ml). The DNA profile was visualized by the gel documentation system.

Statistical analysis

Individual experiments were repeated at least three times. Results were reported as Mean ± SD. Mean data of the results obtained was analyzed using ANOVA and subjected to least significant difference (LSD) for studied parameters comparisons where ever it is applicable.

Results and discussions

Phytochemicals assay of R. tuberosa aqueous extract

Qualitative detection test for important phytochemicals present in the aqueous extract (AE) of R. tuberosa revealed all important components except alkaloids (Table 1). Results of the biochemical assays carried out with the AE exhibited high amounts of flavonoids than phenol (Table 2). The total phenol content was low because the aqueous extract had limit the extraction and solubilisation process of phenols whereas no such hindrance was shown for water soluble flavonoid extraction. Total antioxidant capacity was moderately recorded.

Table 1 Qualitative analysis of phytochemicals in the aqueous extract of Ruellia tuberosa fresh leaves
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Table 2 Quantitative analysis of phytochemicals, total antioxidant property of aqueous extract of Ruelia tuberosa. Results are mean ± standard deviation of three independent experiments
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Phenolic compounds are universally accepted as free radical scavenging phytochemical. And, antioxidant properties of flavonoids express through scavenging or chelating processes (Kessler et al. 2003) due to its hydroxyls group.

Free radical scavenging activity using DPPH. assay

The free radical scavenging activity, determined in terms of total antioxidant contents by DPPH. (stable free radicals) assay. The data presented in Table 2 showed AE had good DPPH• radicals scavenging activity. Gallic acid was used as standard antioxidant in this study. The IC50 values for AE and Gallic acid revealed that DPPH• radicals scavenging ability for later is high. In Fig. 2, Gallic acid and AE of plant show dose-dependent reduction of DPPH radicals. Previous report had also showed radical scavenging ability of R. tuberosa (Cheong et al. 2013). DPPH scavenging activity of AE/Gallic acid is about 0.7 for 10 μg/ml of compound, which indicates effectiveness of AE is close to known antioxidant, gallic acid. The result of the AE in the scavenging assay of DPPH radical additionally established the fact that the extract had an antioxidant potential.

Fig. 2
figure 2

DPPH. radicals scavenging activity of the Ruellia tuberosa aqueous extract in comparison with gallic acid. Results are means of three replica (n = 3)

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Effect of aqueous extract on the in-vitro anti-lipid peroxidation

Membrane damage associated with lipid peroxidation is associated with many patho-physicalogical conditions. The anti-lipid peroxidation activity on goat liver homogenates of the AE is shown in Table 2. The AE showed moderate anti-lipid peroxidation activity. The phenolic and flavonoid contents of AE caused anti-lipid peroxidation activity. Polyphenols extract of many plant are major contributors of antioxidant activity because of their hydrogen donor property (Archana et al. 2005). Flavonoids have been reported to contain antioxidant and free radical scavenging activities in vegetables as well as the ability to protect membrane lipids from oxidation (Amic et al. 2003). Phenols were crucial plant antioxidants that scavenge free radicals and played a pivotal role in stabilizing lipid oxidation.

Effect of aqueous extract on plasmid DNA

The ability of various plant extracts to prevent DNA damage or to stimulate enhancement of DNA repair pathways are being widely studied by researchers from time to time. In our study damage of plasmid DNA (pUC18) by treatment with varying amount of aqueous extract of AE after incubation for 30 min at 37 °C was studied through agarose gel electrophoresis. As visualized by agarose gel electrophoresis, extract conferred a dose-dependent DNA damage (Fig. 3). Agarose gel electrophoresis image of the untreated plasmid DNA showed compact, three form of plasmid (Fig. 3, lane 1). Extract treated DNA gets single strand breaks as evident from the increase in intensity of nicked DNA band and absent of supercoiled form (Fig. 3, lane 2). On increasing dose (lane 3–5), both single and double strand nicks take place. There were both single and double strand breaks at 20 μg/ml of extract (Fig. 3, lane 2). Beyond this concentration, there was rapid increase in fragmentation of all the three forms of plasmid DNA. The DNA fragment of varying sizes was recorded at 100 μg/ml (Fig. 3, lane 5). From the result it is appealing to speculate that experimental extract plays significant role as pro-oxidant at higher doses. Similarly, resveratol (Lastra and Villegas 2007) and phloroglucinols from Garcinia subelliptica (Wu et al. 2008) can induce prooxidant properties, which lead to oxidative breakage of cellular DNA in the presence of transition metal ions such as copper. Ascorbic acid is an essential micronutrient and is considered to have an antioxidant function in living systems. For the past several decades, ascorbic acid has been the subject of considerable interest as an anticancer agent. Several studies have shown that ascorbic acid is cytotoxic to a variety of cancer cells. Ascorbic acid acts as a pro-oxidant and leads to oxidative DNA breakage in lymphocytes. It is further shown that such DNA breakage is inhibited by both iron and copper chelators in cells, whereas in nuclei. It is proposed that the copper-dependent cellular redox status is an important element in the cytotoxic action of ascorbic acid against cancer cells. In chromatin copper is an essential component and can take part in redox reactions. Our plant extract demonstrate similar properties which can be utilize in such condition. The pro-oxidant property of phyto-polyphenols like flavonoids (Rahman et al. 1990), tannins (Singh et al. 2001), caffeic and chlorogenic acid (Yasuko et al. 2002) and curcumin (Ahsan and Hadi 1998) had been reported in earlier literatures.

Fig. 3
figure 3

Effects of Ruellia tuberosa aqueous extract on plasmid (pUC18) DNA as monitored by electrophoretic mobility. Lane 1, plasmid DNA; lanes 2–5, plasmid DNA and aqueous extracts (20–100 μg/ml). The DNA profile shows pro-oxidant property of aqueous extract

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Extract-copper interaction

Metal chelating activity of the AE was tested by monitoring the change of the UV-Visible absorption spectra upon addition of Cu2+ ions at pH 7.4 (Fig. 4). The effect of Cu2+ on the spectral showed characteristics shifting nature of bands of the AE. The change in spectral nature of extract is attributed to the formation of a chelate complex of AE- Cu2+ ions. EDTA, a well-known chelating agent for most of the metal ions was used to confirm the formation of extract-Cu2+ ions chelate. Generally, upon addition of EDTA, the complex formed between extract and Cu2+ breaks, since EDTA is strong chelating agent, itself get bound to Cu2+. But in present study, the AE showed strong chelating activity and EDTA could not break the complex even at high concentration (150 μM). It has been reported that chelating agents are effective as secondary antioxidants because they reduce the redox potential, thereby stabilising the oxidised form of the metal ions. Chelators referred to a particular way in which molecule of a compound makes bond with metals ions. The chemistry implicates formation of 2 or 3 distinct co-ordinate bonds between a ligand molecules (here plant phytochemicals) and a single central metallic atom. Metal chelating agents assemble tissue metal by making soluble, stable complexes which are finally excreted through the urine and/or faeces. Metal ions in excess may lead to many dysfunctions in human body by helping in generation of ROS through Fenton reaction. Alzheimer’s and Parkinson’s diseases are neurodegenerative dysfunction of man due to excess of transition metal (Aparadh et al. 2012). Metal chelators are being applied to neutralize excess of iron in Thalassemia and other anaemia’s patients (Ebrahimzadeh et al. 2008). Hence, metal chelating property of plant extracts prove to be more effective in chelation therapy in human rather than synthetic compounds which have few side effects.

Fig. 4
figure 4

Effect of Cu (II) & EDTA on absorption spectra of Ruellia tuberosa aqueous extract. a Extracts (100 μg/ml) with/without1 Cu (II) incubated for 10 min. b After this EDTA (100–150 μM) was added. c Summary of experiments depicting high metal chelating activity of test extract compared to EDTA. All the spectra were recorded in 10 mM PBS (pH 7.4)

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Conclusions

From the present study, we can conclude that an aqueous extract of Ruellia tuberosa leaf aqueous extract was rich in important phytochemicals. Antioxidant and free radical scavenging activities were also positively exhibited. Metal chelating activity of the plant is much more than EDTA (Fig. 4). This might be due to intrinsic property of the extract since its shown very good antioxidant also. Thus, the plant contains natural antioxidant which might be helpful in inhibiting the advancement of oxidative stress in the living system. High dose of same extract had shown single and double strands breakage of plasmid DNA which reflects its pro-oxidant property. If analysis of our aqueous extracts can be performed in depth it may bear assurance for cures of many deadly diseases such as aging, cancer, diabetics, etc. Being non-synthetic they are free from side effects causing minimum damage to the endogenous system compared to that of synthetic drugs.