PHYTOREMEDIATION OF REACTIVE TURQUOISE BLUE H5G USING IN VITRO CULTURES OF SOLANUM VIRGINIANUM (L.)

Solanum virginianum (L.) belonging to family Solanaceae selected for decolourisation of Reactive Turquoise Blue H5G dye. In vitro grown cultures of S. virginianum were able to remove more than 50% dye concentration up to 110mg/l. comparative analysis of biochemical and antioxidant study showed more activity in treated plants as compared to untreated plants. The phytotoxicity study demonstrated the non-toxic nature of degraded metabolites. Use of such non-edible yet medicinal plant for phytoremediation is discussed.


INTRODUCTION
Textile wet processing is considered as one of the worst industrial sectors in the terms of water consumption and water pollution. There are many textile industries in the country pollute the water bodies with the dyes in the effluent released. Hazardous chemicals are used for various processing in the textile industries as for scouring, desizing, bleaching, printing, dyeing and finishing. These chemicals include inorganic compounds, elements or polymers and organic products. More than 8000 chemical products are associated with the dying processes listed in colour Index (Society of Dyers and Colourists, 1976) whereas, 7X10 5 metric tons of dyestuff is produced every year (Zollinger, 1987). Dyes are carcinogenic and mutagenic, toxic to the flora and fauna (Hu et al., 2009). These dyes include several structural varieties of dyes such as acidic, reactive, basic, disperse, azo, diazo, anthraquinone based and metal complex dyes. Colour, first contaminant of waste water has to be removed before releasing waste water into primary water bodies or on land. Apart from this as less than 1 ppm of the dye concentration is highly visible, it has magnified negative effect on aesthetics, transparency, gas solubility and overall the physicochemical properties of water bodies (Banat et al., 1996). Dye containing effluent have a high chemical oxygen demand, biological oxygen demand, suspended solids and other toxic compounds. To mitigate these problems several physical, chemical and biological clean-up technologies are coming forward. The use of microbes (bioremediation) and plants (phytoremediation) for breaking down hazardous textile dyes is becoming a best choice to detoxify or render harmless environmental pollutants (Kagalkar et al. 2009;Patil et al. 2009). The costs of conventional physical and chemical treatment methods are highly expensive, have low efficacy, are labourers and though they assure decontamination, secondary pollutants may be second parallel problem hence, substitute for the method of decontamination is the need of an hour.
Compared to the physical and chemical technologies, bioremediation is an effective technique which is cost effective (Singh et al., 2008). Among the sources of bioremediation, the use of plants has opened up a new facet to clean up the contaminated soil. Researchers have realized that though the knowledge about detoxification and hyperaccumulation in plants is not well understood, plants are armored with amazing metabolic and absorption capabilities as well as transport systems that can uptake contaminants from soil and water and behave as a good remedy.
Use of plants is safe, easy to operate and is less troublesome (Cunningham and Berti, 2000) because plant physiology and metabolism works in a way that mutations in the system will not spread as in case of microorganisms. Plants have extraordinary potential to concentrate and accumulate elements and compounds as well as organic contaminants and metabolize them in various molecules in their tissues. The plants have developed an advanced regulatory mechanism to co-ordinate effective metabolic activities (Salt et al., 1998). Dye decolorization ability of plants is mainly dependent upon the enzymes they consist of. Never the less the employment of plants for reclamation of highly polluted sites (water bodies or land) has come up. property before and after treatment of dye was successfully carried out in the present investigation.
The studies were confirmed with phytotoxicity analysis. Plant cell cultures offer several advantages to understand accurately the complexity of physiological and biochemical mechanism involved in the degradation of dyes, toxic chemicals and other pollutants (Doran, 2009). The present study indicates that the plant does not lose their potential even after treated with textile dyes and hence the plant can be successfully used in phytoremediation.
As mentioned above, plants are safe to handle. The selected plant shows many advantages such as, it is fast growing, has well developed root system, it is non-edible, non-hazardous and no special care is required. S. virginianum is used in traditional herbal medicines. Non-edible plant is chosen for phytoremediation because there is no risk of mixing experimental material in the routine food chain. In this regards S. virginianum would be an ideal system.

In vitro regeneration of plant:
Morphologically healthy seeds were selected as an explant. All the fruits were washed and surface sterilized with 0.1% HgCl2, followed by three successive washes of sterilized distilled water. Surface sterilization was carried out in laminar air flow. Fruits were blotted and dissected out. The seeds were then blotted on sterile tissue paper, dried and inoculated on ½ MS (Murashige and Skoog) (1962) basal medium aseptically. After germination they was subcultured on MS medium supplemented with different concentrations (0.5mg/l, 0.75mg/l and 1mg/l) of BAP (6 Benzylaminopurine).

Treatment of in-vitro plantlets with RTB H5G
Experiments were conducted to evaluate the efficiency of S. virginianum (L.) in removal/degradation of textile dyes Reactive Turquoise Blue H5G (RTB H5G). RTB H5G (synthetic dye) was procured from local market of Kolhapur. Different concentrations (10mg/l, 30mg/l, 50mg/l, 70mg/l, 90mg/l, 110mg/l, 130mg/l and 150mg/l) of dye were added to MS medium and a set of three Erlenmeyer flasks (100ml capacity) was prepared for each concentration. In vitro grown plantlets of S. virginianum were placed in all the treatment flasks.
Additional two sets of three Erlenmeyer flasks each were prepared for abiotic control (Dye dissolved in distilled water instead of MS) and biotic control (MS Basal) all these flasks were incubated at 27±2 ºC for a period of six days under rotary shaking (110rpm). After day six aliquots (2ml from each flask separately) were collected and centrifuged at 8000rpm for 15min. The clear supernatant was used for measuring the residual dye (%) at 618nm, the absorbance readings were used for calculating degradation (decolourisation) of dye by the formula, % Decolourisation = (Initial absorbance -Final absorbance)/ Initial absorbance x100 The aliquots were collected after every 4-day period. Aseptic conditions were followed throughout the experiments.

Phytochemical analysis:
After 21 days of decolourisation experiment, plants from all the different treatments were collected and plant extracts (1%) were prepared on fresh weight bases. Six different solvent systems, methanol, ethanol, acetone, chloroform, n-Hexane and distilled water were used for extraction.

Quantification of total phenolic content (TPC):
The total phenolic contents of S. virginianum extracts were determined by using modified spectrophotometric Folin-Ciocalteu method (Wolfe et al., 2003). The reaction mixture was prepared by mixing an aliquot of extracts (0.125 ml) with Folin-Ciocalteu reagent (0.125 ml) and 1.25 ml of saturated Na2CO3 solution. Reaction mixture was further incubated for 90 min at room temperature and absorbance was measured at 760nm. The samples were prepared in triplicates for each analysis and the mean value of absorbance was recorded. Results were expressed as of mg gallic acid equivalents (GAE)/g fresh weight of samples of S. virginianum. All the experiments were expressed as mean ± SE of triplicate measurements.

Quantification of total flavonoid content (TFC):
The total flavonoids were estimated by using modified colorimetric method (Luximon-Ramma et al., 2002). The reaction mixture had 1.5 ml of extract to 1.5 ml of 2% methanolic AlCl3.
The mixture was incubated for 10 minutes at room temperature and absorbance was measured at 368nm against 2% AlCl3, which served as blank. The samples were prepared in triplicates for each analysis and the mean value of absorbance was obtained. The optical density (OD) measurements of samples were compared to standard curve of rutin and expressed as mg of rutin equivalent (RE)/100g fresh weight of plant S. virginianum. All the experiments were performed in triplicates and expressed as mean ± Standard Error (SE).

Quantification of total alkaloid content (TAC):
Total alkaloid content of S. virginianum was assessed using phenanthroline method described by Singh et al., (2004). The assay mixture was prepared and incubated for 30 minutes in water bath maintained at 70±2 0 C and the absorbance was measured at 510nm. Distilled water served as blank. The samples were prepared in triplicates for each analysis and the mean value of absorbance was recorded. The optical density (OD) measurements of samples were compared to standard curve of quercetin as mg of quercetin equivalent (QE)/100g fresh weight of S.
virginianum. All the experiments were expressed as mean ± SE.

Ferric reducing antioxidant power assay (FRAP)
The ferric ion reducing capacity was calculated by using assay described by Pulido et al. (2000). To 100 μl plant extract, 3 ml of FRAP reagent [300 mM sodium acetate buffer at pH 3.6, was added. The reaction mixture was incubated at 37°C for 15 min. The absorbance was measured at 595nm. The value of FRAP was expressed as milligrams of ascorbic acid equivalents per 100 gram of Fresh weight.
Plant extract (25 μl) was mixed with 3 ml of DPPH methanolic solution (25 mM). The reaction mixture was incubated in dark at room temperature for 30 min. The absorbance was measured at 517nm against blank. Results were expressed as percentage of inhibition of the DPPH radical and percent antioxidant activity of plant extract was calculated using the following formula:

Ferrous ion chelating activity (FICA)
Ferrous ion chelating activity was measured by following method described by (Dinis et al., 1994). Assay mixture contained 0.1 ml of 2 mM FeCl2 and 0.3 ml of 5 mM ferrozine and mixed with 1 ml of plant extract. The mixture was incubated for 10 min at room temperature and absorbance was measured at 562nm spectrophotometrically. The ability of sample to chelate ferrous ion was calculated as the percent inhibition of Fe +2 to ferrozine complex. Percentage antioxidant activity of plant extract was calculated using the following formula: % Ferrous ion inhibition = [Control (abs) -Sample (abs)] ×100 /Control (abs)
Germination studies were carried out by watering the seeds with respective degraded solution every day. Seeds with radical (≥ 1mm) were considered germinated (Wu et al., 2007). The length of plumule (shoot), radical (root), fresh weight, rate of germination (%) was recorded after eight days. Germination percentage was calculated by the formula: Germination (%) = (No. of seeds germinated / Total No. of seeds) X 100 4. RESULTS:

In vitro regeneration of plant:
Seed germination was achieved on ½ Murashige and Skoog basal medium (1962) ( Table   1). Shoot multiplication, was carried out using in vitro nodal explants subcultured on MS medium supplemented with different concentrations of BAP (0.5mg/l, 0.75mg/l and 1mg/l). Highest shoot number (18.1±0.17, Table 2) was observed on MS medium supplemented with BAP (0.75mg/l).

Decolourisation of textile dye Reactive Turquoise Blue H5G (RTB H5G)
Dyes used in textile industries are with various chemical constituents. concentrations 10mg/l, 30mg/l, 50mg/l and 70mg/l showed decolourisation between 85% -94% within 6-12 days (Table 3, Fig. 1). While the concentrations 50mg/l and 70mg/l showed more than 50% decolourisation. Lowest decolourisation percentage (15.62%) was obtained in 150mg/l dye concentration in 21 days (Table 3, Fig. 1). Variation in the time required and efficiency of decolourisation may probably be due to molecular complexity of dyes and the enzymes produced during decolourisation (Sanghi et al., 2006). Slower rate of decolourisation higher is molecular weight and presence of inhibitory groups like nitro (-NO2) and sulphite (-SO3) in dyes (Hu and Wu, 2001). Maximum decolourisation was observed in shaking conditions as compared to static conditions. This could be due to better oxygen transfer and nutrient distribution as compared to stationary cultures (Kaushik et al., 2009). Hence, subsequent dye decolourisation experiments were carried out under shaking condition.

Biochemical analysis of in vitro untreated plant of S. virginianum
Biochemical analysis and antioxidant potential of plant is determined to verify its medicinal properties. This investigation deals with a remedy for reclamation of polluted sites due to textile dyes. Preliminary studies on decolourisation potential of S. virginianum was tested.
Medicinal constituents of the plant (TPC, TFC, TAC) and antioxidant capacity (FRAP,FICA,SOAS, PMo and DPPH) of treated and untreated plants were evaluated to confirm that plant remains unaltered after treatment with textile dye Reactive Turquoise Blue H5G. This supports the fact that the plant can be safely used for remediation. This type of study is the first report where the plant is evaluated for its medicinal properties before and after treatment.

4.2.1Total Phenolic Content (TPC):
The total phenolic content of S. virginianum was evaluated using extracts of fresh plant material in different solvent systems. The total phenolic content was varying between 10.86±0.26 to 179.58±0.35 mg of Gallic Acid Equivalent/100g of Fresh Weight (Table 4). From the results it was observed that, lowest activity was found in acetone extract and the highest phenolic content was found in distilled water extract.

Total Flavonoid Content (TFC):
The total flavonoid content of S. virginianum was evaluated using extracts of fresh plant material in different solvent systems. The total flavonoid content was varying between 0.96±0.01 to 15.76±0.007 mg of Rutin Equivalent/100g of Fresh Weight (Table 4)

Total Alkaloid content:
The total alkaloid content of S. virginianum was evaluated using extracts of fresh plant material in different solvent systems. The total alkaloid content varies between 39.16±0.05 to 64.12±0.11 mg of Quercitin Equivalent/100g of Fresh Weight (Table 4). From the results it was found that, the lowest activity was found in n-hexane extract and the highest alkaloid content was

1Total Phenolic Content (TPC)
The total phenolic content of S. virginianum was evaluated using extracts of in vitro grown plantlets treated with different concentrations of the dye RTB H5G in different solvent systems.
In 10mg/l, the total phenolic content was varying between 9.89±0.02 to 21.48±0.07 mg of GAE/100g of FW (Table 5). From the results it was observed that, the lowest content was found in distilled water extracts and the highest phenolic content was found in chloroform extract. In 30mg/l, the total phenolic content was varying from 95.73±0.02 to 132.91±0.02 mg of GAE/100g of FW (Table 5). From the results it was found that, n-hexane extracts showed lowest content and distilled water extracts showed highest content. In 50mg/l, the total phenolic content was varying between 55.48±0.11 to 78.55±0.14 mg of GAE/100g of FW (Table 5). From the results it was found that, lowest content was observed in n-hexane extracts and the highest content was observed in acetone extracts. In 70mg/l, the total phenolic content was varying from 60.09±0.21 to 115.48±0.50 mg of GAE/100g of FW (Table 5). From the results it was found that, the lowest content was found in distilled water extracts and methanol extracts while the highest content was observed in ethanol extracts. In 90mg/l, the total phenolic content was varying from 65.73±0.25 to 200.35±0.58 mg of GAE/100g of FW (Table 5). From the results it was found that, methanol extracts shows lowest phenolics whereas distilled water extracts shows highest phenolics. In 110mg/l, the total phenolic content was varying from 16.76±0.17 to 55.48±0.51 mg of GAE/100g of FW (Table 5). From the results it was observed that, the lowest content was found in acetone extracts and the highest phenolics were found in ethanol extracts. In 130mg/l, the total phenolic content was varying from 20.86±0.17 to 36.76±0.32 mg of GAE/100g of FW (Table 5). From the results it was observed that, methanol extracts shows lowest phenolics while chloroform extracts shows highest phenolics. In 150mg/l, the total phenolic content was varying from 217.53±0.20 to 364.45±0.79 mg of GAE/100g of FW (Table 5). From the results it was observed that, lowest content was observed in acetone extracts and the highest content was found in chloroform extracts.

Total Flavonoid Content (TFC)
The Flavonoid Content of S. virginianum was evaluated using extracts of fresh plant material treated with different concentration of the dye in different solvent systems. In 10mg/l, the total flavonoid content was varying between 6.65±0.04 to11.24±0.14 mg of RE/100g of FW (Table   5). From the results it was observed that, the lowest content was found in n-hexane extract and the highest flavonoid content was found in acetone extract. In 30mg/l, the total flavonoid content was varying between 1.67±0.06 to 9.50±0.27 mg of RE/100g of FW (Table 5). From the results it was observed that, n-hexane extract shows lowest flavonoids while acetone extract shows highest content. In 50mg/l, the total flavonoid content was varying between 1.56±0.06 to 4.07±0.04 mg of RE/100g of FW (Table 5). It was observed that, lowest content was found in distilled water extract while the highest content was found in chloroform extract. In 70mg/l, the total flavonoid content was varying between 0.84±0.09 to 11.81±0.04 mg of RE/100g of FW (Table 5). The results revealed that, lowest content was observed in acetone extract while the highest flavonoids were found in ethanol extract. In 90mg/l, the total flavonoid content was varying between 0.74±0.04 to 7.97±0.01 mg of RE/100g of FW (Table 5). From the results it was observed that, lowest content was observed in acetone extract and the highest content was found in ethanol extract. 110mg/l, the total flavonoid content was varying between 0.94±0.03 to 9.56±0.01 mg of RE/100g of FW (Table   5). From the results it was observed that, n-hexane extract shows lowest flavonoids whereas methanolic extracts shows highest flavonoids. 130mg/l, the total flavonoid content was varying between 0.59±0.04 to 3.73±0.04 mg of RE/100g of FW (Table 5). From the results it was observed that, n-hexane extract shows lowest content while distilled water extract shows highest content. In 150mg/l, the total flavonoid content was varying between 0.61±0.32 to 53.75±0.02 mg of RE/100g of FW (Table 5). From the results it was observed that, ethanol extract shows lowest flavonoids and the chloroform extract shows highest flavonoids.

Total Alkaloid Content
The Alkaloid Content of S. virginianum was evaluated using extracts of fresh plant material treated with different concentration of the dye in different solvent systems. In 10mg/l, the total alkaloid ranges between 36.10±0.04 to 60.90±0.17 mg of QE/100g of FW (Table 5). From the results it was found that, n hexane extracts show lowest results whereas acetonic extracts shows highest. In 30mg/l it was found that lowest content was observed in n-hexane extracts and distilled water extracts shows highest alkaloid content and it varies between 25.76±0.15 to 54.55±0.33 mg of QE/100g of FW (Table 5). In 50mg/l, the alkaloid content varying between 30.49±0.06 to 58.95±0.51 mg of QE/100g of FW (Table 5), lowest content was observed in n-hexane extracts and the highest results were observed in methanol extracts. In 70mg/l, it was observed that, distilled water extracts show lowest alkaloid content while the methanolic extracts shows highest alkaloid content and it varies between 6.05±0.06 to 57.16±0.06 mg of QE/100g of FW (Table 5). In 90mg/l, it was seen that, lowest alkaloids were seen in n-hexane extracts whereas methanolic extract shows highest alkaloid content, it ranges between 39.18±0.01 to 55.51±0.23 mg of QE/100g of FW (Table   5). In 110mg/l, the alkaloid content ranges from 25.73±0.02 to 51.40±0.18 mg of QE/100g of FW (Table 5), the lowest results were observed in chloroform extracts and the highest results were obtained from distilled water extracts. In 130mg/l, it was observed that, n-hexane extracts show lowest alkaloid content while the distilled water extract shows highest results, it varies from 25.55±0.01 to 52.49±0.08 mg of QE/100g of FW (Table 5). In 150mg/l, the alkaloid content varies from 21.47±0.02 to 52.94±0.24 mg of QE/100g of FW (Table 5), the lowest results were seen in n-hexane extracts whereas the highest results were observed in methanol extracts.
In the present study fluctuations in the phenolic, flavonoid and alkaloid content was seen with different concentration of dye and with the different solvents used for extraction. This may be due to the stress experienced by the plant because of the dye concentrations.
The PMo activity (Table 5) was found highest in ethanol extract (62.55%) and lowest in acetone extract (28.27%), DPPH activity (Table 5) was observed highest in methanol extract (43.88%) and lowest in n-hexane extract (10.88%). The present study indicates that the plant does not lose their potential even after treated with textile dyes and hence the plant can be successfully used in phytoremediation.

Phytotoxicity study:
Phytotoxicity study was performed to assess the toxicity of dye effluent on common   Fig. 2a).
The degraded products from all the treatment flasks was collected separately and used for watering the seeds of Vigna radiata. Ten seeds per treatment were placed in petriplates with blotting papers and the lethality of degraded solution was tested by watering the seeds regularly with the respective degraded products. The germination results were noted down ( Table 7, Fig. 2c) and it was observed that the degraded solution 10mg/l, 30mg/l, 50mg/l, 70mg/l, 90mg/l and 110mg/l showed 100% seed germination. Degradation product collected from 130mg/l and 150mg/l treatments showed lowered germination as compared to control (Fig. 2b)  compounds in addition to this it can also uptake contaminants from the environment and they have evolved an advanced regulatory mechanism to metabolize them into various molecules in their organs and tissues (Salt et al., 1998). Plants help to clean up various pollutions that include metals, pesticides, explosives and oil, also help to prevent carrying of pollutants and products of contaminants away from sites to other areas by wind, rain and groundwater (Cluis, 2004