The Healthy Plants Of Crotalaria Retusa Biology Essay

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The healthy plants of Crotalaria retusa, Crotalaria prostrata and Crotalaria medicaginea were collected from Rushikonda, Visakhapatnam, India, and the plants Herbarium was deposited in Andhra University, Andhra Pradesh, India, where the voucher numbers was A.U. (B.D.H.) 5527, A.U. (B.D.H.) 2359 and A.U. (B.D.H.) 1850 respectively. Some of the collected plants of three species were grown in the garden of Department of Biotechnology, GITAM University, Visakhapatnam, Andhra Pradesh, which are maintained at 300 - 400 with natural daylight and irrigated water as required. Actively growing plants were used as a source for further studies.

Chemicals

The chemicals used during the course of the current investigation were of analytical grade. Inorganic salts obtained from Qualigens, HiMedia, Merck and Fischer Scientific Limited. Agar and sucrose were obtained from HiMedia, India.

Glassware

Test tubes (2.5X15 cm), petriplates (55mm and 85mm diameter), Erlenmeyer flasks and Beakers (50, 100, 250, 500, 1000 ml capacity), pipettes (1, 2, 5 and 10 ml capacity) and measuring cylinders of all capacities (10-1000 ml) were purchased from "Borosil" India. In addition glass screw capped tissue culture tubes (6X15 cm) were used for culturing.

Plastic ware

Micro tips (2-200 µl and 100-1000 µl) were obtained from "Tarson Product Pvt. Ltd." (India) and plastic beakers of various capacities (50, 100, 250, 500 and 1000 ml) were obtained from "Borosil" India.

Maintenance of Glassware

Glassware was soaked in dilute Hydrochloric acid overnight and rinsed with tap water followed by washing thoroughly with 2% Tween-20. Then they were washed thrice with distilled water followed by tap water. The glassware was then oven dried at 1000C overnight. In order to recycle, the glassware that had contaminated tissue or media prior to washing, the culture containing glassware were decontaminated by autoclaving without opening the closures. Cleaned and dried glassware was finally stored in closed racks until use.

Plant tissue culture

The present investigation is aimed at the in vitro propagation and regeneration of Crotalaria prostrata, Crotalaria medicaginea and Crotalaria retusa using standard protocols (Bhojwani and Razdan, 1996; Hussain et al., 2008). The induced calli and shoots were used as explants for further comparative analysis.

Laboratory conditions

Glassware was soaked in dilute Hydrochloric acid (HCl) overnight. They were rinsed with tap water followed by washing with 2% teepol. They were then washed with tap water followed by 2 - 3 rinses with distilled water. The glassware was oven dried (150 oC) and wrapped with aluminum foil.

The floor of the laminar air flow (Klenzaids) were cleaned with alcohol and tissue culture components like media, sterilized blade, forceps, scalpel, petridishes, beakers, cotton and aluminum foil were placed in the laminar air flow. The UV light and airflow was switched on earlier for at least one hour to maintain aseptic conditions in the inoculation area.

Media preparation

Nutritional requirements for an optimal growth of a tissue under in vitro conditions may vary with the species under investigation. Even tissues from various parts of a plant may have different requirements for a satisfactory growth. Hence in present investigation, the formulations of Murashige and Skoog medium were used and tabulated (Table - 16) (Murashige and Skoog, 1962).

Inorganic and organic salts

The macro and micronutrients are mixed in appropriate concentrations from the prepared stocks (10X and 100X respectively). The organic supplements were mixed from a stock solution (100X). All the stocks were stored in refrigerator except iron stocks. In order to avoid any problem with iron solubility, it was prepared as chelated form. It was prepared by dissolving FeSO4.7H2O and Na2EDTA.2H20 separately by heating and finally two solutions were mixed and made up the final volume to 100 ml with distilled water and stored at room temperature in amber colored bottle.

Plant growth regulators

The stock solutions of various growth regulators such as auxins (2,4-D, IAA, IBA and NAA), cytokinins (BAP and Kinetin) and gibberellins were prepared at 1mg/ml concentration. These stocks were prepared by dissolving in few drops of 1N NaOH or 1N HCl and made up to 100 ml with distilled water in a volumetric flask each and stored in refrigerator at 200C (Table - 17).

Table - 16: Composition of Murashige and Skoog (MS) medium used in in vitro propagation of Crotalaria species (Murashige and Skoog, 1962; Balakrishnan et al., 2009)

Sl. No.

Medium Composition

Concentration (mg/l)

1.

Macronutrients (10X)

NH4NO3

1650.00

KNO3

1900.00

CaCl2.2H2O

440.00

MgSO4.7H2O

180.69

KH2PO4

170.00

2.

Micronutrients (100X)

MnSO4.4H20

16.90

H3BO3.H20

6.20

KI

0.83

Na2MoO4.2H2O

0.25

ZnSO4.7H2O

8.60

CuSO4.5H20

0.025

CoCl2.6H2O

0.025

3.

Iron - salts (100X)

FeSO4.7H2O

27.80

Na2EDTA.2H20

37.3

4.

Vitamins (100X)

Thiamine HCl

0.10

Pyridoxine HCl

0.50

Nicotinic acid

0.50

Glycine

2.00

5.

Myoinositol

100.00

6.

PVP

50.00

7.

Sucrose

30.0 g/l

8.

Agar

8.0 g/l

9.

pH

5.6 ± 0.2

Table - 17: Preparation of stock solutions for various growth regulators, used in present investigation (Balakrishnan et al., 2009).

Growth regulator

Concentration (µM)

Weight for 100 ml (mg)

Solvent used

Auxins

2,4-D

4.52

100

1N NaOH

IAA

5.70

100

1N NaOH

IBA

4.92

100

1N NaOH

NAA

5.37

100

1N NaOH

Cytokinins

BAP

4.43

100

1N NaOH

Kin

4.64

100

1N NaOH

Gibberillic acid

2.88

100

1N NaOH

Carbon source

The growth of plants under in vitro conditions requires a carbon source, which were supplemented in the form of carbohydrates (Glucose, sucrose, maltose and galactose etc.). In present investigation, sucrose was used as a carbon source in all the experiments in different concentration.

Solidifying agent

Bacteriological agar (Himedia, India) at 0.8% was used for solidification in almost all the experiments except rooting experiments (0.6%).

Preparation of 1 liter medium for in vitro propagation

500 ml of double distilled water was taken in Erlenmeyer flask for the preparation of 1 liter plant tissue culture medium. Macro and micronutrient stock solutions were added sequentially followed by the addition of freshly weighted sucrose in desired concentrations. The desired plant growth regulators were added in required quantities at this stage. The pH was adjusted to 5.6 ± 0.2 using 1N NaOH or 1N HCl after the addition of all required constituents for plant growth. The medium was then made up to 1 liter with double distilled water. Gelling agent (Agar) was added as per the requirement and the media was then kept in microwave oven to melt the gelling agent. It was then dispensed into culture tubes (15 ml) and autoclaved at 1210C at 15 lbs/inch2 for 20 minutes. All the plant growth regulators used during the course of present investigation were added before autoclaving the medium. All the culture tubes with media after sterilization were kept in slanting or in vertical position as per our requirement.

Preparation of sterile containers and small equipment

Glass culture vials and vessels were mostly sterilized along with the medium whereas the glassware used for pre-sterilized nutrient medium preparation was sterilized by dry heating at 1800C for 3 hours in hot air oven.

The equipment such as scalpels, forceps and surgical blades were sterilized by dipping in 95% ethanol followed by flaming with Bunsen burner, which is referred as flame sterilization.

Maintenance of aseptic conditions

Prior to commencing the tissue culture experiments, the hands are washed with anti-septic soap followed by swabbing with 95% ethanol. The laminar flow hood (Klenzflow) was cleaned by spraying and swiping with 95% ethanol and all paraphernalia used in inoculation such as media, scalpel, surgical blade, forceps and petriplates etc., were sterilized using autoclave (15 lbs/inch square pressure for 15 mins) and transferred to laminar air flow hood and UV light was switched on for 45 - 60 minutes. Then the UV light was switched off and air blower was switched on (flow rate = 27±3 m/sec). The work in laminar flow was started after 15 minutes of airflow, so as to remove the O3, formed during UV sterilization. In order to minimize the contamination, hands and inoculating area are wiped with alcohol frequently, followed by flame sterilization of scalpel and forceps.

Explant source

The central section of Crotalaria retusa, Crotalaria prostrate and Crotalaria medicaginea leaves were used as explants for in vitro propagation and regeneration experiments.

Explant sterilization

The leaf explants are washed thoroughly with 2% (volume/volume) Tween-20 (Merck, India) followed by running tap water for 10 min and then washed thrice with distilled water. The explants were then dipped in 70% ethanol for 1 min, followed by surface sterilization using 0.1% mercuric chloride for 3 min and finally rinsed thrice with sterile distilled water. The effect of different surface sterilants such as HgCl2, H2O2 and NaOCl on explants for decrease of contamination was studied (Table - 18). The explants were surface-dried on sterile filter paper and used for inoculation on Murashige and Skoog (MS) basal medium supplemented with different concentrations of plant growth regulators (Hussain et al., 2008).

Inoculation

The inoculation was carried out in the vicinity of Bunsen burner. Sterilized explants were transferred on to the sterilized petridish containing filter papers. The explants were dissected and damaged peripheral ends were removed using forceps and scalpel, which were blotted on sterile filter paper. The central section of leaf acts as explant. These explants were inoculated on 15 mL Murashige and Skoog (MS) medium. The mouth of the culture tubes were then covered with sterilized aluminium foil and tied with rubber bands. Cultures tubes were labeled providing the details of the experiment i.e., name of the explant, date of inoculation etc. All the experiments were triplicated.

Incubation

Plant cultures are greatly influenced by physical factors includes: light, temperature and relative humidity. All the cultures were incubated at 25±20C for 16 hours photoperiod and 8 hours dark period with a light supplied by cool-white fluorescent lamps at an intensity of 50-60 µmol m-2s-1.

Callus and Multiple shoots induction

The leaf explants (1 cm) of all three species of Crotalaria were cultured on Murashige and Skoog (MS) medium fortified with BAP (2.21-17.75 µM), Kn (0.46-14.84 µM), NAA (0.53-16.12 µM) and 2, 4-D (2.26-15.83 µM) alone or in combination for callus induction and multiple shoots induction (Table - 19-27). Thereafter, the clumps of shoots produced were separated and divided into single shoots after 30 days. The explants inoculated on Murashige and Skoog medium lack of growth regulators served as control.

Maintenance of callus and induced shoots

The morphogenic callus was selected during subculture for every 3 weeks based on nature of appearance of calli for regeneration i.e., organogenic. Small pieces of tissue were transferred to regeneration medium supplemented with 2,4-D (13.57 µM) and BAP (2.21 µM) for shoot development (Table - 27). All these cultures were incubated at 262oC for 16 hours photoperiod with a light supplied by cool-white fluorescent lamps at an intensity of 50-60 µmol m-2s-1. The aroused shoots directly from explants were then sub-cultured on regeneration medium for shoot elongation and then transferred to MS medium supplemented with BAP+NAA(13.31+2.15 µM) and various concentrations of GA3 (0.29-5.76 µM) for multiple shoots formation (Table - 28). Further, these aroused shoots were sub-cultured on Murashige and Skoog (MS) medium containing various concentrations of IBA & NAA for rooting leading to plantlet development.

In vitro rooting

The aroused shoots were cultured on half-strength Murashige and Skoog (MS) medium supplemented with different concentrations of IBA (2.46 - 19.68 µM) and NAA (2.68 - 21.50 µM) for rooting (Table - 29).

Acclimatization

Aroused plantlets were gently extracted from the glass vessels and washed thoroughly with tap water to remove adhered agar and traces of the medium in order to avoid contamination. These plantlets were given a final wash with distilled water for 5 min and transferred to a plastic cups (8 cm in diameter) containing garden soil subsequently transferred to pot. The pots were maintained in a polyethylene chamber in the culture room with a diffused light (16/8 hrs photoperiod) and irrigated every alternative day with a solution of half-strength Murashige and Skoog (MS) medium. Polyethylene covers were removed gradually, and then the plants were transferred to garden soil for further growth in a greenhouse. All the above experiments were repeated thrice and recorded the observations and results. Some of the completely regenerated plants were used for further comparative experiments conducted with field grown (wild) plants and in vitro propagated plants of Crotalaria species.

Statistical analysis:

The culture tube with one explant on medium with a specified composition of growth regulators was considered as one replicate. Each treatment in each set of experiments consists of 5 replicates and each experiment was repeated thrice. The data shown represent the mean ± standard error of five independent experiments. The data was statistically analyzed by one-way analysis of variance (ANOVA) and the means were assessed by Duncan's Multiple Range Test (DMRT) at 0.05% level of significance (P<0.05) using Statistical Package for Social Sciences (SPSS version 17.0).

Phytochemistry

Biochemical studies

Estimation of chlorophyll pigments

The chlorophyll content was estimated using the standard protocol (Lichtenthaler and Buschmann, 2001). 50 gms of field grown, induced callus and in vitro propagated leaves of Crotalaria species were weighed separately and were grounded the tissue completely in ceramic crucible with liq. Nitrogen, to which 3 ml of cold acetone was added to each sample for the chlorophyll extraction. Each extracted sample was filtered through a Buchner funnel lined with Whatmann no. 1 filter paper which was previously moistened with cold acetone. The filtrates were collected in 100 ml volumetric flasks for each sample separately. Each sample filtrate was made up to 100 ml using cold acetone. These samples were then subjected to spectrophotometeric analysis which is calibrated with acetone. The absorbances of chlorophyll a and chlorophyll b were read at 622 nm and 645 nm respectively using pure acetone as blank. The estimated chlorophyll content for each sample was repeated five times and the data was recorded (Table - 30). The chlorophyll content for each of the samples was estimated using the following equations and their values are expressed in μg/g of dry weight:

Chla = 11.24 A622 - 2.04 A645

Chlb = 20.13 A645 - 2.04 A622

Chltotal = Chla + Chlb

Estimation of Metabolites

About 1 g each of field grown, friable callus and tissue culture grown plant materials were taken separately and homogenized with 80% ethanol in homogenizer and then made the final volume to 100 ml with 80% ethanol. The homogenate obtained were used for the quantitative estimation of metabolites.

Estimation of total sugars (Pushpa and Lakshman, 2011)

The total sugars in field grown, friable callus and tissue culture grown plant materials was estimated using standard Phenol-sulphuric method. 1 g of each samples were macerated with 5 ml of phosphate buffer in mortar and pestle and the homogenates were centrifuged at 8000 rpm for 20 minutes. The supernatants from each of the samples were collected in sterile test tubes. The extraction process was repeated for 4-5 times and made the final volume to 50 ml with distilled water.

1 ml of phenol reagent was added to all the sample aliquots followed by the rapid addition of 5 ml of concentrated sulphuric acid (H2SO4) using a burette. The contents were then mixed thoroughly. The absorbance of developed yellowish-orange colored samples against the phenol reagent blank was measured and recorded at 490 nm in UV-Visible spectrophotometer. The total sugar content was calculated by constructing the calibration curve by plotting the sugar concentration on X - axis and absorbance on Y - axis.

Calculation

The amount of total sugar present in each of the samples was expressed in mg/g of sample dry weight.

Estimation of Protein (Pushpa and Lakshman, 2011)

The total protein in field grown, friable callus and tissue culture grown plant materials was estimated using standard Bradford method (Bradford method, 1976). 1 g of each samples were macerated with 5 ml of phosphate buffer in mortar and pestle and the homogenates were centrifuged at 8000 rpm for 20 minutes. The supernatants from each of the samples were collected in sterile test tubes. The extraction process was repeated for 4-5 times and made the final volume to 50 ml with distilled water.

Preparation of Bradford reagent: 100 mg of Coomassie Brilliant Blue G-250 was dissolved in 50 ml of 95% ethanol and 100 ml of 85% phosphoric acid was added sequentially. The final volume of this mixture was made up 1000 ml with distilled water. The reagent was filtered through Whatmann no. 1 filter paper to remove undissolved particles and stored in cold condition.

0.1 ml of sample solutions were taken and made up to 1 ml with 0.1 M phosphate buffer (pH 7.5). The aliquots of bovine serum albumin (BSA) were pipetted out into the test tubes for the preparation of standard curve and their volumes were made up to 1 ml with 0.1 M phosphate buffer (pH 7.5). 5 ml of Bradford reagent was added to all the tubes and mixed thoroughly. A tube with 1 ml of water instead of protein sample served as blank. The absorbance of colored samples and standard was measured and recorded at 595 nm in UV-Visible spectrophotometer against the blank. The total protein was estimated from the standard curve by plotting the protein concentration on X - axis and absorbance on Y - axis.

Calculation

The amount of total sugar present in each of the samples was expressed in mg/g or 100g of sample dry weight.

Estimation of total phenols

The total phenolic content in field grown, friable callus and tissue culture grown plant materials was estimated using standard Folin-Dennis method (Schanderi, 1970). 1 g of each samples were homogenized with acetone and kept overnight in a flask. The supernatants from each of the samples were collected in sterile test tubes and the residues left were extracted with acetone. The mixture was then filtered and centrifuged at 8000 rpm for 20 minutes. The supernatant obtained was made the final volume to 50 ml with distilled water and used for the estimation of phenolics.

Preparation of Folin-Dennis reagent: 100 g of sodium tungstate and 20 g phosphomolybdic acid was dissolved in 750 ml distilled water and 50 ml phosphoric acid was added sequentially. The mixture was refluxed for 2 h and made up to one liter with distilled water.

0.5 ml of all the sample extracts was made up to 7 ml with distilled water and 0.5 ml of Folin-Dennis reagent was added sequentially. 0.5 ml Folin-Dennis reagent was made up to 7.5 ml with distilled water acts as blank. 1 ml of sodium carbonate solution was then added and incubated for an hour for color development. The absorbance of colored sample mixtures was measured and recorded at 720 nm in UV-Visible spectrophotometer against the blank. The total phenolic content was estimated from the standard curve prepared and expressed in mg/g.

Statistical analysis

The extraction and estimation of metabolite content for each treatment in each set of experiments consists of 5 replicates and each experiment was repeated thrice. The data shown in Table - 30 represent the mean ± standard error of five independent experiments. The data was statistically analyzed by one-way analysis of variance (ANOVA) and the means were assessed by Tukey's test at 0.05% level of significance (P<0.05) using Statistical Package for Social Sciences (SPSS version 17.0).

Extraction

The field grown (wild), induced callus and in vitro regenerated plant materials of three Crotalaria species were shade dried for 7 days. About 50 g dried plant material was weighed for each of C.retusa, C. prostrate and C. medicaginea, which were powdered separately using mechanical grinder into fine powder. These powders were subjected to cold extraction with 98% petroleum ether (Merck, India), 98% chloroform (Merck, India), 95% ethanol (Merck, India) and distilled water for about 18-24 h each in the order of increasing polarity. The percentage extractives of the test samples were performed as per the conventional procedure (Prabhu et al., 2011). The condensed extracts were used for quantitative, qualitative and pharmacognostic evaluations.

Quantitative estimation of Secondary Metabolites

The presence of secondary metabolites in wild, callus and in vitro propagated plants of Crotalaria species were quantitatively determined by adopting standard protocols. Alkaloids were estimated using Ikan's method (Ikan, 1981), flavonoids using Swain and Hillis method (Swain and Hillis, 1959), phenols using Bray and Thorpe method (Akharaiyi and Bolatito, 2010), tannins using Folin-Denis method (Schanderi, 1970) and saponins using Sanchez method (Nishanthi et al., 2012).

Qualitative estimation of Secondary Metabolites

All the extracts were tested with suitable reagents to unfold the diverse classes of chemical constituents present, and then the results were tabulated (Table - 31). Non-polar solvent extracts (petroleum-ether) were tested for the presence of phyto-sterols, triterpenes, and the polar solvent fractions were tested for the presence of alkaloids, phenolic compounds such as flavonoids, procyanidine, tannins and phenolic glycosides, saponins and free reducing sugars. The extracts of all the three samples of Crotalaria were subjected to preliminary phytochemical analysis using appropriate chemicals and reagents followed by thin layer chromatographic screening (Prabhu et al., 2011; Harborne, 2005).

Test for Phenolics (Harborne, 2005)

1 ml of each of the concentrated extracts were heated to remove the solvent and the residues were taken in a little of aqueous methanol (Merck, India). 0.5% ferric chloride solution was added to the methanolic solution and the change in color was marked in alcoholic extract indicating the presence of phenolic compounds.

Test for Sterols and Tritepenes (Abdullahi et al., 2010)

10 ml of each of the concentrated extracts were evaporated to dryness under vaccum and the residue was saponified by refluxing with 0.5 N alcoholic potassium hydroxide (Qualigen, India) for two and half hours. Alcohol was evaporated, the residue diluted with excess of water and the contents were extracted with ether several times. The combined ether extracts were washed freely with distilled water, dried over fused calcium chloride (Himedia, India) and filtered. The ether was distilled off completely and the residues were subjected to Salkowski reaction.

Test for Flavonoids (Trease and Evans, 2002)

Shinoda test

The extracts were added with few magnesium turnings and concentrated hydrochloric acid drop wise, pink scarlet, crimson red or occasionally green to blue color appeared after a few minutes indicates the presence of flavonoids.

Ferric chloride test

The extracts were added with few drops of 10% ferric chloride solution. The presence of phenolic compounds was identified by the development of green-blue or violet colour.

Lead ethanoate test

The extracts were added with 3 ml of lead ethanoate solution. The presence of phenolic compounds was identified by the formation of buff-coloured precipitate.

Test for Glycosides (Ayoola et al., 2008)

5 ml of each of the concentrated extracts were evaporated to dryness, the residue treated with hot water and filtered. 2 ml of 20 % (w/v) aqueous lead acetate solution was added for the precipitation of tannins. The excess of lead from the filtrate was removed by passing hydrogen sulphide gas (Himedia, India). The precipitate of lead sulphide was removed by filtration and excess of hydrogen sulphide was removed by heating the clear filtrate. The filtrate was tested with Fehling's solution for the presence of reducing sugars.

Test for Saponins (Harborne, 1973)

Froth formation test

2 ml of each test sample was placed in a test tube containing water, shaken well, stable froth (foam) appeared and stable for about 30 min indicates the presence of saponins.

Test for Tannins (Ayoola et al., 2008)

A few drops of 0.1% ferric chloride solution (Sigma-Aldrich, India) was added to the extracts and observed for brownish green or a blue-black coloration, which refluxes the presence of tannins.

Test for Alkaloids (Prabhu et al., 2011; Sofowora, 1993; Mattocks and Jukes, 1987)

About 1 ml of each of the concentrated extracts was evaporated to dryness at a controlled temperature and then the residue was treated with 5% hydrochloric acid (Merck, India) and filtered. The filtrates were tested with different reagents such as Mayer's, Dragendroff's, Wagner's and Ehrlich's reagents.

Preparation of chromatographic plates

30 g of Silica gel - G (Fischer Scientific Pvt Ltd) was dissolved in 100 ml of distilled water and mixed thoroughly. The prepared slurry was used for coating a thoroughly cleaned TLC (Thin Layer Chromatography) plates (5 X 15 cm) having a thickness of 0.01 - 0.02 inches using a spreader. The prepared TLC plates were subjected to activation by heating them at 1000C for 30 minutes in hot air oven before commencing the separation. The sample was spotted on TLC plates using capillary tube.

Separation of Secondary Metabolites by thin layer chromatography (TLC)

The secondary metabolites extracted from Crotalaria were qualitatively separated by TLC using Silica gel - G (Fischer Scientific Pvt Ltd) coated on glass plate.

TLC study of alkaloids (Wagner and Bladt, 1996)

The extracted fractions of wild, callus and in vitro propagated Crotalaria species were wetted with half diluted ammonium hydroxide (NH4OH) and lixiviated with ethyl acetate (EtOAc) for 24 hours at room temperature. The organic phase is separated from the acidified filtrate and basified with NH4OH (pH 11.0-12.0). These phytochemicals were extracted with chloroform (3X), condensed by evaporation and then used for chromatography. The alkaloid spots were separated using the solvent mixture chloroform and methanol (3:1). The colour and hRf values of the separated alkaloids were recorded under both ultraviolet (345 nm) and visible light after spraying Dragendorff's reagent.

TLC study of flavonoids (Wagner and Bladt, 1996)

The methanolic extracts of all the samples of Crotalaria species were condensed by evaporation and used for chromatography. Later the flavonoid spots were separated using chloroform and methanol (9:1) solvent mixture. The colour and hRf values of these spots were recorded under ultraviolet (345 nm) light.

TLC study of phenols (Harborne, 1998)

The methanolic extracts of three Crotalaria species were used for chromatography. The phenols were separated using chloroform and methanol (27:0.3) solvent mixture. The colour and hRf values of these phenols were recorded under visible light after spraying Folin-Ciocalteu's reagent at 80oC/10min).

TLC study of saponins (Wagner and Bladt, 1996)

The aqueous extracts of all the samples of Crotalaria species were enriched with saturated n-Butanol, and thoroughly mixed. The n-Butanol fraction of the mixture was condensed and used for chromatography. The saponins were separated using chloroform: acetone (1:1) solvent mixture. The colour and hRf values of these spots were recorded by exposing chromatogram to the iodine vapors.

TLC study of Tannins (Porter et al., 1986)

All the Condensed extracts of Crotalaria species were determined by oxidation of Condensed-Tannin with n-Butanol-Hydrochloric acid reagent in the presence of iron and the tannins was estimated.

Chromatogram Development

The solvent system used for pyrrolizidine alkaloid separation was methanol: chloroform: liq. Ammonia (8:1:0.5). The crude samples were loaded on Silica gel - G TLC plates and placed vertically in the TLC chamber containing suitable solvent mixture. The developed chromatogram plates was removed from the glass chamber and air-dried after 3/4th run of plate length. The Rf values of the developed chromatogram was recorded and compared with the reference crotaline (Sigma-Aldrich, USA) (Table - 32-33). These Rf values were calculated using the following formulae:

Distance traveled by the solute

Rf = ___________________________ X 100

Distance traveled by the solvent

Statistical analysis

All the qualitative screening of the metabolites was conducted in 5 replicates and each experiment was repeated thrice. The data was recorded and the means were assessed by Tukey's test at 0.05% level of significance (P<0.05) using Statistical Package for Social Sciences (SPSS version 17.0).

Pharmacological studies

Antimicrobial activity disc diffusion method (Murugesan et al., 2011)

The extracts of both in vivo and in vitro grown plantlets were tested in present investigation for antibacterial (Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris, Bacillus subtilis and Eschericia coli) activities by disk diffusion method.

Preparation of Nutrient Agar (NA)

5.0 g - Peptone, 3.0 g - Beef Extract, 5.0 g - NaCl, g and 15.0 g - Agar was dissolved in 1000 ml of distilled water and sterilized in autoclave at 1210C at 15 lbs pressure for 20 minutes. The sterilized nutrient agar was cooled to 450C and dispensed into 5 small conical flasks. The inoculums of 5 different pathogen cultures were added into 5 conical flasks separately. These inoculated NAs contain an inoculum size of 106 colony-forming units (CFU)/mL each was poured into sterile petriplates separately.

Now the filter paper discs (6mm in diameter), individually impregnated with 25 µL of extract at concentration of 100 mg/mL was placed on the agar plates previously inoculated with test microorganisms. Similarly, each plate carried a blank disc by adding methanol solvent alone in the center to serve as a negative control and antibiotic discs (6 mm in diameter) of 30 µ g/mL of Rifampicin (Sigma-Aldrich, India) was used as positive control. All the plates were incubated at 37 ± 2oC for 24 h for bacterial growth. The diameters of the inhibition zones were measured in millimeters and recorded (Table - 34). The sensitivity of the microorganisms to the extract was determined by measuring the size of inhibitory zones on the agar surface around the discs.

Determination of antioxidant activity (Ayoola et al., 2008; Sangeetha et al., 2008)

The quantitative measurement of free radical scavenging activities of the extracts of Crotalaria species was carried out in a universal bottle. Each reaction mixture contained 50 µL of test sample with concentration ranging from 20, 40, 60, 80 and 100 µg/ml in methanol and 5 mL of 0.004% (w/v) of 2,2- diphenyl-1-picrylhydrazyl (DPPH) radical solution (Sigma-Aldrich, India) in methanol. The Vitamin C (Sigma-Aldrich, India) was used as a positive control. The discoloration as measured at 517 nm after the incubation for 30 min in the dark condition. The 80% (v/v) methanol was used as a blank and DPPH (in 80% MeOH) used as control. Measurements were taken in triplicate and recorded (Table - 35). The DPPH radical concentration was calculated using the following equation:

DPPH scavenging effect (%) = Ao - A1/Ao X 100

Where 'Ao' was the absorbance of the control and 'A1' was the absorbance of the tested sample (crude extracts) in DPPH. The degree of discoloration indicates the free radical scavenging efficiency of the substances.

Statistical analysis

All the pharmacognostic evaluations were conducted thrice. The data was recorded as mean ± S.E and the means were assessed by Tukey's test at 0.05% level of significance (P<0.05) using Statistical Package for Social Sciences (SPSS version 17.0).

Purification of Alkaloid

As the previous experiments conducted represented that Crotalaria retusa is prominent source in higher accumulation of pyrrolizidine alkaloids, we are aimed to purify and analyze the putative constituent through the analytical techniques. In the above experiment these were qualitatively purified through TLC. The purified phyto chemical fractions of Crotalaria retusa were analyzed and identified by HPLC, LC-MS and NMR spectrometry.

Plant material

Leaves of Crotalaria retusa L. were collected in Rushikonda area, Visakhapatnam, India, in March 2012, and identified by Dr. Lakshminarayana, Andhra University. A voucher specimen (A.U. (B.D.H.) 5527) was deposited at the Herbarium of the Department of Botany, Andhra University, India.

Extraction

Aerial parts of Crotalaria retusa (Fabaceae) were obtained and cut into small pieces of 0.5 cm in size and air dried for a week. Air-dried plant material was then ground into coarse powder in a cross beater mill equipped with a 1 mm sieve. An aliquot (500g) powdered plant material was extracted with HPLC grade methanol (3 X 1000 mL) in an air tight bottle for 72 hrs at room temperature with occasional shaking. The extract was then filtered using Whatmann's filter paper. The filtrate was condensed through evaporation under reduced pressure at 35-400C. The crude extract (28.9g) obtained was dissolved in 10 ml methanol and then filtered before subjecting to HPLC for the purification of desired putative compound.

HPLC analysis

All the chromatographic separations were performed using Hypersil C8 column chromatography on silica gel (250 X 4.6 mm, 5 μm for HPLC-MS) at room temperature. The mobile phase consisted of methanol, acetonitrile and water. The elution profile was 0-30 min, linear gradient from 30% - 70% and the system was let to stabilize for 10 min between consecutive injections. The flow rate was adjusted to 5 mL/min with an injection volume of 500 µL and the effluent was monitored at 254 nm. Fractions of 100 mL were collected and monitored by TLC with respect to standard Crotaline (Rt = 24.2 min). The specified fractions of 5-7 (500-700 mL, 3.5g) were combined and separated on a silica gel column.

Analytical HPLC

The purified alkaloid fraction was analyzed through analytical HPLC to check their level of purity. The initial conditions for HPLC analysis were maintained accordingly (Amakura et al., 2000). Analysis was carried out with FD 2010 (Shimadzu, Japan) series using 4.6 mm X 250 mm C18 column having a particle size of 5µm with a flow rate of 0.8 mL/min with an injection volume of 5 µL. The gradient system was maintained for eluting the sample was methanol: acetonitrile in 7:3 (pH 5.6) and these effluents were monitored at 254 nm with Photo Diode Array (PDA) detector. The mobile phase was filtered (0.22 µm) and degassed with helium before use. The eluates were collected using an automated fraction collector. The confirmatory analysis was conducted with 99.0 % pure standard Crotaline injection. The structural properties of pyrrolizidine alkaloid were confirmed using LC-MS and 1H and 13C NMR spectroscopic analysis.

Preparation of standard sample

An analytical sample was prepared by diluting an aliquot (80 µL) of the stock sample with an aliquot of (20µL) of a solution of Bromine (5µg/ml) as an internal standard to normalize the injection process for LCMS. An aliquot (2µL) of the analytical sample was injected on to the HPLC column.

Analysis of HPLC- Electro Spray Ionization (ESI) MS experiments

This was performed on Shimadzu HPLC auto sampler system coupled to Shimadzu LCMS-2010A ESI (Electron Spray Ionization) Mass spectrometer (Shimadzu, Japan). The sheath and auxillary nitrogen gas acts as nebulizer gas with flow ratio of 70:30. The Shimadzu HPLC instrument equipped with a Luna C18 reverse phase column (250mm X 4.6mm, 5µ), an LC-20AT pump and UV/Visible detector SPD-20A (254 nm), for the analysis. A 20 µl Hamilton injection syringe was engaged with the system. The system was controlled by a PE Sciex Mass Chrom data system (version 1.1.1). The sample was analyzed with operational conditions for the ion spray interface having MS mode with 200-1000 u, step size with 10 u and dwell time 5 min. LC-ESI-MS experiments were performed online directly after LC separation. The MS spectra were considered to be significance for signal-to-noise ratio higher than 5.0. The separation of crude sample was performed at a flow rate of 1 ml/min under uniform conditions. MS spectra were recorded in positive ionization mode and analyzed with a capillary temperature and voltage of 2250C and 43 V respectively in second spectral scan. The selected daughter ions from initial MS fragmentation were selected for further fragmentation.

Nuclear Magnetic Resonance spectrometry (NMR)

The HPLC purified fraction was dissolved in 7mL of 99.9% CDCl3 (Deuteriated Chloroform) for recording of NMR spectra using Bruker Avance 400 Ultra-shield - AV 400 (Bruker BioSpin, Germany) NMR spectrometer with 400MHz magnet with QNP probe (5mm) for the determination of structure for the putative compound using 1H and 13C NMR. The solvent and Tetramethylsilane (TMS) were used as the internal standards for 13C and 1H signals respectively. The chemical shift values for both 13C and 1H NMR signals were recorded for the elucidation of the structure for the targeted putative compound.

Statistical analysis

All the data was recorded thrice for each experiment and evaluated through the Linear regression analysis (R2 ≤ 1).

In silico toxicological studies

The structure of the putative compound was deduced and identified as monocrotaline by comparison with the literature. The confirmed monocrotaline structure was subjected to structure based in silico toxicological studies using standard protocols.

In silico Toxicological studies (Farhan et al., 2009)

The structure of putative compound determined through above analytical techniques (LC-MS and NMR) was subjected to in silico toxicological studies because of their toxicity. The present deciphered structure mimics the standard Crotaline (Sigma-Aldrich). The present investigation focused on predictions for the toxicity and medicinal properties of the deciphered putative molecule.

The putative pyrrolizidine alkaloid structure depiction was done by 2D drawing system using Chemaxon's Marvin Sketch java applet as chemical drawing plug-in in UM-BBD online tool (http://umbbd.msi.umn.edu/predict/). The structure was modified without loosing their biological properties for the reduction of toxicity. Nearly 25 analogues were evaluated for different parameters viz., activity and toxicity using Bioinformatics/ In silico tools.

OECD QSAR Tool Box 2.0 (Milon, 2005)

The main objective of the Toolbox is to allow the user to apply QSAR methodologies to group chemicals into categories and to fill data gaps by read-across, trend analysis and QSARs. The Toolbox has multiple functionalities allowing the user to perform a number of operations, which include:

Identifying the analogues for chemical retrieve experimental results available for those analogues and fill data gaps by read-across or trend analysis.

Categorizing large inventories of chemicals according to mechanisms or modes of action.

Filling data gaps for any chemical by using library of QSAR models.

Evaluating the robustness of a potential analogue for read-across.

Evaluating the appropriateness of a QSAR model for filling a data gap for a particular target chemical.

Building QSAR models.

OECD QSAR Tool box is employed to predict different properties (bioavailability, bioaccumulation, biodegradation fragments etc.,) of all the modified analogues of the structurally predicted putative toxic molecule.

LAZAR (Christoph, 2006)

LAZAR (http://lazar.in-silico.de/predict/) derives its predictions from databases with experimentally determined toxicity data. To make a prediction for a new structure, LAZAR searches in one of these databases for compounds with similar structures (neighbors) and calculates the prediction from their measured activities. The most important feature of LAZAR is that the chemical similarities are always determined with respect to a given toxic activity. The confidence index and activity of the modified analogues of the putative toxic molecule is predicted with LAZAR.

ToxPredict (Ekins et al., 2002)

ToxPredict (http://apps.ideaconsult.net:8080/ToxPredict/) estimates the chemical hazardness of structures. It relies on OpenTox API-v1.1 compliant RESTful web services. Users can either search the OpenTox prototype database, which includes currently quality labeled data for 186912 chemicals 580651 structures, grouped in 7988 datasets, or upload their own chemical structure data. ToxPredict provides access to 19 ready to use models, addressing 24 different endpoints. ToxPredict estimates the hazardness of chemical structures and different stoichiometric values like the xlogP, acute toxicity, pKa, cell permeability and molecular weight. Predictive toxicology studies of analogues were carried to check the chemical hazardness by ToxPredict.

PASS online (Lagunin et al., 2011)

Prediction of Activity Spectra for Substances (PASS) online (http://www.pharmaexpert.ru/passonline/) is designed to evaluate the general biological potential of an organic drug-like molecule. It provides simultaneous predictions of many types of biological activities based on the structure of organic compounds. The Biological Activity Spectrum of a chemical compound is the set of different types of biological activity that reflect the results of the compound's interaction with various biological entities. PASS online gives various facets of the biological action of a compound.

Pa (probability "to be active") estimates the chance that the studied compound is belonging to the sub-class of active compounds.

Pi (probability "to be inactive") estimates the chance that the studied compound is belonging to the sub-class of inactive compounds.

PASS gives hits in the following:

Finding most probable new leads with required activity spectra among the compounds from in-house and commercial data bases.

Revealing new effects and mechanism of action for the old substances in corporate and private data bases.

Determining the assays that are more relevant for a particular compound.

PASS online predicts the biological activity spectrum for the modified analogues on the basis of its structural formula, along with different descriptors like antineoplastic. hepatotoxic etc., so it is possible to estimate if new compounds have a particular effect.

OSIRIS (Sangamwar et al., 2007)

Toxicity risk assessment (http://www.organic-chemistry.org/prog/peo/): while drawing a structure, the toxicity risk predictor will start looking for potential toxicity risks as long as the currently drawn structure is a valid chemical entity. Toxicity risk alerts are an indication that the drawn structure may be harmful concerning the risk category specified. Risk alerts are by no means meant to be a fully reliable toxicity prediction nor should be concluded from the absence of risk alerts that a particular substance is completely free of any toxic effect.

The prediction process relies on a precomputed set of structural fragment that gives rise to toxicity alerts, in case they are encountered in structure currently drawn. The OSIRIS toxicity predictions resulted for mutagenicity, tumorigenicity, irritability, reproductive effectiveness, cLogP value, druglikeness and drug-score of each analogue.

UM-BBD Pathway Prediction System (Hou et al., 2003)

The UM-BBD Pathway Prediction System (http://umbbd.msi.umn.edu/predict/) predicts microbial catabolic reactions using substructure searching, a rule-base, and atom-to-atom mapping. The system is able to recognize organic functional groups found in a compound and predict transformations based on biotransformation rules. The biotransformation rules are based on reactions found in the UM-BBD database or in the scientific literature. Biotransformations are assigned aerobic likelihood by two or more biodegradation experts. Standard conditions assumed for aerobic biotransformations are: exposed to air, in moist soil or water, at neutral pH, 25°C with no competing or toxic other compounds.

PPS predictions are most accurate for compounds that are:

Similar to compounds whose biodegradation pathways are reported in the scientific literature

In environments exposed to air, in moist soil or water, at moderate temperatures and pH, with no competing chemicals or toxins

The sole source of energy, carbon, nitrogen, or other essential element for the microbes in these environments, rather than present in trace amounts.

All the analogues including the structurally predicted putative toxic molecule are subjected to UM-BBD Pathway Prediction System to predict the plausible pathways for microbial degradation of chemical compounds.

Statistical analysis

The data shown represent the mean ± standard error of five independent experiments. The data was statistically analyzed by one-way analysis of variance (ANOVA) and the means were assessed by Duncan's Multiple Range Test (DMRT) at 0.05% level of significance (P<0.05) using Statistical Package for Social Sciences (SPSS version 17.0).

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