The plant material consists of dried powdered seeds of Abrus precatorius Linn. Fabaceae and leaves of Acyranthus aspera Linn.Amaranthaceae.
The seeds of Abrus precatorius Linn. and leaves of Acyranthus aspera Linn. were collected from Pachai hills, Salem, Tamilnadu, India during the month of June 2010. The plant was identified and authenticated by Mr. G.V.S. Murthy, Joint Director, C-I/C, Botanical survey of India, Tamil Nadu Agricultural University Campus, Coimbatore bearing the reference number BSI/SRC/5/23/10-11/Tech-708 and BSI/SRC/5/23/10-11/Tech-595 respectively
EXTRACTION PROCEDURE: Abrus precatorius The fresh seeds were shade dried, broken into small pieces and powered coarsely. About 600 gm of air dried powdered material was extracted with 99.9% of ethanol in a soxhlet extractor for 7 days. The extract was concentrated to dryness under reduced pressure and controlled temperature (40-50áµ’C).
Achyranthes aspera 25 g of shade dried, powder of plant materials were macerated separately with 250 ml of sterile distilled water at room temperature for 24 hrs after then using a mechanical shaker for 4 h. The extract was filtered through muslin cloth and the marc was again soaked with the same volume of water for 12 h and then further using a mechanical shaker for 4h and filtered. The filtrates were then combined concentrated under reduced pressure and evaporated at 400C.
DRUGS AND CHEMICALS:
Glucose and vitamin E were obtained from SD fine chemicals, Mumbai. 1-amino 2-naphthol-4-sulfonic acid, 5, 5-dithiobis-(-2-nitrobenzoic acid) (DTNB), Nitroblue tetrazolium chloride (NBT), Nicotinamide adenine dinucleotide reduced salt (NADH), Nicotinamide adenine dinucleotide phosphate reduced tetra sodium salt (NADPH), Oxidised glutathione, Reduced glutathione, Adenosine-5'-triphophate (ATP) were obtained from Himedia Laboratories Ltd., Mumbai. Goat lenses were obtained from the slaughterhouse Coimbatore. All other chemicals used in the study were obtained commercially and were of analytical grade.
PHYTOCHEMICAL SCREENING
Chemical tests were carried out for the extracts of seeds of Abrus precatorius Linn. and leaves of Acyranthus aspera Linn. for the presence of phytochemical constituents (Trease and Evans, 2002).
TEST FOR TANNINS AND PHENOLICS
To the solution of the extract, 0.1% ferric chloride was added in drops and then observed for a formation of a bluish black or brownish green colouration.
TEST FOR SAPONINS
About 10 ml of the plant extract and 5 ml of water and shaken vigorously for a constant froth formation. About 3 drops of olive oil was further added o the frothing, mixed well and t with 3 drops of olive oil and then observed for the formation of an emulsion.
TEST FOR FLAVONOIDS
i) To a portion of the extract, concentrated Sulphuric acid was added. A yellow colouration observed indicates the presence of flavonoids. The yellow coloration disappeared on standing.
ii) Few drops of 1% Aluminum chloride solution was added to a portion of extract. A yellow coloration indicates the presence of flavonoids.
iii) A portion of the dried extract was heated for 3 min with 10ml ml of ethyl acetate and filtered. To 4ml of the filtrate around 1ml of dilute NH3 solution was added and shaken vigorously. A yellow coloration indicates a positive test for flavonoids.
TEST FOR TERPENOIDS
About 5 ml of the plant extract was mixed with 2 ml of Chloroform and 3 ml concentrated H2SO4 was carefully added test tube to form a layer. A reddish brown coloration of the interface formed shows the presence of terpenoids.
TEST FOR ALKALOIDS
A small portion of the extract was stirred with few drops of dil.Hydrochloric acid and filtered.
i) To the filtrate, Dragendorff's reagent (potassium bismuth iodide solution) was added and an orange brown precipitate indicates the presence of alkaloids.
ii) To the filtrate, Mayer's reagent was added and a cream precipitate indicates the presence of alkaloids.
IN VITRO EXPERIMENTAL MODEL OF CATARACT MECHANISM OF CATARACT FORMATION:
Polyol pathway
Under physiological conditions, the glucose is metabolized through the glycolytic pathway. An excess amount of glucose is converted to sorbitol by enzyme AR via polyol pathway. The glucose conversion into sorbitol by utilizing NADPH results in the reduction of NADPH/NADP+. Moreover, sorbitol undergoes oxidation to fructose by using sorbitol dehydrogenase(SD). Sorbitol does not easily cross cell membrane. Intra lenticular accumulation of sorbital, it leads to the lens damage.
As, the lens begins to swell in response to the hyperosmotic effects of polyol accumulation, membrane permeability changes results in an increase of lenticular sodium and decrease the levels of lenticular potassium, reduced glutathione, ATP and free amino acids. The overall antioxidant status of the lens should be decreased because of depletion of GSH.
GLUCOSE INDUCED CATARACT:
Fresh goat eyeballs were obtained from slaughterhouse. The lenses were removed by extra capsular extraction and incubated in artificial aqueous humor (NaCl 140 mM, KCl 5 mM, MgCl2 2 mM, NaHCO3 0.5mM, NaH(PO4)2 0.5 mM, CaCl2 0.4 mM and Glucose 5.5 mM) at 37°C and pH 7.8 for 72 h. Glucose at a concentration of 55 mM was used to induce cataract . penicillin 32mg% and streptomycin 250% were added to the culture media to prevent bacterial contamination (Langade 2006).
EXPERIMENTAL PROTOCOL:
A total of 42 goat lenses were used and divided into seven experimental groups (6 in each group)
GROUP I : Artificial aqueous humor alone (solvent control) GROUP II : Glucose 55mM alone (negative control) GROUP III : Plant seed extract (100µg/ml) +glucose55mM GROUP IV : Plant seed extract (200µg/ml) +glucose55mM GROUP V : Plant leaves extract (100µg/ml) + glucose55mM GROUP VI : Plant leaves extract (200µg/ml) + glucose55mM GROUP VII : Vitamin E (100µg/ml) + glucose55mM
CALCIUM INDUCED CATARACT:
Fresh goat eyeballs were obtained from slaughterhouse. In order to induce lens opacity in vitro, fresh lenses were incubated for 16 hours at 37C in 10mM Cacl2 solution made in Tris-HCL buffer,0.01mM; pH-7.4. Penicillin 32 mg% and streptomycin 250 mg% were added to the culture media to prevent bacterial contamination (Rai DV, Kohli KS, 2006).
MECHANISM OF CATARACT FORMATION:
Calcium transport pathway
Increased levels of lenticular calcium have been activates calcium dependent proteases. The activated proteases could hydrolyze cytoskeletal proteins and lens crystallins. Crystalline cleavage would form lower molecular weight peptides that could, in turn, aggregate to form higher molecular weight proteins.
EXPERIMENTAL PROTOCOL:
A total of 42 goat lenses were used and divided into seven experimental groups (6 in each group)
GROUP I : Artificial aqueous humor alone (solvent control) GROUP II : Cacl210mM alone (negative control) GROUP III : Plant seed extract (100µg/ml) + Cacl210mM GROUP IV : Plant seed extract (200µg/ml) + Cacl210mM GROUP V : Plant leaves extract (100µg/ml) + Cacl210mM GROUP VI : Plant leaves extract (200µg/ml) + Cacl210mM GROUP VII : Vitamin E (100µg/ml) + Cacl210mM
PREPARATION OF LENS HOMOGENATE:
After incubation, homogenate of lenses (10%w/v) was prepared in Tris buffer (0.23mM, pH 7.8) containing 0.25x10-3M EDTA. The homogenate was centrifuged at 10,000 g for 1 h and the supernatant was used for estimation of total protein, malondialdehyde and lipid hydroperoxides and antioxidants activities.
METHODS:
INHIBITION OF Cu2+ INDUCED LIPOPROTEIN DIENE FORMATION:
Lens homogenate was diluted to 0.67% in phosphate buffered saline. Control experiments consisted of identical assay conditions but without the sample. Oxidation was initiated immediately after addition of sample by the addition of 12 μM final concentration of Cu2+ added as CuSO4_5H2O dissolved in deionized distilled water. Oxidation was determined by measuring the absorbance at 234 nm using a UV-Visible Spectrophotometer. Absorbance was taken after 120 mins at 37áµ’C.The lipoprotein diene formation was measured from the absorbance at a time. The absorbance provides an indication of protection of tissue lipoprotein against oxidation (Hodyson et al.,1999).
ASSAY OF Ca2+-ATPASE ACTIVITY:
To added 0.1 ml of the lens homogenate (10% (w/ v) in 0.25 M sucrose) and add 0.2 ml of the substrate, ATP and incubated for 30 min. The enzyme activity is blocked by adding 2 ml of 10% TCA. Then 0.2 ml of ATP is added and the same hold in ice bath for 20 min. Every tube was centrifuged at 2500 rpm for 10 min and the supernatant collected. The protein free supernatant was analyzed for inorganic phosphate. For that 3 ml of the supernatant was mixed with 1 ml of ammonium molybdate and 0.4 ml 1-amino 2-naphthol-4-sulfonic acid (ANSA). The colour solution was read at 680 nm after 20 min and the inorganic phosphate value expressed as nm five parallel experiments were conducted (Kleinzeller, 1974).
ESTIMATION OF PROTEIN VALUE:
To 0.1 ml of lens homogenate, 4.0ml of alkaline copper solution was added and allowed to stand for 10min. Then, 0.4ml of phenol reagent was added very rapidly and mixed quickly and incubated in room temperature for 30mins for colour development. Reading was taken against blank prepared with distilled water at 610 nm in UV-visible spectrophotometer. The protein content was calculated from standard curve prepared with bovin serum albumin and expressed as µg/mg lens tissue (Lowry et al., 1951).
ESTIMATION OF LIPID HYDROPEROXIDES:
About 0.1ml of lens homogenate was treated with 0.9ml of Fox reagent (7.6 mg xylenol orange, 9.8 ammonium ion sulphate, 188 mg butylated hydroxytoluene(BHT) and were treated into 90ml of methanol and 10ml 250mM sulphuric acid) and incubated for 3 min. The colour developed was read at 560nm using a colorimeter. The values are expressed as nmoles/ mg lens protein (Nieshus and Samuelsson, 1986).
ESTIMATION OF MELONDIALDEHYDE (MDA)
Lenses were homogenized in10% (w/v) 0.1 M Tris-HCl buffer (pH 7.5). One milliliter of the lens homogenate was mixed with 2 ml of TCA-TBA-HCl reagent. This reagent contain 15% trichloroacetic acid (TCA) and 0.375% thiobarbituric acid (TBA) in 0.25 N HCl and boiled for 15 min. Precipitate was separate out after cooling by centrifugation at 1000g for 10 min and absorbance of the sample was read at 535 nm against a blank without lens homogenate. The values are expressed as nmoles of MDA/ min/ mg lens protein (Niehaus and Samuelsson, 1968).
DETERMINATION OF ENZYMATIC ANTIOXIDANTS:
ASSAY OF SOD ACTIVITY:
The assay mixture having 1.2 ml sodium pyrophosphate buffer (0.052 M, pH 8.3), 0.1 ml of 186 μM Phenazonium methosulphate (PMS), 0.3 ml of 300μM NBT, 0.2 ml of 780μM NADH, 1.0 ml homogenate (lens were homogenized in10% (w/v) 0.25 M sucrose buffer) and distilled water to a final volume of 3.0 ml. Reaction was intiated by the addition of NADH and kept at 30áµ’C for 1 min. The reaction was blocked by the addition of 1.0 ml glacial acetic acid and the solution stirred vigorously. 4.0 ml of n-butanol was added into the solution and stirred well. The soution kept in 10 min, centrifuged, the butanol layer separate out and the absorbance was taken at 560 nm against a butanol blank. A system devoid of enzyme serve as the control and five parallel experiments were conducted (Kakkar et al., 1984).
ASSAY OF CATALASE ACTIVITY:
The reaction mixture contained 2.0 ml of homogenate (lens were homogenized in 10% (w/v) 50 mM phosphate buffer, pH 7.0) and 1.0 ml of 30 mM hydrogen peroxide (in 50 mM phosphate buffer, pH 7.0). A system devoid of the substrate (hydrogen peroxide) served as the control. Reaction was initiated by the addition of the substrate and decrease in absorbance monitored at 240 nm for 30 s at 25áµ’C. The difference in absorbance per unit time was expressed as the activity and five parallel experiments were conducted. One unit is defined as the amount of enzyme required to decompose 1.0 mol of hydrogen peroxide per minute at pH 7.0 and 25áµ’C (Aebi, 1984).
ESTIMATION OF GLUTATIONE REDUCTASE:
The enzyme activity was determined spectrophotometrically by the reduce in absorbance of NADPH at 340 nm. The reaction mixture contained 2.1 ml of 0.25 mM, potassium phosphate buffer pH 7.6, 0.1ml of 0.001 M NADPH, 0.2ml of 0.0165M oxidised glutathione and 0.1 ml (10 mg/ml) of bovine serum albumin(BSA). The reaction was taking place by the addition of 0.02 ml of lens homogenate with mixing and the decrease in absorbance at 340nm was measured for 3 min against a blank. Glutathione activity was expressed as nmoles NADPH oxidized/min/ mg lens protein at 30áµ’C (Carlberg and Racker,1955).
ESTIMATION OF GLUTATIONE PEROXIDASE:
The reaction mixture consists of 0.2 ml of 0.4 M Tris buffer, 0.1 ml of sodium azide, 0.1 ml of hydrogen peroxide, 0.2 ml of glutathione and 0.2ml of lens homogenate supernatant incubated at 37áµ’C for 10 min. The reaction was stoped by the addition of 10% TCA and the absorbance was taken at 340nm. Activity was expressed as nmoles/min/mg lens protein (Tappel et al.,)
DETERMINATION OF NON ENZYMATIC ANTIOXIDANT:
ESTIMATION OF GSH CONTENT:
Lenses were homogenized in10% (w/v) cold 20 mM EDTA solution on ice. After deproteinization with 5% TCA, an aliquot of the supernatant was mixed with 150 μM DTNB [5, 5-dithiobis-(-2-nitrobenzoic acid)]. The product was identified and quantified spectrophotometrically at 416 nm. Pure GSH was used as a standard for finding the calibration curve and five parallel experiments were conducted (Ellman's reation, 1959).
STATISTICAL ANALYSIS:
Statistical analysis was carried out by using one-way analysis of variance (ANOVA) followed by Dunnett's test. Results are expressed as mean ± SEM of six lenses in each group. P values < 0.05 were considered significant.
