materials and methodsshodhganga.inflibnet.ac.in/bitstream/10603/37414/9/09... · 2018. 7. 2. ·...

Materials & methods

MATERIALS AND METHODS

1. ANIMALS

Male albino Wistar rats (8-12 week old), weighing 180-250 g, were procured from the Central Animal House Facility, Hamdard University, New Delhi. The animals were kept in polypropylene cages under standard laboratory conditions (12 h light and 12 h dark cycles) and had a free access to commercial pellet diet (Ashirwad Laboratory Animal feed, manufactured by Ashirwad Industries, Mohali, Punjab) and water ad libitum. The animal house temperature was maintained at 25 ± 2°C. The protocol was approved by tlie Institutional Animal Ethics Committee (lAEC) of

Hamdard University (Project no. 471, year: 2008), New Delhi.

2. DRUGS AND CHEMICALS

Amlodipine besylate

Enalapril raaleate

Propranolol hydrochloride

Trifluperazine hydrochloride

Acorus calamus Linn.

Cissampelos pareim Linn.

Asarone (~99%)

Bebeerine (-95%)

Bovine serum albumin

Bovine brain calcineurin

Glutathione reductase

Isoproterenol hydrochloride

L-thyroxine

Ranbaxy Laboratory, Gurgaon, India

Medley Pharmaceuticals LTD., Jammu, India

Cipla LTD., Solan, India

Torrent Phaniiaceuticals Limited, Baddi, India

Rawal HerMed Consultants, New Delhi, India

Rawal HerMed Consultants, New Delhi, India

Aldrich Sigma, St. Louis, LO, USA




Aldrich Sigma, St, Louis, LO, USA



All other chemicals used in this study were of analytical grade29

Materials & methods

3. BIGNOSTIC KITS

Angiotensin II

Tumor necrosis factor-a

Nitric oxide

Lactate dehydrogenase

BioVendor R & D, Brno, Czech Republic

R & D Systems, Inc., Minneapolis, USA

iNtRON biotechnology, Joongwon-Ku, Korea

Reckon diagnostics P.Ltd,, Baroda, India

4. EQUIPMENTS/APPARATUS

Electronic Balance

ELISA reader

Homogenizer

Incubator

Laboratoiy Centrifuge

Micropipette

Motic Digital Microscope

Noninvasive Blood Pressure 200A

pH meter

Spectrophotometer

Ultracentrifuge

Ultra low temperature freezer

UV Spectrophotometer

Water bath shaker

Mettler-Toledo India Private Ltd., Mumbai, India

Electronic Corporation of India Limited, Hyderabad, India

Remi, Mumbai, India

Hicon, Grover Enterprises, Delhi, India

Remi, Mumbai, India

Eppendrof, Hamburg, Germany

Motic, Barcelona, Spain

Biopac system, Inc., California, USA

Mettler-Toledo, Zurich, Switzerland

Systronics, New Delhi, India

Remi, Mumbai, India

New Brunswick Scientific, England, U.K.

Shimadzu, Tokyo, Japan

Remi, Mumbai, India

30

Materials & methods

S. PRELIMINARY STUDIES

Plant materials

The C. pareira roots and A. calamus rhizomes were procured from Rawal HerMed

Consultants, Ashok Vihar, New Delhi, India. The plant material was authenticated by Dr. H. B. Singh, Scientist F and Head, Raw Materials Herbarium and Museum Division, National Institute of Science Communication and Information Resoiu'ces

(NISCAIR) and a voucher specimen (Ref. No. NISCAIR/RHMD/Consult/2008-

09/1095/126 and 2010-11/1412/10) of collected samples were obtained. Samples were also deposited at NISCAIR, Pusa, New Delhi, India.

Preparation o f extracts

1. Ethanolic extract of Cissampelospareira

The roots of C. pareia were washed with distilled water to remove dirt and soil then dried and pulverized into diy powder. The root extract was prepared by refluxing it

with 70% ethanol (54°C for 72 h) in a Soxhlet apparatus, The extract was first distilled then evaporated on water batli to dryness. The dried extract was kept in a desiccator at

room temperature and expressed in tenns of dry weight. The dried extract contained 9.23 g/100 g of the starting crude material. The extract was resuspended in water and used in this study.

2. Ethanolic extract of A corus calamus

The rhizomes of A. calamus were washed with distilled water to remove dirt and soil then dried and pulverized into dry powder. The rhizome extract was prepared by refluxing it with 70% ethanol (54°C for 72 h) in a Soxhlet apparatus. The extract was

first distilled then evaporated on water bath to dryness. The dried extract was kept in a desiccator at room temperature and expressed in terms of dry weight. The dried extract contained 16.44 g/100 g of the starting crude material. The extract was resuspended in water and used in this study.

31

Materials & methods

Phytochemical analysis

The following phytochemical tests were performed to analyze the constituents present in the extract of herbal drugs (Trease and Evans, 1989):

Tests for alkaloids: Plant material (extract) 500 mg was treated with 500 ml of methanol for 20 min on a water bath. The extract was then filtered off and allowed to

cool. The extract was dispensed in 2 ml portions into four different test tubes. Dragendorffs, Hager's, Mayer's and Wagner's alkaloidal reagent were added separately to each tube; the presence or absence of any precipitate was noted in each test tube.

Test for amino acid: To 0.5 ml of drug extract a few drops of 1% ninhydrin, prepared in alcohol, were added. The development of blue or violet color indicated the presence of amino acids.

Borntrager's test for anthraquinone derivatives; To the extract of the herbal drug,1 ml of dilute ammonia (10%) was added and the mixture was shaken. Any change in color was recorded. A pink-red color in the ammoniacal (lower) layer showed antln'aquinone derivatives.

Test for essential oil; To 0.5 ml of herbal extract a few drops of 1 M alcoholic potassium diclaromate and phenolphthalein were mixed in a clean test tube. Formation

of soap indicated the presence of essential oil.

Fehling's test for reducing sugars (in glycosides): The residue (herbal drug extract) was redissolved in water on the water bath. In a test tube, 2 ml of this solution and 1 ml each of Fehling's solutions A and B were added. The mixture was then shaken and heated in a water bath for 10 min. Brick-red-colored precipitate formed, indicating the

presence of reducing sugars.

Test for flavonoids: The herbal drug extract was reduced to dryness on the boiling water bath. The residue was treated with dilute sodium hydroxide (NaOH), followed by addition of dilute hydrochloric acid (HCl); solubility and color was noted, A

yellow solution with NaOH, which turned colorless with dilute HCl, confirmed the

presence of flavonoids.

32

Materials & methods

Frothing test for saponins; one gram of the sample (extract of herbal drug) was weighed in a conical flask in which 10 ml of sterile distilled water was added and boiled for 5 min. The mixture was filtered and 2.5 ml of the filtrate was added to 10 ml of sterile distilled water in a test tube. The test tube was stoppered and shaken vigorously for about 30 sec. It was then allowed to stand for 30 min. Honeycomb froth indicated the presence of saponins.

Salwasld test for sterols: To 2 ml of ethanolic extract, a few drops of concentrated sulfuric acid (H2SO4) were added. Formation of a purple ring at the upper surface of

the solution indicated the presence of sterols.

Test for steroids; Two milliliters of acetic anhydride was added to 0.5 g of the herbal extract with 2 ml of H2SO4. The color changed from violet to blue or green, which indicated the presence of steroids.

Ferric chloride solution test for tannins: The extract was treated with 15% ferric chloride test solution. The resultant color was noted. Development of blue color indicated the presence of condensed tannins and green color indicated the presence of hydrolysable tannins.

Salkowski test for terpenoids; To 0,2 g of herbal drug extract 2 ml of chloroform and concentrated H2SO4 were carefully added to form a layer. A reddish-brown color of the interface formed, which indicated the presence of terpenoids.

High performance thin layer chromatographic analysis

A stock solution (1 mg/ml) of C. pareira root extract was prepared in methanol and analyzed by HPTLC (Camag). Bebeerine (-95%) which is the principal constituent of C. pareira root, was used as standard and the solution (0.1 mg/ml) was prepared in

methanol. Calibration curve was obtained for 10, 20, 30 and 40 ng concentrations of

bebeerine. The solvent system used was n-butanoI;glacial acetic acid.’water (6:2:2). The plates were scanned using TLC Scanner 3 (Camag) at 366 nm (fluorescence/reflectance mode).

33

Materials & methods

High performance liquid chromatographic analysis

C. pareira root extract was analyzed by HPLC (Shimadzu LC-10 AD VP) with photodiode array detector (SPD-MIOA VP). The chromatographic conditions were as follows; Column; Zorbax Eclipse XDB-C18 (3.5 p,m, 4.6 x 150 mm), guard column: Zorbax Eclipse XDB-C18 (5 4.6 x 12.5 mm). Then, 10 ia.1 of the sample wasinjected into SIL-IOAD VP auto sampler. The solvent system used was 68% 30 mM

ammonium acetate, 14 mM triethylamine (TEA) pH 4.85 and 32% acetonitrile. The flow rate was kept constant at 1 ml/min and mn time was 17 min. The column temperature was 3G°C and wave length 230 nm. Samples were prepared by dissolving1 mg of the dried material in 1 ml of acetoniti'ile and filtering through a 0.45 |am (nylon) filter.

Gas chromatography coupled to mass spectrometry (GC-MS) analysis

A. calamus rhizome extract was analyzed by gas chromatography equipped with mass spectrometry (GC-MS-Agilent). The chromatographic conditions were as follows:

Column: Zebron ZB-1 (length 30.0 m, diameter 0.25mm, film thickness 0.25 um). The

conditions for the GC were initial oven temperature of 40°C, injector 250°C, transfer line 280°C, a solvent delay of 2 min, the temperature was ramped at 10°C/min to a final temperature of 140°C and held for 1 rain.

6. EXPERIMENTAL DESIGN

Prepration o f drug solutions■ C. pareira'. Weighed amount of the extract was dissolved in doubled distilled

water.■ A. calamus'. Weighed amount of the extract was dissolved in doubled distilled

water.■ Amlodipine besylate: The drug solution was prepared by dissolving the

weighed amount of it in doubled distilled water.■ Propranolol hydrochloride; The drug solution was prepared by dissolving the

weighed amount of it in doubled distilled water.■ Enalapril maleate: The drug solution was prepared by dissolving the weighed

amount of ft in doubled distilled water.34

Materials & methods

Isoproterenol hydrocholoride: The weighed amount of drug was dissolved in nonmal saline.

L-thyroxine: The weighed amount of drug was dissolved in O.OI mol/L sodium hydroxide (prepared in normal saline) solution.

Experimental protocol

Model I

Protocol I: Influence of C. pareira extract on isoproterenol-induced cardiac hypertrophy in rats.

Groups Treatment given

C Normal saline (0.5 ml/kg/day, i.p., single dose) was given for 30 days and served as control

ISO Isoproterenol (5 mg/kg/day, i.p., single dose) was given for 30 days

CIS50 C. pareira (50 mg/kg/day, by gavage, single dose) alone was given for 30 days

CIS 100 C. pareira (100 mg/kg/day, by gavage, single dose) alone was given for 30 days

CIS200 C. pareira (200 mg/kg/day, by gavage, single dose) alone was given for 30 days

AML Amlodipuie (9 mg/kg/day, by gavage, single dose) alone was given for 30 days

CIS50+ISO C. pareira (50 mg/kg/day) was given along with isoproterenol for 30 days

CISIOO + ISO C. pareira (100 mg/kg/day) was given along with isoproterenol for 30 days

CIS200 + ISO C. pareira (200 mg/kg/day) was given along with isoproterenol for 30 days

AML + ISO Amlodipine (9 mg/kg/day) was given along with isoproterenol for 30 days

No. of animals in each group was 8 except ISO group which contained 10.

35

Materials & methods

Protocol II: Influence of A. calamus extract on isoproterenol-induced cardiac hypertrophy in rats.

Groups Treatment givenAC050 A. calamus (50 mg/ltg/day, by gavage, single dose) alone was given

for 30 days

ACOlOO A. calamus (100 rag/kg/day, by gavage, single dose) alone was given for 30 days

AC0200 A. calamus (200 mg/kg/day, by gavage, single dose) alone was given for 30 days

AC050 + ISO A. calamus (50 rag/kg/day) was given along with isoproterenol for 30 days

ACOlOO + ISO A. calamus (100 mg/kg/day) was given along with isoproterenol for 30 days

ACO200 + ISO A. calamus (200 mg/kg/day) was given along with isoproterenol for 30 days

No. of anijTiflls in each group was 8.

Model II

Protocol III: Influence of C. pareira extract on thyroxine-induced cardiac hypertrophy in rats.


C Normal saline (0,5 ml/kg/day, i.p., single dose in 0.01 mol/L sodium hydroxide) was given for 30 days and served as control

T4 Thyroxine (0.1 mg/kg/day, i.p., single dose) was given for 30 days

CIS50 + T4 C. pareira (50 mg/kg/day) was given along with thyroxine for 30 days



AML + T4 Amlodipine (9 mg/kg/day) was given along with thyroxine for 30 days

No. of animats in each group was 8 except T4 group which contained 10.

36

Materials & methods

Protocol IV: Influence A. calamus extract on thyroxine-induced cardiac hypertrophy in rats.


AC050 + T4 A. calamus (50 mg/kg/day) was given along with thyroxine for 30 days



No. of animals in each group was 8.

Protocol V: Influence of propranolol and enalapril on thyroxine-induced cardiac hypei'trophy in rats.


C Normal saline (0.5 ml/kg/day, i,p., single dose in 0.01 mol/L sodium hydroxide) was given for 30 days and served as control

T4 Thyroxine (0.1 mg/kg/day, i.p., single dose) was given for 30 days

PRO Propranolol (10 mg/kg/day, by gavage, single dose) alone was given for 30 days

ENA Enalapril (10 mg/kg/day, by gavage, single dose) alone was given for 30 days

PR0 + T4 Propranolol (10 mg/kg/day) was given along with thyroxine for 30 days

ENA + T4 Enalapril (10 mg/kg/day) was given along with tliyroxine for 30 days

No. of animals in each group was 6.

37

Materials & methods

7. METHODOLOGY

PARAMETERS ASSESSED

S. NO. PARAMETERS SAMPLE REFERENCE

HEMODYNAMIC MEASUREMENTS

1. Mean arterial pressure (MAP) - -

2. Heart rate (HR) - -

HYPERTROPHY ASSESSMENT

3. Heart weigh/body weight (HW/BW) ratio

- -

BIOCHEMICAL PARAMETERS

4. Angiotensin II (Ang II) Plasma Vollandetal., 1999

5. Tumor necrosis factor-a (TNF-a) Serum Seckinger et al., 1989

6. Calcineurin (CaN) activity Serum Padma and Subramanyam, 1999

7. Nitric oxide (NO) Serum Tracey et al., 1995

8. Lactate dehydrogenase (LDH) Senim Bergmeyer, 1965

9. NaVlC'-ATPase (NKA) Myocardium Akagawa and Tsukada, 1979

LIPID PEROXIDATION AND ANTIOXIDANT ACTIVITY

10. Thiobarbituric acid reactive substance (TEARS)

Serum Wright etal., 1981

11. Total antioxidant capacity (TAC) Serum Koracevic et al., 2001

12. Reduced glutathione (GSH) Serum Jollowetal,, 1974

13. Glutathione peroxidase (GPx) Myocai’dinm Mohandas etal., 1984

14. Glutathione reductase (OR) Myocardium Carlberg and Mannervik, 1975

15. Glutthione-S-transferase (GST) Myocardium Habig et al., 1974

16. Catalase (CAT) Myocardium Clairborae, 1985

17. Superoxide disrautase (SOD) Myocardium Marklund and Marklund, 1974

HISOPATHOLOGICAL ANALYSIS

38

Materials & methods

Pharmacological evaluations

Hemodynamic measurements

Mean arterial pressure (MAP) and heart rate (HR) of rats were recorded by tail-cuff method using Non-Invasive BP Instrument. The animals were trained in restraiaer for 15 min each day for 14 days before completion of treatment. Instrument was set according to the descriptions given in the manual. Before recording MAP, animals’ tail were cleaned and then kept in heating chamber at 37“C for 20 min. The rat was kept in restrainer and tail-cuff was placed on the tail. Pressure and pulse was recorded 7 times for each animal and mean was taken. MAP and HR were calculated by using computerized data acquisition system. MAP and HR were recorded 24 h after last dose of treatment.

Sample prepration

■ Serum prepration

The blood collected from each animal (approx. 2V4 times the volume needed for use) was incubated in an upright position at room temp for 1 h. Once the clot had retracted, the samples were centrifuged at 3,000 g for 20 min. The serum thus, obtained was

stored at -20®C till further use,

■ Plasma preparation (for angiotensin II)

The blood samples were collected in tubes kept on ice at 4°C, usually containing o- phenanthroline 0.44 mM, EDTA 25 mM, p-hydroxy-mercuribenzoic acid ImM and pepstatin A 0.12 mM. The samples were centrifuged at 3000 g for 20 min at 4®C. The

plasma thus, obtained was stored at -20°C,

■ Cardiac tissue homogenatepreparation

Immediately after the sacrifice, the hearts were excised out, washed in ice-cold normal saline and weighed. Subsequently, the weighed cardiac tissue from each group was homogenized (10% w/v) in ice-chilled phosphate buffer (50 mM or 0.1 M, pH 7.4), tris-HCl buffer (75 mM, pH 7.4) or potassium chloride (0.15 M) and centrifuged at

39

Materials & methods

10,000 g for 20 min in high speed coohng centrifuge at -4°C. Clear supernatant was

used for assaying different biochemical parameters according to the procedure.

Biochemical estimations

ESTIMATION IN PLASMA

■ Estimation o f angiotensin I I

Method: Volland et al., 1999

Principle: A specific monoclonal anti-angiotensin II is immobilized on a 96 well plate. After immunological reaction with Ang II and washing, the trapped molecule is

covalently linked to the plate by glutaraldehyde via amino groups. After washing and

denaturing treatment, Ang II can react again with the acetylcholinesterase (AChE)- labelled monoclonal antibody (mAb) used as tracer. The plate is then washed and substrate solution (enzymatic substrate for AChE and chromogen) is added to the wells. The AChE tracer acts on to fonn a yellow compound. The intensity of the color,

which is detennined by spectrophotometry, is proportional to the amount of tracer

bound to the well and is proportional to the amount of angiotensin II.

Reagents;

1. Dilution buffer: Reconstituted one vial with 50 ml of distilled or deionized water. Allowed it to stand 5 min until completely dissolved and then mixed thoroughly by gentle inversion. Stability at 4°C; 1 month.

2. Angiotensin II standai'd: Reconstituted the vial with 1 ml of distilled or deionized water in a polypropylene tube. Allowed it to stand 5 min until completely dissolved and then mixed thoroughly by gentle inversion. The concenQ-ation of the first standard was 125 pg/ml. Prepared seven propylene tubes (for the seven other standards) and added 500 (xl of dilution buffer into each tube. Added 500 nl of the first tube (containing the first standard) to the second tube. Continued this procedure for the other tubes. Thus, standard concentrations were: 125 (SI), 62.5 (S2), 31.25 (S3). 15.63 (S4), 7.81 (S5),

3.91 (S6), 1.95 (S7) and 0.98 pg/ml (S8), respectively.

40

Materials & methods

3. Quality control: Reconstituted one vial with 1 ml of distilled or deionized

water. Allowed it to stand 5 min until completely dissolved and then mixed thoroughly by gentle inversion.

4. Conjugate solution; Reconstituted one vial with 10 ml of dilution buffer.

Allowed it to stand 5 min until completely dissolved and then mixed thoroughly by gentle inversion. Stability at 4®C: 1 month.

5. Glutaraldehyde: Prepared 5 ml of diluted buffer by adding 0.125 ml of the wash solution concentrated in distilled or deionized water. Just before use added 100 of glutaraldehyde then mixed thoroughly by gentle inversion. Stability at 4°C; 24 h.

6. Borane trimethylamine: It was reconstituted with 5 ml of 2 N HCl/methanol (50/50, v/v) and then vortexed until completely dissolved.

7. Substi'ate solution: It was reconstituted with 1 ml of wash solution concentrated and 49 ml of distilled or deionized water and then mixed thoroughly. Stability at 4°C and in the dark: 4 days.

8. Wash buffer: Diluted 2 ml of wash solution concentrated to 0.8 L with distilled or deionized water. Added 400 jil of tween 20 and then mixed by using a magnetic stiiring. Stability at 4°C: 1 week.

Procedure:

1. The first column was left empty for blanking substrate solution.

2. Dispensed 100 |.il of dilution buffer in second column to non specific binding (NSB) wells.

3. Dispensed 100 |jl of Ang II standard of each of the eight standai-ds (SI to S8) in duplicate to appropriate wells (third and foxuth column). Started with tlie

lowest concentration standard (S8) and equilibrated the tip in the next higher standard before pipetting.

41

Materials & methods

4. Dispensed 100 jj,! of sample in duplicate to appropriate wells. Highly concentrated samples were diluted with dilution buffer.

5. Incubated the plate for 1 h at room temperature with gentle agitation.

6. Dispensed 50 |il of glutaraldehyde to each well except blank (B) wells and incubated for 5 min at room temperature with gentle agitation.

7. Added 50 fxl of borane-trimethylamine to each well except blank (B) wells and incubated for 5 min at room temperature with gentle agitation.

8. Washed each well 5 times with the wash buffer (300 fj,l/well), then removed the liquid from the wells by inverting tlie plate and shaking vigorously. Dispensed 100 1 of conjugate solution to each well except blank (B) wells, then covered the plate with a plastic film and incubated overnight at +4°C.

9. Washed each well 5 times with the wash buffer (300 ^1/well), then removedthe liquid from the wells by inverting the plate and shaking vigorously. Added 300 of wash buffer.

10. Incubated the plate for 10 min at room temperature with gentle agitation.

11. Washed each well 5 times with the wash buffer (300 (il/well), then emptied the plate by turning over and shaking, dispensed 200 |xl of substrate solution to 96

wells.

12. Incubated the plate in the dark (plate covered with an aluminium sheet) at room temperature and then optimal development was obtained using an orbital shaker. The plate was read between 405 and 414 nm (yellow color).

13. A standard curve was created by using curve-fitting software or by plotting a curve between the absorbance of each standard point (y axis) versus the concentration of tliat point (x axis). Concentration of samples were calculated by interpolation from the standard curve

42

Materials & methods

ESTIMATIONS IN SERUM

■ Estimation o f tumor necrosis factor-a

Method: Seckinger etal., 1989

Principle: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for rat TNF-a has been pre-coated onto a microplate. Standards, control and samples are pipetted into the wells and any rat TNF-a present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for rat TNF-a is

added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells. The enzyme reaction yields a blue product tliat turns yellow when the stop solution is added. The intensity of the color measured is in proportion to the amoimt of rat TNF-a bound in the initial step. The sample values are then read from tlie standard curve.

Reagents;

1. Rat TNF-ct kit control: Reconstituted the kit control with 1 ml distilled or deionized water. Assayed the control undiluted.

2. Wash buffer: 25 ml of wash buffer concentrate was diluted with distilled or deionized water up to 625 ml.

3. Substrate solution: Color reagents A and B were mixed together in equal

volumes within 15 min of use and protected from light. 100 |xl of the resultant

mixture was required per well.

4. Rat TNF-a standard: Reconstituted the rat TNF-a standard with 2 ml of calibrator diluent RD5-17. This reconstitution produced a stock solution of 800 pg/ml. Allowed the standard to sit for 5 min with gentle mixing prior to

making dilutions.

5. Polypropylene tubes: Pipetted 200 |j.1 of calibrator diluent RD5-17 into each tube. The stock solution was used to produce a dilution series 800 jig/ml, 400 |.ig/mi, 200 p.g/ml, 100 p.g/ml, 50 ng/ml, 25 |ig/ml, 12.5 Jig/ml, and 0 ^g/ml.

43

Materials & methods

Mixed each tube thoi'oughly before the next transfer. The undiluted rat TNF-a standard was served as the high standard (800 pg/ml). Calibrator diluent RD5- 17 was sei-ved as the zero standard (0 pg/ral).

Procedure:

1. Dispensed 50 p,l of assay diluent RDl-41 to each well.

2. Added 50 nl of standard, control and sample to each well and then mixed by gently tapping the plate frame for 1 min. Covered the plate with the adhesive strip and then incubated for 2 h at room temperature.

3. Aspirated each well and washed by filling each well with wash buffer (400 il) using a squirt bottle, manifold dispenser or autowasher, repeated the process 4

times for a total of 5 washes. Completely removed the liquid by aspirating or by inverting the plate and blotted it against clean paper towels.

4. Added 100 jj.1 of rat TNF-a conjugate to each well, covered with a new adhesive strip and then incubated the plate for 2 h at room temperature.

5. Aspirated each well and washed with wash buffer (400 nl), repeated the process 4 times for a total of 5 washes. Removed the liquid by aspirating or by

inverting the plate and blotted it against clean paper towels.

6. Added 100 |il of substrate solution to each well and then incubated for 30 min

at room temperature. The plate was protected firom light.

7. Added 100 |il of stop solution to each well. Gently taped the plate to ensure

thorough mixing.

8. Optical density of each well was detennined at 450 nm within 30 min, using a microplate reader (wavelength correction was set at 540 nm or 570 mn).

9. Averaged the duplicate readings for each standard, control and sample, and subtracted the average zero standard optical density. A standard curve was created by reducing the data using computer software capable of generating a

four parameter logistic (4-PL) curve-fit or by plotting a curve between the

44

Materials & methods

mean absorbance of each standard (y axix) versus the concentration (x axis).

Concentrations of samples were calculated by interpolation from the standard curve.

■ Estimation o f calcineutin activity

Method: Padma and Subramanyam, 1999

Principle: -calmodulin dependent phosphatase activity of calcineurin present inthe samples was assayed in presence and in absence of 150 jiM trifluoperazine.

Reagents:

1. Trifluoperazine (150 |aM): 6.66 mg of trifluoperazine was dissolved in 100 ml of ultra pure water.

2. Tris buffer (25 mM, pH 7.2): 302.50 mg of tris buffer was dissolved in 100 ml

of ultra pure water. pH adjusted to 7.2.

3. 2N-[Morpholino] ethane sulfonic acid (MES) (25 mM, pH 7.0): 488 mg of MES was dissolved in ICO ml of ultra pure water. pH adjusted to 7.0.

4. p-nitrophenyl phosphate (2.4 pM): 0.89076 x 10" mg of p-nitrophenyl

phosphate was dissolved in 1000 ml of ultra piu'e water.

5. Manganese chloride (MnCU) (1 mM): 0.1979 mg of MnCb was dissolved in

1000 ml of ultra pure water.

6. Dipotassium hydrogen phosphate (K2HPO4) (13% w/v): 13 g of K2HPO4 was

dissolved in 100 ml of double distilled water.

Preparation of calibration curve:

1. Prepared bovine brain calcineurin (BBC) stock solution of 0.5 mM

2. Performed serial dilutions of BBC to generate the calcineurin calibration curve (500, 250, 125, 62.5, 31.25, 15.63, 7.31 and 3.65 tiM). The final volume in each well was 30 |xl and the concentration range was 0-500 mM (0,5 mM).

45

Materials & methods

3. Added 25 ixl tris buffer, 25 MBS and 25 fxl MnClz followed by 25 1 p- nitrophenyl phosphate and left the solution at 30°C for 10 min.

4. Reaction was terminated by addition of 10 |j,l K2HPO4.

5. The optical density was measured at 405 nm using an FT ISA reader.

6. The calcineurin calibration curve was obtained by plotting a curve between absorbance (Y) versus concentration (X).

CALCINEURIN CALIBRATION CURVE

y = 0.0013x+0.0411 = 0.9989

♦ Series 1 — Linear (Series 1)

CONCENTRATION (U/dl)

Figure 8. Calibration curve of CaN (U/dl).

Procedure: Assays were conducted in a reaction mixture containing 25 fxl tris buffer

(25 inM, pH 7.2), 25 | 1 MES (25 mM, pH 7.0), 25 [aI p-nitrophenyl phosphate (2.4 HM) and 25 |il of 1 mM MnCk. 30 fil serum was added to the assay mixture. Reaction was initiated by addition of p-nitrophenyl phosphate, followed by incubation at 30°C for 10 min and terminated by addition of 10 jj.1 of 13% (w/v) K2HPO4. Samples were

then transferred to microplates and absorbance measured at 405 nm in an ELISA

reader,

46

Materials & methods

■ Estimation o f nitric oxide

Method: Tracey etal., 1995

Principle: NO is a radical compound produced during the transformation of L-arginine to citralline by NOS. The NO detection kit is based on diazotization (Griess method) assay, can detect in vitro NO’ concentration. The kit accurately detects the NO" concentration by indirectly measuring nitrite (NO2'), which is by-product of NO transfomiation in living tissue. The assay is based on the colorimetric change, which occui's when naphthylethylenediamine is added to the by-product of reaction between sulfanilamide and nitrite.

Reagents:

o N1 buffer (Substrate solution); Sulfanilamide in the reaction buffer

o N2 buffer (Coloring solution): Naphthylethylenediamine in the stabilizer buffer

o Nitiite standai-d, ImM

Preparation of standard curve:

1. Designated triplicates in the 96 well plates for the nitrite standard curve.

2. Dispensed 100 1 of ultra pure water into wells in rows B-H.

3. Added 200 jxl of 1 mM nitrite standard solution to the wells in row A.

4. Performed 6 serial 2-fold dilutions in triplicate to generate the nitrite standai'd reference curve (1000, 500, 250, 125, 62,5, 31.25 and 15.63 (jM). The nitrite solution was not added to the last set of wells, which is negative control (0 |jM). Tlie final volume in each well was 100 [jI and the nitrite concentration range was 0-1,000 [jM (1 mM).

5. Induced prereaction by adding 50 |al of N1 buffer to the samples from step 1 and

incubated for 5-10 min at room temp.

6. Added 50 )ol of N2 buffer and left the solution for 10 min at room temp.

7. The optical density was measured between 520-560 nm using a plate reader.

47

Materials & methods

8. The nitrite standard reference cmve was obtained by plotting the average absorbance value of each concentration of the nitrite standard as a function of “Y” with nitrite concentration as a fiinction of “X”.

0S*1C.{am

SlsMa dCurwa SaiTgtes

mQ

2S0125

3125ISiS

a

m m m o o a a a o a a am m m o o a a a o a a am m m o o o o o o o a o

8SS888888888^ ^ • o o o o o o o o o® ^ « o o o o o o o o o^ # ® o o o o o o o o o

AscDEfGH

Rgu« Ptete m fei tfie Mite standad WNt {Sk seifeil 2-tel diutiins)

NO

Linear (y ■ Ax + B)A»0.0028 B~0.1190, R-SquBre = 0,9739

* Standards♦ --------Suppressed Stds

--------- Standard curve

Figure 9. Calibration curve of NO ()xM).

Procedure of the assay performed:

1, Collected about 100 |jJ of samples and added the samples to wells in duplicate.48

Materials & methodsssssssssasasssssssssssaassBsas

2. Induced prereaction by adding 50 |il of N1 buffer to the samples from step 1 and incubated for 5-10 min at room temp.

3. Added 50 |j.l ofN2 buffer.

4. Left the solution for 10 min at room temp and measui'ed the optical density between 520-560 nra using a plate reader,

■ Estimation o f lactate dehydrogenase

Method: Bergmeyer, 1965

Principle: Lactate dehydrogenase (LDH) catalyses the oxidation of lactate to pyruvate accompanied by the simultaneous reduction of NAD" to NADH. LDH activity in serum is proportional to the increase in absorbance due to tlie reduction of NAD'''.

Reagents:

o 1 LDH (Coenzyme)

o 2 LDH (Buffer Substrate)

Preparation ofworldng reagent:

Reconstituted one vial of 1 LDH with 1.1 ml of 2 LDH, mixed to dissolved and used

after 5 min.

Procedure: 1 ml of working reagent was mixed with 0.05 ml of serum and

absorbance was taken at 340 nm exactly at 1 min and thereafter at 30, 60 and 90

seconds.

Calculation:

LDH activity (lU/L) = AA/min x F

Where,AA/min = change in absorbance per iTiin F = factor = 3376

49

Materials & methods

■ Determination o f total antioxidant capacity

Method: Koracevic et al., 2001

Principle: A standardized solution of Fe-EDTA complex reacts witli hydrogen peroxide by a Fenton type reaction, leading to the fonnation of hydroxyl radicals. These reactive oxygen species degrade benzoate, resulting in the release of tbiobarbituric acid reactive substance (TEARS). Antioxidants from the added sample of human fluid cause

suppression of the production of TBARS. This reaction can be measured spectrophotomefa-ically at 532 nm and the inhibition of color development is defined as

the total antioxidant capacity (TAG).

Reagents:

1. Sodium phosphate buffer (100 inM/L, pH 7.4); SOLUTION A: Dissolved 0.276 g monobasic sodium phosphate monohydrate in 20 ml double distilled water. SOLUTION B; Dissolved 1.42 g dibasic sodium phosphate in 100 ml double distilled water. Mixed 19 ml of solution A with 81 ml of solution B. pH adjusted to 7.4.

2. Sodium benzoate (10 mM/L): Dissolved 144 mg sodium benzoate in 100 ml

double distilled water.

3. NaOH (50 mM/L): Dissolved 500 mg NaOH in 250 ml double distilled water.

4. NaOH (5 mJWL): Dissolved 50 mg NaOH in 250 ml double distilled water.

5. SOLUTION 1: Ethylenediaminetetraacetic acid (2 mM/L in phosphate buffer); 7 mg ethylenediaminetetraacetic acid was dissolved in 11.36 ml phosphate buffer.

6. SOLUTION 2; Ferrous ammonium sulfate (2 mM/L); Dissolved 78.4 mg ferrous

ammonium sulfate in 100 ml double distilled water.

7. Fe-EDTA complex: Prepared freshly by mixing equal volumes of solutions 1 and2, left to stand for 60 min at room temp.

8. Hydrogen peroxide (H2O2) (10 mM/L): Diluted 0.011 ml H2O2 up to 10 ml with

double distilled water.

50

Materials & methods

9. Glacial acetic acid (20%): Diluted 20 ml glacial acetic acid up to 100 ml with double distilled water.

10. Thiobarbituric acid (TBA) (0.8% w/v in 50 mM/L NaOH): Dissolved 0.8 g TBA in 100 ml of 50 itiM/L NaOH.

11. Standard (1 mM/L uric acid in 5 mM/L NaOH); Dissolved 168 mg uric acid in 1000 ml of 5 mM/L NaOH.

Solutions 5-10 were prepared immediately before use. The sodium phosphate buffer and sodium benzoate were kept in a refrigerator (0-4“C) and the uric acid solution in a deep freezer (-20 to -30°C) until use.

Procedure; The steps involved in the estimation were:

Sample Control Standard

Serum 0.01 - -

Uric acid - - 0.01

Buffer 0.49 0.50 0.49

Sodium benzoate 0.50 0.50 0.50

Fe-EDTA 0.20 0.20 0.20

H2O2 0.20 0.20 0.20

Incubated for 60 rain at 37°C, and then added

Acetic acid 1.0 1.0 1.0

TBA 1.0 1.0 1.0

Incubated for 10 ntiin at 100°C and then cooled in an ice bath. Absorbance was measured at 532 mn against deionized water.

Calculation:

TAC (mM/L) = (CUA) (K - A) / (K - UA)

Where,K = absorbance of control A = absorbance of sample UA - absorbance of uric acid solution CUA = concentration of uric acid (in mM/L)

51

Materials & methods

« Estimation o f reduced glutathione

Method; Jollow et al, 1974

Principle: This assay is based on reduction of 5, 5’“Dithiobis-(2-nitrobenzoic acid) by SH groups to form 2-Nitro-5-mercaptobenzoic acid. The nitromercaptobenzoic acid anion has an intense yellow color that is determined spectrophotometrically at 412 n i n .

Reagents:

1. Sulphonic acid (4%): 4 g of sulphonic acid was dissolved in 100 ml of double distilled water.

2. Phosphate buffer (0.1 M, pH 7.4): SOLUTION A: Dissolved 0.276 g monobasic sodium phosphate monohydrate in 20 ml double distilled water. SOLUTION B:

Dissolved 1.42 g dibasic sodium phosphate in 100 ml double distilled water. Mixed 19 ml of solution A with 81 ml of solution B. pH adjusted to 7.4,

3. 5,5'-Dithio-bis(2-nitrobenzoic acid) (DTNB) (100 mM); 990 mg of DTNB was dissolved in 25 ml of absolute methanol.

4. Standard reduced glutathione (GSH) (10 |jM/L): Different dilutions of standard GSH were prepared.


S.No. Cone. (jiM/L) GSH (ml) Phosphate buffer (ml) DTNB (ml)

1. 1 0.1 2.5 0,4

2. 2 0.1 2.5 0.4

3. 3 0.1 2.5 0,4

4. 4 0.1 2.5 0.4

5. 5 0.1 2.5 0.4

6. 6 0.1 2.5 0.4

Mixed and taken optical density at 412 rnn.

52

Materials & methods

GSH CALIBRATION CURVE

CONCENTRATION ([iM/L)

y = 0.1038x+0.0446 R = 0.9998

♦ Series 1Linear (Series 1)

Figure 10. Calibration curve of GSH (jxM/L).

Procedure: 100 |j,l of tlie sample of serum was precipitated with 100 |il of sulphonic acid (4%). The sample was kept at 4°C for 1 h and then centriftiged at 1200 g for 20 min at 4°C. The assay mixture contained 0.1 ml filtered aliquot, 2.5 ml phosphate buffer (0.1 M, pH 7.4) and 0.4 ml DTNB (100 niM) in a total volume of 3 ml. The color developed was read immediately at 412 nin on a Shimadzu spectrophotometer against blank.

■ Estimation of thiobarbituric acid reactive substance

Method: Wright at al., 1981

Principle: Lipid peroxidation is a free radical mediated reaction, 1 mole of malonaldialdehyde also called thiobarbituric acid reactive substance, which is a secondary product of lipid peroxidation, reacts with 2 moles of thiobarbituric acid to

give a pink color, the absorbance of which is detennined at 535 nm.

Reagents:

1. Phosphate buffer (0.1 M, pH 7.4): SOLUTION A: Dissolved 0.276 g monobasic sodium phosphate monohydrate in 20 ml double distilled water. SOLUTION B; Dissolved 1.42 g dibasic sodimn phosphate in 100 ml double distilled water. Mixed 19 ml of solution A with 81 ml of solution B. pH adjusted to 7.4.

53

Materials & methods

2. Ascorbic acid (100 ibM): 1.76 g of ascorbic acid was dissolved in 100 ml of double distilled water.

3. Feme chloride (100 niM): 1.625 g of ferric chloride was dissolved in 100 ml of double distilled water.

4. Trichloroacetic acid (TCA) (10%): 10 g of TCA was dissolved in 100 ml of double distilled water.

5. TBA (0.67%); 0.67 g of TBA was dissolved in 100 ml of double distilled water.

6. Standard tetraethoxypropane (TEP) (10 |aM/L); Different dilutions of standard stock solution of TEP were made.


Cone.(fiM/L)

TEP(ml)

Phosphate buffer (ml)

Ascorbic acid (ml)

Ferric chloride (ml)

1 0.05 0.73 0.2 0.02

2 0.05 0.73 0.2 0.02

3 0.05 0.73 0.2 0.02

4 0.05 0.73 0.2 0.02

5 0.05 0.73 0.2 0.02

6 0,05 0.73 0.2 0.02

Reaction mixture was incubated at 37°C in a water bath for 1 h and then reaction was stopped by the addition of 1 ml TCA. Following addition of 1 ml TBA, the tubes were placed in boiling water-bath for 20 min and then shifted to crushed ice-bath before centrifuged at 2500 g for 10 min. Optical density of supernatant was read at 535 nm.

54

Materials & methods

TBARS CALIBRATION CURVE

§

0

1

y-0 .3413x-0.2433

R = 0.9994

4 Series 1 --— Linear (Series 1)

CONCENTRAITON (fiM/L)

Figure 11. Calibration curve of TBARS (pM/L).

Procedure: In 0.05 ml serum sample, 0.73 ml phosphate buffer (0.1 M, pH 7.4), 0.2 ml ascorbic acid (100 mM) and 0.02 ml ferric chloride (100 mM) were added. Total volume w as 1 ml. Reaction mixture was incubated at 37°C in a water batli for 1 h. Reaction was stopped by adding 1 ml 10% TCA. Following addition of 1 ml of 0.67% TBA, all the tubes were placed in boiling water bath for 20 min and then shifted to crushed ice-bath before being centrifuged at 2500 g for 10 min. The amount of malondialdehyde formed m each of the sample was assessed by measuring optical density of the supernatant at 535 nm using Shimadzu specti-ophotometer against blank.

ESTIMAIONS IN CARDIAC TISSUE

" Estimation of Na*/K^-ATPase

Method: Akagawa and Tsukada, 1979; Fiske and Subbarow, 1925

Principle: The liberation of inorganic phosphorous on incubation of the tissue extract was measured spectrophotometrically at 620 nm.

Reagents:

1. Tris-HCl buffer (75 mM, pH 7.4): 455.55 mg Tris-HCl buffer was dissolved in 50 ml double distilled water and its pH was adjusted to 7.4 using 1 N NaOH.

55

Materials & methods

2. Magnesium sulphate (MgS04) (50 itiM); 150.37 mg MgS04 was dissolved in 25 ml double distilled water.

3. Potassium chloride (KCl) (50 inM): 93.18 mg KCl was dissolved in 25 ml double distilled water.

4. Sodium chloride (NaCl) (600 mM): 876.6 mg NaCl was dissolved in 25 ml double distilled water.

5. EDTA (1 iiiM): 7.30 mg EDTA was dissolved in 25 ml double distilled water.

6. Adenosine triphosphate (ATP) (3 mM); 8.26 mg ATP was dissolved in 5 ml double distilled water.

7. TCA (10%); Dissolved 10 g TCA in 100 ml double distilled water.

8. Ammonium molybdate (2.5%); 2.5 g ammonium molybdate was dissolved in

100 ml of5NH2S04.

9. H2SO4 (5 N): Diluted 6.9 ml of conc. H2SO4 up to 50 ml with double distilled water.

10. Ascorbic acid (2%): Dissolved 2 g ascorbic acid in 100 ml double distilled water.

11. Standard inorganic phosphate (10 n.g/ml): 4.38 mg potassium dihydrogen orthophosphate (KH2PO4) was dissolved in 100 ml double distilled water and this has a conc. of 10 ng/ml inorganic phosphorus (Pi).


Tubes KH2PO4

(ml)Double distUled

water (ml)Ascorbic acid

(ml)Ammonium

molybdate (ml)

1 0.2 3.8 0.5 0.5

2 0.4 3.6 0.5 0,5

3 0.6 3.4 0.5 0.5

4 0.8 3.2 0.5 0.5

5 1.0 3.0 0.5 0.5

Incubated for 30 min and optical density was read at 620 nm

56

Materials & methods

ATPase CALIBRATION CURVE

CONCENTRATION (jig/ml)

y = 0.426x+0.0098 R = 0.9981

♦ Series 1

Linear (Series 1)

Figure 12. Calibration curve of ATPase (jig/ml).

Procedure: Cardiac tissue was homogenized in 75 mM of tris HCl buffer with a ratio

of 1:10 w/v. 100 1 tissue homogenate taken in centrifuge tubes was incubated in a

medium containing 1.5 ml tris HCl buffer and 0.1 ml each of NaCl, KCI, MgS04, EDTA and ATP for 30 min at 37°C. The reaction was arrested by the addition of 1 ml

20% TCA. The precipitates formed after addition of TCA in the tubes were removed by centrifugation and their supernatants were transferred to fresh tubes. The reagent blank contained 1.8 ml tris HCl buffer. To all the above tubes, 0.5 ml ammonium molybdate and 0.2 ml ascorbic acid were added and left for 30 min for the blue color to develop, which was read at 620 nm against the reagent blank using a

spectrophotometer. Na’ /K' -ATPase activity is expressed as nmoles of inorganic phosphorous liberated/min/mg tissue.

■ Glutathione peroxidase

Method; Mohandas et al„ 1984

Principle: The enzyme glutathione peroxidase (GPx) has been reported to reduce hydrogen peroxides to water by using reduced glutathione as a hydrogen donor.

Specrophometric detennination of nicotinamide adenine dinucleotide phosphate oxidized/min reflects enzymatic activity.

57

Materials & methods

Reagents:

1. EDTA (10 mM): Dissolved 37.2 mg EDTA in 10 ml double distilled water,

2. Sodium azide (NaNj) (10 mM): Dissolved 6.5 mg NAN3 in 10 ml double distilled water.

3. Nicotinamide adenine dinucleotide phosphate (NADPH) (0.2 mM); 16.6 mg of NADPH was dissoved in 10 ml of double distilled water,

4. Reduced glutathione (GSH) (1 mM): 37.2 mg of GSH was dissolved in 10 ml of double distilled water.

5. Glutathione reductase (GR); I lU of GR was dissolved in 1 ml of double distilled water

6. Hydrogen peroxide (H2O2) (30%): 3 ml of H2O2 was dissolved in 7 ml of double distilled water.

7. Phosphate buffer (0.1 M, pH 7.4): SOLUTION A: Dissolved 0.276 g monobasic sodium phosphate monohydrate in 20 ml double distilled water.

SOLUTION B: Dissolved 1,42 g dibasic sodium phosphate in 100 ml double distilled water. Mixed 19 ml of solution A with 81 ml of solution B. pH

adjusted to 7.4.

Procedure; The reaction mixture was consisted of 1.49 ml phosphate buffer (0.1 M, pH 7.4), 0.1 ml EDTA (10 mM), 0.1 ml sodium azide (10 mM), 0.05 ml glutathione reductase (1 lU/ml), 0.05 ml GSH (1 mM), 0.1 NADPH (0.2 mM), 0.01 ml H2O2 (0.25 mM) and 0,1 ml PMS (10% w/v) in a total volume of 2 ml. The disappearance of NADPH at 340 nm was recorded at 25“C. The enzymatic activity was calculated as nmol NADPH oxidized/min/mg protein using a molar extinction coefficient of

6.22x10^ M''cm''.

Calculation:

AA/min xvolume of assay x 1000nmol NADPH oxidized/min/mg protein =

6.22 X volume of PMS x mg protein

58

Materials & methods

• Glutathione reductase

Methods: Carlberg and Mannervik, 1975

Principles: The enzyme gluthione reductase (GR) catalyzes the regeneration of reduced glutathione from oxidized gluthione at the expense of NADPH, Spectrophometric determination of NADPH oxidized/min reflects enzymatic activity.

Reagents:

1. EDTA (5 mM); Dissoved 18.6 mg EDTA in 10 ml double distilled water.

2. NADPH (0.1 mM): Dissolved 6.12 mg NADPH in 10 ml double distilled water.

3. Oxidized glutathione (GSSG) (1 mM): Dissolved 6.12 mg GSSG in 10 ml double distilled water.

4. Phosphate buffer (0.1 M, pH 7.4): SOLUTION A: Dissolved 0.276 g

monobasic sodium phosphate raonohydrate in 20 ml double distilled water. SOLUTION B: Dissolved 1.42 g dibasic sodium phosphate in 100 ml double distilled water. Mixed 19 ml of solution A with 81 ml of solution B. pH adjusted to 7.4.

Procedure; The reaction mixture was consisted of 1.65 ml phosphate buffer (G.l M,

pH 7.4), 0.1 ml EDTA (0.5 mM), 0.05 ml GSSG (1 mM), 0.1 ml NADPH (0.1 mM) and O.l ml PMS (10% w/v) in a total volume of 2.0 ml. The disappearance of NADPH at 340 nm was recorded at 25°C. The enzymatic activity was calculated as nmol NADPH oxidized/rain/mg protein using a molar extinction coefficient of 6.22x10^ M"

cm .

Calculation:

AA/min xvolume of assay x 1000 nmol NADPH oxidized/min/mg protein = ____________________________

6.22 X volume of PMS x mg protein

59

Materials & methods

■ GlutationeS-tansferase

Method: Habig at al., 1974

Principle: Assay for the activity is based on the spectrophotometric determination of

a I'Chloro 2, 4-dinitrobenzene conjugate formed in a reduced glutathione coupled

reaction. The conjugate formation is glutathione-S-ti’anasferase catalyzed and therefore is a measure of glutathione-S-tranasferase (GST) activity.

Reagents:

1. Reduced glutathione (GSH) (1 mM): Dissolved 37.2 mg GSH in 10 ml double distilled water

2. 1-Chloro 2, 4-dinitrobenzene (CDNB) (1 mM): 12.12 mg of CDNB was dissolved in 10 ml ethanol (8 ml ethanol mixed with 2 ml double distilled

water)

3. Phosphate buffer (0.1 M, pH 7.4); SOLUTION A: Dissolved 0.276 g monobasic sodium phosphate monohydrate in 20 ml double distilled water. SOLUTION B: Dissolved 1,42 g dibasic sodium phosphate in 100 ml double distilled water. Mixed 19 ml of solution A with 81 ml of solution B. pH

adjusted to 7.4.

Procedure: The reaction mixture was consisted of 1.75 ml phosphate buffer (0.1 M, pH 7.4), 0,1 ml GSH (1 mM), 0.05 ml CDNB (1 mM) and 0.1 ml PMS (10% w/v) in a total volume of 2.0 ml. The change in absorbance was recorded at 340 nm. The enzyme activity was calculated as nmol CDNB conjugate formed/min/mg protein using a molai- extinction coefficient of 9.6 x 10 M"' cm"'.

Calculation:AA/min ><volume of assay x 1000

nmol CDNB conjugate fonned/min/mg protein = ___________________9.6 X volume of PMS x mg protein

60

Materials & methods

■ Catalase

Method: Clairbome, 1985

Principle: Assay of catalase activity depends upon either the decomposition of H2O2

or the liberation of O2. The decomposition of H2O2 denotes the decrease in extinction at 240 nin. The difference in extinction (A E240) per unit time is a measure of the catalase activity.

Reagents:

1. Potassium phosphate buffer (50 mM, pH 7.4): SOLUTION A; Dissolved 0.681 g potassium dihydrogen phosphate in 100 ml double distilled water. SOLUTION B; Dissolved 0.890 g disodium hydrogen phosphate dihydrate in

100 ml double distilled water. Mixed solution A with solution B in the ratio of 1:1.55. pH adjusted to 7.4.

2. H2O2 (30%) (19 mM, pH 7.4): 187 nl of H2O2 was dissolved in 100 ml potassium phosphate buffer.

Procedure: Heart tissue was homogenized in 50 mM/L of potassium phosphate buffer with a ratio of 1:10 w/v. The homogenate was centrifuged at 10,000 g at 4“C in a cooling centrifuge for 20 min. 50 )J.1 of supernatant was added to the cuvette containing 2.95 ml of 19 mM/L solution of H2O2 prepared in potassium phosphate

buffer. The disappeai-ance of I-I2O2 was monitored at 240 nm wavelength at 1 min interval for 3 min.

Calculation;

CAT activity (U/mg protein) = (AA/min x F)/nig protein

Where,AA/inin = change in absorbance per min F=factor= 740.74

61

Materials & methods

■ Superoxide dismutase

Method: Marklund and Marklund, 1974

Principle; Pyrogallol autoxidizes rapidly in aqueous solution, the higher the pH the

faster is autoxidation and several interaiediate products are fonned. Thus the solution fii-st becomes yellow brown witli a spectrum showing a shoulder between 400-425 nm. After a number of minutes the color begins to turn green and finally after a few hours, a yellow color appears. So the rate of autoxidation is studied fi-om the linear increase in absorbance at 420 nm, which is seen for a number of minutes after an

induction period of 10 seconds. Superoxide anion radical (O2*") catalyze the

autoxidaion of pyrogallol. Superoxide dismutase inhibits the autoxidation of pyrogallol by dismutasing superoxide anion radical.

Reagents;

1. Tris HCl buffer (pH 8.5): Dissolved 0.788 g tris HCl buffer and 0.186 gdisodium EDTA in 100 ml double distilled water. pH adjusted to 8,5.

2. Pyrogallol (24 mM): 15.1 mg of pyrogallol was dissolved in 5 ml of 10 inMHCl.

Procedure: 20 mg of heart tissue was homogenized in 2 ml of potassium phosphate buffer (pH 7.4). The homogenate was centrifuged at 10,000 g at 4“C in a cooling centrifuge for 20 min. 100 jul of supernatant was added to 3 ml of Tris HCl buffer, pH 8.5 followed by 25 |j,l of pyrogallol and then mixed thoroughly. The change inabsorbance at 420 nm was recorded at 1 min interval for 3 min.

Calculation:

SOD activity (U/mg protein) = (AA/min ?< F)/mg protein

Where,AA/min = change in absorbance per min F =factor = 8116.6

62

Materials & methods

■ Protein

Principle: The phenolic group of tyrosine and trytophan residues (amino acid) in a protein reacts with the copper [II] ions under alkahne condition and the subsequent reduction of Fohn-Ciocalteau reagent (phosphomolybdicphosphotungstic acid) to produce a blue color complex. Thus, the intensity of color determines the amount of these aromatic acids present in the sample.

Method: Lowary et al., 1951

Reagents:

1. Alkaline sodium carbonate (NajCOs) solution: 100 ml of 0.1 N NaOH solution was prepared by dissolving 400 mg NaOH in double distilled water and the

volmne was made up to 100 ml. Then 2 g Na2C03was dissolved in 100 ml of0.1 N NaOH solution.

2. Copper sulfate (CuS04.5H20)^odium potassium tartrate (Na-K tartrate) solution; 0.5% CuS04.5H20 (0.5 g copper sulfate dissolved in 100 mi

double distilled water) solution was mixed with 1% Na-K tartrate (I g Na-IC tartrate dissolved in 100 ml double distilled water).

3. Alkaline solution: Prepared on the day of use by mixing 50 ml reagent-1 and 1 ml reagent-2.

4. Folin's Ciocalteau phenol reagent; The commercial reagent was diluted with2 volumes of double distilled water before using.

5. Bovine serum albumin (BSA) (1 mg/ml): 5 mg BSA was dissolved in 5 ml double distilled water to get a stock solution of 1 mg/ml.

63

Materials & methods


S.No. Cone.(mg/ml)

BSAsolution

(ml)

Double distilled

water (ml)

Allcalinesolution

(ml)

Folin’sreagent

(ml)

Incubated at37"C

for 30 min and taken 0,D. at 750 nm

1 0.1 0.1 0.9 5 0.5

2 0.2 0.2 0.8 5 0.5

3 0.4 0.4 0.6 5 0.5

4 0.6 0.6 0.4 5 0.5

5 0.8 0.8 0.2 5 0.5

PROTEIN CALIBRATION CURVE

y = 0.782x+0.0196 R^= 0,9962

♦ Series 1 — Linear (Series 1)

CONCENTRATION (mg/ml)

Figue 13. Calibration curve of protein (rjig/ml).

Procedure: Heart tissue was homogenized in 1.17% KCl with a ratio of 1:10 w/v and centrifuged at 10,000 g for 10 min. 1 ml of supernatant was mixed with 5 ml of alkaline copper solution and allowed to stand at room temperature for 10 rain. 0.5 ml of diluted Folin’s reagent (1:2) was then added and the tubes were shaken immediately for a thorough mixing. After 30 min the absorbance was read at 750 nin against the blank. The protein content was expressed in inilligrara.

64

Materials & methods

Assessment of cardiac hypertrophy

The heart was excised, cleaned with ice-cold normal saline and weights of the whole

hearts were measured. The ratio of heart weight to body weight (HW/BW) wascalculated to assess cardiac hypertrophy.

Histopathological studies (Kitagaw et al., 2007)

1. Dehydration; The water content of the tissue was replaced using increasingconcentration of ethanol.

80 % alcohol: 1 hour

95 % alcohol: 1 hour

100% alcohol: 1 hour

2. Clearing: Chloroform was used as a clearing agent for clearing tissue and making it translucent, signifying the completion of process.

3. Impregnation: Complete removal of clearing agent by substitution is done by paraffin, as it penetrates the tissues, hnpregnation was done with 3-

paraffm bath for 3 hrs at 56°-58°C. The tissue was then cast in to blocks of paraffin wax.

4. Sectioning: Sections of the tissue block 3-5|i in thickness were cut with the help of rotary microtome. The section ribbons were made to float on water and then placed on glass slides.

5. Hydration: The sections were hydrated with xylene for 2 min and 70% alcohol for 2 min. Then, they were rinsed with distilled water.

6. Staining; Sections were stained with 1 % heamatoxylin, rinsed in distilled water and then eosin (1% in 90% alcohol) was added for 1 min and the slides were

dried,

7. The stained sections were then covered with glycerin jelly and cover slips were placed carefully on section, taking care that no air bubble should enter.

65

Materials & methods

The slides were observed under the microscope and photographs were taken subsequently.

8. Statistical analysis

Statistical analysis was earned out by using Graphpad Prism 3.0 (Graphpad software; San Diego, California, USA). All results were expressed as Mean ± S.E.M, Groups of data were compared with an analysis of variance (ANOVA) followed by Bonferroni’s test. Values were considered statistically significant at P < 0.05.

66

materials and methodsshodhganga.inflibnet.ac.in/bitstream/10603/37414/9/09... · 2018. 7. 2. ·...

Documents