MATERIALS AND METHODS
4.1. PLANT MATERIAL
4.1.1. COLLECTION OF PLANT
The plant aerial parts of Givotia moluccanawas collected and Authentified by Dr. K. Madhava Chetty, Department of Botany, Sri Venkateswara University, Tirupathi (AP).
4.1.2. PREPARATION OF THE EXTRACT
The dried leaves of G. moluccana was collected, cleaned, dried and powdered in a grinder ­­- mixer to obtain a coarse powder and then passed through 40 mesh sieve. About 1000 gm of powdered drug was extracted with aqueous ethanol by soxhlet apparatus. The extraction was carried out until the drug becomes exhausted. The solvent was recovered from their extract by distillation under reduced pressure. The dried extract thus obtained was kept in a desicator and was used for further experiments.
4.2. EXPERIMENTAL ANIMALS
Healthy adult male wistar rats weighing between 150-200gm were used for the present study. The animals were housed in groups of six and maintained under standard conditions (27±2ºC, relative humidity 44 – 56% and light and dark cycles of 10 and 14 hours respectively) and fed with standard rat diet and purified drinking water ad libitum for 1 week before and during the experiments.
All experiments and protocols described in present study were approved by the Institutional Animal Ethical Committee (IAEC) of P.Rami Reddy Memorial Collage of Pharmacy (1423/PO/a/11/CPCSEA/102/2014).
All the experiments were performed in the morning according to current guidelines for the care of laboratory animals and the ethical guidelines for the investigation of experimental pain in conscious animals (Zimmerman, 1983).
4.3. DRUGS AND CHEMICALS
Epinephrine, DTNB, Triphenyl tetrazolium chloride and isoproterenol were obtained from Sigma-Aldrich, Bangalore. Thiobarbituric acid (TBA), trichloro acetic acid, hydrogen peroxide were obtained from SD fine chemicals Ltd Mumbai. Sodium dihydrogen phosphate, potassium dihydrogen phosphate, tris buffer and all other reagents used were of analytical grade. CK-MB, LDH, SGOT, SGPT, ALP, Total cholesterol, HDL, and triglyceride estimation kits were obtained from Erba diagnostic Ltd. India.
4.4. INSTRUMENTS
Analytical Auto analyzer (MaxLyzer NB-201), UV-Visible spectrophotometer (Shimadzu, Model no: 2203), Electronic balance (Shimadzu, Model no: DS-852 J), Tissue homogeniger (Ever shine, Model no: 607), Remi centrifuge (Remi, Model no: KKLO-9013).
4.5 ACUTE ORAL TOXICITY STUDY
The acute oral toxicity study was done according to OECD 423 guidelines. Wistar albino rats of either sex were selected randomly and divided into six groups (n = 6). The animals were fasted overnight and extract in doses of 100, 250, 500, 1000, 2000 and 5000 mg/kg body weight, were administered orally to II – VI groups. Group I which received vehicle (CMC) served as control. The animals were observed continuously for 2 hr, and then intermittently for 6 hr and at the end of 24 hours, the number of deaths was noted to determine LD50 of the extract (Annie et al., 2004).
4.6. EXPERIMENTAL DESIGN
4.6.1. NEPHROPROTECTIVE ACTIVITY
The experimental animals were randomly divided in to 5 groups (n= 6) and treated for duration of 21 days as per the treatment schedule given in table no: 3. Nephrotoxicity was induced by administration of Gentamycin (80 mg/kg I.P) daily for 7 days. Ethanolic extract of G. moluccanawas freshly suspended in CMC and administered to animals by oral feeding needle.
Table no: 3 Treatment schedule –Evaluation of nephroprotective activity of EEGM against gentamycin induced nephrotoxicity in Wistar Rats.

S.NO

Groups

Treatment (21 days)

Purpose

I

Normal

Vehicle (1% CMC)

To serve as normal animal

II

Control

Gentamycin(80 mg/kg, i.p.)

To serve as control

III

Standard

EEGM (500 mg/kg, p.o.).

To serve as standard

IV

Low dose

Gentamycin (80 mg/kg, i.p.) + EEGM (250 mg/kg, p.o.)

To assess the Nephroprotective activity of EEGM (at low dose)

V

High dose

Gentamycin (80 mg/kg, i.p.) + EEGM(500 mg/kg, p.o.)

To assess the Nephroprotective activity of EEGM (at high dose)

I.P = Intra peritoneal, P.O = Per oral.
 
4.6.2. COLLECTION OF BLOOD AND URINE SAMPLES
The blood samples were collected from the retrorbital venous plexus of rats without any coagulant for the separation of serum, at the regular intervals of the treatment. After collecting the blood in effindraf tubes they were kept for 1 h at room temperature and serum was separated by centrifugation at 2000 rpm for 15 min and stored until analyzed for various biochemical parameters. Urine was collected over 24 hours on the 21st day by keeping the test animals in metabolic cages. The volume of collected urine samples was measured followed by estimation of biochemical parameters, namely urine Creatinine, urine uric acid and urine urea.
4.7. PARAMETERS MONITERED
4.7.1. BIOCHEMICAL ESTMATIONS
i. Estimation of Urea (Berthelot Method)
Principle:
The reaction sequence employed in the assay is as follows:
Urea + H2O Urease 2NH3 + CO2
NH3 + Salicylate +Hypochlorite Nitropruside 2-2-Dicarboxy Indophenol
Urease catalyses the conversion of Urea to Ammonia and Carbondioxide. The ammonia released reacts with a mixture of Slicylate. Hypochlorite and Nitropruside to yield a blue-green colored compound (Indophenol). The intensity of color produced is proportional to the concentration of urea in the sample and is measured photometrically at 578 nm or with yellow filter.
Reagent preparation:
Transfer the entire Enzyme Concentrate (1A) into Urease Reagent (1) with the dropper (or) microtip provided.
Assay Procedure:
Pipette into test tubes labeled Blank (B), Standard(S), Test(T) as follows.

 

Blank(B)

Standard(S)

Test(T)

Working Reagent
Standard (40 mg/dl)
Specimen

1.0 ml 1.0 ml 1.0 ml
_ 10 µl _
_ _ 10 µl

 
 
 
 

Mix and read absorbance for 5 minutes at 37ºC (10 minutes at R.T.)

Alkaline Buffer (2)

1.0 ml 1.0 ml 1.0 ml

Mix and incubate for 5 minutes at 37ºC (10 minutes at R.T.)

 
 

Mix and Read absorbance of Standard (S) and Test (T) against Blank (B) at 578 nm (570-620 nm) or with yellow filter.
The final color is stable for 30 min. at R.T.
Calculations:

Blood urea nitrogen in mg/dl = a X 0.467
Urine Urea in gm/24 hours = a X 24 hrs urine volume in litres.
ii. Estimation of BUN (GLDH-Urease Method)
Methodology : Talke and Schubert, Tiffany et al.
Principle:
The estimation of Urea in serum involves the following enzyme catalyzed reactions:
Urea + H2O Urease 2NH3 + CO2
NH3 + α-KG + NADH GLDH Glutamate + NAD
α-KG : α-Ketoglutarate
GLDH : Glutamate dehydrogenase
The rate of decrease in absorbance is monitored at 340 nm and is directly proportional to urea concentration in the sample.
Procedure:

Pipette into tubes marked

Standard

Test

Working Reagent

1000 µl

1000 µl

Standard

20 µl

–

Test

–

20µl

Mix well, and aspirate standard followed by samples.
Calculation:
Determine absorbance change (ΔA) for the standard and unknown samples by using the formula.
ΔA = A1 – A2
Urea = ΔA of Test Concentration of
(mg/dl) ΔA OF Standard Standard (mg/dl)
iii. Estimation of Uric acid (Uricase/POD)
Principle:
Uric acid is oxidized to Allontoin and hydrogenperoxide by the enzyme uricase. In presence of peroxidase, released hydrogen peroxide is coupled with Aniline derivative and 4-amino antipyrine (4-aap) to form colored chromogen complex. Absorbence of colored dye is measured at 550 nm and is proportional to Uric acid concentration in the sample (Schultz, 1984; Teivedi et al., 1978).
Uric acid + 2H2O Uricase Allontoin + CO2 + H2O2
H2O2 + Aniline derivative + 4-AAP POD Chromogen complex + H2O2
Procedure:

Pipette into tubes marked

Blank

Standard

Test

Serum/Plasma/Urine

–

–

20 ml

Reagent 2

–

20 µl

–

Reagent 1

1000 µl

1000 µl

1000 µl

 
Mix well. Incubate at 37ºC for 5 minutes.
Programme the analyzer as per assay parameters.

Blank the analyzer with reagent blank.
Measure absorbance of standard followed by the test.
Calculate results as per given calculation formula.

Calculations:
Serum/plasma/uric acid = Absorbance of Test 6
(mg/dl) Absorbance of Standard
Urine uric acid = Dilution 24 hours urine volume in dl.
Factor (mg/day)
Conversion factor:
Uric acid concentration in mmol/L = Uric acid in mg/dL 0.059
iv. Estimation of Creatinine (Mod. Jaffe’s Kinetic Method)
Principle:
Picric acid in an alkaline medium reacts with creatinine to form an orange coloured complex with the alkaline picrate. Intensity of the colour formed during the fixed time is directly proportional to the amount of creatinine present in the sample.
Creatinine + Alkaline Picrate Orange Coloured Complex
Procedure:
Pipette into clean dry test tubes labeled as Standard (S) or Test (T):

Addition Sequence

(S) / (T) 300 C / 370 C

Picric Acid Reagent (L1)

0.5 ml

Buffer Reagent (L2)

0.5 ml

Bring reagents to the assay temperature and add

Creatinine Standard (S) / Sample / Diluted Urine

0.1 ml

 
 

Mix well and read the initial absorbance A for the Standard and Test 1 after exactly 30 seconds. Read another absorbance A of the Standard 2 and Test exactly 60 seconds later. Calculate the change in absorbance ΔA for both the Standard and Test.
For Standard Δ AS = A2 S – A1 S
For Test Δ AT = A2 T – A1 T
Calculations:
Creatinine in mg/dl = 2.0
Urine Creatinine in g/L = x 1.0
Urine Creatinine g/24 Hrs. = Urine Creatinine in g/L x Vol. of urine in 24 Hrs.
v. Estimation of Total Protein (Biuret Method )
Methodology:
The peptide bonds of protein react copper ions in alkaline solution to form blue-violet complex, (biuret reaction). Each copper ion complexing with 5 or 6 peptide bonds. Tartarate is added as a stabilizer whilst iodide is used to prevent auto-reduction of the alkaline copper complex. The color formed is proportional to the protein concentration and is measured at 546nm (520-560nm).
Procedure:

Pipette into tubes marked

Blank

Standard

Test

Reagent

1000 µl

1000 µl

1000 µl

Distilled water

20 µl

–

–

Standard

–

20 µl

–

Test

–

–

20 µl

 
Incubate for 10 minutes at 37º C. Read absorbance of the standard and each test at 546 nm( 520-560 nm) against reagent blank.
Calculations:
Calculate the results as follows:
Total Protein = Absorbance of Test Concentration of
(g/dl) Absorbance of Standard Standard (g/dl)
vi. Estimation of Albumin (Bromocresol Green)
Principle:
At pH 3.68, Albumin acts as a cation and binds to the anionic dye Bromocresol Green (BCG),forming a green colored complex. The color intensity of the complex is proportional to Albumin concentration in the sample (Gendler Proteins, 1984; Gustsfsson, 1978).
Albumin + BCG Ph 3.68 Green colored complex.
Procedure:

Pipette into tubes marked

Blank

Standard

Test

Serum/Plasma

–

–

10 µl

Reagent 1

–

10 µl

–

Reagent 2

1000 µl

1000 µl

1000 µl

Mix well. Incubate at Room Temperature (15-30ºC) for 1 minute.
Programme the analyzer as per assay parameters.

Blank the analyzer with reagent blank.
Measure absorbance of standard followed by the test.
Calculate results as per given calculation formula.

Calculations:
Albumin (g/dL) = Absorbance of Test 4
Absorbance of Standard
Globulin = Total Protein – Albumin
Conversion factor:
Albumin concentration in g/L = Albumin concentration in g/dL 10
vii. Estimation of Cholestrol (CHOD-PAP Method)
Methodology: Modified Roeschlau,s Method
Principle:
The estimation of cholesterol involves the following enzyme catalyzed reactions.
Cholestrol ester CE Ckolestrol + Fatty acid
Cholestrol + O2 CHOD Cholest-4-en-3-one + H2O2
2H2O2 + 4AAP + Phenol POD 4H2O + Quinoneimine
CE : Cholestrol esterase
CHOD : Cholestrol Oxidase
4AAP : 4-Aminoantipyrine
Procedure:

Pipette into tubes marked

Blank

Standard

Test

Working Reagent

1000 µl

1000 µl

1000 µl

Distilled Water

20 µl

–

–

Standard

–

20 µl

–

Test

–

–

20 µl

Mix well and incubate at 370C for 10 minutes. Aspirate Blank followed by Standard and Tests. Read the absorbance of standard and each test tube against blank at 505 nm or 505/670 nm on bichromic analyzer.
Calculations:
Cholestrol (mg/dL) = Absorbance of Test Concentration of Standard (mg/dl)
Absorbance of Standard
viii. Estimation of Glucose (GOD – POP Method)
Methodology: Trinder, s Method.
Principle:
Gucose + O2 + H2O Glucose oxidase Gluconic acid + H2O2
H2O2 + 4HBA + 4AAP Peroxidase Quinonemine Dye + 2 H2O
4AAP : 4-Aminoantipyrine
4HBA : 4-Hydroxy benzoic acid.
The intensity of the pink color formed is proportional to the glucose concentration and can be measured photometrically between 500 to 540 nm.
Procedure:

Pipette into tubes marked

Blank

Standard

Test

Working Reagent

1000 µl

1000 µl

1000 µl

Distilled Water

10 µl

–

–

Standard

–

10 µl

–

Test

–

–

10 µl

Mix well and incubate for 10 minutes at 370 C. Read the absorbance of standard and each test tube against reagent blank at 505 nm (500-540nm) or 505/670 nm on bichromic analyzer.
Calculations:
Glucose = Absorbance of Test X Concentration of Standard (mg/dl)
(mg/dL) Absorbance of Standard
ix. Estimation of Bilirubin (BIT & – BID)
Methodology: Diazo Method of Pearlman &Lee
Principle:
Bilirubin reacts with diazotized sulphanilic acid in acidic medium to form pink colored azobilirubin with absorbance directly proportional to Bilirubin concentration. Direct Bilirubin, being water soluble directly reacts in acidic medium. However Indirect or unconjugated Bilirubin is solubilised using a surfactant and then it reacts similar to Direct Bilirubin.
Reagent preparation:

Test

Volume of Working Reagent

Add

Reagent 1

Reagent 2

Reagent 3

Bilirubin
Total

10 ml

10 ml

–

0.2 ml

25 ml

25 ml

–

0.5 ml

50 ml

50 ml

–

1.0 ml

100 ml

100 ml

–

2.0 ml

 
 
 
 
 

Procedure:

Pipette into tubes marked

Blank

Standard

Test

Working Reagent

500 µl

500 µl

500 µl

Distilled Water

25 µl

–

–

Test

–

–

25µl

Mix well and incubate for 5 minutes at 370 C for Total Bilirubin and Direct Bilirubin. Read Absorbance at 546/630 nm against Reagent Blank.
Calculations with Factors:
Total Bilirubin (mg/dl) = Abs. of Test Factor (23).
4.7.2. IN VIVO ANTIOXIDANT PARAMETERS
Preparation of homogenate:
The homogenate of heart was prepared as follows for the remaining animals.
Reagents:

0.25 M sucrose solution: 85.87 g of sucrose was dissolved in 1000 ml of distilled water
10 mM tris buffer solution: 1.2 g of tris was dissolved in 900 ml of distilled water. pH was adjusted to 7.4 with 1M HCl and diluted up to 1000 ml.

Procedure:
Kidneys were excised and chopped with surgical scalp into fine slices and were chilled in the cold 0.25 M sucrose, quickly blotted with filter paper. The tissue was minced and homogenized in ice cold 10 mM tris HCl buffer (to pH 7.4) at a concentration of 10% (w/v) with 25 stokes of tight teflon pestle of glass homogenizer at a speed of 2500 rpm. The prolonged homogenization under hypotonic condition was designed to disrupt as far as possible the ventricular structure of cells so as to release soluble protein and leave only membrane and non-vascular matter in a sedimentable form. It was then centrifuged at 5000 rpm at 20o C temperature and clear supernatant was separated and used to estimate reduced glutathione (GSH), catalase (CAT) and lipidperoxidation (LPO).
a). Catalase (CAT):
Catalase was estimated by the method of Hugo E. Aebi method: hydrogen peroxide: hydrogen-peroxidoreductase.
Principle:
In UV range H2O2 can be followed directly by the decrease in absorbence (O.D 240) per unit time is measure of catalase activity.
H2O2 H2 + O2
RDOH H2O + ROH + A
Decomposition of H2O2 = Decrease in absorbance at 240 nm
Reagents:

Phosphate buffer (50 mM, pH 7.0)

Dissolve 6.81 g KH2PO4 in distilled water and make up to 1000 ml.
Dissolve 8.9 g NaH2PO4. 2H2O in distilled water and make up to 1000 ml.

Mix the solution A and B in proportion 1:15 (v/v)

Hydrogen peroxide (30 mM/I): Dilute 0.34 ml of 30% Hydrogen peroxide with phosphate buffer up to 100 ml.

Procedure:
Dilute homogenate 20 times with Phosphate buffer pH 7.0

Blank

Test

4 ml of homogenate diluted with 2 ml of phosphate buffer PH 7, and take absorbance at 254 nm for 3 min. with 30 sec. interval

2 ml of homogenate diluted with 1 ml of H2O2 (8.5 micro lit. in 2.5 ml phosphate buffer (50mM/l. pH 7.0) and take the absorbance at 254 nm for 3 min. with 30 sec. interval.
(Add H2O2 just before taking O.D)

Calculation:
Log (A / B) × 2297.3
Where,
A: Initial absorbance
B: final absorbance (after 30 second)
Units = µ moles of H2O2 consumed/min/mg
b). Reduced glutathione (GSH):
Reduced glutathione was determined by the method of Moran et al., 1979.
Reagents:

TCA (10% w/v) solution: Accurately weighed 10 g of TCA was dissolved in 100 ml of distilled water.
Phosphate buffer (0.2 M, pH 8)
DTNB reagent (0.6 M): 60 mg of 5,5- dithio bis (2-nitro benzoic acid) was dissolved in 100 ml of 0.2 M sodium phosphate (pH 8).
Standard glutathione: Prepared by dissolving 10 mg of reduced glutathione in 100 ml of distilled water.

Procedure:
To 1 ml of sample, 1 ml of 10% TCA was added. The precipitated fraction was centrifuged and to 0.5 ml supernatant, 2 ml DTNB was added. The final volume was made up to 3 ml with phosphate buffer. The colour developed was read at 412 nm. The amount of glutathione was expressed as µg of GSH/mg protein, reduced glutathione was used as standard (100 µg/ml).

Y – Absorbance of test sample
c). Lipid peroxidation:
Lipid peroxidation was determined by the method of Slater and Sawsyer et al., 1971
Reagents:

Thiobarbituric acid: 0.67% w/v in 1M tris hydrochloride pH -7, 0.67 g of thiobarbituric acid was dissolved in 100 ml of distilled water.
Trichloroacetic acid (20% w/v): 20 g of TCA was dissolved in 100 ml of distilled water.
Standard malondialdehyde (0-25 n.mol)

A stock solution containing 50 mm/ml of 1, 1,3,3-tetra ethoxy propane in tris hydrochloride buffer in pH -7, 10 ml of stock solution was diluted to 100 ml to get a working standard 50 nm malondialdehyde/ml. This was used for preparation of calibration curves.
Procedure:
2 ml of sample was mixed with 2 ml of 20% TCA and kept in ice for 15 min. The precipitate was separated by centrifugation and 2 ml of samples of clear supernatant solution were mixed with 2 ml aq. 0.67% TBA solution. This mixture was heated on a boiling water bath for 10 min. It was cooled in ice for 5 min and absorbance was read at 535 nm. The values were expressed as nm of MDA formed/mg of protein values are normalized to protein content of tissues.

Y – Absorbance differences of final (after 3 min) and initial reading of test sample.