Increase in the incidence of ischemia-reperfusion injury necessitates the development of myocardial salvaging/protection interventions.
Curcuma amada has already reported as an antioxidant (Policegourda et al., 2007) which may play an important role in ischemia-reperfusion injury and protective effect of this plant is not reported.
AIM AND OBJECTIVE
To study the cardioprotective effect of Curcuma amada ethanolic extract in ischemic preconditioning and postconditioning of heart using rat model.
Objectives:
To explore the effect of Curcuma amada ethanolic extract on ischemia-reperfusion injury, ischemic preconditioning and postconditioning of heart.
To compare the cardioprotective effect of ischemic preconditioning and ischemic postconditioning.
EXPERIMENTAL WORK
RHIZOME COLLECTION AND AUTHENTICATION
The dried rhizomes of Curcuma amada are procured from Hari Gokal Sandeep International, New Delhi. The rhizomes are authenticated by NISCAIR (National Institute of Science Communication & Information Resources), New Delhi. The coarsely fine powder of rhizomes is made using pestle and motar and shade dried. For further use it has been stored in a dried airtight container.
EXTRACTION OF PLANT
The coarsely fine dried powder (50g) of Curcuma amada rhizomes have been extracted in Soxhlet apparatus using 95% ethanol at 65-70ã‚œC for 24 hours. The extract is evaporated to dryness and made to a specified volume (Majumdar et al., 2000).
QUALITATIVE CHEMICAL EXAMINATION OF EXTRACT
Preliminary phytochemical evaluation
(i) Detection of alkaloids
Extract is dissolved in dilute hydrochloric acid and filtered. The filtrate is then used to identify the alkaloids.
Mayer's test
Filtrate is treated with Mayer's reagent (saturated solution of potassium mercuric iodide). Formation of yellow cream precipitates indicate the presence of alkaloids.
Wagner's test
Filtrate is treated with Wagner's reagent (saturated solution of iodine in potassium iodide). Formation of reddish brown precipitates indicate the presence of alkaloids.
Dragendorff's test
Filtrate is treated with Dragendorff's reagent (saturated solution of potassium bismuth iodide). Formation of red precipitates indicate the presence of alkaloids.
(ii) Detection of carbohydrates
The plant extract is dissolved in 5 ml distilled water and filtered. The filtrate is then used to test for the presence of carbohydrates.
Molisch's test
Filtrate is treated with 2 drops of alcoholic α-naphthol solution in a test tube. 2 ml of concentrated sulphuric acid was added carefully along the sides of the test tube. Formation of violet coloured ring at the junction indicate the presence of carbohydrates.
Benedict's test
Filtrate is treated with Benedict's reagent and heated on water bath. Formation of orange red precipitates indicate the presence of reducing sugars.
Fehling's test
Filtrate is hydrolysed with dil. hydrochloric acid neutralized with alkali. Then it is heated with Fehling's A and B solutions. Formation of red precipitates indicate the presence of reducing sugars.
(iii) Detection of glycosides
Extract is hydrolyzed with dilute hydrochloric acid and then subjected to test for glycosides.
Modified Borntrager's test
Extract is treated with ferric chloride solution and immersed in boiling water for about 5 minutes. The mixture is cooled and shaken with an equal volume of benzene. The benzene layer was separated and treated with ammonia solution. Formation of rose-pink colour in the ammoniacal layer indicates the presence of anthranol pyridine and methanolic alkali. Formation of pink to blood red colour indicate the presence of cardiac glycosides.
(iv) Detection of saponins
Legal's test
Extract is treated with sodium nitroprusside in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicate the presence of saponins.
Foam test
Small amount of extract is shaken with little quantity of water. The foam produced persisted for ten minutes, indicate the presence of saponins.
(v) Detection of phytosterols
Salkowski's test
Extract is treated with chloroform and filtered. The filtrate is treated with few drops of conc. sulphuric acid, shaken and allowed to stand. Appearance of golden yellow colour indicate the presence of triterpenes.
Tshugajeu test
Extract is treated with chloroform and filtered. Excess of acetyl chloride and a pinch of zinc chloride is added and kept aside for some time till the reaction was complete. After this it is warmed on water bath. Appearance of eosin red colour indicate the presence of triterpenes.
(vi) Detection of fixed oils and fats
Stain test
Small quantities of extract is pressed between two filter papers. An oily stain on filter paper indicate the presence of fixed oil.
(vii) Detection of resins
Acetone-water test
Extract is treated with acetone. Small amount of water is added and shaken. Appearance of turbidity indicate the presence of resins.
(viii) Detection of phenols
Ferric chloride test
Extract is treated with few drops of ferric chloride solution. Formation of bluish black colour indicate the presence of phenols.
(ix) Detection of tannins
Gelatin test
To the extract, gelatin solution (1%) containing sodium chloride is added. Formation of white precipitates indicate the presence of tannins.
(x) Detection of flavonoids
Alkaline reagent test
Extract is treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on addition of dilute acid, indicate the presence of flavonoids.
Lead acetate test
Extract is treated with few drops of lead acetate solution. Formation of yellow colour precipitates indicate the presence of flavonoids.
Shinoda test
To the alcoholic solution of extract, a few fragments of magnesium ribbon and conc. HCl is added. Appearance of magenta colour after few minutes indicate the presence of flavonoids.
Zinc hydrochloric acid reduction test
To the alcoholic solution of extract, a pinch of zinc dust and concentrated HCl has been added. Appearance of magenta colour after few minutes indicate the presence of flavonoids.
(xi) Detection of proteins and amino acids
Ninhydrin test
To the extract, ninhydrin reagent (0.25%) is added and boiled for few minutes. Formation of blue colour indicate the presence of amino acid.
Biuret test The extract is treated with 1 ml of sodium hydroxide solution (10%) and heated. To this a drop of copper sulphate solution (0.7%) is added. Formation of purplish violet colour indicate the presence of proteins.
EXPERIMENAL PROTOCOL
Animals selected: Albino Wistar rats (200-250g)
Grouping of animals
In each group, the isolated rat heart is allowed to stabilize for 20 minutes and has been perfused with Kreb's-Henseleit solution (K-H), during the stabilization period (Bhatti et al., 2008).
Group I: Control group; n=5. The isolated rat heart, after stabilization is perfused with K-H solution then subjected to 30 min of global ischemia followed by 120 min of reperfusion.
Group-II: Curcuma amada (100mg/kg) treated group; n=5. After stabilization, the heart is perfused with K-H solution containing Curcuma amada ethanolic extract for 40 min. Then hearts will be subjected to 30 min ischemia followed by 120 min of reperfusion with K-H solution containing Curcuma amada ethanolic extract (100mg/kg).
Group-III: Preconditioning group; n=5. The isolated rat hearts, after stabilization is subjected to four episodes of ischemia for 5 min and reperfusion for 5 min along with K-H solution to produce ischemic preconditioning. Further, rat hearts has been subjected to 30 min of ischemia followed by 120 min of reperfusion.
Group-IV: Postconditioning group; n=5. The isolated rat hearts, after stabilization is subjected to 30 min of ischemia followed by three episodes of ischemia and reperfusion for 10 sec applied immediately at the onset of 120 min of reperfusion.
Group V: Curcuma amada (100mg/kg) preconditioned group; n=5. The isolated rat hearts after stabilization is subjected to four episodes of ischemia for 5 min and reperfusion for 5 min along with K-H solution containing Curcuma amada ethanolic extract (100mg/kg) to produce preconditioning. Further, rat hearts has been subjected to 30 min of ischemia followed by 120 min of reperfusion.
Group VI: Curcuma amada (100mg/kg) postconditioned group; n=5. The isolated rat hearts, after stabilization is subjected to 30 min of ischemia followed by three episodes of ischemia and reperfusion for 10 sec along with K-H solution containing Curcuma amada ethanolic extract (100mg/kg) to produce postconditioning. These episodes shall be applied immediately at the onset of 120 min of reperfusion.
Isolated rat heart preparation
Rats are heparinized (500 IU, i.p.) about 20 min before sacrificing the animal by cervical dislocation. The heart is rapidly excised and immediately mounted on a Langendorff's apparatus. Isolated heart is then retrogradely perfused at constant pressure of 80mm Hg with Kreb's Heneseleit (KH) buffer (NaCl 118 mM; KCl 4.7 mM; CaCl2 2.5 mM; MgSO4.7H2O 1.2 mM; NaHCO3 25 mM; KH2PO4 1.2 mM; C6H12O6 11mM), pH 7.4, maintained at 37ã‚œC and bubbled with 90% O2. Flow rate is maintained at 7-9 ml/min using Hoffman's screw. The heart is enclosed in a double wall jacket, maintain the temperature of circulating water at 37ã‚œC. Global ischemia is produced for 30 min by blocking the inflow of Kreb's Henseleit solution, followed by reperfusion for 120 min. Four episodes of ischemia and reperfusion after stabilization, each comprising of 5 min occlusion and 5 min reperfusion, are used to produce ischemic preconditioning (in case of preconditioning groups). Coronary effluent is collected immediately, 5min and 30 min after reperfusion for estimation of lactate dehydrogenase (LDH) and creatine kinase, CK (Bhatti et al., 2008).
Assessement of myocardial injury
The myocardial infarct size is measured using the triphenyltetrazolium chloride (TTC) staining method while the level of LDH and CK in coronary effluents are estimated (Garg et al., 2010).
Assessment of myocardial infarct size
The heart is removed from the langendorff apparatus. Then both the auricles and roots of aorta are excised and ventricles are kept overnight at 0ã‚œC. Frozen ventricles are sliced into uniform sections of 2-3 mm thickness. The slices are incubated in 1% triphenyltetrazolium chloride (TTC) at 37ã‚œC in a 0.2 M Tris buffer (pH 7.4) for 20 min. TTC is converted to red formazone pigment by NADH and dehydrogenase enzyme and therefore the viable cells stained deep red. The infracted cells have lost the enzyme and cofactor and thus remained unstained or dull yellow. The ventricular slices are placed between two glass plates. A transparent grid with 100 squares in 1 cm2 is placed over it. Average area of ventricular slice is calculated by counting the number of squares on either side. Similarly, numbers of squares falling over non-stained dull yellow area is counted. Infarct size is expessed as percentage of average ventricular area. Whole of ventricular slices are weighed. Infarct size is expressed as a percentage total ventricular weight (Bhatti et al., 2008).
Estimation of lactate dehydrogenase (King, 1959)
LDH is estimated in samples of coronary effluent collected after stabilization, immediately and 30 min after reperfusion using 2, 4-DNPH method.
Principle
LDH catalyses the following reaction:
Lactate + NAD Pyruvate + NADH
The pyruvate so formed is coupled with 2, 4-dinitrophenylhydrazine (2,4-DNPH) to give correspondence hydrazone which gave a brown colour in alkaline medium. The intensity of this colour is proportional to the amount of LDH activity and is measured spectrophotometrically at 440 nm.
Preparation of reagents:
1) Glycine buffer
Glycine buffer (100 mM) is prepared by dissolving 7.505 g of glycine and 5.85 g of sodium chloride in distilled water. The final volume is adjusted to 1 litre.
2) Buffered lactate substrate
Buffered lactate substrate (pH 10) is prepared by adding 5 ml of sodium lactate solution (70%) to a mixture of 125 ml of glycine buffer and 75 ml of 100 mM sodium hydroxide.
3) NAD+ solution
NAD+ solution is prepared by dissolving 10 mg of NAD+ in 2 ml of distilled water.
4) DNPH colour reagent
200 mg of 2, 4-dinitrophenylhydrazine (DNPH) was dissolved in hot 1 M HC1 and volume is made upto 1 litre with 1 M HC1.
5) Sodium hydroxide solution
400 mM solution of sodium hydroxide is prepared by dissolving 16 g of sodium hydroxide in distilled water and final volume is made upto 1 litre.
6) NADH solution
NADH solution is prepared by dissolving 8.5 mg of NADH in 10 ml of buffered lactate.
7) Standard pyruvate solution
Pyruvate solution (1µM) is prepared by dissolving 11 mg of sodium pyruvate buffered lactate and volume is made to 100 ml.
Procedure
Control (C):
0.1 ml of distilled water is added to 0.5 ml of buffered lactate. The contents are mixed well and incubated at 370C for 20 min. To the above solution, 0.5 ml of DNPH colour reagent and 0.05 ml of coronary effluent (collected immediately after reperfusion) is added. Contents are vortexed and incubated at 370C for 15 min. Then, 5 ml of sodium hydroxide solution is added and the mixture is allowed to stand at room temperature for 5 min.
Standard Curve
Tube No.
1
2
3
4
5
6
7
Enzyme Activity (IU/L)
0
167
333
500
667
833
1000
Pyruvate solution (ml)
000
0.05
0.10
0.15
0.20
0.25
0.30
NADH solution (ml)
000
0.05
0.10
0.15
0.20
0.25
0.30
Buffered lactate (ml)
1.00
0.90
0.80
0.70
0.60
0.50
0.40
NAD+ solution (ml)
000
0.20
0.20
0.20
0.20
0.20
0.20
Distilled water (ml)
0.30
0.10
0.10
0.10
0.10
0.10
0.10
DNPH reagent (ml)
1.00
1.00
1.00
1.00
1.00
1.00
1.00
Mixed and incubated at 37 ï‚°C for 15 min
NaOH solution (ml)
10
10
10
10
10
10
10
All the tubes are vortexed and optical density is measured spectrophotometrical at 440 nm taking tube 1 as blank, standard curve is plotted taking enzyme activity on X-axis and optical density on Y-axis (Figure 3).
Test (T):
0.05 ml of coronary effluent (collected after stabilization of heart, immediately or 30 after reperfusion) is added to 0.05 ml of buffered lactate. Solution is incubated at 370C for 5 min, after vortexing. To the above contents, 0.1ml of NAD+ solution is added, vortexed and incubated at 370C for 15 min. It is followed by addition of 0.5 ml of DNPH colour reagent and contents are incubated at 370C for 15 min. Finally, 5 ml of sodium hydroxide solution is added. Contents are mixed thoroughly and allowed to stand at room temperature for 5 min.
Calculations
Absorbance of test (AT) and control (AC) is measured spectrophotometrically at 440 nm.
Net absorbance of test (AN) = AT - AC
Enzyme activity is calculated from standard plot by marking AN on Y-axis and extrapolating it to corresponding enzyme activity on X-axis.
Estimation of creatine phosphokinase (CK) (Ochei and Kolhatkar, 2006)
CK is measured in samples of coronary effluent after stabilization and 5 min after reperfusion using modified method.
Principle
CK catalyses the following reaction:
Creatine phosphate + ADP Creatine + ATP
At pH 7.4, CK catalyses the forward reaction. The creatine so formed, reacted with diacetyl and α-naphthol in alkaline medium to give pink coloured complex. The intensity of this colour is proportional to enzyme activity and is measured spectrophotometrically at 520 nm. Mg2+ and cysteine are added as activators. P-chloromercuribenzoate is used to stop the reaction by inactivating the enzyme.
Preparation of Reagents
Tris buffer
Tris buffer (pH 7.4) 100 mM is prepared by dissolving 12.1 g of Tris in 820 ml of 100 mM HCl and volume is made upto 1 litre with distilled water.
Sodium hydroxide (0.1N)
Sodium hydroxide (0.1N) is prepared by dissolving 4 g of sodium hydroxide in 1000 ml of distilled water.
Creatine phosphate solution
Creatine phosphate solution is prepared by dissolving 3mg of creatine phosphate in 1ml of distilled water and then 0.4 ml of the Tris buffer and then 2 drops of 0.1N NaOH are added and mixed well. After this 24 mg of cysteine hydrochloride is added to the above solution.
ADP solution
ADP solution is prepared freshly by dissolving 75 mg of ADP in 3ml of distilled water.
p-chloromercuribenzoic acid
a) 1.07 mg of p-chloromercuribenzoic acid is dissolved in 100 ml of 1N NaOH.
b) 25 ml of solution (a) is mixed with 22ml of 1N HCl and 53ml of distilled water.
Alkaline EDTA solution
10 ml of 1N NaOH is dissolved in 1 litre of distilled water. Then 1 g of EDTA is dissolved in above mixture.
Sodium hydroxide (3N)
Sodium hydroxide (3N) is prepared by dissolving 114 g of sodium hydroxide in 1000 ml of distilled water.
Sodium carbonate (6N)
Na2CO3 (6N) is prepared by dissolving 612 g of Na2CO3 in 1000 ml of distilled water.
α-Naphthol solution
Just before use, 50 mg of of α-naphthol is added to a 1.5 ml of 3N NaOH and 1.5 ml of 6N Na2CO3.
Diacetyl solution
Commercially available stock solution of diacetyl is diluted 2000 times.
Standard creatine solution
A stock solution is prepared by dissolving 29.8 mg of creatine hydrate in 100 ml of alkaline EDTA.
Procedure
Blank (B)
To 0.4 ml creatine phosphate, 0.05 ml of distilled water and 0.2 ml of ADP solution is added. Solution is vortexed and incubated at 37°C for half an hour. To the above solution, 0.5 ml of p-chloromercuribenzoic acid, 3.75 ml of EDTA, 1.0 ml of α-naphthol and 0.5 ml of diacetyl solution are added. The solution is thoroughly mixed and incubated in dark (to avoid degradation of α-naphthol) at room temperature for 30 min.
Standard (S)
To 0.4 ml creatine phosphate, 0.2 ml of ADP is added. Solutions are vortexed thoroughly and incubated at 37°C for half an hour. To the above solution, 0.05ml of creatine standard, 0.5 ml of p-chloromercuribenzoic acid, 3.75 ml of EDTA, 1.0 ml of α-naphthol, 0.5 ml of diacetyl solution are added. The solution is thoroughly mixed and incubated in dark (to avoid degradation of α-naphthol) at room temperature for 30 min.
Test (T)
To 0.4 ml of creatine phosphate solution, 0.2 ml of ADP solution and 0.05ml of coronary effluent (collected after stabilization of heart and after 5 min of reperfusion) are added. To the above solution, 0.5 ml of p-chloromercuribenzoic acid, 3.75 ml of EDTA, 1.0 ml of α-naphthol and 0.5 ml of diacetyl solution are added. The solution is thoroughly mixed and incubated in dark (to avoid degradation of α-naphthol) at room temperature for 30 min. After 30 min of incubation in dark, absorbance of test (AT), standard (AS) and blank (AB) are measured spectrophotometrically at 520 nm.
Calculations
AT - AB 2000
CK (1U/L) = X
AS - AB 30
