Exploring Aqueous Extracts Of Aloe Vera Gel Biology Essay

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Diabetes is one of the highest prevailing diseases not only in India but also in rest of the world. Despite of large number of anti-diabetic drugs available, the number of cases of diabetes is still increasing. Moreover the conventional therapy is associated with frustrating side effects, high cost and provide only symptomatic reliefto the patients. This may be due to the reason that the present therapy is oriented towards a particular pathogenic process. So herbal drugs which are having multimodal approach may prove beneficial in the aspects where conventional therapy lacks. Diabetes and its complications are proved to have multifactorial etiology. Oxidative stress is reported as the major contributor of diabetic complications, in association with other factors like inflammation, metabolic disturbances and immune stimulation. Aloe vera, Withania somnifera and Azadirachta indica have established antidiabetic, antioxidant, anti-inflammatory & immunomodulatory but their effect in further vascular complications of diabetes with regard to oxidative stress has not yet been evaluated.

Exploring aqueous extracts of Aloe vera gel, Withania somnifera root and Azadirachta indica leaf against vascular complications in alloxan-induced diabetic rats.

To evaluate the cardioprotective effect of aqueous extracts of Aloe vera gel, Withania somnifera root and Azadirachta indica leaf on cardiac damage in alloxan- induced diabetic rats.

To evaluate the cardioprotective effect of aqueous extracts of Aloe vera gel, Withania somnifera root and Azadirachta indica leaf in isoproterenol induced cardiac damage in alloxan-induced diabetic rats.

To evaluate the protective effect of aqueous extracts of Aloe vera gel, Withania somnifera roots and Azadirachta indica leaf on retinal damage in alloxan-induced diabetic rats.

To evaluate the protective effect of aqueous extracts of Aloe vera gel, Withania somnifera roots and Azadirachta indica leaf on renal damage in alloxan-induced diabetic rats.

Materials and methods

Diabetes is induced in overnight fasted rats with a single intraperitoneal injection of alloxan (150 mg/kg). Blood glucose levels will be estimated 72 hours after collecting blood samples from retro-orbital plexus by using the commercial glucose estimation kit. The blood samples are collected on 0th, 4th, 7th, 14th and at the end of study. The animals having fasting blood glucose level (FBG) above 150mg/dl are considered as diabetic and will be selected for the study. The treatment will be started from 7th day of alloxan injection till the 30th day. The blood parameters to be assessed are blood glucose level, total cholesterol, triglycerides, high density lipoprotein (HDL), low density lipoprotein (LDL), serum creatinine and blood urea nitrogen. The tissue parameters to be assessed are thiobarbituric acid residue (TBARS), glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), lactate dehydrogenase (LDH).

Assessment of blood parameters

Glucose

Total Cholesterol

HDL (High density lipoprotein)

Triglycerides

Creatinine

Urea

Assessment of tissue parameters

TBARS (Thiobarbituric acid residues)

GSH (Glutathione)

CAT (Catalase)

LDH (Lactate dehydrogenase)

CK (Creatinine kinase)

Infract size

Assessment of blood parameters

Rats will be fasted overnight during which they will have free access to water. The animals will then be anaesthesized using diethyl ether and blood is collected from retro-orbital plexus using fine glass capillaries. The blood will be collected in eppendroff tubes and is centrifuged immediately at 3000 rpm for 10 minutes in order to separate the serum. The serum so collected will be used for estimation of glucose, high density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol,triglycerides, urea, creatinine on 0th, 4th, 7th, 14th day and at the end of study (Morani and Bodhankar, 2007).

Estimation of serum glucose level

The blood glucose level is estimated by standard glucose kit.

Principle: The glucose is oxidised to gluconic acid and hydrogen peroxide in the presence of glucose oxidase. In the presence of enzyme released hydrogen peroxide is coupled with phenol and 4-aminoantipyrine to form coloured quinoneimine dye. Absorbance of coloured dye is measured at 505nm and is directly proportional to glucose concentration in the sample (Trinder, 1969).

Glucose + O2 + H2O glucose oxidase gluconic acid + H2O2

H2O2 + phenol + 4-AAP peroxidase quinoneimine dye + H2O

Procedure:

The test tubes will be marked as Blank (B), Standard (S), Test (T).

1500μl working glucose reagent will be added in all the test tubes.

20μl of reagent 3 will be added into (S).

20μl serum will be added in test tube marked (T).

1500μl of purified water is added in all three test tubes.

The above solution will be mixed well and incubated at 370C for 10 minutes.

Absorbance of Standard (S) followed by the Test (T) will be measured at 505 nm using UV spectrophotometer.

Formula used: Serum Glucose (mg/dl) = OD of test/ OD of standardÃ- 100

Estimation of Total Cholesterol and High Density Lipoprotein (One step method)

Principle:.Cholesterol reacts with hot solution of ferric perchlorate, ethyl acetate, and sulphuric acid (Cholesterol reagent) and gives a lavender coloured complex which is measured at 560 nm.

High density lipoprotein (HDL) will be obtained in the supernatant after centrifugation. The cholesterol in HDL fraction is also estimated by this method. HDL and total cholesterol levels will be expressed in mg/ dl (Wybenga and Pileggi., 1970).

Procedure (Total cholesterol):

Test tubes will be marked as Blank (B), Standard (S), Test (T).

3ml Cholesterol reagent will be added in all the three test tubes.

0.015 ml of working cholesterol standard (200 mg%) will added in (S) using micropipette.

0.015 ml of serum will be added in (T).

Contents will be mixed well and keep the tubes immediately in the boiling water exactly for 90 seconds.

Test tubes will be cooled immediately to room temperature under running tap water.

Measure the O.D. of Standard (S) and Test (T) against Blank on UV spectrophotometer at 560 nm.

Formula used: Total Cholesterol in serum= O.D. Test/ O.D. StandardÃ-200

Procedure of HDL estimation

0.2 ml of serum and precipitating agent will be taken, mixed well and kept for 10 minutes at room temperature and then will be centrifuged at 2000 rpm for 10 minutes to obtain a clear supernatant.

Test tubes will be marked as Blank (B), Standard (S), and Test (T).

0.015 ml of working cholesterol standard (200 mg %) will be added in (S).

0.015 ml of supernatant from step (A) will be added in (T).

Contents will be mixed well and the tubes will be kept immediately in the boiling water exactly for 90 seconds.

Test tubes will be cooled immediately to room temperature under running tap water.

Measure the O.D. of Standard (S) and Test (T) against Blank on spectrophotometer at 560nm.

Formula used: HDL in serum= O.D. Test/ O.D. Standard

Estimation of serum triglycerides

Principle: Lipoprotein lipase hydrolyses triglycerides to glycerol and free fatty acids. The glycerol formed with ATP in the presence of glycerol kinase forms glycerol-3-phosphate which is oxidized by enzyme glycerol phosphate oxidase to form hydrogen peroxide. The hydrogen peroxide further reacts with phenolic compound and 4-aminoantipyrine by the catalytic action of peroxidase to form a red coloured quinoneimine dye complex. Intensity of the colour formed is directly proportional to the amount of triglycerides present in the sample.

The serum triglyceride will be estimated by glycerophosphate oxidase peroxidase (GOD-PAP) method using commercially available kit. 1000 µl of enzyme reagent will be added to 10 µl of serum, 10 µl of standard (200 mg/dl) and 10 µl of purified water to prepare test, standard and blank, respectively. All the test tubes will be incubated at room temperature for 15 min. The absorbances of test and standard samples will be noted against blank at 505 nm spectrophotometrically (Godkar & Godkar, 2008).

The serum triglyceride will be calculated using the following formula:

Triglycerides (mg/dl) =

Absorbance of Test

Absorbance of Standard

X 200

Estimation of serum creatinine

The serum creatinine concentration will be estimated by alkaline picrate method using commercially available kit. 2.0 ml of picric acid reagent added to 0.2 ml of serum for deproteinization of specimen. It will be mixed well and centrifuged at 3000 rpm to obtain clear supernatant. For development of colour, 100 µl of buffer reagent will be added to 1.1 ml of supernatant, 0.1ml of standard creatinine and 0.1 ml of purified water to prepare test, standard and blank, respectively. 1.0 ml of picric acid reagent will be added to blank and standard. It will be mixed well and test tubes will be kept at room temperature for 20 minutes. The alkaline picrate reacts with creatinine to form the orange coloured complex. The absorbances of test and standard samples will be noted against the blank at 520 nm spectophotometrically. The serum creatinine concentration will be calculated using the following formula (Godkar & Godkar, 2008):

The serum creatinine concentration (mg/dl) =

Absorbance of test Test

Absorbance of Standard

X 2

Estimation of blood urea nitrogen

The blood urea nitrogen will be estimated by using the commercially available kit. 1000 µl of working reagent (1) will be added to 10 µl of serum, 10 µl of standard and 10 µl of purified water to prepare test, standard and blank, respectively. All the test tubes will be mixed well and incubated for 5 min at 37 °C in incubator. Then 1000 µl of working reagent (2) will be added to all the test tubes. All the test tubes will again be mixed well and incubated for 10 min at 37 °C in incubator. The absorbances of test and standard samples will be noted against blank at 578 nm spectophotometrically (Godkar & Godkar, 2008).

The blood urea will be calculated using the following formula:

Blood urea (mg/dl) =

Absorbance of Test

Absorbance of Standard

X 40

Blood urea nitrogen in mg/dl = blood urea x 0.467

Assessment of tissue parameters

Thiobarbituric acid residues (Ohkawa, 1979; Kartikeyan, 2003)

Preparation of reagents:

8.1% sodium dodecyl sulphate: 810mg of SDS will be dissolved in 10 ml of distilled water to obtain 8.1% of sodium dodecyl sulphate.

20% acetic acid: 20ml of acetic acid will be diluted to 100ml with distilled water and pH- 3.5 will be adjusted with standard sodium hydroxide.

0.8% thiobarbituric acid: Dissolve 400mg of thiobarbituric acid in 50ml of warm water.

15:1 v/v of n-butanol and pyridine mixture: 90ml of n-butanol will be mixed with 6ml of pyridine.

Procedure:

Reaction mixture will be prepared by mixing 0.2ml of tissue homogenate, 0.2ml of 8.1% SDS, 1.5ml of 20% acetic acid, 1.5ml of thiobarbituric acid and 0.6ml of distilled water.

The above mixture will be incubated at 95oC in water bath for 1 hour.

Cool with tap water. Add 1ml of distilled water and mixture of butanol and pyridine (15:1 v/v).

Shake well and centrifuge at 4000 rpm for 10 minutes.

Note the absorbance of organic layer at 532nm.

Formula used:

TBARS= Optical density of organic layer

Extinction coefficient x Protein concentration x Incubation time x volume of sample

Extinction coefficient of chromophore = 1.56X 105 M-1cm-1

Result express as n moles of MDA/min/mg protein.

Glutathione (GSH)

GSH is estimated by method of Ellman. 5, 5 dithiobis (2- nitrobenzoic acid) i.e. DTNB is reduced by -SH group present in glutathione and immense yellow coloured 2-nitro-5-mercapto benzoic acid is formed (Kartikeyan, 2003).

Preparation of reagents:

10% trichloroacetic acid: Dissolve 10g of trichloroacetic acid in 100ml of distilled water.

0.3 M disodium hydrogen phosphate buffer: Dissolve 4.26g of anhydrous disodium hydrogen phosphate in 10ml of distilled water and adjust pH to 8.4 with NaOH.

Ellman reagent: Dissolve 7.92mg of DTNB in 20ml of 1% sodium citrate.

Procedure

Tissue homogenate with 10% trichloroacetic acid is centrifuged at 3000 g for 10 minutes.

The reaction mixture contain 0.5 ml of supernatant, 2 ml of 0.3M of phosphate buffer, 0.4 ml of double distilled water and 0.5 ml of DTNB.

The reaction mixture is incubated for 10 minutes and absorbance is noted at 412 nm.

Formula used

GSH = Optical density

Extinction coefficient x Protein concentration x Volume of sample

Extinction coefficient of chromophore = 13600 M-1cm-1

Result is expressed as n moles of GSH per mg of protein.

Catalase

Catalase level is estimated by method of Aebi (Kartikeyan, 2003).

Preparation of reagents:

50 mM phosphate buffer: 680 mg of potassium dihydrogen phosphate is dissolved in 100 ml of distilled water. 200 mg of sodium hydroxide is dissolved in 100 ml of distilled water. 50 ml of potassium dihydrogen phosphate is mixed with 29.1 ml of sodium hydroxide solution and make volume upto 200ml.

30 mM hydrogen peroxide: 102 mg of H2O2 is dissolved in distilled water and make volume upto 100ml.

Procedure

Tissue homogenate in 50 mM phosphate buffer (pH-7.4) is centrifuged at 3000 g for 10 minute.

Add 50 μl of supernatant to 1.95 ml of 50 mM phosphate buffer and 1.0 ml of 30 mM hydrogen peroxide.

Note absorbance at 240 nm.

Formula used

Catalase = ΔE /min

Extinction coefficient x Protein concentration x Volume of sample

Extinction coefficient= 0.071M-1cm-1

Lactate dehydrogenase (King, 1959)

LDH is estimated in by method of King.

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 was proportional to the amount of LDH activity and was measured spectrophotometrically at 440 nm.

Preparation of reagents

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. Make final volume to 1 litre.

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.

NAD+ solution: NAD+ solution is prepared by dissolving 10 mg of NAD+ in 2 ml of distilled water.

DNPH colour reagent: 200 mg of 2, 4-dinitrophenylhydrazine (DNPH) is dissolved in hot 1 M HC1 and volume is made upto 1 litre with 1 M HC1.

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.

NADH solution: NADH solution is prepared by dissolving 8.5 mg of NADH in 10 ml of buffered lactate.

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 incubate at 370C for 20 min. To the above solution add 0.5 ml of DNPH colour reagent and 0.05 ml of tissue homogenate. 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

Mix and incubate at 37 °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.

Test (T):

Add 0.05 ml of tissue homogenate to 0.05 ml of buffered lactate. Solution is incubated at 370C for 5 min, after vortexing. To the above contents add 0.1ml of NAD+ solution, vortex and incubate 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 allow 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.

Creatine phosphokinase (Ochei and Kolhatkar, 2006)

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:

1.07 mg of p-chloromercuribenzoic acid is dissolve in 100 ml of 1N NaOH.

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 tissue homogenate 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 Ã- 2000

CK (1U/L) = AS - AB 30

Infarct size measurement

Infarct size is measured by macroscopic method. The hearts are removed and both the auricles and the root of the aorta are excised, and the ventricles are frozen. These are then sliced into uniform sections of 2-3 mm thickness and incubated in 1% triphenyltetrazolium chloride (TTC), at 37° C in 0.2M Tris buffer (pH 7.4), for 20 minutes. Triphenyltetrazolium chloride is converted to red formazone pigment by reduced Nicotinamide Adenine Dinucleotide (NADH) and dehydrogenase enzyme and, therefore, stained the viable cells deep red, while the infracted cells remain unstained or dull yellow. The ventricular slices

are placed between two glass plates and a transparent plastic grid with 100 squares in 1 cm2 is placed above it. The average area of each slice is calculated by counting the number of squares on either side and similarly the non stained dull yellow area is counted. The infracted area is expressed as a percentage of the total ventricular area (Bhatti et al., 2008).

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