Bark Of The Anogeissus Latifolia Biology Essay

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Bark of the Anogeissus latifolia was collected near from Siruvani dam, Coimbatore, Tamil Nadu and authenticated by Botanical Survey of India (BSI), Southern circle, Coimbatore, Tamil Nadu. The authentication certificate number is BSI/SC/5/23/09-10/tech-287(annexure). The Bark was separated from adulterants, shade dried and powdered coarsely. It was packed in air-tight container up to the completion of study.

2. EXTRACTION OF PLANT MATERIAL

About 150 gm of air dried powdered plant material was taken in Soxhlet apparatus and extracted with petroleum ether for upto discolouration of solution. After 72 h, the powder was taken out and dried. The Aqueous extract of Anogeissus latifolia (AEAL) was prepared by continuous percolation and concentrated under reduced pressure using rota-evaporator. The concentrated extract was stored in refrigerator at 10°C upto completion of all the pharmacological studies.

3. EXPERIMENTAL ANIMALS

Male and female wistar albino rats (150-200 g) were used for the antidiabetic and anti-inflammatory studies respectively. Male swiss albino mice (25-30 g) were used for the analgesic activity evaluation. In the case of acute toxicity study, female, nalliporious, non-pregnant wistar albino rats (150-200 g) was used. The animals were obtained from Government agricultural university, Mannuthy, Thrissur, Kerala. All animals were housed for at least one week in the laboratory animal room prior to study. The selected animals were housed in polypropylene cages in the standard environmental conditions (20-25°C), 12:12 light: dark cycle, fed with standard rodent diet and water ad libitum. The experiments on animals were conducted in accordance with the international accepted principle for laboratory animal use and the experimental protocols duly approved (KMCRET/Mpharm/4109) by the institutional animal ethical committee (IAEC) of KMCH College of Pharmacy, Coimbatore-48.

4. INSTRUMENTS, CHEMICALS AND DRUGS

Streptozotocin (Himedia, Mumbai).

Propylene glycol (Qualigens fine chemicals, Mumbai).

Acetic acid (0.6 % v/v).

Carrageenan (Himedia, Mumbai) solution (1%) was prepared by dissolving 50 mg in 5 ml normal saline.

Indomethacin (Micro laboratories limited, Himachal Pradesh)

Morphine (Tablets (India) limited, Chennai)

HDL-cholesterol kit (Nicholas Piramal India limited).

Cholesterol kit (Nicholas Piramal India limited).

Triglycerides kit (Nicholas Piramal India limited).

HbA1c kit (Nicholas Piramal India limited).

Haemoglobin kit (Nicholas Piramal India limited).

Creatinine kit (Nicholas Piramal India limited).

Albumin kit (Nicholas Piramal India limited).

SGOT kit (Nicholas Piramal India limited).

SGPT kit (Nicholas Piramal India limited).

Total protein kit (Nicholas Piramal India limited).

Urea Berthodelt kit (Nicholas Piramal India limited).

Uric acid kit (Nicholas Piramal India limited).

Semi autoanalyser photometer 5010 V5+ (Rieles & Nicholas Piramal India limited).

Plethysmometer (INCO, Niviqure systems version 60.1, Ambala.)

Eddy's hot plate method (INCO, Ambala.)

5. PREPARATION OF EXTRACT DOSE

The AEAL extract was suspended in distilled water and administered to the respective groups of animals throughout the duration of the experiment. Each day AEAL was prepared freshly before administration.

6. PRELIMINARY PHYTOCHEMICAL ANALYSIS26

6.1. A. Test for carbohydrates

1. Molische test: The plant extract was treated with few ml of α-napthol and concentrated sulphuric acid along the sides of the test tube. Purple color or reddish violet color ring was produced at the junction between two liquids.

2. Fehling's test: To the extract, equal quantity of fehling's solution A and B was added. Heat gently brick red precipitate was obtained.

3. Benedict's test: To the extract, 5 ml of Benedict's reagent was added. Mix well, boiling the mixture vigorously for two minutes and then cool. Red precipitate was obtained.

4. Barfoed's test: To the extract, Barfoed's solution was added; heat to boiling, formation of red precipitate of copper oxide was obtained.

6.1. B. Test for alkaloids

1. Dragendroff's test: To the extract, add 1 ml of dragendroff's reagent. Orange red precipitate was produced.

2. Wagner's test: To the extract add wagner reagent. Reddish brown precipitate was produced.

3. Mayer's test: To the extract add 1 or 2 ml of mayer's reagent. Dull white precipitate was produced.

4. Hager's test: To the extract add 3 ml of hager's reagent. Yellow precipitate was produced.

6.1. C. Test for steroids and sterols

1. Libermann burchard test: The test sample was dissolved in 2ml of chloroform in a dry test tube. Ten drops of acetic anhydride and 2 drops of conc., sulphuric acid were added. No red colour was obtained.

2. Salkowski test: The test sample was dissolved in chloroform and added equal volume of concentrated sulphuric acid. No bluish red cherry and purple color was noted in chloroform layer.

6.1. D. Test for glycosides

1. Legal's test: Sample was dissolved in pyridine and sodium nitro prusside solution was added to it and made alkaline. Pink red color was produced.

2. Baljet test: To the sample, sodium picrate solution was added. Yellow to orange color was produced.

3. Borntrager test: Add a few ml of dilute of sulphuric acid to the test solution. Boil, filter and extract the filtrate with ether or chloroform. Then organic layer was separated to which ammonia was added, pink, red, or violet color was produced in organic layer.

4. Keller-killiani test: Sample was dissolved in acetic acid containing trace of ferric chloride and transferred to the surface of conc., sulphuric acid. At the junction of liquid reddish brown color was produced which gradually became blue.

6.1. E. Test for saponins

About 1ml of alcoholic sample was diluted separately with distilled water to 20ml, and shaken in graduated cylinder for 15minutes. 1cm layer of foam indicates the presence of saponins.

6.1. F. Test for flavonoids:

Shinoda test: To the sample, magnesium turnings and then concentrated hydrocholic acid was added. Red color was produced.

6.1. G. Test for triterpenoid:

In the test tube, 2 or 3 granules of tin was added, and dissolved in a 2ml of thionyl chloride solution and test solution was added. Pink color was not produced which indicates the absence of triterpenoid.

6.1. H. Test for protein and amino acid

1. Biuret test: 1 ml of 40% sodium hydroxide and 2 drops of 1% copper sulphate was added to the extracts, a violet color indicates the presence of proteins.

2. Ninhydrin test: 2 drops of freshly prepared 0.2% ninhydrin reagent was added to the extracts and heat. Blue color was developed.

3. Xanthoprotein test: 20% of sodium hydroxide or ammonia was added to the extracts. Orange color was produced.

6.2. QUANTIFICATION OF TOTAL FLAVONOIDS AND PHENOLS

Estimation of total flavonoid content 27

Reagents

95% ethanol

10% aluminium chloride

Procedure

The total flavonoid content of the AEAL extract was determined by using aluminium chloride colorimetric method. Quercetin was used to make the calibration curve. Ten milligrams of quercetin was dissolved in 80% ethanol and then diluted to 20, 40, 60, 80 and 100 μg/ml. The diluted standard solutions (0.5 ml) were separately mixed with 1.5 ml of 95% ethanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm. The amount of 10% aluminum chloride was substituted by the same amount of distilled water in blank. Similarly, 0.5 ml of ethanol extracts were reacted with aluminum chloride for determination of flavonoid content as described above. All samples were analysed in triplicate. The mean of three readings was used and the total flavonoid contents were expressed as milligrams of quercetin equivalents per gram extract (Chia-Chi Chang et al., 2002).

6.2.2 Estimation of total phenolic content 28

Reagents

Folin-ciocalteu's

7% sodium carbonate

Procedure

The total phenolic content of the AEAL extract was determined by using the Folin-ciocalteu assay method. An aliquot (1 ml) of extract or standard solution of gallic acid (20, 40, 60, 80 and 100 μg/ml) were mixed with 0.2 ml of Folin-Ciocalteu reagent. After 5 min, 1 ml of 15% Na2CO3 solution and 2 ml of distilled water were added to the above mixture. Reagent blank using distilled water was prepared. After incubation for 90 min at room temperature, the absorbance of the mixture was measured against prepared reagent blank at 750 nm. Samples were analysed in triplicate. The mean of three readings was used and the total phenolic contents were expressed as milligrams of gallic equivalents per gram extract (Marinova et al., 2005).

Statistical Analysis:

The data for various biochemical parameters were analyzed using analysis of variance (ANOVA) Tukey, followed by compared all pairs of column.

6.3. Invitro Anti-oxidant Activity of Aqueous Extract of Anogeissus latifolia

6.3.1. DPPH Radical scavenging activity29

The scavenging activity of aqueous extract of Anogeissus latifolia extract was measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH (Blois, 1958). 0.1Mm solution of DPPH in ethanol was prepared and 1.0ml of this solution was added to 3 ml of extract solution and standard in water at different concentrations (10-100µg/ml). 30 min later absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The capability to scavenge the DPPH radical was calculated using the following equation

Where Aconc was the absorbance of the control reaction and A test was the absorbance in the presence of the sample of the extracts. The mean values were obtained from triplicate experiments. The anti-oxidant activity of the extract was expressed as 1c50.The 1c50 value was 100defined as the concentration (in µg/ml) of extracts that inhibits the formation of DPPH radicals by 50%.

6.3.2. Reducing power 30

The reductive potential of the extract was determined according to the method of Oyaizu (1986). The different concentration of extracts and standard in 1ml of distilled water was mixed with phosphate buffer (2.5ml, 0.2m, pH6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5ml,1% w/v). The mixture was incubated at 50oC for 20min.2.5ml of trichloroaceticacid (10%w/v) was added to the mixture, which was then centrifuged for 10 min at 1000rpm. The 2.5 ml of supernatant was mixed with distilled water (2.5ml) and Fecl3 (0.5ml, 0.1% w/v), and mixed. Absorbance was measured at 700nm in a spectrophotometer. Higher absorbance of the reaction mixture indicated greater reductive potential.

6.3.3. Superoxide anion scavenging activity assay31

The scavenging activity of Anogeissus latifolia towards superoxide anion radicals was measured by the method of Ni-shimiki et.al, (1972) and slightly modified.

About 1ml of nitro blue tetrazolium solution (156µm in 100Mm phosphate buffer, pH 7.4) and 0.1ml of different concentrations of extract and standard in water were mixed. The reaction was initiated by adding 100µl of phenazinemethosulphate(PMS) solution (60µM) in 100Mm phosphate buffer(pH7.4) to the mixture. The reaction mixture was incubated at room temperature for 5 min and the absorbance at 560nm was measured against reagent blank in spectrophotometer. (Rutin was used as standard).The superoxide anion scavenging activity was calculated using the following equation

Where Aconc was the absorbance of the control (without extract) and Atest was the absorbance in the presence of the extract. The experiment was repeated in triplicate.

6.4.4. ABTS radical scavenging activity assay32

The ABTS radical scavenging activity of the extract was measured by Rice-Evans et.al.,(1997). ABTS radical cation (ABTS+) was produced by reacting ABTS solution (7mm) with 2.45 Mm ammonium persulphate and the mixture were allowed to stand in dark at room temperature for 12-16hrs before use. Different concentration (100-350µg/ml) of Aqueous extract and standard (0.5ml) were added to 0.3ml of ABTS solution and the ethanol to make 1ml.Quercetin was used as standard. The absorbance was read at 745nm. The experiment was performed in triplicate. The ABTS radical scavenging activity was calculated using the following equation

Where Aconc was the absorbance of the control (without extract) and Atest was the absorbance in the presence of the extract. The experiment was repeated in triplicate.

7. DETERMINATION OF ACUTE ORAL TOXICITY32

Table 2-Test substance details

Name of the test substance

Aqueous extract of Anogeissus latifolia Bark

Colour of the test substance

Brownish yellow

Nature of the test substance

Slightly Sticky

Table 3-Experimental protocol

Name of the study

Acute toxicity

Guideline followed

OECD 423 method-Acute toxic class method

Animals

Healthy young adult wistar albino rats, nulliparous, non-pregnant

Body weight

150-200 g

Sex

Female

Administration of dose and volume

2000 mg/kg body weight, single dose in 0.5 ml

Number of groups and animals

Two groups, each contains 3 mice were used to study LD50 value. First group was dosed 2000 mg/kg and observed for 24 h. again second group was dosed with 2000 mg/kg for the conformation an observed for 24 h. Both the groups were observed for 14 days to study presence or absence of any toxicity signs.

Route of administration

Oral by using rat oral feeding needle

Vehicle

Distilled Water

Table 4-Housing and feeding conditions

Room temperature

22°C ± 3°C

Humidity

40-60%

Light

12 h : 12h (light : dark cycle)

Feed

Standard laboratory animal food pellets with water ad libitum

Table 5-Study period and observation parameters

Initial once observation

First 30 minutes and periodically for 24 h

Special attention

First 1-4 h after drug administration

Long term observation

Upto 14 days

Direct observation parameters

Tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

Additional observation parameters

Skin and fur, eyes and mucous membrane, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern etc.

The time of death, if any, is recorded. (Complete observations: annexure I). After administration of the drug, food is withheld for a further 1-2 hours.

Study procedure

Acute oral toxicity was performed as per the Organization for Economic Co-operation and Development (OECD) guideline 423 method. The AEAL substance was administered in a single dose by gavage using specially designed rat oral needle. Animals are overnight fasted prior to dosing (food was withheld for 3 h but not water). Following the period of fasting, animals was weighed and test substance was administered. After the AEAL substance administration, food was withheld 2 h in rats. Animals are observed individually after at least once during the first 30 minutes, periodically during the first 24 hrs, with special attention given during the first 4 hrs, and daily thereafter, for a total of 14 days. Those animals which are found dead or in extreme distress if any are removed and humanely killed for animal welfare reasons.

8. PHARMACOLOGICAL PROPERTIES EVALUATION

8.1. ANTIDIABETIC ACTIVITY AND ANTIHYPERLIPEDEMIC ACTIVITY8, 20, 34

8.1.1. Induction of experimental diabetes8, 34

Rats, overnight fasted, were injected with streptozotocin (60 mg/kg, i.p) to induce experimental type 1 diabetes. STZ induces fatal hypoglycemia as a result of massive pancreatic insulin release, the rats were provided with 5% dextrose solution after 6 h of STZ administration for next 24 h to prevent hypoglycemia. Diabetic state was confirmed 72 h after induction of diabetes by measurement of tail vein blood glucose levels with the glucose meter (Glucocard™ 01-mini, Arkray Factory, Inc., Japan) and rats were kept for 14 days under standard laboratory conditions for the stabilization of the blood glucose level. After 14 days, the induction of diabetes was again confirmed by measuring the blood glucose levels. Only those animals with a blood glucose level ≥ 200 mg/dl were selected for the study.

8.1.2. Experimental design for antidiabetic activity8

The diabetic rats were divided into five groups.

Group 1: Control rats received propylene glycol (5 ml/kg).

Group 2: Diabetic rats received propylene glycol (5 ml/kg).

Group 3: Diabetic rats received AEAL 100 mg/kg.

Group 4: Diabetic rats received AEAL 200 mg/kg.

Group 5: Diabetic rats received standard drug glibenclamide (5 mg/kg).

The vehicle, AEAL and glibenclamide were administered to the respective group animals for 28 days. Throughout the study period, AEAL and glibenclamide was freshly suspended in propylene glycol before to the administration. The fasting animal body weight, blood glucose level was estimated on 0, 7th, 14th, 21st and 28th day. At the end of experimental period, rats were fasted overnight and anaesthetized with ketamine (100 mg/kg, i.p.); blood samples was collected through retro-orbital sinus puncture after 90 minutes administration of treatment doses to the respective group animals with or without EDTA containers for the biochemical parameters estimation.

8.1.3. Measurement of biochemical parameters estimation

Blood glucose (BGL), haemoglobin (Hb), glycosylated haemoglobin (HbA1c) was estimated using whole blood. All the above biochemical parameters were estimated using semi-autoanalyzer (Photometer 5010 V5+, Germany) with enzymatic kits procured from Piramal Healthcare limited, Lab Diagnostic Division, Mumbai, India.

8.1.3.1. Estimation of Glucose35

The sample (whole blood) is drawn by capillary action at the tip of the test strip. Glucose in the sample reacts with glucose oxidase and hexaammineruthenium (III) chloride in the test strip, producing hexaammineruthenium (II) chloride. Hexaammineruthenium (II) chloride is produced in proportion to the glucose concentration of the blood sample. Oxidation of the hexaammineruthenium (II) chloride produces an electric current. The meter converts the current to the glucose concentration and displays it as the test result.

B-D-glucose + Hexaammineruthenium (III) chloride GOD D-Glucono-lactone + Hexaammineruthenium (II) chloride

Hexaammineruthenium (II) chloride Hexaammineruthenium (III) chloride + e-

8.1.3.2. Total Cholesterol (TC) 36

Principle

Determination of cholesterol is done after enzymatic hydrolysis and oxidation. The colorimetric indicator is quinoneimine, which is generated from 4-aminoantipyrine and phenol by hydrogen peroxide under the catalytic action of peroxidase (trinder's reaction).

Cholesterol ester + H2O CHE Cholesterol + Fatty acid

Cholesterol + O2 CHO Cholesterol-3-one +H2O2

2H2O2 + 4- Amino antipyrine + Phenol POD Quinonelimine + 4 H2O2

Method

CHOD-PAP: Enzymatic photometric test

Table 6-Reagents

Goods buffer (pH 6.7)

50 mmol/ l

Phenol

5 mmol/l

4-aminoantipyrine

0.3 mmol/l

Cholesterol estrase

> 200 U/l

Cholesterol oxidase

> 100 U/l

Peroxidase

3 KU/l

Standard

(5.2 mmol/l)

Assay procedure

1 ml (1000 µl) of reagent-1 is taken in a 5 ml test tube.

Added 0.01 ml (10 µl) of serum.

Mixed well and incubated at 37â-¦C for 5 min.

Read the test sample.

NORMAL RANGE: < 200 mg/dl in serum.

8.1.3.3. Triglycerides37

Principle

Determination of triglycerides (TG) alters enzymatic splitting with lipoprotein lipase. Indicator is quinoneimine which is generated from 4-aminoantipyrine and 4- chlorophenol by hydrogen peroxidase under the catalytic action of peroxidase.

Triglycerides LPL Glycerol + fatty acid

Glycerol + ATP GK Glycerol-3-phosphate+ ADP

Glycerol-3-phosphate +O2 GPO Dihydroxyaceton phosphate + H2O2

2H2O2 + 4- Amino antipyrine + 4- chlorophenol POD Quinonelimine + HCL + 4H2O2

Method

Colorimetric enzymatic test using glycerol-3-phosphate-oxidase (GPO).

Reagents

Components and concentrations in the test Goods buffer pH 7.2, 50mmol/ l

Table 7-Reagents

4-chloroPhenol

4 mmol/l

ATP

2 mmol/l

Mg2+

15 mmol/l

Glycerokinase

> 0.4 Kµ/l

Peroxidase

> 2 Kµ/l

Lipoprotein lipase

> 4 Kµ/l

4-aminoantipyrine

0.5 mmol/l

Glcerol-3-phosphate- oxidase

> 1.5Kµ/l

Standard

(2.3 mmol/l)

Assay procedure

1 ml (1000 µl) of reagent-1 is taken in a 5 ml test tube.

Added 0.01 ml (10 µl) of serum.

Mixed well and incubated at 37â-¦C for 15 min.

Read the test sample.

Normal Range: < 200 mg/dl in serum.

8.1.3.4. HDL Cholestrol38

Principle

Chylomicrons, VLDL and LDL are precipitated by adding phosphotungstic acid and magnesium ions to the sample. Centrifugation leaves only the HDL in the supernatant. The cholesterol content in it is determined enzymatically.

Method

Phosphotungstic acid precipitation method.

Table 8-Reagents

Phosphotungstic acid

0.55 mmol/l

Magnesium chloride

25 mmol/l

Assay procedure

Preparation of supernatant for the HDL-CHL estimation

Added 200 µl of serum to the 500 µl of HDL-Cholesterol precipitating reagent (from HDL kit) in 1.5 ml centrifuge tube and mixed well. Centrifuged the above solution at 4000 rpm for 10 min.

Preparation of test sample for the estimation of HDL-Cholesterol

Taken 1000 µl of reagent-1 (from cholesterol kit) in a 5 ml test tube.

Added, 100 µl of supernatant from above centrifuged solution

Mixed well and incubated at 37â-¦C for 15 min.

Read the test sample.

Normal Range: > 60 mg/dl in serum.

8.1.3.5. VLDL and LDL cholesterol39

VLDL was calculated by the following equation.

VLDL = Triglycerides / 5.

LDL was calculated by the following equation.

LDL = TC-(HDL+VLDL)

8.1.3.6. Haemoglobin (Hb) 40

Principle

Haemoglobin + Cyanide + Ferricyanide cyanmethemoglobin

Method

Cyanmethemoglobin method.

Reagents

Reagent 1: Buffered cyanide/ Ferricyanide reagent.

Preparation of working reagent

Mix 5 ml of concentrated reagent to 100 ml of distilled water. Mix well and keep in dark.

Assay procedure

5 ml of working reagent was taken in a 10 ml test tube.

To this add, 20 µl of EDTA whole blood.

Mix well and incubate for 5 min at 37°C.

Read the test sample.

Normal Range: 14-18 mg/dl

8.1.3.7. Glycosylated haemoglobin (HbA1c) 41

Principle

Total Hb and HbA1c in hemolyzed blood binds with the same affinity to particles in R1. The amount of binding is proportional to the relative concentration of both substances in the blood. Mouse anti-human HbA1c monoclonal antibody (R2) binds to particle bound HbA1c. Goat anti-mouse IgG poly clonal antibody (R3) interacts with the monoclonal mouse anti-human HbA1c antibody and agglutination takes place. The measured absorbance is proportional to the HbA1c bound to particles, which in turn is proportional to the percentage of HbA1c in the sample.

Method

Particle enhanced immunoturbidimetric test HbA1c is determined directly without measurement of total haemoglobin.

Table 9-Components and concentrations

R1

Buffer

20 mmol/L

Latex

1.5%

R2

Buffer

10 mmol/L

Mouse anti-human Hb1Ac monoclonal antibody

5.5 mg/dl

R3

Buffer

1 mmol/L

Goat anti mouse IgG poly clonal anibody stabilizers

67 mg/dl

Assay procedure

Take 500 µl of lysine solution in 2 ml centrifuge tube. To this, add 10 µl of EDTA whole blood and mix well and wait for 5 min.

Transfer 10 µl of lysed sample from the above solution into another 2 ml centrifuge tube and add 240 µl of reagent-1. Mix well and wait for 2 min.

To this, add 80 µl of reagent-2 and mix well. Wait for 3 min.

To this, add 40 µl of reagent-3 and mix well. Wait for 2 min.

Read the test sample.

Normal Range: 4-6 % (Non -Diabetic)

8.1.3.8. Serum glutamate oxaloacetate transaminase (SGOT) 42

Principle

L-Aspartate + 2-Oxaloglutarate ASAT L-Glutamate + Oxaloacetate

Oxaloacetate + NADH + H⁺ MDH D-Malate + NAD⁺

Addition of pyridoxal-5 phosphate (P-5-P) stabilizes the transaminases and avoids falsely low values in samples containing insufficient endogenous P-5-P, eg. From patients with myocardial infarction, liver diseases and intensive care patients.

Method

Optimized UV test according to the IFCC (International Federation of Clinical Chemistry and Laboratory Medicine).

Reagents

R1 : TRIS pH 7.8 80 mmol/l

L- Aspartate 240 mmol/l

MDH(malate dehydrognase) ≥600 U/l

LDH (lactate dehydrogenase) ≥600 U/l

R2 : 2-Oxaloglutarate 12 mmol/l

NADH 0.18 mmol

Pyridoxal-5-Phosphate FS

Good buffer pH 9.6 0.7 mmol/l

Pyridoxal-5-Phosphate 0.09 mmol/l

Assay procedure

Mixed 800 µl of reagent-1 with 200 µl of reagent-2 in a 5 ml test tube.

To this, added 100 µl of serum.

Mixed well and took the reading immediately.

Normal Range: < 41 U/l

8.1.3.9. Serum glutamate pyruvate transaminase (SGPT) 42

Principle

L-Alanine +2-oxoglutarate ALAT L-Glutamate + Pyruvate

Pyruvate + NADH + H⁺ LDH D-Lactate + NAD⁺

Addition of pyridoxal-5-phosphate (P-5-P) stabilizes the transaminases and avoids falsely low values in samples containing insufficient endogenous P-5-P, e.g. from patients with myocardial infarction, liver diseases and intensive care patients.

Method

Kinetic UV test, according to the International federation of clinical chemistry and laboratory medicine (IFCC).

Reagents

R1 : TRIS pH 7.5 100 mmol/l

L-Alanine 500 mmol/l

LDH (lactate dehydrogenase) ≥1200 U/l

R2 : 2-Oxoglutarate 15mmol/l

NADH 0.18 mmol/l

Pyridoxal -5-Phosphate FS

Good's buffer pH 9.6 0.7mmol/l

Pyridoxal -5-Phosphate 0.09 mmol/l

Assay procedure

Mixed 800 µl of reagent-1 with 200 µl of reagent-2 in a 5 ml test tube.

To this, added 100 µl of serum.

Mixed well and took the reading immediately.

Normal Range: < 41 U/l.

8.1.3.10. Estimation of Urea35

Urea is the nitrogen-containing end product of protein catabolism. States associated with elevated levels of urea in blood are referred to as hyper uremia or azotemia.

Method

Estimation of urea was done by Urease-GLDH: enzymatic UV test.

Principle

Urea + 2H2O Urease 2NH4 + 2HCO3

2- Oxoglutarate +NH4+ +NADH GLDH L- Glutamate +NAD+ + H2O

GLDH: Glutamate dehydrogenase

Reagents

R1 : TRIS pH 7.8 120 mmol/l

2-Oxoglutarate 7 mmol/l

ADP 0.6 mmol/l

Urease ≥ 6 KU/l

GLDH ≥ 1 KU/l

R 2: NADH 0.25 mmol/l

R 3 : Standard 40 mg/dl

Procedure

Take 1000 μl of reagent-1 and 250 μl of reagent-2 in 5 ml test tube.

To this, add 10 μl of serum.

Mix well and immediately read the test sample at 340 nm Hg 334 nm Hg 365 nm optical path 1 cm against reagent blank (2-point kinetic).

And note down the value.

Normal Range: 10 - 50 mg/dl.

8.1.3.11. Estimation of Creatinine43

Method

Estimation of Creatinine by Jaffe Method (modified).

Principle

Creatinine forms a coloured complex with picrate in alkaline medium. The rate of formation of the complex is measured.

Reagents

Reagent 1: Standard Creatinine-2mg/100ml

Reagent 2: Picric acid solution-9 mmol/l

Reagent 3: Sodium hydroxide solution-0.41 mol/l

Procedure

Take 500 μl of reagent-2 and 500 μl of reagent-3 in a 5 ml test tube.

To this, add 100 μl of serum.

Mix well and immediately read the test sample at Hg 492 nm with 1 cm light path and note down the values.

Normal Range: 0.6 -1.1 mg/dl

8.1.3.12. Estimation of Albumin44

Method

Bromocresol-green method (BCG)

Principle

Albumin is bound by BCG dye to produce an increase in the blue-green colour measured at 578 nm. The colour increase is proportional to the concentration of albumin present.

Reagents

Reagent-1: BCG dye solution-0.15 mmol/l

Procedure

Take 3 ml of Reagent-1 in a 5 ml test tube.

To this, add 10 µl of serum.

Mix well and incubate at 30oC for 5 minutes.

Measure the note down the values.

Normal Range: 3.8-4.4 g/100 ml

8.1.3.13. Estimation of Total Protein45

Method

Biuret method

Principle

Protein forms a coloured complex with cupric ions in alkaline medium.

Reagents

Reagent-1: Biuret solution consisting of Cupric sulphate (6 mmol/l) and Potassium iodide (15 mmol/l)

Procedure

Take 1 ml of Reagent-1 in a 5 ml test tube.

To this, add 20 µl of serum.

Mix well and incubate at 30oC for 30 minutes.

Measure the note down the values.

Normal Range: 6.6-8.7 gm/dl

8.1.3.14. Estimation of Uric acid46

Uric acid and its salts are end products of the purine metabolism. In gout, the most common complication of hyperuricemia, i.e. increased serum levels of uric acid lead to formation of monosodium urate crystals around the joints.

Method

Enzymatic photometric test using TOOS (N-ethyl-N-(hydroxyl-3-sulfopropyl)-m-toluidin)

Principle

Uric acid + H2O + O2 URICASE Allantoin + CO2 + H2O2

TOOS + 4-Aminoantipyrine + 2H2O2 POD Indamine + 3H2O

Reagents

R1:

Phosphate buffer pH 7.0 100 mmol/l

TOOS 1 mmol/l

Ascorbate oxidase ≥ 1 kU/l

R2:

Phosphate buffer pH 7.0 100 mmol/l

4-Aminoantipyrine 0.3 mmol/l

K4[Fe(CN)6 10 µmol/l

Peroxidase (POD) ≥ 1 kU/l

Uricase ≥ 50 U/l

Procedure

Take 800 µl of Reagent-1 in a 2ml centrifuge tube.

To this, add 20 µl of serum.

Mix well and incubate at 30oC for 5 minutes.

Then add 200 µl of Reagent 2

Mix well, incubate for 5 min at 37oC

Measure the note down the values.

Normal Range: 1.9-8.2 mg/dl

8.1.4. Preparation of Tissue Homogenate8, 20

The tissue were weighed and 10% tissue homogenate was prepared with 0.025 M Tris -Hcl buffer, pH 7.5.After centrifugation at 10,000 Ã-g for 10 min, the clear supernatant was used to measure thiobarbituric acid reactive substances(TBARS).

For the estimation of non-enzymatic and enzymatic antioxidants, tissue was minced and homogenized (10% w/v) in 0.1 M phosphate buffer (Ph 7.0) and centrifuged for 10 min and the resulting supernatant was used for enzyme assays.

Estimation of lipid peroxidation of rat liver, pancreas and kidneys8, 47, 48

Lipid peroxidation in liver and kidney was estimated calorimetrically by measuring thiobarbituuric acid reactive substances (TBARS) using the method of Fraga et al.(1988). In brief, 0.1 ml of tissue homogenate was treated with 2 ml of TBA-trichloroacetic acid-HCl reagent (0.37% TBA, 0.25 M HCl and 15% TCA, 1:1:1 ratio), placed for 15 min in a water bath and then cooled and centrifuged at 3500 Ã-g for 10 min at room temperature, the absorbance of clear supernatant was measured at 535 nm against a reference blank. Values were expressed as Mm/100 g tissue.

8.1.5. Estimation of proteins45

Principle

Procedure described by Lowry et al (1951) was used for protein estimation. The method is based on the biuret reaction, formation of a protein-copper complex and reduction of phosphomolybadtungstate reagent (Folin-ciocalteu phenol reagent) by tyrosine and tryptophan residues of protein to form a coloured product.

Reagents

Solution A: 1ml CuSO4.5H2O (1%) + 1ml sodium potassium tartarate(2%) + 98 ml 2% Na2CO3 in 0.1N NaOH.

Solution B: Folin Ciocalteu reagent and distilled water mixed in 1:1 ratio just before use.

Procedure

0.01 ml of tissue homogenate (2.5%) was diluted to 1.2 ml and mixed with 6 ml of solution A. The mixture was incubated at room temperature for 10 min and 0.3 ml of solution B was added, it is mixed immediately and kept at room temperature for 30 min. Optical density was taken at 750 nm. The amount of protein was calculated from the standard curve of bovine serum albumin (BSA).

IN VIVO ANTI OXIDANT STUDIES8, 20

A. Enzymatic antioxidant activity

8.1.6. Estimation of Superoxide Dismutase (SOD) activity49

The activity of superoxide dismutase (SOD) was assayed by the method of Kakkar et al based on the oxidation of epinephrine adrenochrome transition by enzyme. The post-mitochondrial rat suspension of rat liver (0.5ml) was diluted with distilled water (0.5).To this chilled ethanol (0.25ml) and chloroform (0.15ml) were added. The mixture was shaken for 1 min and centrifuged at 2000 rpm Ã- g for 10 min. The PMS (0.5ml) was added with PBS buffer (Ph 7.2; 1.5ml). The reaction initiated by the addition of epinephrine(0.4ml) and change in optical density (O.D.,min-1) was measured at 470 nm. SOD activity was expressed as U/mg of protein. Change in O.D (min-1) at 50% inhibition to adrenochrome transition by the enzyme was taken as one enzyme unit.

8.1.7. Estimation of Catalase (CAT) activity50

Catalase (CAT) was estimated by the method of Sinha (1972). The reaction mixture (1.5 ml vol) contained 1.0 ml of 0.01 M phosphate buffer (pH7.0), 0.1 ml of tissue homogenate and 0.4 ml of 2M H2O2. The reaction was stopped by the addition of 2.0 ml dichromate-acetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed in 1:3 ratio). Then the absorbance was measured at 530 nm. CAT activity was expressed as µM of H2O2 consumed/min/mg protein.

8.1.8. Estimation of Glutathione peroxidase (GPx) activity51

Glutathione peroxidase (GPx) was measured by the method described by Rotruck et al.(1973).Briefly, the reaction mixture contained 0.2 ml of 0.4 M phosphate buffer (pH 7.0), 0.1 ml 10 mM sodium azide, 0.2 ml tissue homogenized in 0.4 M , phosphate buffer (pH 7.0), 0.2 ml tissue homogenized in 0.4 M, phosphate buffer (pH 7.0), 0.2 ml reduced glutathione and 0.1 ml 0.2 mM hydrogen peroxide. The contents were incubated for 10 min at 37oC, 0.4 ml 10% TCA was added to stop the reaction and centrifuged at 3200 rpm Ã- for 20 min. The supernatant was assayed for glutathione content using Ellman's reagent (19.8 mg for 5,5'-dithiobisnitrobenzoic acid (DTNB) in 100 ml 0.1% sodium nitrate). The activities were expressed as µg of GSH consumed/ min/mg protein.

B. Non enzymatic anti oxidant activity

8.1.9. Estimation of reduced glutathione (GSH) activity52

Reduced glutathione (GSH) was measured by the method of Ellman (1959).The PMS of rat liver (720 µl) and 5% TCA were mixed to precipitate the protein content of the supernatant.After centrifugation at 10000 rpm Ã- g for 5 min, the supernatant was taken. DTNB (5,5'-dithio-bis(2-nitrobenzoic acid) and Ellman's reagent was added to it and the absorbance was measured at 412 nm. A standard graph was drawn using different concentration of standard GSH solution.GSH contents were calculated in the PMS of rat liver.

8.2. ANTI INFLAMMATORY AND ANALGESIC ACTIVITIES

8.2.1. SCREENING OF ANTI INFLAMMATORY ACTIVITY 22

Experimental method

Anti-inflammatory activity was assessed by carrageenan induced paw edema in rat model.

Experimental design

Wistar albino rats, female, fasted overnight weighed between 150-200 g were used for the study.

Twenty four rats are divided into 4 groups of 6 rats each for various treatments.

Group 1 : Control rats received propylene glycol (5 ml/kg).

Group 2 : Standard drug treated group which received indomethacin (10 mg/kg).

Group 3 : Received AEAL (100 mg/kg).

Group 4 : Received AEAL (200 mg/kg).

Subsequently 30 min after above treatment, 0.1 ml of 1% carrageenan (dissolved in saline) is injected subcutaneously into the sub-planter region of right hind paw to induce oedema. The paw volume was measured initially and at 1st, 2nd, 3rd, 4th, 5th and 6th h after carrageenan injection using plethesmographic method.

8.2.2. SCREENING OF ANALGESIC ACTIVITY22, 23

8.2.2.1. Acetic acid induced writhing method22, 23

Male swiss albino mice (over night fasted) were divided into 4 groups of 6 mice each for various treatments.

Group 1 : Control mice received propylene glycol (5 ml/kg).

Group 2 : Standard drug treated group which received aspirin (25 mg/kg, i.p).

Group 3 : Mice received AEAL 100 mg/kg.

Group 4 : Mice received AEAL 200 mg/kg.

Acetic acid solution 0.6% v/v (10 ml/kg) is injected by intra peritoneal route one hour after treatment and number of writhes (i.e. index of pain reaction against chemical stimuli characterized by abdominal muscle contraction together with turning of trunk and extension of hind limbs) will counted over a period of 20 min.

8.2.2.2. Hot Plate Method23

Eddy's Hot plate was maintained at 55 ± 1°C. Male swiss albino mice were divided in 4 groups of 6 animals each. The animals were placed on the hot plate and the basal reaction time taken to cause a discomfort (licking of paw or jumping response whichever appeared first) was recorded at 0 min. Cut-off period of 15 seconds was established to prevent damage to the paws.

Group 1 : Control mice received propylene glycol (5 ml/kg).

Group 2 : Standard drug treated group which received morphine (5 mg/kg).

Group 3 : Mice received AEAL 100 mg/kg.

Group 4 : Mice received AEAL 200 mg/kg.

The reaction time in seconds was reinvestigated at 30, 60, 120 and 240 min after the treatment. Changes in reaction time were noted.

9. Statistical Analysis

Statistical analysis was carried out using Graph pad prism 5.0 (Graph pad software). All data were expressed as mean ± SEM. Groups of data were compared with an analysis of variance (ANOVA) followed by Dunnett's t test. Values were considered significant, when p < 0.05.

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