Residue Remaining After Incineration Biology Essay

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The species for the proposed study that is Delonix regia Rafin barks were collected in the month of July 2010 from erode district, Tamilnadu. Care was taken regarding the age and the health of the plant to obtain a best condition barks.

ANALYTICAL PARAMETERS

ASH VALUE (Kokate et al, 1985)

Principle

The ash content of a crude drug is generally taken to be the residue remaining after incineration. It usually represents the inorganic salts naturally occurring in the drug and adhering to it, but it may also include inorganic matter added for the purpose of adulteration. There is a considerable difference varies within narrow limits in the case of the same individual drug. Hence an ash determination furnishes a basis for judging the identity and cleanliness of a drug and gives information relative to its adulteration with inorganic matter. Ash standards have been established for a number of official drugs. Usually these standards get a maximum limit on the total ash or on the acid insoluble ash permitted.

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The total ash is the residue remaining after incineration. The acid insoluble ash is the part of the total ash which is insoluble in diluted hydrochloric acid.

The ash or residue yielded by an organic chemical compound is as a rule, a measure of the amount of inorganic matters present as impurity. In most cases, the inorganic matter is present in small amounts which are difficult to remove in the purification process and which are not objectionable if only traces are present. Ash values are helpful in determining the quality and purity of the crude drugs in powder form.

Procedures given in Indian pharmacopoeia were used to determine the different ash values such as total ash and acid insoluble ash.

Total ash

Weighed accurately about 3 gm of air dried powdered drug was taken in a tarred silica crucible and incinerated by gradually increasing the temperature to make it dull red until free from carbon cooled and weighted and then calculated the percentage of total ash with reference to the air dried drug.

Acid insoluble ash

The ash obtained as directed under total ash above was boiled with 25 ml of 2N HCl for 5 minutes. The insoluble matter was collected on ash less filter paper, washed with hot water ignited and weighed, then calculated the percentage of acid insoluble ash with reference to the air dried drug.

Water soluble ash

The total ash obtained was boiled with 25 ml of water for 5 minutes. The insoluble matter was collected on an ash less filter paper, washed with hot water and ignited for 15 minutes at a temperature not exceeding 450ËšC. The weight of insoluble matter was subtracted from the weight of total ash. The difference in weight represents the water soluble ash. The percentage of water soluble ash calculated with reference to the air dried drug.

EXTRACTIVE VALUES

Extractive values of crude drugs are useful for their evaluation, especially when the constituents of a drug cannot be readily estimated by any other means. Further, these values indicate the nature of the constituents present in a crude drug.

Determination of alcohol soluble extractive value

5 gm of the air-dried coarse powder of the barks of Delonix regia was macerated with 100 ml of 90% ethanol in a closed flask for 24 hours, shaking frequently during the first 6 hours and allowing standing for 18hours. Thereafter, it was filtered rapidly taking precautions against the loss of the solvent. Out of that filtrate, 25 ml of the filtrate was evaporated to dryness in a tarred flat bottomed shallow dish, dried at 105ËšC and weighed. The percentage of ethanol soluble extractive value was calculated with reference to the air- dried drug. The results are recorded in the table.

Determination of water soluble extractive value

Weigh accurately 5 gm of coarsely powdered drug and macerate it with 100 ml of chloroform water in a closed flask for 24 hours, shaking frequently during the first 6 hours and allow to standing for 18 hours. Thereafter, it was filtered rapidly taking precautions against loss of the solvent. Then 25 ml of the filtrate was evaporated to dryness in a tarred flat bottomed shallow dish, dried at 105ËšC and weighed. The percentage of water soluble extractive was calculated with reference to the air dried drug. The results are given in the table.

LOSS ON DRYING

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Loss on drying is the loss in weight in percentage w/w determined by means of the procedure given below. It determines the amount of volatile matter of any kind (including water) that can be driven off under the condition specified (Desiccators or hot air oven). If the sample is in the form of large crystals, then reduce the size by quick crushing to a powder.

Procedure

About 1.5 gm of powdered drug was weighed accurately in a tarred porcelain dish which was previously dried at 105ËšC in hot air oven to constant weight and then weighed. From the difference in weight, the percentage loss of drying with reference to the air dried substance was calculated.

PRELIMINARY PHYTOCHEMICAL ANALYSIS

Extraction of Delonix regia Rafin bark:

Methanolic extraction:

About 500g of air dried powdered material was taken in 1000ml soxhlet apparatus and extracted with petroleum ether for 2 days. At the end of second day the powder was taken out and it was dried. After drying it was again packed and extracted by using methanol (Changshu yangyuan chemicals, China) as solvent, till colour disappeared. The temperature was maintained at 55°C-65°C. After that extract was concentrated by distillation and solvent was recovered. The final solution was evaporated to dryness. The colour, consistency and yield of methanolic extract were noted.

Aqueous extraction:

The crude aqueous extract (AESS) of the Samanea saman (Jacq) Merr bark was prepared according to the standard methods (Onyeyili P.A et al., 2001). One hundred grams of the powdered plant material was mixed with 500 ml of distilled water in a 1 L flask and boiled for 1.5 h. It was allowed to cool to 40oC and then filtered using whatman No.1 filter paper. The filtrate was then concentrated in a rotary

Name of extract

Color

Consistency

Yield%W/W

Methanolic extract

Brownish

Sticky mass

22.2

Aqueous extract

Brownish

Sticky mass

8.5Table: 1. Nature and colour of extract of Delonix regia Rafin bark.

CHEMICAL TESTS:

A) Test for carbohydrates

1. Fehling's Test: Equal quantity of Fehling's solution A and B is added. Heat gently, brick red precipitate is obtained.

2. Molisch's Test: It consists of treating the compounds of a-naphthol and concentrated sulphuric acid along the sides of the test tube.

Purple colour or reddish violet colour was produced at the junction between two liquids. (Kokate, C.K et al, 2000)

3. Barfoed's test: To the 5ml of the Barfoed's solution add 0.5ml of solution under examination, heat to boiling, formation of red precipitate of copper oxide is obtained.

4. Benedict's test: To the 5ml of Benedict's reagent, add 8 drops of solution under examination. Mix well, boiling the mixture vigorously for two minutes and then cool. Red precipitate is obtained.

B) Test for Alkaloids

1. Dragendroff's Test: To the extract, add 1ml of Dragendroff's reagent Orange red precipitate is produced.

2. Mayer's Test: To the extract add 1ml or 2ml of Mayer's reagent. Dull white precipitate is produced.

3. Wagner's test: To the extract add Wagner reagent. Reddish brown precipitate is produced.

4. Hager's Test: To the extract add 3ml of Hager's reagent yellow Precipitate is produced.

C) Test for Steroids and Sterols

1. Liebermann Burchard test: Dissolve the test sample in 2ml of chloroform in a dry test tube. Now add 10 drops of acetic anhydride and 2 drops of concentrated sulphuric acid. The solution becomes red, then blue and finally bluish green in colour.

2. Salkowski test: Dissolve the sample of test solution in chloroform and add equal volume of conc. sulphuric acid. Bluish red cherry red and purple color is noted in chloroform layer, whereas acid assumes marked green fluorescence.

D) Test for Glycosides

1. Legal's test: Sample is dissolved in pyridine; sodium nitropruside solution is added to it and made alkaline. Pink red colour is produced.

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

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3. Baljet test: To the drug sample, sodium picrate solution is added. Yellow to orange colour is produced.

4. Killer Killani test: Sample is dissolved in acetic acid containing trace of ferric chloride and transferred to the surface of concentrated sulphuric acid. At the junction of liquid reddish brown color is produced which gradually becomes blue.

E) Test for Saponins

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

F) Test for Flavonoids

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

G) Test for Tri-terpenoids

In the test tube, 2 - 3 granules of tin was added, and dissolved in a 2ml of thionyl chloride solution and test solution is added. Pink colour is produced which indicates the presence of triterpenoids.

H) Tests for Tannins and Phenolic Compounds:

The Phenol content in the raw material of Delonix regia Rafin extract was estimated by spectroscopically method.

To 2-3 ml of extract, add few drops of following reagents:

a). 5% FeCl3 solution: deep blue-black color.

b). Lead acetate solution: white precipitate.

c). Gelatin solution: white precipitate

d). Bromine water: decolouration of bromine water.

e). Acetic acid solution: red color solution

f). Dilute iodine solution: transient red color.

g). Dilute HNO3: reddish to yellow color.

I) Test for Fixed Oils and Fatty acids

a). Spot test:

Small quantity of the extract is placed between two filter papers. Oil stain produced with any extract shows the presence of fixed oils and fats in the extracts.

b). Saponification test:

Few drops of 0.5N alcoholic potassium hydroxide are added to the extract with few drops of phenolphthalein solution. Later the mixture is heated on water bath for 1-2 hours soap formation indicates the presence of fixed oils and fats in the extracts.

J) Test for Gums and Mucilage:

a). Ruthenium red test:

Small quantities of extract are diluted with water and added with ruthenium red solution. A pink colour production shows the presence of gums and mucilage.

K) Test for Proteins and Amino acids

Biuret test: Add 1 ml of 40% sodium hydroxide and 2 drops of 1% copper sulphate to the extract, a violet colour indicates the presence of proteins.

Xanthoprotein test: To the extract, add 20% of sodium hydroxide or ammonia. Orange colour indicates presence of aromatic amino acid.

Ninhydrin test: Add 2 drops of freshly prepared 0.2% Ninhydrin reagent to the extract and heat. A blue colour develops indicating the presence of proteins, peptides or amino acids.

TOXICOLOGICAL EVALUATION

Determination of LD50 value of Delonix regia Rafin

Acute Oral Toxicity Study

The procedure was followed by using OECD guidelines 423 (Acute toxic class method).

Animals:

Male albino mice weighing 20-25g were used in the present study. All rats were kept at room temperature of 22-25°C in the animal house. All the animals were followed the internationally accepted ethical guidelines for the care of laboratory animals. Prior to the experiments, rats were fed with standard food for 1 week in order to adapt to the laboratory conditions. All animal procedures were performed after approval from the institutional ethics committee. The experimental protocol has been approved by institutional animal ethics committee, JKKMMRF College of Pharmacy, B.Komarapalayam, Namakkal Proposal number: JKKMMRFCP/IAEC/2010/001.

Procedure:

Twelve animals Albino mice, (25-30gm) were selected for studies.The acute toxic class method is a step wise procedure with 3 animals of single sex per step. Depending on the mortality and / or moribund status of the animals, on average 2-4 steps may be necessary to allow judgment on the acute toxicity of the test animals while allowing for acceptable data based scientific conclusion.

The method uses defined doses (5, 50, 300, 2000 mg / kg body weight) and the results allow a substance to be ranked and classified according to the Globally Harmonized system (GHS) for the classification of chemical which cause acute toxicity.

Most of the crude extracts possess LD50 value more than 2000 mg. /kg of the body weight of animal used.

Dose volume was administered 0.1 ml / 100 gm body weight to the animal

by oral route. After giving the dose the toxic signs were observed within 3-4 hours.

Body weight of animals before and after administration, onset of toxicity and signs of toxicity like changes in skin and fur, eyes, and mucous membrane and also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behavior pattern, signs of tremors, convulsion, salivation, diarrhoea, lethargy, sleep and coma was also to be noted, if any , was observed.

Observation

No toxicity or death was observed for these given dose levels, in selected and treated animals. So the LD50 of the MEDR as per OECD guidelines-423 is greater than 2000mg/kg (LD50 > 2000mg/kg).

Hence the biological dose was fixed at three levels 100,300 and 500mg/kg body weight for the extract.

PHARMACOLOGICAL EVALUATION:

ANTIDIABETIC ACTIVITY:

ORAL GLUCOSE TOLERANCE TEST (OGTT):

Animals:

Male albino-Wistar rats weighing 150-250g were used in the present study. All rats were kept at room temperature of 22-25°C in the animal house. All the animals were followed the internationally accepted ethical guidelines for the care of laboratory animals. Prior to the experiments, rats were fed with standard food for 1 week in order to adapt to the laboratory conditions. All animal procedures were performed after approval from the institutional ethics committee. The experimental protocol has been approved by institutional animal ethics committee, JKKMMRF College of Pharmacy, B.Komarapalayam, Namakkal Proposal number : JKKMMRFCP/IAEC/2010/001.

Procedure:

The overnight fasted (18 hr) normal rats were taken and divided into five groups and each group consists of six animals. They were provided with drinking water only.

Group I: Normal control rats fed with carboxy methyl cellulose (CMC) ..

Group II:Treated with MEDR 100mg/kg, per oral, dissolved in carboxy

methyl cellulose (CMC).

Group III: Treated with MEDR 300mg/kg, per oral, dissolved in carboxy

methyl cellulose (CMC).

Group IV: Treated with MEDR 500mg/kg, per oral, dissolved in carboxy

methyl cellulose (CMC).

Group V: Treated with standard drug, Glibenclamide 3mg/kg body weight.

Glucose (2g/kg) load was fed 30 minutes after the administration of extracts. Blood was withdrawn from tail vein under mild ether anesthesia at initial, and 30,60, 90,120 minutes after glucose administration (V.Babu et al, 2003) and glucose levels were estimated using glucose strips and a glucometer (Standard diagnostics Ltd). Blood glucose levels were noted and reported.

Statistical analysis

All the values are expressed as mean ± S.E.M for groups of six animals each. Analyzed by one way ANOVA and compared by using Tukey- Kramer multiple comparison test using InStat v.2.02 software (GraphPad Software Inc.). The values are statistically significant at three levels, ***p<0.001. **p<0.01. *p<0.05. But ns if p > 0.05.

SCREENING OF ANTI-DIABETIC ACTIVITY:

Animals:

Male albino-Wistar rats weighing 150-250g were used in the present study. All rats were kept at room temperature of 22-25°C in the animal house. All the animals were followed the internationally accepted ethical guidelines for the care of laboratory animals. Prior to the experiments, rats were fed with standard food for 1 week in order to adapt to the laboratory conditions. All animal procedures were performed after approval from the institutional ethics committee. The experimental protocol has been approved by institutional animal ethics committee, JKKMMRF College of Pharmacy, B.Komarapalayam, Namakkal. Proposal number : JKKMMRFCP/IAEC/2010/001.

Chemicals:

Alloxan monohydrate (LOBA Chemie, Mumbai, India) was purchased, preserved at 25°C and used for this study.

Glibenclamide is an oral antidiabetic preparation with an efficient hypoglycaemic action. Daonil (Glibenclamide) (S.K.Prasad et.al, 2009) manufactured by Aventis Pharma Ltd. Goa, India, was collected from market and preserved at room temperature.

Induction of Experimental Diabetes:

Diabetes is to be induced in overnight fasted adult Wistar albino rats weighing 150-250 gm by single i.p. injection of 140 mg/kg Alloxan monohydrate (M.Amarnath satheesh et al, 2004). Alloxan monohydrate was dissolved in normal saline (pH 4.5). Animals were fed with 5 % glucose solution in order to prevent hypoglycemic shock for 18 hrs. Hyperglycemia is to be confirmed by elevated blood glucose levels in plasma, determined at 72 hrs and then on day 0 after injection. The threshold value of fasting plasma glucose to diagnose diabetes was taken as >200mg/dL Only rats found with permanent diabetes were used for the antidiabetic study.

Experimental Design

Experimental rats were divided into six groups of six animals each and treated for 28 days as follows.

Group I: Normal control rats fed with carboxy methyl cellulose (CMC) for 28 days..

Group II: Diabetic controls rats (Alloxan monohydrate 140 mg/kg body weight of rats, once i.p injection).

Group III: Diabetic rats treated with MEDR 100mg/kg, per oral, dissolved in

carboxy methyl cellulose (CMC) for 28 days.

Group IV: Diabetic rats treated with MEDR 300mg/kg, per oral, dissolved in carboxy

methyl cellulose (CMC) for 28 days.

Group V: Diabetic rats treated with MEDR 500mg/kg, per oral, dissolved in carboxy

methyl cellulose (CMC) for 28 days.

Group VI: Diabetic rats treated with standard drug, Glibenclamide 3mg/kg body weight for 28 days.

Sample collection:

Fasting blood glucose (FBG) of all rats was determined before the start of the experiment. Blood sample was collected at weekly intervals from tail vein puncture till the end of study. In the Continuous 28 days of drug treatment, a blood glucose level of all animals was determined at the 0, 7, 14, 21, 28 day. On day 28, blood was collected by cardiac puncture under mild ether anesthesia from overnight fasted rats. Blood was collected in tubes containing potassium oxalate and sodium fluoride as anticoagulant for estimation of fasting plasma glucose. Plasma and serum were separated by centrifugation. After centrifugation at 2,000 rpm for 10 minutes, the clear supernatant was used for the analysis of various biochemical parameters. The pancreas and liver tissues were excised and rinsed in ice-cold saline and kept in formalin solution for further histopathological studies.

EVALUATION OF PARAMETERS

1. Estimation of changes in body weight, water and feed intake by animals:

Body weight of all rats was measured from starting day (0 day) of the experiment to 28th day of the experiment. Both initial and final body weights were noted and reported. Similarly, water and feed intake by the animals measured from starting day (0 day) of the experiment to final day (28th day) of the experiment

2. Estimation of blood glucose levels

Glucose level in plasma was estimated by glucose oxidase/peroxidase method using a commercial kit from Med source Ozone Biomedicals Pvt Ltd followed by Trinder, P. (1969) Annals.Clin.Bio chem. 6, 24.

Reagents

Enzyme reagent

Buffer solution

Glucose standard (100 mg %)

Procedure

10 l of plasma was added to 1.0 ml of working enzyme reagent, mixed well and incubated at 37C for 15 min. The colour developed was read at 505 nm against blank containing distilled water instead of the sample. A standard was also processed similarly.

The level of glucose is expressed as mg/dL.

3. Estimation of plasma insulin level

Insulin was assayed in plasma using a commercial kit by enzyme linked immunosorbant assay (ELISA) technique.

Reagents

Monoclonal anti-insulin antibody

Enzyme conjugate: Anti-insulin antibodies conjugated to horseradish peroxidase

Standard: Human insulin

Solution A: Buffer solution containing hydrogen peroxide

Solution B: Tetramethylbenzidine

Concentrated wash buffer

Stop solution: 2 N HCL

Procedure

25 l of the plasma was dispensed in micro wells coated with anti-insulin antibody. To this, 100 l of the enzyme conjugate was dispensed into each well, mixed for 5 sec and incubated at 25C for 30 min. The wells were rinsed five times with washing buffer. Then, 100 l of solution A and then 100 l of solution B were dispensed into each well. This was incubated for 15 min at room temperature. The reaction was stopped by adding 50 l of 2 N HCl to each well and read at 450 nm.

The values are expressed as µU/ml plasma.

4. Estimation of glycosylated Haemoglobin (Hb A1c)

Haemoglobin is that portion of blood which carries the oxygen. One gram of hemoglobin is capable of combining with 1.36 ml of oxygen under optimal physiological condition. The normal value of haemoglobin as reported for human blood is 14.5g per cent.

Glycosylated Haemoglobin was estimated by the Bannon's method using a commercial kit of Asritha quality processes. Quality diagnostics Hyderabad followed by Trivelli, L.A., Ranney, H.M. and Lai, H.T., New Eng.J.Med.284, 353(1971).

Reagents

Lysing reagent

Ion exchange resin tube

Distilled water

Procedure

Step A: Hemolysate Preparation

250 µl of Lysing reagent was taken in a test tube, to this 50µl of well mixed whole blood was added, mixed well. Incubated for five minutes at room temperature to allow complete lysis of R.B.C.

Step B: Glyco Hemoglobin (GHb) Separation

0.1 ml of Hemolysate from step A was added in to ion exchange resin tubes. The resin tube was agitated by vortex mixer for 5 minutes. The resin was allowed to settle. Then the supernatant was separated. The absorbance's of the supernatant was measured at 420 nm in a colorimeter.

Step C: Total Hemoglobin (THb) Estimation

5 ml of distilled water was taken in a glass tube. To this 0.02 ml of hemoglysate from Step A was added, mixed well and the absorbance was measured at 420 nm in a colorimeter.

The GHb in % was calculated by the following formula

Abs. of. GHb

GHb = ________________ X 4.61

Abs. of THb

Where, 4.61= Assay factor

From this GHb % (A1), HbA1c (A1c) and Mean blood glucose (MBG) was calculated by the standard chart provided with kit.

The values were expressed as % of haemoglobin.

Lipid extraction for estimation of lipids

The serum were rinsed in cold physiological saline thoroughly and dried by pressing between the folds of filter paper. A known weight of tissues was homogenized with 2.5 ml of ethanol-ether mixture (3:1 v/v) and contents were digested for about 2 h at 60-65ï‚°C. The supernatant was collected 3 ml of ethanol-ether mixture was added to the residue and digested further for a period of 2 h at 60-65ï‚°C. The supernatant was collected. Then 1 ml of chloroform-methanol mixture (1:1 v/v) was added to the residue. It was again digested for 2 h at 50-55ï‚°C and the supernatant was collected. The supernatant was pooled and made to a specified volume. Aliquots of this extract were then used for the estimation of cholesterol, free fatty acids, triglycerides and phospholipids. Serum was also processed similarly.

5. Estimation of total cholesterol

Total cholesterol was estimated by the following method

Reagents

Ferric chloride-acetic acid reagent: 0.05% ferric chloride in acetic acid.

Concentrated sulfuric acid.

Cholesterol stock standard: 1 mg/ml in acetic acid.

Cholesterol working standard: 40 g in ferric chloride-acetic acid reagent.

Procedure

0.1 ml of extract was evaporated to dryness and 5.0 ml ferric chloride- acetic acid reagent was added, mixed and centrifuged. To the supernatant 3.0 ml of concentrated sulfuric acid was added and the absorbance was read after 20 min at 560 nm against a reagent blank. A Standard in the concentration range of 40-200 g was treated similarly.

Values were expressed as mg/dL serum.

6. Determination of triglycerides

Triglycerides were determined by the following method.

Triglycerides are extracted by isopropanol, which upon saponification with potassium hydroxide yield glycerol and soap. The glycerol liberated is treated with meta per iodate, which releases formaldehyde, formic acid and iodide. The formaldehyde released reacts with acetyl acetone and ammonia forming yellow coloured compound, the intensity of which is measured at 420nm.

Reagents

Isopropanol

Activated aluminium oxide (Neutral)

Saponification reagent - 5 g of potassium hydroxide was dissolved in 60 ml of distilled water and 40 ml of isopropanol was added to it

Sodium meta per iodate reagent - 77 g of anhydrous ammonium acetate was dissolved in about 700 ml of distilled water, 60 ml glacial acetic acid was added to it followed by 650 mg of sodium meta periodate. The mixture was diluted to 1 litre with distilled water

Acetyl acetone reagent - 0.75 ml of acetyl acetone was dissolved in 60 ml of distilled water and 40 ml of isopropanol was added to it

Standard triolein solution - 1 g of triolein was dissolved in 100 ml isopropanol. 1 ml of stock standard was diluted to 100 ml to prepare a working standard 100 µg of triolein/ml.

Procedure

An aliquot of serum/lipid extract was evaporated to dryness. 0.1 ml of methanol was added followed by 4 ml of isopropanol. 0.4 g of alumina was added to all the tubes and shaken well for 15 min. Centrifuged and then 2 ml of the supernatant was transferred to labeled tubes. The tubes were placed in a water bath at 65°C for 15 min for saponification after adding 0.6 ml of the saponification reagent followed by 0.5 ml of acetyl acetone reagent. After mixing, the tubes were kept in a water bath at 65°C for 1 h, the contents were cooled and absorbance was read at 420nm. A series of standards of concentrations 8-40 mg triolein were treated similarly along with a blank containing only the reagents. All the tubes were cooled and read at 420nm.The triglyceride content was expressed as mg/dL - serum

7. Cholesterol content in lipoprotein fractions:

Cholesterol in the lipoprotein fractions was also determined by following method as described earlier. HDL cholesterol was analyzed in the supernatant obtained after precipitation of plasma with phosphotungstic acid/Mg2+. LDL cholesterol and VLDL cholesterol were calculated as follows:

VLDL-C = Triglycerides/5

LDL-C = Total cholesterol - (HDL-C + VLDL-C)

The levels of HDL, LDL and VLDL-cholesterol are expressed as mg/dL.

8. Alkaline Phosphatase:

Alkaline phosphatase was estimated in serum by commercial kit (Vital Diagnostics Pvt Ltd) followed by following method.

Reagents:

substrate reagent: alkaline phosphate reagent ( tablet form)

buffer - DEA buffer

Working reagent:

One tablet of substrate reagent was dissolved in 1.1 ml of buffer reagent.

Procedure:

20 µl of serum was added to 1 ml of working reagent mixed well. The absorbance was measured at 0, 30, 60 and 90 seconds in a auto analyzer and the mean absorbance was taken (ΔA)

The ALP was calculated by the following formula

ALP in U/L= ΔA / min X 2713

Where, 2713 = standard factor

The activity of the enzyme is expressed as U / litre of serum.

THE HISTOLOGICAL STUDY

After blood sampling for the biochemical analysis, the animals were sacrificed, quickly dissected and small slices of liver and kidney were taken and fixed in 10% formalin. The specimens were dehydrated in ascending grades of ethanol, cleared in xylene and embedded in paraffin wax. Sections of 6 μm in thickness were prepared and stained with Haematoxylin and Eosin then examined under microscopy. (Pearse AG. 3rd ed, Vol. I)

STATISTICAL ANALYSIS

All the values of body weight, fasting blood glucose level, and biochemical parameter estimations were expressed as mean ± standard error of mean (S.E.M) and was analyzed for significance by ANOVA and groups were compared by Tukey-Kramer multiple comparison test, using InStat v.2.02 software (GraphPad Software Inc.).Differences between groups (p Value) were considered significant at P<0.05 level.

EVALUATION OF WOUND HEALING ACTIVITY

Animals:

Healthy Wistar albino rats of either sex and of approximately the same age, weighing between 150-250 gm were used for the study. They were individually housed, maintained in clean polypropylene cages containing paddy husk bedding and fed with standard diet and water ad libitum in animal house facility and maintained under standard experimental conditions. The experimental protocol has been approved by institutional animal ethics committee, JKKMMRF College of Pharmacy, B.Komarapalayam, Namakkal. Proposal number : JKKMMRFCP/IAEC/2010/001.

Drugs and Chemicals

Formycetin ointment; Brand name- soframycin (Aventis Pharma. Goa) is procured from Deepa pharmacy, Erode and used as std drug. Sodium alginate (2% w/w) is used as ointment base. MEDR ointment is prepared in 3 concentrations (2.5 % w/w, 5%w/w &10% w/w) by trituration method.

Experimental procedure

Excision was inflicted on the rats according to methods described by Morton and Malone (1972) under light ether anesthesia. The dorsal fur of the animals was shaved with an electric clipper. Full skin thickness was excised from the marked area to get a wound measuring about 500 mm2 by using toothed forceps and pointed scissors (Nayak et al.,2007). The animals were divided into five groups of six animals each as mentioned below

Group I- Control group with wound and treated with ointment base.

Group II- Test group with wound and treated with 2.5%w/w of methanolic bark extract of Delonix regia Rafin.

Group III- Test group with wound and treated with 5%w/w of methanolic bark extract of Delonix regia Rafin.

Group IV- Test group with wound and treated with of 10%w/w methanolic bark extract of Delonix regia Rafin

Group V- Standard group with wound and treated with 1 %w/w Framycetin ointment.

All the formulations were applied once a day till the complete epithelialization starting from the day of wounding.

Wound healing property was evaluated by wound contraction percentage and wound closure time. The wound surface area was measured immediately by placing a transparent paper over the wound and tracing it out, area of this impression was calculated using the graph sheet(Table-14). The same procedure is employed every third day until healing was complete (Srinivas et al., 2008 and Manjunatha et al.,2006).

EVALUATION OF PARAMETRS

Epithelialization Period

It was monitored by noting the number of days required for the scar to fall off from the wound surface without leaving a raw wound behind.(Table 15)

Wound Contraction

It was noted by following the progressive changes in wound area planimetrically, excluding the day of wounding. The evaluated surface area was then employed to calculate the percentage of wound contraction, taking the initial size of the wound, 1 cm2 as 100% by using the following equationTable 16)

% of wound contraction = Initial wound size - specific day wound size x 100

Initial wound size

Statistical analysis

All the values are expressed as mean ± S.E.M for groups of six animals each. Analyzed by one way ANOVA and compared by using Tukey- Kramer multiple comparison test using InStat v.2.02 software (GraphPad Software Inc.). The values are statistically significant at three levels, ***p<0.001. **p<0.01. *p<0.05. But ns if p > 0.05.

ANTI INFLAMMATORY ACTIVITY:

Animals:

Healthy Wistar albino rats of either sex and of approximately the same age, weighing between 150-250 gm were used for the study. They were individually housed, maintained in clean polypropylene cages containing paddy husk bedding and fed with standard diet and water ad libitum in animal house facility and maintained under standard experimental conditions. The experimental protocol has been approved by institutional animal ethics committee, JKKMMRF College of Pharmacy, B.Komarapalayam, Namakkal. Proposal number : JKKMMRFCP/IAEC/2010/001.

Drugs and Chemicals:

Carragenan , Indomethacin (Brand:Artisid .Sun Pharma ) capsules obtained from Deepa pharmacy Erode.

Experimental Design

Anti-infllammatory activity was evaluated using the carrageenan induced rat paw edema according to the technique of winter et al. After 16hrs of fasted rats were divided into five groups of six animals each and treated as following.

Group I: Carrageenan control rats fed with vehicles only.

Group II: Carrageenan induced rats treated with MEDR 100mg/kg, per oral, dissolved in carboxy methyl cellulose (CMC) .

Group III: Carrageenan induced rats treated with MEDR 300mg/kg, per oral, dissolved

in carboxy methyl cellulose (CMC).

Group IV: Carrageenan induced rats treated with MEDR 500mg/kg, per oral, dissolved

in carboxy methyl cellulose (CMC) .

Group V: Carrageenan induced rats treated with standard drug, Indomethacin 10mg/kg body weight.

After 1 h, 0.1 ml of 1%w/v carrageenan suspension was injected subcutaneously into the plantar surface of the right hind paw. The paw volume was measured using a plethysmometer immediately and 3 h after carrageenan injection. The results are given in Table 17.

Statistical analysis

All the values are expressed as mean ± S.E.M for groups of six animals each. Analyzed by one way ANOVA and compared by using Tukey- Kramer multiple comparison test using InStat v.2.02 software (GraphPad Software Inc.). The values are statistically significant at three levels, ***p<0.001. **p<0.01. *p<0.05. But ns if p > 0.05.

EVALUATION OF ANTHELMINTIC ACTIVITY

Animals

Anthelmintic activity was carried out on adult earthworm, Pherithema posthuma (Mali et al., 2008). Pherithema posthuma is commonly known as earth-worms were collected (due to its anatomical and physiological resemblance with the intestinal roundworm parasites of human being) from water logged area nearby Komarapalayam, Namakkal district, Tamil Nadu.

Drugs and Chemicals:

Albendazole and Normal Saline were procured for Deepa pharmacy. Erode.

Experimental procedure

Indian adult earthworms (Pherithema posthuma) were collected from moist soil and washed with normal saline to remove all faecal matter and were used for the anthelmintic study. The earthworms of 3-5 cm in length and 0.1-0.2cm in width were used for all the experimental protocol. Ten groups were made as given below, each containing six adult earthworms and it must be of approximately equal size.

Group I - animals served as normal controls.

Group II - received 20mg/ml of methanolic extract of Delonix regia Rafin bark.

Group III - received 40mg/ml of methanolic extract of Delonix regia Rafin bark.

Group IV - received 60mg/ml of methanolic extract of Delonix regia Rafin bark.

Group V - received 20mg/ml of aqueous extract of Delonix regia Rafin bark.

Group VI - received 40mg/ml of aqueous extract of Delonix regia Rafin bark.

Group VII - received 60mg/ml of aqueous extract of Delonix regia Rafin bark.

Group VIII - received 20mg/ml of Albendazole suspension.

Group IX - received 40mg/ml of Albendazole suspension.

Group X - received 60mg/ml of Albendazole suspension.

The solutions of alcoholic extract, aqueous extract and albendazole were made in the concentrations of 20, 40, 60 mg/ml in normal saline as vehicle. Groups of earthworms were released into 10 ml of desired formulations as made above, and one group was treating as control in normal saline. The observation was made for the time taken to cause paralysis and death of individual worms. Time for paralysis was noted when no movement of any sort could be observed except when the worms were shaken vigorously. Death was concluded when the worms lost their motility when dipped in warm water (50oC) followed with fading away of their body colors. The results are given in table 18.

Statistical analysis

All the values are expressed as mean ± S.E.M for groups of six animals each. Analyzed by one way ANOVA and compared by using Tukey- Kramer multiple comparison test using InStat v.2.02 software (GraphPad Software Inc.). The values are statistically significant at three levels, ***p<0.001. **p<0.01. *p<0.05. But ns if p > 0.05.