69

also observed in aspirin induced gastric ulceration and secretion in pyloric

ligated rats.

Rajkapoor et al., (2003) reported the anti-ulcer activity of alcoholic

extract of Bauthinia variegata Linn against gastric ulcer induced by pyloric

ligation and aspirin induced ulcer model in rats. The stomach was incised

along with greater curvature and examined for ulcer. Effect of alcoholic

extract of B. variegata on volume of gastric secretion, total, free acidity and

ulcer index in pylorus ligated and aspirin induced ulcer rat was determined.

Oral administration of alcoholic extract of B. variegata decreased the

volume of gastric secretion, total, free acidity and ulcer index with respect to

control.

3. MATERIALS AND METHODS

Collection of plant materials

The leaves of two variants of Aegle marmelos was collected from

different place of Thiruvarur district (Harithuvaramangalam Village, Town

Amaravathi, Puliyakkudi).

70

Collected specimen was carefully examined and identified with the

help of regional floras (Gamble, 1967; Kirthikar and Basu, 1980; Matthew,

1983; Nair and Hendry, 1983 and Henry et al., 1987). The botanical identity

was authenticated by Dr. M. Jegadeesan, Professor and Head, Dept. of

Environmental and Herbal Sciences, Tamil University, Thanjavur. Specimen

was further confirmed with reference to herbarium sheets available in the

Rapinant Herbarium of St. Joseph’s College, Tiruchirappalli, TamilNadu,

India. A voucher specimen has been deposited in the Department Herbarium

for future reference

(TUH-270, 272).

Preparation of powder (Harborne, 1973)

The leaf parts of Aegle marmelos (L.) was collected and dried under

shade. These dried materials were mechanically powdered, sheaved using

80 meshes and stored in an airtight container. These powdered materials

were used for further physiochemical, phytochemical and fluorescent

analysis.

ANALYTICAL METHODS

The procedures recommended in Indian Pharmacopoeia (Anonymous,

1996) were followed for the determination of total ash, water-soluble ash,

acid- insoluble ash, sulphated ash and loss on drying at 110oC.

Total Ash value

71

5g of plant powder was ignited in an electric furnace at 600oC in silica

crucible until the sample reaches a constant weight.

Water – soluble ash value

Total ash obtained was heated upto 600oC with addition of 25ml

of water for 10 minutes. It was filtered in an ash less filter paper (Whatman

No. 41) and the residue was ignited in the furnace to get a constant weight.

Acid - insoluble ash value

Total ash obtained was heated with addition of 25ml of dil. HCl for 10

minutes. It was filtered in an ash less filter paper (Whatman No.41)

and the residue was ignited in the furnace to get a constant weight.

Sulphated ash value

1g of plant powder was ignited in an electric furnace until the drug

gets charred. The crucible was cooled and the residue was moistened with

1ml of H2SO4, heated gently until the white fumes were no longer evolved

and ignited at 800o C ± 25

o C until all black particles disappear. The crucible

was allowed to cool; few drop of H2SO4 was added and again heated. The

ignition was carried as before, allowed to cool and then weighed. This was

repeated until the sample reaches a constant weight.

SOLUBILITY PERCENTAGE (Kokate, 1994)

72

Alcohol

5g of powdered material along with 100ml of alcohol was shaken well

occasionally for the first 6 hours and kept undisturbed for 18 hours. The

liquefied extract thus obtained was concentrated in a vacuum pump and the

percentage was calculated with the weight of the drug powder taken.

Water

The procedure adopted for the solubility percentage of the plant

powder in alcohol is used with chloroform water instead of alcohol to get the

water solubility percentage.

Preparation of Extracts

Successive solvent extract

100 gm of shade dried powdered plant material was extracted

successively using the following solvents in a soxhlet extractor.

a) Petroleum ether (60oC – 80

oC)

b) Benzene (80oC)

c) Chloroform (60oC)

d) Ethyl alcohol (78oC)

e) Water (100oC)

73

Each time before extracting with next solvent powdered was dried in

an air oven below 50oC. Finally, marc was macerated with chloroform water

for 24 hour to obtain the aqueous extract.

The extract was concentrated by distilling off the solvent and then

evaporating to dryness on a water-bath.

The extract was weighed and its percentage was calculated in terms of

air-dried weight of the plant material.

The colour and consistency of the extract was noted. These extracts

were used for preliminary phytochemical and pharmacological studies.

POWDER ANALYSIS

Fluorescent analysis was carried out by using the method of Chase

and Pratt (1949). Behavior of different chemical reagents was carried out as

mentioned by Kay (1938) and Johansen (1940)

QUALITATIVE PHYTOCHEMICAL ANALYSIS

Qualitative phytochemical analyses were done using the procedures of

Kokate (1994). Alkaloids, carbohydrates, tannins and phenols, flavonoides,

gums and mucilage, fixed oils and fats and saponins were qualitatively

analyzed.

74

Alkaloids

The extracts were dissolved in dil. H2SO4 and filtered. The filtrate

was treated with Mayer’s, Dragendroff’s, Hager’s and Wagner’s reagents

separately. Appearance of cream, orange brown, yellow and reddish brown

precipitates in response to the above reagents respectively indicate the

presence of alkaloids.

Carbohydrates

300mg of 50% alcoholic extracts were dissolved in water and filtered.

The filtrate was treated with con H2SO4 and then with Molisch’s reagent.

Appearance of pink or violet colour indicates the presence of carbohydrates.

The filtrate was boiled with Fehling’s and with Benedict solution.

Formation of brick red precipitate in Fehling’s and Benedict’s solution is the

positive result for reducing sugars and non-reducing sugars respectively.

Tannins and phenols

Small quantity of 50% alcoholic extract was dissolved in water and

5% ferric chloride solution or 1% Gelatin solution or 10% lead acetate

solution was added. Appearance of blue colour with ferric chloride or

precipitation with other reagent indicates the presence of tannins and

phenols.

Flavonoids

75

The extract mixed with few ml of alcohol was heated with magnesium

and then con. HCl was added under cooling. Appearance of pink colour

indicates the presence of flavonoids.

The extract was treated with few ml of aqueous NaOH. Appearance

of yellow and change to colorless with HCl indicate the presence of

flavonoids.

Gum and mucilage

About 10ml of the extract was slowly added to 25ml of absolute

alcohol under constant stirring. Precipitation indicates the presence of gum

and mucilage

Fixed oils and fats

A drop of concentrated extract was pressed in between two filter

papers and kept undisturbed. Oil stain on the paper indicates the presence of

oils and fats.

Saponins

About 1ml of the extract was dissolved in 20ml of water and shake in

a graduated cylinder for 15 minutes. Formations of one cm layer of foam

indicate the presence of saponins.

Phytosterol

76

The extract was treated with Lieberman Burchard under suitable

conditions. Appearance of blue-emerald green indicates the presence of

phytosterol and terpenes.

Quantitative Phytochemical Studies

Estimation of Ascorbic Acid (Vitamin – C)

Ascorbic acid (Vitamin – C) was estimated following the procedure of

AOAC (Anonymous, 1980).

Reagents:

1. Oxalic acid : 4% concentration in water

2. Thiourea : 10% concentration in water.

3. DNPH : 2% concentration was prepared by

dissolving 2g of Dinitrophenyl hydroxine

(DNPH) in 100ml of 0.5N H2SO4 and filtered.

4. H2SO4 : 80% concentration in water.

5. Bromine water : Few drops of liquid bromine was

dissolved in water.

77

6. Standard solution: 100mg of ascorbic acid was dissolved in 100ml of

4% oxalic acid in a standard flask. Working

standard was prepared by dissolving 10ml of

standard solution with 90ml of 4% oxalic acid. The

concentration was 100 mg/ml. It was converted to

de-hydro form by adding bromine water. When it

turns orange in colour, air was blown to remove

the excess of bromine.

Sample preparation

5g of dried plant powder (sample) was ground well in a pestle and

mortar with oxalic acid. Known volume of (10ml) the above was changed to

de-hydroform using the procedure adopted for working standard.

Standard curve

Different aliquot (0.2 to 2ml) of de-hydroform of working standard

was taken in test tubes and their volume was made to 3ml with water. To

each tube was added 1ml of DNPH and 1 to 2 drops of thiourea. The tubes

were incubated at 37oC for 3 hours. After incubation the orange-red

oxazone crystals formed was dissolved by adding 7ml of 80% H2SO4. The

absorbance was measured at 540nm and standard graph was plotted.

Estimation in sample

78

De-hydroform of sample was taken in aliquots and preceded it for

plotting on the standard curve. The absorbance was compared with the

standard graph and the percentage of ascorbic acid was calculated.

ESTIMATION OF TANNINS

Estimation of tannins was carried out by using Folin-Denis reagent

(Anonymous, 1980)

Reagents

1. Folin-Denis reagent: To 750 ml of water, 100 gm of sodium tungstate

was added. 20gm of phosphomolydic acid and 50ml of 85%

phosphoric acid were also added. The whole mixture was refluxed for

2 hours. It was cooled and diluted to 1000ml.

2. Saturated sodium carbonate solution: 35gm of anhydrous sodium

carbonate was dissolved in 10 ml of water at 70-80oC and cooled

overnight. Clear liquid was decant and used.

3. Standard solution: 100 mg of tannic acid was dissolved in 1 liter of

water. Fresh solutions were prepared for each test.

Preparation of Sample

79

5gm of sample was boiled with 400ml of water for 30 minutes. The

extract was cooled and transferred to 500ml flask and made up to the

volume.

Preparation of standard curve

10ml of standard solution was made up to 100ml distilled water. 1 -

10ml aliquots were taken in clear test tubes. 0.5ml of Folin-Denis reagent

and one ml of sodium carbonate solution was added to each tube. Each tune

was made upto 10 ml with distilled water. All the reagents in each tube were

mixed well and kept undisturbed for about 30 minutes and read at 760 nm

against reagent blank.

Estimation of sample

An aliquot of the sample extract containing not more than 0.1mg of

tannic acid was used and the percentage of tannin was determined.

Calculation

Mg. of tannic acid X dilution x 100

Tannin as tannic acid = Mg.of sample taken X weight of sample X 100

for colour developmet taken

Estimation of Total Terpenoid

80

100g of plant powder were taken separately and soaked in alcohol for

24 hours. Then filtered, the filtrate was extracted with petroleum ether; the

ether extract was treated as total terpenoids (Ferguson, 1956).

Estimation of Total Alkaloid

This alcoholic extract of plant sample was treated with 0.1N HCl and

aqueous acidified layer thus obtained was partitioned with chloroform in a

separating funnel. The chloroform layer is rejected. The aqueous layer was

basified with ammonium hydroxide and then partitioned with chloroform.

The chloroform layer was concentrated and tested for alkaloids with alkaloid

testing reagents (Ferguson, 1956).

Isolation of Tannin-Free Total Glycosidal Extract

100g of air-dried powder were extracted with ethanol: water (2:1).

The aqueous ethanol extract thus obtained contains tannins which usually

interfere with the biological activities. Hence, this should be removed by

treating with 5% neutral lead acetate reagent which precipitates the tannins

as lead tannate. The aqueous ethanolic solution was treated with 5% neutral

lead acetate solution and the precipitated lead tannate was filtered off. This

process is repeated with until no more precipitate was obtained. The clear

filtrate now contained the excess un-precipitated lead ions in solution

which were removed by passing H2S gas into the solution. This removed the

lead ions as insoluble complex black lead sulphide. The black precipitate

was filtered and this process was usually repeated until no more black

precipitate was formed and the solution strongly smelled of H2S. The

solution, usually of syrupy consistency, was concentrated over water-

81

bath maintained at 55oC. This procedure also removed the excess of H2S

(Ferguson, 1956).

TLC Studies

TLC plates were prepared by using Silica Gel-G as adsorbent. 100g

silica gel-G was mixed with sufficient quantity of distilled water to make

slurry. The slurry was immediately poured into a spreader and plates were

prepared by spreading the slurry on glass plates of required size. The

thickness of the layer was fixed 1.5mm. Plates were allowed to air dry for

one hour and layer was fixed by drying at 110oC for two hours.

Using a micropipette, about 10µml of 1% w/v solution of extracts

were loaded gradually over the plate. The loaded plated was eluted by

suitable mobile phase like TBA (t-BuOH-AcOH-H2O – 3:1:1 ratio), BAW

(n-BuOH–AcOH–H2O – 4:1:5 ratio- Upper Phase), Forestal (AcOH – Con.

HCl – H2O – 30:3:10 ratio), 60% AcOH and Water. Before elution, the tank

was allowed 30 minutes for saturation with mobile phase. The extracts

showed separation into bands. The chromatograms were observed under

visible light and were photographed. The Rf value of the band can be

obtained by using the following formula.

Distance traveled by the substance (cm)

Rf = -----------------------------------------------------

Distance traveled by the mobile phase (cm)

HPTLC Analysis

High Performance Thin Layer Chromatography (Sethi, 1996)

82

HPTLC was performed on aluminum packed silica gel 60F254 HPTLC

plates (Merck). The mobile phase was acetone - alcohol (1:1) for EtOH

extracts of both the samples. Samples were applied to the plates as sharp

bands by means of Camag Linomat IV samples applicator. After drying the

spots in a current of air the plats were placed in one trough of Camag twin

trough glass chamber. The mobile phase was poured into the chamber left to

equilibrate for 30 min. the plate was then developed until the solvent front

had traveled a distance of 7 cm above the position of sample application.

The plate was removed from the chamber and dried in a current of air.

Detection was performed with a Camag TLC Scanner.

Chromatographic Condition

Stationary Phase : HPTLC Aluminum plate

percolated with silica gel 60F254.

Solvent system : Acetone - Alcohol (1:1)

Separation Technique : Ascending.

Migration distance : 70mm.

Detection : UV.

Wave length : 270nm.

GC-MS Studies

The aqueous alcohol extract was examined in GC-MS for its chemical

composition by GC-MS engine model, GC–Clarus 500; Perkin Elmer and

Computer Mass Library (Wiley 138L) of 80,000 compounds with a GC

column Elite – 1 (100% Methyl Poly Siloxane). The other conditions were

as follows.

83

Injector: GC-Clarus – 500; Perkin Elmer; Carrier gas flow Helium 1

ml/min; Split ratio – 1:25; Sample injected 1µl; Oven temperature – 110deg

– 2 min hold; Upto 270deg at the ratio of 5 deg/min – 4 min hold; Injector

temperature 250oC; Total GC- time 38 min; MS inlet line temperature

200oC; Source temperature 200oC; Electron energy 70eV; Mass Scan 25-

400; MS time 39 min.

TOXICOLOGICAL STUDIES

Preliminary Screening and Estimation of LD50

The study was carried out in the laboratory of PRILS Institute of

Paramedical Science, Pattukkottai. The ethical committee of the institute

approved the study. Twelve groups of rats were selected for the LD50

studies. In each group 4 rats were participated (n=4). Drug Aegle marmelos

were given to the animals orally, at the dose of 100mg, 200mg, 500mg, 1gm,

2gm, and 3gm. The animals were observed for alertness, gait, posture;

tremor and response to touch, pain, sound etc, continuously for first 6 hrs

and later at intervals of 24 hours for 3 days observed any mortality in any

group were noted.

Acute toxicity studies (Turner, 1965 and Miller and Trainter, 1944)

Sub-acute studies (14 days) was carried out in the PRILS- Institute of

Pharmacological Science, Pattukkottai. For the sample there was four groups

of animals and in each group there were 10 animals (n=10). Control group

were fed with normal saline and the other three groups with 100 mg/kg/body

wt, 200mg/kg/body wt, 400mg/kg/body weight dose of drugs. Their physical

84

activity, every day body weight, water and food intake and temperature were

also recorded for all groups of animals. After 14 days, all the animals were

sacrificed and samples of Liver, Stomach and Intestine were taken for study.

The necropsy findings were tabulated.

BIOCHEMICAL AND PHARMACOLOGICAL STUDIES

All Biochemical and Pharmacological experiments involving animals

described in the present work were carried out and get approved by Local

Animal Ethical Committee of Dept. of Pharmacology, Periyar Maniammi

University for women, Thanjavur.

Anti-Ulcer Activity (Ethanol-induced Model)

The gastric ulcers were induced in rats of either sex weighing 150 –

160g by administrating absolute alcohol (8ml/Kg). They were kept in

specially constructed cages to prevent coprophagia during and after the

experiment. The rats were divided into groups of eight groups each

containing six animals and fasted for 24 hours allowing free access to water.

First group receive ethanol orally. The second group receives ethanol and

standard antiulcer drugs Ranitidine (150 mg/Kg). The third, fourth and fifth

groups were given absolute alcohol and ethanol extract of Aegle marmelos

variant-I at a dose of 100, 200 and 300 mg /kg b.w respectively. The sixth,

seventh and eighth groups were given absolute alcohol and ethanol extract of

Aegle marmelos variant-III at a dose of 100, 200 and 300 mg /kg b.w

85

respectively. The drugs were administered orally 30 min prior to the oral

administration of absolute ethanol. The animals were anaesthetized 6 hr later

with either stomachs were incised along the greater curvature, collected the

gastric juice and ulceration was scored.

The samples were analyzed for gastric volume, pH, free and total

acidity, sodium and potassium output as recommended (Pillai and

Santhakumari, 1985; Jeffery et al., 1991). Bio-medical estimations, like

total proteins, total hexoses, hexosamine, fucose, sialic acid and pepsin

(Lowry et al., 1951; Winzler, 1958; Dische and Borentreund, 1950; Dische

and Shettles, 1948; Warren, 1959; Debnath et al., 1974) were also done.

The mucosa was flushed with saline and stomach pinned on a frog board and

scored. The scoring is done as described by Laurence and Bacharach (1964)

in the Table - A.

Table – A. Ulcer Score

Ulcer

Score Descriptive Observation

0 Normal rugal pattern

1 Alteration in normal rugal pattern

2 Scattered haemorrhage lesions

3 Haemorrhage lesions and ulcers

4 Penetrating and perforating ulcers

Estimation of free and total acidity, mucosal glycoproteins and pepsin in

gastric juice (Hawk, 1947; Szabo et al., 1985)

86

Collection of gastric juice

Gastric juice was collected from the pylorus-ligated rats. The gastric

juice thus collected was centrifuged and the volume of gastric juice as well

pH of gastric juice was measured. The sodium (Na+) and potassium (K

+) ion

concentration of gastric juice was carried out in flame photometer (Jeffery et

al., 1991). Then the gastric juice was subjected to bio-chemical estimation

as follows.

Determination of free and total acidity in gastric juice (Hawk, 1947)

1ml of gastric juice was pipetted into a 100ml conical flask; added

10ml of distilled water the pH of this solution was noted using with the help

of pH -Meter, then added 2 to 3 drops of Topfer’s reagent and triturated with

0.01N NaOH ( which was previously standardized with 0.01N of oxalic acid

) until all traces of the red colour disappears and the colour of solution was

yellowish orange. The volume of alkali added was noted. The volume

corresponds to free acidity. Then 2 to 3 drops of phenolphthalein solution

was added and titration was continued until a definite red tinge reappears.

Again the total volume of added was noted. The volume corresponds to

total acidity.

Acidity was calculated by using the formula:-

Volume of NaOH X Normality of NaOH X 100

Acidity = ------------------------------------------------------ meq/l/100g

0.1

87

Estimation of Sodium (Na+) and Potassium (K

+) ion concentration in

gastric juice (Jeffery et al., 1991)

The estimation for sodium and potassium ions was carried out using

Systronics mediflame 127 – flame photometer.

Preparation of stock solution

1. Sodium stock solution was prepared by dissolving 2.542g NaCl in

1 liter of distilled waster. It contains 1mg Na per ml (i.e. 1000

ppm). Stock solution was diluted to give four solutions containing

10, 5, 2.5 and 1 ppm of sodium ions.

2. Potassium stock solution was prepared by dissolving 1.909g KCl

in 1 liter of distilled water. It contains 1mg potassium per ml (i.e.

1000 ppm). Stock solution was diluted to give four solutions

containing 20, 10, 5 and 2 ppm of potassium ions.

Procedure

For sodium and potassium, the flame intensity corresponding to the

concentration of stock solution was noted using appropriate filters. The

88

results were plotted in a graph. The flame intensity of the gastric juice was

noted. The concentration of sodium and potassium ions was calculated from

the graph. The results are expressed in terms of mg / l.

Estimation of total proteins (Lowry et al., 1951)

The dissolved protein in gastric juice was estimated in the alcoholic

precipitate obtained by adding 90% alcohol with gastric juice in 9:1 ratio.

Then 0.1ml of alcoholic precipitate of gastric juice was dissolved in 1 ml of

0.1N NaOH and from this 0.05ml was taken in another test tube, to this 4ml

of alkaline mixture was added and kept for 10 min. Then 0.4ml of phenol

reagent was added and again 10 min was allowed for colour development.

Reading was taken against blank prepared with distilled water at 610nm in

Systronics UV-VIS spectrophotometer-180. The protein content was

calculated from standard curve prepared with bovine albumin and was

expressed in terms of µg/ ml of gastric juice.

Estimation of total carbohydrates (Goel et al., 1985)

The dissolved mucosubstance in gastric juice was estimated in the

alcoholic precipitate obtained by adding 90% alcohol with gastric juice in

9:1 ratio. Briefly the method consists of taking two aliquots of gastric juice

and treated as follows:

A) To 1ml of gastric juice, 9ml of 90% alcohol was added. The

mixture was kept for 10 minutes before it was centrifuged. The

89

supernatant was discarded. The precipitate was dissolved in 0.5ml

of 0.1N NaOH. To this 1.8ml of 6N HCl was added. The mixture

was hydrolyzed in water bath at 100oC for 2 hours. The

hydrolysate was neutralized by 5N NaOH using phenolphthalein

as indicator and the volume was made upto 4.5ml with distilled

water and using for the estimation of total hexoses, hexosamine

and fucose as described below.

B) To the other aliquot of 0.5ml gastric juice, 4.5ml of alcohol was

added. The mixture was shaken for 10 minutes and centrifuged to

obtain precipitate. The precipitate was dissolved in 0.5ml of 0.1N

H2SO4. This reconstituted solution was transferred to glass-

stoppered tubes and then hydrolyzed in a water bath at 100oC for 1

hour. After hydrolysis, the volume restored to 0.5ml; 0.2ml of this

hydrolyzes was used for the estimation of sialic acid.

After obtaining the concentration (µg/ml) of individual carbohydrates

namely hexose, hexosamine, fucose and sialic acid, the total carbohydrate

content was calculated by adding the concentration of individual

carbohydrates. Mucosubstances activity has been expressed as ratio of total

carbohydrates to total proteins.

Estimation of hexoses (Winzler, 1958)

To 0.4ml of hydrolysate, 3.4ml of Orcinol reagent was added. The

mixture was then heated in the boiling water bath 60 oC for 15 minutes. This

was then cooled under running tap water land intensity of the colour was

90

read in Systronics UV-VIS spectrophotometer- 180 at 540nm against the

blank by using distilled water instead of hydrolysate. Total hexoses content

was determined from the standard curve of D(+) – galactose-mannose and

has been expressed in µg/ml of gastric juice.

Estimation of hexosamine (Disch and Borentreund, 1950)

0.5ml of the hydrolysate fraction was taken. To this 0.5ml of acetyl-

acetone reagent was added. The mixture was heated in boiling water bath at

60oC for 20 minutes, and then cooled under running tap water. 1.5ml of

90% alcohol was added and allowed for 30 minutes. The colour intensity

was measured in Systronics UV-VIS spectrophotometer- 180 at 540nm

against blank prepared by using distilled water instead of hydrolysate.

Hexosamine content was determined from the standard curve prepared by

using D(+) – glucosamine hydrochloride and concentration has been

expressed in µg/ml of gastric juice.

Estimation of fucose (Dische and Shettles, 1948)

In this method, three test tubes were taken. In one tube 0.4ml of

distilled water was taken to serve as control and in each of the other two

0.4ml of hydrolysate were taken. To all three tubes 1.8ml of H2SO4: water

(6:1) added by keeping the test tubes in ice-cold water bath to prevent

breakage due to strong exothermic reaction. The mixture was then heated in

boiling water bath for exactly 3 min. The tubes were taken out and cooled.

To the blank and to one of the hydrolysate containing tube (unknown), 0.1ml

of cysteine reagent was added while cysteine regent was not added to the last

test tube containing the hydrolysate (unknown blank). It is then allowed for

91

90 minutes to complete the reaction. The reading was taken in Systronics

UV-VIS spectrophotometer- 180 at 396 and 430nm setting zero with the

distilled water. The optical density for the fucose in the hydrolysate was

calculated from the differences in the reading obtained at 396 and 430nm

and subtracting the values without cysteine. This was read against standard

curve prepared with D(+) – fucose content and was expressed in terms of

µg/ml of gastric juice.

(OD396 – OD430) unknown

– (OD396 – OD430) unknown blank

True optical Density = ----------------------------------------------------------

(OD396 – OD430) water blank

Estimation of sialic acid (Warren, 1959)

To 0.5ml of the hydrolysate in 0.1N H2SO4, 0.2ml of sodium

periodate was added and mixed thoroughly by shaking. A time of 20

min was allowed to elapse before addition of 1ml of sodium arsenite

solution to this mixture. The brown colour produced disappeared after

shaking. Then 3ml of thibarbituric acid was added and the mixture was

heated in boiling water bath for 15 minutes. After cooling the tubes, 4.5ml

of cyclohexanone was added and through shaking was done for 15 seconds

till all the colour was taken up by the cyclohexanone supernatant. The

mixture was centrifuged to get a clear pink layer of cyclohexanone. This

supernatant was pipeted out and intensity of colour was measured in

Systronics UV-VIS spectrophotometer- 180 at 550nm. The sialic acid

content of the sample was determined from the standard curve of sialic acid

and has been expressed in terms of µg/ml of gastric juice.

92

Estimation of pepsin (Debnath et al., 1974)

For each determination four tubes (1) and (2) containing 5ml of

substrate, (3) and (4) containing 10ml TCA was placed in the water bath at

37oC. The gastric juice was mixed with an equal volume of HCl at pH

2.1,

warmed to 37oC and added 1ml of mixture to each tube (1) and (4),

incubated for 15 minutes and at the end mixed the contents of tube (1) with

tube (3) and allowed to stand in the bath for about 4 minutes. Contents of

tube (1) and tube (3) give test and contents of tube (2) and tube (4) gives

blank. Both the contents were filtered after 25-30 minutes, 2ml of filtrate

was pippeted into 10 ml of NaOH, mixed by gentle rotation, then 1ml of

phenol was added and again mixed by gentle rotation. After 30 min,

intensity of colour was measure at 680 nm in Systronics UV-VIS

spectrophotometer- 180.

The difference between test and blank gives a measure of peptic

activity. As standard, mixed 2ml of freshly prepared phenol solution

containing 50µg/ml with 10ml of NaOH and 1ml of phenol reagent was

added. After 5-10 minutes, the colour intensity was measured at 680nm.

Anti-Inflammatory Studies

The most common methods to evaluate the anti inflammatory activity

are hind paw oedema (acute) and cotton pellet granuloma (chronic) methods.

These two methods were used to study the anti inflammatory activity of

Aegle marmelos.

93

Study on acute inflammation- Carregeenin induced hind Paw Oedema

(Winter et al., 1962)

Paw oedema was induced in rats by injecting 0.1ml of percentage

Carrageenan into the right hind paw. Different groups were treated with 100,

200 and 300mg/kg (p.o) of ethylalchol and water extract of Aegle marmelos.

Prior to Carrageenan injection, ibuprofen (20mg/kg. s.c) was administered to

a separate group of animals, 30min prior to carregeenin injection. The paw

volume wash measured 5hrs after carregeenin injection using a

plethysmograph. A significant reduction in the paw volume compared to

control animals was considers as anti-inflammatory response. The

percentage of inhibition was calculated by the following formula.

Control - Treated

Percentage of inhibition = --------------------------------- x 100

Control

Study on chronic inflammation- Cotton pellet implantation in rats

(Winter et al., 1962)

Sterile cotton pellets (10mg) were implanted sub-cutaneously in rats

under light ether anesthesia. The control animals received distilled water.

Ibuprofen (20 mg/kg. s.c) of ethanol and water extracts of Aegle marmelos

(200 and 400 mg/ kg .p.o.) was administered too different groups of animals

24 hours before commencing the experiment and continued for 7 days. All

the animals were sacrificed on the eighth day. Cotton pellets with

surrounding granulomatous tissue were removed, dried at 50oC for 24 hrs

and weighed. The increase in weight over the initial weight was recorded. A

94

significant reduction in the weight of cotton pellets compared to control

groups was considered as anti-inflammatory response.

Antipyretic Activity (Loux et al., 1972 and Lassman et al., 1977)

Hyperpyrexia was induced in rats by subcutaneous injection of 10

ml/kg of a 20% aqueous suspension of dried yeast in the back below the

nape of the rat. The animals were then fasted for the duration of the

experiment, water being made available ad libitum. Control temperatures

were taken 24 hr after the yeast injection to determine the pyretic response to

yeast. Temperatures taken 1 hour prior to drug administration in fevered

animals were served as the pre-drug control. Extract of both (300 mg/kg)

was given orally 12 hr after yeast injection. Paracetamol (150 mg/kg) served

as the reference drug. The temperatures were recorded and compared.

Test

Percentage inhibition= X 100

Control

Antioxidant Activities

In vitro free radical scavenging activities

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Inhibition of lipid peroxidation (Ohkawa et al., 1979)

Rat liver homogenate was used as the source of polyunsaturated fatty

acids for determining extends of lipid per oxidation. Liver was collected

immediately after the sacrifice of the animals by cervical dislocation under

mild ether anesthesia. The liver was homogenized with 40 mM Tris-Hcl

buffer (pH 7.0) and centrifuged at 3000 rpm for 10 min to get a clear

supernatant, HAEGG solution of different concentration (25 – 1000 µg/ml)

and 100 µl of each of 1.5 M KCl, 15 mM FeSo4 and6 mM ascorbic acid was

incubated at 37oC for 1 hour. 1ml of 10% TCA was added to the reaction

mixture and centrifuged at 3000 rpm for 20 min at 4oC to remove the

insoluble proteins. Supernatant was removed and 1 ml of TBA (0.8%) was

added to this fraction followed by heating at 90oC for 20 min in a water bath.

After cooling, the coloured TBA-MDA complex was extracted with organic

solvent (2 ml butanol) and absorbance was measured at 532 nm. Percentage

inhibition was calculated using the formula.

Absorbance of control – Absorbance of test

Percentage inhibition=

Absorbance of control

Nitric oxide Radical Scavenging Activity (Green et al., 1982)

Nitric oxide (NO) radicals were generated from sodium nitroprusside

solution at physiological pH. Sodium nitroprusside (25µl to 1000 µl/ml) was

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mixed with 1 ml of extract of different concentration in phosphate buffer

(pH 7.4). The mixture was incubated at 25oC for 150 min. To 1 ml of the

incubated solution 1 ml of Griess’s reagent (1% sulpanialmide, 2% O-

phosphoric acid and 1% napthyl ethylene diamine dihydrochloride) was

added. Absorbance was read at 546 nm and percentage inhibition was

calculated using the formula.

Absorbance of control – Absorbance of test

Percentage inhibition= X100

Absorbance of control

Glutathione assay (GSH)

The mucosa of glandular stomach was removed by scraping with a

blunt knife and 10% homogenase was prepared. Reduced glutathione (GSH)

in the gastric mucosa was determined by Ellman’s reaction using 5’5’-

dithio-bis-w-nitrobenzoic acid (DTNB) as described (Moron et al., 1979).

Briefly, the homogenate was precipitated with 25% trichloroacetic acid

(TCA) and centrifuged. The supernatant was taken for GSH estimation using

freshly prepared DTNB solution. This intensity of the yellow colour formed

was read at 412 nm in Spectrophotometer.

ANTI-MICROBIAL ACTIVITY

Well Diffusion Assay Method (Bauer et al., 1996)

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The EtOH extract of leaves of Aegle marmelos was tested for their

antibacterial and antifungal studies.

The microbial strains tested against Aegle marmelos extracts were

Escherichia coli, Streptococcus pyogenes, Helicobacter pylori,

Pseudomonas aeruginosa, Aspergillus niger, Candida albicans,

Trichoderma viride and Fussarium spp.

Test against standard controls

The commercially available antibiotics disc was used as standard

controls for all the test micro-organism. The sensitivity patterns were

recorded and the readings were interpreted according to the critical diameter

given by National Committee for Clinical Standards (NCCLS, 1997).

The microbes were obtained from the Microbiology Laboratory, Sea

Horse Hospital Pvt., Tiruchirapalli. The test bacterial strains were seeded

over the Muller Hinton agar plates and Sabouraud’s dextrose agar plates

were prepared for fungi aseptically. Wells were made on the agar surface

with 5mm cork borer. The test drugs (0.5ml) were injected into the well

using a micropipette for all concentration (10, 20 and 30%) separately and it

was compared with the standard drugs Amoxicillin and Clotrimazole for

bacterial and fungal strains respectively. The plates were incubated at 37 ±

2oC for 48 to 72 hrs under microaerophilic contidion. The plates were

observed for the elevating zone around the well. The zone of inhibition was

calculated by measuring the diameter of their inhibition zone around the well

(in mm) including the well diameter. Readings were taken in three different

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fixed directions in all three replicates and the average values were

calculated.

Statistical Analysis (Ghosh, 1984)

The raw data of the present study were subjected to simple statistical

analysis to draw meaningful interpretation and conclusion.

1. The standardization values of study drugs were expressed in

percentage (w/w).

2. For pharmacological studies:

• The mean ± SEM and student’s‘t’ test are computed for all

the biochemical estimations, to find out statistical

significance at 1% and 5% probability levels.

4. RESULTS