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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