27
4 MATERIALS AND METHODS 4.1 MATERIALS 4.1.1 Plant Materials Rhodomyrtus tomentosa (Myrtaceae) leaves
Mallotus philippensis (Euphorbiaceae) leaves
4.1.2 Source of Chemicals and Drugs
S.No Name of the material
1 Trichloroacetic acid - Loba Chemie Indo Austrand Co. Mumbai, India
2 Aspirin – Microlabs, Bangalore, India
3 Omeprazole –Cipla Pvt Ltd, Bangalore, India
4 Ranitidine- Microlabs, Bangalore, India
5 Anaesthetic ether –Sigma solvents and Pharmaceuticals- Mumbai, India
6 Absolute alcohol- Nice – Cochin, India
7 Formalin –Nice–Cochin, India
8 Methanol –Leo chemicals, Bangalore, India
9 Chloroform –S.D. Fine – Chem Ltd, Mumbai, India
10 Heparin –HEP-5, Gland Pharma Ltd, Hyderabad, India
11 KCl –S.D. Fine – Chem Ltd. Mumbai, India
12 NaCl –LeoChem, Bangalore, India
13 EDTA-S.D.Fine Chem Ltd. Mumbai, India
28
14 KH2PO4 –Qualigens fine Chemicals, Mumbai, India
15 K2HPO4 –Leochem, Bangalore, India
16 H2O2 -S.D.Fine Chem Ltd. Mumbai, India
17 Chem. Kit for SGOT estimation –Coral clinical systems, Verna Goa, India
18 Chem. Kit for SGPT estimation –Coral clinical systems, Verna Goa, India
19 Chem. Kit for SALP estimation -Coral clinical systems, Verna Goa
20 D (+) fucose standard – Sigma Chemical Company, California, U.S.A.
21 D (+) Glucosamine hydrochloride- Merck India, India
22 Chem. Kit for Cholesterol estimation -Coral clinical systems, Verna Goa, India
23 Chem. Kit for Triglycerides estimation -Coral clinical systems, Verna Goa, India
24 Chem. Kit for Total protiens estimation -Coral clinical systems, Verna Goa, India
25 2,2-diphenyl-1-picryl hydrazyl (DPPH) - Sigma Chemical Company, California, U.S.A.
26 2,2- azino- bis (3- ethylbenz-thiazoline 6- sulfonic acid )- Sigma Chemical Company, California
27 Rutin- Acros Organics, New Jersey, U.S.A.
28 Naphthyl ethylene diamine dihydrochloride ( NEDD)- Roch- Light Ltd., Suffolk, U.K.
29 Folin- Ciocalteu reagent – S.D. Fine Chem, Ltd., Biosar, India
30 Ascorbic acid- S.D. Fine Chem, Ltd., Biosar, India
31 Butylated Hydroxy anisole – S.D. Fine Chem, Ltd., Biosar, India
32 Adrenaline- Loba Chemie Indo Austrand Co., Mumbai, India
29
33 Sulphanilic acid- Hi- media Laboratories Ltd., Mumbai, India
34 Gallic acid- Loba Chemie Indo Austrand Co. Mumbai, India
36 Thiobarbituric acid- Loba Chemie Indo Austrand Co. Mumbai 4.1.3 Instruments
S.No Name of the Equipment
1 13C NMR: DAM X- 400 m/z- Bruker India PVT Ltd., Hyderabad, India
2 FT IR., Spectrophotometer Perkin Elmer, U.S.A.
3 Proton NMR: DDR X- 500 m/z- Bruker (India) Pvt Ltd., Hyderabad.
4 UV-Visible Spectrophotometer: Shimadzu 160 – Japan
5 Digital balance (ACCULAB – Sartorious group)
6 Rotary flash evaporator -SUPERFIT, Rotary “Vacuum Digital Bath”
7 Elisa Reader: Bio- Rad Laboratories Inc, California, USA, model 550
8 Centrifuge: Remi R-8c, Mumbai, India
9 Autoanalyser (ARTOS, The versatile Autoanalyser)
10 Incubator: Remi, Mumbai, India
11 Deep freezer - BLUE STAR, Model No.- CHE 400
12 CAMAG Linomat 1V, No-022.78.6, twin trough chamber and TLC Scanner-3 Switzerland.
13 Perkin Elmer Clarus 500 Gas Chromatograph -Perkin Elmer, USA.
30
4.1.4 Animals Healthy adult albino rats of Wistar strain weighing 150-200 g were
procured from Sri Venkateshwara enterprises, Bangalore and housed
in Animal House of Dayananda Sagar College of Pharmacy, Bangalore,
India.
12 ± 1 hour day and night schedule with temperature between 15–
25 ± 2 °C and good ventilation was provided in the animal house. The
animals were housed in large spacious hygienic cages during the
course of the experimental period. The animals were fed with rat pellet
feed supplied by Durga feeds, Bangalore, India and water ad libitum.
4.1.5 CPCSEA / IAEC Approval for Animal Studies Approval was obtained from the CPCSEA/IAEC for animal studies
bearing Registration No. DSCP/PhD.Col/ IAEC/08/08-09.
4.2 METHODS
4.2.1 Collection And Authentication Of Plant Materials The plant materials, Rhodomyrtus tomentosa (Myrtaceae) and
Mallotus philippensis (Euphorbiaceae) leaves were collected from the
Nilgiris, Ootacamund, India. The plants were authenticated by Dr.
Rajan, Medicinal Plants Survey and Collection unit, Government Arts
College, Ootacamund, Tamil Nadu, India.
31
4.2.2 Extraction The leaves of Mallotus philippensis were shade dried and
mechanically reduced to a coarse powder. The weight of the coarse
powder was around 800 g. The powder was subjected to hot
continuous successive extraction in a Soxhlet apparatus with solvents
in the increasing order of polarity using petroleum ether, chloroform,
ethyl acetate and methanol under controlled temperature (50-60 °C).
Extractives were concentrated below 40 °C and further drying was
carried out under reduced pressure. The four dried extractives were
stored in a dessicator for further evaluation[65].
The leaves of Rhodomyrtus tomentosa was dried under shade and
mechanically reduced to a coarse powder. The weight of the coarse
powder was around 1.2 kg. The powder was subjected to hot
continuous successive extraction in a Soxhlet apparatus with solvents
in the increasing order of polarity using petroleum ether, chloroform,
ethyl acetate, aqueous alcohol (70%) and water under controlled
temperature (50-60 °C).
Extractives were concentrated below 50 °C and further drying was
carried out under reduced pressure. The five dried extractives were
stored in a dessicator for further evaluation.
32
4.2.3 Percentage Yield The percentage yields of the extractives were calculated using the
formula:
Weight of the extractives
% Yield =------------------------------------ x 100 Weight of the crude drug
4.2.4 Qualitative Phytochemical Screening
The following tests were carried out on the herbal extractives to
detect various phytoconstituents present in them[66], [67].
4.2.4.1 Test for Carbohydrates Molisch’s Test (General test)
To 2-3 ml of each of the extractives, few drops of alpha-naphthol
solution in alcohol was added, mixed and conc. H2SO4 was added
from the sides of the test tube.
Test for Reducing Sugars
Fehling’s Test
1 ml Fehling’s A and Fehling’s B solutions were mixed and boiled
for 1 min. Equal volume of test solution of each extractive was added.
The mixture was heated on a boiling water bath for 5-10 min.
Test for Gums
Test solution of each extractive was hydrolysed using dilute HCl.
Fehling’s or Benedict’s test was performed.
33
Test for Mucilage
Powdered drug material of each extractive was soaked in water or
aqueous KOH.
4.2.4.2 Tests for Proteins Biuret Test (General test)
To 3 ml of test solution from each extractive, 1 ml of 4% NaOH and
few drops of 1% CuSO4 solution was added.
Millon’s Test (for proteins)
3 ml of test solution of each extractive was mixed with 5 ml
Millon’s Reagent. White ppt. formed was warmed.
4.2.4.3 Test for Amino Acids Ninhydrin Test (General test)
To 3 ml of test solution of each extractive, 3 drops of 5% Ninhydrin
solution was added and heated on a boiling water bath for 10 mins.
4.2.4.4 Test for Fats and Oils Each extractive was spotted on the filter paper. A thick section of
each extractive was placed on a glass slide. A drop of Sudan Red III
reagent was added. After 2 min, it was washed with 50% alcohol,
mounted in glycerine and observed under microscope.
34
4.2.4.5 Test for Alkaloids To residue from each extractive, dilute HCl was added. It was
shaken well and filtered. Following tests were performed with filtrate.
Dragendroff’s Test
To 2-3 ml of filtrate from each extractive, few drops of dragendroff’s
reagent was added.
Mayer’s Test
2-3 ml of filtrate from each extractive, few drops of Mayer's reagent
was added.
4.2.4.6 Test for Tannins and Phenolic Compounds FeCl3 Test To 2-3 ml of each test solution, few drops of 5% FeCl3 solution was
added.
Lead acetate Test
To each of the test solution of the extractives 2-3 ml of 10% lead
acetate solution was added.
4.2.4.7 Test for Glycosides Borntrager’s test
2 ml of filtrate hydrolysate was shaken with 3 ml of chloroform,
chloroform layer was separated, and 10% NH3 solution was added.
35
Legal’s Test
About 50 mg of each of the extractive was dissolved in pyridine.
Sodium nitropruside solution was added and made alkaline using
10% NaOH solution.
Test for Cardiac Glycosides - Baljet’s Test
A thick section of each extractive was mixed with sodium picrate.
Test for Saponin Glycosides - Foam Test
The portion of each extractive was shaken vigorously with water.
4.2.4.8 Tests for Flavonoids Shinoda Test
To each extractive, 5 ml of 95 % ethanol, few drops of conc. HCl
and 0.5 g of magnesium turnings were added.
Lead Acetate test
To small quantity of residue of each extractive, lead acetate
solution was added.
4.2.4.9 Test for Steroids Salkowski Reaction
To 2 ml of the test solution of each extractive, 2 ml of chloroform
and 2 ml of conc. H2SO4 was added and shaken well.
36
Liebermann-Burchard Reaction
2 ml of each test solution was mixed with chloroform, 2 ml of acetic
anhydride and conc. H2SO4 was added from the sides of test tube.
4.3 ESTIMATION OF TOTAL PHENOLIC CONTENT Total phenolic content present in the extractives was measured by
Folin-Ciocalteau assay (FC) using Gallic acid as the calibration
standard to express results in mol-1 or Gallic acid equivalents
(GAE)[68].
Principle
FC reagent is a heteropolyacid complex formed of orthophosphoric
acid and molybdotungstic acid. When Gallic acid is treated with FC
reagent in basic medium, the hydroxyl group of Gallic acid brings
about a reduction of 1, 2 or 3 oxygen atoms from tungstic and / or
molybdate in FC reagent thereby producing reduced species having
characteristic intense blue colour exhibiting λmax at 738 nm. This
principle is made use of in estimation of Gallic acid where there is an
increase in the intensity of blue colour with increased concentration.
Selection of Colorimetric Parameters
To obtain an optimum experimental condition, various parameters
were performed, such as:
1. Selection of solvent
2. Selection of standard
3. Selection of wavelength for detection
37
Selection of Solvent
Selection of solvent was initially found out by carrying out the
colorimetric study using different solvents. When the method was
carried out with methanol turbidity was found to develop. However,
when same was repeated with water no turbidity developed. The
optimum solvent was selected as water for the colorimetric study.
Selection of Standard
The colorimetric method was carried out with Gallic acid as
standard. The intensity of colour developed by pyrocatechol method
was very less intense when compared to that of Gallic acid in water.
So finally, Gallic acid in water was selected as the standard for the
estimation of total phenolic content by visible spectrophotometry.
Selection of Wavelength for Detection
The colour developed was measured at different wavelengths such
as 220, 254, 260, 300, 325, 360 and 738 nm. The λmax of the standard
Gallic acid was found to be at 738 nm. So 738 nm was selected as the
detection wavelength for the colorimetric studies.
Optimized Parameters for Colorimetric Studies
Solvent : Water
Detector : Visible spectrophotometry
Temperature : 28 °C ± 2 °C
Standard : Gallic acid
Detection wavelength : 738 nm
38
Chemicals and Reagents
Folin-Ciocalteu Reagent: Folin-Ciocalteu reagent was diluted (1:10)
with distilled water and used.
Sodium Carbonate: 20.25 g of sodium carbonate (Na2CO3 .10 H2O)
was solubilised in 100 ml of distilled water and used.
Methanol: Distilled
Preparation of Test Solutions
10 mg of the extractives was dissolved in 10 ml of methanol to get
1 mg/ml solution. These solutions were diluted with methanol to
obtain lower dilutions.
Preparation of Standard Solution
Gallic acid: 100 mg of Gallic acid monohydrate was dissolved in 100
ml of distilled water to get 1 mg/ml solution. It was serially diluted
with water to obtain solutions ranging from 0.25 g/ml to 250 g/ml.
Procedure
400 l of each of the extractives (1 mg/ml to 0.5 mg/ml) was
separately mixed with 2 ml of Folin-Ciocalteu reagent and 1.6 ml of
sodium carbonate. After shaking, it was kept for 2 h reaction time.
The absorbance was measured at 738 nm in a spectrophotometer.
Using Gallic acid monohydrate, standard curve was prepared.
Total phenol content of the extractives was calculated from the
standard curve and expressed in terms of Gallic acid equivalent in
mg/g or % w/w of the extractives.
39
4.4 IN VITRO ANTIOXIDANT STUDIES 4.4.1 DPPH Method The antioxidant activity of the various extractives of the selected
plants was assessed on the basis of the radical scavenging effect of the
stable DPPH free radical[69]. When DPPH radical reacts with donors of
hydrogen, it gets reduced to a corresponding hydrazine. The purple
DPPH radical upon reaction with donors of hydrogen changes to
yellow colour. It is a decolourising assay which is evaluated by the
addition of the antioxidant to DPPH solution in methanol where the
decrease in absorbance is measured.
Chemicals
DPPH (2, 2 -Diphenyl-1-picryl-hydrazyl), DMSO (dimethyl sulphoxide)
and methanol.
Preparation of test and standard solutions
Each of the extractives and the standards were dissolved in DMSO
to prepare 1000 g/ml solution. From the above stock solution further
dilutions were made to get different dilutions such as 500 g/ml, 250
g/ml, 125 g/ml, 62.5 g/ml, 31.25 g/ml, 15.62 g/ml, 7.81
g/ml, 3.90 g/ml, 1.95 g/ml, 0.97 g/ml, 0.48 and 0.24 g/ml.
Preparation of DPPH stock solution
Weighed 21 mg of DPPH, placed in 100 ml volumetric flask, added
methanol, and made up to the mark.
40
Preparation of DPPH working solution
18 ml of DPPH stock solution was pippeted out into a 100 ml
volumetric flask and diluted with methanol.
Procedure
Sample: Placed 10 l of above dilutions (sample) into 6 wells in
microtiter plate (different dilution in different rows from A to G, where
G row contains lowest dilution and A contains highest). 200 l of
DPPH was added into each well.
Sample blank: Placed 200 l of methanol into all the 6 wells (A-G
row), and added 10 l of different drug solution as above.
Control: Placed 10 l of DMSO in horizontal 5 wells (H row), then
added 200 l of DPPH working solution.
Control blank: Placed 10 l of DMSO and 200 l of methanol in 6th
well of H row. Incubated the microtiter plate at 37 C for 20 min and
reading was taken by using Elisa reader at 517 nm. Calculated the %
inhibition by using the formula,
OD Control – OD Sample % Inhibition =------------------------------------------X 100
OD Control
IC50 was calculated by plotting the graph, taking % inhibition on y,
axis and concentration on x, axis.
41
4.4.2 H2O2 Radical Scavenging Method Hydrogen peroxide is one of the the least reactive molecules among
the reactive oxygen species. Under physiological pH and temperature
it is generally stable in the absence of metal ions. However these
molecules can be generated from superoxide anion by the enzyme
superoxide dismutase through a dismutation reaction[70].
Chemicals: H2O2, Methanol, Phosphate buffer saline.
Preparation of different concentrations of extractives
Each of the extractives was dissolved in methanol to prepare 1000
g/ml solution. From the above solution further dilutions was made to
get different dilutions such as as 500 g/ml, 250 g/ml, 125 g/ml,
62.5 g/ml, 31.25 g/ml, 15.62 g/ml, 7.81 g/ml, 3.90 g/ml, 1.95
g/ml, 0.97 g/ml, 0.48 and 0.24 g/ml.
Preparation of Phosphate buffer saline
Solution A: NaCl 4 g
KCl 0.1 g
Na2HPO4 0.5 g
KH2PO4 0.1 g
Weighed above chemicals and dissolved in 400 ml of distilled water.
Solution B: CaCl2 0.05 g
MgCl2 0.05 g
Weighed above chemicals and dissolved in 100 ml of distilled water.
Mixed solution A and solution B to prepare 500 ml PBS.
42
Preparation of H2O2 solution
To 100 ml of PBS solution, 0.2267 ml of H2O2 was added.
Procedure
Sample: Placed 1 ml of different dilutions of extractives into seven
different vials and added 2 ml of H2O2 to each vial.
Sample blank: Placed 1 ml of different dilutions of extractives into 7
different vials, then added 2 ml of PBS in each vials.
Control: 1 ml of methanol and 2 ml of H2O2 was placed in a vial.
Control blank: 1 ml of methanol and 2 ml of PBS was placed in a vial.
Note: Prepared and transferred the H2O2 solution very quickly and
completed the experiment within 5 min as the half-life of H2O2 very
less.
Absorbance was recorded by using UV-visible spectrophotometer at
230 nm. Using the formula described in DPPH method, the percentage
inhibition was calculated. IC50 was calculated from the % inhibition Vs
concentration graph.
4.4.3 Nitric Oxide Method Nitric oxide is generated spontaneously from sodium nitroprusside
in aqueous solution at physiological pH, which further interacts with
oxygen to produce nitrite ions. This can be measured by spectroscopic
methods[71]. Nitric oxide generated in this manner is converted to
nitric acid and nitrous acids in contact with dissolved oxygen and
43
water. The liberated nitrous acid is estimated using Griess reagent
which forms a purple azodye in the presence of sample under study,
likely to be a free radical scavenger causing a decrease in the amount
of nitrous acid. The degree of decrease in the formation of purple
azodye will reflect the extent of scavenging.
Chemicals and reagents Sodium nitroprusside (SNP)
0.2998 g of SNP was dissolved in distilled water and the volume
was made upto 100 ml in a volumetric flask (10 mM).
Sulphanilic acid (0.33%w/v)
0.33 g of sulphanilic acid was heated with 20% glacial acetic acid
until it dissolves, made up the volume to 100 ml using 20% GAA.
Napthyl ethylene diamine dihydrochloride (NEDD, 0.1%)
0.1 g of NEDD was heated with 60 ml of 50% GAA until it
dissolves, and then volume was made up to 100 ml with the same. All
the above reagents were kept at 37°C overnight before use.
Test and standard dilutions
10 mg of each of the extractives and the standards were dissolved
in 10 ml of distilled DMSO to obtain a solution of 1 mg/ml. Serial
solutions in DMSO was carried out to obtain lower dilutions in the
range of 500 - 0.24 g/ml.
44
Procedure
Sample: 2 ml SNP + 0.5 ml PBS + 0.5 ml test solution.
Sample blank: 2 ml distilled water + 0.5 ml PBS + 0.5 ml test
solution.
Control: 2 ml SNP + 0.5 ml PBS + 0.5 ml DMSO.
Control blank: 2 ml distilled water + 0.5 ml PBS + 0.5 ml DMSO.
The reaction mixture containing the extract in DMSO, sodium
nitroprusside (10 mM, 4 ml) and phosphate buffer saline (PBS, pH
7.4, 1 ml) was incubated at 25 °C for 150 min. After incubation, 0.5
ml of reaction mixture from each reacting vessel was separately taken,
to which was added 1 ml sulphanilic acid, allowed to stand for 5 min
and then 1 ml of NEDD was added, and incubated for 30 min. The
absorbance of the pink chromophore formed was measured at 540 nm
against the corresponding blank. IC50 values were calculated by
plotting the graph, taking % inhibition on y, axis and concentration on
x, axis.
4.4.4 ABTS Radical Cation Decolourisation Assay This method involves the scavenging of ABTS [2, 2’ azino bis (3-
ethylbenz-thiazoline-6-sulfonic acid) diammonium salt] radical cation.
The principle behind the technique involves the generation of ABTS
radical cation, a blue green chromogen which is produced by a
reaction between ABTS and potassium persulphate. The coloured
radical is converted to colourless ABTS in the presence of antioxidant
45
reductant. The absorbance of this colourless ABTS is measured at
734 nm[72].
Preparation of extractives and Standard Solutions
Different concentrations of extractives and the standard (0.2-100
µg/ml) were prepared by dissolving in DMSO.
Generation of ABTS Radical Cations (ABTS+)
A reaction between ABTS solution (7 mM) and ammonium
persulphate (2.45 mM) gave ABTS radical cations (ABTS+). This
reaction mixture was allowed to stand for 12-16 h in the dark room
before use.
Procedure
Sample: 0.3 ml ABTS + 0.5 ml test solution
Sample blank: Ethanol + 0.5 ml solution of extractives
Control: 0.3 ml ABTS + 0.5 ml DMSO + 1 ml with ethanol
Control blank: Ethanol + 0.5 ml DMSO.
0.5 ml of the different concentrations of each of the extractives were
added to 0.3 ml of ABTS solutions and the final volume was made up
to 1ml with ethanol. Absorbance of the solution was read at 745 nm.
Percentage inhibition was calculated by using the formula.
OD Control – OD sample
% Inhibition =------------------------------------------X 100 OD Control
46
The IC50 value was calculated by plotting the graph, taking %
inhibition on y axis and concentration on x axis.
4.4.5 Measurement of Reductive Ability Transformation of Fe3+ to Fe2+ in the presence of extractives under
study was taken as the parameter to study the measurement of the
reductive ability[73].
Chemicals and Reagents
1. Potassium ferricyanide (K3Fe(CN)6, 1%): 1 g of potassium
ferricyanide was weighed and solubilised in distilled water to
make up the volume to 100 ml in a volumetric flask.
2. Trichloro acetic acid (TCA, 10%): 10 g of TCA was weighed
accurately and dissolved in distilled water to make up the
volume to 100 ml in a volumetric flask.
3. Ferric Chloride (FeCl3. 0.1%) solution: 0.1 g of FeCl3 was
weighed accurately and dissolved in distilled water to make up
volume to 100 ml in a volumetric flask.
4. Phosphate buffer pH 6.6: 46 g of potassium dihydrogen
phosphate and 60.5 g of dipotassium hyrodgen phosphate and
disodium edatate (100 ml of 0.02 M) were solubilised in 1 litre
distilled water. A pH of 6.6 was maintained with KH2PO4.
47
Preparation of Test Solutions
Each extract (10 mg) was weighed accurately and dissolved in 10
ml DMSO to get 1 mg/ml solution. These solutions were diluted with
DMSO to obtain 0.1, 0.2, 0.4 and 0.6 mg/ml solutions.
Preparation of Standard Solutions
1. Ascorbic Acid: 10 mg of ascorbic acid was weighed accurately
and dissolved in 10 ml distilled water to get 1 mg/ml solution.
This solution was diluted with water to obtain 0.025, 0.05,
0.075, 0.1 and 0.15 mg/ml solutions.
2. Rutin: 10 mg of rutin was weighed accurately and dissolved in
10 ml methanol to get 1 mg/ml solution. This solution was
further diluted with methanol to get 0.1, 0.2, 0.4 and 0.6 mg/ml
solutions.
Procedure
One ml of the plant extractives, 2.5 ml of phosphate buffer and 2.5
ml of potassium ferricyanide were incubated at 50° C for 20 min and
2.5 ml of trichloroacetic acid were added to the mixture.
Centrifugation of the contents was carried out for 10 min at 3000
rpm.
From the upper part 2.5 ml was diluted with 2.5 ml of distilled
water and shaken with 0.5 ml fresh FeCl3. After 20 min the
absorbance was measured at 700 nm. The blank solution contained
distilled water instead of the samples.
48
4.5 ACUTE TOXICITY STUDY 14 Days single dose oral acute toxicity and gross behavioural
study[74], [75]
Procedure
In the acute toxicity study, using up and down procedure, different
extractives of Mallotus philippensis in 0.1% (w/v) aqueous suspension
of sodium carboxy methyl cellulose (CMC) was administered orally to
female Sprague Dawley rats weighing 160-190 g.
The procedure was divided into two phases, Phase I (observation
made on day one), and Phase II (observed the animals for next 14
days). Four sets of healthy female rats (each set of 3 rats) were used
for the experiment. First set of animals were fasted for 18 h deprived
from food, water withdrawn before 4 hour of the dosing, body weights
were noted before and after dosing. Three rats were dosed at 400
mg/kg and if no mortality or overt toxicity occurred within 48 h
another set of three rats were dosed at 800 mg/kg. In the absence of
mortality a third set of animals were dosed at 2000 mg/kg and if no
evidence of toxicity was observed three additional rats were dosed at
this level to nullify the errors. Dosing volumes were fixed at 10 ml/kg.
A control group of 3 rats received only the vehicle. Individually
animals were observed for 4 h to see any clinical symptoms, any
change in behaviour or mortality. 6 h post dosing again body weights
were recorded. From the next day onwards, each day every 1 hour the
behavioural change, clinical symptoms or mortality was observed in
49
the same animals for next 14 days and animal body weights were
recorded on 8th and 14th day post dosing.
The same procedure was used to study the acute toxicity of the
different leaf extractives of Rhodomyrrtus tomentosa. The studies were
conducted in compliance with OECD Test guidelines 425.
Selection and Preparation of Dose for Pharmacological Screening
The extractives were suspended in 0.1% CMC solution to prepare
two dose levels, 100 and 200 mg/kg b.w. of the animals for acute
studies and three dose levels of 100, 200 and 400 mg/kg b.w. for
chronic models.
4.6 EVALUATION OF ANTI ULCER ACTIVITY IN RATS
4.6.1 Ethanol Induced Ulcers in Rats Ethanol induced ulcer model[76], in rats was studied for all
extractives of both plants to determine the ulcer index and ulcer
inhibition. Albino rats weighing between 160 - 180 g were divided into
12 groups consisting of six animals each. Experimental design and
dosing schedule was as follows.
Group I: Ulcer control - 1ml ethanol/200 g, p.o.
Groups II and III: Petroleum ether extractives of Rhodomyrtus
tomentosa at 100 and 200 mg/kg b.w, p.o for seven days
respectively.
50
Groups IV and V: Chloroform extractives of Rhodomyrtus
tomentosa at 100 and 200 mg/kg b.w. p.o. respectively for seven
days.
Groups VI and VII: Ethyl acetate extractives of Rhodomyrtus
tomentosa at 100 and 200 mg/kg b.w. p.o. respectively for seven
days.
Groups VIII and IX: Aqueous alcoholic (70%) extractives of
Rhodomyrtus tomentosa at 100 and 200 mg/kg b.w, p.o.
respectively for seven days.
Group X and XI: Water extractives of Rhodomyrtus tomentosa
orally at 100 and 200 mg/kg b.w, p.o. respectively for seven days.
Group XII: Omeprazole, orally at 2 mg/kg b.w. p.o. respectively
for seven days.
On the final day of dosing, the animals also received extractives
and the standard drug thirty minutes before administration of 1ml of
ethanol. Animals were sacrificed after one hour and the contents of
the gastric juice in the stomach were aspirated. Later the stomachs
were removed and kept immersed in saline for 5 min. Incisions of the
stomach were performed along the greater curvature and linear
haemorrhagic lesions in the glandular regions were observed. A pair of
dividers was used to measure the length of all the lesions in the
stomachs. The length (mm) of each lesion was determined at 10 x
magnification and summed up per stomach. Ulcer index was the sum
of length of all lesions for each stomach.
51
(UI) Stomachs were immersed in 10% formalin for 24 h to study
the histopathological changes in treated and ulcerated rats.
Photographs of the opened stomachs were taken. The percentage ulcer
inhibition was calculated by the following formula and the results
were tabulated.
Ulcer Index in Control – Ulcer index in Test
% Ulcer protection = ----------------------------------------------------- x 100 Ulcer Index in Control
Histopathological Evaluation of Ethanol induced Ulcers
The stomachs of the all groups of animals were immersed in 10%
formalin to study the histopathological changes. After the standard
processing the wet ulcerated tissues were embedded in paraffin and
cut into thick sections. Haematoxylin–eosin were used to stain the
tissues and mounted with with Canada balsam. Parameters used to
study histopathological changes included shedding of gastric
epithelium, gastric erosions, infiltration of neutrophils, oedema and
inflammation.
Ethanol induced ulcer model was carried out with the different
extractives of Mallotus philippensis based on the previous protocol to
select the extractives with anti ulcer activity for further evaluation on
other anti ulcer models.
Selection of Extractives Based upon the results of ethanol induced ulcer model the
aqueous alcoholic extractives of Rhodomyrtus tomentosa (RTLE) and
52
the methanolic extractives of Mallotus philippensis (MEMP) was
selected for further anti ulcer evaluation studies using Aspirin +
pyloric ligation induced gastric ulceration in rats, Forced swimming
stress induced gastric ulceration in rats and acetic acid induced
chronic ulcer model in rats.
4.6.2 Aspirin Induced Modified Pylorus Ligated Model The selected extractives of both plants were subjected to anti ulcer
studies using Aspirin induced modified pylorus ligated model[76]. Adult
Wistar albino rats of either sex weighing 180-250 g were fasted for 48
h with free access to water and divided into six groups of six animals
each. They were placed in cages with grating floor to avoid
coprophagy. The experimental design and dosing schedule was carried
out as follows.
Group 1: Ulcer control (Solvent) (10 ml/kg) + Aspirin (200 mg/kg)
Group 2: Ranitidine (50 mg/kg)
Group 3: RTLE (100 mg/kg)
Group 4: RTLE (200 mg/kg)
Group 5: MEMP (100 mg/kg)
Group 6: MEMP (200 mg/kg)
In aspirin plus pyloric ligation induced ulcer model, one hour
before pyloric ligation, aspirin at a dose of 200 mg/kg was
administered orally as a suspension in 0.1% CMC. The animals were
orally treated with the extractives at doses of 100 and 200 mg/kg once
53
daily for seven days and 1 hour before administration of aspirin. The
standard group of animals were also treated in the same way.
Pyloric ligations were performed under ether anaesthesia taking
care to avoid damage to the pylorus and the blood vessels. After
ligation the stomachs were replaced and abdominal wall sutured.
Food and water was restricted during the post-operative period of four
h. The animals were sacrificed at the end of four hours using excess
ether anaesthesia. Thereafter the stomachs were opened and the
contents of the gastric juice were collected. The contents were
centrifuged and various biochemical estimations were carried out in
the collected samples of control and treated groups of animals. The
stomach samples were soaked in saline for five minutes and fixed to
boards for morphological examinations of ulcer indices. Photographs
were taken for further reference.
4.6.2.1 Evaluation of Ulcer Index and Inhibition The ulcer index was calculated by counting the lesions with the aid
of hand lens (10 X) and graded as follows.
0 = Normal coloured stomach
0.5 = Red colouration
1 = Spot ulcer
1.5 = Haemorrhagic streaks
2.0 = ulcers > 3 but < 5
3.0 = ulcers > 5
54
Mean ulcer score for each animal was expressed as ulcer index. Ulcer
protection was calculated according to the standard formula[77].
Ulcer Index in Control – Ulcer index in Test
% Ulcer protection =------------------------------------------------------x 100 Ulcer Index in Control
The volume and pH of the collected gastric juice was recorded. Free
acidity and total acidity was calculated. Various bio-chemical
estimations like total proteins, total hexoses, hexosamine, fucose,
sialic acid, total carbohydrate and carbohydrate/protein ratio of the
gastric juice were performed using standard methods.
4.6.2.2 Bio-Chemical Estimations in Gastric Juice The various biochemical parameters like carbohydrate content viz.
fucose, hexosamine, total hexoses, sialic acid and total carbohydrates
were estimated. Gastric volume, pH, free and total acidity and total
proteins were also evaluated[78].
Gastric volume
The gastric juice was centrifuged, allowed to decant, and taken
into a glass syringe of graduation 0.01 ml. The volume of gastric juice
was measured.
Determination of pH
Using the pH meter the pH of the gastric juice was measured.
55
Determination of free acidity and total acidity
0.01 N NaOH was standardised using oxalic acid as the primary
standard. Free acidity was estimated by titrating 1 ml of gastric juice
with 0.01 N Sodium hydroxide using topfer’s reagent as indicator until
the red colour changes to yellowish orange. The volume of sodium
hydroxide consumed was noted which corresponded to free acidity.
Titration with 0.01N NaOH was continued using phenolphthalein as
indicator until the yellowish orange colour changed to red. The
amount of NaOH consumed was noted and corresponded to total
acidity.
Acidity was calculated by using the following formula, and
expressed as mEq/l/100g.
Volume of NaOH x Actual Normality of NaOH X 100 Acidity = -------------------------------------------------------------------- 0.1
Estimation of Total Proteins[79]
An alcoholic precipitate was prepared by adding 9.0 ml of 90%
alcohol to 1 ml of gastric juice. To 0.1 ml of this protein mixture, 1 ml
of 0.1N NaOH was added. 0.4 ml phenol reagent was added to 0.05
ml of the reaction mixture and was kept for ten minutes to complete
the reaction. Absorbance was measured at 610 nm against blank in a
spectrophotometer. The amount of protein present in the gastric juice
was calculated from standard curve prepared with bovine albumin
and was expressed in term of µg/ml of gastric juice.
56
Estimation of Total Carbohydrates
An alcoholic precipitate containing the dissolved mucosubstances
was prepared by adding 9 ml of 90% alcohol to 1 ml of gastric juice.
The mixture was allowed to decant and the supernatant layer was
discarded. The precipitate containing the mucosubstances was
dissolved in 0.5 ml of 0.1N sodium hydroxide and 1.8 ml of 6N HCl
was added. This mixture was boiled on a water bath, neutralised and
diluted with distilled water to get a final volume of 4.5 ml. This
solution was used for the estimation of carbohydrates like total
hexoses, hexosamine, sialic acid and fucose as follows.
Estimation of Fucose
Reagents
Cysteine reagent: 600 mg of cysteine hydrochloride was dissolved
in 20 ml of distilled water. It was stored in refrigerator until use.
D (+) Fucose: Standard
Procedure
The blank and the sample tubes containing 0.4 ml of distilled
water and 0.4 ml of hydrolysate was mixed carefully in a water bath
with 1.8 ml sulphuric acid mixture. The reaction was allowed to take
place for 3 minutes by heating on a water bath. After cooling the tubes
0.1 ml of cysteine reagent was added to the blank and to one of the
tubes containing the hydrolysate (unknown) while cysteine reagent
was not added to the test-tube containing the hydrolysate (unknown
blank). The reaction was allowed to continue for 90 min. The
57
absorbance was read spectrophotometrically at 396 and 430 nm using
distilled water as blank.
The optical density for the fucose in the hydrolysate was
calculated. The readings, which were taken at 396 and 430 nm, were
noted and the difference calculated. Then the values without cysteine
were subtracted from this and evaluated. Standard curve was
prepared with D (+) – fucose.
(OD396 – OD430)unknown – (OD396 – OD430)unknown blank
True optical density =----------------------------------------------------------- (OD396 – OD430)water blank
The fucose content was expressed in µg/ml of gastric juice.
Estimation of Hexosamine
Reagents
Acetyl acetone reagent: 0.3 ml acetyl acetone and 9.7 ml of 1.5N
sodium carbonate (anhydrous) were mixed just before use.
Ethanol: It was dehydrated.
Ehrlich’s reagent: It was prepared by mixing 1.6 g of p-dimethyl amino
benzaldehyde in 30 ml of 90 % ethanol and then adding 30 ml of
Conc. HCl. The reagent was then stored in a refrigerator. D (+)
Glucosamine hydrochloride was used as standard.
Procedure
0.5 ml of acetyl-acetone reagent was added to 0.5 ml of the
hydrolysate fraction. The reaction was allowed to take place by
heating for 20 min in a boiling water bath. The mixture was allowed
58
to cool and 1.5 ml of 90% alcohol and 0.5 ml of Ehrlich’s reagent was
added. After 30 min the intensity of colour development was measured
spectrophotometrically at 530 nm against blank. The amount of
hexosamine present in the sample was estimated from the standard
curve prepared by using D (+) glucosamine hydrochloride and
concentration was expressed in µg/ml of gastric juice.
Estimation of Total Hexoses
Reagents
Orcinol reagent: 1.6 g of orcinol was dissolved in 100 ml of distilled
water. This was prepared fresh before use.
Sulphuric acid: 150 ml of conc. sulphuric acid was mixed with 100 ml
of distilled water.
D (+) Galactose- Mannose- Standard
Orcinol- Sulphuric acid reagent: One volume of orcinol was mixed
with 7.5 ml of sulphuric acid. This was prepared fresh before use.
Procedure
The reaction mixture containing 0.4 ml of hydrolysate and 3.4 ml
of orcinol reagent was heated for 15 min. in the boiling water bath.
The tubes were cooled to room temperature and the colour developed
was measured at 540 nm against the blank. The amount of hexoses
present in the sample was determined from the standard curve of D
(+) galactose - mannose and has been expressed in µg/ml of gastric
juice.
59
Estimation of Sialic Acid
Reagents
Sodium arsenite: 0.5 M sodium sulphate was prepared in 0.1N
sulphuric acid and to 100 ml of this solution 10 g of sodium arsenite
was added and stored in glass stoppered bottle.
Thiobarbituric acid: 0.5 M sodium sulphate was prepared and to
100 ml of this solution 600 mg of thiobarbituric acid was added and
stored.
Sialic acid- Standard
Procedure
A reaction mixture containing 0.5 ml of the hydrolysate in 0.1N
H2SO4 and 0.2 ml of sodium periodate was mixed and allowed to
stand for 20 min. 1 ml of sodium arsenite solution was added and
mixed by shaking. 3 ml of thiobarbituric acid was added and the
mixture was heated on a boiling water bath for 15 min. After cooling,
4.5 ml of cyclohexanone was added, thoroughly shaken and
centrifuged. The pink colour formed in the supernatant layer was
pipetted out and intensity of colour was measured
spectrophotometrically at 550 nm. A standard curve was prepared
using sialic acid and the amount of sialic acid present in the sample
was determined expressed in µg/ml of gastric juice.
4.6.3 Acetic Acid Induced Chronic Ulcer Model In Rats The selected plant extractives of both plants were subjected to
Acetic acid induced Chronic Ulcer Model in rats[80]. Adult Wistar
60
albino rats weighing 160-220 g of both sexes were selected. Six groups
of ten animals each were formed with these rats. To avoid coprophagy
they were placed in cages with grating floor. The rats were fasted for
24 h, but allowed free access of water.
Group I - Control (Non-ulcerated) (10 ml/kg, 0.1% CMC p.o.)
Group II - Solvent control (Ulcerated) (10 ml/kg, 0.1% CMC p.o.)
Group III - Animals received Omeprazole (20 mg/kg p.o.)
Group IV - Animals received MEMP (100 mg/kg p.o.)
Group V - Animals received MEMP (200 mg/kg p.o.)
Group VI - Animals received MEMP (400 mg/kg p.o.)
Albino rats weighing 160-220 g were fasted for 24 h and abdomen
was opened under light ether anaesthesia. A cylindrical plastic mould
was placed near the region of the lesser curvature of the stomach and
50 µl of 50% glacial acetic acid was administered upon the wall of the
stomach corpus. The stomach wall was wiped with cotton wool soaked
in saline. Povidone iodine was applied to the abdominal stitches for
the next few days to avoid infection. Thereafter the animals were fed
with normal diet and access to water.
Group I served as nonulcerated control and received only the
vehicle while Group II served as the ulcerated control. Rats of Group
III served as standard and was administered Omeprazole at 20 mg/kg
while groups IV, V and VI were treated with 100, 200 and 400 mg/kg
of MEMP respectively as a suspension in 0.1% CMC. The treatment
61
with the standard drug and the extractives were carried out for
21 days. During the remaining length of the experimental period of
21 days the amount of food and water consumed by the animals were
noted. On the final day of the experiment, blood was withdrawn and
the blood cell count of all group of animals were estimated by
standard methods. The animals were sacrificed by excess anaesthesia
and Ulcer area of all groups of animals was calculated using the
standard formula. Ulcer area was calculated as the product of length
and width of ulcer (mm2).
The stomachs were then processed for estimation of mucous
barrier and antioxidant enzymes. The stomach samples of the treated
and the control group of animals were stored in formalin for
histopathological studies.
The rate of healing of ulcers were calculated by comparing the
ulcer index of extractives and Omeprazole treated rats with those of
the ulcerated controls.
4.6.3.1 Estimation of Anti Ulcer Potential Gastric Mucosal Defensive Factors - Estimation of Mucous
Barrier
The glandular portion of the stomach was collected and soaked in
10 ml of 0.1% alcian blue 8GX dissolved in 0.16 M sucrose which was
previously buffered with 0.05 M sodium acetate adjusted to pH 5.8
62
with HCl. After 24 h the uncomplexed part was removed by
successively washing with 0.25 M sucrose for 15-20 mins. Dye
complexed with mucous was separated by immersing for 2 h in 10 ml
aliquots of 0.5 M magnesium chloride. The resulting blue coloured
solution was mixed with diethyl ether of equal volume and the optical
density of aqueous phase was measured at 605 nm. The barrier
mucous was expressed in terms of microgram of alcian blue dye/g of
wet stomach glandular tissues.
Mucous barrier [microgram of alcian blue dye/g of wet stomach
glandular tissues (g)]
Absorbance x 105 = ---------------------------------------------------------------
E 1%1cm x wt. of glandular tissues E 1%1cm for alcian blue = 189
4.6.3.2 In Vivo Anti Oxidant Parameters A Potter Elvehjem glass homogenizer was used to homogenize the
stomach samples in 5 ml of ice cold physiological saline. The
homogenates were centrifuged at 800 x g for 10 min followed by
centrifugation of the supernatant at 2000 rpm for 10 min in a cooling
centrifuge.
i) Catalase (CAT)
The catalysis of H2O2 to H2O in an incubation mixture adjusted to
pH 7.0 was recorded at 254 nm. The reaction mixture containing 0.1
63
ml of tissue homogenate and 2.6 ml of 25 mM potassium phosphate
buffer, pH 7.0 was incubated at 37 °C for 15 min. The reaction was
started with the addition of 0.1 ml of 10 mM H2O2. The time required
for the absorbance to decrease from 0.45 to 0.4 was used for the
calculation of enzymatic activity. One unit of catalase activity was
defined as the amount of enzymes causing the decomposition of µM
H2O2/mg protein/min[81].
ii) Super Oxide Dismutase (SOD)
Super oxide dismutase measurement was carried out based on the
ability of SOD to inhibit spontaneous oxidation of adrenaline to
adrenochrome. 2.78 ml of sodium carbonate buffer (0.05 mM) and 100
μl of stomach homogenate or sucrose (blank) were incubated at 30 °C
for 45 min. The absorbance was adjusted to zero with sample. 100 μl
of adrenaline solution (9.0 mM) was added and the reaction was
started. For a period of 8-12 min the change in the absorbance was
recorded at 480 nm in a semi autoanalyzer. The SOD activity was
expressed as units/mg tissue protein[82].
iii) Estimation of Lipid Peroxide
Total amount of lipid peroxidation products as thiobarbituric acid
reacting substances in the tissue homogenates were measured[83].
Procedure
The reaction mixture containing 0.1 ml of tissue homogenate (Tris-
HCl buffer, pH 7.5) and 2.0 ml of TBA-TCA-HCl reagent (thiobarbituric
64
acid 0.37%, 0.25 N HCl and 15% TCA mixed in 1:1:1 ratio) was mixed
and heated on a boiling water bath for 15 min. The resultant solution
was cooled and centrifuged for 10 min at 1000 rpm. Supernatant layer
was separated and the absorbance of the same was measured against
reference blank at 535 nm. Since 99% of TBARS is MDA the
concentrations of the samples were calculated based on the extinction
co-efficient of MDA (1.56×105 mmol−1cm−1).
4.6.3.3 Short Term Toxicity Evaluation Short term toxicity of the extractives was measured by observing
the initial and final day RBC count, WBC count, changes in body
weight, food and water intake, stool structure and any behavioural
changes during the course of experiments.
I. Estimation of Red Blood Cell Count (RBC)[84]
Procedure
The blood was sucked up to the mark 0.5 in the pipette. The
pipette was held in a horizontal position and by placing the tip of the
pipette against the index finger and blowing gently on the rubber tube,
adjusted the blood so that it was exactly on the 0.5 mark. Dipped the
pipette in red blood cell diluting fluid and sucked to '101' mark. The
pipette was held horizontally and shaken for 10 s for thorough mixing.
Raised ridges of the Neubauer Chamber on both the sides of the
counting ruled areas so, as to form a chamber of 0.1mm depth after
keeping the cover slip in position.
65
The pipette containing diluted blood was thoroughly shaken and
the first 4 to 5 drops was discarded. The neubauer slide was charged
with one or two drops of diluted blood taking care to avoid air
bubbles. Blood was placed on the platform of microscope in such a
way that bright light covers the ruled area. The central square of area
1 sq. mm is subdivided into 25 sub squares. The four corner and one
middle sub squares are used for Red blood cell counting. The RBC’s
were counted by visualizing them on computer screen.
1 The area of each section = -------- Square millimeter = 0.04 sq. mm. 25 Counting of RBC’s
Counting chamber was adjusted so as to focus first section which
was composed of 16 small squares under high resolution. The absence
of thick crossed triple lines to the left and above the 'R' section was
identified as the first section. Then the cells were counted in the first
section as per following guidelines:
i) Started the counting from the small square at upper left hand
comer and follow the straight path to right hand side. ii) A cell which
touched any of the triple lines which border the left side and top of the
section was counted. iii) A cell which touched any one of the triple
lines which border the right side and bottom of section was not
counted. iv) The cells were counted in each small square of the other
four sections.
66
Formula for Regular Calculations
Red blood cell count = Total number of R.B.C in 5 sections x
Total Correction Factor
= Total number of R.B.C in 5 sections x (50 x 200) or 10000.
II. Estimation of White Blood Cell Count (WBC)
Procedure
The blood was sucked up to the mark 0.5 in the pipette. The
pipette was held in horizontal position and by placing the tip of the
pipette against the index finger and blowing gently on the rubber tube,
adjusted the blood so that it was exactly on the 0.5 mark. Dipped the
pipette in white blood cell diluting fluid and sucked to '11' mark. The
pipette was held horizontally and shaken for 10 s for thorough mixing.
Raised ridges of the Neubauer Chamber on both the sides of the
counting ruled areas so, as to form a chamber of 0.1mm depth after
keeping the cover slip in position. The pipette containing diluted blood
was thoroughly shaken and the first 4 to 5 drops was discarded. Then
2 drops of the diluted blood from the pipette was placed between the
cover slip and ruled areas without any air bubbles. The neubauer
slide was charged with diluted blood and was placed on the platform
of microscope in such a way that bright light covers the ruled area.
The four corner squares of area 1 square mm, which was composed of
16 small squares, each (1 mm x 1 mm), were used for white blood cell
count. Counting of WBC was done by following the same rules as in
RBC counting.
67
Formula for Regular Calculations
White blood cell count = Total number of white blood cells in
4 sections x Total Correction Factor
= Total number of white blood cells in 4 sections x 50.
III. Clinical Signs and Symptoms, Food and Water Intake, Body Weights and Stool Structure During the Chronic Anti ulcer study every day morning the treated
group animals were observed for any clinical signs and symptoms of
toxicity. Food and water intake, change in body weight and mortality
to deduce any toxicity due to the treatment was also observed.
4.6.3.4 Histopathology After sacrificing the animals at the end of the experiments, whole
blood was removed from the body by retro orbital puncture. Then
stomachs were collected from the animals of each group and stored in
10% formalin for histopathological studies.
The acetic acid induced chronic ulcer studies as per the previous
experimental design was carried out with the selected aqueous
alcoholic extractives of Rhodomyrtus tomentosa (RTLE) at three
different doses based on the following grouping.
Group I - Control (Non-ulcerated), (10 ml/kg 0.1% CMC p.o)
Group II - Solvent control (Ulcerated) (10 ml/kg 0.1% CMC p.o.)
Group III - Animals received Omeprazole 20 mg/kg.
Group IV -Animals received RTLE (100 mg/kg p.o)
68
Group V - Animals received RTLE (200 mg/kg p.o.)
Group VI - Animals received RTLE (400 mg/kg p.o)
4.6.4 Forced Swimming Stress Induced Gastric Ulceration In Rats
The selected extractives of both plants were subjected to Anti ulcer
studies using Forced Swimming stress induced Gastric ulceration in
rats[85]. Adult Wistar albino rats of either sex weighing 180-250 g were
fasted for 24 h and divided into six groups of ten animals each. They
were allowed free access to water.
Group 1: Served as solvent control (stress control) (10 ml/kg)
Group 2: Received Ranitidine (50 mg/kg)
Group 3: Received RTLE (100 mg/kg)
Group 4: Received RTLE (200 mg/kg)
Group 5: Received MEMP (100 mg/kg)
Group 6: Received MEMP (200 mg/kg)
The vehicle, extractives and standard drug were administered p.o
to groups of rats (200-220g) for 7 days prior to and one hour before
subjecting them to stress. The rats were then made to swim in
cylindrical jars containing 9” water at 25 ±1ºC for 7 h continuously.
At the end of seven hours the animals were patted dry and then
sacrificed by ether anaesthesia. The stomachs of the control and the
treated group of animals were opened and macroscopic observations
were carried out with the help of a hand lens to grade the degree of
69
ulceration. The ulcerogenic indices were calculated based on the
following scoring pattern.
Criterion Score
Shedding of epithelium 10
Petichial and frank hemorrhages 20
One or two ulcers 30
Many Ulcers 40
Perforated Ulcers 50
The score of each group of 6 rats were then expressed as mean ±
SEM. The scores of the treated groups were compared with of those of
stress control group.
Blood was withdrawn from animals of control and treated groups
by retro-orbital puncture. Serum was separated by centrifuging the
samples at 6000 rpm for 10 min in a cooling centrifuge. Alkaline
phosphatase activity was estimated. Protein content was estimated in
blood and stomach homogenates.
The following antioxidant parameters was estimated in blood and
stomach homogenates based on the previous protocol
1. Superoxide dismutase
2. Catalase
3. Thiobarbituric acid reactive substances
70
4.6.4.1 Estimation of Alkaline Phosphatase (ALP) Alkaline phosphatase activity was estimated as per the standard
procedure prescribed by the manufacturer’s instruction manual
provided in the kit (Coral clinical systems, Verna Goa, India) using
Semi Autoanalyser (ARTOS).
Principle
In the presence of an oxidizing agent Serum alkaline phosphatase
(ALP) hydrolyses p-nitro phenyl phosphate to para nitrophenol. The
absorbance measured in the reaction is a direct measure of ALP
activity.
p-nitro-phenyl phosphate + H2O ALP, Mg+2 p-nitrophenol + Phosphate
Procedure
Addition Sequence
Sample (µl)
Working reagent 1000
Sample 20 Mixed well and aspirated.
4.6.4.2 Biochemical Estimations (i) Cholesterol (ii) SGOT (iii) SGPT (v) Triglycerides (vi) Proteins
The biochemical parameters were estimated as per the standard
procedure prescribed by the manufacturer’s instruction manual
71
provided in the kit. (Coral clinical systems, Verna Goa, India) using
Semi Autoanalyser (ARTOS).
1) Estimation of Serum SGPT (UV- Kinetic method)
Principle
The transfer of amino group from L- Alanine to 2-oxo glutarate
with the formation of pyruvate and L-glutamate is catalyzed by SGPT.
The pyruvate so formed is allowed to react with NADH to produce L-
lactate. An indicator reaction coupled with LDL in the presence of
NADH (nicotinamide adenine dinucleotide) regulates this rate of
reaction. A decrease in the absorbance of NADH at 340 nm is a direct
measure of the oxidation of NADH in this reaction which in turn is
proportional to the activity of SGPT.
L- alanine +2- oxoglutarate ALT pyruvate +L glutamate
Pyruvate + NADH LDH L- Lactate + NAD
Where, ALT: Alanine amino transferase
LDH: Lactate dehydrogenase
Procedure
Addition Sequence
Sample (µl)
Working reagent 1000
Sample 100
Mixed well and aspirated.
72
2) Estimation Of Serum SGOT (UV- kinetic method)
Principle
The oxidation of NADH in the following reaction is measured as a
decrease in the absorbance of NADH at 340 nm, which is proportional
to SGOT activity.
L- Aspartate + 2- oxoglutarate AST oxaloacetate + L glutamate
Pyruvate + NADH MDH L- Malate + NAD
Sample + NADH LDH L- Lactate + NAD
Where: AST: Aspartate amino transferase
Procedure
Addition Sequence
Sample (µl)
Working reagent 1000
Sample 100 Mixed well and aspirated.
3) Estimation of Serum Cholesterol
Principle
Cholesterol esterase activates the dissociation of cholesterol esters
to cholesterol and fatty acids. Cholesterol oxidase then converts the
cholesterol into hydrogen peroxide and cholesterone. A reaction takes
place between hydrogen peroxide and 4-amino antipyrine and phenol
in the presence of peroxidase to form a quinoneimine dye.
73
Estimation of cholesterol involves the following enzymatic reaction.
Cholesterol esters cholesterol esterase Cholesterol +fatty acids
Cholesterol + O2 cholesterol oxidase Choles-4-en-3-one + H2O2
H2O2 + 4- Amino antipyrine + phenol POD Quinoneimine + H2O2
Where, POD: peroxidase
The absorbance of quinoneimine was measured spectrometrically
at 505 nm and was proportional to cholesterol concentration in the
specimen.
Procedure
Addition Sequence
Blank
(µl)
Standard
(µl)
Sample
(µl) Working reagent
1000
1000
1000
Distilled water
20
----
----
Standard
----
20
----
Sample
----
----
20
The reaction mixture was mixed well and incubated at 37 °C for 10
min. Aspirated blank followed by standard and tests and then
absorbance of the sample and standard were measured against blank
at 510/630 nm.
74
4) Estimation of Serum Triglycerides (Enzymatic method)
Principle
Triglycerides + H20 Glycerol + Free fatty acids
Glycerol + ATP Glycerol-3-phosphate+ ATP
Glycerol-3- phosphate +O2 DHAP +H2O2
2H2O2 + 4 AAP Quinoneimine dye + 4H2O
The intensity of chromogen quinoneimine formed is measured at
510 nm and is proportional to the concentration of triglycerides in the
sample.
Procedure
Addition sequence
Blank
(µl)
Standard
(µl)
Sample
(µl)
Working reagent
1000
1000
1000
Distilled water
20
----
---
Standard
----
20
---
Sample
----
----
20
Mixed well, incubated at 37 °C for 10 min. The absorbance of
standard and sample was measured against blank within one hour.
5) Estimation of Proteins
Estimation of protein content was carried out as per the standard
procedure prescribed by the manufacturer’s instruction manual
75
provided in the kit. (Coral clinical systems, Verna Goa, India) using
Semi Autoanalyser (ARTOS).
Principle
Cu+2 ions in alkaline solution undergoes biuret reaction with the
peptide bond in proteins to form a bluish coloured complex. In this
reaction each of the copper ions undergoes complexation with 5 or 6
peptide bonds. Tartarate was added as a stabilising agent.
In order to prevent auto reduction of alkaline copper complex,
iodine was added. The color developed is measured at 546 nm and is
proportional to the protein concentration in the sample.
Procedure
The reaction mixture was incubated at 37ºC for 10 mins.
Absorbance of standard and each sample was read at 546 nm against
blank.
Addition Sequence
Blank
(µl)
Standard
(µl)
Sample
(µl) Working reagent
1000
1000
1000
Distilled water
20
----
---
Standard
---
20
---
Sample
----
----
20
76
4.6.4.3 Histopathological Studies
Processing of Isolated Stomach
The animals were sacrificed and the stomach of each animal was
isolated and was cut along the greater curvature, preserved and fixed
in 10 % formalin for two days. Then the stomach piece was washed in
running water for about 12 h to remove the formalin and was followed
by dehydration with isopropyl alcohol of increasing strength (70%,
80% and 90%) for 12 h each. Then finally, dehydration was done
using absolute alcohol with about three changes for 12 h each.
Dehydration was performed to remove all traces of water. Further
alcohol was removed by using chloroform and chloroform removed by
paraffin infiltration. After paraffin infiltration the stomachs were
subjected to automatic tissue processing unit.
Embedding in Paraffin Vaccum
Hard paraffin was melted and the hot paraffin was poured into L-
shaped blocks. The stomachs were then dropped into the molten
paraffin quickly and allowed to cool.
Sectioning
The blocks were cut using microtome to get sections of thickness
of 5 . The sections were taken on a micro slide on which egg albumin
i.e., sticking substance was applied. The sections were allowed to
remain in an oven at 60 °C for 1 h. Paraffin melts and egg albumin
denatures, thereby fixing tissue to slide.
77
Staining
Eosin is an acid stain, hence it stains all the cell constituents pink
which are basic in nature i.e., cytoplasm. Haematoxylin is a basic
stain which stains the acidic cell components blue i.e. DNA in the
nucleus.
4.6.5 Statistical Analysis Values were expressed as mean ± SEM. The experimental mean
values were compared statistically with that of vehicle control mean
values using One Way Analysis of Variance (ANOVA). This was
followed wherever applicable by Dunnett’s test or Tukey’s multiple
comparison test. The analysis was carried out using Graph Pad Prism
software V.4. p values less than 0.5 were considered to be statistically
significant.
4.7 ISOLATION OF PHYTOCONSTITUENTS BY COLUMN CHROMATOGRAPHY
About 15 g of the dried aqueous alcoholic extractives of
Rhodomyrtus tomentosa (RTLE) was defatted using n-hexane for 6 h
for removal of fatty materials. The material was then dried and
dissolved in 100 ml of methanol.
About 500 g of silica gel of column chromatography grade was
activated by keeping in an oven for 1 h at 105 °C. It was kept in a
dessicator for 1 h.
78
Materials
Chromatographic column, cotton, silica gel (60-120 mesh particle
size), toluene and ethyl acetate
Stationary phase
Silica gel (60- 120 mesh particle size)
Mobile phase
Toluene and ethyl acetate
Method
The extractives was mixed well and dried by continuous stirring
with activated silica gel and was used for performing column
chromatography. A column of length 60 cm and diameter 1.5 cm was
used for performing column chromatrography. The column was
packed upto 2/3 portions with silica gel by wet packing procedure.
The column was then eluted with a mixture of toluene and ethyl
acetate in different ratios (10, 9:1, 7:3). Every 30 ml fractions were
collected in different boiling tubes and similar fractions were clubbed
together after analysing with TLC. First 30 ml was discarded. Different
fractions were collected in upto 60 test tubes. Each tube was kept for
concentrating on a water bath. Further TLC of each of the fractions
collected in the tubes was performed to analyse the purity of the
isolated fractions. TLC profiles were developed using Toluene:
chloroform: methanol (8:2:1)
79
Fractions with the same bands and similar Rf values were clubbed
together. Fractions 15-18, 22-25 was clubbed together and
concentrated. The solubility of the compound was checked in various
solvents like acetone, benzene, hexane, chloroform and methanol. The
compound in tubes 15-18 was named Compound 1. The compound in
tubes 22-25 showed two spots in TLC and hence further separation
and purification of compounds was carried out by preparative TLC as
follows.
Preparative TLC
The clubbed fractions in tubes 22-25 showed two spots in the TLC.
Further separation and purifications were carried out using
preparative TLC.
The second compound was spotted in the preparative TLC glass
plate (20x20). Toluene: Ethyl acetate (7:3) was used as the solvent
system. The two major bands were scraped. The scraped material 1
was dissolved in methanol and kept at zero degrees for crystallization
and was named compound II.
The second compound was also dissolved in methanol and kept in
room temp for crystallization and was named compound III. The
solvent system used for identifying the individual purified compounds
was Methanol: Water: Glacial acetic acid (7:2:1) and visualized in
Visible, UV 254 and UV 365 nm and also after derivatisation with
Anisaldehyde-sulphuric acid reagent.
80
4.8 GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) ANALYSIS OF RTLE
GC-MS analysis was carried out with a Perkin Elmer Clarus 500
gas chromatograph (Perkin Elmer, USA) based on the following
chromatographic conditions.
1. GC Programme
Column: Elite-1(100 % dimethyl poly siloxane), 30 x 0.25mm x 1 µmdf
Equipment: GC Clarus 500 Perkin Elmer
Carrier gas: Helium
Flow rate: 1ml/min, Split: 10:1
Detector: Mass detector Turbo mass gold- Perkin Elmer
Software: Turbomass 5.2
Sample injected: Aqueous alcoholic extractives (2 µl) of Rhodomyrtus
tomentosa was injected at 250 °C.
Oven Temperature programme
110°C – 2 min hold
Upto 200 °C at the rate of 10 °C/min. - No hold
Upto 280 °C at the rate of 5 °C/min. - 9 min hold
Injector temperature – 250 °C
Total running time of GC - 36 min.
2. MS Programme
Library used NIST Version- Year 2005
Inlet line temperature: 200 °C
Source temperature: 200 °C
81
Electron energy: 70 eV
Mass scan m/z: 45-50
Solvent delay: 0-2 min.
MS running time: 36 min.
4.9 ESTIMATION OF BETULIN IN MEMP BY HPTLC HPTLC is a modern, automated and sophisticated form of
separation technique widely used for evaluation of quality of herbal
materials. Raw materials and finished products can be assayed and
evaluated with respect to reference materials.
HPTLC provides the means not only for flexible screening
procedures and qualitative analysis but also for quantitative
determinations. The present studies suggest the antioxidant and anti
ulcer potential of Mallotus philippensis leaves.
The genus Mallotus contains betulin which is reported to possess
antioxidant properties. HPTLC separation of the methanolic
extractives of Mallotus philippensis leaves and its comparison to
standard betulin revealed the presence of betulin in the extractives.
Literature review suggested that, there is no HPTLC method available
for the estimation of betulin in MEMP. Hence, an attempt was made to
establish a HPTLC method for the quantification of betulin content in
MEMP.
82
Chromatography conditions Applicator: Camag Linomat 5
Scanner: Camag TLC scanner 3
Software: Wincat 4.3.2
Photo documentation unit: Camag Reproster 3
Adsorbent: TLC aluminium plate pre- coated with silica gel - 60 F254
Mode of TLC development: Ascending mode
Solvent run upto: 70 mm
Scanning wavelength: 254 nm
Solvent system: Methanol: Toluene: Acetone: Acetic acid (2.5:1:0.5:0.2)
Estimation of Betulin in MEMP The methanolic extractives (1g) was filtered through Whatman
(Maidstone, England) qualitative filter paper No.1 pore size 11 µm and
dissolved in ethanol in a volumetric flask and the final volume was
adjusted to 100 ml in a volumetric flask and used for HPTLC analysis.
Chromatography was performed on 20 cm x 20 cm aluminium packed
HPTLC plates coated with 0.2 mm layers of silica gel - 60 F254
previously washed with methanol. A working standard of 1 mg/ml
solution of betulin was prepared and used as the reference standard.
With a help of a Camag Linomat IV applicator, 5 µl of solution was
applied on the plates. The chromatogram was developed upto 70 mm
under chamber saturation conditions using Methanol: Toluene:
Acetone: Acetic acid, 2.5:1:0.5:0.2 (v/v) as a solvent system in Camag
twin trough chamber. The plates were developed and dried and Rf was
83
heated for 5 min to evaporate the solvents. The sample solution (5 µl
and 10 µl) was applied along with the standard solution. Spots with
values of 0.71 visible were scanned at 254 nm. Rf values were
recorded using a software. The developed plates were photo
documented at 254 nm. After chromatography the amount of betulin
present in MEMP was quantified using the standard formula.