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.

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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)

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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.

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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

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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.

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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

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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.