chapter 4: materials and methods -...

Chapter 4 Materials & Methods

Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 86

CHAPTER 4: MATERIALS AND METHODS

4.1 STANDARDIZATION OF SELECTED PLANT MATERIALS

The roots of Bauhinia variegata Linn and bark of Tectona grandis Linn were

collected from local areas of Kolhapur (Maharashtra) & Nipani (Karnataka). The fruits

of Schrebera swietenioides were collected from Medicinal Plant Conservation Area,

Forest Department Sandoor, Bellary (Karnataka). These plant parts were authenticated

from Dr. (Prof). S.R. Yadav, Department of Botany, Shivaji University, Kolhapur

(Maharashtra). The certificate of authentication is given in Annexure. Selected medicinal

plant parts (Crude drug) were cut into small pieces, cleaned and shade dried at room

temperature then subjected to physical evaluation with different parameters. Then these

selected medicinal plant parts were subjected to size reduction to get coarse powder,

separately, in a mechanical grinder and then passed through sieve no.40 to get desired

particle size and stored in well-closed glass jars. Then uniform powder was subjected to

standardization with different parameters.

Procedure for Different Parameters:212,213

a) Determination of Alcohol-soluble Extractive:

Macerated 5g of the air dried drug coarsely powdered with 100 ml of ethanol of the

specified strength in a closed flask for twenty-four hours, shaking frequently during six

hours and allowed to stand for 18 hrs. Filtered rapidly taking precautions against loss of

ethanol. Evaporated to dryness in a tared flat bottom shallow dish, dried at 1051C and

weighed. Calculated the percentage of alcohol-soluble extractive with reference to the air

dried drug.

b) Determination of Water Soluble Extractive:

Followed as directed for the determination of alcohol-soluble extractive using

chloroform water instead of ethanol.



c) Determination of Pet. Ether Soluble (40-600C) Extractive:

Proceeded as directed for the determination of alcohol-soluble extractive, used

petroleum ether (40-600C) instead of ethanol.

d) Loss on Drying (LOD):

About 2-3 g of powder is accurately weighed in a China dish and kept in a hot air

oven maintained at 1101C for four hours. After cooling in a desicator, the loss in

weight was recorded. This procedure was repeated till constant weight was obtained.

Loss on drying (%) LOD = Loss in weight x 100

Weight of the drug in gms

e) Determination of Ash:

About 2-3 g weighed crude drug powder in a tared silica dish was ignited and

weighed. Scattered the powder drug on the bottom of the dish, incinerated by gradually

increasing the heat not exceeding dull red heat until free from carbon, cool and weighed.

Total ash value of sample % = (z – x) x 100

y

Where, z = weight of the dish + ash (after complete incineration)

x = weight of empty dish

y = weight of drug taken

f) Determination of Acid Insoluble Ash:

Boiled the ash for 5-10 min with 25 ml of diluted hydrochloric acid, collected the

insoluble matter in a Gooch crucible washed with hot water, ignited and weighed.

Calculated the percentage of acid insoluble ash with reference to the air dried drug.

Acid insoluble ash value of the sample % = a x 100

y

Where, a = weight of the residue

y = weight of air dried drug



g) Determination of Water-soluble Ash:

Boil the ash for 5-10 min with 25 ml of water. Collected the insoluble matter in a

Gooch crucible, washed with hot water and ignited to constant weight at a low

temperature. Subtracted the weight of insoluble matter from the weight of ash. The

difference in weight represents the water soluble ash. Calculated the percentage of water

soluble ash with reference to the air dried drug.

Water soluble ash = Weight of ash – Weight of insoluble matter

Percentage of water soluble ash = a x 100

b

Where, a = water soluble ash

b = air dried drug

4.2 Extraction of Bauhinia variegata roots, Tectona grandis bark and Schrebera

swietenioides fruits:

There is increasing interest in the extraction and isolation of secondary metabolites

from plants. Extraction methods are applied to obtain a crude plant extract. The classical

chemical procedure for obtaining organic constituents from dried plant tissues was

continuous Soxhlet extraction with a range of solvents. Extraction of volatile

components from plant needs special precautions. The method to obtain the water

soluble components from fresh plant material was called as maceration214

.

a) Preparation of aqueous extract:

About 100gm powder of these selected medicinal plants was subjected to cold

maceration with water in a 1 liter conical flask for about 7 days at room temperature. The

flask was securely plugged with adsorbent cotton and was shaken periodically till

complete maceration. After maceration the marc was placed in a muslin cloth and the

filtrate was concentrated to residue at low temperature.



b) Preparation of ethanolic & petroleum ether extracts:

An about 50 gm powder of these selected medicinal plants was subjected to

extraction by using the ethanol (95%) and 40-60ºC petroleum ether solvent in a Soxhlet

extractor. The extraction was continued until the solvent in the thimble became clear.

After the effective extraction, solvents were concentrated at room temperature in

reduced pressure using a rotary evaporator (Gucchi rota evaporator) and water was

removed by heating on water bath and the extract obtained with each solvent was

weighed. Its percentage was calculated. The color and consistency of the extract was

noted. These extracts were stored in amber colored glass jar in refrigerator.

These obtained extracts were subjected to chemical investigation. The dried

extracts of selected medicinal plants were mixed with required quantity of distilled water

and 1% Gum acacia for preparation of suspension. This suspension was administered

orally to rats by intragastric tube for evaluating pharmacological activity like acute

toxicity study, antidiabetic and hepatoprotective activity.

4.3 PRELIMINARY PHYTOCHEMICAL INVESTIGATION OF EXTRACTS:

Qualitative chemical tests of aqueous, ethanolic and petroleum ether extracts of

selected medicinal plants i.e. Bauhinia variegata roots, Tectona grandis bark and

Schrebera swietenioides fruits were conducted to identify the various phytoconstituents.

The various tests and reagents used are given below and observations are recorded in the

Table No.7, 8 and 9.

1. Tests for Carbohydrates:

Molisch's test (General test):

To 2-3 ml test solution (T.S.), added few drops of -naphthol solution in alcohol,

shaken and added concentrated H2SO4 from sides of the test tube was observed for violet

ring at the junction of two liquids.



For Reducing Sugars:-

a) Fehling's test: 1 ml Fehling's A and 1 ml Fehling's B solutions was mixed and

boiled for one minute. Added equal volume of T.S. Heated in boiling water bath

for 5-10 min was observed for a yellow, then brick red precipitate.

b) Benedict's test: Equal volume of Benedict's reagent and T.S. in test tube were

mixed. Heated in boiling water bath for 5 min. Solution may appear green,

yellow or red depending on amount of reducing sugar present in T.S.

Tests for Monosaccharides:

Barfoed's test: Equal volume of Barfoed's reagent and T.S. were added. Heated

for 1-2 min, in boiling water bath and cooled. Observed for red precipitate.

Tests for Hexose Sugars:

Cobalt-chloride test: 3 ml of T.S. was mixed with 2 ml cobalt chloride, boiled

and cooled. Added FeCl3 drops on NaOH solution. Solution observed for

greenish blue (glucose), purplish (Fructose) or upper layer greenish blue and

lower layer purplish (Mixture of glucose and fructose).

Tests for Non-Reducing Sugars:

a) T.S. does not give response to Fehling's and Benedict's test.

b) Tannic acid test for starch: With 20% tannic acid, test solution was observed for

precipitate.

2. Tests for Proteins:

a) Biuret test (General test): To 3 ml T.S. added 4% NaOH and few drops of 1%

CUSO4 solution observed for violet or pink color.

b) Million's test (for proteins): Mixed 3 ml T.S. with 5 ml Million's reagent, white

precipitate obtained. Precipitate warmed turns brick red or precipitate dissolves

giving red color was observed.



c) Xanthoprotein test (For protein containing tyrosine or tryptophan): Mixed 3 ml

T.S. with 1 ml concentrated H2SO4 observed for white precipitate.

d) Test for protein containing sulphur: Mixed 5 ml T.S. with 2 ml 40% NaOH

and 2 drops 10% lead acetate solution. Solution was boiled it turned black or

brownish due to PbS formation was observed.

e) Precipitation test: The T.S. gave white colloidal precipitate with following

reagents:

i) Absolute alcohol

ii) 5% mercuric chloride solution

iii) 5% cupric sulphate solution

iv) 5% lead acetate

v) 5% ammonium sulphate

3. Tests for Steroid:

a) Salkowski Reaction: To 2 ml of T.S., 2 ml chloroform and 2 ml concentrated

H2SO4 was added. Shake-well, whether chloroform layer appeared red and acid

layer showed greenish yellow fluorescence was observed.

b) Libermann-Burchard Reaction: Mixed 2 ml T.S. with chloroform. Added 1-2

ml acetic anhydride and 2 drops of conc.H2SO4 from the side of test tube

observed for first red, then blue and finally green color.

c) Libermann's reaction: Mixed 3 ml T.S. with 3 ml acetic anhydride. Heated and

cooled. Added few drops concentrated H2SO4 observed for blue color.

4. Tests for Amino Acids:

a) Ninhydrin test (General test):- 3 ml T.S. and 3 drops 5% Ninhydrin solution were

heated in boiling water bath for 10 min. Observed for purple or bluish color.



b) Test for tyrosine: Heated 3 ml T.S. and 3 drops Million's reagent. Solution

observed for dark red color.

c) Test for tryptophan: To 3 ml T.S. added few drops glycoxalic acid and

concentrated H2SO4 observed for reddish violet ring at junction of the two layers.

5. Tests for Flavonoids:-

a) Shinoda test: To dried powder or T.S., added 5 ml 95% ethanol, few drops

concentrated HCl and 0.5 g magnesium turnings. Pink color was observed.

b) To small quantity of residue, added lead acetate solution observed for Yellow

colored precipitate.

c) Addition of increasing amount of NaOH to the residue whether showed yellow

coloration, which was decolorized after addition of acid was observed.

d) Ferric chloride test: T.S, added few drops of ferric chloride solution observed

for intense green color.

6. Tests for Alkaloids:-

a) Dragendroff's test: To 2-3 ml T.S. added few drops Dragendroff's reagent

observed for orange brown precipitate.

b) Mayer's test: 2-3 ml T.S. with few drops Mayer's reagent observed for precipitate.

c) Hager's test: 2-3 ml T.S. with Hagers reagent observed for yellow precipitate.

d) Wagner's test: 2-3 ml T.S. with few drops of Wagner's reagent observed reddish

brown precipitate.

7. Tests for Tannins and Phenolic Compounds:-

To 2-3 ml T.S, added few drops of whether showed following was observed-

a) 5% Ferric chloride solution: Deep blue-black colored.

b) Lead acetate solution: White precipitate.

c) Gelatin solution: White precipitate.



d) Bromine water: Discoloration of bromine water.

8. Test for Vitamins:

a) Test for Vitamin A: Dissolved a quantity equivalent to 10-15 units in 1ml

chloroform and add 5 ml of antimony trichloride solution, a transient blue colour

is produced immediately.

b) Test for vitamin C (Ascorbic acid): Diluted 1 ml of 2% w/v solution with 5 ml

of water and added 1 drop of freshly prepared 5% w/v solution of sodium

nitroprusside and 2 ml dilute NaOH solution. Added 0.6 ml of hydrochloric acid

dropwise and stirred, the yellow color turns blue.

c) Test for Vitamin D: Dissolved a quantity equivalent to about 100 units of

Vitamin D, activating in chloroform and added 10 ml of antimony tricohloride

solution, a pinkish-red color appeared at once.

9. Tests for Glycosides:-

General test for Glycosides:

Part A: To 2-3 ml of T.S. added dil. H2SO4 and heat on a water bath for 1-2 mins.

Neutralise with 10% NaOH, check with litmus paper and to resulting solution

add Fehling's A & B. Increased red precipitate shows glycosides are present.

Part B: To 2-3 ml of T.S. added water and heat. According to NaOH added for

neutrlaisation added equal quantity of water. To the resulting solution added

Fehling's A & B. Increased red precipitate showes glycosides are absent.

Compare Part A and B.

Tests for Cardiac Glycosides:-

a) Baljet's test: A T.S. observed for yellow to orange colour with sodium picrate.

b) Legal's test (For cardenoloids): To T.S, added 1 ml pyridine and 1 ml sodium

nitroprusside observed for pink to red color.



c) Test for deoxysugars (Kellar Killani test): To 2 ml T.S. added glacial acetic acid,

one drop of 5% FeCl3 and concentrated H2SO4 observed for reddish brown colour

at junction of the two liquid and upper layers bluish green.

d) Libermann's test (For bufadenolids): Mixed 3 ml T.S. with 3 ml acetic anhydride.

Heated and cooled. Added few drops concentrated H2SO4 observed for blue

color.

Tests for Saponin Glycosides:-

a) Foam test: The T.S. was shaken vigorously with water. Persistent foam was

observed.

b) Haemolytic test: Added T.S. to one drop of blood placed on glass slide.

Haemolytic zone whether appeared was observed.

Tests for Coumarin Glycosides:

T.S. when made alkaline, observed for blue or green fluorescence.

10. Test for Triterpenoids:

a) Salkowaski test: When a few drops of conc. sulphuric acid is added to the test

solution, shaken and allowed to stand, lower layer turns yellow.

b) Liebermann Burchardt test: The T.S. treated with acetic anhydride, mixed well

and conc. sulphuric acid is added from the sides of the test tube. Deep red

colour forms.

4.4 ANTIOXIDANT ACTIVITY STUDY

Antioxidant activity can be measured by using in vitro methods and animal studies.

The chemistry responsible for these effects is ready donation of electrons to reactive

oxygen species (ROS) by antioxidants, which then quenches the ROS and produces more

stable and thus less damaging species.



In vitro measurement

The interpretation of results obtained from in vitro measurements of antioxidant

activity of a compound or of a crude plant extract, must be dealt with caution as the

antioxidative effect of a tested compound may vary considerably with the method and

conditions used, thus selection of the appropriate assay to be used should be based on the

intended application of the antioxidant.

Reaction with 1-1 –Diphenyl-2-Picryl-Hydrazyl (DPPH):215

DPPH is a molecule containing a stable free radical. In the presence of an

antioxidant which can donate an electron to DPPH, the purple color which is typical to

free DPPH radical decays and the change in absorbency at 517nm is followed either

spectrophotometrically. This simple test can provide information on the ability of a

compound to donate a hydrogen atom, on the number of electrons a given molecule can

donate, and on the mechanism of antioxidant action. In cases where the structure of the

electron donor is not known (eg. a plant extract), this method can afford data on the

reduction potential of the sample, and hence can be helpful in comparing the reduction

potential of unknown materials, A similar method, using a different stable radical

(galvinoxyl) was introduced.

Free Radical Scavenging Capacity on DPPH Radical215-217

Mechanism:

Free radical scavenging potentials of the extracts were tested against a methanolic

solution of 1, 1-diphenyl-2-picryl hydrazyl (DPPH). Antioxidants react with DPPH and

convert it to 1-1-diphenyl -2-picryl hydrazine. The degree of discoloration indicates the

scavenging potential of the antioxidant extract. The change in the absorbance produced

at 517nm has been used as a measure of antioxidant activity.



Procedure

The free radical scavenging activity of all plants different solvent extracts was

measured by 1, 1-diphenyl-2-picryl-hydrazyl (DPPH). For DPPH assay, the method of

Blois218

was adopted. The capacity of all plants different solvent extract to scavenge the

lipid-soluble DPPH radical was monitored at an absorbance of 517 nm. Aqueous,

ethanolic & petroleum ether solvent extracts (1 ml) of plant Bauhinia variegata, Tectona

grandis and Schrebera swietenioides, at several concentrations ranging from 10-1000 μg

/ ml was allowed to react with DPPH. Thirty minutes later, the absorbance was measured

at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical

scavenging. The percentage inhibition of absorbance was calculated for each

concentration relative to a blank absorbance using the spectrophotometer. The DPPH

scavenging capacity of the extract is compared with that of BHT (Butylated

hydroxytoluene). All determinations were carried out three times, and in triplicate.

Percentage inhibition was calculated as DPPH radical scavenging activity.

DPPH radical Scavenging effect (%) = (Abs control – Abs sample) × 100

(Abs control)

Where, Abs control is the absorbance of initial conc. of DPPH radical

Abs sample is the absorbance of DPPH radical + sample Extract / standard



From the obtained DPPH radical Scavenging effect (%), the IC50 were calculated,

which represents the concentration of the scavenging compound that caused 50%

neutralization.

Screening of the potent antioxidant activity exhibiting solvent extract of selected

medicinal plants i.e. ethanolic extract of Bauhinia variegata root, Tectona grandis bark

and Schrebera swietenioides fruit was subjected for further antidiabetic &

hepatoprotective activity study.

4.5 ANIMAL SELECTION:

Male Albino rats weighing 150-200 g breed in the animal house, Sri.

Venkateshwara enterprises, Bangalore, were used in this study. They were employed for

assessing acute oral toxicity, antidiabetic and hepatoprotective activity study. The

animals were allowed free access to commercial rat pallet diet (Lipton India ltd.,

Mumbai, India) and water ad libitum. Rats were housed in a group of six in clean cages

at 25C and 12 hours photoperiod with relative air humidity of 30 to 60%. The bedding

material of the cages was changed everyday. All the experimental procedures were

carried out accordance with committee for the purpose of control and supervision of

experiments on animal (CPCSEA) guidelines. The experimental procedures were

approved by the institutional animal ethical committee (IAEC) (CPCSEA/1/15/2007).

The certificate of approval of the research project is given in Annexure.

4.5.1 Toxicity Studies219

Acute oral toxicity – Acute toxic class method:

The acute oral toxicity was carried out as per the guidelines set by Organization for

Economic Co-operation and Development (OECD), revised draft guidelines 423,

received from Committee for the Purpose of Control and Supervision of Experiments on

Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India.



The principle is based on a stepwise procedure with the use of a minimum number

of animals per step to obtain sufficient information on the acute toxicity of the test

substance to enable its classification. The substance is tested using a stepwise procedure,

each step using three animals of either sex. Absence or presence of compound related

mortality of the animals will determine the next step of,

- No further testing is required

- Dosing of three additional animals with the same dose

- Dosing of 3 animals at the next higher or the next lower dose level

The method enables judgment with respect to classifying the test substance to one of

the series of toxicity classics defined by fixed LD50 cut off values.

Description of the method:

1) Selection of animal species:

Healthy young albino rats of either sex weighing 150-200 g (8 to 12 weeks old)

were used for acute toxicity study to determine LD50 of ethanolic extract of selected

medicinal plants i.e. Bauhinia variegata roots, Tectona grandis bark and Schrebera

swietenioides fruits. Totally there were three groups.

2) Housing and feeding condition:

The temperature in the experimental room was around 25C. Lighting was natural

sequence being 12 hours dark, 12 hours light. The conventional laboratory diet was fed

with adequate supply of drinking water.

3) Preparation of Animals:

The animals were randomly selected, marked to permit individual identification

and kept in polypropylene cages for one week prior to dosing to allow acclimatization of

them to laboratory conditions.



4) Preparation of doses:

All the extracts were prepared as a suspension by triturating with water and 1%

gum acacia.

5) Administration of doses:

The test substances are administered in a single dose by gauge using a stomach

tube. Prior to dosing, animals were kept for 12 h of fasting. Then animals were weighed

and test substance was administered. After the dose was administered food was withheld

for a further 3-4 h.

6) Number of animals and dose levels:

In each step three animals were used in each group. Study was begun at 2000

mg/kg body weight. The procedure of dose selection and finalizing LD50 cut off values is

as,

Sr. No. Name of Extract LD50 Cut off

mg/kg body wt. Vehicle

1. Ethanolic extract of Bauhinia

variegata roots 2000 mg

1% Gum

acacia

2. Ethanolic extract of Tectona

grandis bark 2000 mg

1% Gum

acacia

3. Ethanolic extract of Schrebera

swietenioides fruits 2000 mg

1% Gum

acacia

1/10th

of this lethal dose was taken as effective dose (therapeutic dose) for

subsequent anti-diabetic and hepatoprotective activity.

Observations:

Animals were observed initially after dosing at least once during the first 30

minutes, periodically during the first 24 h. In all cases death was observed within first 24

h. Additional observations like changes in skin and fur, eyes and mucous membranes,

and also respiratory, circulatory, autonomic and central nervous system and somatomotor



activity and behavior pattern. Attention was also given to observation of tremors and

convulsions.

4.6 ANTIDIABETIC ACTIVITY STUDY

Screening of antidiabetic activity of these selected medicinal plant extracts was

done in rats by conducting glucose tolerance test (GTT) study and evaluating their

effects (Single dose and Multidose treatment study) on blood glucose level, serum lipid

profiles and histology of livers in alloxan diabetic rats.

Chemicals and drugs

Glibenclamide was obtained from Hoechst India as in form of Daonil tablet

Alloxan (Sigma Aldrich Co., USA)

Cholesterol, HDL, Triglycerides kits were procured from ERBA diagnostics

Manheim Ltd. India.

Glucometer (Sugar-check, Wockhardt Ltd, Mumbai, India).

Instruments

Micro Pipettes (Erba Bio Medicals Ltd.)

Star 21 Blood Chemistry – semi auto analyzer

Micro Centrifuge Model MC-2 RPM- 10,000 (Genei Pvt. Ltd, Bangalore).

UV-visible Spectrophotometer -1800 (Shimadzu)

A) Oral glucose tolerance test (OGTT)

Fasting blood glucose level of each rat was determined at zero time after

overnight fasing with free access to water. Rats were divided into five groups containing

six rats each.

Design of the study:

The rats were divided into 5 groups of six animals each.



They were grouped as:

1) Group I: Control group-1 ml of 1% gum acacia suspension orally

2) Group II: Standard drug- Glibenclamide (2.5 mg/kg)

3) Group III: Bauhinia variegata ethenolic extract (200 mg/kg)

4) Group IV: Tectona grandis ethenolic extract (200 mg/kg) and

5) Group V: Schrebera swietenioides ethenolic extract (200 mg/kg)

These animals received their doses by oral route using an orogastric tube. Glucose

(2 g/kg) was orally administered 30 min. after the administration of extracts or

Glibenclamide or gum acacia suspension. Blood samples were collected from the tail

vein under ether anaesthesia just prior to and 30, 60, 120 and 240 min after glucose

loading. Glucose levels were estimated using glucose-oxidase-peroxidase reactive strips

and a glucometer.

B) Effects of extracts on blood glucose levels in alloxan induced diabetic rats [Single

dose (Acute) treatment]

The animals were selected and weighed then marked for individual identification.

The rats were injected with alloxan monohydrate in saline (0.9% NaCl) at a dose of 120

mg/kg body weight intrperitonally to induce diabetes in overnight fasted male wistar

albino rats weighing 170-200 g. After one hour of alloxan administration the animals

were given feed ad libitum. A 5% dextrose solution was given in feeding bottle for a day

to over come the early hypoglycemic phase. After 72 h, animals with blood glucose

levels higher than 250 mg/dl were considered diabetic and were included in the study.

Rats were divided into six groups containing six rats each.






1) Group I: Normal control group-1 ml of 1% gum acacia suspension orally

2) Group II: Diabetic control group-1 ml of 1% gum acacia suspension orally

3) Group II: Diabetic rats receiving standard drug- Glibenclamide (2.5 mg/kg)

orally

4) Group III: Diabetic rats receiving Bauhinia variegata ethanolic extract (200

mg/kg) orally,

5) Group IV: Diabetic rats receiving Tectona grandis ethanolic extract

(200 mg/kg) orally and

6) Group V: Diabetic rats receiving Schrebera swietenioides ethanolic extract

(200 mg/kg) orally.

Blood samples were collected from the tail vein prior to and at 2, 4, 6 and 8 h

intervals after the administration of the extract and blood glucose levels were estimated

using glucometer.

C) Effects of extracts on blood glucose levels in alloxan induced diabetic rats [Multi

dose (sub acute) treatment]

The animals were selected and weighed, then marked for individual identification.

The rats were injected with alloxan monohydrate in saline (0.9% NaCl) at a dose of 120

mg/kg body weight intrperitonally to induce diabetes in overnight fasted male wistar

albino rats weighing 170-200 gm. After one hour of alloxan administration the animals

were given feed ad libitum. A 5% dextrose solution was given in feeding bottle for a day

to over come the early hypoglycemic phase. After 72 h, animals with blood glucose

levels higher than 250 mg/dl were considered diabetic and were included in the study.

Rats were divided into six groups containing six rats each.






1) Group I: Normal control group-1 ml of 1% gum acacia suspension orally

2) Group II: Diabetic control group-1 ml of 1% gum acacia suspension orally

3) Group II: Diabetic rats receiving standard drug- Glibenclamide (2.5 mg/kg)

orally

4) Group III: Diabetic rats receiving Bauhinia variegata ethenolic extract (200

mg/kg) orally

5) Group IV: Diabetic rats receiving Tectona grandis ethenolic extract

(200 mg/kg) orally and

6) Group V: Diabetic rats receiving Schrebera swietenioides ethenolic extract

(200 mg/kg) orally.

These rats were given the same doses of the extract once daily for 15 days in this

study. Blood samples were collected from the tail vein of nonfasted rats on days 0, 5, 10

and 15 of extract administration and blood glucose levels were recorded. Serum lipid

profiles on day 15 were measured by an autoanalyzer, pancreas- histopathological

examination was performed after sacrificing the animal under anesthesia on 15th

day and

body weight measurement were carried out on days 0, 5, 10 and 15 of study.

a) Measurement of biochemical parameters:

The triglycerides, total-cholesterol, HDL-cholesterol, LDL-cholesterol and

VLDL-cholesterol concentrations were measured using commercial kits by enzymatic

photo colorimetric method.

i) Estimation of triglycerides220

Triglyceride was estimated by using method of Wako by using a standard kit

obtained from ERBA diagnostics Manheim Ltd.

Kit Contents:



Reagent 1: Triglycerides DES reagent

Lipoprotein Lipase, Glycerol kinase, Glycerol-3-Phosphate Oxidase, Peroxidase, 4-

aminoantipyrine and ATP., 3-5 D H B S and buffer ( pH 7.0) Mg2+, GPO.

Reagent 2: Triglycerides standard (200 mg/dl)

Preparation of the working reagent: Dissolved content of one bottle of reagent with the

amount of AQUA- 4 indicated on the label. Swirl to dissolve, allowed to stand for 10

minutes at room temperature.

Principle:

Serum triglycerides are hydrolyzed to glycerol and fatty acids by lipase. In the

presence of ATP and glycerol-kinase, glycerol is converted into glycerol-3-phosphate

and ADP. Glycerol-3-PO4 oxidase dissociates glycerol-3-phosphate into dihydroxy-

acetone phosphate and hydrogen peroxide. In the presence of peroxidase, hydrogen

peroxide reacts with ESPAS (N-Ethy-N-sulfopropyl-n-methoxyaniline) and 4-

aminoantipyrine to form a colored complex. The intensity of the colour developed is

proportional to the triglycerides concentration and is measured photometrically at 505nm.

Procedure:

Pipette into test tubes Blank Standard Test

Working Reagent 1.0ml 1.0ml 1.0ml

Standard -- 10μl --

Sample -- -- 10μl

Distilled water 10μl -- --

Mixed and incubated at 370C for 10 min. Readed the absorbances of standard and each

sample at 505 nm on auto analyzer against reagent blank.



ii) Estimation of total cholesterol (CHOD-PAP METHOD)221

Total cholesterol was estimated by using end point method by using a standard kit

obtained from ERBA diagnostic Mannheim Gmbh Ltd.

Kit Contents:

Working solution: (cholesterol oxidase, cholesterol esterase, Peroxidase, 4-amino

antipyrine, sodium phenolate, phosphate buffer, lipid clearing agent).

Standard cholesterol. 200 mg/dl

Principle:

Cholesterol esters are hydrolysed by enzyme cholesterol esterase to free cholesterol

and fatty acids. Free cholesterols are oxidized to cholest 4 en-3 one and H2O2 is liberated.

This then couples with 4-aminotipyrine and phenol in the presence of peroxidase to form

a coupled compound. The test tubes are gently shaken to mix the components and

incubated at 37°C for 10 min. At the end of 10 min, test, standard and blank solutions are

measured in auto analyzer and direct readings are obtained.

Procedure:

Pipette into tubes marked Blank Standard Test

Working Reagent 1000 µl 1000 µl 1000 µl

Distilled water 20 µl -- --

Standard -- 20 μl --

Test -- -- 20 μl

Mixed well, incubated at 370C for 10 min. aspirate blank followed by standard and

tests. Readed the absorbance of standard and each test tube against blank at 505 nm on

semi auto analyzer.

iii) Estimation of serum High Density Lipoproteins (HDL): 10% (Phosphotungstic

acid method)222



Chylomicrons LDL and VLDL are precipitated from serum by phosphotungstate in

the presence of divalent cations such as magnesium. The HDL Cholesterol remains

unaffected in the supernatant and is estimated using ERBA cholesterol reagent.

Procedure:

Pipette into tubes

marked

Blank Standard Test

Working reagent 0.5 ml 0.5 ml 0.5 ml

Standard - 50 μl -

Sample - - 50 μl

iv) Estimation of serum Very Low Density Lipoprotein Cholesterol and v) Low

Density Lipoprotein223,224

Serum Very Low Density Lipoprotein cholesterol (VLDL) and Low Density

Lipoprotein (LDL) cholesterol is estimated as per Friedevald’s equation.

VLDL-cholesterol = serum triglyceride

5

LDL-cholesterol= serum total-cholesterol− VLDL-cholesterol−HDL-Cholesterol

Results were expressed in mg/dl.

b) Measurement of body weight:

Body weight of all the experimental animals was recorded on zero day,

fifth day, Tenth day and final day i.e. fifteenth day using a digital weighing

scale. The percentage change in body weight was calculated using the formula



Body weight on 5/10/15th

day - Initial weight X 100

Initial body weight on

c) Histopathology of pancreas225

i) Processing of isolated organs:

The whole pancreas from each animal was removed after sacrificing the animal

under anesthesia. A small cut was given to avoid any cell degeneration during storage

and preserved in formalin (10% solution) for at least 2 days. The pancreas pieces were

washed in running water for about 12 hours. This was followed by dehydration with

alcohol of increasing strength (70, 80 and 90%) for 12 hours each. Then the final

dehydration was done using absolute alcohol 3 times for 12 hours each. Again the tissue

is cleaned by using xylene 2 times for 15 to 20 min. each. After cleaning the organ pieces

were subjected to paraffin infiltration in automatic tissues processing unit.

ii) Embedding in paraffin:

Hard paraffin was melted and was poured into square-shaped blocks. The pancreas

pieces were then dropped into the liquid paraffin quickly and allowed to cool.

iii) Sectioning:

The blocks were cut using microtome to get sections of thickness 5 microns. The

sections were then taken on a microscopic slide on which egg albumin (sticky substance)

was applied. The sections were allowed to remain on the sticky substance for three days

till it sticks firmly onto the slide. The section should be dried completely before staining.

iv) Staining:

Eosin is an acidic stain and Hemotoxylin is a basic stain, which are used for staining.

v) Observation:

All the slides were observed for changes in histopathological characteristics and

photographs were taken.



Statistical analysis

Values are presented as mean ± S.E.M. Statistical difference between treatments

and the controls were tested by one-way analysis of variance (ANOVA), followed by

Dunnett’s multiple comparison test using the “Stat” statistics computer program. A

difference in the mean values of P<0.05 was considered to be statistically significant.

4.7 HEPATOPROTECTIVE ACTIVITY STUDY

Hepatoprotective activity was carried out by using albino rats. The animals were

divided into six groups of six rats in each group226

Chemicals and drugs

All chemicals and solvents used were of analytical grade are procured from

Merck Ltd., Mumbai, India and Sigma Aldrich Co., USA.

Liv 52 syrup was obtained from Himalaya Drug Company, India. [Composition-

Capparis spinosa (34 mg), Cichorium intybus (34 mg), Solanum nigrum (16 mg),

Terminalia arjuna (16 mg), Achillea millefolium (8 mg), Tamarix gallica (8 mg)

and Cassia occidentalis (8 mg)]

Instruments

Micro Pipettes (Erba Bio Medicals Ltd.)

Star 21 Blood Chemistry – semi auto analyzer

Micro Centrifuge Model MC-2 RPM- 10,000 (Genei Pvt. Ltd, Bangalore).

UV-visible Spectrophotometer -1800 (Shimadzu)






1) Group I: Normal control group-1 ml of 1% gum acacia suspension orally up

to 10 days

2) Group II: CCl4 intoxicated control group-1 ml of 1% gum acacia suspension

orally up to 10 days

3) Group II: CCl4 intoxicated rats receiving standard drug- Liv 52 syrup

(1 ml/kg) orally up to 10 days

4) Group III: CCl4 intoxicated rats receiving Bauhinia variegata ethanolic

extract (200 mg/kg) orally up to 10 days

5) Group IV: CCl4 intoxicated rats receiving Tectona grandis ethanolic extract

(200 mg/kg) orally up to 10 days and

6) Group V: CCl4 intoxicated rats receiving Schrebera swietenioides ethanolic

extract (200 mg/kg) orally up to 10 days.

Except group I (Normal control group), all remaining groups were received

Carbon tetrachloride at a dose of 0.7 ml/kg, on 3, 6 & 10th

day by intraperitoneal route.

On 10th

day, 1 h after last dose of Carbon tetrachloride, animals were sacrificed by

cervical dislocation & the blood was collected from the carotid artery, serum is separated

& used for estimation of biochemical parameters.

A) The biochemical parameters includes

Serum glutamate pyruvate transaminase (SGPT)

Serum glutamate oxaloacetate transaminase (SGOT)

Serum alkaline phosphatase (ALP)

Serum total bilirubin

a) Estimation of serum SGPT (UV Kinetic method)227

Principle



SGPT catalyses the transfer of amino group from L-alanine to 2-oxoglutarate with

the formation of pyruvate and L-glutamate. The pyruvate so formed is allowed to react

with NADH to produce L-lactate. The rate of this reaction is monitored by an indicator

reaction coupled with LDH in the presence of NADH (nicotinamide adenine

dinucleotide). The oxidation of NADH in this reaction is measured as a decrease in the

absorbance of NADH at 340 nm, which is proportional to SGPT activity.

Reaction

ALT

L-Alanine + 2-Oxoglutarate Pyruvate + L-Glutamate

LDH

Pyruvate + NADH L-Lactate + NAD

ALT : Alanine aminotransferase

LDH : Lactate dehydrogenase

Procedure


Working Reagent 0.5 ml 0.5 ml 0.5 ml

Standard -- 50 µl --

Sample -- -- 50 µl

b) Estimation of SGOT (IFCC method)228

Principle

SGOT catalyses the transfer of amino group from L-aspartate to 2-oxoglutarate

forming oxaloacetate and L-glutamate. The rate of this reaction is monitored by an

indicator reaction coupled with malate-dehydrogenase (MDH) in which the oxaloacetate

formed is converted to malate in the presence of reduced nicotinamide adenine

dinucleotide (NADH). The oxidation of NADH in this reaction is measured as a decrease

in absorbance of NADH at 340 nm, which is proportional to SGOT activity.



Reaction

ALT

L-Aspartate + 2-Oxoglutarate Oxaloacetate + L-Glutamate

MDH

Oxaloacetate + NADH Malate + NAD

LDH

Sample Pyruvate + NADH L-Lactate + NAD

AST : Aspartate aminotransferase.

MDH : Malate dehydrogenase

LDH : Lactate dehydrogenase

Procedure:




Sample -- -- 50 µl

c) Estimation of serum alkaline phosphate (ALP)229

Principle

Serum alkaline phosphatase hydrolyses p-nitrophenyl phosphate into p-nitrophenol

and phosphate in the presence of oxidizing agent Mg2+

. This reaction is measured as

absorbance is proportional to the ALP activity.

Reaction

Mg2+

ALP

P-Nitro phenyl phosphate + H2O P-Nitrophenol + Phosphate

Procedure:




Sample -- -- 50 µl



d) Estimation of serum Bilirubin (Diazo method, end point)

230

Principle

Bilirubin reacts with diazotised sulphanilic acid in acidic medium to form pink

colored azobilirubin with absorbance directly proportional to bilirubin concentration.

Direct bilirubin, being water soluble directly reacts in acidic medium. However, indirect

bilirubin is solubilised using a surfactant and then it react similar to direct bilirubin.

Procedure




Sample -- -- 50 µl

B) Histopathology of Liver231

i) Processing of isolated organs:

The liver from each animal was removed after sacrificing the animal under

anesthesia. Liver tissues from rats of all groups were carefully removed of adipose tissue.

A small cut was given to avoid any cell degeneration during storage and preserved in

formalin (10% solution) for at least 2 days. The liver pieces were washed in running

water for about 12 hours. This was followed by dehydration with alcohol of increasing

strength (70, 80 and 90%) for 12 hours each. Then the final dehydration was done using

absolute alcohol 3 times for 12 hours each. Again the tissue is cleaned by using xylene 2

times for 15 to 20 min. each. After cleaning the organ pieces were subjected to paraffin

infiltration in automatic tissues processing unit.

ii) Embedding in paraffin:



Hard paraffin was melted and was poured into square-shaped blocks. The liver

pieces were then dropped into the liquid paraffin quickly and allowed to cool.

iii) Sectioning:

The blocks were cut using microtome to get sections of thickness 5 microns. The

sections were then taken on a microscopic slide on which egg albumin (sticky substance)

was applied. The sections were allowed to remain on the sticky substance for three days

till it sticks firmly onto the slide. The section should be dried completely before staining.

iv) Staining:

Eosin is an acidic stain and Hemotoxylin is a basic stain, which are used for staining.

v) Observation:

All the slides were observed for changes in histopathological characteristics and

photographs were taken.

Statistical analysis

Values are presented as mean ± S.E.M. Statistical difference between treatments

and the controls were tested by one-way analysis of variance (ANOVA), followed by

Dunnett’s multiple comparison test using the “Stat” statistics computer program. A

difference in the mean values of P<0.05 was considered to be statistically significant.

4.8 ISOLATION OF ACTIVE PRINCIPLES

A number of chromatographic techniques have been developed into important

tools in the field of analytical and preparative natural product chemistry associated with

the characterization of biologically active compounds. The separation and purification of

plant constituents is mainly carried out using different chromatographic techniques like

Thin layer chromatography (TLC), High performance thin layer chromatography



(HPTLC), Column chromatography (CC) etc. The selection of technique depends upon

solubility properties and volatilities of the compounds to be separated.

The selected extracts of medicinal plants were subjected for various

fractionations with different solvents according to polarity in the column

chromatography technique for separating active phytoconstituents. The separated

fractions were subjected for characterization of active phytoconstituents by qualitative

chemical method and TLC method.

Column chromatography:

In column chromatography, the stationary phase, a solid adsorbent i.e. crystalline

silica gel 60-120 # (LR), is placed in a vertical glass (usually) column and the mobile

phase, polar or non polar liquid, is added to the top and flows down through the column

(by either gravity or external pressure). Column chromatography is generally used as a

purification technique: it isolates desired compounds from a mixture.

150 g of silica gel for column chromatography (laboratory grade) was activated

in hot air oven at 110C for one hour. The appropriate solvent was used to build the

silica gel in the glass column. The glass wool was fixed at the bottom. The activated

silica gel was charged into the column in small portions with gentle tapping after each

addition in order to ensure uniform packing. The small quantity of solvent system was

allowed to remain on the top of the column (about 2 cm). The air bubbles present in the

column were removed by gentle tapping to get a uniform bed of adsorbent.

Afterwards the extract was dissolved in a small quantity of solvent (5 ml) and

adsorbed on silica gel and then charged into the column. The chromatogram was

allowed to develop overnight, taking care to prevent the drying of the column by

plugging the open end with adsorbent cotton. The elution of solvent system was started

after complete saturation of the column.



The details of column chromatography are as under:

Adsorbent

Silica gel for column chromatography

activated at 110C for 1 h

Length of the column 41 cm.

Length of the adsorbent 22 cm.

Diameter of the column Outer 3 cm, inner 2.8 cm.

Rate of elution 10-15 drop /min.

Volume of the elute collected 25 ml each

Total volume of each mixture

elute collected

100 ml.

Elution Appropriate solvents

Totally around 10 fractions were collected and each fraction was subjected to qualitative

chemical test and thin layer chromatography for the identification of phytoconstituents.

Table No. 3: The solvent system for column chromatography:

Phyto-constituents Stationary phase Solvent system

Flavonol Glycosides Silica gel Toluene: Dioxan: Acetic acid (90:25:5)

Triterpenoids Silica gel Chloroform/ acetone (8:2)

Anthra-quinones Silica gel Ethyl acetate: Methanol: Water

(100:13.5:10)

Sterols Silica gel Petroleum ether : Acetone (85:15)



4.9 CHARACHTERIZATION OF ACTIVE PRINCIPLES

The obtained column fractions of extract were subjected to qualitative test and thin

layer chromatography for identification of phytoconstituents.

A) Characterization of isolated compound by qualitative chemical tests:

Different qualitative chemical tests of isolated column fractions were performed for

identification of phytoconstituents such as flavonoids, anthraquinones, triterpenoids,

sterols etc.

B) Characterization of isolated compound by TLC:

Thin layer chromatography is done exactly as it says - using a thin, uniform layer

of silica gel or alumina coated onto a piece of glass, metal or rigid plastic. The silica gel

(or the alumina) is the stationary phase. The mobile phase is a suitable liquid solvent or

mixture of solvents.

TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick

answer as to how many components are in a mixture. TLC is also used to support the

identity of a compound in a mixture when the Rf of a compound is compared with the Rf

of a known compound (preferably both run on the same TLC plate). The retention factor,

or Rf, is defined as the distance traveled by the compound divided by the distance

traveled by the solvent.

Rf value = Distance travelled by solute (cm)

Distance travelled by solvent (cm)

Preparation of TLC Plate:

TLC was performed using silica gel as adsorbent. Slurry of silica gel was prepared

in distilled water. The slurry was applied to get a thin layer of 0.3 mm thickness over a

clean and dry glass plate of 10 × 20 cm size by pouring method. The plate was activated

at 110ºC for an h.



Table No. 4: Mobile phases for TLC of isolated fractions of extract for

identification of different phytoconstituents

Phyto-

constituents

Stationary

phase Solvent system Visualizing reagent

Flavonol

Glycosides Silica gel

Toluene: Dioxan: Acetic

acid (90:25:5) Anisaldehyde-H2SO4

Triterpenoid

Saponins Silica gel chloroform/ acetone (8:2)

Acetic anhydride- H2SO4 /

ethanol (1:1).

Anthra-

quinones Silica gel

Ethyl acetate: methanol:

water (100:13.5:10) 10% Ethenolic KOH

Sterols

Silica gel

Petroleum ether : Acetone

(85:15) Liebermann Burchard reagent

chapter 4: materials and methods -...

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