Chapter – III Materials and Methodsshodhganga.inflibnet.ac.in/bitstream/10603/8870/13/14_chapter...

Chapter ndash III Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods

64

31 Plant Material Collection And Authentication

The flowers of Nerium oleander L were collected from Kota district of Rajasthan in

India and authenticated by routine pharmacognostic procedures by Dr SN Sharma

senior scientist botany division of Indian Institute of Integrative Medicine (IIIM)

Jammu India A voucher specimen was retained and deposited at the crude drug

repository of the herbarium of IIIM vide CDR accession no21869

32 Preparation Of Extracts

The flowers (2 kg) were dried in shade and coarsely powdered The powder was

extracted with petroleum ether (40-60 ordmC) for defatting and then macerated with ethanol

water (11) with constant stirring (1200 rpm 3h) The solvent containing extract was

filtered and the marc was completely drained this process was repeated thrice for the

complete extraction of the phytochemicals The extracts obtained during the three cycles

were independently combined and reduced to 18th of its original volume under rota

evaporator at 45 degC and then lyophilized (Vis Tis Advantage freeze Dryer 20 EL-85) to

obtained a yield of 11 (ww)

33 Animals

Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams as well as

male and female Swiss albino mice (8-12 weeks old) weighing 18-20 g were purchased

from Central Animal House Facility IMTECH Chandigarh 551999 CPCSEA The

protocol for CNS studies was approved by the Institution Animals Ethics Committee

(IAEC) Of ASBASJSM College of Pharmacy Bela (Ropar) Punjab (Registration No

72402aCPCSEA-29102002) The animals were housed under standard laboratory

conditions with temperature (23plusmn1degC) relative humidity (55plusmn10) 1212 h light dark

cycles and fed with standard pellet diet (Ms Ashirwad Industries Mohali) and purified

water ad libitum

34 Preparation of Drugs

Test doses of various fractions were freshly prepared as a homogenized suspension of N

oleander flowerrsquos extract in doses of 100 200 and 400 mgkg except chloroform extract

(25 50 and 100 mgkg) administered orally to Swiss albino mice once during the

experiment Chemicals reagents and testing kits etc were procured from the central

stores of the ASBASJS Memorial College of Pharmacy Bela (Rupnagar) Punjab

(India)


65

35 Ascertaining Margin Safety by Acute Oral Toxicity Studies in Rats And Mice

(OECD Guideline 423)

Whenever an investigator administers a chemical substance to a biological system

different types of interactions can occur and a series of dose-related responses result In

most cases these responses are desired and useful but there are a number of other effects

which are not advantageous These may or may not be harmful to the patients The types

of toxicity tests which are routinely performed by pharmaceutical manufacturers in the

investigation of a new drug involve acute sub-acute and chronic toxicity Acute toxicity

is involved in estimation of LD50 (the dose which has proved to be lethal (causing death)

to 50 of the tested group of animals) Determination of acute oral toxicity is usually an

initial screening step in the assessment and evaluation of the toxic characteristics of all

compounds

Category 1

gt 0-5

Category 2

gt 5 ndash 50

Category 3

gt 50 - 300

Category 4 gt 300 - 2000

Category 5

gt 2000 - 5000

Category 5

Or unclassified

5 25 30 50 200 300 500 1000 2000 2500 5000 infin

5mgkg 3animals

2-3

300mgkg 3animals

50mgkg 3animals

50mgkg 3animals

5mgkg 3animals

2000mgkg 3animals

300mgkg 3animals

2000mgkg 3animals

2-32-32-3 2-3

2-3 2-3 2-3

0-1 0-1

0-1

0-1

0-1 0-1

0-1

0-1

0

0

3 2other

3(at 300)At 1st stepother

13 (at 50)At 1st step

0

Test Procedure with a Starting Dose of 300 mgkg Body weight

Acute toxicity study according to OECD guideline 423

Figure 5 Scheme of acute toxicity study according to OECD guideline 423


66

36 Preliminary Exploration of the CNS Activity Of 50 Hydroalcoholic Extract

(HyE) of N oleander Flowers in Mice

361 Spontaneous motor activity

The animals were divided into 4 groups with each group constituting 6 mice

Group-1 Control

Group-2 Standard (Diazepam) 4 mgkg

Group-3 HyE of N oleander 100 mgkg


Each Group of animal constitutes 6 mice In this experiment Group-I served as control

Group-II as standard (Diazepam 4 mgkg ip) while Group-III to Group-IV were

administered with drug (100 and 200 mgkg) po respectively For the spontaneous motor

activity individual mouse was introduced into actophotometer (INCO) and its spontaneous

locomotor activity was measured in 10 min duration

362 Pentobarbitone-induced sleeping time

The animals were divided into three new groups (n=6) Group-I was treated orally with

pentobarbital (50 mgkg ip) Group-II and Group-III received N oleander orally at the

dose of 100 and 200 mgkg respectively Pentobarbital (50 mgkg ip) was injected 30 min

before oral administration for all groups The time elapsed between loss and recovery of

the righting reflex was noted and taken as sleep time

Group-1 pentobarbital (50 mgkg ip) Control



363 Explored the muscle relaxant property of HyE in mice using Rota rod

apparatus (Motor coordination)

Before performing the experiment the fresh animals were trained to remain for 3 min on

rotarod apparatus the rod rotating at a speed of 25 rpm After training all mice were

divided into three groups (n=6) Group-I served as control Group-II and Group-III as N

oleander treated 100 and 200 mgkg respectively and the effect on motor coordination was


67

assessed by rotarod apparatus The fall-off time from the rotarod was noted for each

mouse

Group-1 Control



364 Determined the anti anxiety effect of HyE in mice using elevated plus maze

(EPM)

To conduct the EPM test 30 mice were taken and divided into 4 groups with each group

consisting 6 mice The plus maze consists of two open arms and two closed arms (50 x 10

x 50 cm each) elevated to a height Thirty minutes post treatment each mouse was placed

in turn in the centre of the maze facing one of the closed arms The cumulative times spent

by each mouse in the open and closed arms of the maze were recorded for 5 min

Group-1 Control




365 Explored the anticonvulsant property of HyE against Pentylenetetrazol (PTZ)

induced convulsions in mice

Three groups of mice (n=10) were used Group-I served as vehicle control Group-II and

Group-III as drug treated 100 and 200 mgkg po respectively After 2 h of treatment PTZ

was administered (60 mgkg ip) in all three groups Each group was observed for 30 min

and the onset of convulsion and duration of convulsion showed was counted

Group-1 Control + PTZ (60 mgkg ip)

Group-2 HyE of N oleander 100 mgkg + PTZ (60 mgkg ip)



68

366 Explored the anticonvulsant property of HyE against maximal electroshock

(MES) induced convulsions in mice

Group-I served as vehicle control Group-II and III were treated orally with N oleander

dose 100 and 200mgkg po respectively After 2 h from treatment the above mention

groups were given current of 12 mA for 02 seconds in electroconvulsiometer (INCO) by

using ear electrode The onset of convulsion duration of tonus was noted 100 protection

considered when hind limb tonic extension was completely abolished

Group-1 Control + MES (12 mA for 02 seconds)

Group-2 HyE of N oleander 100 mgkg + MES (12 mA for 02 seconds)


37 Successive solvent extraction of the flowers of Nerium oleander L

The flowers of Nerium oleander were dried in shade and coarsely powdered Coarsely

powdered flower (5 kg) was successively extracted in the Soxhlet apparatus using

petroleum ether chloroform ethyl acetate methanol and water as solvent for the complete

extraction of the phytochemicals The five extracts were dried in rotary evaporator at 45 degC

and the dried extracts were stored in vacuum desiccators containing anhydrous silica gel

All the five extracts were subjected to acute toxicity studies as per the OECD guidelines

Figure 6 The complete scheme of the extraction and fractionation of dried flowers of N oleander


69

38 Dose Determination of Successive Solvent Extracts and Ascertaining Margin

Safety by Acute Oral Toxicity Studies in Rats and Mice (OECD guideline 423)

The maximum tolerable dose determination was performed using OECD (Organization for

Economic Corporation and Development) guideline 423 The study is performed as a

stepwise procedure to explore toxicity at dose level 5 50 300 and 2000 mgkg po with 3

animals at each dose level Three female Swiss mice (18-22 g) were used for the study

and were not fed for 3-4 h prior to the experiment They were individually administered

various extract of N oleander (2000 mgkg po) on day 1 of the experiment Each animal

was continuously monitored during first 30 min and then they were monitored on an

hourly basis for next 4 hrs Subsequently they were observed after a four hour interval

After this they were under observation for 14 days to monitor any abnormal signs and

symptoms depicting toxicity These animals were humanely killed for animal welfare

reasons after termination of the experiment The animals were observed for any change in

skin and fur eyes and mucous membranes respiratory circulatory and autonomic and

central nervous systems somatosensory activity and behavioral pattern Attention was

given to observation like tremors convulsions salivation diarrhoea lethargy sedation

hypnosis and coma

39 Preliminary Phytochemical Screening

The successive solvent extracts were subjected to preliminary phytochemicals screening

for the detection of various phytoconstituents such as alkaloids steroids flavonoids

glycosides tannins phenolic compounds carbohydrates proteins amino acids and fats

The following tests were carried out to identify the various phytoconstituents present in the

extracts (Harborne 1998)


70

Phyto

Chemical

Test Reagent Reaction Results

1

Alkaloids

A small quantity

sample + 5 ml of

15 vv HCl and

filtered

These filtrates (TFl) were then used

for testing alkaloids with following

reagents

Mayerrsquos

test

Mayerrsquos reagent

(MR) 136 gm

HgCl2 + 60 ml H2O

(dil upto 100 ml

H2O)

To a little of the TFl (taken in a

watch glass) + a few drops of the

MR

Cream coloured

precipitate

Dragen-dorffs

test

Sol A (17 gm of

bismuth subnitrate

+ 200 gm tartaric

acid + 800 ml H2O)

Sol B (160 gm

potassium iodide +

4 ml H2O)

Soln A + Sol B (11

vv proportion)

Working standard (WS) was

prepared by taking 50 ml of this

soln (A+B) + 100 gm of tartaric acid

( upto 500 ml H2O)

WS was sprayed on whatmann No

1 filter paper and the paper was

dried The TFl (basification) +

dilute ammonia was extracted with

chloroform and the CE was applied

on the filter paper impregnated

with WS with the help of a

capillary tube

Orange red colour

Wagnerrsquos test 127 g of I + 2 gm

of KI + 5 ml H2O

(dil upto 100 ml

H2O)

Few drops reagent + TFl Brown flocculent

precipitate

Hagerrsquos

reagent

A saturated aq Sol

of picric acid was

employed for this

test

When the test filtrate was treated

with this reagent

an orange yellow

ppt was obtained

(alkaloids)

2

Saponins

Froth

test

A few mg sample taken in a test

tube and shaken vigorously with a

small amount of NaHCO3 +H2O

Honeycomb like

froth is obtained

3

Sterols

Salkowask

i reaction

Few mg sample + 2 ml CHCl3 + 2

ml conc H2SO4 was added from the

side of the test-tube The test-tube

was shaken for few minutes

red colour in the

chloroform layer

(sterols)

Lieberman

nrsquos test

Few mg sample + few ml Ac2O

gently heated The contents of the

test-tube were cooled + Few drops

conc H2SO4 from the side of the

test-tube

A blue colour

(sterols)


71

Lieberman

n

Burchardrsquos

reaction

Few mg sample + CHCl3 + few

drops of Ac2O + conc H2SO4 from

the side of the tube

Red blue

green

4

Carbohydr

ates

Molischrsquos

test

2-3 ml of extract + few drops 95

α-naphthol sol + alcohol shaken

and conc H2SO4 was added from

side of the test tubes

Red brown ring at

the junction of the

liquids (presence of

carbohydrates)

Fehlingrsquos

solution

test

1 ml of fehlingrsquos A

and B sol were

mixed and boiled

for one minute

Equal volume of extract was added

and heated on boiling water bath for

5-10 min

yellow brick red

(reducing sugars)

Benedictrsquos

solution

test

Equal volume of Benedictrsquos reagent

+ extract were mixed in test tube


5 min

red solution

(reducing sugars)

5

Tannins

The test residue of each extract was

taken separately in water warmed

and filtered Tests were carried out

with the filtrate using following

reagents

Ferric

chloride

reagent

A 5 wv sol of FeCl3 + 90

alcohol was prepared Few drops of

this sol + little of the above filtrate

Dark green or Deep

blue colour (tannins

present)

Lead

acetate

test

10 wv sol of basic lead acetate +

H2O + test filtrate

If ppt is obtained

tannins present

6

Proteins

and amino

acid

Ninhydrin

test

The Ninhy drin reagent is 01 wv

solution of ninhydrin in n-butanol

A little of this reagent was added to

the test extract

Violet or Purple

colour if amino

acids present

Millonrsquos

test

Millonrsquos reagent 3

ml of mercury in 27

ml of fuming nitric

acid

This sol was then diluted with

equal quantity H2O Aq Sol of the

residue was taken + 2 to 3 ml of

Millonrsquos reagent

The white

precipitate which

slowly turns to

pink is obtained if

proteins are present

7

Tests for

flavonoids

Shinoda

test

A small quantity to test residue was

dissolved in 5 ml ethanol (95 vv)

and reacted with few drops of

concentrated hydrochloric acid and

05 gm of magnesium metal

Pink crimson or

magenta colour is

developed within a

minute or two

(flavonoids)

Ammonia

test

Few mg sample in 1 ml of 95 vv

ethanol and apply a drop of this

solution on filter paper then expose

this paper to ammonia vapors

Yellow color

(flavonoids)


72

Lead

acetate sol

test

To small quantity of extract + lead

acetate solution

Yellow coloured

ppt (flavonoids)

8

Triterpenoi

ds

Libermann

-Burchard

test

Extract treated with few drops of

acetic anhydride boil and cool

concentrated sulphuric acid is

added from the side of the test tube

A brown ring at the

junction of two

layers and the

(upper layer turns

green) sterols and

deep red colour

triterpenoids

Salkowskirsquo

s test

Treat extract in chloroform with

few drops of concentrated

Sulphuric acid shake well and

allow to stand for some time

lower layer red

colour (sterols) and

yellow coloured

(triterpenoids)

310 Preliminary In Vitro Screening of the Extracts

3101 Antioxidant activity

31011 DPPH radical scavenging activity of all the successive solvent extracts

Free radicals contribute in some severe chronic diseases in humans including

atherosclerosis arthritis cardiovascular diseases ischemia and reperfusion injury of many

tissues central nervous system injury neurological disorders gastritis cancer and AIDS

ROS including superoxide anion radical hydroxyl radical and hydrogen peroxide are

generated under physiological and pathological stresses in human body Oxidation process

is one of the most important routs for producing free radicals in food drugs and even

living systems Antioxidants played an important role in lowering oxidative stresses

caused by ROS and protect against these complex diseases through scavenging free

radicals and reducing hydrogen peroxide Currently available synthetic antioxidants like

butylated hydroxy anisole (BHA) butylated hydroxy toluene (BHT) tertiary butylated

hydroquinon and gallic acid esters have been suspected to cause or prompt negative health

effects Hence strong restrictions have been placed on their application and there is a trend

to substitute them with naturally occurring antioxidants Moreover these synthetic

antioxidants also show low solubility and moderate antioxidant activity An easy rapid

and sensitive method for the antioxidant screening of plant extracts is free radical

scavenging assay using 1 1-diphenyl-2-picryl hydrazyl (DPPH) stable radical

spectrophotometrically In the presence of an antioxidant DPPH radical obtains one more

electron and the absorbance decreases We have also found the relationship of total


73

flavonoid and phenol contents with antioxidant activity In the longer term plant species

(or their active constituents) identified as having high levels of antioxidant activity in vitro

may be of value in the design of further studies to unravel novel treatment strategies for

disorders associated with free radicals induced tissue damage Therefore it increases

interest in natural antioxidant products for use as medicines and food additives Vitamin C

vitamin E and carotenoids are some of these widely used natural antioxidants (Shimada et

al 1992)

The free radical scavenging activity of extract was measured by DPPH radical using the

method described previously (Shimada et al 1992) A 01mM solution of DPPH radical

in ethanol was prepared and 1ml of this solution was added to 3ml of extract solution in

water at different concentrations (50-250 microgmL)The mixture was shaken vigorously and

allowed to stand at room temperature for 30 min Then the absorbance was measured at

517 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Lower absorbance of the

reaction mixture indicated higher free radical scavenging activity The percent DPPH

scavenging effect was calculated using the following equation

DPPH radical scavenging effect () = 100 minus [(A0 ndash At) A0) times 100] (01)

Where A0 was the absorbance of the control reaction and At was the absorbance in the

presence of the standard sample or extract All the tests were performed in triplicate and

graph was plotted with the mean plusmn SD values BHA was used as standard antioxidant

compound

31012 Reducing power assay

The reducing power of extract was determined according to the method described

previously (Oyaizu M 1986) The different concentrations of extract (50-250 microgmL) in

1ml of distilled water was mixed with phosphate buffer (25 ml 02 M pH 66) and

potassium ferricyanide [K3Fe(CN)6] (25 ml 1) The mixture was incubated at 50 degC for

20 min A portion (25 ml) of trichloroacetic acid (10) was added to the mixture which

was then centrifuged for 10 min at 3000 rpm The upper layer of solution (25 ml) was

mixed with distilled water (25 ml) and FeCl3 (05 ml 01) and the absorbance was

measured at 700 nm in a spectrophotometer (UV -1601 Shimadzu Japan) Higher

absorbance of the reaction mixture indicated greater reducing power α-Tocopherol was

used as standard antioxidant compound


74

31013 Total antioxidant activity in linoleic acid emulsion system

The total antioxidant activity of the extract was determined according to the thiocyanate

method (Mitsuda et al 1996) 10 mg of extract was dissolved in 10 ml water Different

concentration of extract (50-250 microgmL) or standard samples in 25 ml of potassium

phosphate buffer (004 M pH 70) was added to linoleic acid emulsion (25 ml) in

potassium phosphate buffer (004 M pH 70) 5 ml linoleic acid emulsion consists of 175

g Tween-20 155 microl linoleic acid and 004 M potassium phosphate buffer (pH 70) On

the other hand 50 ml control consists of 25 ml linoleic acid emulsion and 25 ml

potassium phosphate buffer (004 M pH 70) The mixed solution was incubated at 37 degC

in a glass flask and in the dark After the mixture was stirred for 3 min the peroxide value

was determined by reading the absorbance at 500 nm in a spectrophotometer (UV -1601

Shimadzu Japan) after reaction with ferrous chloride (FeCl2) and thiocyanate at intervals

during incubation During the linoleic acid oxidation peroxides are formed These

compounds oxidize Fe2+ to Fe3+ The latter Fe3+ ions form complex with SCNminus which had

maximum absorbance at 500 nm Therefore high absorbance indicates high linoleic acid

oxidation The solutions without extract or standards were used as blank samples All data

about total antioxidant activity are the average of triplicate analyses The inhibition of lipid

peroxidation in percentage was calculated by following equation

Inhibition () = (A0 ndash At) A0 times 100 (02)


presence of the sample All the tests were performed in triplicate and graph was plotted

with the mean plusmn SD values α-Tocopherol were used as standard antioxidant compound

31014 2 2rsquo-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical

decolorization assay

ABTS was dissolved in water to make a concentration of 7 mM ABTS radical was

produced by reacting the ABTS stock solution with 245 mM potassium persulfate (final

concentration) and allowing the mixture to stand in the dark at room temperature for 12ndash

16 h before use For the test of samples the ABTS stock solution was diluted with

phosphate-buffered saline 5 mM (pH 74) to an absorbance of 070 at 734 nm After the

addition of 10 ml of diluted ABTS to 20 microl of sample the absorbance reading was taken


75

5 min after the initial mixing (Re R et al 1998) This activity is given as percent ABTS

radical scavenging that is calculated as follows

ABTS radical scavenging activity = [Control absorbance ndash Sample absorbance] times 100

[Control absorbance]

31015 Superoxide anion scavenging activity

The assay was based on the capacity of the methanolic extract to inhibit blue formazon

formation Superoxide radical were generated in riboflavin-light-NBT (Nitroblue

tetrazolium) system (Beauchamp and Fridovich 1971) The total volume of the reactant

mixture was 3 ml Each 3 ml of this reaction mixture contained 50 mM sodium phosphate

buffer (pH 76) 20 microg riboflavin and 12 mM EDTA and 01 mg NBT and 1 ml sample

solution Reaction was started by illuminating the reaction mixture with different

concentrations of the methanolic plant extract (50-250 microgml) for 90 sec After

illumination the absorbance was measured at 590 nm The reaction assembly was

enclosed in a aluminium foil lined box Unilluminated identical tubes containing reaction

mixture served as blank The percentage inhibition of superoxide anion generation was

calculated using the equation 01 Ascorbic acid was used as standard compound

31016 Hydroxyl radical scavenging activity

The reaction mixture containing 2-deoxy-d-ribose (1 mM) phenyl hydrazine (02 mM) (in

phosphate buffer pH 74) and different concentration of the test samples (50-250 microgml)

were incubated for 4 h at 37 degC The reaction was stopped by the addition of 28 (wv)

trichloroacetic acid solution followed by centrifugation at 5000 rpm (for 10 min) The

supernatant was mixed with aqueous 1 (wv) thiobarbituric acid (TBA) The TBA

reactive product thus formed was directly measured at 532 nm (Halliwell B et al 1987)

31017 Metal chelating activity

The chelating of ferrous ions by the methanolic plant extract was measured by the method

described previously (Dinis et al 1994 Kumaran and Joel Karunakaran 2006)

Different concentrations of the extract (50-250 microgml) were added to a solution of FeCl2

(005 ml 2 mM) The reaction was initiated by addition of 5 mM ferrozine (02 ml) The

reaction mixture was then shaken vigorously and allowed to stand at room temperature for


76

10 min The absorbance of the solution was then measured at 562 nm The percentage

inhibition of ferrozine ndashferrous complex formation was calculated by using the equation

01 EDTA (Ethylenediaminetetraacetic acid) was used as standard chelating compound

3102 Cyclooxygenase -1 (COX-1) and Cyclooxygenase -2 (COX-2) assays

The COX-1 assay was performed according to the method described elsewhere (Redl K et

al 1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M

Tris-HCL 18 microl of L-adrenaline-D-hydrogentartrate and 10 microl of hematine COX-1 (02

units) was added to the mixture and incubated for 5 min and then arachidonic acid (5 microl)

was added After 20 min 10 of formic acid (10 microl) was added to stop the incubation at

37 degC and then PGE2 enzyme-immunoassay (R and D systems) was used to measure the

concentration of PGE2

The COX-2 assay was performed according to the method describes earlier (Redl K et al

1994 Aguilar JL et al 2002) Sample solution (10 microl) was added to 190 microl of 01 M Tris-

HCL buffer 18 microl of L-adrenaline-D-hydrogentartrate 10 microl disodium edetate (Na2-

EDTA) and hematine (10 microl) Then 02 units of COX-2 was added to the mixture and pre-

incubated for 5 min Afterwards 5 microl arachidonic acid were added The incubation at 37 oC

was arrested after 20 min by addition of 10 microl of formic acid (10) At the end PGE2-

enzyme-immunoassay (R and D systems) was used to measure the concentration of PGE 2

3103 Antibacterial activity

31031 Microorganisms

The microbial strains used for testing antimicrobial activities included the Gram positive

bacteria Bacillus subtilis Staphylococcus aureus and Gram negative bacteria Escherichia

coli and Salmonella typhi The bacteria were cultured on nutrient agar slants The cultures

were maintained by subculturing periodically and were stored at 4 degC prior to use

31032 Screening for antibacterial activity

Antibacterial activity was tested by the agar-well diffusion method Different

concentrations of the extracts (50 and 100 mgml) were prepared by reconstituting the

extract in dimethyl sulphoxide (DMSO) The test microorganisms were seeded into the

medium by gently mixing 05 ml (For determination of antibacterial activity bacterial


77

cultures were adjusted to 05 McFarland turbidity standards) of the 24 hrs fresh cultures

with 20 ml sterile melted agar cooled to about 45 ordmC in sterile Petri plates After

hardening four 6 mm diameter wells were made using a sterile borer The wells were

filled with 100 microl of the sample extracts or solvent blanks The antibacterial assay plates

were incubated at 37 ordmC for 24 h The standard antibiotic streptomycin (500 microgmL) served

as positive antibacterial control The diameter of the zones of inhibition around each of the

wells (well diameter included) was taken as measure of the antimicrobial activity Each

experiment was carried out in triplicate and the mean diameter of the inhibition zone was

recorded (Mukherjee PK et al 1995)

31033 Determination of Minimum Inhibitory Concentration (MIC)

The extracts which showed antibacterial activity in the agar-well diffusion method were

subjected to the MIC assay The minimum inhibitory concentration (MIC) of the extracts

was determined for each of the test organisms in triplicates 05 ml of varying

concentrations of the extracts (200 180 150 100 80 50 10 05 005 and 0005

mgml) 2 ml of nutrient broth was added (so the extracts were dilute by a factor of 5)

Therefore the final concentrations were 4 36 3 2 16 1 02 01 001 0001 and 0

mgml as a control) and then a loopful of the test organism previously adjusted to 05

McFarland turbidity standard for bacteria was introduced to the tubes The procedure was

repeated on the test organisms using the standard antibiotic (Streptomycin 500 microgmL) A

tube containing nutrient broth only was seeded with the test organisms as described above

to serve as control Tubes containing bacterial cultures were then incubated at 37 degC for 24

hr period After incubation the tubes were then examined for microbial growth by

observing the turbidity present in the tubes (Salama HMH et al 2009)

311 Neuropsychopharmacological Profiling of the Extracts


The animals were divided into 14 groups with each group constituting 6 mice For

spontaneous motor activity every mouse was introduced into the actophotometer (INCO)

and its score of locomotor was measured for 10 min duration


78

Group-1 Control

Group-2 Standard (Dia) 4 mgkg

Group-(3-5) Petroleum ether Extract (PE) (100 200 and 400 mgkg po)

Group-(6-8) Chloroform Extract (CE) (25 50 and 100 mgkg po)

Group-(9-11) Ethyl Acetate Extract (EA) (100 200 and 400 mgkg po)

Group-(12-14) Methanol Extract (MENO-F) (100 200 and 400 mgkg po)

In this study all extract were administrated orally at three selected dose level

3112 Elevated plus maze test

To conduct the EPM test 66 mice were taken and divided into 11 groups each group

contain 6 mice The plus ndash maze consists of two open arms and two closed arms (50 x 10 x

40 cm each) elevated to a height 30 min post treatment each mouse was placed in turn in

the centre of the maze facing one of the closed arms The cumulative times spent by each

mouse in the open and closed arms of the maze were recorded upto 5 min

Group-1 Control

Group-2 Dia 2 mgkg

Group-(3-5) CE (25 50 and 100 mgkg po)

Group-(6-8) EA (100 200 and 400 mgkg po)

Group-(9-11) MENO-F (100 200 and 400 mgkg po)

3113 EPM model for Nootropic activity

The animals were placed individually on the EPM 30 min after extract administration the

animal was placed at the end of the open arms facing away from centre of the maze and

the time to move from the open arm to the closed arm was recorded as transfer latency

(TL) The recording was done on the first day and after 24 hrs for 90 sec TL on the first

day served as a measure of acquisition learning and TL after 24 hrs for retrieval or explicit

learning (Banji Otilia et al 2007)


79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg

Group-(3-5) PE (100 200 and 400 mgkg po)




3114 Anti-convulsant potential of EA of N oleander flowers against PTZ induced

convulsions in mice

Five groups of mice (n=10) were used Group-I served as positive control Group-II served

as standard Group-III Group-IV and Group-V served as drug treated 100 200 and 400

mgkg po respectively After 2 h of treatment PTZ was administered (60 mgkg ip) in all

five groups Each group was observed for 30 min and the onset of convulsion and duration

of convulsion showed was counted

Experimental design


Group-2 Dia (5 mgkg po) + PTZ (60 mgkg ip)

Group-3 EA of N oleander 100 mgkg + PTZ (60 mgkg ip)



3115 Anti-convulsant property of EA against MES induced convulsions in mice

Animals were divided into five groups containing 10 animals each Group-I served as

positive control Group-II served as standard Group-III Group-IV and Group-V were

treated orally with N oleander at doses of 100 200 and 400 mgkg po respectively After


80

2 h from treatment the above mention groups were given current of 12 mA for 02 sec in

electroconvulsiometer (INCO) by using ear electrode The onset of convulsion duration of

tonus was recorded 100 protection considered when hind limb tonic extension was

completely abolished

Experimental design


Group-2 Diazepam (5 mgkg po) + MES (12 mA for 02 sec)

Group-3 EA of N oleander 100 mgkg + MES (12 mA for 02 sec)



312 Exclusion and Inclusion Criteria for Further Biological Activity Selection

In vitro preliminary screening demonstrated that the MENO-F was showed strong inhibitor

of lipid peroxidation as found by the results of total antioxidant activity This indicated the

possible effectiveness of the methanolic extract as a hepatoprotective In the following

hepatoprotective activity of the methanolic extract of the flowers of N oleander was

evaluated In vitro cyclooxygenase inhibitory activity of the methanolic extract directed

the study towards evaluating the anti-inflammatory potential of the extract

Neuropsychopharmacological profiling of all the successive solvent extracts revealed that

the methanolic extract consistently and dose dependently enhanced the cognition As the

methanolic extract proved to be a consistent cognition enhancer in elevated plus maze

model for Nootropic activity further the methanolic extract was evaluated for

neuroprotective effect in dementia related to AD


81

Figure 7 Flow chart representing schematically selection criteria for biological activities


82

313 Evaluation of Hepatoprotective Potential of the MENO-F

3131 Test Animals

Wistar rats (180ndash240 g) of either sex procured from the central animal house ASBASJSM

College of Pharmacy Bela (Ropar)-140111 (Punjab) were used for the study The animals

were housed in large clean polypropylene cages in a temperature controlled room (22 plusmn 2

degC) with relative humidity (44ndash55) under 12 h light and dark cycles All the animals

were acclimatized to laboratory environment for a week prior to experiments Animals

were provided with a standard rodent pellet diet and clean drinking water ad libitum The

care and use of laboratory animals were strictly in accordance with the guidelines

prescribed by the Institutional Animal Ethical Committee constituted under the guidelines

of Committee for the Purpose of Control and Supervision of Experiments on Animals

(CPCSEA) India

3132 Experimental design

A total of 36 rats were divided into 6 groups of 6 rats each

bull Group I served as normal control and received only the vehicle (1 mlkgday of 1

CMC po)

bull Group II received CCl4 1 mlkg (11 of CCl4 in olive oil) ip once daily for 7 days

bull Group III received CCl4 1 mlkg (11 of CCl4 in olive oil) ip and silymarin 100

mgkg orally (po) for 7 days

bull Groups IV V VI were administered MENO-F at 100 200 and 400 mgkg body

weight po respectively and dose of 1 mlkg ip of CCl4 (11 of CCl4 in olive oil)

for 7 days

All rats were sacrificed by cervical dislocation after 24 h of the last treatment Just before

sacrifice blood was collected from the retro-orbital sinus plexus under mild ether

anesthesia Collected blood was allowed to clot and serum was separated at 3500 rpm for

15 min for carrying out further biochemical investigations One part of liver was dissected

out and used for biochemical and histopathological studies


83

3133 Measurement of serum biochemical parameters

The activities of serum aspartate transaminase (AST) alanine transaminase (ALT)

alkaline phosphatase (ALP) and total bilirubin were determined using the Hitachi 912

clinical chemistry automatic analyzer (Roche Diagnostic GmbH Mannheim Germany)

3134 Assessment of lipid peroxidation and superoxide dismutase (SOD)

In chilled normal saline excised livers were perfused to remove all the blood cells Then

they were cut down into small pieces placed in 01M phosphate buffer (pH 74) and

homogenized using remi homogenizer to obtain 20 homogenate The homogenate thus

obtained was centrifuged at 3000 rpm for 15 min and the supernatant was collected in an

Eppendorf tube This supernatant was again centrifuged at 12000 rpm for 30 min The

final supernatant was used for the determination of malonaldehyde (MDA) as a lipid

peroxidation marker (Nourooz-Zadeh et al 1995) Superoxide dismutase (SOD) was also

assayed by the method described previously (Misra and Fridovich 1972)

3135 Histopathology

The liver tissue was dissected out and fixed in 10 formalin solution and then dehydrated

in ethanol (50-100) cleared in xylene and embedded in paraffin wax Afterwards thick

sections (5ndash6 mm) were made and then stained with hematoxylin and eosin dye for

photomicroscopic observation Scoring on scale of 1-4 was done for the liver sections

under microscope as given below (Hirayama et al 1979 Ala-Kokko et al 1987)

bull 0 = Normal liver histology

bull 1 = Tiny and short septa of connective tissue without influence on the structure of

hepatic lobules

bull 2 = Large septa of connective tissue flowing together and penetrating into the

parenchyma Tendency to develop nodules

bull 3 = Nodular transformation of the liver architecture with loss of structure of

hepatic lobules

bull 4 = Excessive formation and deposition of connective tissue with subdivision of

the regenerating lobules and with development of scars


84

3136 Statistical analysis

The data were expressed as mean plusmn SD Statistical differences at p lt 0001 between the

groups were analyzed by one-way ANOVA followed by Turkey as post hoc using

GraphPad Instat software package The IC50 values were calculated graphically by linear

regression analysis

314 Neuroprotective Appraisal of MENO-F in Dementia Related to AD

3141 Test animals

Adult male Sprague Dawley rats (3-4 months of age) weighing 200-250 grams were used

for the experiment Animals were procured from the central animal house facility

IMTECH Chandigarh The animals were housed under standard laboratory conditions

with light dark cycle of 1212 temperature (23 plusmn 2 oC) and relative humidity of 55 plusmn 10

The animals were maintained on standard pellet diet and purified water ad libitum The




(CPCSEA) India



Group 1 Served as normal control and received normal drinking water ad libitum and

standard laboratory feed

Group 2 Served as positive control and received metals (Al and Cu in micro mol) in water ad

libitum (PC)

Group 3 Served as standard and received metals (Al and Cu in micro mol) in water ad libitum

plus vitamin E (100 mgkg)

Group 4 Served as test and received (Al and Cu in micro mol) in water ad libitum plus

MENO-F (200 mgkg)


MENO-F (400 mgkg)


85

Metals (M) in drinking water ad libitum to induce Alzheimer disease

Figure 8 A schematic diagram of the experimental setup depicted

3143 Radial 8 arm maze test

The maze consisted of eight arms extending radially from a central area (287 cm in

diameter) The doors 9 cm high will be placed between each arm and the central platform

The floor of arms and central area were painted black The apparatus was placed 40 cm

above the floor and surrounded by various extramaze cues such as a laboratory bench

posters and a clock The extramaze cues were placed in the same positions during the

study In order to investigate the spatial memory each arm was numbered from 1-8 outside

in the training period At the end of each arm (Nos 1 3 5 and 7) there was a food cup that

held a few mg of food pellet Prior to the performance of the maze task the animals were

kept on a restricted diet Before the actual training began the animals were shaped for 4

days to run to the end of the arms and consume the bait in groups of four The bait was

initially available throughout the maze but was gradually restricted to the food cup

Following this shaping period each animal was placed individually in the center of the

maze and subjected to the maze training The rats received five training trials every day for

5 days with a 5-min inter-trial interval The trial was continued until all four baits in the

food cups had been consumed or until 5 min has elapsed The radial arm maze was cleaned

with 70 ethanol and dried before each trial Each animal was placed individually in the

center of the maze where the same four arms (Nos 1 3 5 and 7) were baited for each

daily training trial The other four arms (Nos 2 4 6 and 8) were never baited An arm

1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days

Day 1st to 35th metals (in micro mol) in water ad libitum)

Day 35th to 42nd treated with Standard (Vit-E) and Extract (Nerium oleander)

Day 33rd to 35th training on radial eight arm maze

Body weight (weekly) Food and Water intake (average of every 7 days)

Blood collection (lipid profile) Remove Brain for (Oxidative parameter and Histopathology)


86

entry was counted when all four limbs of the rat were within an arm Measurements were

made of the number of reference memory errors (entering an arm that does not contain

food) and working memory errors (entering an arm containing food) The number of entry

into the baited and non-baited arms that were previously visited was calculated as memory

errors The pattern in the arm entries until all four baits had been consumed was also

recorded After a 5-day training period (total 25 trials) the rats were maintained with one

trial per day The rats that fulfilled the criteria (no more than one error per trial and 2 or

less over three consecutive trials) were used for behavioral and pharmacological

experiment

Figure 9 Radial eight arm maze

3144 Biochemical estimations

31441 Determination of lipid profile

For determination of lipid profile blood was collected from the rats after 12 h of fasting

and 24 h after the last dose of drug The blood was collected from the retro orbital plexus

under ether anesthesia and collected in non heparinsed eppendorf tubes The blood samples

were allowed to clot for 45 min at room temperature Serum was separated from the

clotted blood by centrifuging at 5000 rpm for 20 min Triglycerides were analyzed by

using Necpath triglyceride testing kit (GodPod method) according to manufacturer

instructions Serum total cholesterol was determined using Necpath Total cholesterol test

kit (Chod-pap endpoint method) and HDL-Cholesterol was estimated by PTA method

using Enzopak HDL- Cholesterol test kit


87

VLDL and LDL were calculated as

VLDL cholesterol = TG5

LDL cholesterol = TC ndash (VLDL + HDL cholesterol)

The values of lipid profile are expressed in mgdl

31442 Preparation of brain homogenate

The rats were sacrificed by decapitation and brains were collected rinsed in ice cold

normal saline followed by 015 M Tris-HCl (pH 74) blotted dry and weighed A 10 wv

of homogenate was prepared in 015 M Tris-HCl buffer and processed for the estimation of

lipid peroxidation by the method described previouisly (Ohkawa H et al 1979) A part of

homogenate after precipitating proteins with trichloroacetic acid (TCA) was used for

estimation of glutathione by the method of The rest of the homogenate was centrifuged at

15000 rpm for 15 min at 4 degC The supernatant thus obtained was used for the estimation

of superoxide dismutase (SOD) by the method described previously (Kakkar P et al

1984)

31443 Estimation of superoxide dismutase (SOD)

SOD activity of the brain tissue was analyzed by the method described previously

(Kakkar P et al 1984) Assay mixture contained 01 ml of sample 12 ml of sodium

pyro-phosphate buffer (pH 83 0052 M) 01 ml phenazine methosulphate (186 microM) 03

ml of 300 microM nitroblue tetrazolium 02 ml NADH (750 microM) Reaction was started by

addition of NADH After incubation at 30degC for 90 s the reaction was stopped by the

addition of 01 ml glacial acetic acid Reaction mixture was stirred vigorously with 40 ml

of n-butanol Mixture was allowed to stand for 10 min centrifuged and butanol layer was

separated Color intensity of the chromogen in the butanol layer was measured at 560 nm

spectrophotometrically and concentration of SOD was expressed as unitsmg protein

31444 Protein estimation

The protein content was measured according to the method described previously (Lowry

OH et al 1951) using bovine serum albumin as standard


88

31445 Estimation of GSSG

To measure Glutathione-oxidized (GSSG) levels in brain homogenate GSSG stock

solution was prepared in 5-sulfosalicylic acid (5-SSA) 5 and used as standard for GSSG

assays Stock solution was diluted with 5-SSA 5 to give final concentrations in a range

of 12ndash003 mM Brain samples were successively diluted with 5-SSA 5 and the pH for

both of samples and standards was adjusted to 74 by addition of triethanolamine solution

11 in water (TEAM solution) Samples and standards were kept on ice The reaction

mixture (freshly prepared) were mixed at room temperature in a reservoir 1ml of 10 mM

5 50-dithio-bis(2-nitrobenzoic acid) 10 ml of phosphate buffer and 30 Unitmg protein of

glutathione reductase The absorbance was read at UV spectrophotometer at 505 nm

Results were expressed in micromoles of GSSGmg protein

31446 Estimation of GSH

To measure the reduced glutathione (GSH) level the tissue homogenate (in 01 M

phosphate buffer pH 74) was taken The procedure was followed initially as described

previously (Ellman GL 1959) The homogenate was added with equal volume of 20

trichloroacetic acid (TBA) containing 1 mM EDTA to precipitate the tissue proteins The

mixture was allowed to stand for 5 min prior to centrifuge The supernatant (200 microl) was

then transferred to a new set of test tubes and added 18 ml of the Ellmans reagent (5 5-

dithio bis-2-nitrobenzoic acid) (01mM) was prepared in 03M phosphate buffer with 1

of sodium citrate solution) Then all the test tubes make upto the volume of 2 ml After

completion of the total reaction solutions were measured at 412 nm against blank

Absorbance values were compared with a standard curve generated from standard curve

from known GSH

31447 Nitrite estimation

Nitrite is the stable end product of NO in vitro system Accumulation of nitrite was

measured in cell-free supernatants from brain homogenate by spectrophotometer assay

based on Greiss reaction Briefly the supernatant of brain homogenate was mixed with

equal volume of Greiss reagent (1 sulphanilamide 01 naphthylethylenediamine

dihydrochloride 25 phosphoric acid) and incubated at room temperature for 10 min to

yield a chromophore Absorbance was read at 543 nm spectrophotometrically The nitrite

concentration was calculated from a standard curve and expressed as micromolar per

milliliter (Green LC et al 1982)


89

31448 Myeloperoxidase activity

Myeloperoxidase (MPO) activity was determined by modified technique described

elsewhere (Bird JE et al 1988) The supernatant collected from homogenate was mixed

with O-phenylenediamine (660 mgml in phosphate buffer) and 300 mM H2O2 was added

to it initiate the reaction Absorbance was measured at 492 nm at an interval of 30 s for 2

min Change in optical densityminute was calculated and results were expressed as

percentage myeloperoxidase activity considering 100 myeloperoxidase activity in the

control group

31449 Estimation of acetylcholinesterase (AChE) activity

Brain Homogenates were prepared in 10 ml of 01 M phosphate buffer pH 80 (50 mgml)

for 5 min in ice bath and used as enzyme source Acetyl cholinesterase activity was

measured by the method described previously (Ellman GL et al 1961) Enzyme activity

was measured at 25 oC in a 10 mm path length cuvette using an incubation mixture

consisting of 001 ml of (05times 105 M) freshly prepared acetylthiocholine (ACh) as

substrates 005 ml of the chromogenic agent (DTNB) (55-diothio-bis-2-nitrobenzoate)

(125times104 M DTNB dissolved in 01 M phosphate buffer pH 70) 005 ml of enzyme

containing supernatant and 145 ml of 01 M phosphate buffer pH 80 controls contained

005 ml of buffer in place of tissue homogenate Hydrolysis of the substrate produced

yellow colour which was measured at 412 nm

3145 Histopathology

At the end of the experiment the animals were sacrificed by an overdose of diethyl ether

and brain from the animals were collected and placed in 40 vv neutral buffered

formalin Histopathology of the brain was performed by the pathologist at Guru Angad

Dev Veterinary Science University (GADVSU) Ludhiana (Punjab) Hematoxylin and

eosin dyes were used for the staining of tissue The stained slides were observed by the

pathologist for the pathological changes and results were interpreted


Results were expressed as mean plusmn SEM The results were analyzed by using Graph pad

prism software Statistical analysis was carried out for one way analysis of variance

(ANOVA) followed by Dunnettrsquos test Minimum significant value was set as P le 005


90

315 Anti-Inflammatory Activity Screening of the Menthanolic Extract of Flowers of

Nerium Oleander L

3151 Test Animals




with light and dark cycle of 1212 temperature 23 plusmn 2 oC and relative humidity of 55 plusmn

10 The animals were maintained on standard pellet diet and purified water ad libitum

The care and use of laboratory animals were strictly in accordance with the guidelines



(CPCSEA) India

3152 Carrageenan induced rat paw oedema

The animals were divided into four groups (n=6) Group I served as control and received

vehicle only (1 Tween 80 10 mlkg po) Group II served as standard and received

Indomethacin (5 mgkg po) Group III group IV and group V served as test group which

received methanolic fraction (RG-M) at doses of 100 200 and 400 mgkg per orally

Anti-inflammatory activity of the methanolic fraction was evaluated by Carrageenan

induced rat paw oedema model as described earlier (Winter CA et al 1962 Yam MF et

al 2009) The animals were first treated with methanolic fraction (RP-M) or

Indomethacin After one hour the animals were injected with 01 ml of 1 Carrageenan (in

1 Tween 80) solution in the sub-plantar region of the right hind paw The volume of the

paw was measured by using a volume displacement technique using a plethysmometer

immediately and thereafter at 1 3 and 5 h after the stimulus The decrease in paw volume

as compared to the control group (vehicle only treated) was considered as an anti-

inflammatory response

3153 Exudative inflammation

A total of 24 rats were used in the experiment They were divided into four groups of six

rats each RG-M (200 and 400 mgkg ip) acetylsalicylic acid (100 mgkg ip) and


91

control vehicle (normal saline ip) were administered to different groups of animals

Thereafter peritoneal exudates were tested for total leukocyte count and total protein

content (using Bradford reagent) 3 h following intraperitoneal administration of 005 N

acetic acid (Chaudhuri AKN et al 2005)


Results were expressed as Mean plusmn SD The results were analyzed by using Graph pad

prism software Statistical analysis was carried out for One way analysis of variance

(ANOVA) followed by Turkey as post hoc using GraphPad Instat software package

Minimum significant value was set as P le 005


92

REFERENCES

Aguilar JL Rojas P Marcelo A Plaza A Bauer R Reininger E Klaas CA Merfort I

Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae) J

Ethnopharmacol 2002 81 271-276

Ala-Kokko L Stenback F Ryhanen L Preventive effect of malotilate on carbon

tetrachloride induced liver damage and collagen accumulation in the rat Biochem J 1987

246 503-509

Beauchamp C Fridovich I Superoxide dismutase improved assays and an assay

applicable to acrylamide gels Anal Biochem 1971 44 276-277

Bird JE Milhoam K Wilson CB Young SG Mundy CA Parthasarathy S Al E Ischemic

acute renal failure and antioxidant therapy in rats J Clin Invest 1988 31 1630-1638

Chaudhuri AKN Karmakar S Roy D Pal S Pal M Sen T Antiinflammatory activity of

Indian black tea (Sikkim variety) Pharmacol Res 2005 51 169-175

Dinis TCP Madeira VMC Almeida LM Action of phenolic derivatives (Acetaminophen

Salicylate and 5-Aminosalicylate) as inhibitors of membrane lipid peroxidation and as

peroxyl radical scavengers Arch Biochem Biophy 1994 315 161-169

Ellman GL Courtney KD Jr VA Featherstone RM A new rapid colorimetric

determination of acetylcholinesterase activity Biochem Pharmacol 1961 88-95

Ellman GL Tissue sulphydryl groups Arch Biochem Biophys 1959 82 70-77

Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Analysis of nitrate

nitrite and [15 N] nitrate in biological fluids Anal Biochem 1982 126 131-138

Halliwell B Gutteridge JM Aruoma OI The deoxyribose method a simple btest-tubeQ

assay for determination of rate constants for reactions of hydroxyl radicals Anal Biochem

1987 165 215- 219

Hirayama C Morotami I Hiroshige K Quantitative and metabolic changes of hepatic

collagens in rats after carbon tetrachloride poisoning Biochem J 1979 118 229-232

Kakkar P Das B Viswanathan PN A modified spectrophotometric assay of superoxide

dismutase Ind J Biochem Biophys 1984 21 131-132


93

Kumaran A Joel Karunakaran R Antioxidant activities of the methanol extract of

Cardiospermum halicacabum Pharmaceut Biol 2006 44 146-151

Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the folin

phenol reagent J Biol Chem 1951193 265-275

Misra HP Fridovich I Role of superoxide anion in the autoxidation of epinephrine and a

simple assay for superoxide dismutase J Biol Chem 1972 247 3170

Mitsuda H Yuasumoto K Iwami K Antioxidation action of indole compounds during the

autoxidation of linoleic acid Eiyo to Shokuryo 1996 19 210-214

Mukherjee PK Balasubramanium R Saha K Saha BP Pal M Antibacterial efficiency of

Nelumbo nucifera (Nymphaeaceae) rhizomes extract Indian Drugs 1995 32 274-276

Nourooz-Zadeh J Tajaddini-Sarmadi J Mccarthy S Betteridge DJ Wolff SP Elevated

levels of authentic plasma hydroperoxides in NIDDM Diabetes 1995 44 1054-1058

Ohkawa H Onishi N Yagi K Assay for lipid peroxidation in animal tissue by

thiobarbituric acid reaction Anal Biochem 1979 95 351-358

Oyaizu M Studies on product of browning reaction prepared from glucose amine Jpn J

Nutr 1986 44 307-315

Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant

activity applying an improved ABTS radical cation decolorization assay Free Radical Biol

Med 1998 72 1231-1237

Redl K Breu W Davis B Bauer R Anti-inflammatory active polyacetylenes from Bidens

campylotheca Planta Medica 1994 60

Salama HMH Marraiki N Antimicrobial activity and phytochemical analysis of

Polygonum aviculcare L (Polygonaceae) naturally growing in Egypt Aust J Basic and

Appl Sci 2009 3(3) 2008-2015

Shimada K Fujikawa K Yahara K Nakamura T Antioxidative properties of xanthin on

autoxidation of soybean oil in cyclodextrin emulsion J Agric Food Chem 1992 40 945-

948


94

Winter CA Risley EA Nus GV Carrageenan-induced edema in hind paw of the rat as an

assay for anti-inflammatory drug Proc Soc Exp Biol Med 1962 111 544minus547

Yam MF Ang LF Ameer OZ Salman IM Aziz HA Asmawi MZ Anti-inflammatory

and analgesic effects of Elephantopus tomentosus ethanolic extract J Acupunct Meridian

Stud 2009 2 280minus287


65

35 Ascertaining Margin Safety by Acute Oral Toxicity Studies in Rats And Mice

(OECD Guideline 423)

Whenever an investigator administers a chemical substance to a biological system

different types of interactions can occur and a series of dose-related responses result In

most cases these responses are desired and useful but there are a number of other effects

which are not advantageous These may or may not be harmful to the patients The types

of toxicity tests which are routinely performed by pharmaceutical manufacturers in the

investigation of a new drug involve acute sub-acute and chronic toxicity Acute toxicity

is involved in estimation of LD50 (the dose which has proved to be lethal (causing death)

to 50 of the tested group of animals) Determination of acute oral toxicity is usually an

initial screening step in the assessment and evaluation of the toxic characteristics of all

compounds

Category 1

gt 0-5

Category 2

gt 5 ndash 50

Category 3

gt 50 - 300

Category 4 gt 300 - 2000

Category 5

gt 2000 - 5000

Category 5

Or unclassified

5 25 30 50 200 300 500 1000 2000 2500 5000 infin

5mgkg 3animals

2-3

300mgkg 3animals

50mgkg 3animals

50mgkg 3animals

5mgkg 3animals

2000mgkg 3animals

300mgkg 3animals

2000mgkg 3animals

2-32-32-3 2-3

2-3 2-3 2-3

0-1 0-1

0-1

0-1

0-1 0-1

0-1

0-1

0

0

3 2other

3(at 300)At 1st stepother

13 (at 50)At 1st step

0

Test Procedure with a Starting Dose of 300 mgkg Body weight

Acute toxicity study according to OECD guideline 423

Figure 5 Scheme of acute toxicity study according to OECD guideline 423


66





Group-1 Control

























67


mouse

Group-1 Control




(EPM)






Group-1 Control














68




















69

















hypnosis and coma








70

Phyto

Chemical


1

Alkaloids

A small quantity

sample + 5 ml of

15 vv HCl and

filtered



reagents

Mayerrsquos

test

Mayerrsquos reagent

(MR) 136 gm

HgCl2 + 60 ml H2O

(dil upto 100 ml

H2O)



MR

Cream coloured

precipitate

Dragen-dorffs

test

Sol A (17 gm of

bismuth subnitrate

+ 200 gm tartaric

acid + 800 ml H2O)

Sol B (160 gm

potassium iodide +

4 ml H2O)

Soln A + Sol B (11

vv proportion)




( upto 500 ml H2O)








capillary tube

Orange red colour


of KI + 5 ml H2O

(dil upto 100 ml

H2O)


precipitate

Hagerrsquos

reagent

A saturated aq Sol

of picric acid was

employed for this

test


with this reagent

an orange yellow

ppt was obtained

(alkaloids)

2

Saponins

Froth

test




Honeycomb like

froth is obtained

3

Sterols

Salkowask

i reaction





red colour in the

chloroform layer

(sterols)

Lieberman

nrsquos test





test-tube

A blue colour

(sterols)


71

Lieberman

n

Burchardrsquos

reaction




Red blue

green

4

Carbohydr

ates

Molischrsquos

test





Red brown ring at

the junction of the


carbohydrates)

Fehlingrsquos

solution

test


and B sol were

mixed and boiled

for one minute



5-10 min

yellow brick red

(reducing sugars)

Benedictrsquos

solution

test




5 min

red solution

(reducing sugars)

5

Tannins





reagents

Ferric

chloride

reagent




Dark green or Deep


present)

Lead

acetate

test


H2O + test filtrate

If ppt is obtained

tannins present

6

Proteins

and amino

acid

Ninhydrin

test




the test extract

Violet or Purple

colour if amino

acids present

Millonrsquos

test


ml of mercury in 27

ml of fuming nitric

acid





The white

precipitate which

slowly turns to

pink is obtained if


7

Tests for

flavonoids

Shinoda

test






Pink crimson or

magenta colour is

developed within a

minute or two

(flavonoids)

Ammonia

test





Yellow color

(flavonoids)


72

Lead

acetate sol

test


acetate solution

Yellow coloured

ppt (flavonoids)

8

Triterpenoi

ds

Libermann

-Burchard

test





A brown ring at the

junction of two

layers and the

(upper layer turns

green) sterols and

deep red colour

triterpenoids

Salkowskirsquo

s test





lower layer red


yellow coloured

(triterpenoids)























73







al 1992)













compound













74
































75































76































77






























78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


66





Group-1 Control

























67


mouse

Group-1 Control




(EPM)






Group-1 Control














68




















69

















hypnosis and coma








70

Phyto

Chemical


1

Alkaloids

A small quantity

sample + 5 ml of

15 vv HCl and

filtered



reagents

Mayerrsquos

test

Mayerrsquos reagent

(MR) 136 gm

HgCl2 + 60 ml H2O

(dil upto 100 ml

H2O)



MR

Cream coloured

precipitate

Dragen-dorffs

test

Sol A (17 gm of

bismuth subnitrate

+ 200 gm tartaric

acid + 800 ml H2O)

Sol B (160 gm

potassium iodide +

4 ml H2O)

Soln A + Sol B (11

vv proportion)




( upto 500 ml H2O)








capillary tube

Orange red colour


of KI + 5 ml H2O

(dil upto 100 ml

H2O)


precipitate

Hagerrsquos

reagent

A saturated aq Sol

of picric acid was

employed for this

test


with this reagent

an orange yellow

ppt was obtained

(alkaloids)

2

Saponins

Froth

test




Honeycomb like

froth is obtained

3

Sterols

Salkowask

i reaction





red colour in the

chloroform layer

(sterols)

Lieberman

nrsquos test





test-tube

A blue colour

(sterols)


71

Lieberman

n

Burchardrsquos

reaction




Red blue

green

4

Carbohydr

ates

Molischrsquos

test





Red brown ring at

the junction of the


carbohydrates)

Fehlingrsquos

solution

test


and B sol were

mixed and boiled

for one minute



5-10 min

yellow brick red

(reducing sugars)

Benedictrsquos

solution

test




5 min

red solution

(reducing sugars)

5

Tannins





reagents

Ferric

chloride

reagent




Dark green or Deep


present)

Lead

acetate

test


H2O + test filtrate

If ppt is obtained

tannins present

6

Proteins

and amino

acid

Ninhydrin

test




the test extract

Violet or Purple

colour if amino

acids present

Millonrsquos

test


ml of mercury in 27

ml of fuming nitric

acid





The white

precipitate which

slowly turns to

pink is obtained if


7

Tests for

flavonoids

Shinoda

test






Pink crimson or

magenta colour is

developed within a

minute or two

(flavonoids)

Ammonia

test





Yellow color

(flavonoids)


72

Lead

acetate sol

test


acetate solution

Yellow coloured

ppt (flavonoids)

8

Triterpenoi

ds

Libermann

-Burchard

test





A brown ring at the

junction of two

layers and the

(upper layer turns

green) sterols and

deep red colour

triterpenoids

Salkowskirsquo

s test





lower layer red


yellow coloured

(triterpenoids)























73







al 1992)













compound













74
































75































76































77






























78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


67


mouse

Group-1 Control




(EPM)






Group-1 Control














68




















69

















hypnosis and coma








70

Phyto

Chemical


1

Alkaloids

A small quantity

sample + 5 ml of

15 vv HCl and

filtered



reagents

Mayerrsquos

test

Mayerrsquos reagent

(MR) 136 gm

HgCl2 + 60 ml H2O

(dil upto 100 ml

H2O)



MR

Cream coloured

precipitate

Dragen-dorffs

test

Sol A (17 gm of

bismuth subnitrate

+ 200 gm tartaric

acid + 800 ml H2O)

Sol B (160 gm

potassium iodide +

4 ml H2O)

Soln A + Sol B (11

vv proportion)




( upto 500 ml H2O)








capillary tube

Orange red colour


of KI + 5 ml H2O

(dil upto 100 ml

H2O)


precipitate

Hagerrsquos

reagent

A saturated aq Sol

of picric acid was

employed for this

test


with this reagent

an orange yellow

ppt was obtained

(alkaloids)

2

Saponins

Froth

test




Honeycomb like

froth is obtained

3

Sterols

Salkowask

i reaction





red colour in the

chloroform layer

(sterols)

Lieberman

nrsquos test





test-tube

A blue colour

(sterols)


71

Lieberman

n

Burchardrsquos

reaction




Red blue

green

4

Carbohydr

ates

Molischrsquos

test





Red brown ring at

the junction of the


carbohydrates)

Fehlingrsquos

solution

test


and B sol were

mixed and boiled

for one minute



5-10 min

yellow brick red

(reducing sugars)

Benedictrsquos

solution

test




5 min

red solution

(reducing sugars)

5

Tannins





reagents

Ferric

chloride

reagent




Dark green or Deep


present)

Lead

acetate

test


H2O + test filtrate

If ppt is obtained

tannins present

6

Proteins

and amino

acid

Ninhydrin

test




the test extract

Violet or Purple

colour if amino

acids present

Millonrsquos

test


ml of mercury in 27

ml of fuming nitric

acid





The white

precipitate which

slowly turns to

pink is obtained if


7

Tests for

flavonoids

Shinoda

test






Pink crimson or

magenta colour is

developed within a

minute or two

(flavonoids)

Ammonia

test





Yellow color

(flavonoids)


72

Lead

acetate sol

test


acetate solution

Yellow coloured

ppt (flavonoids)

8

Triterpenoi

ds

Libermann

-Burchard

test





A brown ring at the

junction of two

layers and the

(upper layer turns

green) sterols and

deep red colour

triterpenoids

Salkowskirsquo

s test





lower layer red


yellow coloured

(triterpenoids)























73







al 1992)













compound













74
































75































76































77






























78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


68




















69

















hypnosis and coma








70

Phyto

Chemical


1

Alkaloids

A small quantity

sample + 5 ml of

15 vv HCl and

filtered



reagents

Mayerrsquos

test

Mayerrsquos reagent

(MR) 136 gm

HgCl2 + 60 ml H2O

(dil upto 100 ml

H2O)



MR

Cream coloured

precipitate

Dragen-dorffs

test

Sol A (17 gm of

bismuth subnitrate

+ 200 gm tartaric

acid + 800 ml H2O)

Sol B (160 gm

potassium iodide +

4 ml H2O)

Soln A + Sol B (11

vv proportion)




( upto 500 ml H2O)








capillary tube

Orange red colour


of KI + 5 ml H2O

(dil upto 100 ml

H2O)


precipitate

Hagerrsquos

reagent

A saturated aq Sol

of picric acid was

employed for this

test


with this reagent

an orange yellow

ppt was obtained

(alkaloids)

2

Saponins

Froth

test




Honeycomb like

froth is obtained

3

Sterols

Salkowask

i reaction





red colour in the

chloroform layer

(sterols)

Lieberman

nrsquos test





test-tube

A blue colour

(sterols)


71

Lieberman

n

Burchardrsquos

reaction




Red blue

green

4

Carbohydr

ates

Molischrsquos

test





Red brown ring at

the junction of the


carbohydrates)

Fehlingrsquos

solution

test


and B sol were

mixed and boiled

for one minute



5-10 min

yellow brick red

(reducing sugars)

Benedictrsquos

solution

test




5 min

red solution

(reducing sugars)

5

Tannins





reagents

Ferric

chloride

reagent




Dark green or Deep


present)

Lead

acetate

test


H2O + test filtrate

If ppt is obtained

tannins present

6

Proteins

and amino

acid

Ninhydrin

test




the test extract

Violet or Purple

colour if amino

acids present

Millonrsquos

test


ml of mercury in 27

ml of fuming nitric

acid





The white

precipitate which

slowly turns to

pink is obtained if


7

Tests for

flavonoids

Shinoda

test






Pink crimson or

magenta colour is

developed within a

minute or two

(flavonoids)

Ammonia

test





Yellow color

(flavonoids)


72

Lead

acetate sol

test


acetate solution

Yellow coloured

ppt (flavonoids)

8

Triterpenoi

ds

Libermann

-Burchard

test





A brown ring at the

junction of two

layers and the

(upper layer turns

green) sterols and

deep red colour

triterpenoids

Salkowskirsquo

s test





lower layer red


yellow coloured

(triterpenoids)























73







al 1992)













compound













74
































75































76































77






























78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


69

















hypnosis and coma








70

Phyto

Chemical


1

Alkaloids

A small quantity

sample + 5 ml of

15 vv HCl and

filtered



reagents

Mayerrsquos

test

Mayerrsquos reagent

(MR) 136 gm

HgCl2 + 60 ml H2O

(dil upto 100 ml

H2O)



MR

Cream coloured

precipitate

Dragen-dorffs

test

Sol A (17 gm of

bismuth subnitrate

+ 200 gm tartaric

acid + 800 ml H2O)

Sol B (160 gm

potassium iodide +

4 ml H2O)

Soln A + Sol B (11

vv proportion)




( upto 500 ml H2O)








capillary tube

Orange red colour


of KI + 5 ml H2O

(dil upto 100 ml

H2O)


precipitate

Hagerrsquos

reagent

A saturated aq Sol

of picric acid was

employed for this

test


with this reagent

an orange yellow

ppt was obtained

(alkaloids)

2

Saponins

Froth

test




Honeycomb like

froth is obtained

3

Sterols

Salkowask

i reaction





red colour in the

chloroform layer

(sterols)

Lieberman

nrsquos test





test-tube

A blue colour

(sterols)


71

Lieberman

n

Burchardrsquos

reaction




Red blue

green

4

Carbohydr

ates

Molischrsquos

test





Red brown ring at

the junction of the


carbohydrates)

Fehlingrsquos

solution

test


and B sol were

mixed and boiled

for one minute



5-10 min

yellow brick red

(reducing sugars)

Benedictrsquos

solution

test




5 min

red solution

(reducing sugars)

5

Tannins





reagents

Ferric

chloride

reagent




Dark green or Deep


present)

Lead

acetate

test


H2O + test filtrate

If ppt is obtained

tannins present

6

Proteins

and amino

acid

Ninhydrin

test




the test extract

Violet or Purple

colour if amino

acids present

Millonrsquos

test


ml of mercury in 27

ml of fuming nitric

acid





The white

precipitate which

slowly turns to

pink is obtained if


7

Tests for

flavonoids

Shinoda

test






Pink crimson or

magenta colour is

developed within a

minute or two

(flavonoids)

Ammonia

test





Yellow color

(flavonoids)


72

Lead

acetate sol

test


acetate solution

Yellow coloured

ppt (flavonoids)

8

Triterpenoi

ds

Libermann

-Burchard

test





A brown ring at the

junction of two

layers and the

(upper layer turns

green) sterols and

deep red colour

triterpenoids

Salkowskirsquo

s test





lower layer red


yellow coloured

(triterpenoids)























73







al 1992)













compound













74
































75































76































77






























78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


70

Phyto

Chemical


1

Alkaloids

A small quantity

sample + 5 ml of

15 vv HCl and

filtered



reagents

Mayerrsquos

test

Mayerrsquos reagent

(MR) 136 gm

HgCl2 + 60 ml H2O

(dil upto 100 ml

H2O)



MR

Cream coloured

precipitate

Dragen-dorffs

test

Sol A (17 gm of

bismuth subnitrate

+ 200 gm tartaric

acid + 800 ml H2O)

Sol B (160 gm

potassium iodide +

4 ml H2O)

Soln A + Sol B (11

vv proportion)




( upto 500 ml H2O)








capillary tube

Orange red colour


of KI + 5 ml H2O

(dil upto 100 ml

H2O)


precipitate

Hagerrsquos

reagent

A saturated aq Sol

of picric acid was

employed for this

test


with this reagent

an orange yellow

ppt was obtained

(alkaloids)

2

Saponins

Froth

test




Honeycomb like

froth is obtained

3

Sterols

Salkowask

i reaction





red colour in the

chloroform layer

(sterols)

Lieberman

nrsquos test





test-tube

A blue colour

(sterols)


71

Lieberman

n

Burchardrsquos

reaction




Red blue

green

4

Carbohydr

ates

Molischrsquos

test





Red brown ring at

the junction of the


carbohydrates)

Fehlingrsquos

solution

test


and B sol were

mixed and boiled

for one minute



5-10 min

yellow brick red

(reducing sugars)

Benedictrsquos

solution

test




5 min

red solution

(reducing sugars)

5

Tannins





reagents

Ferric

chloride

reagent




Dark green or Deep


present)

Lead

acetate

test


H2O + test filtrate

If ppt is obtained

tannins present

6

Proteins

and amino

acid

Ninhydrin

test




the test extract

Violet or Purple

colour if amino

acids present

Millonrsquos

test


ml of mercury in 27

ml of fuming nitric

acid





The white

precipitate which

slowly turns to

pink is obtained if


7

Tests for

flavonoids

Shinoda

test






Pink crimson or

magenta colour is

developed within a

minute or two

(flavonoids)

Ammonia

test





Yellow color

(flavonoids)


72

Lead

acetate sol

test


acetate solution

Yellow coloured

ppt (flavonoids)

8

Triterpenoi

ds

Libermann

-Burchard

test





A brown ring at the

junction of two

layers and the

(upper layer turns

green) sterols and

deep red colour

triterpenoids

Salkowskirsquo

s test





lower layer red


yellow coloured

(triterpenoids)























73







al 1992)













compound













74
































75































76































77






























78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


71

Lieberman

n

Burchardrsquos

reaction




Red blue

green

4

Carbohydr

ates

Molischrsquos

test





Red brown ring at

the junction of the


carbohydrates)

Fehlingrsquos

solution

test


and B sol were

mixed and boiled

for one minute



5-10 min

yellow brick red

(reducing sugars)

Benedictrsquos

solution

test




5 min

red solution

(reducing sugars)

5

Tannins





reagents

Ferric

chloride

reagent




Dark green or Deep


present)

Lead

acetate

test


H2O + test filtrate

If ppt is obtained

tannins present

6

Proteins

and amino

acid

Ninhydrin

test




the test extract

Violet or Purple

colour if amino

acids present

Millonrsquos

test


ml of mercury in 27

ml of fuming nitric

acid





The white

precipitate which

slowly turns to

pink is obtained if


7

Tests for

flavonoids

Shinoda

test






Pink crimson or

magenta colour is

developed within a

minute or two

(flavonoids)

Ammonia

test





Yellow color

(flavonoids)


72

Lead

acetate sol

test


acetate solution

Yellow coloured

ppt (flavonoids)

8

Triterpenoi

ds

Libermann

-Burchard

test





A brown ring at the

junction of two

layers and the

(upper layer turns

green) sterols and

deep red colour

triterpenoids

Salkowskirsquo

s test





lower layer red


yellow coloured

(triterpenoids)























73







al 1992)













compound













74
































75































76































77






























78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


72

Lead

acetate sol

test


acetate solution

Yellow coloured

ppt (flavonoids)

8

Triterpenoi

ds

Libermann

-Burchard

test





A brown ring at the

junction of two

layers and the

(upper layer turns

green) sterols and

deep red colour

triterpenoids

Salkowskirsquo

s test





lower layer red


yellow coloured

(triterpenoids)























73







al 1992)













compound













74
































75































76































77






























78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


73







al 1992)













compound













74
































75































76































77






























78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


74
































75































76































77






























78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


75































76































77






























78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


76































77






























78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


77






























78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


78

Group-1 Control













Group-1 Control

Group-2 Dia 2 mgkg












79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


79

Experimental design

Group-1 Control

Group-2 Dia 2 mgkg






convulsions in mice






Experimental design











80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


80





Experimental design



















81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


81



82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


82


3131 Test Animals










(CPCSEA) India




CMC po)






for 7 days







83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


83














3135 Histopathology








hepatic lobules




hepatic lobules




84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


84





regression analysis


3141 Test animals









(CPCSEA) India






libitum (PC)




MENO-F (200 mgkg)


MENO-F (400 mgkg)


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


85






















1-7

days

8-14

days

15-21

days

21-28

days 29-35 days

36-42 days







86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


86









experiment














87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


87














1984)















88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


88























from known GSH











89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


89








control group












3145 Histopathology












90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


90


Nerium Oleander L

3151 Test Animals









(CPCSEA) India



















91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


91











92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


92

REFERENCES






246 503-509

















1987 165 215- 219






93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


93
















Nutr 1986 44 307-315



Med 1998 72 1231-1237





Appl Sci 2009 3(3) 2008-2015



948


94





Stud 2009 2 280minus287


94





Stud 2009 2 280minus287

Chapter – III Materials and Methodsshodhganga.inflibnet.ac.in/bitstream/10603/8870/13/14_chapter...

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