methodology and experimental detailsshodhganga.inflibnet.ac.in/bitstream/10603/4393/12/12_chapter...
TRANSCRIPT
50
METHODOLOGY AND
EXPERIMENTAL DETAILS
51
MATERIALS AND METHODS
Chemical used:
Methanol (SD Fine Chemical Ltd. Mumbai)
Anaesthetic ether (Sigma)
Carbon tetrachloride (SD Fine Chemical Ltd. Mumbai)
Paracetamol (Strides Arcolabs Ltd. Bangalore)
Thioacetamide (SD Fine Chemical Ltd. Mumbai)
Silymarin (Micro Labs Bangalore)
Liquid paraffin (CDH, Mumbai)
Nitroblue tetrazolium NBT (Loba Chemical, Mumbai)
EDTA (Nice Chemicals, Cochin)
Hydroxylamine hydrochloride (Nice Chemicals, Cochin)
Sodium carbonate (SD Fine Chemical Ltd. Mumbai)
Folin Cioalteau Reagent (Loba Chemical, Mumbai)
Copper sulphate (SD Fine Chemical Ltd. Mumbai)
Sodium hydroxide (SD Fine Chemical Ltd. Mumbai)
90% Alcohol (SD Fine Chemical Ltd. Mumbai)
Standard bovine albumin (SD Fine Chemical Ltd. Mumbai)
Pottasium dihydrogen orthophosphate (SD Fine Chemical Ltd. Mumbai)
Disodium hydrogen orthophosphate (SD Fine Chemical Ltd. Mumbai)
Hydrogen peroxide (SD Fine Chemical Ltd. Mumbai)
52
Kits used:
ASAT kit (Crest Biosystems, Coral Clinical Systems, Goa)
ALAT kit (Crest Biosystems, Coral Clinical Systems, Goa)
ALP kit9 (Crest Biosystems, Coral Clinical Systems, Goa)
Bilirubin kit (Crest Biosystems, Coral Clinical Systems, Goa)
Equipment used:
Analytical balance (Schimadzu/Japan)
Semi analyzer (Qualigen, AR 601 Mumbai)
53
4.1 STUDY SAMPLES
The leaves and bark of Buchanania lanzan were supplied by Yucca
Enterprises Mumbai. These were shade dried and used. Muppu and
Vidhakanachoornam were supplied by Dr. Jai Prakash Intergrated
Research Centre for Siddha Medicines Bangalore.
4.2 PLANT EXTRACTION
The leaves and bark of the plant were shade-dried and extracted using
various solvents in order of increasing polarity and extracts were
subjected for preliminary phytochemical investigation.
4.3 PHYTOCHEMICAL SCREENING 52
Standard phytochemical test were used to screen the extract for the
presence of different constituents. The various extracts of both the bark
and mature leaves of Buchanania lanzan were subjected to qualitative
analysis for the phytoconstituents like alkaloids, carbohydrates,
glycosides, steroids, tannins, proteins, amino acids and flavonoids as per
the standard procedures. The results are tabulated as shown in.
A. Tests for Alkaloids:
Hager’s test:- Extract treated with a few drops of Hager’s reagent
(saturated solution of picric acid) – yellow precipitates indicate presence
of alkaloids.
54
Mayer’s test:- Extract treated with Mayer’s reagent (potassium mercuric
iodide solution) – formation of cream precipitates indicate presence of
alkaloids.
Dragendorff’s test:- Extract treated with Dragendorff’s reagent
(potassium bismuth iodide solution) - orange precipitates.
Wagner’s test:- Extract treated with Wagner’s reagent (iodine,
potassium–iodine solution) –reddish brown precipitates.
B. Tests for carbohydrates:
Molish’s test:- Extract treated with Molish’s reagent (alpha naphthol in
95% ethanol) and a few drops of concentrated sulphuric acid were added
by the sides of the test tubes. A violet ring at the junction is formed.
Fehling’s test:- Extract treated with Fehling’s reagent (Fehling’s reagent
A-copper sulphate in water and Fehling’s reagent B-sodium potassium
tartarate) followed by heating. Red coloured precipitates indicate
presence of reducing sugars.
Barford’s test:- Extract treated with Barford’s reagent (copper acetate
in water and glacial acetic acid) - red colouration.
Benedict’s test:- Extract treated with Benedict’s reagent (copper
sulphate, sodium citrate and sodium carbonate in water)- red coloured
precipitates.
55
C. Tests for Steroids, Terpenoids and Cardiac Glycosides:
Liebermann-Burchard test:- 10 g of extract was dissolved in 1 ml of
chloroform, 1 ml of acetic anhydride was added followed by the addition
of 2 ml of concentrated sulphuric acid from the sides of the test tube.
Formation of reddish brown colour at the junction indicates the presence
of steroids/terpenoids/cardiac glycosides.
Salkowski test:- 1 ml of concentrated sulphuric acid was added to 10 g
of the extract dissolved in 1 ml of chloroform. A reddish brown layer
exhibited by the chloroform layer and green fluorescence by the acid
layer suggests the presence of steroids.
Noller’s test:- 5 g of the extract was dissolved in 2 ml of 0.01%
anhydrous stannic chloride in pure thionyl chloride .Yellow colour
indicates the presence of triterpenoids.
Legal’s test:- The extract was treated with sodium nitroprusside in
pyridine and methanolic alkali. The formation of pink colour indicates
the presence of cardiac glycosides.
Balget’s test:- 1 ml of the extract solution was treated with a few drops
of sodium picrate reagent, yellowish orange colour indicates the presence
of cardiac glycosides.
Killer Killani’s test:- 5 g of the extract was treated with 1 ml of glacial
acetic acid and a few drops of ferric chloride solution. 2 ml of
concentrated sulphuric acid were carefully added from the sides of the
test tube. Formation of a reddish brown colour at the junction of the two
56
layers and formation of bluish green colour in the upper layer indicates
the presence of deoxy sugar in the carbohydrates.
D. Tests for Saponins:
Diluted 1 ml of the extract with distilled water to 10 ml and shaken in
a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam
indicates the presence of saponins.
Haemolysis test:- 2 ml of 1.8% sodium chloride solution was taken in
two test tubes. To one test tube 2 ml of distilled water were added and to
another 2 ml of 1% extract were added. Blood is obtained by pricking the
thumb and 5 drops of blood were added to each tube, the contents were
gently mixed and observed under the microscope. Haemolysis indicates
the presence of saponins.
E. Tests for Tannins:
Ferric chloride test:- Extract treated with ferric chloride solution–blue
colour is formed.
Gelatin test:- Extract treated with gelatin solution–white precipitates
appear.
Lead acetate test:- Extract treated with lead acetate solution–yellow
precipitates appear.
57
F. Tests for Proteins and Amino Acids:
Millon’s test:- Extract treated with Millon’s reagent (mercuric nitrate in
nitric acid)-red colour is formed.
Biuret test:- Extract treated with sodium hydroxide and copper
sulphate solution drop wise –violet colour.
Ninhydrin test:- Extract treated with ninhydrin reagent and ammonia
and heated –violet colour is formed.
G. Tests for Flavonoids:
Ferric chloride test:- To the extract add a few drops of neutral ferric
chloride solution- blackish red colour is formed.
Lead acetate test:- To the extract add lead acetate solution-yellow
precipitates.
Magnesium ribbon test:- To the extract add few fragments of
magnesium ribbon and concentrated hydrochloric acid along the sides of
the test tube-magenta colour is formed is formed.
Zinc–hydrochloride test:- To the extract, a pinch of zinc dust was added
followed by addition of concentrated hydrochloric acid along the sides of
the test tubes-magenta colour is formed.
58
4.4 IN VITRO ANTIOXIDANT ACTIVITY
4.4.1 IN VITRO SCRENING OF BUCHANANIA LANZAN
The in vitro antioxidant screening was carried out using the following
models.
1. In vitro DPPH assay
2. Scavenging of ABTS
3. Scavenging of hydroxyl radical
2,2-Diphenyl-I-picryl hydrzyl (DPPH) Assay53
DPPH assay is based on the measurement of the scavenging ability of
antioxidant towards the stable DPPH radical. The free radical DPPH is
purple in colour in methanol and is reduced to the corresponding
hydrazine, which is yellow in colour, when it reacts with a hydrogen
donor.It is a discoloration assay, which is evaluated by the addition of
the antioxidant to 2,2-diphenyl-1-picryl hydrzyl (DPPH) solution in
ethanol or methanol and the decrease in the absorbance is measured .
The free radical-scavenging activity of the extracts was measured in
terms of hydrogen donating or radical-scavenging ability using the stable
radical DPPH.
NO2N N
Ph
Ph
NO2
NO2
+ R- OHO2N N
Ph
Ph
NO2
NO2
NH
+ R-O
DPPH radical Stable phenoxy radical
(Purple) (Yellow)
59
DPPH solution (100 μM): 22 mg of DPPH was accurately weighed and
dissolved in 100 ml of methanol. From this stock solution, 18 ml was
taken and diluted to 100 ml with methanol to obtain 100 μM DPPH
solutions.
Preparation of Test and Standard Solution
The extracts and the standards, ascorbic acid and rutin (21 mg each)
were separately dissolved in 5 ml of freshly distilled DMSO. These
solutions were serially diluted with freshly distilled DMSO to obtain the
lower dilutions.
Procedure
The assay was carried out in a 96 well microtitre plate. To 200 μl of
DPPH solution, 10 μl of various concentrations of the extract or the
standard solution was added separately in wells of the microtitre plats.
The plats were incubated at 370C for 30 min. Absorbance was measured
at 517 nm using ELISA reader. Thirty minutes later, the absorbance was
measured at 517 nm. Lower absorbance of the reaction mixture indicates
higher free radical-scavenging activity. Ascorbic acid and rutin were
used as a standard drugs.
Scavenging of 2, 2’-azino-bis (3-ethylbezothiazoline-6-sulfonic acid)
diammonium salt (ABTS) radical cation assay54
ABTS assay is relatively recent one, which involves a more drastic
radical, chemically produced and, is often used for screening complex
60
antioxidant mixture such as plant extracts, beverages and biological
fluids. The solubility in both the organic and aqueous media and the
stability in a wide pH range raised the interest in the use of ABTS radical
for the estimation of the antioxidant.
Preparation of test and standard solution:
13.5mg of each of the extracts and the standards, ascorbic acid and
rutin were accurately weighed and separately dissolved in 2 ml of DMSO.
These solutions were serially diluted with DMSO to obtain the lower
dilutions.
Procedure:
ABTS (54.8 mg) was dissolved in 50 ml of distilled water to 2 mM
concentration and potassium persulphate (17 mM, 0.3 ml) was added.
The reaction mixture was left to stand at room temperature overnight in
dark before use. To 0.2 ml of various concentrations of the extracts or
standards, 1.0 ml of distilled DMSO and 0.16 ml of ABTS solution was
added to make a final volume of 1.36 ml. Absorbance was measured
spectrophotometrically, after 20 min at 734 nm.
Scavenging of Hydroxyl Radical by Deoxyribose Method55
The sugar deoxyribose (2-deoxy-D-ribose) is degraded on exposure to
hydroxyl radical generated by irradiation or by Fenton systems. If the
resulting complex mixture of products is heated under acid conditions,
malondialdehyde (MDA) is formed and may be detected by its ability to
react with thiobarbituric acid (TBA) to form a pink chromogen.
61
Preparation of test and standard solutions:
16 mg of each of the extracts and BHA were accurately weighed and
separately dissolved in 2 ml of DMSO. These solutions were serially
diluted with DMSO to obtain the lower dilutions.
Procedure:
To the reaction mixture containing deoxyribose (3 mM, 0.2 ml), ferric
chloride (0.1 mM, 0.2 ml), EDTA (0.1 mM, 0.2 ml), ascorbic acid (0.1 mM,
0.2 ml) and hydrogen peroxide (2 mM, 0.2 ml) in phosphate buffer (pH,
7.4, 20 mM), 0.2 ml of various concentrations of extracts or standards in
DMSO were added to give a total volume of 1.2 ml. The solutions were
then incubated for 30 min at 370C. After incubation, ice-cold trichloro
acetic acid (0.2 ml, 15% w/v) and thiobarbituric acid (0.2 ml, 1% w/v), in
0.25 N HCl were added. The reaction mixture was kept in a boiling water
bath for 30 min, cooled and the absorbance was measured at 532 nm.
4.4.2 IN VITRO ANTIOXIDANT ACTIVITY OF VIDAKANACHOORNAM
AND MUPPU
The formulations Muppu and Vidakanachoornam were evaluated by
Superoxide radical scavenging activity
Hydroxyl radical scavenging activity
Nitric oxide method
62
Superoxide Radical Scavenging Activity56
All the solutions were prepared in 100 mM phosphate buffer (pH 7.4).
1ml of nitroblue tetrazolium (NBT, 156 μM), 1 ml of reduced
nicotinamide adenine dinucleotide (NADH, 468 μM) 3 ml of HEPD
solution of different concentrations (50, 100, 200, 400, 800, 1000 1200
μg/ml) were mixed. The reaction was initiated by adding 100 μl of
phenazine methosulphate (PMS, 60 μM). The reaction mixture was
incubated at 25°C for 5 min, followed by measurement of absorbance at
560 nm.
Hydroxyl Radical Scavenging Activity57
Stock solutions of EDTA (1mM), FeCl3(10 mM), ascorbic acid (1 mM),
H2O2 (10 mM), deoxyribose (10 mM), were prepared in distilled deionized
water.The assay was performed by adding 0.1 ml FDTA, 0.01 ml of FeCl3,
0.1 ml H2O2, 0.36 ml of deoxyribose, 1.0 ml of the extract of different
concentration (200, 400. 800, 1000, 1200 mg/ml) dissolved in distilled
water, 0.33 ml of phosphate buffer (50 mM, PH-7.4), 0.1 ml of ascorbic
acid in sequence. The mixture was then incubated at 37°C for 1 hr. A 1.0
ml portion of the incubated mixture was mixed with 1.0 ml of 10% TCA
1.0 ml of 0.5% TBA (in 0.025 M NaOH containing 0.025% BHA) to
develop the pink chromogen measured at 532 nm. The hydroxyl radical
scavenging activity of the extract is reported as % inhibition of
deoxyribose degradation is calculated by using following equation.
63
Percentage inhibition = (1– absorbance of test/absorbance of control) ×
100
Nitric oxide Method58–60
In aqueous solution at physiological pH, sodium nitroprusside
generates nitric oxide, which interacts with oxygen to produce nitrite
ions. 1 ml of 10 mM sodium nitroprusside was mixed with 1 ml of test
solution of different concentrations (80, 160, 320, 500, 800, 1000 μg/ml)
in phosphate buffer (pH 7.4) and the mixture was incubated at 25°C for
150 min. From the incubated mixture, 1 ml was taken out 1 ml of Griess’
reagent (1% sulphanilamide, 2% o-phosphoric acid 0.1% naphthyl
ethylene diamine dihydrochloride was added to it. Absorbance of the
chromophore formed by the diazotization of nitrite with sulfanilamide
subsequent coupling with naphthyl ethylene diamine dihydrochloride
was read at 546 nm. Percentage inhibition was calculated by comparing
the results of the test with those of the control using following equation;
Percentage inhibition = (1– absorbance of test/absorbance of control) ×
100
64
4.4.3 COMPARATIVE IN VITRO ANTIOXIDANT ACTIVITY OF MUPPU,
BUCHANANIA LANZAN AND VIDAKANACHOORNAM
The comparative in vitro antioxidant activity of Vidakanachoornam ,
Muppu and plant Buchanania lanzan was carried out using DPPH
method.
4.5 IN VIVO ANTIOXIDANT ACTIVITY
4.5.1 Acute oral toxicity studies61,62
The acute oral toxicity study was performed according to the OPPTS
(Office of prevention of pesticides and toxic substance) Guidelines
following the up and down procedure62.Male albino mice (30-35 g) were
maintained under controlled standard animal house conditions with
access to food and water ad libitum.The mice were acclimatized for 5 days
and fasted overnight, with access for drinking water. Animals were
weighed; limit and main test were performed. The limit test is carried out
first at 5000 mg /kg body weight for one animal and if animal dies, main
test is performed. If the animal survives two more animals are dosed, if
both survive the test should be terminated. The main test is performed
with an initial dose of 175mg/kg body weight. The following sequence is
followed. 175, 550, 1750, and 5000 mg/kg body weight.First one animal
is dosed with 175 mg/kg body weight. If animal dies a much lower dose
is tested. If animal survives, then two more animals are dosed, after 48
65
hours observation of the first animal. If survives, then the main test
should be terminated .if animal dies, two more animals are dosed and
observed.
The dosing is stopped when one of the following stopping criteria is met.
Three consecutive animals survive at the upper bound.
5 reversals occur in any 6 consecutive animals tested
At least 4 animals have followed the first reversal and the specified
likelihood ratios exceed the critical value.
The control mice received vehicle (Tween 80 1%p.o) only.
4.5.2 IN VIVO ANTIOXIDANT STUDIES OF BUCHANANIA LANZAN
Test animals
Animal house was well maintained under standard hygienic
conditions. Rats were housed in groups of 6 in polypropylene cages
(32x24x16). They were provided with commercial food pellets [containing
(%w/w) protein 22.10, oil 4.13, fibre 3.15, ash 5.15, sand (silicia)1.12]
and water ad libitium. Cleaning and sanitation work was done on
alternate days. Paddy husk was provided as bedding material, which was
changed everyday. Wistar albino rats and mice of either sex were used
for the study of the crude extracts. The animals were housed at central
animal house under standard conditions of temperature (27 ± 2°C),
relative humidity (44-56%) and light and dark cycles of 10 and 14 h
respectively, for 1 week before and during the experiments. Animals were
66
provided with standard diet (Lipton, India) and water ad libitum. The food
was withdrawn at 18 to 24 h before the start of the experiment. All the
experiments were performed in the morning according to the ethical
guidelines for the care of the laboratory animals61.
In vivo antioxidant study of the Plant extract was carried out using
Biochemical estimation of the enzyme level in the liver homogenate.
1. S.O D estimation
2. Estimation of catalase
3. Estimation of total protein
4. Liver weight estimation
5. Histopathological studies
Experimental Design43
After seven days of acclimatization, the rats were divided into six
groups, each group consisting of six animals, (n=6). Treatment was done
for 8 days as follows:
Group I: Normal control (0.9% normal saline; 1 ml/kg p.o .) daily for 8
days and received liquid paraffin (2 mL/kg, s.c.) on last day. Group II
(CCl4) received (0.9% normal saline; 1 ml/kg p.o.) once daily for 8 days
and received CCl4: liquid paraffin (1:1, 2 mL/kg body weight, s.c.) on last
day. Group III received standard drug ascorbic acid (250 mg/kg, p.o.)
67
once daily for 8 days and received CCl4: liquid paraffin (1:1, 2 mL/kg
body weight, s.c.) on last day.Group IV received standard drug ascorbic
acid (500 mg/kg, p.o.) once daily for 8 days, and received CCl4: liquid
paraffin (1:1, 2 mL/kg body weight, s.c.) on last day. Group V received
methanolic extract of Buchanania lanzan (250 mg/kg, p.o.) once daily for
8 days and received CCl4: liquid paraffin (1:1, 2 mL/kg body weight, s.c.)
on last day . Group VI received the methanolic extract of Buchanania
lanzan (500 mg/kg, p.o.) once daily for 8 days and received CCl4: liquid
paraffin (1:1, 2 mL/kg body weight, s.c.) on last day. Animals were
sacrificed 24 h after the last treatment.
Blood was collected, allowed to clot and serum was separated at 2500
rpm for 15 min and biochemical investigations were carried out. Liver
was dissected out and used for histopathological studies.
Preparation of Liver Homogenate
The liver samples used for homogenate preparations were rinsed in
sterile ice cold saline and then homogenized in ice –cold 0.25 M sucrose
1:10 w/v solution using a mortar and pestle .Then, the obtained
homogenate was centrifuged at 5000 rpm for 15 minutes, the
supernatant was decanted and taken for estimation of antioxidants and
enzyme levels using kits.
Preparation of 0.25M Sucrose Solution
This solution was prepared by dissolving 342.30 gms of sucrose in
1000 ml of distilled water.
68
Estimation of Super Oxide Dismutase (SOD)6
Estimation of S.O.D is done by autooxidation of hydroxylamine
hydrochloride at pH 10.2 yielding nitrite, which was measured
colorimetrically. Ò2 can also be generated during auto oxidation of
hydroxylamine. Accompanying the autoxidation of hydroxylamine, NBT is
reduced and nitrite is produced in the presence of EDTA, which can be
detected colorimetrically at 560 nm. One enzymatic unit of S.O.D is the
amount in the form of proteins present in 100 µl of 5% tissue
homogenate required to inhibit the reduction of 24 mM NBT by 50% and
is expressed as units/mg of protein.
Reagents
Sodium carbonate solution: 1.06 gms of sodium carbonate was dissolved
in 100 ml of distilled water.
Nitroblue Tetrazolium (NBT) (Loba chemie, Mumbai, India ) 10 mg of NBT
was dissolved in 100 ml of distilled water .
EDTA solution (Nice chemicals, Cochin):37 mg of EDTA di-sodium salt
was dissolved in 100 ml of distilled water.
Hydroxyl amine hydrochloride (Nice chemicals, Cochin): 16.5 mg of
hydroxylamine hydrochloride was dissolved in 100 ml of distilled water.
Experimental Procedure
100 µl of 5% tissue homogenate in 0.2 M sucrose in phosphate
buffer (0.25 M pH 7.4) was taken in a test tube and to this a
mixture containing 1ml of sodium carbonate, 0.4 ml of NBT and
69
0.2 ml of EDTA were added and zero minute reading was taken at
560 nm.
The reaction was initiated by the addition of 0.4 ml of 1 mM
hydroxylamine hydrochloride to the test tube.
The reaction mixture was incubated at 25 C for 5 minutes; the
reduction of NBT was measured at 560 nm.
A parallel control without the tissue homogenate was also treated
in a similar manner as the test.
One enzymatic unit of S.O.D is the amount in the form of proteins
present in 100 µl of 5% tissue homogenate required to inhibit the
reduction of 24 mM NBT by 50% and is expressed as units/mg of
protein.
The concentration of enzyme is calculated using the formula
Units /mg=1000/ µgmof enzyme resulting in ½ max.inhibiton
Estimation of Catalase64
Estimation of catalase activity was done by determining the
decomposition of H2O2 at 240 nm in an assay mixture containing
phosphate buffer (0.25 M, pH 7).The estimation was done
spectrophotometrically following the decrease in absorbance at 240 nm.
The reaction mixture contained 0.01 M phosphate buffer (pH 7.0), 2 mM
H2O2 and the enzyme extract. The specific activity of catalase was
expressed in terms of units/gram of liver tissue. One international unit
70
of catalase utilized is that amount which catalyzes the decomposition of
1 micromole hydrogen peroxide per minute per mg of protein 370C.
Catalase activity was calculated using millimolar extinction coefficient of
0.07 and is expressed in terms of micromole per minute per milligram of
protein.
Reagents
Phosphate buffer (0.25M, pH7): 3.22gm potassium dihydrogen
orthophosphate and 7.268 gm of di-sodium hydrogen orthophosphate
was dissolved in distilled water and the volume was made upto 1000 ml.
Hydrogen peroxide: (0.042 ml=12.3 M) 0.34 ml of 30% hydrogen peroxide
was diluted to100 ml with distilled water.
Experimental Procedure:
100µl of 5% tissue homogenate in 0.15 M KCl buffer was added
to1.9ml of phosphate buffer (0.25 M, pH 7) and the absorbance was
measured at 240 nm. To the above reaction mixture 1 ml of hydrogen
peroxide was added and the absorbance measured after allowing it to
stand for 1 minute at 240 nm using phosphate buffer as blank solution.
One international unit of catalase utilized is that amount which catalyzes
the decomposition of 1 micromole of hydrogen peroxide per minute per
milligram of protein at 370C and is expressed in terms of units/mg of
protein.
The catalase concentration is calculated using the formula
71
Units/mg= Abs X1000/43.6 X mgof enzyme/ml reaction mixture.
Estimation of Proteins65
The method is based on the formation of a coloured complex of
proteins with Folin Ciocalteau reagent which can be measured
calorimetrically at 610 nm. Bovine serum was used as a standard.
Reagents
1. Alkaline reagent: 2 g of sodium carbonate was dissolved in 100 ml of
0.1 N NaOH solution.
2. Alkaline mixture: To 100 ml of alkaline reagent, 1 ml of 4% aqueous
copper sulphate solution was added. This was freshly prepared.
3. Phenol reagent (Folin Ciocalteau reagent) (Loba chemie, Mumbai)
commercially available reagent was diluted by adding equal volume of
distilled water before use and stored in the refrigerator.
4. 0.1 N NaOH solution.
5. 90% alcohol.
6. Preparation of standard bovine albumin solution: 8mg (0.08%)of
standard bovine albumin (S.D Fine chemicals, Mumbai) was dissolved
in 10 ml of distilled water.
72
Experimental Procedure
1. To 0.1 ml of the homogenate, 0.9 ml of 90% alchohol was added
and centrifuged at 3000 rpm for 10 minutes. The supernatant was
discarded and the precipitate was dissolved in 1ml of 0.1N NaOH.
2. 0.05 ml of the above solution was taken in a test tube, to which 4
ml of freshly prepared alkaline mixture was added and allowed to
stand for 10 minutes.
3. To the above reaction mixture 0.4 ml of phenol reagent was added
and allowed to stand for further 10 minutes for the reaction to
complete.
4. The absorbance was measured at 610 nm using distilled water as
blank.
5. 0.1 ml of standard was taken in a test tube and processed as
mentioned in step 2 and 3.
Histopathological Studies
The liver tissue was dissected out and fixed in 10% formalin,
dehydrated in gradual ethanol (50 to 100%), cleared in xylene and
embedded in paraffin. Sections were prepared and then stained with
hematoxylin and eosin (H to E) dye for photomicroscopic examination.
73
Estimation of Liver Weight
All the surviving animals were sacrificed after 48 hrs of CCl4
treatment; the liver was collected for weight determination and the mean
change in weight for each group was calculated and compared with CCl4
control.
4.5.3 IN VIVO ANTIOXIDANT ACTIVITY OF MUPPU AND
VIDAKANACHOORNAM
Experimental Design
After seven days of acclimatization, the rats were divided into six
groups (n=6). Treatment was done for 8 days as follows:Group I: Normal
control (0.9% normal saline; 1 ml/kg p.o), Group II: CCl4 control (CCl4:
liquid paraffin (1:2); 1ml/kg s.c.), Group III: CCl4 + Liquid Extract (LE-
100 mg/kg/day; p.o), Group IV: CCl4 + Liquid Extract (LE-200
mg/kg/day; p.o), Group V: CCl4 + Formulation (FR-100 mg/kg/day; p.o).
Group VI: CCl4 + Formulation (FR-200 mg/kg/day; p.o), Group II-IV:
CCl4 in liquid paraffin (1:2) (1.0 ml/kg s.c.) was given once every 72 h.
TISSUE SAMPLE PREPARATION FOR ESTIMATION OF MDA, GSH,
CAT
74
After 8 days dosing, animals were sacrificed by cervical dislocation.
Liver samples were dissected out and washed immediately with ice cold
saline to remove as much blood as possible. Liver homogenates (5% w/v)
were prepared in cold 50 mM potassium phosphate buffer (pH 7.4) using
a Remi homogenizer. The unbroken cells and cell debris were removed by
centrifugation at 1000 rpm for 10 minutes using a Remi refrigerated
centrifuge. The supernatant was used for the estimation of GSH66,
malondialdehyde MDA67, and catalase.
Reduced Glutathione (GSH)66
Reduced Glutathione was estimated spectrophotometrically by
determination of DTNB (Dithiobis-(2-nitrobenzoic acid) reduced by SH-
groups, expressed as μmol/mg wet tissue. The sulphahydryl groups
present in the glutathione forms a coloured complex with DTNB,which is
measured colorimetrically at 412nm.The amount of glutathione was
determined using its molar extinction coefficient of 13600/m/cm and is
expressed in terms of micro mol per mg of protein . To 0.1 ml of different
tissue samples, 2.4 ml of 0.02 M EDTA solution was added and kept on
ice bath for 10 min. Then 2 ml of distilled water and 0.5 ml of 50 %w/v
TCA were added. This mixture was kept on ice for 10-15 min, then
centrifuged at 3000 rpm for 15 min. To 1 ml of supernatant, 2.0 ml of
Tris buffer (0.4M) was added. Then 0.05 ml of DTNB solution (Ellman’s
reagent; 0.01M DTNB in methanol) was added and vortexed thoroughly.
75
Optical density (OD) was read (within 2-3 min after the addition of DTNB)
at 412 nm in spectrophotometer against a reagent blank.
Lipid Peroxidation67
To 1ml of tissue homogenate, 1ml of normal saline (0.9% w/v) and 2.0
ml of 10% TCA were added and mixed well. The mixture (3000 g) was
then centrifuged at room temperature for 10 min to separate proteins. 2
ml of supernatant was taken, 0.5 ml 1.0% TBA was added to it followed
by heating at 95°C for 60 min. to generate the pink coloured MDA. OD of
the samples was measured at 532 nm using Biotek Synergy 4
spectrophotometer. The levels of lipid peroxides were expressed as nM of
MDA/mg wet tissue using extinction co-efficient of 1.56 x105 M-1 cm-1.
Malondialdehyde is a secondary product of lipid peroxidation, reacts with
thiobarbituric acid at pH 3.5. The red pigment produced estimated by
measuring the absorbance at 532 nm.
4.5.4 STUDY OF COMPARATIVE IN VIVO ANTIOXIDANT ACTIVITY OF
VIDAKANACHOORNAM, MUPPU AND BUCHANANIA LANZAN 68
Experimental Design
Male albino mice 10–12 weeks old and weighing 18–22 g obtained, in
groups of six were employed for the study. These were maintained at a
room temperature of 22 ± 2 °C with 12 h light/dark cycle and free access
76
to pellet food and water. Group I: Naïve Control animals received (saline,
p.o.),Group II:Stresc ontrol.,Group III: Animals received standard
ascorbic acid at the dose of 100 mg/kg, p.o.,Group IV: Animals received
Muppu at the dose of 100 mg/kg, p.o.,Group V: Animals received Muppu
at the dose of 200 mg/kg, p.o.,Group VI: Animals received Muppu at the
dose of 400 mg/kg, p.o.,Group VII: Animals received Vidakanachoornam
at the dose of 100 mg/kg, p.o.,Group VIII: Animals received
Vidakanachoornam at the dose of 200 mg/kg, p.o.,Group IX: Animals
received Vidakanachoornam at the dose of 400 mg/kg p.o.,Group X
Buchanania lanzan leaf extract at the dose of 100 mg/kg, p.o. Group XI:
Animals received Buchanania lanzan leaf extract at the dose of 200
mg/kg, p.o.,Group XII: Animals received Buchanania lanzan leaf extract
at dose of 400mg/kg,p.o.Stress was induced in Mice by restraining
animals in well-ventilated horizontal 50-ml conical polypropylene tubes
for 12 h during the dark cycle (2000–0800 h) during experimental
periods of 14 days, One group of mice was taken as a non-treated naive
control group without any restrained conditions and was given full
access to food and water (naive-controll69. one group served as the
stress control to which drug was not administered but was subjected to
restrained stress (, treated groups were subjected to restrain stress as
well as drug administration for 14 consecutive days. Serum is separated
from whole blood which is taken from retro orbital plexus. After
microcentifugation for10min at 2000 rpm, RBCs and other cells are
77
settled down and supernatant straw coloured fluid is Serum. The serum
is separated by micropipettes in a separate micro-centrifuge tube and the
procedure is repeated thrice for complete removal
of any remaining RBCs and used blood of stressed animals for TBARS
and GSH Estimation.
Estimation of Blood Glutathione 70
0.2 mL fresh serum was collected from each animal and 1.8 mL
distilled water were added to it and 3 mL of precipitating solution were
added to the mixture. The mixture was then allowed to stand for
approximately 5 min and then filtered. 2.0 ml of filtrate were added to
8.0 ml of phosphate solution in cuvette and 1.0 mL DTNB reagent was
added to cuvette and the optical density (OD) was measured at 412 nm.
Estimation of Thiobarbituric Acid Reaction Substances (TBARS) 71
Mouse serum (0.2 ml) was pipetted out in a test tube, followed by
addition of 0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 30% acetic
acid (pH 3.5), 1.5 ml of 0.8% of thiobarbituric acid and the volume was
made up to 4 ml with distilled water. The test tubes were incubated for 1
h at 950C and allowed to cool down at room temperature and 1 ml of
distilled water and 5 ml of n-butanol-pyridine mixture (15:1 v/v) were
78
added. The tubes were centrifuged at 4000 rpm for 10 min. The
absorbance of developed pink colour was measured
spectrophotometrically (Shimadzu 1700, Singapore) at 532 nm. A
standard calibration curve was prepared using 1-10 nM of 1,1,3,3-
tetramethoxypropane. The TBARS value was expressed as nanomoles per
mg of protein.
Histopathological Studies
For histopathology the following steps were involved:
Processing of the Isolated Liver
The animals were euthanized using anaesthic ether and their liver
was dissected out. The isolated organ was sliced and fixed in neutral
formalin (10%) solution for at least 3 days. Liver pieces were then washed
under running water for about 12 hours. This was followed by
dehydration with alcohol of increasing strength (70%, 80%, 90%) for 12
hours each. Final dehydration was carried out using absolute alcohol
with about three changes at 12 hours interval. Clearing was done with
xylene with 2 changes at 15 to 20 min interval. After cleansing, the organ
pieces were subjected to paraffin infiltration in automatic tissue
processing unit. The organ pieces were washed under running water to
remove the formalin completely. Alcohol of increasing strength was used
as a dehydrating agent. Alcohol was removed by using xylene, and the
xylene was removed by paraffin infiltration.
Embedding in Paraffin
79
Hard paraffin was melted and hot paraffin was poured into L–shaped
blocks. The liver pieces were then dropped into liquid paraffin quickly
and allowed to cool.
Sectioning
The blocks were cut using microtome to get sections of 5 microns
thickness. The sections were taken on micro slide on which egg albumin
(sticky substance) was applied. The sections were allowed to dry
completely before staining on a hot plate.
Staining
Eosin is an acid stain. It stains all the basic cytoplasm cells
constituents. Hematoxylin is a basic stain, which stains all the acidic cell
components blue (DNA, RNA, and nucleus).
Preparation of the Slide
The sections were deparaffinized by washing with xylene for about
15 minutes. The sections were dehydrated by washing in alcohol of
decreasing strengths (100%, 90%, 80%, and 70%).
The sections were finally washed with water.
The sections were stained with Hematoxylin for 15 minutes.
It was rinsed with tap water.
Acid alcohol was differentiated by 3 to 10 quick dips. The
differentiation with a microscope showed that the nuclei were
distinct light or colourless.
80
The slide was washed with tap water very briefly.
The section was dipped in ammonia water, lithium carbonate until
sections became bright blue by 3 to 5 dips.
The slide section was washed in running tap water for 10-20
minutes. If washing is inadequate, eosin will not stain evenly.
The slide was stained with eosin for 15 seconds to 2 minutes,
depending on the age of the eosin and the depth of the counter
stain desired. For even staining results, slides were dipped in
several times before allowing them to set in the eosin for the
desired time.
Slides were rehydrated in 95% alcohol and absolute alcohol until
excess eosin was removed. Two changes of 2 min interval were
performed of different treatments (95% alcohol, absolute alcohol
and xylene)
The section was then mounted in DPX (diphenylxylene) mountant.
Staining results showed blue coloured nucleus and cytoplasm with
various shades of pink with change in different tissue components.
Statistical Analysis
The data were expressed as mean ± S.E.M. The differences among
means were analyzed by one-way ANOVA. A value of P < 0.05 was
considered as statistically significant. The significance of difference
among groups was assessed using one way analysis of variance (ANOVA)
81
followed by Tukey-Karmer multiple comparison test between the data of
control and treated groups. The values were expressed as mean ± SEM,
where* p<0.05 was considered as significant, **p<0.01 was considered
very significant and ***p<0.001was considered extremely significant.
4.6 EVALUATION OF ANTISTRESS/ADAPTOGENIC ACTIVITY
4.6.1.FORCED SWIMMING TEST 72
Briefly, the animals were forced to swim individually in a glass jar (25
× 12 × 25 cm) containing water of 15 cm height at a temperature of 25 ±
3°C for a period of 15 min in a training session on day 1 and for 5 min on
day 2 of the experiment. Each animal made vigorous attempts to get out
of water bath during first couple of minutes and thereafter surrendered
to experimental conditions and assumed a typical immobile posture with
occasional escape attempts. This phenomenon is a measurement of
acute stress followed by depression. The total duration of immobility was
recorded. Animals were removed from water, dried and kept in their
respective cages.
Experimental Design
The animals were individually weighed and divided into eleven groups,
each group comprising of six animals. All the animals were fasted 3 h prior
to commencement of experiment but water was provided ad libitum. The
grouping of animals was done as follows:
82
Group I: Control animals received (saline, p.o.),Group II: Standard
Imipramine 15 mg/kg, p.o.,Group III: Animals received Muppu at the
dose of 100 mg/kg, p.o.,Group IV: Animals received Muppu at the dose
of 200 mg/kg, p.o.,Group V: Animals received Muppu at the dose of 400
mg/kg, p.o.,Group VI: Animals received Vidakanachoornam at the dose
of 100 mg/kg, p.o.,Group VII: Animals received Vidakanachoornam at
the dose of 200 mg/kg, p.o.,Group VIII: Animals received
Vidakanachoornam at the dose of 400 mg/kg, p.o.,Group IX: Animals
received Buchanania lanzan leaf extract at the dose of 100 mg/kg, p.o.
Group X: Animals received Buchanania lanzan leaf extract at the dose of
200 mg/kg, p.o.,Group XI: Animals received Buchanania lanzan leaf
extract at the dose of 400 mg/kg, p.o. After 1 hr post-administration of
the drugs, the animals were placed in the jar one by one for despair swim
test (Forced Swim test). The immobility time in seconds was noted .
4.6.2 SWIMMING ENDURANCE TEST 73
The mice were divided into five groups of six animals each n=6. Group
I Normal control (0.9% normal saline; 1 ml/kg p.o.), Group II received
Impramine 15 mg/kg, p.o., Group III received Muppu at the dose of 250
mg/kg, p.o., Group IV received Vidakanachoornam 250 mg/kg, p.o., and
Group V received Buchanania lanzan leaf extract 250 mg/kg, p.o.The
drugs once daily for 14 days. On the fourteenth day all groups were
allowed to swim till exhaustion in separate cylindrical containers (bucket)
83
filled with water (maintained at 250C). Death due to drowning was taken
as the end point. Swimming time for individual animal was noted down
and the mean swim survival time for each group was calculated. Mean
swim time of drug treated groups were compared with that of the control
group.
4.6.3. ANOXIC STRESS TOLERANCE IN MICE 42
The mice were divided into five groups of six animals each n=6. Group
I Normal control received (0.9% normal saline; 1 ml/kg p.o.), Group II
received Impramine 15 mg/kg, p.o., Group III received Muppu at the
dose of 250 mg/kg, p.o., Group IV received Vidakanachoornam 250
mg/kg, p.o., and Group V with Buchanania lanzan leaf extract 250
mg/kg, p.o. Conical flasks of 250 ml capacity were used for the study.
The flasks were made airtight by using rubber cork before the beginning
of the experiment. Mice of equal weight were used in the experiments.
Each mouse served as its own control. The anoxic stress tolerance time
was measured as follows: Each animal was kept in the airtight vessel
and the time was noted by using a stop watch. The moment animal
showed its first convulsion, it was removed immediately from the vessel
and resuscitated if needed. The time duration from the entry of the
animal in a hermetic vessel to the appearance of the first convulsion was
taken as the time of anoxic stress tolerance. The appearance of
convulsion is a sharp end point, as delay of even one minute in removal
kills the animal. The anoxic stress tolerance time was determined for
84
each animal individually, before subjecting the animals to the drug
treatment and after one week, two weeks and third week of the drug
treatments.
4.6.4 RESTRAINT STRESS INDUCED CHANGES IN RATS 74
The mice were divided into six groups of six animals each n=6. Group
I Normal control (0.9% normal saline; 1 ml/kg i.p.), Group II Stress
control (0.9% normal saline; 1 ml/kg i.p.)and restraint stress, Group III
received 15 mg/kg, p.o., Impramine, Group IV received Muppu at the
dose of 250 mg/kg, p.o., Group V received Vidakanachoornam 250
mg/kg, p.o., and Group VI with Buchanania lanzan leaf extract 250
mg/kg, p.o.
Animals were pretreated with the drugs for eight days. They were
deprived of food 24 hrs before the commencement of experiment with
water ad libitum. After one hour of administration of the last dose, all
four limbs of the animal were tied and kept in separate chambers
(cylindrical vessels, 50 cm high and 40 cm wide) at 40C in the refrigerator
for two hours. Blood was collected by retro-orbital puncture method
using heparinised capillary tubes. The blood samples were analysed for
corticosterone estimation. Animals were sacrificed using cervical
dislocation. Adrenal gland was collected for assessing any changes in the
weight, when compared to the control group.
Plasma Corticosterone Estimation75
85
Plasma corticosterone was estimated by sulphuric acid induced
fluorescence measurement method.
Materials Required:
Flourescent reagent: Absolute alcohol and concentrated sulphuric
acid mixed in ratio of 3:7 and cooled.
Dichloromethane
Distilled water
Hydrocortisone standard (LOBA chemicals)
Two ml of plasma was taken in a separating funnel and 15 ml of
dichloromethane were added to it and shaken well, it was allowed to
stand, two layers are formed. The upper layer is separated out and is
discarded. 10 ml of the lower layer is treated with 5 ml of the Flourescent
reagent (as prepared by mixing 3 ml of absolute alcohol with 7 ml
concentrated of sulphuric acid, following by cooling) and shaken well.
Again the lower layer is separated and reading is taken at 530 nm using
photoflourimeter, as mentioned below:
(A) Blank; 2 ml of triple distilled water;
(B) Standard solution: 2 ml of hydrocortisone standard solution of
known strength (100mcg/100ml);
(C) Test solution: 2 ml of the lower layer obtained from the above
procedure.
The concentration of the plasma cortisol was calculated as follows:
86
Concentration of the plasma cortisol =reading of the test / reading of the
standard X100
4.6.5 ESTIMATION OF URINARY VMA AND ASCORBIC ACID LEVELS
76
Ascorbic Acid Estimation77, Urine VMA78
Experimental design
Rats of either sex weighing between 150-250 g were divided into six
groups (I, II, III, IV, V, and VI) each comprising of six animals. The 24 h
urine sample from each group was collected into two different beakers,
one containing 5 ml of 10% oxalic acid for spectrophotometric
determination of ascorbic acid at 550 nm and the other containing 0.5
ml of 6 N hydrochloric acid for the determination of vanillylmandellic
acid (VMA) spectrometrically at 360 nm. The experimental protocol was
divided into four phases. In the first phase of the experiment, 24 h urine
samples were collected in all the six groups and subjected to analysis for
both VMA and ascorbic acid and the normal values were recorded for
seven consecutive days. In the second phase, the animals in each group
were subjected to fresh water swimming stress individually. In this
method, rats were forced to swim until exhausted (three to four minutes)
in cylindrical vessel of 60 cm height and 45 cm diameter containing
water at room temperature (280C). Water depth was always maintained
at 40 cm. The 24 h urinary levels of VMA and ascorbic acid under
87
stressed conditions were determined again as described above daily for
seven consecutive days. Third phase of experiment consists of
administration of Methanolic Extract (ME) to same groups of animals
after having recovered completely to normal condition. Groups II, III, IV,
V and VI were administered orally with Methanolic Extract at daily doses
of 10, 20, 30, 40, and 50 mg/kg body weight, respectively for seven
consecutive days while group I serving as control. The 24 h urine
samples were collected and the levels of both VMA and ascorbic acid
were determined. The final phase of the experiment consisted of
administrated of Methanolic Extract (ME) to the same groups of animals
after a recovery period of one week. Groups II, III, IV, V, VI were
administered orally with Methanolic Extract at doses of 10, 20, 30, 40
and 50 mg/kg body weight, respectively, and hour prior to the daily
induction of stress for seven consecutive days while group I serving as
control. The 24 h urine sample were collected and analyzed for VMA and
ascorbic acid for seven consecutive days to study the influence of the
Methanolic Extract on the stress induced biochemical change.
4.6.6 ESTIMATION OF BLOOD GLUTATHIONE AND TBARS LEVELS
Male albino mice 10–12 weeks old and weighing 18–22 g obtained, in
groups of six were employed for the study. These were maintained at a
room temperature of 22 ± 2 °C with 12 h light/dark cycle and free access
to pellet food and water. Group I: Naïve Control animals received (saline,
88
p.o.),Group II:Stresc control.,Group III: Animals received standard
ascorbic acid at the dose of 100 mg/kg, p.o.,Group IV: Animals received
Muppu at the dose of 100 mg/kg, p.o.,Group V: Animals received Muppu
at the dose of 200 mg/kg, p.o.,Group VI: Animals received Muppu at the
dose of 400 mg/kg, p.o.,Group VII: Animals received Vidakanachoornam
at the dose of 100 mg/kg, p.o.,Group VIII: Animals received
Vidakanachoornam at the dose of 200 mg/kg, p.o.,Group IX: Animals
received Vidakanachoornam at the dose of 400 mg/kg p.o.,Group X
Buchanania lanzan leaf extract at the dose of 100 mg/kg, p.o. Group XI:
Animals received Buchanania lanzan leaf extract at the dose of 200
mg/kg, p.o.,Group XII: Animals received Buchanania lanzan leaf extract
at dose of 400mg/kg,p.o.Stress was induced in Mice by restraining
animals in well-ventilated horizontal 50-ml conical polypropylene tubes
for 12 h during the dark cycle (2000–0800 h) during experimental
periods of 14 days, One group of mice was taken as a non-treated naive
control group without any restrained conditions and was given full
access to food and water (naive-controll69. One group served as the
stress control to which drug was not administered but was subjected to
restrained stress , treated groups were subjected to restrain stress as
well as drug administration for 14 consecutive days. Serum is separated
from whole blood which is taken from retro orbital plexus. After
microcentifugation for10min at 2000 rpm, RBCs and other cells are
settled down and supernatant straw coloured fluid is Serum. The serum
89
is separated by micropipettes in a separate micro-centrifuge tube and the
procedure is repeated thrice for complete removal
of any remaining RBCs and used blood of stressed animals for TBARS
and GSH Estimation.
Estimation of Blood glutathione 70
In which 0.2 mL fresh blood was collected from each animal and1.8
mL distilled water was added to it and 3 mL of precipitating solution was
added to mixture. The mixture was then allowed to stand for
approximately 5 min and then filtered. 2.0 mL of filtrate was added to 8.0
mL of phosphate solution in cuvette and 1.0 mL DTNB reagent was
added to cuvette and the optical density (OD) was measured at 412 nm.
Estimation of TBARS71
The quantitative measurement of thiobarbituric acid reactive
substances (TBARS), an index of lipid peroxidation in mouse serum
.Mouse serum (0.2 ml) was pipetted out in a test tube, followed by
addition of 0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 30% acetic
acid (pH 3.5), 1.5 ml of 0.8% of thiobarbituric acid and the volume was
made up to 4 ml with distilled water. The test tubes were incubated for 1
h at 95 0C and allowed to cool down at room temperature and 1 ml of
distilled water and 5 ml of n-butanol-pyridine mixture (15:1 v/v) were
added. The tubes were centrifuged at 4000 rpm for 10 min. The
absorbance of developed pink colour was measured
spectrophotometrically (Shimadzu 1700, Singapore) at 532 nm. A
90
standard calibration curve was prepared using 1-10 nM of 1, 1, 3, 3-
tetramethoxypropane. The TBARS value was expressed as nanomoles per
mg of protein.
4.7 HEPATOPROTECTIVE ACTIVITY 79–81
4.7.1 EVALUATION OF HEPATOPROTECTIVE ACTIVITY OF MUPPU
Experimental design
Carbon Tetrachloride-induced Hepatotxicity in Rats
Rats were divided into four groups of six each: control, hepatotoxin,
positive control and test groups. The control group received oral vehicle
treatment at 0, 24 and 48 h. The animals in hepatotoxin-treated groups
received vehicle 0 h and at 24 h following by carbon tetrachloride diluted
in liquid paraffin (1:1, s.c.) at a dose of 1.25 ml/kg, while at 48 h these
animal received only vehicle. The test groups received the first dose of
extracts at 0 h, second dose of extracts at 24 h, which was followed by a
dose of extracts at 0 h, second dose of extracts at 24 h, which was
following by a dose of carbon tetrachloride and at 48 h the third dose of
extracts. The positive control group has received the first dose of LIV 52
(0.125 mg/kg body wt) 5 at 0 h, at 24 h the second dose of LIV 52
followed by a dose of carbon tetrachloride and at 48 h the third dose of
LIV 52. After 72 h blood was collected from all the groups, and allowed to
clot for the separation of serum. The serum was used for estimation of
biochemical parameters.
91
4.7.2 EVALUATION OF THE HEPATOPROTECTIVE ACTIVITY OF
VIDAKANACHOORNAM AND BUCHANANIA LANZAN
Biochemical Determinations
The biochemical parameters like serum enzymes: aspartate
aminotransferase (AST); alanine transaminase (ALT) alkaline
phosphatase (SALP); and total bilirubin were assayed using assay kits
supplied by Span Diagnostics, Surat.
4.7.3 EVALUATION OF THE HEPATOPROTECTIVE ACTIVITY OF
VIDAKANACHOORNAM AND BUCHANANIA LANZAN AND MUPPU
Drug-Induced Potentiation of Pentobarbitone Sleeping Time82
Drug treatment was continued for seven days.Group I:Vehicle control
received saline p.o;GroupII received groups received carbon tetrachloride
diluted in liquid paraffin (1:1, s.c.) at a dose of 1.25 ml/kg, Group III: N
animals received Liv -52 p.o,Group IV: Animals received
Vidakanachoornam at the dose of 250 mg/kg, p.o.,Group V: Animals
received Buchanania lanzan leaf extract at the dose of 250 mg/kg,
p.o.,Group VI: Animals received Muppu at the dose of 250 mg/kg, p.o.,
All the animals were deprived of food for 24 hrs before the experiment
with free access to water. The experiment was conducted in a quite and
temperature of room was maintained at 250C in the afternoon between
92
12 and 5 pm. Sleeping time was measured before and after the drug
treatment by injecting single dose of pentobarbitone 45 mg/kg body
weight i.p to all the mice. Sleeping time was taken using a stop watch
and the corresponding to the time elapsed between the loss of righting
reflex and the recovery of the same reflex. This time was expressed in
minutes. The mean narcosis time of the drug treatment was compared to
control group.
CARBON TETRACHLORIDE–INDUCED PROLONGATION OF
PENTOBARBITONE SLEEPING TIME AND MORTALITY IN ALBINO
MICE
After determining the effect of the drug treatment on narcosis time,
the treatment was continued till 14 days. On the fourteenth day, after
one hour of the last dose administration, all the mice were treated with
carbon tetrachloride 1ml/kg body weight of mice along with equal
volume of liquid paraffin.
After 30 minutes of carbon tetrachloride administration, sodium
pentobarbitone was injected at a dose of 45 mg/kg body weight by i.p
route. The mean sleeping time was calculated for each group. 48-hour
mortality was assessed and compared with the control group.
4.8 EVALUATION OF ANTIINFLAMMATORY OF BUCHANANIA
LANZAN 83,84
93
4.8.1 EVALUATION OF ANTIINFLAMMATORY USING CARRAGEENAN
INDUCED RAT PAW OEDEMA
Experimental Design
Rats weighing 150-250 g were used in the experiments. The animals
were housed at room temperature (20 ± 20C) in standard cages with
standard pellet food and kept under controlled environment following the
standard operating procedures of animal house with the approval of
institutional animal ethics committee.
Carrageenan (Sigma), acetylsalicylic acid, CMC (Carboxymethyl
cellulose) and DMSO (Dimethyl sulfoxide) were employed; all the
compounds were dissolved in mixture of CMC and DMSO (9:1).
In this method, rats were divided into seven groups of six animals
each. The animals were pretreated with drug and Methanolic Extracts
(ME) in concentration of (ME-10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg,
and 50 mg/kg) were given by i.p route 30 minutes before carrageenan
injection (phlogistic agent) of 0.1 ml dose. Carrageenan was injected into
the sub plantar tissue of left hind paw of each rat. Swellings of
carrageenan-injected foot were measured at 30 min, 60 min, 120 min,
180 min using plethysmometer. The right hind paw was injected with 0.1
ml of vehicle. The animals received the standard drug aspirin (20 mg/kg,
i.p.) which served as reference standard. The experimental groups were
as follows (n=6);,Group 1:Control (CMC+DMSO), i.p;,Group 2:
Acetylsalicylic acid (20 mg/kg), i.p;,Group 3: ME (10 mg/kg), i.p;,Group
94
4: ME (20 mg/kg), i.p;,Group 5: ME (30 mg/kg), i.p;,Group 6: ME (40
mg/kg), i.p;,Group 7: ME (50 mg/kg), i.p;
Statistical Analysis
Results were expressed as mean ± S.E.M.
Percentage of inhibition (Ι %) = [(1-(dt/dc)] x 100.
Where dt is the difference in paw volume in drug-treated group and dc
the difference in paw volume.
4.9 ISOLATION AND CHARACTERIZATION
4.9.1 HPTLC FINGER PRINTING OF BUCHANANIA LANZAN
4.9.2 ISOLATION OF PHYTOCONSTITUENTS FROM LEAVES OF
BUCHANANIA LANZAN
Column chromatography
Preparation of the extract
Shade-dried powdered leaves (5 kg) of B. Lanzan were extracted with
methanol in a Soxhlet apparatus. The extract was filtered, and
concentrated under reduced pressure to give dark brown mass. The yield
of the extract was 10%. The extract was then subjected to preliminary
phytochemical analysis using standard methods.
95
Chemicals: The chemicals for isolation were obtained from Merck, and
SD Fine Chemicals, Mumbai.
Spectral analysis: was done at II Sc Bangalore and Quest Lab.
Isolation: The methanolic extract (30 gm) was subjected to column
chromatography on silica gel using solvents of increasing polarities
starting from petroleum ether, chloroform, ethyl acetate and methanol to
yield several fractions (181 fractions in total Elution was monitored by
then layer chromatography (TLC) ).
Fractions 32-36 eluted with chloroform: petroleum ether (1:1 mixture)
were combined together due to similarities in TLC pattern. The solvent
system [toluene: ethyl acetate 1:1] showed a major spot at Rf 5.2. This
fraction was re-chromatographed on a column of silica gel, elution being
carried out using different ratios of petroleum ether and chloroform.
Fractions of 10 ml were collected. Fraction ninth showed a single spot on
TLC. This fraction was recrystalised and dried to get 0.43 mg of
compound which was coded as SKM-L-1.
Fractions 89-112, eluted with methanol: ethyl acetate (4:6 mixture)
when subjected to TLC, using the solvent system toluene: ethyl acetate
7:3, showed a two major spots at Rf value of 0.4 and 0.7. This combined
fraction was subjected rechromatography on a column of silica gel and
also eluting with ethyl acetate and methanol in different ratios. Thirty six
96
fraction of 5 ml each were collected and monitored by TLC. The fractions
(17-19) of the second column were mixed and recrystallise to get 0.40 mg
of the pure compound which was coded as SKM-L-2. Fractions 26 and
27 of the second column showed a single spot on TLC. These fractions
were mixed and left over night to obtain 0.51 mg of SKM-L3.
4.9.3 ISOLATION OF PHYTOCONSTITUENTS FROM BARK OF
BUCHANANIA LANZAN
5.9.3 CHEMICAL STUDIES ON THE BARK OF BUCHANANIA LANZAN
Bark of Buchanania lanzan was dried in shade and was rendered into
moderately coarse powder. The bark powder (1kg) was extracted with
methanol in a soxhlet apparatus till the last drops of the extract did not
show any residue on evaporation of the solvent. The extract was filtered
and the solvent removed under reduced pressure to obtain a semisolid
dark brown resinous residue (150 gm). The residue was refluxed for 1 hr
with a mixture of chloroform and ethanol (1:1), and filtered. This was
repeated one more time. The solvent from the combined extract was
removed under reduced pressure to give a brownish–yellow residue (40
g). The residue was dissolved in a mixture of chloroform and ethanol (1:1)
and silica gel for chromatography (100-200) mesh size (200 g) was slowly
added to the solution and stirred thoroughly directing hot air from a
blower to it. When the silica gel coated with the residue became free
97
flowing it was loaded on the column of silica gel (600 g) which served as a
stationary phase. Gradient elution with the solvents of increasing
polarity using petroleum ether (60-80): chloroform
(100:0 to 10:90); pure chloroform, chloroform: ethyl acetate (100:10 to
10:90); ethyl acetate (100:0 to 10:90); ethyl acetate pure; ethyl acetate:
methanol (95:5 to 50:50) was carried out. The eluate was collected in
250-ml fractions, solvent removed and the residue weighed after drying
the residue in vacuum desiccator.
Thin Layer chromatography:
The fractionation was monitored by thin layer chromatography on
TLC glass plates (520 cm) coated with silica gel G (0.25 mm thick).
The plates were activated at 1100C for 30 min before use. The
solvent system used were:
Toulene: Ethyl acetate (80:20)
Petroleum ether: Ethyl acetate : Methanol (5:3:2)
Chloroform: Ethyl acetate (5:5)
Detection of the spots was done by exposing the developed plate
under iodine vapors. Fractions with similar Rf values were pooled
together. The work up yielded residues which were crystallized
individually and were found homogenous on TLC, each showing a single
spot.