Post on 17-Mar-2018
3. MATERIALS AND METHODS
3.1. PHARMACOGNOSY
3.1.1. Collection of plant materials
Roots of Tephrosia purpurea Pers. and Tephrosia villosa Pers. were
collected in and around Thanjavur.
3.1.2. Plant Identifications
Collected specimen were carefully examined and identified with the help
of regional Floras (Gamble, 1967; Kirthikar and Basu, 1980; Mathew,
1983; Nair and Hendry, 1983 and Henry et al., 1987). The botanical
identity was authenticated by Dr. M. Jegadeesan, Professor and Head,
Dept. of Environmental and Herbal Sciences, Tamil University,
Thanjavur. Specimens were further confirmed with reference to
Herbarium sheet available in the Botanical Survey of India, Southern
Circle, and Coimbatore. A voucher specimen has been deposited at Tamil
University Herbarium for future reference (TUH 277-278).
3.1.3. Taxonomy
Systematic position of the plant specimen has been assigned as per the
angiosperm taxonomic classification of Bentham and Hooker (1862-
1883).
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3.1.4. Anatomical and histochemical studies (Krishnamoorthy, 1988)
Free hand section of roots of T. purpurea and T. villosa were taken. Then
these sections were stained with different chemical reagents for the
localization of alkaloids, phytosterol, lipids, tannins and carbohydrates.
Destaining was made by washing with respective solvents of the stains.
Sections were mounted on slides using glycerin and covered with cover
glass and margins were sealed with nail polish and observed under
microscope.
3.1.5. Preparation of powder (Harborne, 1973)
The root was separated from the selected species of Tephrosia and dried
under shade. These dried materials were mechanically powdered, sheaved
using 80 meshes and stored in an airtight container. These powdered
materials were used for further physicochemical, phytochemical and
fluorescent analysis.
3.1.6. Analytical studies
The procedures recommended in Indian Pharmacopoeia (Anonymous,
1966; 1985; 1996) were followed for the determination total ash, water-
soluble ash, water-insoluble ash, acid- insoluble ash, sulphated ash and
loss on drying at 110oC.
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a) Total ash value
5 g of plant powder was ignited in an electric furnace at 600oC in silica
crucible until the sample reaches a constant weight. Percentage of total
ash value was calculated.
b) Water – soluble ash value
Total ash obtained was heated upto 600oC with addition of 25 ml of water
for 10 minutes. It was filtered in an ashless filter paper (Whatman No. 41)
and the residue was ignited in the furnace to get a constant weight.
c) Acid - insoluble ash value
Total ash obtained was heated with addition of 25 ml of dil. HCl for 10
minutes. It was filtered in an ash less filter paper (Whatman No.41) and
the residue was ignited in the furnace to get a constant weight.
d) Sulphated ash value
1g of plant powder was ignited in an electric furnace until the drug gets
charred. The crucible was cooled and the residue was moistened with 1ml
of H2SO4, heated gently until the white fumes were no longer evolved and
ignited at 800oC ± 25
oC until all black particles disappear. The crucible
was allowed to cool; few drop of H2SO4 was added and again heated. The
ignition was carried as before, allowed to cool and then weighed. This
was repeated until the sample reaches a constant weight.
3.1.7. Solubility percentage (Kokate, 1994)
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a) Alcohol
5 g of powdered material along with 100 ml of alcohol was shaken well
occasionally for the first 6 hours and kept undisturbed for 18 hours. The
liquefied extract thus obtained was concentrated in a vacuum pump and
the percentage was calculated with the weight of the drug powder taken.
b) Water
The procedure adopted for the solubility percentage of the plant powder in
alcohol is used with chloroform water instead of alcohol to get the water
solubility percentage.
3.1.8. Powder analysis
Fluorescent analysis was carried out by using the method of Chase and
Pratt, 1949. Behavior of different chemical reagents was carried out as
mentioned by Kokoshi et al. (1958)
3.1.9. Qualitative phytochemical analysis
Qualitative phytochemical analyses were done using the procedures of
Kokate (1994). Alkaloids, carbohydrates, tannins and phenols,
flavonoides, gums and mucilage, fixed oils and fats and saponins were
qualitatively analyzed.
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a) Alkaloids
The extracts were dissolved in dil. H2SO4 and filtered. The filtrate was
treated with Mayer’s, Dragendroff’s, Hager’s and Wagner’s reagents
separately. Appearance of cream, orange brown, yellow and reddish
brown precipitates in response to the above reagents respectively indicate
the presence of alkaloids.
b) Carbohydrates
300 mg of 50 per cent alcoholic extracts were dissolved in water
and filtered. The filtrate was treated with con H2SO4 and then with
Molisch’s reagent. Appearance of pink or violet color indicates the
presence of carbohydrates. The filtrate was boiled with Fehling’s and
with Benedict solution. Formation of brick red precipitate in Fehling’s
and Benedict’s solution is the positive result for reducing sugars and non-
reducing sugars respectively.
c) Tannins and phenols
Small quantity of 50 per cent alcoholic extract was dissolved in water and
5 per cent ferric chloride solution or 10 per cent lead acetate solution was
added. Appearance of blue color with ferric chloride or precipitation with
other reagent indicates the presence of tannins and phenols.
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d) Gum and mucilage
About 10 ml of the extract was slowly added to 25 ml of absolute alcohol
under constant stirring. Precipitation indicates the presence of gum and
mucilage.
e) Fixed oils and fats
A drop of concentrated extract was pressed in between two filter papers
and kept undisturbed. Oil stain on the paper indicates the presence of oils
and fats.
f) Saponins
About 1ml of the extract was dissolved in 20 ml of water and shake in a
graduated cylinder for 15 minutes. Formations of one cm layer of foam
indicate the presence of saponins.
g) Phytosterol
The extract was treated with Lieberman Burchard under suitable
conditions. Appearance of blue-emerald green indicates the presence of
phytosterol and terpenes.
3.1.10. Preparation and concentration of Extracts (Successive)
(Anonymous, 1966).
The root powders of T. purpurea and T. villosa were extracted
successively using Soxhlet apparatus with petroleum ether 60-80°C,
benzene 60°C, chloroform 60°C, alcohol 78°C. Each time before
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extracting next solvent powdered materials was dried in hot air oven
below 50°C. The extracts dried over anhydrous sodium sulphate, stored in
sealed vials in a refrigerator (5-8°C). Finally mark was macerated with
chloroform water for 24 hrs to obtain the aqueous extract. The extract was
concentrated by distilling of the solvent and then evaporation to dryness
on a water bath.
3.1.11. Cold Extraction Technique
The air-dried material was coarsely powdered to aid the extraction. They
were soaked in alcohol and kept for 48 hours. The extract thus obtained
was decanted and filtered. The clear extract was subsequently
concentrated using rotary vacuum evaporator. This method was done to
save the active principles if any that would have been otherwise
inactivated by a heating process that usually involved in any concentration
process. Pilot biological studies conducted using this extract and when
compared its effects with the heated extracts, the active principles
withstood the heating up to 65oC for least 24 hours. So subsequently the
extracts were concentrated over a boiling water bath on glass Petri-dishes
by free evaporation.
3.1.12. Preparation of Extract
50 per cent alcohol extract was prepared according to the methodology of
Indian Pharmacopoeia (Anonymous, 1996). The 50 per cent alc. extract
was subjected to pharmacological studies and GC/MS.
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3.2. PHYTOCHEMISTRY
3.2.1. Quantitative phytochemical studies
a) Estimation of total terpenoid
100 g of plant powder were taken separately and soaked in alcohol for 24
hours. Then filtered, the filtrate was extracted with petroleum ether; the
ether extract was treated as total terpenoid (Ferguson, 1956)
b) Estimation of total alkaloid
This alcoholic extract of plant sample was treated with 0.1N HCl and
aqueous acidified layer thus obtained was partitioned with chloroform in a
separating funnel. The chloroform layer is rejected. The aqueous layer
was basified with ammonium hydroxide and then partitioned with
chloroform. The chloroform layer was concentrated and tested for
alkaloids with alkaloid testing reagents (Ferguson, 1956)
c) Estimation of Tannin-free total glycoside
100 g of air-dried powder were extracted with ethanol; water (2:1). The
aqueous ethanol extract thus obtained contains tannins which usually
interfere with the biological activities. Hence, this should be removed by
treating with 5 per cent neutral lead acetate reagent which precipitates the
tannins as lead tannate. The aqueous ethanolic solution was treated with
5per cent neutral lead acetate solution and the precipitated lead tannate
was filtered off. This process is repeated with until no more precipitate
was obtained. The clear filtrate now contained the excess un-precipitated
lead ions in solutions which were removed by passing H2S gas into the
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solution. This removed the lead ions as insoluble complex black lead
sulphide. The black precipitate was filtered and this process was usually
repeated until no more black precipitate was formed and the solution
strongly smelled of H2S. The solution, usually of syrupy consistency, was
concentrated over water-bath maintained at 55oC. This procedure also
removed the excess of H2S (Ferguson, 1956)
d) Estimation of total flavonoid
Isolation of flavonoid from the ethanol extract of root was carried out on
the basis of solubility. For isolation, distilled water (100 ml) was added to
the concentrated ethanol extract (50 ml). After about 1 hour precipitation
was observed. This precipitate was recovered by filtration. Further, the
precipitate was dissolved in chloroform (100 ml) by shaking for 15 min
and heated gently for 5 min and filtered in hot state. The chloroform
soluble fraction was discarded and insoluble fraction, left on filter paper
was dissolved in ethyl acetate soluble fraction was discarded and insoluble
fraction, left on filter paper was crystallized with methanol, thereafter the
residue obtained. For characterization positive result for Shinoda test
which is characteristic of flavonoids. (Jain et al., 2006)
e) Estimation of ascorbic acid (vitamin – C) (Ganguly, 1948)
Titration with Iodine (or) Chloramines T 2
Principle
For determination of ascorbic acid, 0.1N iodine solution (or)
chloramines-T solution have been recommended by Leonhardt and
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Moeser Both these volumetric solution oxidize ascorbic acid to dehydro
ascorbic acid quantitatively. However Chloramine-T is prepared because
of it is better ability to retain it is titrate with iodine, the end point is not
well defined and starch solution used as indicator retards the reaction.
Titration with iodine is the U.S.P procedure.
Reagents
Iodine solution, 0.1N
14.085 g of reagent is dissolved in water to make 100 ml
Petroleum ether boiling ranges 40C Potassium Iodide. Reagent grade
Hydrochloric acid (5 g.1.122 - 1.124). Starch solution. A 1 per cent
aqueous solution.
Procedure
A measured volume of assay solution containing 75-250 mg of ascorbic
acid is transferred to a conical flask and diluted to 75 ml with water 1ml
of hydrochloric acid, 1 g of Potassium iodide and a few drops of starch
solution as indicator are added and the solution is titrated with 0.1 N
chloramines-T until a blue color appear in the titrating flask.
The solution can also be titrated using 0.1 N iodine solutions. Ascorbic
acid in Pharmaceutical preparations containing alcohol, glycerol and syrup
is determined by titration with iodine 12 vitamin A&B interfere with the
determination.
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Calculation
Each milliliter of 0.1 N Chloramine -t T or 0.1N iodine solution
corresponds 8.806
3.2.2. GC-MS Studies
The 50 per cent alc. extract was examined in GC-MS for its chemical
composition by GC-MS engine model, GC–Clarus 500; Perkin Elmer and
Computer Mass Library (Wiley 138L) of 80,000 compounds with a GC
column Elite – 1 (100per cent Methyl Poly Siloxane). The other
conditions were as follows.
Injector: GC-Clarus – 500; Perkin Elmer; Carrier gas flow Helium 1
ml/min; Split ratio – 1:25; Sample injected 1µl; Oven temperature –
110deg – 2 min hold; Up to 270deg at the ratio of 5 deg/min – 4 min hold;
Injector temperature 250oC; Total GC- time 38 min; MS inlet line
temperature 200oC; Source temperature 200
oC; Electron energy 70eV;
Mass Scan 25-400; MS time 39 min.
3.2.3. TLC studies
TLC studies on 50 per cent alcoholic extracts of the powdered
drugs of T. purpurea and T. villosa were carried out.
TLC plates were prepared by using Silica Gel-G as adsorbent. 100 g
silica gel-G was mixed with sufficient quantity of distilled water to make
slurry. The slurry was immediately poured into a spreading the slurry on
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glass plates of required size. The thickness of the layer was fixed 1.5 mm
plates were allowed to air dry for one hour and layer was fixed by drying
at 110oC for two hours.
Using micropipette, about 10 ml of 1 per cent w/v solution of extraction
were loaded gradually over the plate. The loaded plate was eluted by
suitable mobile phase like TBA (t-BuOH – AcOH – H2O - 3:1:1 ratio)
BAW (n-BuOH- AcOH-H2O – 4:1:5 ratio – Upper phase), Ferostal
(AcOH – Con.HCl – H2O – 30:3:10 ratio) and MWF (Methanol-Water-
Formic acid- 40:57:3, v/v/v) reported by Jain et al., 2009. Before the
elution, the tank and extract were allowed 30 minutes for saturation with
mobile phase. The extracts showed separation into bands. The
chromatograms were observed under visible light and were photographed.
The Rf value of the band can be obtained by using the following formula.
Rf =
Distance traveled by the substance (cm)
Distance traveled by the mobile phase (cm)
3.3. PHARMACOLOGY
Pharmacological experiments involving animals described in the present
work were carried out and get approved by Local Animal Ethical
Committee of Dept. of Pharmacology, Periyar College of Pharmacy for
Women, Trichy.
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3.3.1. Toxicological Studies
Gross behavioural and acute oral toxicity studies
Gross behavioural and acute toxicity studies of the extracts were
determined as suggested by Turner (1965). The mice were divided into 6
groups and 6 mice in each, 50per cent ethanolic extract was administered
orally at different dose levels of 1, 2 and 3 g/Kg B.W. to the overnight
fasted animals. The group receiving Tween 80 (1 ml/Kg B.W.) was kept
as control. The animals were subjected to primary screening studies at ½,
1, 2 and 4h respectively was recorded. Behaviour of the animals and any
other toxic symptoms were also observed for 24, 48 and 72 h and the
animals were kept under observation upto 14 days after drug
administration to find our delayed mortality if any (Miller and Trainter,
1944; Gosh, 1991).
3.3.2. Anti-ulcer study (Ethanol induced model)
The gastric ulcer was induced in rats of either sex weighing between 130-
140 g by administrating absolute ethanol (8 ml/Kg B.W.). They were kept
in specially constructed cages to prevent coprophagia during and after
the experiment. The rats were divided in to nine groups each containing
six animals and fasted for 24 hrs and allowed free access to water only
and second group received ethanol orally. The third group received
ethanol and standard antiulcer drugs Ranitidine (150 mg/Kg B.W.). The
fourth, fifth, sixth, seventh, eighth and ninth groups were given absolute
alcohol and 50 per cent alc. extracts of T. purpurea and
T. villosa at a dose of 5, 10, 20 mg/Kg B.W. respectively. The drugs were
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administered orally 30 min prior to the oral administration of absolute
ethanol. The animals were anesthetized 6 h later with ether and stomachs
were incised along the greater curvature, collected the gastric juice and
ulceration was recorded.
The collected gastric juice were analyzed for gastric volume, pH, free
acidity (Jeffery et al., 1992). Biochemical estimation like total protein,
hexose, hexosamine (Dische and Schettles, 1948; Dische and
Borentreund, 1950; Lowery et al., 1951; Winzler, 1958; Warren, 1959;
Debnath et al., 1974 and Goel et al., 1985) were also done. The mucosa
was fleshed with saline and stomach pinned on a frog board and scored.
The number of ulcer and length of each ulcer were measured.
3.3.2.1. Biochemical estimation
Estimation of free and total acidity, mucosal glycoproteins and pepsin
in gastric juice (Hawk, 1947 and Szabo et al., 1985)
a) Collection of gastric juice
Gastric juice was collected from the pylorus-ligated rats. The gastric juice
thus collected was centrifuged and the volume of gastric juice as well pH
of gastric juice was measured. The sodium (Na+) and potassium (K
+) ion
concentration of gastric juice was carried out in flame photometer (Jeffery
et al., 1991). Then the gastric juice was subjected to bio-chemical
estimation as follows.
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b) Determination of free and total acidity in gastric juice (Hawk,
1947)
1 ml of gastric juice was pipette into a 100ml conical flask; add 10ml of
distilled water. Note the pH of this solution using with the help of pH -
Meter, then added 2 to 3 drops of Topfer’s reagent and triturated with
0.01N NaOH ( which was previously standardized with 0.01N of oxalic
acid ) until all traces of the red color disappears and the color of solution
was yellowish orange. The volume of alkali added was noted. The
volume corresponds to free acidity. Then 2 to 3 drops of phenolphthalein
solution was added and titration was continued until a definite red tinge
reappears. Again the total volume of added was noted. The volume
corresponds to total acidity.
Acidity was calculated by using the formula:-
Volume of NaOH x Normality of NaOH x 100
Acidity = meq/l/100g
0.1
c) Estimation of Sodium (Na+) and Potassium (K
+) ion concentration
in gastric juice (Jeffery et al., 1991)
The estimation for sodium and potassium ions was carried out using
Systronics mediflame 127 – flame photometer.
Preparation of stock solution
1. Sodium stock solution was prepared by dissolving 2.542 g NaCl
in 1 liter of distilled water. It contains 1mg Na per ml (i.e. 1000
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ppm). Stock solution was diluted to give four solutions
containing 10, 5, 2.5 and 1 ppm of sodium ions.
2. Potassium stock solution was prepared by dissolving 1.909 g KCl
in 1 liter of distilled water. It contains 1mg potassium per ml
(i.e. 1000 ppm). Stock solution was diluted to give four solutions
containing 20, 10, 5 and 2 ppm of potassium ions.
Procedure
For sodium and potassium, the flame intensity corresponding to the
concentration of stock solution was noted using appropriate filters. The
results were plotted in a graph. The flame intensity of the gastric juice was
noted. The concentration of sodium and potassium ions was calculated
from the graph. The results are expressed in terms of mg / l.
d) Estimation of pepsin (Debnath et al., 1974)
For each determination four tubes (1) and (2) containing 5ml of substrate,
(3) and (4) containing 10ml TCA was placed in the water bath at 37oC.
The gastric juice was mixed with an equal volume of HCl at pH 2.1,
warmed to 37oC and added 1ml of mixture to each tube (1) and (4),
incubated for 15 minutes and at the end mixed the contents of tube (1)
with tube (3) and allowed to stand in the bath for about 4 minutes.
Contents of tube (1) and tube (3) give test and contents of tube (2) and
tube (4) gives blank. Both the contents were filtered after 25-30 minutes,
2ml of filtrate was pippeted into 10 ml of NaOH, mixed by gentle
rotation, then 1ml of phenol was added and again mixed by gentle
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rotation. After 30 min, intensity of color was measure at 680 nm in
Systronics UV-VIS spectrophotometer- 180.
The difference between test and blank gives a measure of peptic activity.
As standard, mixed 2 ml of freshly prepared phenol solution containing 50
µg/ml with 10 ml of NaOH and 1 ml of phenol reagent was added. After
5-10 minutes, the color intensity was measured at 680 nm.
e) Estimation of total proteins (Lowry et al., 1951)
The dissolved protein in gastric juice was estimated in the alcoholic
precipitate obtained by adding 90 per cent alcohol with gastric juice in 9:1
ratio. Then 0.1 ml of alcoholic precipitate of gastric juice was dissolved
in 1 ml of 0.1N NaOH and from this 0.05 ml was taken in another test
tube, to this 4 ml of alkaline mixture was added and kept for 10 min.
Then 0.4 ml of phenol reagent was added and again 10 min was allowed
for color development. Reading was taken against blank prepared with
distilled water at 610 nm in Systronics UV-VIS spectrophotometer-180.
The protein content was calculated from standard curve prepared with
bovine albumin and was expressed in terms of µg/ ml of gastric juice.
f) Estimation of total carbohydrates (Goel et al., 1985)
The dissolved mucosubstance in gastric juice was estimated in the
alcoholic precipitate obtained by adding 90 per cent alcohol with gastric
juice in 9:1 ratio. Briefly the method consists of taking two aliquots of
gastric juice and treated as follows:
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A) To 1 ml of gastric juice, 9 ml of 90 per cent alcohol was added.
The mixture was kept for 10 minutes before it was centrifuged.
The supernatant was discarded. The precipitate was dissolved in
0.5 ml of 0.1N NaOH. To this 1.8 ml of 6N HCl was added. The
mixture was hydrolyzed in water bath at 100oC for 2 hours. The
hydrolysate was neutralized by 5N NaOH using phenolphthalein
as indicator and the volume was made up to 4.5 ml with distilled
water and using for the estimation of total hexoses, hexosamine
and fucose as described below.
B) To the other aliquot of 0.5 ml gastric juice, 4.5 ml of alcohol was
added. The mixture was shaken for 10 minutes and centrifuged
to obtain precipitate. The precipitate was dissolved in 0.5 ml of
0.1N H2SO4. This reconstituted solution was transferred to glass-
stoppered tubes and then hydrolyzed in a water bath at 100oC for
1 hour. After hydrolysis, the volume restored to 0.5 ml; 0.2 ml of
this hydrolyzes was used for the estimation of sialic acid.
After obtaining the concentration (µg/ml) of individual carbohydrates
namely hexose, hexosamine, fucose and sialic acid, the total carbohydrate
content was calculated by adding the concentration of individual
carbohydrates. Mucosubstances activity has been expressed as ratio of
total carbohydrates to total proteins.
g) Estimation of hexoses (Winzler, 1958)
To 0.4 ml of hydrolysate, 3.4 ml of Orcinol reagent was added. The
mixture was then heated in the boiling water bath 60oC for 15 minutes.
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This was then cooled under running tap water land intensity of the color
was read in Systronics UV-VIS spectrophotometer- 180 at 540 nm against
the blank by using distilled water instead of hydrolysate. Total hexoses
content was determined from the standard curve of D (+) – galactose-
mannose and has been expressed in µg/ml of gastric juice.
h) Estimation of hexosamine (Disch and Borentreund, 1983)
0.5 ml of the hydrolysate fraction was taken. To this 0.5 ml of acetyl-
acetone reagent was added. The mixture was heated in boiling water bath
at 60oC for 20 minutes, and then cooled under running tap water. 1.5 ml
of 90per cent alcohol was added and allowed for 30 minutes. The color
intensity was measured in Systronics UV-VIS spectrophotometer- 180 at
540 nm against blank prepared by using distilled water instead of
hydrolysate. Hexosamine content was determined from the standard
curve prepared by using D (+) – glucosamine hydrochloride and
concentration has been expressed in µg/ml of gastric juice.
i) Estimation of fucose (Dische and Schettles, 1948)
In this method, three test tubes were taken. In one tube 0.4 ml of distilled
water was taken to serve as control and in each of the other two 0.4 ml of
hydrolysate were taken. To all three tubes 1.8 ml of H2SO4: water (6:1)
added by keeping the test tubes in ice-cold water bath to prevent breakage
due to strong exothermic reaction. The mixture was then heated in boiling
water bath for exactly 3 min. The tubes were taken out and cooled. To the
blank and to one of the hydrolysate containing tube (unknown), 0.1 ml of
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cysteine reagent was added while cysteine regent was not added to the last
test tube containing the hydrolysate (unknown blank). It is then allowed
for 90 minutes to complete the reaction. The reading was taken in
Systronics UV-VIS spectrophotometer- 180 at 396 and 430 nm setting
zero with the distilled water. The optical density for the fucose in the
hydrolysate was calculated from the differences in the reading obtained at
396 and 430 nm and subtracting the values without cysteine. This was
read against standard curve prepared with D (+) – fucose content and was
expressed in terms of µg/ml of gastric juice.
(OD396 – OD430) unknown
– (OD396 – OD430) unknown blank
True optical Density =
(OD396 – OD430) water blank
j) Estimation of sialic acid (Warren, 1959)
To 0.5 ml of the hydrolysate in 0.1N H2SO4, 0.2 ml of sodium periodate
was added and mixed thoroughly by shaking. A time of 20 min was
allowed to elapse before addition of 1 ml of sodium arsenite solution to
this mixture. The brown color produced disappeared after shaking. Then 3
ml of thibarbituric acid was added and the mixture was heated in boiling
water bath for 15 minutes. After cooling the tubes, 4.5 ml of
cyclohexanone was added and through shaking was done for 15 seconds
till all the color was taken up by the cyclohexanone supernatant. The
mixture was centrifuged to get a clear pink layer of cyclohexanone. This
supernatant was pippeted out and intensity of color was measured in
Systronics UV-VIS spectrophotometer- 180 at 550 nm. The sialic acid
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content of the sample was determined from the standard curve of sialic
acid and has been expressed in terms of µg/ml of gastric juice.
3.3.3. Anti-inflammatory studies
The anti-inflammatory effects exerted by different doses of 50per cent alc.
extract of T. purpurea and T. villosa were evaluated by conducting the
following methods:
a) Carrageenan induced rat-paw oedema method.
b) Cotton-Pellet granuloma method.
a) Acute Anti-inflammatory Studies: Carrageenan Induced Rat-Paw
Oedema Method (Winter et al., 1962)
Carrageenan induced rat paw oedema method (Winter et. al., 1962) was
followed for acute anti-inflammatory study. The carrageenan, which was
used to provoke rat-paw oedema, was of Sigma grade with the following
particulars: carrageenan (gelatin, vegetable, Irish moss No. 1013; type-I)
believed to be a natural mixture of approximately 80per cent and 20 per
cent, κ and λ carrageenan respectively.
The rat paw oedema was provoked by sub-plantar injection of 0.1 ml 1 per
cent aqueous suspension of carrageenan in 0.9 per cent NaCl in the left
hind-paw. The hind-paw volume was measured by dipping the foot in the
mercury bath of the Plethysmograph – apparatus up to the anatomical
hairline on lateral malleolus (Goldenberg and Ilse, 1977). The
displacement of mercury made a rise in the colored fluid in the thin limb
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of the Plethysmograph. Before commencing the experiment, the initial
level of the colored fluid in the thin limb was adjusted to zero. So the
increase in reading directly gave the volume of the foot.
Wistar albino rats weighing about 125-150 g were separated into 8 groups
of 6 in each. The animals were allowed with food and water ad libitum
(Chaudari et al., 1977). Each animal in a group was recognised by a mark
of dye on the fur. The initial paw volume was measured and recorded. 1st
group of animals were served as negative control and given 0.75per cent
CMC (5 ml/Kg B.W.). 2nd
group of animals were served as positive
control, which were given Dichlofenac sodium (5 mg/Kg B.W.).The
extracts were administered orally one hour before commencing the
experiments at a dose of 5, 10, 20 mg/Kg body weight. 1per cent
carrageenan suspension in normal saline was prepared 1 hour before use.
0.1 ml was injected under the plantar aponeurosis. 3 hours after the
carrageenan injection the hind-paw volumes were recorded. Thus these
studies were rather indicative of acute anti-inflammatory effect.
The differences between the initial and final paw volume indicated the
oedema volume due to inflammation. The percentage inhibitions produced
by the drugs or principles were calculated which are directly indicative of
the anti-inflammatory activity exerted. The paw volume was measured at
0, 1, 3, 5 hours after injection of carrageenan. Drug pre-treatment was
given one hour before the injection of carrageenan. Mean increase in paw
was measured and per cent inhibition was calculated.
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100 – Odema Volume in the treated
% of Inhibition = X 100
Odema Volume in the Control
(Chu and Kovacs, 1977)
b) Sub Acute Anti-inflammatory studies: Cotton–Pellet Granuloma
Model
Sub-acute inflammation was produced by the method described by Winter
and Portar (1957) and Turner (1965). This method or technique rather
reveals the chronic (sub-acute) anti-inflammatory effects of a test
substance when it was administered for 7 days in seven divided doses
(Meir et al., 1950 and Winter and Porter, 1957). This method used here
was adopted from Sheth et al. (1972) with a slight modification of using
only male rats (Lassman et al., 1977)
Wistar albino rats weighing about 125 -150 g were divided into 6 groups
of 6 animals in each and were kept in separate cages one or two days
before commencing the experiments.
Using sensitive monoplane balance, which reads the weights to an
accuracy of 0.1 mg, Cotton pellets weighing exactly 10 mg each were
made. Each cotton pellet was rolled to an identical size. The pellets were
sterilized in an autoclave for 45 minutes under 15lbs/ inch 2 of steam
pressure.
The ventral portions of animals were shaved with scissors and swabbed
with alcohol. The animals were anaesthetized with anesthetic ether. 2 or
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1cm skin incisions were made one on the mid thorax and the other on the
mid abdominal region. Using a pair of blunt artery forceps, a small
channel was made bilaterally and one cotton pellet was placed in each
channel, 1 in the axilla, one in each and 2 in the groin, one in each
subcutaneously. All the air in the channels was removed by gentle
pressing the incisions were closed with the stitches.
Treatment to the control, positive control and the test groups were
initiated a soon as the animals were recovered from anesthesia and
continued daily for 7 days. The dosage, treatment and grouping were done
as described in acute anti-inflammatory studies.
On the 8th
day the animals were sacrificed and the cotton pellets were
removed. They were weighed immediately after separating the adjoining
and masking subcutaneous fascia. Then they were dried in a hot air oven
maintained at 60oC for at least 24 hours and weighed. By subtracting dry
weight of granuloma cotton with initial by weight, the percentage
inhibition of granuloma was calculated.
The weight of the cotton pellet before implantation was subtracted from
the weight of dried granuloma pellets. The values were expressed as mean
± SEM.
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3.3.4. Anti-histamine activity
3.3.4.1. Instruments
1. Kymograph and smoked drum
2. Frontal lever
3. L. stand
4. T-rod
5. X-blocks
6. Screw clip
7. Marriotte bottle
8. Rubber tubes
9. Tuberculin’s syringe 26 no needle
10. Droppers
11. Thermometer
12. Thread and needle (non-stretch nylon)
13. Surgical gloves
14. Acrylic board
15. Dissection Kit: Scalpel, Forceps, Scissors, Dissection Pins, Tape
16. Microscope
17. Petridish
3.3.4.2. Chemical used
Histamine was purchased from Sigma-Aldrich, St. Louis, USA. Other
chemicals used include Sodium chloride (NaCl), Potassium Chloride
(KCl) Calcium Chloride (CaCl2), Magnesium Chloride (MgCl2) Sodium
bicarbonate (NaHCO3), Sodium dihydrogen phosphate (NaH2PO4) and
Glucose from Hi-Media laboratory Pvt Ltd, Mumbai, India.
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3.3.4.3. Extract preparation
60 mg portion of both 50per cent alc. extracts were scraped off
from the bottom of the container and placed in a mortar and pestle. To this
added 2ml of distilled water and triturated well. This mixture was then
made up with 6ml of distilled water. This process gave a stock solution of
10 mg / ml. This solution was tested against the guinea pig ileum
preparation.
3.3.4.4. Histamine
A stock solution of 5µg/ml was made with tyrode solution. This
concentration was added to the bath and used as a standard drug.
3.3.4.5. Selection of animal’s species
The Healthy Adult male guinea pigs (460 g; Hartley strain) were
kept separately in individual polypropylene cage with stainless steel
hopper. The females were nulliparous and non-pregnant.
3.3.4.6. Housing and feeding
The temperature in the experimental animal room was 220 C ± 30
0 C.
Although the relative humidity was 30 per cent and preferably not
exceeding 70 per cent other than during room cleaning and the aim was
50-60 per cent. Lighting used artificially, the sequence being 12 hours
light and 12 hours dark. The animal was housed individually. For feeding,
conventional laboratory diets was used with an unlimited supply of
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drinking water. The study was performed under CPCSEA guidelines and
IAEC.
3.3.4.7. Preparation of animals
The animals were uniquely identified and kept in their cages for five days
prior to dosing for acclimatized to the laboratory conditions. During
acclimatization the animals were observed for ill health.
3.3.4.8. Perfusion Apparatus (Morgan et al., 1961)
In this system the tissue was suspended in a 2 X 20 cm (internal
dimensions) water-jacketed chamber with a coarse sintered glass filter
disk sealed into the lower portion. A mixture of moistened O2:CO2 (95:5)
was delivered by small diameter tubing to the lower portion of the
chamber by the aerator.
a) Methods
For the preparation of tissues, adult male guinea pigs (460 g; Hartley
strain) were killed by a blow to the head and exanguinated. The abdomen
region was opened and identified ileo-cecal junction. The lumen of ileum
were removed, the intact tissues and rubbed preparations in which the
blood had been removed by vigorously rubbing the luminal surface with
filter paper. A piece of ileum was excised (approximately 3-4 cm) by
using surgical suturing needle tied a thread at each end. One end of the
thread was tied to the hook of the aeration tube and the other to frontal
writing lever. The ileum was mounted in 30 ml organ bath under a load of
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500mg. The tissues were allowed to equilibrate for 90min in Tyrode
solution (composition in mM): NaCl 139.2, KCl 2.7, CaCl2 1.8, and
MgCl2 0.49, NaHCO3 11.9, NaH2PO4 0.4, glucose 5.5, pH 7.4 and gassed
with 5per cent CO2 and 95per cent O2 at 37°C. During the equilibration
period the bath fluid was exchanged every 10 min with fresh Tyrode
solution. All protocols were applied to both intact and rubbed
preparations. (Braunstein et al., 1988)
b) Drug injected order
Stabilization--Histamine (500ng) --Washing--Histamine (1µg)--Histamine
(2 µg) --Washing-Histamine (4µg) –Washing-- Sample (2mg) +Histamine
(500ng) --Washing--Sample (4mg) +Histamine (500ng)--Washing--
Sample (8mg) + Histamine (500ng) --Washing—
3.3.5. Analgesic activities
a) Acetic acid induced model (Chemical stimulus)
Mice were divided in to 8 groups (6). Group wise animal received T.
purpurea and T. villosa of 50per cent alc. extracts at a dose level of 5, 10,
20 control groups received normal saline and the reference group received
400 mg/Kg B.W. aspirin. Drug pretreatment was given one hr before i.p
injection of 0.6per centv/v acetic acid (10 ml/Kg B.W.). The severity of
pain response (writhing) was assessed by counting the number of writhes
(construction of abdomen) in mice (Koster et al., 1959). Number of
writhes per animal was counted during 15 minutes series beginning 5
minutes after injection of acetic acid.
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Analgesic activity was calculated as maximum possible effect (per cent
MPE) using the following relation.
100 x (Mean of writhes in control group-Mean of writhes
in treated group)
% MPE =
Mean of writhes in control group
b) Tail immersion method (Thermal stimulus)
All the mice were screened by expose to thermal stimulation. Those
showing positive response were divided into groups of six animals each.
Normal saline (control) 5, 10, 20 mg/Kg B.W. of the selected extracts
respectively and 1mg/Kg B.W. Fortral (Pentazocine) were administered
i.p. The time taken to withdraw the tail clearly out of the water was
considered as the reaction time with cut off time being 60 seconds. The
reading was immediately after administration of the drugs, and 60 min
later (Awe et al., 1998)
3.3.6. Anti-diarrhoeal studies (Castor oil induced)
The method described by Awounters et al. (1978) was followed.
Healthy albino rats of the either sex (160-190g) were divided into 8
groups of 6 animals each. They were fasted for 18hours prior to the test,
with free access to water. Group-1 received the vehicle (0.2 ml of 5per
cent Tween 80) and served as the control group. Groups 2 received
standard drug (Loperamide 3 mg/Kg B.W.), the third, fourth, fifth, sixth,
seventh and eight groups were given 50per cent alc. extract of the
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T. purpurea and T. villosa root at a dose of 5, 10 and 20 mg/Kg B.W.
respectively.
Thirty minutes after the drug treatment, each rat was administered
1ml of castor oil orally and housed separately in metabolic cages, with
special provision to separate urine and faeces. The diarrheal episodes were
observed for 4 hours. Percent inhibition of diarrhoea was calculated using
the mean stool weight. Anti-diarrheal activity was determined in terms of
percentage protection.
3.3.7. Antimicrobial studies
3.3.7.1. Media preparation
a) Bacterial medium (Muller Hinton Agar)
36 g of Muller Hinton Media (Hi-Media) was mixed with distilled water
and then sterilized in autoclave at 15 lb pressure for 15 minutes. The
sterilized media were poured into petri dishes. The solidified plates were
pored with 6 mm dia. cork borer. The plates with wells were used for the
antibacterial studies.
b) Fungal medium (Potato Dextrose Agar)
200 gm of potato slices were boiled with distilled water. The potato
infusion was used as water source of media preparation. 20 g of dextrose
was mixed with potato infusion and the pH was adjusted to 5.6. 20 g of
agar was added as a solidifying agent. These constituents were mixed and
autoclaved. Streptomycin sulphate of 50 µg/ml was added when pouring
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into petriplates at molten condition. The solidified plates were pored with
6mm dia. cork borer.
3.3.7.2. Test against standard controls
Commercially available antibiotic disc Oflaxacin (20 µg) and
Chloramphenicol (30 µg) were used as standard control for the entire test
microorganism. The sensitivity patterns were recorded and the readings
were interpreted according to the critical diameter given by National
Committee for Clinical Laboratory Standards (NCCLS, 1997).
3.3.7.3. Bacterial inoculums
Bacterial inoculums were prepared by inoculating a loopful of test
organisms in 5 ml nutrient broth and incubated at 370C for 5 to 8hrs till a
moderate turbidity was developed. The turbidity was matched with 0.5
McFarland standard (WHO drug information, 1993) and the culture was
diluted with sterile distilled water if necessary which corresponds to the
cell density of 1.5 x 108 (cfu/ml).
3.3.7.4. Fungal inoculums
The in vitro method proposed by National Committee for Clinical
Laboratory Standard for testing Molds (NCCLS, 1997) was followed for
the present study. The fungal stock inoculum suspensions were prepared
from two day old culture grown on PDA medium. The fungal colonies
were covered with 10 ml of sterile distilled water and suspensions were
made by gently probing the surface with the tip of the Pasteur pipette.
93
The resulting measure of conidia and hyphal fragments were withdrawn
and transferred to a sterile tube. Heavy particles were allowed to settle for
5-20 minutes, and the homogenous suspension were collected and mixed
with a vortex mixture. The density of these suspensions was adjusted with
a spectrophotometer at a wave length of 530 nm for 80-85per cent to
obtain the standard inoculums.
3.3.7.5. Antimicrobial assay (Bauer et al., 1996)
a) Antibacterial activity of the plant extract
Antibacterial and antifungal activity of the plant extract was tested using
well diffusion method (Bauer et al., 1996). The prepared culture plates
were inoculated with different selected strains of bacteria and fungi using
streak plate method. Wells were made on the agar surface with 6mm cork
borer. The extracts were loaded into the well using sterile syringe. The
plates were then kept in refrigerator for 15min. to allow diffusion of the
extracts into the media. The plates were incubated 24 hours at 37 ± 2oC
and 48 hours at 29 ± 1oC bacterial and fungal plates respectively. The
plates were observed for inhibition zone formation around the well. The
zone of inhibition was calculated by measuring the diameter of the
inhibition zone around the well (in mm) including the well diameter. The
readings were taken in three different fixed directions in all 3 replicates
and the average values were tabulated.
The 50 per cent alc. extract of T. purpurea and T. villosa was used
throughout the study. The extract of 10, 25 & 50 µ1 were tested against
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different bacterial pathogens such as Bacillus subtilis, Citrobacter
divergens, Klebsiella pneumonia, Staphylococcus aureus,
Staphylococcus epidermis for their antimicrobial activity. It was
demonstrated by well diffusion assay.
b) Antifungal activity of the plant extract
One ml volume of the inoculum was uniformly seeded on PDA plates
consist of 50 µg/ml of streptomycin sulphate. The in vitro antifungal
activity of the plant extracts were assessed by well diffusion method.
After 10 minutes, three wells of 6 mm size were made with the help of
cork borer and each concentration of the test drugs was aseptically loaded.
The plates were incubated at 29 ± 10C and were examined for the
appearance of fungal colony with inhibition zone.
The 50 per cent extract of 10, 25 & 50 µ1 were tested against different
fungal pathogens such as Aspergillus flavus, Aspergilus niger, Candida
albicans, Ganoderma lucidum, Mucor indicus for their antifungal
activity. It was demonstrated by well diffusion assay.
3.3.8. Statistical Analysis (Ghosh, 1984)
The raw data of the present study were subjected to simple statistical
analysis to draw meaningful interpretation and conclusion.
1. The standardization values of study drugs were expressed in
percentage (w/w).
2. For pharmacological studies:
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• Student’s ‘t’ test are computed for all the biochemical
estimations, to find out statistical significance at 1 per cent and
5 per cent probability levels.