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Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 118
CHAPTER 5: RESULTS
5.1. A) Standardization result of selected plant materials:
Table No 5: Standardization result of selected plant materials
Sr.
No. Parameters
Observation Observation Observation
Bauhinia
variegata roots
Tectona grandis
Bark
Schrebera
swietenioides
fruits
I.
Physical Tests
Nature Coarse Powder Coarse Powder Coarse Powder
Color Brown Brown Brown
Odor No Particular Odor No Particular
Odor Characteristics
Taste No Taste No Taste Bitter
II.
Extractive Value
Aqueous 23% 20% 12%
Alcohol 12% 14% 7%
Ether 4% 4% 3%
III. Loss on Drying 10% 13% 11%
IV.
Ash values
Total ash 11% 12% 8%
Acid insoluble ash 1% 2% 3%
Water soluble ash - - 0.8% 1%
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 119
5.1. B) Results of % yield of extracts:
Table No.: 6 Result of % yield of extracts
Sr.
No. Extracts
Nature of
Extract
Color of
Extract
% Yield
(w/w)
01 Bauhinia variegata root
aqueous extract
Semisolid
viscous Brown 14.8%
02 Bauhinia variegata root
ethanolic extract
Semisolid
viscous Brown 11.6 %
03 Bauhinia variegata root
petroleum ether extract
Semisolid
viscous Brown 4.5%
04 Tectona grandis bark
aqueous extract
Semisolid
viscous Brown 12.3%
05 Tectona grandis bark
ethanolic extract
Semisolid
viscous Brown 8.4 %
06 Tectona grandis bark
petroleum ether extract
Semisolid
viscous Brown 6.8%
07 Schrebera swietenioides
fruits aqueous extract
Semisolid
viscous Brown 14.2%
08 Schrebera swietenioides
fruits ethanolic extract
Semisolid
viscous Brown 10.2. %.
09 Schrebera swietenioides
fruits petroleum ether extract
Semisolid
viscous Brown 3.4%
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 120
5.1. C) PRELIMINARY PHYTOCHEMICAL INVESTIGATION OF -
A) Bauhinia variegata root different solvent extract
Table No.7
SR.
NO. NAME OF THE TEST
OBSERVATION
Aqueous
Extract
Ethanolic
Extract
Pet.Ether
Extract
I.
Test for Carbohydrates
a) Molisch's test
b) Fehling's test
c) Benedict's test
d) Barfoed's test
e) Cobalt chloride test
f) Test for non-reducing sugars
+
+
+
+
+
-
+
+
+
+
+
-
-
-
-
-
-
-
II.
Test for Proteins
a) Biuret test
b) Million's test
c) Xanthoprotein test
d) Test for protein containing
sulphur
e) Precipitation test
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
III.
Test for Steroids
a) Salkowski reaction
b) Liebermann – Burchard
c) Liberman's reaction
+
+
+
+
+
+
+
+
+
IV.
Test for Amino Acids
a) Ninhydrin test
b) Test for Tyrosine
c) Test for Tryptophan
-
-
-
-
-
-
-
-
-
V.
Test for Flavonoids
a) Shinoda test
b) Lead acetate test
c) Alkaline solution test
d) Ferric chloride test
+
+
+
+
+
+
+
+
-
-
-
-
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 121
SL.
NO. NAME OF THE TEST
OBSERVATION
Aqueous
Extract
Ethanolic
Extract
Pet. Ether
Extract
VI
Test for Alkaloids
a) Dragendroff's test
b) Mayer's test
c) Hager's test
d) Wagner's test
-
-
-
-
-
-
-
-
-
-
-
-
VII.
Test for Tannins
e) Lead acetate test
f) 5% FeCl3 test
g) Gelatin solution
h) Bromine water
-
-
-
-
-
-
-
-
-
-
-
-
VIII.
Test for Glycosides
a) Part A test
b) Part B test
c) Baljet's test
d) Legal's test
e) Keller Killani test
f) Libermann's test
g) Foam test
h) Hemolytic test
i) Fluorescence test
+
+
-
-
-
-
-
-
-
+
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
IX.
Test for Triterpenoids
a) Salkowaski test
b) Liebermann Burchardt test
-
-
-
-
-
-
The result of qualitative chemical investigation of the extracts has indicated the
presence of “Sterols” in aqueous, ethanolic and petroleum ether extract, “Flavanoidal
glycosides” in ethanolic and petroleum ether extract and “Carbohydrates” in aqueous and
ethanolic extract of Bauhinia variegata roots.
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 122
Preliminary phytochemical investigation of-
B) Tectona grandis bark different solvent extracts
Table No. 8
SR.
NO. NAME OF THE TEST
OBSERVATION
Aqueous
Extract
Ethanolic
Extract
Pet. Ether
Extract
I.
Test for Carbohydrates
a) Molisch's test
b) Fehling's test
c) Benedict's test
d) Barfoed's test
e) Cobalt chloride test
f) Test for non-reducing sugars
+
+
+
+
+
-
+
+
+
+
+
-
-
-
-
-
-
-
II.
Test for Proteins
a) Biuret test
b) Million's test
c) Xanthoprotein test
d) Test for protein containing
sulphur
e) Precipitation test
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
III.
Test for Steroids
a) Salkowski reaction
b) Liebermann – Burchard
c) Liberman's reaction
+
+
+
+
+
+
+
+
+
IV.
Test for Amino Acids
a) Ninhydrin test
b) Test for Tyrosine
c) Test for Tryptophan
-
-
-
-
-
-
-
-
-
V.
Test for Flavonoids
a) Shinoda test
b) Lead acetate test
c) Alkaline solution test
d) Ferric chloride test
-
-
-
-
-
-
-
-
-
-
-
-
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 123
SR.
NO. NAME OF THE TEST
OBSERVATION
Aqueous
Extract
Ethanolic
Extract
Pet. Ether
Extract
VI
Test for Alkaloids
a) Dragendroff's test
b) Mayer's test
c) Hager's test
d) Wagner's test
-
-
-
-
-
-
-
-
-
-
-
-
VII.
Test for Tannins
a) Lead acetate test
b) 5% FeCl3 test
c) Gelatin solution
d) Bromine water
+
+
+
+
-
-
-
-
-
-
-
-
VIII.
Test for Glycosides
a) Part A test
b) Part B test
c) Baljet's test
d) Legal's test
e) Keller Killani test
f) Libermann's test
g) Foam test
h) Hemolytic test
i) Fluorescence test
+
+
-
-
-
-
-
-
-
+
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
IX.
Test for Triterpenoids
a) Salkowaski test
b) Liebermann Burchardt test
-
-
-
-
-
-
The result of qualitative chemical investigation of the extracts has indicated the
presence of “Sterols” in aqueous, ethanolic and petroleum ether extract, “Glycosides and
Cabohydrates” in aqueous and ethanolic extract whereas “Tannins” in aqueous extract of
Tectona grandis bark.
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 124
Preliminary phytochemical investigation of
c) Schrebera swietenioides root different extract
Table no. 9
SR.
NO.
NAME OF THE TEST
OBSERVATION
Aqueous
Extract
Ethanolic
Extract
Pet. Ether
Extract
I.
Test for Carbohydrates
a) Molisch's test
b) Fehling's test
c) Benedict's test
d) Barfoed's test
e) Cobalt chloride test
f) Test for non-reducing sugars
+
+
+
+
+
+
-
-
-
-
-
-
-
-
-
-
-
-
II.
Test for Proteins
a) Biuret test
b) Million's test
c) Xanthoprotein test
d) Test for protein containing
sulphur
e) Precipitation test
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
III.
Test for Steroids
a) Salkowski reaction
b) Liebermann – Burchard
c) Liberman's reaction
-
-
-
-
-
-
-
-
-
IV.
Test for Amino Acids
a) Ninhydrin test
b) Test for Tyrosine
c) Test for Tryptophan
-
-
-
-
-
-
-
-
-
V.
Test for Flavonoids
a) Shinoda test
b) Lead acetate test
c) Alkaline solution test
d) Ferric chloride test
-
-
-
-
-
-
-
-
-
-
-
-
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 125
SR.
NO.
NAME OF THE TEST
OBSERVATION
Aqueous
Extract
Ethanolic
Extract
Pet. Ether
Extract
VI
Test for Alkaloids
a) Dragendroff's test
b) Mayer's test
c) Hager's test
d) Wagner's test
-
-
-
-
-
-
-
-
-
-
-
-
VII.
Test for Tannins
a) Lead acetate test
b) 5% FeCl3 test
c) Gelatin solution
d) Bromine water
-
-
-
-
-
-
-
-
-
-
-
-
VIII
.
Test for Glycosides
a) Part A test
b) Part B test
c) Baljet's test
d) Legal's test
e) Keller Killani test
f) Libermann's test
g) Foam test
h) Hemolytic test
i) Fluorescence test
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
IX.
Test for Triterpenoids
a) Salkowaski test
b) Liebermann Burchardt test
+
+
+
+
-
-
The result of qualitative chemical investigation of the extracts has indicated the
presence of "Triterpenoids" in ethanolic and aqueous extract whereas
“Carbohydrates” in aqueous extract of Schrebera swietenioides fruit.
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 126
5.2 ANTIOXIDANT ACTIVITY
Antioxidant activity / free radical scavenging activity of selected medicinal plants
different solvent extracts were measured by using DPPH-stable free radical. In the
presence of an antioxidant which can donate an electron to DPPH, the purple color which
is typical to free DPPH radical decays and the change in absorbency was observed
spectrophotometrically at 517 nm. For DPPH assay, the method of Blois was adopted.
The DPPH scavenging capacity of the extract was compared with BHT (Butylated
hydroxytolune). All determinations were carried out three times, and in triplicate.
Percentage inhibition was calculated as DPPH radical scavenging activity.
From the obtained DPPH radical Scavenging effect (%), the IC50 value were
calculated, which represents the concentration of the scavenging compound that caused
50% neutralization. The observation of absorbance of selected medicinal plants different
solvent extracts and percentage scavenging capacity with IC50 value of various extracts
and standard are given in Table No-10 and 11 and as a histogram in figure No 13, 14 and
15.
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 127
Table No. 10
Evaluation of selected medicinal plants different solvent extract for free radical scavenging activity on DPPH radical
I) Absorbance of different solvent extracts (Values are mean of 3 replicates)
Conc.
(µg/ml)
Aq.
B.V.
Eth.
B.V.
Pet. Eth
B.V.
Aq.
S.S.
Eth.
S.S.
Pet. Eth
S.S.
Aq.
T.G.
Eth.
T.G.
Pet. Eth
T.G.
Stand.
BHT
1000 0.173 0.123 0.236 0.134 0.177 0.223 0.119 0.103 0.209 0.024
500 0.268 0.214 0.367 0.262 0.223 0.360 0.243 0.197 0.313 0.014
250 0.397 0.303 0.403 0.376 0.304 0.385 0.304 0.238 0.360 0.049
100 0.436 0.398 0.428 0.434 0.381 0.402 0.363 0.332 0.402 0.203
50 0.494 0.438 0.463 0.473 0.402 0.461 0.407 0.401 0.428 0.311
10 0.523 0.492 0.471 0.501 0.483 0.482 0.497 0.463 0.463 0.402
Blank 0.539 0.503 0.499 0.539 0.503 0.499 0.539 0.503 0.499 0.503
Where- Aq. B.V. : Aqueous Bauhinia variegata extract
Eth. B.V. : Ethanolic Bauhinia variegata extract
Pet. Eth B.V. : Petroleum ether Bauhinia variegata extract
Aq.S.S. : Aqueous Schrebera swietenioides extract
Eth. S.S. : Ethanolic Schrebera swietenioides extract
Pet.Eth S.S. : Petroleum ether Schrebera swietenioides extract
Aq. T.G. : Aqueous Tectona grandis extract
Eth. T.G. : Ethanolic Tectona grandis extract
Pet. Eth T.G. : Petroleum ether Tectona grandis extract
BHT : Butylated hydroxytoluene
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 128
Table No.11
Evaluation of selected medicinal plants different solvent extract for free radical scavenging activity on DPPH radical
II) % DPPH radical scavenging activity ( INHIBITION %) (Values are mean of 3 replicates)
Conc.
(µg/ml)
Aq.
B.V.
Eth.
B.V.
Pet. Eth
B.V.
Aq.
S.S.
Eth.
S.S.
Pet. Eth
S.S.
Aq.
T.G.
Eth.
T.G.
Pet. Eth
T.G.
Stand.
BHT
1000 67.90% 75.54% 52.70% 75.13% 64.18% 55.31% 77.92% 79.52% 58.11% 95.22%
500 50.27% 67.45% 26.45% 51.39% 55.66% 27.85% 54.91% 60.83% 37.27% 97.21%
250 26.34% 59.76% 19.23% 30.24% 39.56% 22.84% 43.59% 52.68% 27.85% 90.25%
100 19.10% 20.87% 14.22% 19.48% 24.25% 19.43% 32.65% 33.93% 19.43% 47.71%
50 08.34% 12.92% 07.21% 12.24% 20.07% 07.16% 24.48% 20.01% 14.22% 38.17%
10 02.96% 02.18% 05.61% 07.05% 03.97% 03.40% 07.79% 07.95% 07.21% 20.07%
IC 50 490 µg
/ml
211 µg
/ml
920 µg
/ml
473 µg
/ml
423 µg
/ml
887 µg
/ml
417 µg
/ml
238 µg
/ml
897 µg
/ml
107 µg
/ml
Where- Aq. B.V. : Aqueous Bauhinia variegata extract
Eth. B.V. : Ethanolic Bauhinia variegata extract
Pet. Eth B.V. : Petroleum ether Bauhinia variegata extract
Aq.S.S. : Aqueous Schrebera swietenioides extract
Eth. S.S. : Ethanolic Schrebera swietenioides extract
Pet.Eth S.S. : Petroleum ether Schrebera swietenioides extract
Aq. T.G. : Aqueous Tectona grandis extract
Eth. T.G. : Ethanolic Tectona grandis extract
Pet. Eth T.G. : Petroleum ether Tectona grandis extract
BHT : Butylated hydroxytoluene
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 129
Fig. No. 13.Histogram showing % anti radical activity of different solvent extracts
of Bauhinia variegata on DPPH antioxidant model
Figure No. 14.Histogram showing % anti radical activity of different solvent
extracts of Tectona grandis on DPPH antioxidant model
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 130
Figure No.15.Histogram showing % anti radical activity of different solvent
extracts of Schrebera swietenioides on DPPH antioxidant model
Table No.12: Comparison of IC50 values of selected medicinal plants different
solvent extract for % antiradical activity on DPPH radical
IC 50 value (μg /ml )
Solvent extract
Bauhinia
variegata root
Tectona grandis
bark
Schrebera
swietenioides fruit
Aqueous
490 µg /ml 417 µg /ml 473 µg /ml
Ethanolic
211 µg /ml 238 µg /ml 423 µg /ml
Petroleum
ether
920 µg /ml 897 µg /ml 887 µg /ml
The above data, (Table No. 12) indicates that antioxidant activity exerted by ethanolic
extract of these selected medicinal plants exhibited maximum percentage inhibition of
free radicals in less amount of concentration.
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 131
5.3 ACUTE ORAL TOXICITY STUDIES:
The acute oral toxicity study was carried out on selected medicinal plants extract
i.e. ethanolic extract of Bauhinia variegata root, Tectona grandis bark and Schrebera
swietenioides fruit on albino rats following OECD guidelines (No. 423). Selected
medicinal plants were found safe (no mortality) even when given at the dose of 2000
mg/kg body weight with no signs of acute oral toxicity at respective dose.
Hence, 1/10th
of this lethal dose was taken as effective dose (therapeutic dose) for
subsequent antidiabetic and hepatoprotective activity i.e., 200 mg/kg b. w. p. o.
5.4 ANTIDIABETIC ACTIVITY:
Screening of antidiabetic activity of these selected medicinal plant extracts was
done in rats by conducting glucose tolerance test (GTT) study and evaluating their
effects (Single dose and Multidose treatment study) on blood glucose level, serum lipid
profiles and histology of pancreas in alloxan diabetic rats.
5.4.1 Oral glucose tolerance test (OGTT)
All extracts significantly (P<0.01) improved the glucose tolerance test up to 4 h
(Table No 13 and Figure No 16). Ethanolic extract of Bauhinia variegata root showing
15 and 29%, ethanolic extract of Tectona grandis bark showing 15 and 20% whereas
ethanolic extract of Schrebera swietenioides fruit showing approximately 22 and 14%
reduction in blood glucose level from control values at the end of 2 h and 4 h
respectively. The Glibenclamide also improved the glucose tolerance test up to 4 h.
5.4.2 The effect of different extracts on the blood glucose levels in alloxan-diabetic
rats (Single dose treatment/Acute study)
The administrations of selected medicinal plant extracts were found to reduce
blood glucose level in alloxan induced diabetic rats in single dose study. These extracts
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 132
exhibited significant (P<0.05) antihyperglycemic efficacy from 1 h after its oral
administration, the effect lasted up to 6 hrs when compared with normal rats and diabetic
control rats. Blood glucose lowering potential percentage of Bauhinia variegata root
ethanolic extract was 21%, Tectona grandis bark ethanolic extract was 16% and
Schrebera swietenioides fruit ethanolic extract was 13% at 6 hr after administration,
while the standard drug Glibenclamide (2.5 mg/kg) caused 20% reduction of blood
glucose at the same time interval when compared with diabetic control rats (Table No. 14
and figure No. 17).
5.4.3 Multidose treatment / Sub acute study:
A) The effect of different extracts on blood glucose levels in alloxan induced
diabetic rats
During the Multidose treatment period, administration of ethanolic extract of
Bauhinia variegata root (200 mg/kg/day) caused a significant decrease of 29%, 38% and
52%, Tectona grandis bark (200 mg/kg/day) caused a significant decrease of 28%, 38%
and 51% whereas Schrebera swietenioides fruit (200 mg/kg/day) caused a significant
decrease of 19%, 24% and 37% in blood glucose levels on 5th
, 10th
and 15th
day
intervals, respectively, when compared with diabetic control group. When comparison
between initial blood glucose level and 15th
day blood glucose level was made in the
same group animals, it was found that, there is a reduction of 55.50% in ethanolic extract
of Bauhinia variegata root, 53.91% in ethanolic extract of Tectona grandis bark, 42.19%
in ethanolic extract of Schrebera swietenioides fruit whereas 47.10% reduction was
observed in standard drug- Glibenclamide (2.5 mg/kg) treated animals (Table No. 15 and
figure No. 18).
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 133
B) The effect of different extracts on body weight measurement in alloxan induced
diabetic rats
The change in the body weight of animals in different groups is compared at the
end of study on 15th
day. Normal control animals were showed increase in their body
weight by 6.65%, but diabetic control rats showed significant reduction in the body
weight i.e. 28.74%, whereas Bauhinia variegata root extract (200 mg/kg/day), Tectona
grandis bark extract (200 mg/kg/day) and standard drug-Glibenclamide (2.5 mg/kg)
treated group shown highly significant prevention of reduction in body weight (P <
0.001) when compared with initial body weight of same animals (Table No. 16).
C) The effect of different extracts on serum lipid profiles in alloxan induced
diabetic rats
Observations of hypoglycemic effect of ethanolic extract of Bauhinia variegata
root (200 mg/kg/day), Tectona grandis bark (200 mg/kg/day) and Schrebera
swietenioides fruit (200 mg/kg/day) in alloxan-induced hyperglycemic rats serum lipid
profiles is shown in Table No. 17 and Figure No. 19. These Serum levels were measured
on 15th
day of treatment with different extract treated groups. These levels were
expressed as mg/dl and were given in mean S.E.M.
1. Triglyceride (TG):
There was a significant increase in triglyceride in the diabetic control animals
(123.5±2.4 mg/dl) compared to that of normal control animals (85.25±1.5 mg/dl).
Triglyceride level in ethanolic extract of Bauhinia variegata root 200 mg/kg (94.00±9.3
mg/dl) showed a highly significant (p<0.01) reduction; Tectona grandis bark 200 mg/kg
(99.00±4.7 mg/dl) showed a significant (p<0.05) reduction whereas Schrebera
swietenioides fruit 200 mg/kg (104.0±3.0 mg/dl) showed slight reduction in triglyceride
level when compared to that of diabetic control triglyceride level (123.5±2.4 mg/dl).
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 134
Glibenclamide (2.5 mg/kg) (92.25±8.0 mg/dl) also showed highly significant (p<0.01)
reduction in the triglyceride level in the serum compare to that of diabetic control
animals (123.5±2.4 mg/dl).
2. High Density Lipoprotein Cholesterol: (HDL-C):
There was a significant decrease in HDL cholesterol in the diabetic control
animals (30.00±1.4 mg/dl) compared to that of normal control animals (37.00±1.5
mg/dl). HDL cholesterol in ethanolic extract of Bauhinia variegata root 200 mg/kg
(45.66±1.5 mg/dl) showed a highly significant (p<0.01) rise; Tectona grandis bark
200 mg/kg (40.83±1.9 mg/dl) also shown a highly significant (p<0.01) rise whereas
Schrebera swietenioides fruit 200 mg/kg (35.23±2.1 mg/dl) showed a significant
(p<0.05) rise in HDL cholesterol level when compared to that of diabetic control
HDL cholesterol level (30.00±1.4 mg/dl). Glibenclamide (2.5 mg/kg) (51.50±1.9
mg/dl) also showed highly significant (p<0.01) rise in the HDL cholesterol level in
the serum compare to that of diabetic control animals (30.00±1.4 mg/dl).
3. Very Low Density Lipoprotein (VLDL-C):
There was a significant increase in very low density lipoprotein cholesterol level
in the diabetic control animals (27.75±1.3 mg/dl) compared to that of normal control
animals (19.00±0.73 mg/dl). Very low density lipoprotein cholesterol level is
reduced significantly (p<0.05) only in ethanolic extract of Bauhinia variegata root
200 mg/kg. Glibenclamide (2.5 mg/kg) (21.00±2.6 mg/dl) also showed significant
(p<0.05) reduction in very low density lipoprotein cholesterol level in the serum
compare to that of diabetic control animals (27.75±1.3 mg/dl).
4. Low Density Lipoprotein (LDL-C):
There was a significant increase in low density lipoprotein cholesterol levels in
the diabetic control animals (34.42±3.7 mg/dl) compared to that of normal control
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 135
animals (16.25±0.4 mg/dl). Low density lipoprotein cholesterol levels in ethanolic
extract of Bauhinia variegata root 200 mg/kg (23.00±4.9 mg/dl) showed a significant
(p<0.05) reduction, Tectona grandis bark 200 mg/kg (22.00±5.3 mg/dl) also shown a
significant (p<0.05) reduction in low density lipoprotein cholesterol levels when
compared to that of diabetic control low density lipoprotein cholesterol levels
(34.42±3.7 mg/dl). Glibenclamide (2.5 mg/kg) (19.00±1.9 mg/dl) also showed
significant (p<0.05) rise in the low density lipoprotein cholesterol levels in the serum
compare to that of diabetic control animals (34.42±3.7 mg/dl).
5. Total cholesterol (TC):
There was a significant increase in total cholesterol levels in the diabetic control
animals (83.00±2.00 mg/dl) compared to that of normal control animals (55.50±1.6
mg/dl). Total cholesterol levels in ethanolic extract of Bauhinia variegata root 200
mg/kg (58.50±2.7 mg/dl) showed a highly significant (p<0.01) reduction whereas
Tectona grandis bark 200 mg/kg (61.00±5.5 mg/dl) also showed a highly significant
(p<0.01) reduction in total cholesterol level when compared to that of diabetic
control total cholesterol level (83.00±2.00 mg/dl). Glibenclamide (2.5 mg/kg)
(58.50±2.7 mg/dl) also showed highly significant (p<0.01) reduction in the total
cholesterol level in the serum compare to that of diabetic control animals (83.00±2.00
mg/dl).
D) The effect of different extracts on histopathology of pancreas in alloxan induced
diabetic rats
Histopathological examination of pancreas of these animals showed comparable
regeneration of Islets of Langerhans and β cells by ethanolic extract of Bauhinia
variegata, Tectona grandis, Schrebera swietenioides plant and Glibenclamide
standard drug, which were earlier, necroses by alloxan.
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Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 136
Group I: Normal control (1% gum acacia): Normal acini and normal cellular
population of the islets of Langerhans. (Figure No. 20)
Group II: Diabetic control: Extensive damage of the islets of Langerhans and
they appeared to be irregular (Figure No. 21)
Group III: Glibenclamide (2.5 mg/kg): Moderate expansion of cellular
population and size of islet cells (Figure No. 22)
Group IV: Bauhinia variegata ethanolic extract (200 mg/kg): Partial
restoration of normal cellular population and size of islet cells (Figure No. 23)
Group V: Tectona grandis ethanolic extract (200 mg/kg): Partial restoration of
normal cellular population and size of islet cells (Figure No. 24)
Group VI: Schrebera swietenioides ethanolic extract (200 mg/kg): Partial
restoration of normal cellular population and size of islet cells (Figure No. 25)
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 137
Table No.13
The effect of different extracts on the blood glucose levels in glucose loaded rats (OGTT)
Exp.Group
(n= 6) Treatment
Blood glucose concentration (mg/dl) (mean ± S.E.M.)
In fasting 30 min 60 min 120 min 240 min
I Normal control
(1% gum acacia) 93.50 ± 3.4 162.2 ± 4.5 163.3 ±2.2 160.5 ± 3.9 131.3 ± 3.8
II Glibenclamide (2.5 mg/kg) 95.00 ±2.8 142.0 ±1.7 * 145.5 ±1.1 * 138.0 ±2.8 * 99.50 ±1.6 *
III Bauhinia variegata ethanolic
extract (200 mg/kg) 91.00 ±5.2 137.0 ±3.2 * 136.5 ±2.3 * 117.5 ±4.2 * 97.83 ±2.5 *
IV Tectona grandis ethanolic
extract (200 mg/kg) 87.50 ±4.2 118.5 ±3.1 * 107.2 ±1.6 * 101.0 ±1.7 * 95.33 ±2.7 *
V Schrebera swietenioides
ethanolic extract (200 mg/kg) 98.33 ±14.2 131.0 ±2.7 * 132.7 ±3.5 * 125.7 ±3.2 * 114.0 ±1.4 *
*P < 0.01. Significant, compared to normal control
n= no of animals in each group
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 138
Fig. No. 16 Histogram showing the effect of extracts on the blood glucose levels in
glucose loaded rats (GTT)
Where, BVE-Bauhinia variegata roots ethanolic extract
TGE- Tectona grandis bark ethanolic extract and
SSE-Schrebera swietenioides fruit ethanolic extract.
The effect of extracts on the blood glucose
levels in glucose loaded rats (GTT)
0
20
40
60
80
100
120
140
160
180
0 min 30 min 60 min 120 min 240 min
Time (Min)
BG
L (
mg
/dl)
Normal control
Glibenclamide (2.5
mg/kg)
BVE (200 mg/kg)
TGE(200 mg/kg)
SSE (200 mg/kg)
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 139
Table No. 14:
The effect of different extracts on the blood glucose levels in alloxan-diabetic rats (Single dose treatment/Acute study)
Exp.
Group
(n= 6)
Treatment
Blood glucose concentration (mg/dl) (mean ± S.E.M.)
0 h 30 min 60 min 120 min 240 min 360 min
I Normal control
(1% gum acacia) 85.25±2.6 86.25±1.8
# # 87.50±2.0
# # 89.75±1.8
# # 92.25±2.4
# # 93.50±3.5
# #
II Diabetic control 287.5±5.0 294.8±5.1* 294.5±5.2* 290.5±4.1* 292.0±3.5* 293.3±4.1*
III Glibenclamide (2.5 mg/kg) 297.3±7.2 287.0±8.0* 274.0±9.7* 258.8±12.9* # 252.0±13.1*
# # 235.8±12.6*
# #
IV Bauhinia variegata ethanolic
extract (200 mg/kg) 275.5±7.7 270.0±5.9*
# 263.0±4.1*
# # 262.0±5.3* 248.3±6.1*
# # 233.8±7.2*
# #
V Tectona grandis ethanolic
extract (200 mg/kg) 273.3±5.0 268.8±5.5*
# 259.8±6.0*
# # 262.0±6.2* 254.5±6.6*
# # 246.8±6.3*
# #
VI Schrebera swietenioides
ethanolic extract (200 mg/kg) 279.5±6.3 271.0±6.5*
# 265.8±8.1*
# 262.8±9.9* 258.8±9.8*
# 255.0±10.8*
#
*P < 0.01 Significant, compared to normal, #P < 0.05 &
# #P < 0.01 Significant, compared to diabetic control.
n= no of animals in each group
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 140
Fig. No.17.Histogram showing the effect of the different extracts on the blood
glucose levels in alloxan-diabetic rats (Single dose treatment/Acute study)
Where, BVE-Bauhinia variegata roots ethanolic extract
TGE- Tectona grandis bark ethanolic extract and
SSE-Schrebera swietenioides fruit ethanolic extract.
The effect of different extracts on the blood glucose
levels in alloxan-diabetic rats (single dose
treatment/acute study)
0
50
100
150
200
250
300
350
0hour 30 min 60 min 120
min
240
min
360
minTime (Min)
BG
L(m
g/d
l)
Normal control
Diabetic control
Glibenclamide (2.5 mg/kg)
BVE (200 mg/kg)
TGE (200 mg/kg)
SSE (200 mg/kg)
Chapter 5 Results
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Table No. 15:
The effect of different extract on the blood glucose levels in alloxan-diabetic rats (Multidose treatment /Sub acute study)
Exp.Group
(n= 6)
Treatment
Fasting blood glucose concentration (mg/dl) (mean ± S.E.M.)
0th
Day 5th
Day 10th
Day 15th
Day
I Normal control
(1% gum acacia) 85.25±2.6 85.75±1.8
# # 85.25±1.3
# # 87.25±1.1
# #
II Diabetic control 287.5±5.2 274.3±7.1** 264.3±5.3** 255.8±5.1**
III Glibenclamide (2.5 mg/kg) 299.3±6.9 217.3±14.3** # #
188.3±13.8** # #
158.3±15.3** # #
IV Bauhinia variegata ethanolic
extract (200 mg/kg) 275.5±7.7 194.2±16.0**
# # 164.4±10.6**
# # 122.6±9.4*
# #
V Tectona grandis ethanolic
extract (200 mg/kg) 270.3±6.0 199.0±5.2**
# # 164.5±9.4* *
# # 124.5±7.3*
# #
VI Schrebera swietenioides
ethanolic extract (200 mg/kg) 277.3±6.7 223.3±12.5**
# 200.3±6.5**
# # 160.3±6.4**
# #
*P < 0.05 & **P < 0.01 Significant, compared to normal, #P < 0.05 &
# #P < 0.01 Significant, compared to diabetic control.
n= no of animals in each group
Chapter 5 Results
Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 142
Figure No. 18. Histogram showing the effect of different extracts on the blood
glucose levels in alloxan-diabetic rats (Multidose treatment/sub acute study)
Where, BVE-Bauhinia variegata roots ethanolic extract
TGE- Tectona grandis bark ethanolic extract and
SSE-Schrebera swietenioides fruit ethanolic extract.
The effect of different extract on the blood glucose levels in
alloxan-diabetic rats (Multidose treatment /sub acute study)
0
50
100
150
200
250
300
350
0th Day 5th Day 10th Day 15th DayDays
BG
L(m
g/d
l)
Normal control
Diabetic control
Glibenclamide (2.5 mg/kg)
BVE (200 mg/kg)
TGE (200 mg/kg)
SSE (200 mg/kg)
Chapter 5 Results
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Table No. 16:
The effect of different extracts on the body weight measurement in alloxan-diabetic rats (Multidose treatment /Sub acute study)
Exp.
Group
(n= 6)
Treatment
Body weight of the animal (gm) (mean ± S.E.M.) % Change in
body weight 0th
Day 5th
Day 10th
Day 15th
Day
I Normal control
(1% gum acacia) 195.4 ±2.75 200.3 ± 5.31.8 204.8 ± 2.8 208.4 ±2.89 6.65
II Diabetic control 203.50 ± 2.8 172.00 ± 2.59 158.00 ± 2.51 145.00 ± 1.72 - 28.74
III Glibenclamide (2.5 mg/kg) 206.5 ± 2.84 203.00 ± 2.64 # 196.50 ± 2.02
# 192.00 ± 1.90
# - 7.02
IV Bauhinia variegata ethanolic
extract (200 mg/kg) 206.50 ± 2.4 200.00 ± 2.07
# 194.00 ± 1.69
# 187.0 ± 1.50
# - 9.44
V Tectona grandis ethanolic
extract (200 mg/kg) 205.50 ± 2.39 198.00 ± 2.15
# 191.50 ± 1.89
# 182.50 ± 1.24
# - 11.19
VI Schrebera swietenioides
ethanolic extract (200 mg/kg) 206.00 ± 2.79 178.00 ± 2.41 163.00 ± 2.32 152.00 ± 1.72 - 26.21
#P < 0.001 Significant,compared to diabetic control.
n= no of animals in each group
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Table No. 17:
The effect of different extracts on the serum profile in alloxan -diabetic rats after 15 days of treatment
Exp.Group
(n= 6) Treatment TGL mg/dl HDL mg/dl VLDL mg/dl LDL mg/dl
Total Cholesterol
mg/dl
I Normal control
(1% gum acacia) 85.25±1.5 ** 37.00±1.5 * 19.00±0.73 * 16.25±0.4 * 55.50±1.6 **
II Diabetic control 123.5±2.4 30.00±1.4 27.75±1.3 34.42±3.7 83.00±2.0
III Glibenclamide (2.5 mg/kg) 92.25±8.0 ** 51.50±1.9 ** 21.00±2.6 * 19.00±1.9 * 58.50±2.7 **
IV Bauhinia variegata ethanolic
extract (200 mg/kg) 94.00±9.3 ** 45.66±1.5 ** 22.00±2.9 * 23.00±4.9 * 60.00±6.1 **
V Tectona grandis ethanolic
extract (200 mg/kg) 99.00±4.7 * 40.83±1.9 ** 25.00±1.9 22.00±5.3 * 61.00±5.5 **
VI Schrebera swietenioides
ethanolic extract (200 mg/kg) 104.0±3.0 35.23±2.1 * 23.00±0.73 24.00±5.3 68.00±5.1
*P < 0.05 & **P < 0.01 Significant, compared to diabetic control.
Results are expressed as mean ± S.E.M., n= no of animals in each group.
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Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 145
Fig. No. 19.Histogram showing the effect of different extracts on the serum profile
in alloxan-diabetic rats after 15 days of treatment
Where, BVE-Bauhinia variegata roots ethanolic extract
TGE- Tectona grandis bark ethanolic extract and
SSE-Schrebera swietenioides fruit ethanolic extract.
The effect of different extracts on the serum profile in
alloxan -diabetic rats after 15 days of treatment
0
20
40
60
80
100
120
140
TGL HDL VLDL LDL TC Serum Profiles
mg
/dl
Normal control
Diabetic control
Glibenclamide (2.5
mg/kg)
BVE (200 mg/kg)
TGE (200 mg/kg)
SSE (200 mg/kg)
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Histopathology of Pancreas:
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5.5 HEPATOPROTECTIVE ACTIVITY STUDY
The hepatic enzymes viz; SGPT, SGOT, SALP and Total Bilirubin level and histology of
livers were assessed in groups of rats treated with Normal control group (1% gum
acacia), CCl4 control, CCl4 + Standard drug-Liv 52 Syrup (1 ml/kg), CCl
4 + Bauhinia
variegata root ethanolic extract (200 mg/kg), CCl4
+ Tectona grandis bark ethanolic
extract (200 mg/kg) and CCl4
+ Schrebera swietenioides fruit ethanolic extract (200
mg/kg).
5.5.1 The effect of different extracts on serum enzymes and total bilirubin level in
CCl4 toxicated rats
Observations of effect of ethanolic extract of Bauhinia variegata root (200
mg/kg/day), Tectona grandis bark (200 mg/kg/day) and Schrebera swietenioides fruit
(200 mg/kg/day) on serum enzymes and total Bilirubin level in CCl4 toxicated rats is
shown in Table No.18. These Serum levels and total bilirubin level were measured on
10th
day of treatment with different extract treated groups.
1. SGPT: These levels were expressed as U/L and were given in mean S.E.M.
There was a significant increase in SGPT enzyme level in the CCl4control animal
(217.0±28.7 U/L) compared to that of normal control animals (131.00±20.5 U/L). SGPT
level in ethanolic extract of Bauhinia variegata root 200 mg/kg (149.0±27.1 U/L),
Tectona grandis bark 200 mg/kg (160.0±7.4 U/L) and Liv 52 syrup-1 ml/kg (140.0±19.1
U/L) showed significant reduction (p<0.05) whereas Schrebera swietenioides fruit 200
mg/kg (175.0±10.7 U/L) showed slight reduction in SGPT level when compared to that
of CCl4 control SGPT level (217.0±28.7 U/L).
2. SGOT: These levels were expressed as U/L and were given in mean S.E.M.
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Dept. Of Pharmacognosy & Phytochemistry KLE University’s College of Pharmacy, Belgaum. 149
There was a significant increase in SGOT enzyme level in the CCl4control animal
(340.0±21.0 U/L) compared to that of normal control animals (86.00±17.1 U/L). SGOT
level in ethanolic extract of Bauhinia variegata root 200 mg/kg (198.5±4.4 U/L),
Tectona grandis bark 200 mg/kg (173.0±34.4 U/L), Schrebera swietenioides fruit 200
mg/kg (238.0±17.2 U/L) and Liv 52 syrup-1 ml/kg (182.0±5.1 U/L) showed highly
significant (p<0.01) reduction in the SGOT level in the serum compare to that of CCl4
control (340.0±21.0 U/L).
3. ALP: These levels were expressed as U/L and were given in mean S.E.M.
There was a significant increase in ALP enzyme level in the CCl4control animal
(385.0±27.3 U/L) compared to that of normal control animals (161.0±15.2 U/L). ALP
level in ethanolic extract of Bauhinia variegata root 200 mg/kg (292.0±38.4 U/L),
Tectona grandis bark 200 mg/kg (323.3±24.5 U/L) and Schrebera swietenioides fruit
200 mg/kg (275.0±8.4 U/L) showed significant (p<0.05) reduction whereas Liv 52
syrup-1 ml/kg (219±12.2 U/L) showed highly significant (p<0.01) reduction in the ALP
level in the serum compare to that of CCl4 control (385.0±27.3 U/L).
4. Serum total bilirubin: These levels were expressed as mg/dl and were given in mean
S.E.M.
There was a significant increase in total bilirubin level in the CCl4control animal
(2.123±0.1 mg/dl) compared to that of normal control animals (0.700±0.07 mg/dl). Total
bilirubin level in ethanolic extract of Bauhinia variegata root 200 mg/kg (0.856±0.1
mg/dl), Tectona grandis bark 200 mg/kg (0.900±0.1 mg/dl), Schrebera swietenioides
fruit 200 mg/kg (0.976±0.2 mg/dl) and Liv 52 syrup- 1ml/kg (0.800±0.1 mg/dl) showed
highly significant (p<0.01) reduction in the total bilirubin level in the serum compare to
that of CCl4 control (2.123±0.1 mg/dl).
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5.5.2 The effect of different extracts on histopathology of liver in CCl4 induced
hepatotoxic rats
Histopathological examination of liver of these animals has shown following
observations.
Group I: Normal control (1% gum acacia): Normal hepatic cells with well
preserved cytoplasm, prominent nucleus and nucleolus and well brought out central
vein. (Fig. 26)
Group II: CCl4 control: Massive fatty changes, necrosis, hemorrhagic foci in
hepatic parenchyma, ballooning degeneration and broad infiltration of the
lymphocytes and the loss of cellular boundaries. (Fig. 27)
Group III: Liv 52 syrup (1 ml/kg): Normal lobular pattern with a mild degree of
fatty change, necrosis and lymphocyte infiltration almost comparable to the normal
control. (Fig. 28)
Group IV: Bauhinia variegata ethanolic extract (200 mg/kg): Normal lobular
pattern with a mild degree of fatty change, necrosis and lymphocyte infiltration
almost comparable to the normal control. (Fig. 29)
Group V: Tectona grandis ethanolic extract (200 mg/kg): Normal lobular pattern
with a mild degree of fatty change, necrosis and lymphocyte infiltration almost
comparable to the normal control. (Fig. 30)
Group VI: Schrebera swietenioides ethanolic extract (200 mg/kg): Normal lobular
pattern with a mild degree of fatty change, necrosis and lymphocyte infiltration. (Fig.
31)
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Table No. 18
The effect of different extracts on serum enzymes & total bilirubin
Exp.Group
(n= 6) Treatment
SGPT
(U/L)
SGOT
(U/L)
ALP
(U/L)
TOTAL
BILIRUBIN
(mg/dl)
I Normal control
(1% gum acacia) 131.0±20.5* 86.00±17.1* * 161.0±15.2* * 0.700±0.07 * *
II CCl4 control 217.0±28.7 340.0±21.0 385.0±27.3 2.123±0.1
III LIV 52 syrup (1 ml/kg) 140.0±19.1* 182.0±5.1* * 219.0±12.2* * 0.800±0.1 * *
IV Bauhinia variegata ethanolic
extract (200 mg/kg) 149.0±27.1* 198.5±4.4* * 292.0±38.4* 0.856±0.1 * *
V Tectona grandis ethanolic
extract (200 mg/kg) 160.0±7.4* 173.0±34.4* * 323.3±24.5* 0.900±0.1 * *
VI Schrebera swietenioides
ethanolic extract (200 mg/kg) 175.0±10.7 238.0±17.2** 275.0±8.4* 0.976±0.2 * *
*P < 0.05 & **P < 0.01 Significant, compared to CCl4 control.
Results are expressed as mean ± S.E.M., n= no of animals in each group.
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Histopathology of liver
Chapter 5 Results
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Chapter 5 Results
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5.6 SEPARATION AND CHARACHTERIZATION OF ACTIVE
PHYTOCONSTITUENTS OF EXTRACTS WITH CHROMATOGRAPHIC
TECHNIQUES
The selected medicinal plant extracts were subjected for fractionations with solvents
according to polarity in the column chromatography technique for separating active
phytoconstituents. Totally around 10 column fractions were collected from each extract
and each column fraction was subjected to qualitative chemical test (flavonoids, sterols,
anthraquinone glycosides and triterpenoids) and thin layer chromatography for the
identification of same phytoconstituents.
a) Qualitative chemical test for column fractions: (Table No.: 19)
Fraction No.
Bauhinia variegate
root ethanolic
extract
Tectona grandis
bark ethanolic
extract
Schrebera
swietenioides fruit
ethanolic extract
1 - - -
2 - - -
3 Flavonoids Glycosides -
4 - - -
5 - - Triterpenoids
6 - - -
7 Sterols/steroids - -
8 - Sterols/steroids -
9 - - -
10 - - -
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Qualitative chemical investigation results revealed presence of following
phytoconstituents:
sterols & flavonoids in Bauhinia variegata root ethanolic extract,
quinones & sterols in Tectona grandis bark ethanolic extract &
triterpenoids in Schrebera swietenioides fruit ethanolic extract
b) TLC method:
The results of qualitative chemical investigation for presence of phytoconstituents were
further confirmed by thin layer chromatography. The collected fractions of selected
medicinal plants when co-chromatographed with reference standard compounds like
quercetin, β-sitosterol and oleonilic acid on TLC, the development of thin layer
chromatogram has revealed the presence of one spot. The TLC profile of collected
fractions (Table No-20 and TLC plate fig.no.32-36) confirms the presence of different
phytoconstituents in these selected medicinal plants.
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Table No. 20. TLC profile comparism of column fractions of extracts and standards
Phyto-constituents
Solvent system
Visualizing reagent Rf values
Figure no.
Flavonol Glycosides
1. Bauhinia variegata root ethanolic extract
column fraction no.3
2. Quercetin
Toluene: Dioxan:
Acetic acid (90:25:5)
Anisaldehyde-
H2SO4
0.83 (Yellow)
0.85 (Yellow)
32
Sterols
1. Bauhinia variegata root ethanolic extract
column fraction no 7
2. Tectona grandis bark ethanolic extract column
fraction no 8
3. β-sitosterol
Petroleum ether : Acetone
(85:15)
Liebermann
Burchard reagent
0.47 (Brown)
0.43 (Yellowish
Brown)
0.45 (Brown)
33 & 35
Anthra-quinones
Tectona grandis bark ethanolic extract column
fraction no 3
Ethyl acetate: methanol:
water (100:13.5:10)
10% Ethenolic
KOH
0.81 (Yellowish
green)
34
Triterpenoid Saponins
1.Schrebera swietenioides fruit ethanolic extract
Column fraction no 5
2.Oleanolic acid
Chloroform: acetone
(8:2)
Acetic anhydride-
H2SO4 / ethanol (1:1).
0.74 (Brown color)
0.75(Brown)
36
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TLC plate:
Fig. No.32 TLC of Flavonoid glycosides (Bauhinia variegata column fraction) and
Standard
Fig. No.33 TLC of Sterols (Bauhinia variegata column fraction) and standard
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Fig. No.34 TLC of Quinone (Tectona grandis column fraction)
Fig. No.35 TLC of Sterols (Tectona grandis column fraction) and standard
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Fig. No. 36 TLC of Triterpenoids (Schrebera swietenioides column fraction) and
standard