CHAPTER 5: RESULTS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/5943/15/15_chapter 5.pdfCHAPTER 5: RESULTS 5.1. A ... Test for Flavonoids a) Shinoda test b) Lead acetate

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


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%


ethanolic extract

Semisolid

viscous Brown 11.6 %


petroleum ether extract

Semisolid

viscous Brown 4.5%

04 Tectona grandis bark

aqueous extract

Semisolid

viscous Brown 12.3%


ethanolic extract

Semisolid

viscous Brown 8.4 %


petroleum ether extract

Semisolid

viscous Brown 6.8%

07 Schrebera swietenioides

fruits aqueous extract

Semisolid

viscous Brown 14.2%


fruits ethanolic extract

Semisolid

viscous Brown 10.2. %.


fruits petroleum ether extract

Semisolid

viscous Brown 3.4%

Chapter 5 Results


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


SL.


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


Preliminary phytochemical investigation of-

B) Tectona grandis bark different solvent extracts

Table No. 8

SR.


OBSERVATION

Aqueous

Extract

Ethanolic

Extract

Pet. Ether

Extract

I.


a) Molisch's test

b) Fehling's test

c) Benedict's test

d) Barfoed's test



+

+

+

+

+

-

+

+

+

+

+

-

-

-

-

-

-

-

II.

Test for Proteins

a) Biuret test

b) Million's test



sulphur


-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

III.

Test for Steroids




+

+

+

+

+

+

+

+

+

IV.


a) Ninhydrin test



-

-

-

-

-

-

-

-

-

V.

Test for Flavonoids

a) Shinoda test




-

-

-

-

-

-

-

-

-

-

-

-

Chapter 5 Results


SR.


OBSERVATION

Aqueous

Extract

Ethanolic

Extract

Pet. Ether

Extract

VI

Test for Alkaloids


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


f) Libermann's test

g) Foam test

h) Hemolytic test


+

+

-

-

-

-

-

-

-

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

IX.


a) Salkowaski test


-

-

-

-

-

-


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


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.


a) Molisch's test

b) Fehling's test

c) Benedict's test

d) Barfoed's test



+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

II.

Test for Proteins

a) Biuret test

b) Million's test



sulphur


-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

III.

Test for Steroids




-

-

-

-

-

-

-

-

-

IV.


a) Ninhydrin test



-

-

-

-

-

-

-

-

-

V.

Test for Flavonoids

a) Shinoda test




-

-

-

-

-

-

-

-

-

-

-

-

Chapter 5 Results


SR.

NO.

NAME OF THE TEST

OBSERVATION

Aqueous

Extract

Ethanolic

Extract

Pet. Ether

Extract

VI

Test for Alkaloids


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


f) Libermann's test

g) Foam test

h) Hemolytic test


-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

IX.


a) Salkowaski test


+

+

+

+

-

-


presence of "Triterpenoids" in ethanolic and aqueous extract whereas

“Carbohydrates” in aqueous extract of Schrebera swietenioides fruit.

Chapter 5 Results


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


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


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


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


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


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


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


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


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


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.

Chapter 5 Results


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


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


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


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.


Chapter 5 Results


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)




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


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** # #


extract (200 mg/kg) 275.5±7.7 194.2±16.0**

# # 164.4±10.6**

# # 122.6±9.4*

# #


extract (200 mg/kg) 270.3±6.0 199.0±5.2**

# # 164.5±9.4* *

# # 124.5±7.3*

# #


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.


Chapter 5 Results


Figure No. 18. Histogram showing the effect of different extracts on the blood

glucose levels in alloxan-diabetic rats (Multidose treatment/sub acute study)




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


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


extract (200 mg/kg) 206.50 ± 2.4 200.00 ± 2.07

# 194.00 ± 1.69

# 187.0 ± 1.50

# - 9.44


extract (200 mg/kg) 205.50 ± 2.39 198.00 ± 2.15

# 191.50 ± 1.89

# 182.50 ± 1.24

# - 11.19


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.


Chapter 5 Results


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 **


extract (200 mg/kg) 94.00±9.3 ** 45.66±1.5 ** 22.00±2.9 * 23.00±4.9 * 60.00±6.1 **


extract (200 mg/kg) 99.00±4.7 * 40.83±1.9 ** 25.00±1.9 22.00±5.3 * 61.00±5.5 **


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.

Chapter 5 Results


Fig. No. 19.Histogram showing the effect of different extracts on the serum profile

in alloxan-diabetic rats after 15 days of treatment




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)

Chapter 5 Results


Histopathology of Pancreas:

Chapter 5 Results


Chapter 5 Results


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.

Chapter 5 Results


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


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


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).

Chapter 5 Results


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)

Chapter 5 Results


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 * *


extract (200 mg/kg) 149.0±27.1* 198.5±4.4* * 292.0±38.4* 0.856±0.1 * *


extract (200 mg/kg) 160.0±7.4* 173.0±34.4* * 323.3±24.5* 0.900±0.1 * *


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.

Chapter 5 Results


Histopathology of liver

Chapter 5 Results


Chapter 5 Results


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 - - -

Chapter 5 Results


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.

Chapter 5 Results


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

Chapter 5 Results


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

Chapter 5 Results


Fig. No.34 TLC of Quinone (Tectona grandis column fraction)

Fig. No.35 TLC of Sterols (Tectona grandis column fraction) and standard

Chapter 5 Results


Fig. No. 36 TLC of Triterpenoids (Schrebera swietenioides column fraction) and

standard

Documents

CHAPTER 5: RESULTS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/5943/15/15_chapter 5.pdfCHAPTER 5: RESULTS 5.1. A ... Test for Flavonoids a) Shinoda test b) Lead acetate