EXPERIMENTAL WORK - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/8698/14/14_chapter...

Characterization of Isolated Compounds

267

EXPERIMENTAL WORK

The process that leads the plant to isolate pharmacologically active pure

constituents is very long and tedious and requires a multidisciplinary collaboration of

botanists, phytochemists and pharmacologists. The project involves

Collection and Authentication of the Plant Material

Extraction of the selected plant

Preliminary screening of extracts

Isolation and purification of compounds

Structure elucidation of compound

Extraction and Phytochemical Screening of Flemingia wightiania

A. Collection and Authentification of Flemingia wightiania

The whole plant Flemingia wightiania widely found in the South India. The plant

was collected from the Chittoor district (Andhra Pradesh), identified and authenticated by

Dr. K. Madhava Chetty, Asst. Professor, Dept. of Botany. Sri Venkateswara University,

Tirupathi.

B. Extraction Process

The whole plant was shade dried at room temperature and was chopped into small

pieces. Dried plant were powdered and packed in air tight container.

The coarse material was subjected to successive soxhlet extraction by using

different solvents. Solvents are used based on their increasing order of polarity i.e., n-

Hexane (0.1), Ethyl acetate (4.4), Methanol (5.1) and Water (10.2). The extraction is

carried with Methanol, n-Hexane and Ethyl acetate. The aqueous extraction was carried

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268

out by cold maceration process. The extracts were concentrated under reduced pressure

and stored in desiccators.

Table 1: Extractive Yield and Percentage Yield of Flemingia wightiania

Sl. No. Extracts Yield in gm Percentage Yield

1. n-Hexane 42.34 8.46%

2. Ethyl acetate 39.00 7.8%

3. Methanol 53.20 10.64%

4. Aqueous 64.46 6.44%

Preliminary Qualitative Phytochemical Studies:

Table 2: Preliminary Phytochemical Screening of Flemingia wightiania Extracts

Sl. No. Test n-Hexane

Extract

Ethyl

acetate

Extract

Methanolic

Extract

Aqueous

Extract

i Alkaloids

A. Mayer’s Test

B. Dragandroff’s Test

C. Wagner’s Test

D. Hager’s test

-

+

-

-

-

-

-

-

+

+

+

+

+

-

-

+

ii Carbohydrates

A. Molish’s Test

B. Barfoed’s Test

C. Bendict’s Test

D. Fehling,s Test

+

+

-

-

-

-

-

-

+

+

-

-

-

+

+

+

iii Flavonoids

A. Shinoda Test

B. Ferric chloride Test

C. Alkaline Test

D. Lead acetate Test

-

-

-

-

-

-

-

-

+

+

-

+

-

+

-

+



269

iv Steroids

A. Salkowski Test

B. Liebermann Buchard’s Test

C. Sulphur Test

-

+

+

+

+

+

+

+

+

-

+

+

v Glycosides

A. Balget’s Test

B. Keller- Killani Test

C. Bromine water Test

D. Legal’s Test

+

+

-

+

-

+

-

-

+

+

+

+

+

-

-

-

Vi Triterpinoids

A. Libermann-Burchard’s Test

B. Salkowski’s Test

+

-

-

-

-

-

-

-

vii Proteins

A. Millon’s Test

B. Ninhydrin Test

C. Xanthoproteic Test

D. Biret Test

-

-

+

-

-

-

-

-

-

+

-

-

-

+

+

Viii Tannins

A. Ferric chloride Test

B. Gelatin Test

-

-

+

-

+

-

+

-

ix Saponins

A. Foam Test

B. Haemolysis Test

-

-

-

-

+

-

-

-

- Absent + Presence

Characterisation of Isolated Compounds:

The isolated compounds were characterized using physical properties, chemical

tests, Rf value and spectral data especially IR, Mass and NMR.



270

Compound 1: (FW-1)

Eluent : 75% ethyl acetate in Hexane

Physical Properties : Pale yellow crystalline compound

Chemical Test : It gave positive test for FeCl3, lead acetate, test for Schinoda of

flavanoids.

TLC : Green color spot

It was collected from 279-343 fractions, as pale green colored crystallized needles

from 75% ethyl acetate in hexane fraction.

IR Spectrum indicated bands at 3398 (OH), 2921(C-H stretch), 1630(C=O) cm-1

.

1H NMR Data:

1H

NMR spectrum (400MHz, CDCl3, δ ppm) showed the

presence of a 1 hydroxyl (δ 13.501,1H, S), a downfield methylene proton (δ 6.708, 1H, s)

characteristic of H-3 of flavonoids , (δ 6.410, 6.389 1H,s) assigned to H-6 of ring A of

5,7-dihydroxy flavonoids. The 1H NMR spectrum also showed the presence of a signal at

δ 7.358(1H, J = 8 Hz), δ 7.260 (1H, J = 1.6 Hz), (δ 7.056,7.036,7.016 2H,J=8.34,t) and δ

7.975 (1H, J = 6.4 Hz), (δ 6.410,6.389 1H,J=8.34,d) (δ 8.014 (1H, J = 4.2 Hz),

characteristic of 1, 4 – disubstituted phenyl unit.

Mass Data: The [MH]¯ion at M/Z=421.2 was observed in mass spectroscopy.

The above data is well related to Flemingin D except for having methoxy at

instead of hydroxyl group. Basing on the above data the compound was found to be a

flavanoid with molecular formula



271

OH

OH

OH O

O

CH3CH3

Figure 1: Flemingin D

Figure 2: IR Spectrum of FW-01



272

Figure 3: MASS SPECTRUM OF FW-01

Figure 4: 1H NMR Spectrum of FW-01



273

Compound 2: (FW-2)

The isolated compound were characterized using physical properties, chemical

tests, Rf value and spectral data especially IR, Mass and NMR.

Eluent : 60% Ethyl acetate in Hexane

Physical Properties : Pale yellow amorphous compound

Chemical Tests : It gave positive test for FeCl3, lead acetate, test for Schinoda of

flavonoids.

TLC : Greenish yellow color spot

It was collected from 244-278 fractions, as pale yellow colored amorphous from

60% ethyl acetate in hexane fraction and had molecular formula of C15H10O7

IR Spectrum indicated bands at 3398 (OH), 2921(C-H stretch), 1630(C=O)cm-1

.

1H NMR Data: The

1H NMR spectrum showed the presence of a signal at δ

1H

NMR (DMSO, 300MHz): δ 6.17 (1H, d, J= 2.0 Hz, H-6), 6.37 (1H, d, J= 2.0 Hz, H-

8), 6.87 (1H, d, J= 8.0 Hz, ), 7.62(1H, dd, J= 2.0, 7.5 Hz), 7.73 (1H, d, J = 2.0 Hz)

Mass Data: The [MH]¯ion at M/Z=302.1 was observed in mass spectroscopy.

The above data is tallied well with that of quercetin compound and was confirmed

by comparison with an authentic sample.



274

O OH

OHOH

O

OH

OH

2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one

Figure 5: Quercetin

Figure 6: IR Spectrum of FW-02



275

Figure 7: MASS Spectrum of FW-02

Figure 8: 1H NMR Spectrum of FW-02


Material and Methods

276

MATERIAL AND METHODS

Anti-Microbial Activity1:

Anti-Bacterial Activity:

Method: Agar diffusion method was used to carry out this study.

Preparation of Test Solutions:

Test samples of the plant extract were prepared in DMSO (Dimethyl Sulfoxide)

and the final volume made with distilled water to get 25 and 50 mg/ml concentrations.

Preparation of Standard Solutions:

Ciprofloxacin was the reference standard drugs prepared in distilled water to get

75 l/ml, 50 l/ml, 25 l/ml, 10 l/ml, and 5 l/ml.

Test Organisms:

1. Enterococcus faecalis gram positive

2. Staphylococcus aereus

3. Bacillus subtilis

4. Shigella sonnei

5. Klebsiallea pneumonia gram negative

6. Escherichia coli

7. Salmonella typhi

8. Proteus vulgaris

Mueller Hinton media was prepared using following ingredients:

1. Casein hydrolysate (enzymic) 17.5 g

2. Beef infusion 30.0 g

3. Soluble starch 1.5 g



277

4. Agar 20.0 g

5. Distilled water Q.S. to 1000ml

The final pH of the medium after sterilization at 1.1 kg/cm2 to 7.4 ± 0.2 (at 25

ºC),

(121 º C) for 15 minutes was adjusted

Nutrient Agar

1. Beef extract 3.0 g

2. Peptone 5.0 g

3. Sodium chloride 5.0 g

4. Agar 5.0 g


pH 7.2

The above were dissolved in one liter distilled water and sterilized at 121 ºC for

15 minutes.

Method:

The antibacterial activity of the extract was assayed using the disc diffusion

method. For inoculum preparation and assay of antibacterial activity, Muller-Hinton agar

was used. The bacteria were sub cultured and routinely maintained on both nutrient agar

and Muller-Hinton agar. Antimicrobial activity was evaluated using the agar diffusion

technique in Petri dishes. Each extract was loaded on sterile filter paper discs and air

dried. Indicator microbes were spread on Muller-Hinton agar plates with sterile effusion

the discs were placed on plates. After incubation for 24 hours at 30 °C, a clear zone

around a disc was evidence of antimicrobial activity. Discs loaded with the extracting

agents were tested as controls.



278

Anti-Fungal Activity:

Method: Agar diffusion method was used to carry out this study.

Preparation of Test Solutions:

Test samples of the plant extract were prepared in DMSO (Dimethyl Sulfoxide)

and the final volume made with distilled water to get 25 and 50 mg/ml concentrations.

Preparation of Standard Solutions:

Fluconazole was the reference standard drugs prepared in distilled water to get

75 l/ml, 50 l/ml, 25 l/ml, 10 l/ml, and 5 l/ml.

Test Organisms:

1. Candida albicans

2. Aspergillus fumigates

3 .Dreschlera turcica

4. Fusarium verticillioides

Preparation of potato dextrose broth:

1. Peeled potato 200-300g

2. Dextrose 5g

3. Agar 20.0 g


The final pH of the medium after sterilization at 1.1 kg/cm2

to 7.4 ± 0.2 (at 25ºC),

(121ºC) for 15 minutes was adjusted.

Nutrient Agar

1. Beef extract 3.0 g

2. Peptone 5.0 g



279

3. Sodium chloride 5.0 g

4. Agar 5.0 g


pH 7.2

The above were dissolved in one liter distilled water and sterilized at 121 ºC for

15 minutes.

Method

The antibacterial activity of the extract was assayed using the disc diffusion

method. For inoculum preparation and assay of antibacterial activity, Muller-Hinton agar

was used. The bacteria were sub cultured and routinely maintained on both nutrient agar

and Muller-Hinton agar. Antimicrobial activity was evaluated using the agar diffusion

technique in Petri dishes. Each extract was loaded on sterile filter paper discs and air

dried. Indicator microbes were spread on Muller-Hinton agar plates with sterile effusion

the discs were placed on plates. After incubation for 24 hours at 30°C, a clear zone

around a disc was evidence of antimicrobial activity. Discs loaded with the extracting

agents were tested as controls.


Results and Discussion

280

RESULTSAND DISCUSSION

Anti Microbial Studies Reports:

The disc diffusion method was used to determine zones of inhibition of Flemingia

wightiania. extracts (n-Hexane, Ethyl acetate, Methanol and Aqueous). The MEFW and

AEFW showed significant activity against Staphylococcus aureus, Bacillus subtilis in

gram positive and Escherichia coli, Proteus vulgaris in gram negative at the

concentration of 75 µgms. HEFW and EAEFW extracts showed less significant activity.

In Anti fungal activity the MEFW and AEFW showed significant activity against

Candida albicans, Asperigillus fumigates Dreschlera turcica and Fusarium verticillioides

at the concentration of 75 µgms.

HEFW and EAEFW extracts showed less significant activity. These extracts were

compared with Fluconazole which is used as a standard.



281

Table 1: Anti bacterial Activity and Extracts of Flemingia wightiania:

Staphylococcus aureus Enterococcus faecalis Klebsiella pneumonia Escherichia coli

Concentration

in µgms

75 50 25 10 5 75 50 25 10 5 75 50 25 10 5 75 50 25 10 5

n-Hexane 14 12 11 9.5 - 12 9 - - - 10 - - - - 15 12 9.5 - -

Ethyl acetate 15 13.5 12.5 10 - 10 - - - - 9.5 - - - - 13 10 9 - -

Methanol 20 17 15.5 12 11 16 13 11 10 9.5 17 15.5 13.5 11 10 19 17 16 14 12

Aqueous 17 14 12 10 9 15 13 9 - - 16 12.5 10.5 9.5 - 14 12 11 10 9

Bacillus subtilis Shigella sonnei Salmonella typhi Proteus vulgaris

Concentration

in µgms

75 50 25 10 5 75 50 25 10 5 75 50 25 10 5 75 50 25 10 5

n-Hexane 16 14 12 10 - 13 10 9 - - 11 9 - - - 16 12.

5

10 - -

Ethyl acetate 15.

5

13 12 10 - 11 10 - - - 9 - - - - 14 10 - - -

Methanol 19 16 14 10 9 17 15.

5

12 10 9.5 18 16 13 11 10 18 13 10 9 1

Aqueous 18 16 15 12 10 15 12 9 - - 16 12 10.

5

9 - 15 12 10 9 -

Standard

(Ciprofloxacin)

22 20 18 17 14 21 20 18 16 15 22 19.

5

17 15 13 23.

5

19.

5

17 16 14



282

Table 2: Anti fungal Activity and Extracts of Flemingia wightiania:

Candida albicans Aperigillus fumigatus Dreschlera turcica Fusarium verticillioides

Concentration

in µgms

75 50 25 10 5 75 50 25 10 5 75 50 25 10 5 75 50 25 10 5

n-Hexane 14 12.5 10 - - 16 14.5 13 10 - 15 11 9 - - 15 13 12 10 -

Ethyl acetate 10 - - - - 13 10 - - - 12 10 - - - 14 11 9 - -

Methanol 18 15.5 13.5 12 10 17 16 14 11 9 17 14 11 10 - 17 15.5 12 10 9

Aqueous 16 14 12 11 9 15 12 11 9.5 - 17 13 11 10 9 15.5 13 11.5 9 -

Standard

(Flucanazole)

20.5 17 15.5 12.5 11 20 17 16 13 10 20.5 17 15.5 12.5 11 20 17 16 13 10



283



283


Animals Used In Study

Albino wistar rats weighing 150-200 g and Albino mice 20-30 g was procured

from Biogen, Bangalore. They were maintained in the animal house of Gautham College

of Pharmacy. Animals were maintained under controlled condition of temperature at

27 ± 2o

C and 12 hour light-dark cycles. They were housed in polypropylene cages and

had a free access to standard pellets (Amruth) and water ad libitum. The animals housing

and handling were in accordance with CPCSEA guidelines.

All the studies conducted were approved by the Institutional Animal Ethical

Committee (IAEC) of Gautham College of Pharmacy, Bangalore, Karnataka. (REF-

IAEC/012/12/2010) according to prescribed guidelines of Committee for the Purpose of

Control and Supervision of Experiments on Animals (Reg No: 491/01/c/CPCSEA), Govt.

of India.

Analgesic Activity

i. Acetic acid Induced Writhing in Mice2:

Albino mice weighing 20-30 mg/kg were divided into six groups of six in each

group. One hour after the administration of the test drug and diclofenac sodium

(10 mg/kg i.p), the mice were given intraperitoneal injection of 0.7%v/v acetic acid

solution (volume of injection 0.1ml 10g), the mice were placed individually into glass

beakers and 5 minutes, were allowed to elapse. The number of writhes produced in these

animals was counted for 15 minutes. For scoring purposes, a writhe is indicated by

stretching of the abdomen with simultaneous stretching of at least one hind limb. The

formula for computing present inhibition was;



284

Group-I: Distilled water will be supplied and served as control.

Group-II: Animals received a dose of 10 mg/kg of Diclofenac sodium i.p. and served

as standard

Group-III: Animals received a dose of 300 mg/kg of HEFW p.o.

Group-IV: Animals received a dose of 300 mg/kg of EAEFW p.o.

Group-V: Animals received a dose of 300 mg/kg of MEFW p.o.

Group-VI: Animals received a dose of 300 mg/kg of AEFW p.o.

ii. Tail Flick Method3:

Albino wistar rats weighing 150-230 mg/kg were divided into six groups of six in

each group. The tail flick latency was assessed by analgesiometer. A light beam is

focused (exerting radiant heat) to the proximal third of the tail. The rat tries to pull the tail

away and rotates the head this reaction is known as escape reaction. The reaction time is

recorded ½, 1, 2, 3, 4, 5 and 6 hours following intra peritoneal administration of the

standard and oral administration of the test compounds. The strength of the current

passing through the naked nichrome wire was kept constant at 6 amperes. The distance

between the heat source and tail skin was 1.5 cm. The site of application of the radiant

heat in the tail was maintained at 2.5 cm measured from the root of the tail. The cutoff

reaction time was fixed at 10 seconds to avoid tissue damage.


Group-II: Group-II: Animals received a dose of 10 mg/kg of Diclofenac sodium i.p.

and served as standard





285



iii. Hot plate Method4:

Albino mice weighing 20-30 mg/kg were divided into six groups of six in each

group. The temperature is controlled for 55 ± 1ºC. The animals were placed into the

Perspex cylinder on the heated surface and the time (sec) to discomfort reaction ( licking

paws or jumping) was recorded as response latency, period to and 30, 60, 90, 120 and

180 minutes following intra peritoneal administration of the standard and oral

administration of the test compounds. A latency period of 15 seconds was identified as

complete analgesia and the measurement was terminated if it exceeded the latency period

in order to avoid injury.


Group-II: Animals received a dose of 10 mg/kg of Pentazocine lactate i.p. and served

as standard







286

RESULTS AND DISCUSSION

Analgesic Activity

i. Acetic acid Induced Writhing in Mice

Control and various treated groups were tested for analgesic activity against acetic

acid induced writhing, which is nothing but the painful reaction. Thirty minutes after the

treatment each mouse was 0.1 ml 0.7% v/v aqueous solution of acetic acid injected i.p.

The number of abdominal constrictions was cumulatively counted from 0 - 10 minutes.

The % reduction of writhing in standard Diclofenac sodium 10 mg/kg treated group was

found to be 55.20% against control.

The mean response of control and standard was 41.50 ± 1.25 and 18.59 ± 092

respectively. The respective test compounds HEFW, EAEFW, MEFW and AEFW in its

300mg/kg dose, showed mean writhing responses as 25.59 ± 1.43, 28.33 ± 1.66, 20.67 ±

1.11 and 23.83 ± 1.30. In terms of percentage inhibition of writhing by Diclofenac

sodium was 55.20% while with the test compound it was 38.33%, 31.73%, 50.19% and

42.57% respectively. The values are tabulated in the Table 3 and shown in Figure 1.

Acetic acid-acid-induced writhing model represents pain sensation by triggering

localized inflammatory response. Such pain stimulus leads to the release of free

arachidonic acid from tissue phospholipids5. The acetic acid induced writhing response is

a sensitive procedure to evaluate peripherally acting analgesics. The response is thought

to be mediated by peritoneal mast cells acid sensing ion channels

and the prostaglandin

pathway6-8

.



287

Table 3: Effect of Flemingia wightiania Plant Extracts on Acetic acid Induced

Writhing in Mice

Groups Treatment Mean no of

writhing ±SEM

% Inhibition of

writhes

Group-I Saline 41.50 ± 1.25 -

Group-II Diclofenac sodium

(10mg/kg)

18.59 ± 092 55.20%

Group-III HEFW (300mg/kg) 25.59 ± 1.43 38.33%

Group-IV EAEFW (300mg/kg) 28.33 ± 1.66 31.73%

Group-V MEFW (300mg/kg) 20.67 ± 1.11 50.19%

Group-VI AEFW (300mg/kg) 23.83 ± 1.30 42.57%

Values are Mean ± SEM (n=6) one way ANOVA followed by Dunnett’s test. Where, ***

P<0.001, ** P<0.01, * P<0.05 and ns represents Not significant.

HEFW - n-Hexane extract of Flemingia wightiania

EAEFW - Ethyl acetate extract of Flemingia wightiania

MEEW - Methanolic extract of Flemingia wightiania

AEFW - Aqueous extract of Flemingia wightiania

Gro

up I

Gro

up II

Gro

up III

Gro

up IV

Gro

up V

Gro

up VI

0

10

20

30

40

50

Nu

nb

er o

f w

rit

hes

Figure 1: Effect of Flemingia wightiania Plant Extracts on Acetic acid Induced

Writhing in Mice



288

ii. Tail Flick Method in Rats

In the tail flick method, the increase in latency period at different time points

significantly differed (P<0.001) compared to baseline values within the same drug treated

groups. The extracts and diclofenac sodium caused significant increase (P<0.001) in the

percentage reaction time whilst the control and dose of extracts (300 mg/kg). The

percentage increase in reaction time was dose dependent. At all the specified time

intervals, the percentage of tail flick elongation time differed significantly (P<0.001)

between the extracts and diclofenac sodium at the doses of plant extracts, being greater

for diclofenac sodium. At the peak of activity, MEFE and AEFW extracts showed

(P<0.001) and significantly of tail flick elongation time respectively, whilst diclofenac

sodium gave (P<0.001) elongation of tail flicking time. The values are tabulated in the

Table 4 and shown in Figure 2.



289

Table 4: Effect of Flemingia wightiania Plant Extracts on Tail Flick Method Test in Rats

Groups Treatment Reaction Time (Sec)

0 hr ½ hr 1 hr 2 hr 3 hr 4 hr 5 hr 6 hr

Group-I Saline 4.51 ± 0.29 4.48 ± 0.23 5.05 ± 0.34 5.30 ± 0.28 5.70 ± 0.38 5.76 ± 0.36 4.86 ± 0,57 5.65 ± 0.51


(10mg/kg)

4.71 ± 0.31 8.86 ±

0.39***

10.05 ±

0.45***

11.52 ±

0.98***

12.32 ±

0.77***

13.17 ±

1.19***

14.28

±0.90***

13.62 ±

0.63***

Group-

III

HEFW

(300mg/kg)

4.50 ± 0.27 7.10 ±

0.62ns

7.86 ±

0.80*

9.13 ±

0.68**

9.33 ±

0.57**

10.13 ±

0.81**

11.12 ±

0.82***

9.48 ±

0.84**

Group-

IV

EAEFW

(300mg/kg)

4.71 ± 0.48 6.95 ±

0.86ns

7.71 ±

0.74ns

8.61 ± 1.01* 8.71 ± 0.63* 9.80 ±

0.75**

10.90 ±

0.90***

8.86 ± 0.82*

Group-

V

MEFW

(300mg/kg)

4.43 ± 0.44 8.51 ±

0.99**

9.71 ±

0.87***

10.58 ±

0.56***

11.25 ±

0.86***

12.02 ±

0.87***

13.07

±0.65***

11.13 ± 0.69

***

Group-

VI

AEFW

(300mg/kg)

4.70 ± 0.24 7.60 ±

0.83*

8.60 ±

0.97**

9.86 ±

0.96**

10.47 ±

0.66***

11.33 ±

0.63***

12.58 ±

0.74***

9.93 ±

0.73***

Values are Mean ± SEM (n=6) one way ANOVA followed by Dunnett’s test. Where, *** P<0.001, ** P<0.01, * P<0.05 and ns

represents Not significant.







290

0 hr

1/2

hr1

hr2

hr3

hr4

hr5

hr6

hr

0

5

10

15

20

Group I Group II Group III Group IV

Group V Group VI

Reacti

on

tim

e (

Sec)

Figure 2: Effect of Flemingia wightiania Plant Extracts on Tail Flick Method test in mice



291

iii. Hot plate Method in Mice

The standard pentazocine lactate (10 mg/kg) was given i.p., n-Hexane, Ethyl

acetate, Methanol and Aqueous extracts given orally, in a dose of 300 mg/kg, elicited a

significant analgesic activity in the hot plate method as evidenced by increase in latency

time in seconds as compared with vehicle control. The increase in latency time was dose

dependant. Latency time was noted 30, 60, 90,120 and 180 minutes after administration

of vehicle, standard and plant extracts. The values are tabulated in the Table 5 and shown

in Figure 3.

To evaluate the analgesic activity, hot plate method was chosen. In this method

pentazocine lactate (10 mg/kg) was used as reference standard. The HEFW, EAEFW,

MEFW and AEFW extracts of Flemingia wightiania produced antinociception against

thermal induced pain stimuli in mice at various time points of post treatment. The hot

plate test is considered to be selective for opioid like compounds, which are centrally

acting analgesic in several animal species. The hot plate method has been found to be

suitable for evaluation of centrally acting analgesic9,10

. The HEFW, EAEFW, MEFW

and AEFW (300 mg/kg p.o.) increase the reaction time in dose dependent manner to the

thermal stimulus.



292

Table 5: Effect of Flemingia wightiania Plant Extracts on Hot plate Method test in Mice

Groups Treatment Reaction time (Sec)

0 min 30 min 60 min 90 min 120 min 180 min

Group-I Saline 2.66 ± 0.33 2.33 ± 0.21 2.83 ± 0.30 3.66 ± 0.49 4.16 ± 0.60 3.16 ± 0.30

Group-II Pentazocine

lactate (10mg/kg)

2.50 ± 0.22 5.50 ± 0.42*** 7.16 ± 0.60*** 9.33 ± 0.66 *** 12.17 ± 0.30*** 14.33 ± 0.33***

Group-III HEFW

(300mg/kg)

2.16 ± 0.16 3.83 ± 0.47* 5.00 ± 0.36** 6.50 ± 0.42** 7.16 ± 0.30*** 9.83 ± 0.47***

Group-IV EAEFW

(300mg/kg)

2.83 ± 0.30 3.33 ± 0.42ns 4.66 ± 0.33* 6.16 ± 0.40** 6.66 ± 0.42** 9.66 ± 0.61***

Group-V MEFW

(300mg/kg)

3.16 ± 0.47 4.66 ± 0.33** 6.16 ± 0.40*** 8.15 ± 0.47*** 11.33 ± 0.49*** 13.00 ± 0.36***

Group-VI AEFW

(300mg/kg)

3.00 ± 0.36 4.50 ± 0.42** 5.50 ± 0.22*** 7.33 ± 0.49*** 10.50 ± 0.50*** 12.17 ± 0.47***









293

0 min

30 min

60 min

90 min

120 min

180 min

0

5

10

15

20


Group V Group VI

Rea

ctio

n t

ime

(S

ec)

Figure 3: Effect of Flemingia wightiania Plant Extracts on Hot plate Method test in Mice



294



294


Anti-Inflammatory Activity

A. Acute Anti inflammatory Activity

i. Formalin-induced Paw Oedema in Rats 11

Acute inflammation was induce by injecting formalin (0.1 ml of 1% suspension in

0.9% saline) in sub-plantar region and paw volume was measured 0, 1, 2, 3, 4 and 5 hours

with the help of Plethysmometer.

All the treatment compounds compound were administered 30 minutes prior to

formalin. Acute inflammation was induced in right hind paw. A mark was put on the leg

second at the leg at the mallaleous region to facilitate the dipping of the leg to the same

level at the second and subsequent times.

The initial reading was taken at 0th

hour, i.e., immediately after injecting formalin

and the procedure was repeated at 1, 2, 3, 4 and 5 hours after formalin injection. The

difference between 0 hour reading and one of the subsequent reading provides the actual

edema volume at the time. The mean paw volume at different times was calculated and

compared with the control and the percentage inhibition was then calculated by using the

formula;



as standard






295


B. Chronic Anti inflammatory Activity

i. Formalin Induced Paw Oedema12

Albino wistar rats weighing 170-230 g were divided into six groups of six in each

group. All these animals were fasted for 18 hours before the beginning of the experiment

and water was given ad libitum. In animals of all the groups chronic inflammation was

produced by sub plantar injection of 20µl of freshly prepared 2% suspension of formalin

in normal saline in right hind paw of rat was used as the oedematogenic agent. Animals

were treated with drugs for 6 consecutive days.

The paw volume was measured using a plethysmometer before and 6 days after

formalin challenge in each group. The increase in paw volume and percent of inhibition

was calculated.



as standard







296


Anti inflammatory Activity

A. Acute Anti inflammatory Activity

i. Formalin-induced paw Oedema in Rats

All the test compounds were tested with the diclofenac sodium as a standard drug

in the dose of 10 mg/kg for the anti-inflammatory activity.

Presently diclofenac sodium showed significant 87.14 % inhibition of

inflammation at 5th

hour (0.18 ± 0.01) when compared with control (1.40 ± 0.05)

respectively.

The test compounds showed maximum percentage of inhibition of oedema at 5th

hour significantly in respective dose level i.e., at 300 mg/kg the test compounds HEFW,

EAEFW, MEFW and AEFW showed 77.14%, 73.57 %, 85.00% and 83.57%. The values

are tabulated in the Table 6 and shown in Figure 4.

It is well known that inhibition of formalin-induced pedal oedema in rats is

one of the most suitable test procedures to screen anti-arthritic and anti-inflammatory

agents as it closely resembles human arthritis13

. Injection of formalin subcutaneously

into hind paw of rats produces localized inflammation and pain. The nociceptive

effect of formalin is biphasic, an early neurogenic component followed by a later

tissue mediated response14

. Thus formalin-induced arthritis is a model used for the

evaluation of an agent with probable anti-proliferative activity. This experiment is

associated with the proliferative phase of inflammation. Results with Flemingia

wightiania of HEFW, EAEFW, MEFW and AEFW are showed quite compatible with

those of the standard drug diclofenac sodium. Therefore, the test drug appears to be

effective against formalin-induced arthritis.



297

Table 6: Effect of Flemingia wightiania Plant Extracts on Formalin-induced Paw Oedema in Rats

Groups Treatment

0 hr 1hr 2hr 3hr 4 hr 5 hr % Inhibition

Group-I Saline 0.66 ± 0.01 0.78 ± 0.05 0.99 ± 0.05 1.20 ± 0.06 1.25 ± 0.07 1.40 ± 0.05 -


(10mg/kg)

0.15 ± 0.01 0.37 ± 0.02*** 0.55 ± 0.04*** 0.36 ± 0.2*** 0.30 ± 0.03*** 0.18 ± 0.01*** 87.14 %

Group-III HEFW

(300mg/kg)

0.13 ± 0.01 0.60 ± 0.04* 0.76 ± 0.05** 0.63 ± 0.03*** 0.48 ± 0.02*** 0.32 ± 0.05*** 77.14%

Group-IV EAEFW

(300mg/kg)

0.14 ± 0.02 0.67 ± 0.06ns 0.79 ± 0.03* 0.61 ± 0.02*** 0.51 ± 0.02*** 0.37 ± 0.01 *** 73.57 %

Group-V MEFW

(300mg/kg)

0.15 ± 0.02 0.52 ± 0.03** 0.61 ± 0.03*** 0.46 ± 0.02*** 0.36 ± 0.02*** 0.21 ±0.02*** 85.00%

Group-VI AEFW

(300mg/kg)

0.13 ± 0.01 0.56 ± 0.03** 0.65 ± 0.03*** 0.50 ± 0.02*** 0.37 ± 0.02*** 0.23 ± 0.03*** 83.57%









298

0 hr

1 hr

2 hr

3 hr

4 hr

5 hr

0.0

0.5

1.0

1.5

2.0


Group V Group VI

Paw

Oed

em

a V

olu

me

Figure 4: Effect of Flemingia wightiania Plant Extracts on Formalin-induced Paw Oedema in Rat



299

B. Chronic Anti inflammatory Activity

i. Formalin-induced paw Oedema in Rats

Formalin induced paw oedema is one of the most suitable test procedure to screen

chronic anti-inflammatory agents. The results obtained as mean increase in paw volume

(ml) and % inhibition are represented in Table 7.

The mean response of standard was 82.40% inhibition of increase in paw

thickness after 6 days respectively. In this model at 300 mg/kg dose level of HEFW,

EAEFW, MEFW and AEFW extracts showed 40.77%, 39.48%, 62.23% and 54.93%

inhibition of increase in paw thickness after 6 days, However at MEFW, AEFW extracts

showed 62.23% and 54.93% inhibition of increase in paw thickness after 6 days. All the

results were compared with solvent control and diclofenac sodium reference drug control.

Table 7: Effect of Flemingia wightiania Plant Extracts on Formalin-induced Paw

Oedema in Rats

Groups

Treatment

Initial Paw

Volume

Paw

Volume After

6 Days

Increase in

Paw Volume

% of

Inhibition

Group-I Saline 1.28 ± 0.07 3.61 ± 0.12 2.33 ± 0.06 -


(100 mg/kg)

1.23 ± 0.04 1.65 ± 0.05 0.41 ± 0.07 82.40%

Group-III HEFW (300mg/kg) 1.30 ± 0.05 2.85 ± 0.13 1.38 ± 0.22 40.77%

Group-IV EAEFW (300mg/kg) 1.30 ± 0.07 2.86 ± 0.14 1.41 ± 0.19 39.48%

Group-V MEFW (300mg/kg) 1.23 ± 0.07 2.08 ± 0.14 0.88 ± 0.11 62.23%

Group-VI AEFW (300mg/kg) 1.26 ± 0.07 2.26 ± 0.16 1.05 ± 0.14 54.93%

Results are expressed on Mean + SEM from four observations Paw Volume was

measured after 6 days.



300

EXPERIMENTAL WORK


Animals Used In Study

Albino wistar rats weighing 150-200 g and Albino mice 20-30 g was procured

from Biogen, Bangalore. They were maintained in the animal house of Gautham College

of Pharmacy. Animals were maintained under controlled condition of temperature at

27 ± 2º C and 12 hour light-dark cycle. They were housed in polypropylene cages and had

a free access to standard pellets (Amruth) and water ad libitum. The animals housing and

handling were in accordance with CPCSEA guidelines.

All the studies conducted were approved by the Institutional Animal Ethical

Committee (IAEC) of Gautham College of Pharmacy, Bangalore, Karnataka. (REF-

IAEC/012/12/2010) according to prescribed guidelines of Committee for the Purpose of

Control and Supervision of Experiments on Animals (Reg No: 491/01/c/CPCSEA), Govt.

of India.

Determination of Acute Toxicity (LD50)15

Animals

Female Albino rats of weighing 150-220 g were used for the study. They were

nulliparous and non-pregnant. These were acclimatized to laboratory condition for one

week prior to start of dosing.

Preparation of Dose

n-Hexane, Ethyl acetate extracts (HEFW, EAEFW) was dissolved in tween 80,

Methanol and Aqueous extracts of Flemingia wightiania (MEFW, AEFW) were



301

dissolved in saline/water, to prepare a dose of 2000 mg/kg. The doses were selected

according to the OECD guideline No. 425.

Procedure

The procedure was divided into two phases. Phase I (observation made on day

one) and Phase II (observed the animals for next 14 days of drug administration). Two

sets of healthy female rats (each set of 3 rats) were used for this experiment. First set of

animals were divided into three groups, each of one in a group. Animals were fasted

overnight with water ad libitum. Animals received a single dose of 2000 mg/kg was

selected for the test, as the test item was a source from herb. After administration of

extract, food was withheld for 3-4 hours. If the animal dies, conduct the main test to

determine the LD50. If the animal survives, dose four additional animals sequentially so

that a total of five animals are tested. However, if three animals die, the limit test is

terminated and the main test is performed. The LD50 is greater than 2000 mg/kg, if three

or more animals survive. If an animal unexpectedly dies late in the study, and there are

other survivors, it is appropriate to stop dosing and observe all animals to see if other

animals will also die during a similar observation period. Late deaths should be counted

the same as other deaths. The same procedure was repeated with another set of animals to

nullify the errors.

Antidiabetic Activity

A. Single Dose Study in Normal Rats16

Albino wistar rats weighing 150-200 mg/kg were divided into six groups of six in

each group. Animals were fasted overnight for 16 hours prior to the experiment. The

blood glucose levels were measured just prior to and 1, 2, 4, 8, 12 and 24 hours after drug



302

administration. The blood glucose levels were measured from the tail vein by using

Sugarcheck glucometer manufactured by Wockhardt.


Group-II: Animals received a dose of 5 mg/kg of Glibenclamide p.o. and served as

standard





B. Oral Glucose Tolerance Test in Normal Rats (OGTT) 17

The oral glucose tolerance test was performed in rats weighing 150-200 g. The

animals were fasted for 16 hr before the experiment but allowed free access to water.

These Rats were divided into six groups, six in each group. Rats of all groups were

loaded with glucose 2 g/kg p.o 30 minutes after drug administration. Blood samples were

collected from the tail vein prior to drug administration and at 1, 2 and 3 hour of glucose

administration.

Group-I: Animals received distilled water and after 30 minutes a glucose load of

2 g/kg is administered p.o. which was served as control.

Group-II: Animals received a dose of 5 mg/kg of Glibenclamide p.o. and after 30

minutes a glucose load of 2 g/kg is administered p.o which was served as standard.

Group-III: Animals received a dose of 300 mg/kg of HESP p.o. and after 30 minutes a

glucose load of 2 g/kg is administered p.o.



303

Group-IV: Animals received a dose of 300 mg/kg of EAEFW p.o. and after 30

minutes a glucose load of 2 g/kg is administered p.o.

Group-V: Animals received a dose of 300 mg/kg of MEFW p.o. and after 30 minutes a

glucose load of 2 g/kg is administered p.o.

Group-VI: Animals received a dose of 300 mg/kg of AEFW p.o. and after 30 minutes

a glucose load of 2 g/kg is administered p.o.

C. Effect of Extracts in Streptozotocin Induced Diabetic Rats18, 19

Substances used in study

Streptozotocin (Sigma-Aldrich laboratories)

Citrate buffer pH 4.5 (I.P)

Unless otherwise specified all the chemicals and reagents used are of analytical

grade.

Preparation of Streptozotocin (STZ) Solution

STZ was dissolved in ice-cold citrate buffer of pH 4.5 and a single intraperitoneal

injection was administered within five minutes to avoid degradation.

Preparation of Buffers and Solutions

Preparation of Citrate Buffer

Solution-A was prepared by dissolving 1.92 g of Citric Acid in 100 ml of distilled water

to give 0.1 M citric acid solution.

Solution-B was prepared by dissolving 2.94 g of Tri-sodium Citrate in 100 ml of distilled

water to give 0.1M Sodium citrate solution.



304

To 28 ml of solution-A, 22 ml of solution-B was added and diluted to 100 ml with

distilled water to give citrate buffer of pH 4.4. The pH of this solution was adjusted to 4.5

with 0.1 M sodium citrate solution.

Experimentally Induced Diabetes Mellitus20

Female Wistar rats weighing 150-200 g were used for this study. The animals

were overnight fasted for 16 hours before the induction of diabetes. Diabetes was induced

by a single dose of 45 mg/kg body weight of streptozotocin by intraperitoneal route.

After a period of 3 days blood glucose levels were checked by snipping the tail of STZ

treated fasted rats. Rats showing the blood glucose levels more than 300 mg/dl is taken

into the study21

.

Experimental Procedure

Diabetes was induced in fasted female Albino wistar rats (150-200g) by intraperitoneal

injection of 45mg/kg body weight of STZ except Group I. After 72 hours, animals with

fasting blood glucose levels higher than 300 mg/dl were selected and used.

Group-I: Animals received distilled water only and served as normal control.

Group-II: Animals received distilled water only and served as diabetic control

Group-III: Animals received a dose of 5 mg/kg of Glibenclamide p.o. and served as

standard

Group-IV: Animals received a dose of 300 mg/kg of HEFW p.o.

Group-V: Animals received a dose of 300 mg/kg of EAEFW p.o.

Group-VI: Animals received a dose of 300 mg/kg of MEFW p.o.

Group-VII: Animals received a dose of 300 mg/kg of AEFW p.o.



305

The study was carried out for 21 days. Fasting blood glucose levels were

measured before the administration of extracts. It was recorded as 0 day. The doses of

the extracts (n-Hexane, Ethyl acetate, Methanol and Aqueous) along with the standard

(Glibenclamide) were given daily to the animals for 21 days. The blood glucose levels

were checked on 0, 7, 14 and 21 day of the treatment period. Blood was collected from

snipping of the rat tail. Blood glucose levels were measured by using the glucometer

Sugarcheck.

Analytical Method Used in the Study

Collection of Blood and Serum Samples:

At the end of the experiment blood was collected by cardiac puncture from each

rat under mild ether anaesthesia. Some part of blood samples were used for the estimation

of haemoglobin levels and remaining was allowed to clot for 30 minutes at room

temperature and they were centrifuged at 3000 rpm for 10 minutes. The serum was used

for the study of biochemical parameters.

Collection of Tissue:

The animals were scarified pancreas and liver were collected. Pancreas was used

for the histopathological study and liver was used for the estimation of SOD, GSH and

TBARS.

Physical Parameters:

A. Determination of Body Weight:

Body weight of the entire animal in each group was noted on the 0, 7, 14 and 21

day of the experiment period. The weight difference was calculated.



306

B. Determination of Wet Liver Weight:

Animals were sacrificed and livers were isolated and washed with saline and

weights determined by using an electronic balance. The liver weights were expressed

with respect to its body weight i.e. gm/100gm.

Estimation of Biochemical Parameters

A. Estimation of Serum Albumin Levels22,23

Principle

Albumin binds with Bromocresol green (BCG) at pH 4.2 causing a shift in

absorbance of the yellow BCG dye. The blue green colour formed is proportional to the

concentration of albumin present, when measured photometrically between 580-630 nm

with maximum absorbance at 625 nm.

Albumin + Bromocresol Green Green albumin BCG complex

Procedure:

Pipette into tubes marked Blank Standard Test

Albumin Reagent 1000μl 1000µl 1000μl

Distilled Water 10µl -- --

Standard -- 10μl --

Test -- -- 10µl

Mix well, read the absorbance of standard and each test at 630 nm (580-630 nm) against

reagent blank, after one minute incubation at 37 ºC.

Calculation:

Albumin (g/dl) = l



307

B. Estimation of Serum Urea24,25

Principle

Urease breaks down urea into ammonia and carbon dioxide in alkaline medium.

Ammonia liberated from the breakdown of urea reacts with hypochlorite and salicylate to

form dicarboxyindophenol. This reaction is catalysed by the presence of Nitroprusside.

The intensity of the colour produced by the reaction is directly proportional to

concentration of urea present in the sample and it is measured photometrically at 600 nm.

(600 - 630 nm.)

Reaction:

Urea + H2O 2NH3 + CO2

NH3 + Salicylate + CIO 2, 2 dicarboxylindophenol

Procedure:

Reagent Blank Standard Test

Working reagent 1 ml 1ml 1ml

Standard - 10µl -

Sample - - 10µl

Mix well and incubate 37 ºC For 5 min.

Step 2

Reagent 2

1ml 1ml 1ml

Mix well and incubate at 37 ºC for 5 minutes. Measure the absorbance of standard

and sample against the reagent blank at 600 nm (580-630 nm) within 60 minutes.

Calculation:

Urea Conc. =

Sodium

Nitroprusside

Urease

e



308

C. Estimation of Serum Total Proteins26,27

Principle

The peptide bond of proteins reacts with CU+2

ions in alkaline solution to from a

blue violet complex (Biuret reaction), each copper ion complexing with 5 or 6 peptide

bonds. Tarterate is added as stabilizer while iodine is used to prevent auto reduction of

alkaline copper complex. The colour formed is proportional to the protein concentration

and is measured at 546 nm.

Proteins + Cu++

Blue violet coloured complex

Procedure:

Pipette into tubes marked Blank Standard Test

Reagent 1000µl 1000μl 1000µl

Distilled water 20μl - -

Standard 20µl

Test - - 20μl

Incubate for 10 minutes at 37 ºC. Read absorbance of the Standard and each test at

546 nm (520-560 nm) against reagent blank.

Calculation:

Total Protein

D. Estimation of Serum Triglycerides28-30

Principle

Triglycerides + H2O Glycerol + Free fatty acids

Glycerol + ATP Glycerol-3-phosphate + ADP

Glycerol 3 phosphate + O2 DAP + H2O2

H2O2 + 4-AAP + TOOS Quinoneimine dye + H2O

Gk

Mg +2

GPO

Peroxidase



309

Procedure:

Pipette in tubes marked Blank Standard Test

Working reagent 1000μl 1000μl 1000µl

Distilled water 10μl -- --

Standard -- 10µl --

Sample -- -- 10µl

Mix and incubate for 10 mins at 37 ºC read the absorbance of standard and

each sample at 546/670 nm on bichromatic analyzers against reagent blank.

Calculation:

Triglycerides (mg/dl) = )

E. Estimation of Serum Cholesterol31-33

Principle

The cholesterol esters are hydrolysed by enzyme cholesterol esterase to give free

cholesterol and fatty acid molecules. This free cholesterol gets oxidized in presence of

cholesterol oxidase to liberate cholset-4-en-3-one and H2O2. Liberated H2O2 by this

reaction combines with phenol and 4 aminoantipyrine in presence of peroxidase to form

red colour quinonimine complex, the intensity of which is measured at 505 nm. (490-

530) it is directly proportional to cholesterol conc. present in sample.

Reaction:

Cholesterol esters + H2O Cholesterol +fatty acids

Cholesterol + O2 Choles-4-en-3-one + H2O2

2H2O2 + Phenol + 4- Amino antipyrine Quinoneimine +4H2O

Cholesterol Estarase

Cholesterol Oxidase

Esterase Peroxidase



310

Procedure:

Reagents Blank Standard Test

Enzyme reagent 1ml 1ml 1ml

Standard - 10µl -

Sample - - 10µl

Mix well and incubate for 5 minutes at 37 ºC. Mix well and measure the absorbance

of standard and test against the reagent blank at 505 nm (490-530).

Calculation:

Cholesterol Conc. (mg/dl) =

F. Estimation of Serum HDL- Cholesterol

Preparation:

Take 0.5 ml of serum/plasma in to glass tube. Add 50 µl precipitating reagent.

Mix well, leave it at R.T. For 10 minutes. Centrifuge at 3000 r.p.m. for 10 minutes. Take

the clear Supernatant for HDL Cholesterol estimation.

Procedure:

Reagents Blank Standard Test

Enzyme reagent 1ml 1ml 1ml

Standard - 10µl -

Supernatant Sample - - 10µl

Mix well and incubate for 5 minutes at 37 ºC Measure the absorbance of HDL and

Std. at 510 nm.

Calculation:

HDL Cholesterol mg % =



311

Low density lipoprotein (LDL) and very low density lipoprotein (VLDL) values

were calculated by using Friedewalds formula as given below102

.

VLDL= TG/5

LDL= TC – (HDL + VLDL)

G. Estimation of Haemoglobin Levels34

Principle

Haemoglobin levels are estimated by using a simple method described by

Sahali’s. The haemoglobin contained in a known quantity of blood is converted into acid

haematin by means of HCl. This colour is then compared with a standard tube containing

acid haematin.

Haemoglobin + 0.1 N HCl Acid haematin

Procedure

Place 5 drops of 0.1N HCl in the graduated tube (Shale tube) and place it beside

the colour comparator. Pipette out the blood with the help of micropipette up to the mark

20 mm. transfer it immediately into the graduated tube mix it well. Add distilled water,

drop by drop, each time mixing the solution with the stirring rod. Keep adding water and

mixing until the colour of the solution matches the standard on either side. It is compared

under the natural light. Note the readings on the tube.

Estimation of Antioxidant Activity

Livers of the animals were homogenized with ice-chilled 10% Phosphate buffer

and centrifuge at 2000 rpm to 10 minutes. The supernatant liquid is used for the

estimation of following parameters.



312

A. Superoxide Dismutase Levels35

Principle

The enzyme is necessary for survival in all oxygen metabolizing cells. It is found

in the cytosol and inter membrane space of mitochondria of eukaryotic cells. It contains

copper and zinc. In normal cells, this radical alone is the precursor of hydrogen peroxide.

Superoxide dismutase scavenges the super oxide (O-2

) and thus provides a first

line defense against free radical damage. SOD’s are a family of metalloenzyme that

catalyze the dismutation of super oxide anion (O-2

) to hydrogen peroxide and molecular

oxygen in the following manner.

2H2O2 + 2O- → 2H2O + O2.

In the erythrocytes, the super oxide anion (O-2

) interacts with peroxides to form

hydroxyl radicals (OH-), which causes heamolyses in the absence of SOD activity. SOD

measurement was carried out on the ability of SOD to inhibit spontaneous oxidation of

epinephrine to adrenochrome.

Procedure

0.2 ml of the supernatant was added to 2.5 ml of 0.05M carbonate buffer

(pH 10.2) equilibrated in the spectrophotometer for 2-3 minutes. The reaction was then

initiated by the addition of 0.3 ml of freshly prepared 0.3 mM adrenaline as substrate to

the buffered-supernatant mixture which was quickly mixed by inversion and the

absorbance taken. The reference corvette contained 2.5 ml of the carbonate buffer, 0.3 ml

of the substrate and 0.2 ml of distilled water. The increase in absorbance at 420 nm due to

the adenochrome formed was monitored every 30 seconds for 120 seconds. 1 unit of

SOD activity was given as the amount of SOD necessary to cause 50% inhibition of the



313

auto-oxidation of adrenaline to adenochrome during 120 seconds. The results are

expressed as unit (U) of SOD activity per mg of tissue.

Calculation

SOD=

Unit: Units/ mg Protein.

B. Thiobarbituric Acid Reactive Substances Assay36

Procedure

The assay is based on the reaction of thiobarbituric acid (TBA) with

malondialdehyde (MDA), a breakdown product derived from many oxidized molecules.

The resulting chromogen formed is measured at its absorbance maximum 532 nm.

Briefly, to a test tube containing 0.1 ml homogenate, 1 ml of TBA reagent containing

equal proportions of 0.375% TBA, 15% TCA and 0.25 N HCl was added and placed in a

boiling water bath for 30 minutes. Then the mixture was placed in crushed ice for

10 minutes followed by centrifugation at 6000 rpm for 5 minutes. The absorbance of the

clear pink color supernatant was measured at 532 nm against appropriate blank. TBARS

concentrations of the samples were calculated using the extinction co-efficient of MDA

which is 1.56×105mmol

−1cm

−1 as 99% of TBARS is MDA.

Calculation

LPO= -

Unit: nmol MDA / min X mg protein.



314

C. Glutathione Estimation37

GSH + DNTB GS-TNB + TNB.

The assay is based on the principle of Ellman’s reaction. The sulfhydryl group of

glutathione reacts with DTNB (5,5’-dithiobis-2-nitrobenzoic acid) and produces a yellow

coloured 5-thio-2-nitrobenzoic acid (TNB). Measurement of the absorbance of TNB at

412nm provides an accurate estimation of glutathione in a sample. Briefly, 0.5ml of

homogenate is mixed with 0.1 ml of 25% TCA to precipitate proteins and centrifuged at

4000 rpm for 5 minutes. Then 0.3 ml of the supernatant was mixed with 0.5 ml of 0.1M

phosphate buffer (pH 7.4) and 0.2ml of 10 mM DTNB. This mixture was incubated for

10 minutes and the absorbance was measured at 412 nm against appropriate blanks. The

extinction coefficient (εmM

) for TNB is 14.15.

Enzyme in units/ml =-

Histopathological Study38

Processing of Isolated Pancreas

The animals were sacrificed and the pancreas of each animal was isolated. The

isolated pancreas was cut into small pieces and preserved and fixed in 10% formalin for

two days. Following this was the washing step where by the pancreas pieces were washed

in running water for about 12 hours. This was followed by dehydration with isopropyl

alcohol of increasing strength (70%, 80% and 90%) for 12 hours each. Then the final

dehydration is done using absolute alcohol with about three changes for 12 hours each.

The clearing was done by using chloroform with two changes for 15 to 20

minutes each. After clearing, the pancreas pieces were subjected to paraffin infiltration in

automatic tissue processing unit.



315

The pancreas pieces were washed with running water to remove formalin

completely. To remove the water, alcohol of increasing strengths was used since it is a

dehydrating agent. Further alcohol was removed by using chloroform and chloroform

removed by paraffin infiltration.

Embedding in Paraffin Vacuum:

Hard paraffin was melted and the hot paraffin was poured into L-shaped blocks.

The pancreas pieces were then dropped into the molten paraffin quickly and allow

cooling.

Sectioning:

The blocks were sectioned by using microtome to get sections of thickness of 5 .

The sections were taken on a micro slide on which a egg albumin (sticking substance)

was applied. The sections were allowed to remain in an oven at 60 ºC for 1 hour. Paraffin

melts and egg albumin denatures, thereby fixes tissues to slide.

Staining:

Eosin is an acid stain. Hence it stains all the cell constituents pink which are basic

in nature, eg: Cytoplasm. Haematoxylin basic stain which stains all the acidic cell

components blue eg: DNA in the nucleus.

Procedure:

1. Deparaffinized the sections by washing with chloroform for about 15 minutes.

2. Hydrate the sections by washing in isopropyl alcohol of decreasing strength

(100%, 90%, 80% and 70%).

3. Finally washed with water.

4. Stained with haematoxylin for 15 minutes.



316

5. Rinsed in tap water.

6. Differentiated in 1% acid alcohol by 10 quick dips. Checked the differentiation

with a microscope. Nuclei were distinct and the back ground was very light (or

colourless).

7. Washed in tap water.

8. Dipped in (Lithium carbonate) until sections become bright blue (3-5 dips).

9. Washed in running tap water for 10 to 20 minutes, if washing is inadequate eosin

will not stain evenly.

10. Stained with eosin for 15 seconds – 2 minutes depending on the age of the eosin

and the depth of the counter stain desired. For even staining results, dip slides

several times before allowing them to set in the eosin for the desired time.

11. Dehydrated in 95% isopropyl and absolute isopropyl alcohol until excess eosin is

removed, 2 changes of 2 minutes each (check under microscope).

12. An absolute isopropyl alcohol 2 changes of 3 minutes each.

13. Chloroform 2 changes of 2 minutes each.

14. Mounted in DPX (Desterene dibutyl phthalate xylene).

Results:

Nuclei - Blue colour

Cytoplasm - Various shades of pink identifying different tissue components.

All the sections of the tissues were examined under microscope for the analyzing

the altered architecture of the pancreas tissue due to Streptozotocin treatment and

improved pancreas architecture due to pretreatment with test extracts and standard drug.



317

Statistical Analysis:

The values are expressed as Mean ± SEM. The data was analysed by using one

way ANOVA followed by Dunnett’s test using Graph pad prism software. Statistical

significance was set at P ≤ 0.05.



318


Acute Toxicity Studies (LD50)

In both phase I and Phase II procedures, none of the animals show any toxicity

upon the single administration of HEFW, EAEFW, MEFW and AEFW (2000 mg/kg).

Hence the procedure is repeated by increasing the dose of extracts (3000 mg/kg). None of

the animals had shown any toxicity. Thus, 1/10th

of maximum dose (300 mg/kg) tested

was selected for the present study.

Anti diabetic activity

Single Dose Study in Normal Rats

Hypoglycaemic activity of HEFW, EAEFW, MEFW and AEFW were studied on

normal rats and the results were tabulated in Table 8 and showed in Figure 5.

HEFW shows its action at time interval of 2 and 4 hour (P<0.05) and a much

reduced activity at the time interval of 8, 12, 24 hours (P <0.01). But it has no significant

activity at the time interval of 1st hour.

EAEFW shows its action at time interval 4, 8 and 12th

hr (P<0.05) and a much

reduced activity at the time interval 24 hr (P <0.01). But it has no significant activity at

the time interval of 1st and 2

nd hours when compared to control.

MEFW shows a significant reduction in the blood glucose levels at 2, 4, 8, and 12

and 24 hours, onset of action is shown at 1hr after the treatment. Less significant

reduction in blood glucose levels was shown at 1st hour (P<0.05). A maximum blood

glucose reduction was shown at 24th

hour (P<0.001) compared to the normal untreated

group.

AEFW shows a significant action in reducing the blood glucose levels at the time

interval of 2 hour (P<0.05) after the treatment. Less significant reduction in blood



319

glucose levels was shown at 4th

hour (P<0.01). It also show a marked decrease in the 8,

12 and 24 hours (P<0.001). But it has no significant activity at the time interval of 1 hour

when compared to control.

Glibenclamide showed it effect from 1 hour after treatment the onset of

Glibenclamide starts from 1 hour after the treatment. It reduces maximum blood glucose

levels at 12 hours (P<0.001). Glibenclamide significantly reduced the blood glucose

levels after treatment in normal rats. All the blood glucose levels of treated group were

compared with the normal control group animals.

Single dose study for 24 hours was carried out in normoglycemic rats. MEFW and

AEFW showed maximum decrease in blood glucose levels at 24th

hour compared to

normal group. MEFW also showed a significant decrease from the 1st hour of the drug

administration. AEFW showed a significant decrease in blood glucose level at 24th

hour

compared to normal levels. It also showed its activity from the 2nd

hour after the drug

administration when compared with the normal control.

Glibenclamide (5mg/kg) showed a maximum decrease of blood glucose levels in

normoglycemic rats at 24th

hour of our study. But HEFW and EAEFW did not show its

effect for 24 hours when compared with the normal rats. It may produce hypoglycemia in

normal animals by stimulating the pancreatic beta-cells to produce more insulin and by

increasing the glycogen deposition in the liver39

.



320

Table 8: Effect of Flemingia wightiania Plant Extracts on Blood Glucose Levels on Single Dose Study in Normal Rats

Groups Treatment Blood Glucose Levels (mg/dl)

0 1 2 4 8 12 24

Group-I Saline 94.33 ± 3.63 94.33 ± 4.08 91.50 ± 2.79 89.67 ± 2.29 84.67 ± 3.93 81.33 ±

3.11

78.83 ±

2.04

Group-II Glibenclamide

(5mg/kg) 89.50 ± 4.53

72.50 ±

1.97***

54.17 ±

4.96***

56.83 ±

2.35***

46.50 ±

4.27***

47.33 ±

4.12***

43.00 ±

4.12***

Group-III HEFW

(300mg/kg) 84.17 ± 3.88 82.67 ± 3.77

ns 76.33 ± 3.91* 77.00 ± 2.87*

69.00 ±

1.31**

65.83 ±

2.1**

63.00 ±

2.35**

Group-IV EAEFW

(300mg/kg) 82.67 ± 2.96 82.00 ± 1.48

ns 85.67 ± 4.49

ns 78.83 ± 2.57*

73.00 ±

2.80*

69.00 ±

2.62*

61.50 ±

2.62**

Group-V MEFW

(300mg/kg) 82.33 ± 4.89 79.17 ± 3.76*

71.33 ±

2.43**

68.67 ±

2.57***

63.33 ±

2.18***

60.67 ±

1.85***

58.67 ±

1.54***

Group-VI AEFW

(300mg/kg) 86.83 ± 5.90 81.50 ± 4.93

ns 76.17 ± 4.14* 73.83 ± 3.91**

66.67 ±

2.43***

63.33 ±

2.43***

59.33 ±

2.30***

Values are Mean ± SEM (n=6) one way ANOVA followed by Dunnett’s test. Where, *** P<0.001, ** P<0.01, * P<0.05 and

ns represents Not significant.







321

Fastin

g1

hr2

hr4

hr8

hr

12 h

r

24 h

r

0

50

100

150


Group V Group VI

Time in Hours

Blo

od

Glu

cose

Lev

els

(mg

/dl)

Figure 5: Effect of Flemingia wightiania Plant Extracts on Blood Glucose Levels on Single Dose Study in Normal Rats



322

Oral Glucose Tolerance Test in Normal Rats

The effect of HEFW, EAEFW, MEFW and AEFW on glucose tolerance test was

tabulated in the Table 9 and shown in Figure 6.

HEFW show a significant decrease in blood glucose levels, when administration

30 minutes before glucose loading. It showed a significant activity at the time intervals of

2 and 3 hour (P< 0.01). Significant reduction was more at 3rd

hour when compared with

the 2nd

hour.

Most significant decrease in blood glucose levels was observed when the MEFW

was administered 30 minutes before glucose loading. Where as a most significant

reduction was more at 3rd

hour (P<0.001) when compared with the 1 and 2nd

hour.

Where as AEFW also show a significant decrease blood glucose levels, when

administration 30 minutes before glucose loading. It showed a very less significant

activity at the time intervals of 1and 2nd

hour (P< 0.01) when compared with 3rd

hour (P<

0.001).

But EAEFW showed a much reduced activity (P<0.05) compare with the

Methanol, Aqueous and n-Hexane extract. It showed its effect at 30 minutes after glucose

administration.

Glibenclamide showed its potent antidiabetic activity in normal rats it bring backs

the elevated blood glucose levels to normal levels compared to normal control group at

3rd

hour (P<0.001).

Overall the different extracts of Flemingia wightiania had showed a significant

decrease in the blood glucose levels when compared with the normal control group rats at

time intervals 1, 2 and 3rd

hour.



323

Oral glucose tolerance test was studied on the normal rats. The lowering of

glucose can be better seen in assay of glucose tolerance40

. The fasting blood glucose

levels decreases in glibenclamide, MEFW and AEFW treated rats. HAFW and EAEFW

shows a reduced activity. Such a phenomenon was already seen in the indigenous plants

and reported41,42

. The lowering of glucose levels is may be due to the inhibition of

intestinal absorption, or it may act by potentiating the secretion of insulin and by increase

in the utilization of glucose levels in muscles43

.



324

Table 9: Effect of Flemingia wightiania Plant Extracts on Blood Glucose Levels on Oral Glucose Tolerance Test in

Normal Rats

Groups Treatment

Blood Glucose Levels (mg/dl) and Time in hr

0 1 2 3

Group-I Saline 87.17 ± 5.12 133.0 ± 4.38 109.7 ± 3.97 95.17 ± 6.15

Group-II Glibenclamide (5mg/kg) 88.50 ± 2.83 103.3 ± 2.67*** 79.50 ± 2.55*** 60.00 ± 3.55***

Group-III HEFW (300mg/kg) 79.67 ± 2.23 118.0 ± 5.43ns

90.17 ± 4.66** 75.00 ± 3.38**

Group-IV EAEFW (300mg/kg) 79.00 ± 2.51 120.5 ± 4.07ns

97.33 ± 3.47ns

78.00 ± 4.78*

Group-V MEFW (300mg/kg) 81.67 ± 2.14 108.0 ± 3.88** 83.33 ± 4.06*** 67.50 ± 1.76***

Group-VI AEFW (300mg/kg) 80.33 ± 3.36 112.2 ± 5.08** 88.17 ± 4.32** 69.00 ± 3.72***









325

0 hr

1 hr

2 hr

3 hr

0

50

100

150


Group V Group VI

Time in Hours

Blo

od

Glu

cose

Lev

els

(mg

/dl)

Figure 6: Effect of Flemingia wightiania Plant Extracts on Blood Glucose Levels on Oral Glucose Tolerance Test in

Normal Rats



326

Effect of Extracts in Streptozotocin Induced Diabetic Rats

A chronic study of 21 days was done in STZ induced diabetic rats with Flemingia

wightiania plant extracts and the results of blood glucose levels are tabulated in the

Table 10 and they are also shown in Figure 7.

Blood glucose levels on day zero showed no significant intra group variation.

Administration of streptozotocin (45 mg/kg, i.p.) in 0.01M citrate buffer (pH 4.5) showed

a significant increase in fasting blood glucose levels (387.2 ± 28.41mg/dl). After 21 days,

diabetic control rats exhibited significantly higher blood glucose levels (331.7 ± 10.19

mg/dl) as compared to the normal control rats (106.0 ± 5.13 mg/dl).

Diabetic rats treated with the HEFW also shown a significant activity when

compared with the diabetic control rats. Blood glucose levels on 21 day was 159.8 ±

11.47 which is less when compared with the initial blood glucose level 392.8 ± 25.04 on

0 day of treatment.

Diabetic rats treated with a MEFW for 21 days lowers the blood glucose levels

from 392.3 ± 17.28 to 123.5 ± 5.23 (P<0.001). It also shows a significant activity in

decreasing the blood glucose levels on 7th

and 14th

day. (317.8 ± 13.52 and 218.0 ± 9.76).

Similarly diabetic rats treated with the AEFW also shown a significant activity

when compared with the diabetic control rats. Blood glucose levels on 21 day was 133.0

± 9.14 which is less when compared with the initial blood glucose level 376.8 ± 16.45 on

0 day of treatment.

A daily treatment of EAEFW for a period of 21 days lowers the blood glucose

levels in diabetic treated rats. Blood glucose levels on 7th

and 14th

day (341.2 ± 9.76 and



327

270.2 ± 12.75 respectively) of treatment also show a significant reduce in blood glucose

levels when compared with the diabetic control group of animals.

Glibenclamide showed its potent antidiabetic activity and reduced the blood

glucose levels of diabetic rats to the level significantly (399.8 ± 25.04 to 109.0 ± 6.14) at

day 21. It elevated blood glucose levels on 7th

and 14th

day were 294.0 ± 23.90 and 194.0

± 13.06 respectively. Which is having a high significant activity P<0.001.

Streptozotocin (STZ), a broad spectrum antibiotic has been widely used for

inducing the diabetes mellitus in animals. STZ enters the β-cell via a glucose transporter

(GLUT2) and cause alkylation of DNA. DNA damage induces activation of poly ADP-

ribosylation leads to depletion of cellular NAD+

and ATP. Increased ATP-

dephosphorylation causes release of superoxide radicals, consequently hydrogen peroxide

and hydroxyl radicals are also generated. And also a toxic amount of nitric oxide44

was

liberated after the treatment of STZ which inhibits the aconitase activity and participates

in DNA damage. As a result of STZ action, β-cells undergo the destruction by necrosis45

.

As it was explained, that STZ damages β-cells in pancreas there is increased

blood glucose levels in STZ treated rats. A prolonged treatment (21 days) of diabetic rats

with the HAFW, EAEFW, MEFW and AEFW showed a significant reduction in the

blood glucose levels than the untreated rats. This hypoglycaemic activity may be due to

the stimulation of surviving β-cells to release more insulin. Flemingia wightiania may act

by inhibiting hepatic gluconeogenesis or inhibiting α-glucosidase enzyme in the intestine,

which is the enzyme helpful for breakdown of disaccharides to form glucose46

.



328

Table 10: Effect of Flemingia wightiania Plant Extracts on Blood Glucose Levels in STZ Induced Diabetic Rats

Groups Treatment Blood Glucose Levels (mg/dl)

0 day 7 day 14 day 21 day

Group-I Saline 95.83 ± 5.58 92.67 ±5.62*** 98.50 ± 5.81*** 106.0 ± 5.13***

Group-II Saline + STZ (45mg/kg) 387.2 ± 28.41 398.7 ± 22.90 366.5 ± 12.30 331.7 ± 10.19

Group-III Glibenclamide (5mg/kg) + STZ (45mg/kg) 399.8 ± 25.04 294.0 ± 23.90*** 194.0 ± 13.06*** 109.0 ± 6.14***

Group-IV HEFW(300mg/kg) + STZ (45mg/kg) 392.8 ± 25.04 329.2 ± 14.75* 244.2 ± 12.75*** 159.8 ± 11.47***

Group-V EAEFW (300mg/kg) + STZ (45mg/kg) 383.8 ± 11.49 341.2 ± 9.76ns

270.2 ± 12.75*** 192.2 ± 9.76***

Group-VI MEFW (300mg/kg) + STZ (45mg/kg) 392.3 ± 17.28 317.8 ± 13.52** 218.0 ± 9.76*** 123.5 ± 5.23***

Group-VII AEFW (300mg/kg) + STZ (45mg/kg) 376.8 ± 16.45 324.0 ± 15.10* 232.3 ± 17.00*** 133.0 ± 9.14***



All the values are compared with the diabetic control group.







329

0 Day

7 Day

14 D

ay

21 D

ay

0

100

200

300

400

500


Group V Group VI Group VII

Blo

od

Glu

co

se L

evels

(m

g/d

l)

Figure 7: Effect of Flemingia wightiania Plant Extracts on Blood Glucose Levels in STZ Induced Diabetic Rats



330

Effect of extract on Physical Parameters of STZ induced diabetic rats:

i. Body Weight

There is a significant change seen in the body weight of animals after the

treatment inducing diabetes with STZ. The decreased body weight of the animals were

significantly regains when compare with the diabetic control animals after treatment for

21 days with the extract. And also the body weight of normal control group was

significantly increased compared to initial body weight. The changes in body weight of

the animals during 0, 7, 14 and 21 days was tabulated in the Table 11 and shown in

Figure 8.

ii. Wet Liver Weight

Rats treated with STZ shown a decrease in the liver weight of untreated diabetic

rats, where as in treated rats there is a significant restoration of wet liver weight which is

near to the normal levels. The values of the wet liver weight were tabulated in the

Table 11 and shown in Figure 9.

Induction of diabetes with STZ is associated with a characteristic reduce in body

weight than the normal rats, this may be due to the wasting and loss of tissue protein.

Whereas, diabetic rats treated with HAFW, EAEFW, MEFW and AEFW showed an

improved result when compared with normal diabetic control. Which may be due to the

protective effect in controlling muscle wasting i.e., reversal of gluconeogenesis and may

also be due to the improvement of glycemic control47

.

A decrease in the liver weight observed in diabetic animals, this is may be due to

insufficient release of insulin may causes decrease in the storage of glucose as glycogen



331

in liver. After 21 days of treatment with Flemingia wightiania in diabetic animals, an

increased in the liver weight is observed than the untreated rats. A less significant

increase in liver weight is seen in HEFW treated diabetic rats. EAEFW shows did not

show significant increase in liver weight is seen in treated diabetic rats. This result is may

be the extract is having insulin like action which is reported as by Viswanathan et al48

.



332

Table 11: Effect of Flemingia wightiania Plant Extracts on Body Weight and Liver Weight in STZ Induced Diabetic Rats

Groups Treatment

Body Weight (gm) Liver Weight (gm)

0 Day 7 Day 14 Day 21 Day Wet liver

weight

Weight/100gm

body Wt

Group-I Saline 181.7 ± 5.11 185.0 ± 3.35*** 190.7 ± 4.42*** 194.7 ± 4.16*** 7.35 ± 0.42*** 3.77 ± 0.19***

Group-II Saline + STZ

(45mg/kg)

170.0 ± 3.89 143.0 ± 2.89 141.3 ± 0.76 145.3 ± 2.57 3.79 ± 0.17 2.54 ± 0.12

Group-III Glibenclamide

(5mg/kg) + STZ

(45mg/kg)

178.2 ± 2.58 169.0 ± 3.64*** 180.5 ± 4.10*** 186.2 ± 2.30*** 6.68 ± 0.33*** 3.59 ± 0.20***

Group-IV HEFW(300mg/kg) +

STZ (45mg/kg)

172.5 ± 4.91 158.3 ± 3.82ns

166.5 ± 3.99** 172.0 ± 3.38*** 5.39 ± 0.15** 3.13 ± 0.06ns

Group-V EAEFW (300mg/kg)

+ STZ (45mg/kg)

176.2 ± 5.14 157.8 ± 5.33ns

164.2 ± 6.04** 166.0 ±6.85** 4.94 ± 0.20* 3.03 ± 0.14ns

Group-VI MEFW (300mg/kg) +

STZ (45mg/kg)

173.3 ± 4.46 164.5 ± 3.67** 172.7 ± 3.53*** 180.5 ± 4.10*** 6.22 ± 0.28*** 3.46 ± 0.22**

Group-VII AEFW (300mg/kg) +

STZ (45mg/kg)

177.7 ± 5.28 161.5 ± 5.05* 170.2 ± 5.00*** 176.0 ± 3.98*** 5.50 ± 0.66*** 3.10 ± 0.16ns


represents Not significant. All values are compared with diabetic control.



333

0 Day

7 Day

14 D

ay

21 D

ay

120

140

160

180

200

220



Bo

dy w

eig

ht

(gm

s)

Figure 8: Effect of Flemingia wightiania Plant Extracts on Body Weight and Liver Weight in STZ Induced Diabetic



334

Gro

up I

Gro

up II

Gro

up III

Gro

up IV

Gro

up V

Gro

up VI

Gro

up VII

0

2

4

6

8

10

Liv

er W

eig

ht

(gm

)

Figure 9: Effect of Flemingia wightiania Plant Extracts on Liver Weight of STZ

Induced Diabetic Rats

Effect of extract on Biochemical Parameters of STZ induced diabetic rats:

i. Serum Albumin levels

Serum albumin levels were decreased in the diabetic animals, as compared with

the normal control animals. Whereas albumin levels in the diabetic control group is

1.74 ± 0.11 g/dl. But albumin levels after treatment with the Flemingia wightiania Plant

extracts shows an increased the serum albumin levels. The values of the albumin levels

are mentioned in the Table 12 and shown in Figure 10.



335

Table 12: Effect of Flemingia wightiania Plant Extracts on Serum Albumin Levels in

STZ Induced Diabetic Rats

Groups Treatment Serum Albumin Levels

(g/dl)

Group-I Saline 3.63 ± 0.09***

Group-II Saline + STZ (45mg/kg) 1.74 ± 0.11

Group-III Glibenclamide (5mg/kg) + STZ (45mg/kg) 3.32 ± 0.11***

Group-IV HEFW(300mg/kg) + STZ (45mg/kg) 3.01 ± 0.22***

Group-V EAEFW (300mg/kg) + STZ (45mg/kg) 2.52 ± 0.10**

Group- VI MEFW (300mg/kg) + STZ (45mg/kg) 3.32 ± 0.09***

Group- VII AEFW (300mg/kg) + STZ (45mg/kg) 3.268 ± 0.16***



All values are compared with diabetic control.

Gro

up I

Gro

up II

Gro

up III

Gro

up IV

Gro

up V

Gro

up VI

Gro

up VII

0

1

2

3

4

Alb

um

in L

evels

mg

/d

Figure 10: Effect of Flemingia wightiania Plant Extracts on Serum Albumin Levels

in STZ Induced Diabetic Rats

ii. Serum Urea Levels

Diabetic rats showed an increased in the levels of serum urea. Treatment of these

rats with the extract and glibenclamide showed a decrease in the urea levels when

compared with the normal animals. The urea levels in the diabetic rats are 47.98 ± 2.46



336

g/dl, where it is decrease to 31.65 ± 1.09g/dl in treated group and 29.41 ± 0.98 g/dl in

glibenclamide treated rats. Serum Urea levels of treated and Normal rats are expressed in

the Table 13 and shown in Figure 11.

Table 13: Effect of Flemingia wightiania Plant Extracts on Serum Urea Levels in


Groups Treatment Serum Urea Levels

(mg/dl)




Group-IV HEFW(300mg/kg) + STZ (45mg/kg) 39.22 ± 1.20**







Gro

up I

Gro

up II

Gro

up III

Gro

up IV

Gro

up V

Gro

up VI

Gro

up VII

0

20

40

60

Ure

a L

evels

mg

/dl

Figure 11: Effect of Flemingia wightiania Plant Extracts on Serum Urea Levels in




337

iii. Serum Total Protein

Serum protein levels are decreased (5.21 ± 0.20 g/dl) in the untreated diabetic rats

compares to the normal control rats (8.64 ± 0.31 g/dl). After treatment with the

Glibenclamide, MEFW, AEKT and HEFW showed a significant increase in the serum

protein levels compared with the diabetic control animals. EAEFW showed a less

significant activity in increasing the protein levels. The values of serum total protein

levels are shown in the Table 14 and Figure 12.

Table 14: Effect of Flemingia wightiania Plant extract on Serum Total Protein levels

in STZ induced diabetic rats

Groups Treatment Serum Total Protein

Levels (mg/dl)













338

Gro

up I

Gro

up II

Gro

up III

Gro

up IV

Gro

up V

Gro

up VI

Gro

up VII

0

2

4

6

8

10

Seru

m P

roti

en

Levels

mg

/dl

Figure 12: Effect of Flemingia wightiania Plant extracts on Serum Total Protein

levels in STZ induced diabetic rats

A marked reduction in the levels of total protein and albumin levels was observed

in the diabetic rats. The decrease in the albumin levels is due to the increased protein

catabolism. Present study shows that the treatment of diabetic rats with the MEFW,

AEFW and glibenclamide showed a significant increase in the levels of albumin and

protein levels compared to the normal untreated diabetic rats.

An increased plasma urea levels was found in the diabetic rats when compared

with the respective control group rats. While after the treatment with Flemingia

wightiania were significantly decreased. Similar observations are also reported in the

D-400 herbal formulation49

.

iv. Haemoglobin Levels

A daily dose of the extracts for a period of 21 days showed an increased in the

haemoglobin level of diabetic rats. But the EAEFW showed a less significant activity

when compared with the diabetic control rats. Glibenclamide restores the haemoglobin



339

levels to normal levels after treatment. The values are tabulated in the Table 15 and

shown in Figure13.

The haemoglobin levels of the diabetic group of rats were found to be reduced

significantly as against the normal haemoglobin levels of the normal group of rats; this is

due to an increased formation of its glycosylated form. During hyperglycaemic condition

protein synthesis is attenuated or reduced in all the tissues and thus the synthesis of

haemoglobin also reduced50

.

Table 15: Effect of Flemingia wightiania Plant Extracts on Haemoglobin Levels in


Groups Treatment Haemoglobin

(mg/dl)







Group- VII AEEW (300mg/kg) + STZ (45mg/kg) 11.80 ± 0.27***






340

Gro

up I

Gro

up II

Gro

up III

Gro

up IV

Gro

up V

Gro

up VI

Gro

up VII

0

5

10

15

Hae

mo

glo

bin

(m

g/d

l)

Figure 13: Effect of Flemingia wightiania Plant Extracts on Haemoglobin Levels in


v. Serum Lipid Profile

The lipid profile was evaluated by estimating triglycerides (TG), total cholesterol

(TC), HDL-Cholesterol (HDL-C), LDL-Cholesterol (HDL-C), VLDL-Cholesterol

(VLDL-C) in normal and diabetic animals. The STZ diabetic animals showed a

significant increased in the TG, TC, LDL-C and VLDL-C levels and suppression of

HDL-C levels compared to control group (Table 16 and Figure 14). But after treatment

with the root extracts and glibenclamide diabetic animals Showed decrease in the TG,

TC, LDL-C and VLDL-C levels and increase in the HDL-C levels compared to untreated

diabetic rats.

Under normal condition, insulin activates enzyme lipoprotein lipase and

hydrolyses triglycerides. Insulin deficiency results in failure to activate the enzymes there

by causing hypertriglyceridemia51

. This altered lipid metabolism leads to diabetic

complications. Practically it has been observed that there is an altered in levels of serum

cholesterol and triglycerides levels in STZ treated rats, causes hypercholesterolemia and

hyper-triglyceridemia52

.



341

Table 16: Effect of Flemingia wightiania Plant Extracts on Serum Lipid Profile of STZ Induced Diabetic Rats

Groups Treatment Serum Lipid Profile mg/dl

TC TG HDL-C LDL-C VLDL-C

Group-I Saline 62.92 ± 1.08*** 74.22 ± 4.69*** 23.48 ± 0.42*** 24.59 ± 0.90*** 14.84 ± 0.92***

Group-II Saline + STZ (45mg/kg) 120.4 ± 3.15 139.8 ± 10.03 15.77 ± 0.72 76.69 ± 3.21 27.96 ± 2.00

Group-III Glibenclamide (5mg/kg) +

STZ (45mg/kg)

74.85 ± 0.81*** 75.29 ± 4.91*** 22.23 ± 0.82*** 37.56 ± 0.62*** 15.06 ± 0.98***

Group-IV HEFW(300mg/kg) + STZ

(45mg/kg)

96.77 ± 2.48*** 92.44 ± 5.24*** 19.36 ± 0.48*** 59.98 ± 2.47*** 18.49 ± 1.05***

Group-V EAEFW (300mg/kg) +

STZ (45mg/kg)

97.66 ± 2.66*** 112.7 ± 4.545* 18.19 ± 0.27* 56.92 ± 3.03*** 22.54 ± 0.90*

Group- VI MEFW (300mg/kg) + STZ

(45mg/kg)

75.97 ± 1.82*** 85.14 ± 5.95*** 21.47 ± 0.42*** 37.39 ± 2.64*** 17.03 ± 1.91***

Group- VII AEEW (300mg/kg) + STZ

(45mg/kg)

81.25 ± 2.71*** 85.23 ± 2.04*** 19.53 ± 0.47*** 44.64 ± 2.95*** 17.05 ± 0.41***






342

Total C

holest

erol

Trigly

cerid

es

HDL-C

LDL-C

VLDL-C

0

50

100

150

200



Ser

um

Lev

els

(mg

/dl)

Figure 14: Effect of Flemingia wightiania Plant Extracts on Serum Lipid Profile of STZ Induced Diabetic Rats



343

Effect of Extracts on Antioxidant Levels

i. Superoxide Dismutase Levels

Diabetic rats exhibited significant lower SOD (8.38 ± 0.46) as compared to those

of control rats (13.70 ± 0.39) treatment with the root extract significantly elevated the

reduce SOD levels. MEFW, AEFW, HEFW, EAEFW and Glibenclamide showed a

marked decrease in the SOD levels (P< 0.001) compared to the diabetic control. These

values are tabulated in the Table 17 and Figure 15.

Superoxide dismutase is an enzymatic antioxidant which reduces superoxide

radical to hydrogen peroxide and oxygen. A decrease in the antioxidant activity in liver

results in the accumulation of free radicals (hydroxyl radical) in diabetic rats.

Administration of the HEFE, EAEFE, MEFW, AEFW and glibenclamide increased the

activity of SOD levels to a significant level of P<0.001. While the SOD levels of

untreated diabetic control rats having lowered levels. The Flemingia wightiania may act

by either directly scavenging the reactive oxygen metabolites or by increasing the anti-

oxidant molecules.

Table 17: Effect of Flemingia wightiania Plant Extracts on SOD Levels in STZ


Groups Treatment SOD U/mg

protein





Group-V EAEFW (300mg/kg) + STZ (45mg/kg) 10.96 ± 0.32***





344




Gro

up I

Gro

up II

Gro

up III

Gro

up IV

Gro

up V

Gro

up VI

Gro

up VII

0

5

10

15

Su

pero

xid

e D

ism

uta

se

(U/m

g P

ro

tein

)

Figure 15: Effect of Flemingia wightiania Plant Extracts on SOD Levels in STZ


ii. Thiobarbuturic Acid Reactive Substance Levels (TBARS)

Rats treated with STZ had a TBARS level of 3.67 ± 0.26 nmoles of MDA/ 100

mg of tissue when measured on day 21. This was significantly higher when compared to

levels in normal control rats of 0.93 ± 0.05 nmoles of MDA/ 100 mg of tissue.

Diabetic rats treated with MEFW had a lipid peroxidation levels of 1.49 ± 0.09

nmoles of MDA/ 100 mg of tissue and also rats treated with the AEFW, HEFW and

EAEFW treated rats having a TBARS levels of 1.74 ± 0.16, 1.91 ± 0.11 and 2.12 ± 0.14

nmoles of MDA/ 100 mg of tissue. Where as in glibenclamide treated rats the levels are

restored to normal levels of 1.27 ± 0.08 nmoles of MDA/ 100 mg of tissue. These values

are expressed in Table 18 and shown in Figure 16.



345

In diabetes, lipid peroxidation is one of the characteristic features of chronic

diabetes. The increased free radicals react with the polyunsaturated fatty acids in cell

membrane leading to lipid peroxidation. This in turn results in the development of free

radicals. Low levels of lipoxygenase peroxides stimulate the release of insulin. But, if the

concentration of this peroxidase increases it results in uncontrolled release of lipid

peroxidation. The most commonly used indicator of lipid peroxidation is TBARS. There

was a significant elevation of TBARS in liver tissue in diabetic control animals when

compared to the normal rats. Administration of HEFE, EAEFE, MEFW, AEFW and

glibenclamide significantly reduce the TBARS levels. The effect shown is may be due to

prevention of potential glycation of anti-oxidant enzymes.

Table 18: Effect of Flemingia wightiania Plant Extracts on TBARS Levels in STZ


Groups Treatment

TBARS (nmoles of

MDA/ 100 mg of

tissue)

Group-I Saline 0.93 ± 0.05 ***


Group-III Glibenclamide (5mg/kg) + STZ (45mg/kg) 1.27 ± 0.08 ***

Group-IV HEFW(300mg/kg) + STZ (45mg/kg) 1.91 ± 0.11 ****

Group-V EAEFW (300mg/kg) + STZ (45mg/kg) 2.12 ± 0.14 ***








346

Gro

up I

Gro

up II

Gro

up III

Gro

up IV

Gro

up V

Gro

up VI

Gro

up VII

0

1

2

3

4

5

nm

ol o

f M

DA

/ 100 m

g o

f T

issu

e

Figure 16: Effect of Flemingia wightiania Plant Extracts on TBARS Levels in STZ


iii. Glutathione Estimation

Rats treated with STZ had a tissue GSH level of 29.46 ± 2.80 mM/ 100 mg of

tissue when measured on day 21. Treatment with MEFW, AEFW, HEFW and EAEFW

show a decrease GSH levels in STZ treated rats. These values are having a significant

higher (P<0.001) when compared to GSH levels in diabetic control rats. The values are

tabulated in the Table 19 and shown in Figure 17.

Glutathione which is a tripeptide normally present at high concentrations

intracellularly. Glutathione is helpful for reducing the toxic effects of lipid peroxidation.

Decreased level of GSH in liver during diabetes represents its increased utilization due to

oxidative stress. Significant increased levels of GSH were shown in the diabetic rats

treated with the HEFE, EAEFE, MEFW, AEFW and glibenclamide.



347

Table 19: Effect of Flemingia wightiania Plant Extracts on GSH Levels in STZ


Groups Treatment GSH (mM/ 100 mg of

tissue)

Group-I Saline 42.88 ± 0.77 ***


Group-III Glibenclamide (5mg/kg) + STZ (45mg/kg) 41.06 ± 0.61 ***

Group-IV HEFW(300mg/kg) + STZ (45mg/kg) 37.10 ± 1.03 ****

Group-V EAEFW (300mg/kg) + STZ (45mg/kg) 36.23 ± 0.074 ***






Gro

up I

Gro

up II

Gro

up III

Gro

up IV

Gro

up V

Gro

up VI

Gro

up VII

0

10

20

30

40

50

mM

/ 100 m

g o

f T

issu

e

Figure 17: Effect of Flemingia wightiania Plant Extracts on GSH Levels in




348

Histopathological Study of Pancreas

i. Group –I (Normal Control + Saline)

Section studied shows pancreatic lobules separated by connective tissue septa.

The pancreatic lobules consist largely of the exocrine acini and their intralobular ducts.

Most of the lobules show small, round, light-staining islets of langerhans. The centre of

islet cells consist of aggregates of small Beta-cells (70%, Short-arrow) having basophilic

granules, while the periphery comprises of large Alpha-cells (25%, Long-arrow) having

eosinophilic granules. Intervening these cells are seen thin walled capillaries.

ii. Group –II (Diabetic Control + Streptozotocin 45 mg/kg)


The number of islets appears reduced in number. The center of islet cells consist of

quantitative decrease in Beta-cells (30%, Long-arrow) having basophilic granules, while

the periphery comprises of large Alpha-cells (65%, Short-arrow) having eosinophilic

granules. Also seen are some degenerated beta cells and lymphocytic infiltration amidst

these islet cells.

iii. Group –III (Streptozotocin [45mg/kg] + Glibenclamide [5mg/kg])


Most of the lobules show large areas of light-staining islets of langerhans. The center of

islet cells consist of Beta-cells (60%, long-arrow) having basophilic granules, while the

periphery comprises of Alpha-cells (35%, short-arrow) having eosinophilic granules.

Also seen are few congested vascular spaces amidst these islet cells.



349

iv. Group – IV (Streptozotocin [45mg/kg] + n-Hexane extract [300mg/kg])


The pancreatic lobules consist largely of the exocrine acini and their intralobular ducts.

Most of the lobules show small, round, light-staining islets of langerhans. The center of

islet cells consist of aggregates of small Beta-cells (60%, Short-arrow), while the

periphery comprises of large Alpha-cells (35%, Long-arrow). Intervening these cells are

seen thin walled capillaries.

v. Group – V (Streptozotocin [45mg/kg] + Ethyl acetate extract [300mg/kg])


The number of islets appears reduced in number. The center of islet cells consist of

quantitative decrease in Beta-cells (55%, long-arrow), while the periphery comprises of

large Alpha-cells (40%, short-arrow). Also seen are some degenerated beta cells.

vi. Group – VI (Streptozotocin [45mg/kg] + Methanol extract [300mg/kg])


Most of the lobules show large areas of light-staining islets of langerhans. The center of

islet cells consist of quantitative increase in Beta-cells (70%, short-arrow), while the

periphery comprises of Alpha-cells (25%, long-arrow). Also seen are congested vascular

spaces amidst these cells.

vii. Group – VI (Streptozotocin [45mg/kg] + Aqueous extract [300mg/kg])


The center of islet cells consist of small Beta-cells (65%, Short - arrow), while the

periphery comprises of large Alpha-cells (30%, Long - arrow).



350

The histological evidence shown the authenticated injury caused by STZ and the

protection offered by HEFE, EAEFE, MEFW, AEFW and glibenclamide in pancreatic

cells are shown. Microscopically examination revealed loss of architecture and cell

necrosis with inflammatory collections in the central zone in STZ induced rats.

Histopathological study shows that Flemingia wightiania has the capacity to increase

islet cell mass. However, the expansion was better with MEFW and AEFW.

Glibenclamide a sulfonylurea derivative shows its action by stimulating the

pancreatic β-cells. This drugs act sensitive potassium (K ATP) channel present on the β-

cell membrane causes depolarization and triggers the opening of voltage dependent

calcium channels. This influx causes release of insulin from the stored granules in β-islet

cells of pancreas. In the same way the plant Flemingia wightiania also may have a similar

action in treatment of diabetes53

.

Group I Group II

Group III Group IV



351

Group V Group VI

Group VII

Figure 18: Histopathology of Pancreas

Group –I (Normal control- Saline)

Group –II (Diabetic control-Streptozotocin 45 mg/kg)

Group –III (Streptozotocin [45mg/kg] + Glibenclamide [5mg/kg])

Group – IV (Streptozotocin [45mg/kg] + HEFW [300mg/kg])

Group – V (Streptozotocin [45mg/kg] + EAEFW [300mg/kg])

Group – VI (Streptozotocin [45mg/kg] + MEFW [300mg/kg])

Group – VII (Streptozotocin [45mg/kg] + AEFW [300mg/kg])


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Summary

358

SUMMARY

Chapter I

The general introduction of natural chemistry and importance of present research

work was given and also brief introduction on phyto constituents were presented.

Introduction to plants sources of drugs and some important medicinal plants used drugs

and their traditional use in treating diseases.

Chapter II

In this chapter gives detailed literature survey that is made on the selected plats

Mollugo oppositifolia, Smilax Perfoliata and Flemingia wightiania. A careful study of

literature revealed that on work is done on the whole plant of Mollugo oppositifolia. A

brief review on smilax species which includes phytochemical constituents, biological

activities and uses previously reported from other species and also literature review on

smilax perfoliata were included literature. Regarding the plant Flemingia wightiana the

literature study revealed that phytochemical constituents, biological activities and uses

previously reported from the species.

Chapter III

Chapter-III deals with the present work on Mollugo oppositifolia. The process of

extraction from the dried plant material of Mollugo oppositifolia, isolation and

characterization of the compounds from the methanolic extract of whole plant of Mollugo

oppositifolia are documented in this chapter.


Summary

359

Compound MO-01 was obtained as white needles from 25% ethylacetate-hexane

fraction and has a molecular formula C35H60O6, m.p. 280-282 ºC. It gave positive result

for LB test for sterols. MO-01 was characterized as β-sitosterol glucoside.

Compound MO-02 was obtained as Flourescent yellow powder from 75% ethyl

acetate–methanol fraction and has a molecular formula C21H19O10, m.p – 236 ºC. It gave

positive result for Schinoda test for flavonoids. MO-02 was characterized as vitexin.

Compound MO-03 was obtained from 35% ethyl acetate –methanol fraction. It

was obtained as Dark brownish Crystals, having melting point 260-262 ºC. It showed

positive for Molisch’test, Saponin’s test. MP-03 was not characterized due to paucity of

sample.

Chapter IV

The n-Hexane, Ethyl acetate, Methanol and Aqueous extracts of Mollugo

oppositifolia shows significant zone of inhibition against bacteria and fungi.

In this study the n-Hexane, Ethyl acetate, Methanol and Aqueous extracts of

Mollugo oppositifolia is evaluated for its Analgesic activity of acetic acid induced

writhing in mice, tail flick method in rats and hot plate method in mice. The Methanol

and Aqueous extracts produced significantly analgesic activity. n-Hexane and Ethyl

acetate extracts showed less activity.

Anti-inflammatory activity evaluated was acute and chronic models of

inflammation. The acute inflammatory property the formalin induced paw oedema in rats

was employed and for acute anti-inflammatory activity, formalin-induced paw oedema in

rats was used. The result of study showed that methanol and aqueous extracts of Mollugo


Summary

360

oppositifolia has showed significantly acute anti inflammatory activity. It also showed

significantly chronic anti inflammatory activity. n-Hexane and Ethyl acetate extracts

showed a less effect compare to the other two extracts.

The effect of extracts was investigated in normal rats and glucose over loaded

rats. The extracts were subjected to comparative evaluation of antidiabetic activity in

STZ (45 mg/kg) induced diabetic rats for a period of 21 days. The extracts showed a

significant glycemic control effect in normal rats and glucose over loaded rats. The

methanol and aqueous extracts showed a significant reduced in the blood glucose levels,

lipid profile and serum biomarkers in diabetic rats. n-Hexane and ethyl acetate extracts

showed a less effect compare to the other two extracts. The extracts also showed a

correspondent effect in enzymatic and non-enzymatic levels in diabetic rats. The whole

plant of Mollugo oppositifolia extracts showed an increased β-cell mass in islets of

pancreas.

Overall the whole plant extracts of Mollugo oppositifolia are having anti-

microbial, analgesic, anti-inflammatory and anti-diabetic effect and also antioxidant

activity in diabetic rats.

Chapter V

In this chapter phytochemical screening of the ethyl acetate extract of the entire

plant of Smilax perfoliata and the experimental work on Smilax perfoliata were

described. The extraction, isolation and characterization of the compounds from the ethyl

acetate extract of this plant are documented in this chapter.


Summary

361

From the experimental work column chromatography afforded two compounds

designated as SP-01 and SP-02. These compounds were identified as 1, 3, 6 and 8 tetra

hydroxy anthraquinone and Rutin by spectral studies and by comparison with

authenticated sample.

Chapter VI

The n-Hexane, ethyl acetate, methanol and aqueous extracts of Smilax Perfoliata

shows significant zone of inhibition against bacteria and fungi.

In this study the n-Hexane, ethyl acetate, methanol and aqueous extracts of Smilax

Perfoliata is evaluated for its Analgesic activity of acetic acid induced writhing in mice,

tail flick method in rats and hot plate method in mice. The aqueous and methanol extracts

produced significantly analgesic activity. n-Hexane and ethyl acetate extracts showed less

activity.




rats was used. The result of study showed that aqueous and methanol extracts of Smilax

Perfoliata has showed significantly acute anti inflammatory activity. It also showed

significantly chronic anti inflammatory activity. n-Hexane and Ethyl acetate extracts

showed a less effect compare to the other two extracts.





Summary

362


aqueous and methanol extracts showed a significant reduced in the blood glucose levels,

lipid profile and serum biomarkers in diabetic rats. n-Hexane and Ethyl acetate extracts



plant of Smilax Perfoliata extracts showed an increased β-cell mass in islets of pancreas.

Overall the whole plant extracts of Smilax Perfoliata are having antimicrobial,

analgesic, anti-inflammatory and anti-diabetic effect and also antioxidant activity in

diabetic rats.

Chapter VII

In this chapter phytochemical examination of the extract of the entire plant

Flemingia wightiana and the experimental work on Flemingia wightiana were described.

The extraction, isolation and characterization of the compounds from the Ethyl acetate

extract of this plant are documented in this chapter. From the experimental work on

column chromatography afforded two compounds designated as FW1, FW-2. These

compounds were identified neo Flemingin –D, quercetin by spectral studies and by

comparison with authenticated sample.

Chapter VIII

The n-Hexane, ethyl acetate, methanol and aqueous extracts of Flemingia

wightiania shows significant zone of inhibition against bacteria and fungi.


Summary

363

In this study the n-Hexane, Ethyl acetate, Methanol and Aqueous extracts of

Flemingia wightiania is evaluated for its Analgesic activity of acetic acid induced

writhing in mice, tail flick method in rats and hot plate method in mice. The Methanol

and Aqueous extracts produced significantly analgesic activity. n-Hexane and Ethyl

acetate extracts showed less activity.




rats was used. The result of study showed that methanol and aqueous extracts of

Flemingia wightiania has showed significantly acute anti inflammatory activity. It also

showed significantly chronic anti inflammatory activity. n-Hexane and ethyl acetate

extracts showed a less effect compare to the other two extracts.





methanol and aqueous extracts showed a significant reduced in the blood glucose levels,

lipid profile and serum biomarkers in diabetic rats. n-Hexane and ethyl acetate extracts



plant of Flemingia wightiania extracts showed an increased β-cell mass in islets of

pancreas.


Summary

364

Overall the whole plant extracts of Flemingia wightiania are having anti-

microbial, analgesic, anti-inflammatory and anti-diabetic effect and also antioxidant

activity in diabetic rats.


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