229

6. PHARMACOLOGICAL ACTIVITY OF SYNTHESIZED COMPOUNDS

6.1. Materials and methods:

6.1.1. Chemicals and reagents:

All the analytical grade chemicals were purchased from SD Fine Ltd., Mumbai.

6.1.2. Laboratory animals:

Healthy, adult Wistar albino rats of both sexes (150-220g) were obtained from the

central animal house facility RVS College of Pharmaceutical Sciences, Sulur, C

oimbatore, Tamilnadu. The animals were kept in a well ventilated room and the

animals were exposed to 12 hrs day and night cycle with a temperature between

20±3ºC.The animals were housed in large spacious, hygienic polypropylene cages

during the course of the experimental period. The animals were fed with water and

rat feed ad libitum, supplied by this institution. The animal experiments were

carried out as per Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA) guidelines and after the Institutional Animal

Ethics Committee (IAEC) (No: IAEC-1013/C/06/CPCSEA) approval.

6.1.3. Acute toxicity studies:

The acute toxicity test was carried out according to the Organization for Economic

Co-operation and Development (OECD) guidelines 423 (ATCM -Acute oral

Toxicity Classic Method). Albino rats of either sex weighing between 150 and 220

g were grouped into 20 groups of six animals each, starved for 24 h with water ad

libitum prior to test. On the day of the experiment animals were administered with

different compounds to different groups in an increasing dose of 10, 20, 100, 200,

230

1000 and 2000 mg/kg body weight orally. The animals were then observed

continuously for 3 h for general behavioral, neurological, autonomic profiles and

then every 30 min for next 3 h and finally for next 24 h or for a total of 14 days.

6.1.4. Evaluation of anti-inflammatory activity170

The albino rats were divided into 22 groups of six animals each as follows.

Group 1: Solvent control 1ml/kg 0.3% (w/v) Carboxy methyl cellulose (CMC).

Group 2: Animals received Indomethacin 200 mg/kg in 0.3% CMC orally.

Group 3-22: Animals received test compounds 6a-e, 7a-j, 8a-e (200 mg/kg) in 0.3%

CMC orally.

In vivo acute anti-inflammatory activity was evaluated using carrageenan-induced

rat paw edema assay model of inflammation for the compounds prepared. Male

albino rats (170–220 g) were fasted with free access to water at least 12 hour prior

to experiments and were divided randomly into 22 groups of six each. Control

group received 0.1 ml of 1% sodium carboxymethyl cellulose (sodium CMC),

standard group received 200 mg/kg of Indometacin and test groups received 200

mg/kg of synthesized compounds. The rats were dosed orally, 1 hr later; a

subplantar injection of 0.1 ml of 1% solution of carrageenan in sterile distilled

water was administered to the left hind footpad of each animal. The paw volume

was measured by using plethysmometer at 0, 1, 2, 3, and 4 hrs after carrageenin

challenge. The percent increase in the edema (paw volume) was calculated by

comparing it with zero minute reading.

231

Table No. 6.1.1. Inhibitory effects of test compounds 6a-e, 7a-j and 8a-e on

carrageenan-induced edema of the hind paw in rats

Values are expressed in terms of mean ± S.E.M (n=6), Values are expressed in terms of mean ± S.E.M,

Significance was calculated by using one way ANOVA with Dunnet’s t-test. The difference in results was

considered significant when p < 0.05. * p < 0.05 Vs control at 200 mg/kg b.w. ** p < 0.01 Vs control at 200

mg/kg b.w, *** p < 0.001 Vs control at 200 mg/kg b.w

S. No. Group Treatment Swelling volume (mL)

1hr 2hr 3hr 4hr

1. Control 1mL/kg 0.43±0.07 1.20±0.00 2.19±0.03 3.91±0.08

2. Indomethacin 200mg/kg 0.44±0.05 0.76±0.00 1.06±0.01 1.66±0.013***

3. 6a 200mg/kg 0.46±0.21 1.18±0.00 1.00±0.01*** 2.35±0.006**

4. 6b 200mg/kg 0.42±0.19 0.67±0.16 1.35±0.16** 1.74±0.003*

5. 6c 200mg/kg 0.47±0.78 0.85±0.01 1.07±0.00 1.76±0.021**

6. 6d 200mg/kg 0.36±0.03 0.91±0.01 1.13±0.00 1.88±0.024*

7. 6e 200mg/kg 0.45±0.02 0.87±0.01 0.86±0.01* 1.94±0.044

8. 7a 200mg/kg 0.44±0.01 1.03±0.01 1.22±0.00* 1.94±0.006*

9. 7b 200mg/kg 0.42±0.00 0.91±0.00 1.12±0.00*** 1.92±0.013**

10. 7c 200mg/kg 0.40±0.01 0.84±0.00 1.03±0.00 1.98±0.013

11. 7d 200mg/kg 0.41±0.02 0.89±0.00 1.00±0.01** 1.89±0.011

12. 7e 200mg/kg 0.42±0.00 0.92±0.01 1.22±0.00 2.05±0.037*

13. 7f 200mg/kg 0.41±0.01 0.85±0.01 1.14±0.00*** 1.72±0.013**

14. 7g 200mg/kg 0.45±0.01 1.12±0.01 1.33±0.00 2.14±0.013

15. 7h 200mg/kg 0.45±0.01 0.80±0.02 1.09±0.00** 1.69±0.030**

16. 7i 200mg/kg 0.43±0.01 0.69±0.00 1.02±0.00*** 1.57±0.017**

17. 7j 200mg/kg 0.43±0.01 0.96±0.00 1.16±0.01 1.92±0.023

18. 8a 200mg/kg 0.43±0.01 0.92±0.02 1.22±0.00 2.12±0.035*

19. 8b 200mg/kg 0.44±0.01 1.01±0.00 1.16±0.01** 2.23±0.046

20. 8c 200mg/kg 0.44±0.01 0.73±0.01 1.09±0.00** 1.67±0.030*

21. 8d 200mg/kg 0.43±0.01 0.93±0.01 1.06±0.00 2.02±0.026

22. 8e 200mg/kg 0.44±0.01 0.94±0.01 1.08±0.00* 2.10±0.020

232

Table No. 6.1.2. Percentage inhibition of test compounds 6a-e, 7a-j and 8a-e on carrageenan-

induced edema of the hind paw in rats

Values are expressed in terms of mean ± S.E.M, Significance was calculated by using one way

ANOVA with Dunnet’s t-test. The difference in results was considered significant when p < 0.05. * p < 0.05 Vs control at 200 mg/kg b.w.

** p < 0.01 Vs control at 200 mg/kg b.w,

*** p < 0.001 Vs

control at 200 mg/kg b.w

S. No. Group Treatment Paw volume(ml) as measured

by mercury displacement at

4 hr

Percentage inhibition

of paw edema at 4 hr

1. Control 1mL/kg 3.91±0.08 -

2. Indomethacin 200mg/kg 1.66±0.013*** 57.54

3. 6a 200mg/kg 2.35±0.006** 39.89

4. 6b 200mg/kg 1.74±0.003* 55.49

5. 6c 200mg/kg 1.76±0.021** 54.98

6. 6d 200mg/kg 1.88±0.024* 51.92

7. 6e 200mg/kg 1.94±0.044 50.38

8. 7a 200mg/kg 1.94±0.006* 50.89

9. 7b 200mg/kg 1.92±0.013** 49.36

10. 7c 200mg/kg 1.98±0.013 50.13

11. 7d 200mg/kg 1.89±0.011 51.66

12. 7e 200mg/kg 2.05±0.037* 47.57

13. 7f 200mg/kg 1.72±0.013** 56.01

14. 7g 200mg/kg 2.14±0.013 45.27

15. 7h 200mg/kg 1.69±0.030** 56.78

16. 7i 200mg/kg 1.57±0.017** 59.85

17. 7j 200mg/kg 1.92±0.023 50.89

18. 8a 200mg/kg 2.12±0.035* 45.78

19. 8b 200mg/kg 2.23±0.046 42.97

20. 8c 200mg/kg 1.67±0.030* 57.28

21. 8d 200mg/kg 2.02±0.026 52..43

22. 8e 200mg/kg 2.10±0.020 46.29

233

The percentage inhibition of edema was calculated at 4th

hour assuming 100%

Inflammation in vehicle group.

% Anti-inflammatory activity = (Vc-Vt / Vc) x 100

Where, Vt- mean increase in paw edema volume in the drug treated group,

Vc - mean increase in paw edema volume in control group.

6.2. Ulcerogenic Activity:

Albino rats of either sex were divided into control, standard and different test

groups of six animals each group (170–250 g). They were starved for 48 h (water

ad libitum) prior to drug administration. Control group received only 0.5%

sodium CMC solution, standard group was orally administered with Indomethacin

in sodium CMC solution and test compounds 7f, 7h, 7i and 8c were administered

orally at the dose of 200 mg/kg and 200 mg/kg, respectively. Six hours later the

animals were sacrificed using excess ether anesthesia. The stomach was excised

carefully, opened along the greater curvature; the luminal contents were removed.

The mucosa was flushed with saline and the stomach pinned on a frog board.

The ulcer index was calculated according to the method171

. The lesions were

counted with the aid of hand lens (10X) and each given a severity rating as

follows

Ulcer Score Descriptive Observation

0 Normal

1 Less than 1mm (Pin point)

2 1-2 mm

3 Greater than 2 mm and above

234

Statistical analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA)

followed by Dunnett’s t-test for multiple comparisons of all compounds in various

pharmacological assays. Datas are expressed as mean ±SEM. The significance of

difference was accepted at p < 0.05.

(a) (b)

(c) (d)

235

(e)

Fig. No. 6.2.1. Ulcerogenic activity of a) compound 7f, b) compound 7h, c)

compound 7i, d) control, and e) Indomethacin

Table 6.2.1. Ulcerogenic activity of selected compounds in comparison with

Indomethacin

S.No Compound Ulcer index (±SEM)

1 Control 0

2 7f 7.33 ± 0.35**

3 7h 10.67 ± 0.14*

4 7i 15.82 ± 0.18*

5 8c 12.76 ± 0.22

6 Indomethacin 31.28 ± 0.48**

The results are expressed as mean ± SEM (n=6). Data analyzed by one-way

ANOVA followed by Dunnett’s t-test. *p< 0.05 significant from control;

**p<0.01 significant from control.

236

6.3. Anti Bacterial Activity:

6.3.1. In-vitro antibacterial activity:

All the twenty newly synthesized compounds were screened for their in-vitro

antibacterial activity against Gram-positive organisms, Bacillus subtilis (ATCC

6633) and Staphylococcus aureus, (ATCC 25923) and Gram-negative organisms,

Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853))

by cup-plate method172

. Antimicrobial activity is measured in vitro in order to

determine a) the potency of an antibacterial agent in solution b) the sensitivity of a

given microorganism to know concentrations of the synthesized drug.

6.3.2. Procedure for In-vitro antibacterial study:

The in-vitro antibacterial activity of the synthesized compounds was evaluated

against two gram positive and two gram negative bacterias. N, N –Dimethyl

sulfoxide (DMSO) were used to prepare stock solution of standard and

synthesized drugs (0.5 mg/ml). Normal saline was used to prepare inoculum of

the bacteria to be used for in vitro antimicrobial study. In order to ensure that the

solvent had no effect on bacteria growth, an inoculated control test was performed

with only DMSO and found inactive in culture media.

6.3.3. Cup plate method:

Cup-plate method is used for the study of antimicrobial activity. A suspension of

the test organism was well mixed with 25 ml of sterile liquid nutrient agar media,

at a temperature between 40-500 C and poured immediately in to a pre-sterilized

petri-dishes. After congealing the seeded agar was punched out to make holes (10

mm in diameter) at spaced out positions on the petri dish. The cavity was filled

237

with drug solution at a concentration of 500 µg/ml. After adding drugs agar

plates were set a side in refrigeration for 15 mins, and incubated for 24 hrs at 370

C. After incubation zones were measured at mm and reported in Table no 6.3.1.

Table 6.3.1. Antimicrobial activity of synthesized compounds and standard

(500 µg/ml) [Zone of inhibition (mm)]

Compound

Gram positive Gram negative

B.subtilis S.aureus E.coli P.aeruginosa

6a --- 20 18 10

6b 22 27 29 22

6c 25 18 10 20

6d 25 30 25 19

6e 25 27 27 23

7a 23 18 21 15

7b 14 23 19 21

7c 25 27 20 18

7d 28 -- 29 22

7e 25 28 22 --

7f 21 08 -- 12

7g -- 25 27 08

7h 22 23 17 21

7i 16 18 21 17

7j 20 27 22 21

8a 08 -- 06 15

8b 25 30 25 20

8c 16 14 28 17

8d 26 25 28 22

8e 18 21 24 16

Ampicillin 32 34 32 29

238

Fig. No. 6.3.1. Anti bacterial activity of synthesized compounds 7d, 8d and 6d

239

Fig. No. 6.3.2. Anti bacterial activity of Standard drug Ampicillin

240

6.3.2. DETERMINATION OF MIC:

6.3.2.1. Media Composition:

Muller-Hinton agar is a microbiological growth medium that is commonly used

for antibiotic susceptibility testing. It contains:

300 gms beef infusion

17.5 gms casein hydrolysate

1.5 gms starch

Water make up to 1000 ml

Finally, pH was adjusted to neutral at 250

C.

6.3.2.2. In vitro Evaluation of Antibacterial Activity of Compounds :

Broth microdilution method173

using Mueller-Hinton agar medium was employed to

study the preliminary antibacterial activity of synthesized compounds against Gram-

positive (Bacillus subtilis (ATCC 6633) and Staphylococcus aureus,(ATCC 25923))

and Gram-negative (Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa

(ATCC 27853)) bacteria. The antibacterial activity of the test compounds was

compared with ampicillin. Twofold serial dilutions of the test compounds and

reference drugs were prepared in Muller-Hinton agar. Test compounds, standard drug

ampicillin were dissolved in dimethylsulfoxide (DMSO, 1 ml) and the solution was

diluted with distilled water (9 ml). Further progressive serial dilutions with melted

Muller-Hinton agar were performed to obtain the required concentrations from 5-

1000 μg/ml. The test tubes were inoculated with selected microorganisms and

incubated at 37 ˚C for 24 h. The minimum inhibitory concentration (MIC) was the

lowest concentration of the tested compound that yields no visible growth on the

241

plate. To ensure that the solvent had no effect on the bacterial growth, a control was

performed with the test medium supplemented with DMSO at the same dilutions as

used in the experiments.

Table No. 6.3.2. Anti bacterial activity of the synthesized compounds 6(a-e),

7(a-j) and 8(a-e)

Comp.

Minimum inhibitory concentrations (µg/ml)

B. subtilis S. aureus E. coli P. aeruginosa

6a -- 250 500 > 500

6b 125 31.2 15.6 125

6c 62.5 500 > 500 250

6d 500 125 250 500

6e 62.5 15.6 15.6 125

7a 125 500 250 > 500

7b > 500 62.5 500 125

7c 62.5 31.2 250 500

7d 31.2 -- 31.2 125

7e 62.5 31.2 125 --

7f > 500 > 500 -- > 500

7g -- 62.5 31.2 > 500

7h 125 125 > 500 250

7i > 500 500 250 > 500

7j 250 31.2 125 250

8a > 1000 -- > 1000 > 500

8b 62.5 15.6 62.5 250

8c 62.5 > 500 15.6 > 500

8d 31.2 7.8 15.6 31.2

8e 500 62.5 62.5 > 500

Ampicillin 1.56 1.56 3.13 3.13

242

6.4. Anti-Fungal Activity:

The synthesized compounds were screened for their antifungal activity against the

organism Aspergillus niger and Saccharomyces cerevisiae by cup-plate method as

described for the evaluation of antibacterial activity except incubation period (48

hrs at 250C). The study revealed that synthesized compounds possess significant

antifungal activity.

Table No. 6.4.1 Antifungal activity of compounds and standard

(500 µgm/mL) Zone of inhibition (mm)

Compound

Organism

Aspergillus niger Saccharomyces

cerevisiae

6a -- 18

6b 20 --

6c 08 24

6d 22 21

6e 25 22

7a 12 16

7b 24 21

7c 22 19

7d -- 17

7e 19 24

7f 12 18

7g 17 14

7h 19 --

7i 22 24

7j 24 18

8a 18 06

8b 25 10

8c 26 24

8d 22 26

8e 14 27

Griseofulvin) 34 35

243

Figure 6.4.1. Anti fungal activity of synthesized compounds and standard

drug

244

Table No. 6.4.2. Antifungal activity of the synthesized compounds

6(a-e), (a-j) and 8(a-e)

Compound

Minimum inhibitory concentrations (µg/ml)

Aspergillus niger Saccharomyces

cerevisiae

6a -- 500

6b 250 --

6c > 500 62.5

6d 62.5 125

6e 31.2 62.5

7a > 500 > 500

7b 62.5 125

7c 125 500

7d -- > 500

7e 500 31.2

7f > 500 500

7g > 500 > 500

7h 500 --

7i 125 62.5

7j 31.2 500

8a 500 > 500

8b 31.2 > 500

8c 15.6 31.2

8d 125 15.6

8e > 500 250

Standard

(Griseofulvin)

3.46 1.25

245

6.5. In vitro methods employed in antioxidant studies:

6.5.1. Reducing power ability

The reducing power ability of the synthesized compounds was measured by

according to the method of Oyaizu174

. Different concentrations of the synthesized

compounds (50-250 μg/ml) in DMSO (1 ml) were mixed with 2.5 ml of

phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1 % potassium ferricyanide and

incubated at 500 C for 30 min. After that 2.5 ml of trichloroacetic acid (10%) was

added to the mixture and then centrifuged for 10 min at 1000 g. 2.5 ml of the

upper layer of solution was diluted with 2.5 ml of distilled water and shaken with

0.5 ml fresh 0.1% ferric chloride. The absorbance was measured at 700 nm using

UV- spectrophometer. The reference solution was prepared as above, but

contained DMSO instead of the samples. Increased absorbance of the reaction

mixture indicates increased reducing power. All experiments were done in

triplicate using ascorbic acid as positive control.

6.5.2. Hydrogen peroxide scavenging activity

The principle of this method depended on the decrease in absorbance of H2O2

upon its oxidation. Hydrogen peroxide solution (2 mM/l) was prepared with 0.1

M phosphate buffer (pH, 7.4). Then, compounds (25-400 g/ml) in 5 ml

phosphate buffer were added to hydrogen peroxide solution (0.6 ml). Absorbance

of hydrogen peroxide at 230 nm was determined spectrophotometrically after 10

min against a blank solution containing phosphate buffer without hydrogen

peroxide. The percentage scavenging of hydrogen peroxide of both synthesised

246

compounds 6 (a-e), 7(a-j) and 8 (a-e) and standard compound were determined175

.

The percentage of scavenged [H2O2]:

[H2O2] = [(Ac-At)/Ac] X 100

Where Ac was the absorbance of the control and At was the absorbance in the

presence of the standard sample or synthesized compounds.

Calculation of 50% Inhibitory Concentration (IC50)

The concentration (mg/ml) of the various compounds required to scavenge 50%

of the radicals was calculated by using the percentage scavenging activities at five

different concentrations of the compound. Percentage inhibition (I %) was

calculated using the formula,

I % = (Ac-As) x 100

Ac

Where Ac is the absorbance of the control and As is the absorbance of the sample.

Statistical analysis

Results were statistically evaluated by analysis of variance (ANOVA)

followed by Dennett’s multiple comparison test, P<0.05 was considered to be

statistically significant.

247

Table No. 6.5.1. Reductive ability of synthesized compounds

Comp Concentration in μg/ml

50 100 150 200 250

6a 0.212±0.05 0.432±0.00 0.575±0.01 0.744±0.01 1.212±0.01

6b 0.427±0.00 0.615±0.00 0.849±0.00 1.432±0.00 1.878±0.00

6c 0.442±0.00 0.793±0.00 0.952±0.00 1.268±0.00 1.734±0.00

6d 0.351±0.00 0.549±0.00 0.828±0.00 1.094±0.00 1.525±0.00

6e 0.394±0.00 0.687±0.00 0.857±0.00 1.353±0.00 1.839±0.00

7a 0.381±0.01 0.492±0.00 0.531±0.00 0.876±0.00 1.282±0.00

7b 0.442±0.01 0.638±0.00 0.816±0.00 1.217±0.00 1.497±0.00

7c 0.492±0.00 0.680±0.00 0.911±0.00 1.232±0.00 1.479±0.00

7d 0.591±0.00 0.797±0.00 0.940±0.00 1.491±0.00 1.714±0.00

7e 0.462±0.00 0.690±0.00 1.078±0.00 1.216±0.00 1.421±0.00

7f 0.282±0.005 0.439±0.005 0.622±0.006 0.811±0.005 0.916±0.004

7g 0.309±0.001 0.427±0.004 0.592±0.002 0.781±0.003 0.906±0.005

7h 0.277±0.002 0.396±0.003 0.622±0.001 0.807±0.002 0.953±0.003

7i 0.291±0.003 0.414±0.005 0.659±0.003 0.847±0.004 0.953±0.003

7j 0.371±0.003 0.488±0.002 0.722±0.005 0.925±0.001 1.037±0.003

8a 0.381±0.002 0.690±0.001 0.797±0.004 1.283±0.003 1.749±0.002

8b 0.334±0.002 0.611±0.002 0.844±0.003 1.263±0.002 1.839±0.001

8c 0.577±0.004 0.783±0.003 0.957±0.002 1.486±0.002 1.852±0.002

8d 0.488±0.001 0.622±0.001 0.933±0.004 1.232±0.002 1.479±0.003

8e 0.289±0.003 0.426±0.004 0.684±0.002 0.855±0.006 0.983±0.004

STD 0.601±0.003 0.713±0.005 0.839±0.003 0.911±0.008 1.190±0.11

Values shown are mean ± SEM for four tests p<0.01, as compared to

control

248

Table No. 6.5.2. Hydrogen peroxide radical assay for compound 6 (a-e)

Compound Concentration

(µg/ml) Absorbance at 230 nm % inhibition IC 50 µg/ml

Control 0.735

6a

25

50

100

200

400

0.615 ± 0.001

0.527 ± 0.008

0.441 ± 0.003

0.297 ± 0.001

0.197 ± 0.002

16.32 ± 0.22

28.29 ± 0.23

40.04± 0.11

59.59 ± 0.13

73.19 ± 0.16

186

6b

25

50

100

200

400

0.538 ± 0.002

0.412 ± 0.005

0.326 ± 0.001

0.269 ± 0.005

0.141 ± 0.002

26.04 ± 0.55

43.94 ± 0.23

55.64 ± 0.53

63.40 ± 0.07

80.81 ± 0.25

132

6c

25

50

100

200

400

0.311 ± 0.001

0.213 ± 0.005

0.188 ± 0.008

0.092 ± 0.005

0.072 ± 0.006

57.68 ± 0.07

71.02 ± 0.05

74.42 ± 0.03

87.48 ± 0.09

90.20±0.096

76

6d

25

50

100

200

400

0.458 ± 0.002

0.313 ± 0.008

0.297 ± 0.005

0.196 ± 0.002

0.118 ± 0.008

37.68 ± 0.07

57.41 ± 0.12

59.59 ± 0.21

73.33 ± 0.07

83.94±0.127

86

6e

25

50

100

200

400

0.322 ± 0.002

0.242 ± 0.008

0.192 ± 0.005

0.102 ± 0.002

0.094 ± 0.008

56.19 ± 0.48

67.07 ± 0.57

73.87 ± 0.48

86.12 ± 0.26

87.21 ± 0.51

66

Values shown are mean ± SEM for four test p<0.01, as compared to

control

249

Table No. 6.5.3. Hydrogen peroxide radical assay for compound 7 (a-e)

Compound Concentration (µg/ml) Absorbance at 230 nm % inhibition IC 50 µg/ml

Control 0.735

7a

25

50

100

200

400

0.627 ± 0.002

0.535 ± 0.003

0.455 ± 0.001

0.298 ± 0.005

0.199 ± 0.006

14.69 ± 0.51

27.21 ± 0.21

38.09 ± 0.34

59.40 ± 0.46

72.90 ± 0.41

171

7b

25

50

100

200

400

0.618 ± 0.002

0.527 ± 0.006

0.447 ± 0.001

0.299 ± 0.004

0.197 ± 0.003

15.91 ±0.22

28.29 ±0.37

39.18 ±0.43

59.31 ±0.27

73.10 ±0.34

152

7c

25

50

100

200

400

0.312 ± 0.002

0.215 ± 0.004

0.189 ± 0.005

0.093 ± 0.002

0.075 ± 0.006

57.55 ±0.38

70.74 ±0.14

74.28 ±0.35

87.34 ±0.26

89.79 ±0.18

82

7d

25

50

100

200

400

0.617 ± 0.002

0.528 ± 0.003

0.448 ± 0.002

0.298 ± 0.004

0.196 ± 0.005

16.05 ±0.43

28.16 ±0.23

39.04 ±0.53

59.45 ±0.09

73.33 ±0.36

142

7e

25

50

100

200

400

0.457 ± 0.004

0.314 ± 0.001

0.295 ± 0.004

0.198 ± 0.002

0.117 ± 0.005

37.82 ±0.43

57.27 ±0.17

59.86 ±0.09

73.06 ±0.17

84.08 ±0.33

84

Values shown are mean ± SEM for four test p<0.01, as compared to

control

250

Table No. 6.5.4. Hydrogen peroxide radical assay for compound 7 (f-j)

Compound Concentration

(µg/ml)

Absorbance at 230

nm

%

inhibition

IC 50

µg/ml

Control 0.735

7f

25

50

100

200

400

0.309 ± 0.003

0.206 ± 0.001

0.180 ± 0.004

0.088 ± 0.006

0.071 ± 0.001

57.95 ± 0.22

71.97 ± 0.09

75.51 ± 0.15

76.53 ± 0.07

90.34 ± 0.42

93

7g

25

50

100

200

400

0.610 ± 0.002

0.525 ± 0.001

0.437 ± 0.001

0.292 ± 0.005

0.194 ± 0.002

17.0 ± 0.14

28.57 ± 0.06

40.54 ± 0.24

60.27 ± 0.37

73.60 ± 0.41

184

7h

25

50

100

200

400

0.452 ± 0.003

0.310 ± 0.002

0.292 ± 0.004

0.194 ± 0.001

0.116 ± 0.007

38.50 ± 0.11

57.82 ± 0.25

60.02 ± 0.31

73.6 ± 0.08

84.21 ± 0.34

125

7i

25

50

100

200

400

0.619 ± 0.008

0.522 ± 0.002

0.435 ± 0.002

0.290 ± 0.001

0.191 ± 0.004

17.14 ± 0.42

28.97 ± 0.32

40.81 ± 0.26

60.54 ± 0.17

74.01 ± 0.43

165

7j

25

50

100

200

400

0.457 ± 0.001

0.311 ± 0.004

0.298 ± 0.002

0.198 ± 0.002

0.120 ± 0.003

37.80 ± 0.24

57.68 ± 0.36

59.21 ± 0.11

73.06 ± 0.28

83.67 ± 0.26

108

Values shown are mean ± SEM for four test p<0.01, as compared to

control

251

Table No. 6.5.5. Hydrogen peroxide radical assay for compound 8 (a-e)

Compound Concentration

(µg/ml)

Absorbance at 230

nm

%

inhibition

IC 50

µg/ml

8a

25

50

100

200

400

0.612 ± 0.002

0.520 ± 0.003

0.430 ± 0.006

0.288 ± 0.004

0.190 ± 0.003

16.70 ± 0.12

29.25 ± 0.08

41.49 ± 0.11

60.81 ± 0.01

74.14 ± 0.08

169

8b

25

50

100

200

400

0.310 ± 0.001

0.208 ± 0.002

0.181 ± 0.002

0.089 ± 0.006

0.066 ± 0.005

57.80 ± 0.21

71.70 ± 0.04

75.37 ± 0.06

87.89 ± 0.02

91.02 ± 0.02

82

8c

25

50

100

200

400

0.456 ± 0.001

0.312 ± 0.002

0.294 ± 0.001

0.195 ± 0.003

0.112 ± 0.001

37.95 ± 0.19

57.55 ± 0.22

60.00 ± 0.35

73.46 ± 0.41

84.76 ± 0.06

96

8d

25

50

100

200

400

0.308 ± 0.002

0.206 ± 0.004

0.180 ± 0.003

0.086 ± 0.005

0.067 ± 0.003

58.09 ± 0.26

71.97 ± 0.35

75.51 ± 0.05

88.29 ± 0.26

90.88 ± 0.21

90

8e

25

50

100

200

400

0.611 ± 0.001

0.519 ± 0.003

0.427 ± 0.003

0.286 ± 0.002

0.192 ± 0.001

16.89 ± 0.08

29.38 ± 0.49

41.90 ± 0.41

61.08 ± 0.16

73.87 ± 0.28

168

α-tocopherol

(standard)

25

50

100

200

400

0.232 ± 0.708

0.197 ± 0.008

0.176 ± 0.001

0.091 ± 0.002

0.071 ± 0.007

68.43 ± 0.10

73.46 ± 0.15

76.05 ± 0.06

87.61 ± 0.10

90.34 ± 0.05

42

Values shown are mean ± SEM for four test p<0.01, as compared to control

252

Reference:

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