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A COMPARATIVE STUDY ON GENOTOXIC ACTIVITY OF CURCUMA AROMATICA Salisb AND CURCUMA ZEDOARIA (Christm.) Roscoe RHIZOMES.

Results and Discussions

Department of Pharmaceutical Biotechnology, JSS College of Pharmacy, Udhagamandalam.

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6. RESULTS AND DISCUSSION

6.1. Results

6.1.1. Phyto constituent’s analysis

50 % hydro alcoholic extract of Curcuma aromatica salisb found to contain alkaloids,

tannin and flavonoids where as Curcuma zedoaria (Christm.) Roscoe contain alkaloids

and flavonoids. Total phenol content was found to be 77.88095±1.7 and

38.08095±1.37 for Curcuma aromatica and Curcuma zedoaria respectively which was

represented as mg/g of gallic acid. Total flavonol content was found to be

459.5333±8.68 and 21.33333±1.11 for Curcuma aromatica and Curcuma zedoaria

respectively which was represented as mg/g of quercetin. Results are tabulated in Table

No.1.




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Table No.1: Qualitative and quantitative analysis of phytoconsituents of Curcuma

aromatica and Curcuma zedoaria.

Constituents

Curcuma aromatica salisb

Curcuma zedoaria

(Christm.) Roscoe

Alkaloids Present Present

Carbohydrates Absent Absent

Glycosides Absent Absent

Saponins Absent Absent

Tannin Present Absent

Protein and amino acids Absent Absent

Flavonoids Present Present

Steroids Absent Absent

Total phenol content (mg/g

of gallic acid)

77.88095±1.7 38.08095±1.37

Total flavonol content

(mg/g of quercetin)

459.5333±8.68 21.33333±1.11

6.1.2. In vitro- antioxidant studies

In vitro- antioxidant studies were carried out for 50 % hydro alcoholic extract of

Curcuma aromatica and Curcuma zedoaria and Curcumin by DPPH method, Nitric

oxide method, deoxyribose method, lipid peroxidation method, reducing power and

total antioxidant capacity method. Ascorbic acid and rutin were used as the standard for

DPPH method, Nitric oxide method and reducing power assay. α-tocopherol was used

as standard for lipid peroxidation method, β-hydroxy aniline was added as standard for

Deoxyribose method. The results are tabulated in Table No.2.




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Table No.2: In vitro antioxidant methods

Test

compounds

DPPH

method

IC50

(µg/ml)

Nitric

oxide

method

IC50

(µg/ml)

Deoxyribose

method

IC 50

(µg/ml)

LPO

assay

IC 50

(µg/ml)

Reducing

Power

(µg/ml)

TAC(mm

equivalent

to

ascorbic

acid)

Curcuma

aromatica

Salisb

37.45±

2.500

372.27±

3.230

302.85±

2.680

420±

0.869

4.717±

0.065

107.2±

3.923

Curcuma

zedoaria

227.8±

4.875

>1000 829±

0.650

630.2±

1.230

12.726±

0.066

283±

3.000

Curcumin 15.75±

1.820

108.47±

2.720

95.56±

1.643

110±

2.105

4.521±

0.014

58±

4.379

Ascorbic

acid

6.0±

1.000

- - - 3.51±

0.007

-

Rutin 11.75±

0.480

88.47±

2.540

- - - -

α-

tocopherol

- - - 100.5±

0.120

- -

Β-hydroxy

aniline

- - 75.6±

0.060

- - -

6.1.2.1. DPPH method

When compared to the ascorbic acid and rutin which showed the IC50 value 6.0±1.0 and

11.75±0.48 respectively in DPPH method. Curcumin showed potent antioxidant




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activity among all the test compounds which showed the IC50 value 15.75±1.82 where

as Curcuma aromatica salisb and Curcuma zedoaria (Christm.) Roscoe showed IC50

value 37.45±2.5, 227.8±4.875 respectively. IC50 values were represented in µg/ml. The

results are represented in the Graph No.2.

6.1.2.2. Nitric oxide method

In Nitric oxide method, rutin was used as standard which showed the IC50 value at the

concentration of 88.47±2.54 µg/ml. Curcumin showed the most potent activity in

Nitric oxide method and its IC50 value was found to be 108.47± 2.72 where as IC50

value for Curcuma aromatica salisb was found to be 372.27±3.23 µg/ml. Even at 1000

µg/ml concentration Curcuma zedoaria fails to answer for the Nitric oxide method. The

result is represented in Graph No.3

6.1.2.3. Reducing power assay

Ascorbic acid was used as standard which showed the activity at the concentration of

3.51±0.007µg/ml. Curcumin and Curcuma zedoaria showed potent reducing power at

the concentration of 4.521± 0.014 and 12.726±0.066 µg/ml and Curcuma aromatica

showed reducing power at the concentration of 4.717±0.065 µg/ml. The result is

represent in Graph No.4

6.1.2.4. Total antioxidant capacity

Total antioxidant capacity for curcumin, Curcuma aromatica and Curcuma zedoaria

was found to be 58±4.379, 107.2±3.923, 283±3.00 respectively. The results were

represented as mM equivalent to ascorbic acid. The result is represented in Graph No.5.




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6.1.2.5. Scavenging of hydroxyl radical by deoxyribose method

In this method β-hydroxy aniline was used as standard or reference compound. β-

hydroxy aniline showed the hydroxyl radical scavenging activity with the IC50 value

of 75.6±0.06. When compared to the standard β-hydroxy aniline, curcumin was found

to be potent and it showed the hydroxyl radical scavenging activity with IC50 value of

95.56±1.643. Curcuma aromatica scavenged the hydroxyl radical with the IC 50 value

of 302.85±2.68 and Curcuma zedoaria showed the hydroxyl radical scavenging activity

with IC 50 value of 829±0.650. The result is represented in Graph No.6

6.1.2.6. LPO assay

In this method α-tocopherol was used as standard compound. It showed the lipid

peroxidation activity with the IC50 value of 100.5±0.12 µg/ml. When compared to α-

tocopherol, curcumin was found to be potent in the LPO assay with the IC 50 value of

110.±2.15 µg/ml, Curcuma aromatica showed the IC 50 value of 420±0.869 µg/ml and

Curcuma zedoaria showed the IC 50 value 630.2±1.23 µg/ml. The result is represented

in Graph No.7

6.1.3. Genotoxic Studies

6.1.3.1. Ames Reverse mutation assay

In this assay, two strains were used. They are TA 98 and TA 100. TA 98 is used to

determine frame shift mutation that is caused by the test compounds. TA100 is used to

determine base pair substitution. Both the strains were obtained from Amala Cancer

Research Institute, Thirussur, Kerala.




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6.1.3.1.1. TA98

This strain is used to determine frame shift mutation that is caused by the test

compounds.

Control

To determine the frame shift mutation, in the absence of S9 factor, 2- nitrofluorene and

in the presence of S9 factor Benzo[a]pyrene was used as the positive control which

produced the number of revertant colonies such as 682±86, and 510±44 respectively at

the concentration of 50µg/ml. In the negative control plate the revertant colonies

produced were 12±1, 16±2 in the absence and presence of S9 respectively. In the

presence of S9 factor revertant colonies found to decrease in the case of positive control

where as for the test compounds, revertant colonies found to increase in the presence of

S9 factor.

In the absence and presence of S9 factor

Revertant colonies were found to be more at the concentration of 50 μg/ml for

Curcuma aromatica in the absence of S9 factor 14±2 and in the presence of S9 factor

the number of revertant colonies were 18±3. For Curcuma zedoaria, in the absence of

S9 factor 10±1 and in the presence of S9 factor the number of revertant colonies was

16±1. The number of revertant colonies produced by curcumin was found to be 23±1 in

the absence of S9 factor and 26±3 in the presence of metabolic activation factor. The

results are tabulated in Table No.3 and represented in Graph No.8.




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Table No.3: Ames test result for the Strain TA98

Test compounds

Mean ±SEM (-S9) Mean ±SEM (+S9)

Concentrations Concentrations

50µg/ml 50µg/ml

Positive control 682±86 510±44

Negative control 12±1 16±2

Curcuma aromatica salisb 14±2 18±3

Curcuma zedoaria (Christm.) Roscoe 10±1 16±1

Curcumin 23±5 26±3

6.1.3.1.2. TA 100

TA100 was used to determine base pair substitution that is produced by the test

compounds.

Control

To detect the base pair substitution type of mutation, in the absence of S9 factor sodium

azide and in the presence of S9 factor 2- aminoanthracene were used as positive control

which gave the number of revertant colonies such as 1501 ±132 and 308±20 at the

concentration of 50 µg/ml. In the negative control plates the number of revertant

colonies was 14±2 and 78±18 in the absence and presence of S9 factor respectively.

In the Absence and presence of S9 factor

In the absence of S9 factor, the number of revertant colonies was found to be 16±3 for

Curcuma aromatica at the concentration of 50 µg/ml. At the concentration of 50 µg/ml




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the numbers of revertant colonies were 18±1 for Curcuma zedoaria and 25±2 for

Curcumin.

In the presence of S9 factor the revertant colonies were 64±10 for Curcuma aromatica

at the concentration of 50 µg/ml respectively. At the concentration of 50 µg/ml, the

numbers of revertant colonies were 80±8 for Curcuma zedoaria and 86±7 for

curcumin.

When compared to positive control the numbers of revertant colonies were found to be

more or less equivalent to negative control which confirms the absence of genotoxic

effect of test compounds.

In the presence of S9 factor Benzo [a] pyrene was used as the positive control, 2-

nitrofluorene was used positive control in the absence of S9 factor. The results are

tabulated in Table No.4 and represented in Graph No.9.

Table No.4: Ames test result for TA 100

Test compounds

Mean ±SEM (-S9) Mean ±SEM (+S9)

Concentrations Concentrations

50µg/ml 50µg/ml

Positive control 1501±132 308±20

Negative control 14±2 78±18

Curcuma aromatica salisb 16±3 64±10

Curcuma zedoaria (Christm.) Roscoe 18±1 80±8

Curcumin 25±2 86±7




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6.1.3.2. Chromosomal aberration test

This test was performed to investigate the potential of Curcuma aromatica salisb,

Curcuma zedoaria (Christm.) Roscoe and curcumin in inducing the chromosomal

aberration in cultured human peripheral blood lymphocytes. This assay was performed

both in the presence and absence of metabolic activation system. Each concentration

included negative and reference items and they were tested in duplicate cultures. In the

absence of metabolic activation, Mitomycin C at the concentration 0.3µg/ml was used

as reference compound. In the presence of metabolic activation cyclophosphamide

monohydrate at the concentration of 40 µg/ml was used as reference compound.

6.1.3.2.1. Curcuma aromatica salisb

The hydro alcoholic extract of Curcuma aromatica salisb was tested at the following

concentrations: 312.5, 156.25, and 78.125 µg/ml both in the absence and presence of

metabolic activation system (1% v/v S9).

6.1.3.2.1.1. Pretest

In solubility test, Dimethyl sulfoxide (DMSO) was selected as the solvent for

treatment. Based on the results of solubility, precipitation and pH, 2500 µg/ml of

hydro alcoholic extract of Curcuma aromatica was selected as the highest

concentration for pretest both in the absence and presence of metabolic activation

system (1% v/v S9). The Curcuma aromatica extract was tested at the following

concentrations:2500,1250,625,312.5 µg/ml in pretest. The results are tabulated in Table

No. 5.




1

Table No. 5: Result for pH and Precipitation Test for Curcuma aromatica

Concentration

Recorded pH

Precipitation Test

0 hour 4 hour

2500 µg/ml 7.21 7.29 No precipitation

1250 µg/ml 7.22 7.26 No Precipitation

625 µg/ml 7.25 7.22 No Precipitation

312.5µg/ml 7.20 7.28 No Precipitation

Dimethyl sulfoxide 7.23 7.30 No Precipitation

The cytotoxicity due to treatment with Curcuma aromatica extract was assessed based

on reduction(>50%) in the percent mitotic index. The cytotoxicity experiment was

conducted at the concentrations of 2500, 1250, 625, 312.5 µg/ml under the specified

conditions The reduction in the percent mitotic index observed was 83.58, 77.61,

67.16, 56.71 in the absence and 80.4, 76, 60, 52 in the presence of metabolic activation

system at the concentrations of 2500, 1250, 625, 312.5 µg/ml of hydro alcoholic extract

of Curcuma aromatica salisb respectively and the results are tabulated in Table No.6.




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Table No. 6: Percentage Mitotic Index- Pretest for Curcuma aromatica salisb

Concentra

tion (µg/ml)

R Mitotic Index

In the Absence of metabolic

activation

In the Presence of metabolic

activation

Percent

age

Mitotic

index

Mean

percent

age MI

Reduct

ion in

MI

(%)

Percentag

e

Mitotic

index

Mean

percent

age MI

Reducti

on in

MI (%)

Negative

control

1 3.4 3.35 NA 3.9 3.75 NA

2 3.3 3.6

312.5(T1) 1 1.4 1.45 56.71 1.6 1.8 52.0

2 1.5 2.0

625 (T2) 1 1.2 1.1 67.16 1.56 1.5 60.0

2 1.0 1.44

1250(T3) 1 0.8 0.75 77.61 0.86 0.9 76

2 0.7 0.94

2500(T4) 1 0.5 0.55 83.56 0.79 0.735 80.4

2 0.6 0.68

R-Replicate T- Treatment group NA- Not applicable MI- Mitotic index

Based on the results of pretest 312.5 µg/ml of Curcuma aromatica extract was selected

as the highest treatment concentration both in the absence and presence of metabolic

activation system (1% v/v S9) for the main study.




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6.1.3.2.1.2.Main study

In the main study, the cultures were exposed to hydro alcoholic extract of Curcuma

aromatica for a short period of time (4 hours) both in the absence and presence of


The mean number of percent aberrent cells were 1.5, 2.5, 3.0, 4.5 and 16 at doses 0.0

(negative control), 78.125 (T1), 156.25 (T2), 312.5 (T3) µg/ml and 0.3 µg/ml of

Mitomycin C (reference item) respectively, in the absence of metabolic activation

system.

In the presence of metabolic activation system the mean number of percent aberrent

cells were 1.5, 1.5, 1.5, 3.0 and 13.5 at doses 0.0 (negative control), 78.125 (T1),

156.25 (T2), 312.5 (T3) µg/ml and 40 µg/ml of cyclophosphamide monohydrate

(Reference item) respectively.

Reduction(>50%) in the percent mitotic index was observed in the cultures treated at

the dose 312.5 µg/ml both in the absence and presence of metabolic activation system

(1% v/v S9) when compared with the negative control.

Dose dependent increase in the percent aberrant cells was not observed in the cultures

treated upto the dose level of 312.5 µg/ml both in the absence and presence of

metabolic activation system when compared with the negative control, where the

reference item caused a significant increase in percent aberrant cells both in the absence

and presence of metabolic activation system (1% v/v S9). Dose dependent or

significant increse in the percent aberrant cells was not observed in all the tested doses

both in the absence and presence of metabolic activation system. Statistical analysis

(ANOVA and t-test) was performed using statplus 2009 professional 5.7.8 A




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statistically significant increase (p-level <0.05) in aberrent cells was not observed in

ANOVA (comparing negative control and test item doses) both in the presence and

absence of metabolic activation system, where statistically significant increase (P value

<T-criter value ) in aberrent cells was observed in T test (comparing means of negative

control and reference item) both in the presence and absence of metabolic activation

system. Biological and statistical relevance of the result was considered for evaluation.

The results are tabulated in Table No. 7,8 and represented in Graph No. 10,11,12 and

13.




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Table No.7: Percentage Mitotic index and Chromosomal aberrations for Curcuma

aromatica in the Absence of metabolic activation.

Concentration

Culture

No

Percentage

Mitotic

index

Frequencies of

aberrations

Total

Number of

Aberration

Percentage

of

Aberrant

cells

Negative

control

1 4.0 1R,1CLg 2 1

2 4.20 2Cb 2 2

78.125 (T1) 1 2.0 1Cg,1D, 1Di 3 2

2 3.0 1F,2CLb, 1

CLg

4 3

156.25 (T2) 1 2.1 1Cb, 1P, 1D, 3 3

2 2.4 2CLb, 2Cb 4 4

312.5(T3) 1 1.95 1CLb, 1Di,

2D,2Cg

6 4

2 1.83 1F,1Cb,1Cg,

2D,1E

6 5

RI 1 1.07 5CLb,3F,1CLg,

6D,4Cg,2E,2D

24 17

2 2.00 6F,2CLb,1D,

3Di,4Cg,3Cb,

2D,2R

23 15

CLg-Chromosomal gap, Cg- Chromatid gap, Cb- chromatid break, CLb- chromosomal

break, D- Deletion, Di- dicentric R- ring P- Ploidy, F- Fragement E- Exchange RI-

Reference item T1- Lowest concentration T3- Highest concentration.




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Table No.8: Percentage Mitotic index and chromosomal aberration for Curcuma

aromatica in the presence of metabolic activation

Concentra

tion

Culture

No

Percentage

Mitotic

index

Frequencies of

aberrations

Total

Number of

Aberration

Percentage

of

Aberrant

cells

Negative

control

1 3.5 1Cb 1 1

2 4.0 1 Cb, 1D 2 2

78.125 (T1) 1 3.2 1 CLb,1D 2 2

2 3.0 1F 1 1

156.25 (T2) 1 2.0 1Di 1 1

2 2.5 1 CLb, 2D 1 1

312.5(T3) 1 1.8 1Cg,2Cb 3 2

2 1.32 1F,1CLb,

1Cb,1Di

4 4

RI 1 2.0 2CLb,2Cb,4D,

3F,2Di,2R,2E,

3Clg,1P,2Cg

23 16

2 1.8 3CLb,2Cb,4CLg,

4D,2Di,1R,1E,2F

19 11







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6.1.3.2.2. Curcumin

The curcumin was tested at the following concentrations 40, 20, 10 µg/ml both in the

absence and presence of metabolic activation system (1% v/v S9).

6.1.3.2.2.1. Pretest


treatment. Based on the results of solubility, precipitation and pH, 312.5 µg/ml of

curcumin was selected as the highest concentration for pretest both in the absence and

presence of metabolic activation system (1% v/v S9). The results are tabulated in Table

No.9.

Table No.9: Result for pH and Precipitation Test for Curcumin

Concentration

Recorded pH

Precipitation Test

0 hour 4 hour

2500 µg/ml NA NA Precipitation



312.5 µg/ml 7.25 7.30 No precipitation




Negative control 7.18 7.25 No precipitation

Note: Dimethyl sulfoxide (DMSO) was used for negative control.

NA=Not Applicable.




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The cytotoxicity due to treatment with curcumin was assessed based on reduction

(>50%) in the percent mitotic index. The cytotoxicity experiment was conducted at the

concentrations of 312.5,156.25,78.125,39.062 µg/ml under the specified conditions.

The reduction in the percent mitotic index observed was 85.71, 62.86, 57.14, 47.14 in

the absence and 87.5, 73.33, 57.5, 47.22 in the presence of metabolic activation (1%

v/v S9) respectively. Hence, 40µg/ml was selected as the highest concentration both in

the presence and absence of metabolic activation system for the main study. The results

are tabulated in Table No.10.

.




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Table No. 10: Percentage Mitotic Index- Pretest for Curcumin

Concentra

tion (µg/ml)

R Mitotic Index


activation


activation

Percent

age

Mitotic

index

Mean

percent

age MI

Reduct

ion in

MI

(%)

Percentag

e

Mitotic

index

Mean

percent

age MI

Reducti

on in

MI (%)

Negative

control

1 4.0 3.5 NA 3.9 3.6 NA

2 3.0 3.3

312.5(T1)

1 1.5 1.85 47.14

1.7 1.9 47.22

2 2.2 2.1

625 (T2)

1 1.6 1.5 57.14 1.42 1.53 57.5

2 1.4 1.64

1250(T3)

1 1.4 1.3 62.86 0.7 0.96 73.33

2 1.2 1.22

2500(T4)

1 0.4 0.5 85.71 0.3 0.45 87.5

2 0.6 0.6

Key: CON. = Concentration, R= replicate, T= Treatment Group, NA= Not applicable,

MI= Mitotic Index.

Note: % Reduction = Mean MI of Negative control group – Mean MI of Treatment

group/Mean MI of negative control group x 100.

.




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6.1.3.2.2.2. Main study

In the main study, the cultures were exposed to Curcumin for a short period of time (4

hours) both in the absence and presence of metabolic activation system (1% v/v S9).

The mean number of percent aberrent cells were 1.5, 5.0, 4.5, 5.5 and 16 at doses 0.0

(negative control), 10 (T1), 20 (T2), 40 (T3) µg/ml and 0.3 µg/ml of Mitomycin-C

(reference item) respectively, in the absence of metabolic activation system.

In the presence of metabolic activation system the mean number of percent aberrent

cells were 1.5, 5.5, 5.5, 6.5 and 13.5 at doses 0.0 (negative control), 10 (T1), 20 (T2),

40 (T3) µg/ml and 40 µg/ml of Cyclophosphamide monohydrate (reference item),

respectively.

Reduction(>50%) in the percent mitotic index was not observed in the cultures treated

up to the dose 40 µg/ml both in the absence and presence of metabolic activation

system (1% v/v S9) when compared with the negative control.

Increase in the percent aberrant cells was not observed in the cultures treated upto the

dose level of 40 µg/ml both in the presence and absence of metabolic activation system

when compared with the negative control, where the reference item caused a significant

increase in percent aberrant cells both in the absence and presence of metabolic

activation system (1% v/v S9). Statistical analysis (ANOVA and t-test) was performed

using statplus 2009 professional 5.7.8 A statistically significant increase (p-level

<0.05) in aberrent cells was not observed in ANOVA (comparing negative control and

test item doses) in the presence of S9 and a statistically significant increase (p-level

<0.05) was observed in aberrent cells in the absence of metabolic activation system.

Where statistically significant increase (T-criter value>P value) in aberrent cells was




Page 111

observed in T test (comparing means of negative control and reference item) both in the

presence and absence of metabolic activation system. Biological and statistical

relevance of the result was considered for evaluation. The results are tabulated in

Table No.11,12 and represented in Graph No. 14,15,16,17.




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Table No.11: Percentage Mitotic index and Chromosomal aberrations for

Curcumin in the Absence of metabolic activation.

Concentrati

on

Culture

No

Percentage

Mitotic

index

Frequencies of

aberrations

Total

Number of

Aberra

tion

Percentage

of

Aberrant

cells

Negative

control

1 3.8 1Cb, 1CLg 2 1

2 3.4 1R,1D 2 2

10(T1)

1 3.2 2CLb, 1E, 1P,

1D, 1F

6 4

2 3.00 1F,1CLb,2CLg,

2Cb,2D

8 6

20(T2)

1 3.2 1Cb,1CLb,2Di,1P

,1D

6 4

2 1.5 1CLb,2Cg, 1D,

1E,1F,1Di

7 5

40(T3)

1 2.5 1CLg, 2D, 1E,

1Di, 1CLb

6 5

2 1.62 2F,1Cb,1Cg,2D,

1CLg, 1CLb

8 6

RI

1 1.07 5CLb,3F,1CLg,6

D, 4Cg,2E, 3Di

24 17

2 2.00 6F,2CLb,1D,3Di,

4Cg, 3Cb, 2P, 2R

23 15



Reference item T1- Lowest concentration T3- Highest concentration




Page 113

Table No.12: Percentage Mitotic Index and Chromosome Aberrations for

Curcumin in the Presence of Metabolic Activation.

Concentrat

ion

Cultu

re No

Percentage

Mitotic

index

Frequencies

of

aberrations

Total

Number

of

Aberrati

on

Percenta

ge of

Aberran

t cells

Negative

control

1 3.5 1Cb, 1CLg 2 1

2 4.1 1CLb, 1D 2 2

10(T1) 1 3.1 1CLb, 1E, 2D, 1F, 2Cb 7 7

2 2.8 2CLg,2R,1Cb, 2CLb 7 4

20(T2) 1 2.7 1Cb,2CLb,2D, 1E 6 6

2 2.6 2CLb,1Cg, 1P, 2F,1Di 7 5

40(T3) 1 2.4 2CLg, 2D, 1E, 2CLb,

1Di

8 6

2 1.5 1F,2Cb,2Cg,2D, 1CLg,

2CLb

10 7

RI 1 2.0 2CLb,2Cb,4D,3F,2Di,2R

,2E,3CLg, 1P,2Cg

23 16

2 1.3 3CLb, 2Cb, 4CLg, 4D,

2Di, 1R, 1E,2F

19 11







Page 114

6.1.3.2.3. Curcuma zedoaria (Christm.) Roscoe

The Curcuma zedoaria (Christm.) Roscoe was tested at the following concentrations:

156.25, 78.12, 39.06 µg/ml both in the absence and presence of metabolic activation

system (1% v/v S9).

6.1.3.2.3.1.Pretest


treatment. Based on the results of solubility, precipitation and pH 156.25 µg/ml of

hydro alcoholic extract of Curcuma zedoaria was selected as the highest concentration

for pretest both in the absence and presence of metabolic activation system (1% v/v

S9). The hydro alcoholic extract of Curcuma zedaoria was tested at the following

concentrations: 156.25, 78.12, 39.06, 19.53µg/ml in pretest. The results are tabulated in

Table No.13.




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Table No.13: Result for pH and Precipitation Test for Curcuma zedoaria

Concentration

Recorded pH

Precipitation Test

0 hour 4 hour





Negative control 7.20 7.26 No precipitation

Note: Dimethyl sulfoxide (DMSO) was used for negative control.

NA = Not Applicable.

The cytotoxicity due to treatment with Curcuma zedoaria extract was assessed based

on reduction(>50%) in the percent mitotic index. The cytotoxicity experiment was

conducted at the concentrations of 156.25, 78.125, 39.062 and 19.531 µg/ml under the

specified conditions. The reduction in the percent mitotic index(>50%) was not

observed at any of the tested concentrations of test item 156.25, 78.125, 39.062 and

19.531 µg/ml both in the absence and presence of metabolic activation (1% v/v S9).

Hence, 156.25 µg/ml was selected as the highest concentration for the main study both

in the absence and presence of metabolic activation system (1% v/v S9) for the main

study. The results are tabulated in Table No.14.




Page 116

Table No. 14: Percentage Mitotic Index- Pretest for Curcuma zedoaria

Concentrati

on (µg/ml)

R Mitotic Index


activation


activation

Percent

age

Mitotic

index

Mean

percent

age MI

Reduct

ion in

MI

(%)

Percentag

e

Mitotic

index

Mean

percent

age MI

Reducti

on in

MI (%)

Negative

control

1 4.0 3.75 NA 4.0 3.7 NA

2 3.5 3.4

19.531(T1) 1 3.0 3.4 9.33 3.4 3.3 10.81

2 3.8 3.2

39.062 (T2) 1 2.8 3.1 17.33 3.0 3.125 15.54

2 3.4 3.25

78.125 (T3) 1 2.7 2.95 21.33 3.0 3.05 17.56

2 3.2 3.1

156.25 (T4) 1 2.7 2.9 22.66 3.0 3.1 16.22

2 3.1 3.2

Key: CON. = Concentration, R= replicate, T= Treatment Group, NA= Not applicable,

MI= Mitotic Index.

Note: % Reduction = Mean MI of Negative control group – Mean MI of Treatment

group/Mean MI of negative control group x 100.




Page 117

6.1.3.2.3.2. Main study

In the main study, the cultures were exposed to hydro alcoholic extract of Curcuma

zedoaria for a short period of time (4 hours) both in the absence and presence of


The mean number of percent aberrent cells were 1.0, 1.5, 2.0, 2.0 and 13.5 at doses 0.0

(negative control), 39.062 (T1), 78.125 (T2), 156.25 (T3) µg/ml and 40 µg/ml of

Cyclophosphamide monohydrate (reference item) respectively, in the presence of

metabolic activation system.

In the absence of metabolic activation system the mean number of percent aberrent

cells were 1.0, 2.0, 2.5, 6.5 and 16.0 at doses 0.0 (negative control), 39.062 (T1),

78.125 (T2), 156.25 (T3) µg/ml and 0.3 µg/ml of Mitomycin-C (reference item),

respectively.

Reduction(>50%) in the percent mitotic index was not observed in the cultures treated

upto the dose of 156.25 µg/ml both in the absence and presence of metabolic activation

system (1% v/v S9) when compared with the negative control.

Dose dependent increase in the percent aberrant cells was not observed upto 78.125

µg/ml but the aberration frequency was more at the highest concentration treated

156.25 µg/ml in the absence of metabolic activation system when compared with the

negative control. Where the Dose dependent increase in the percent aberrant cells was

not observed at all the tested doses in the presence of metabolic activation system.

Reference item caused a significant increase in percent aberrant cells both in the

absence and presence of metabolic activation system (1% v/v S9). Statistical analysis

(ANOVA and t-test) was performed using statplus 2009 professional 5.7.8. A

statistically significant increase (p-level <0.05) in aberrent cells was observed in




Page 118

ANOVA (comparing negative control and test item doses) in the absence of metabolic

activation system where statistically significant increase (p-level <0.05) in aberrent

cells was not observed, in the presence of metabolic activation. Statistically significant

increase (P value <T-criter value) in aberrent cells was observed in T test (comparing

means of negative control and reference item) both in the presence and absence of

metabolic activation system. Biological and statistical relevance of the result was

considered for evaluation. The results are tabuated in Table No.15, 16 and represented

in the Graph No. 18,19.20,21.




Page 119

Table No.15: Percentage Mitotic index and Chromosomal aberrations for

Curcuma zedoaria in the Absence of metabolic activation.

Concentration

Culture

No

Percentage

Mitotic

index

Frequencies of

aberrations

Total

Number of

Aberra

tion

Percent

age of

Aberrant

cells

Negative

control

1 3.8 1 CLg 1 0

2 3.4 1Cg, 1D,1Cb 3 2

39.062 (T1)

1 3.2 1Cb, 1Cg 2 1

2 3.00 1CLb,2CLg,

2Cb

5 3

78.125 (T2)

1 2.9 2Cg,2CLb,

1P,1D

6 3

2 2.6 1CLb,2CLg,

1D

4 2

156.25 (T3)

1 2.8 1CLg,1Cg,

4D,1 Di, 1CLb,

1Cb ,1P

10 7

2 2.6 2Cg, 4 CLb,

2Cb, 3D

11 6

RI

1 1.07 5CLb,3F,1CLg,

6D, 4Cg,2E,

3Di

24 17

2 2.00 6F,2CLb,1D,3

Di, 4Cg, 3Cb,

2P, 2R

23 15







Page 120

Table No.16: Percentage Mitotic Index and Chromosome aberrations for

Curcuma zedoaria in the Presence of Metabolic Activation.

Concentrat

ion

Culture

No

Percentage

Mitotic

index

Frequencies

of

aberrations

Total

Number

of

Aberrati

on

Percent

age of

Aberra

nt cells

Negative

control

1 3.9 1 CLb, 1D 2 2

2 3.7 1 Cg 1 0

39.062 (T1)

1 3.8 1CLg, 1Cg 2 0

2 3.3 2Cb, 2CLg, 1D 5 3

78.125 (T2)

1 3.6 1Cg,2CLg, 1D 4 1

2 3.2 2Cg,2CLb, 1D 5 3

156.25 (T3)

1 2.97 1CLg,2Cg, 1Di, 1CLb,

1Cb

6 3

2 3.62 2 Cg, 1CLg, 1D, 1 P 5 1

RI

1 2.0 2CLb,2Cb,4D,3F,2Di,2

R,2E,3CLg, 1P,2Cg

23 16

2 1.3 3CLb, 2Cb, 4CLg, 4D,

2Di, 1R, 1E,2F

19 11







Page 121

6.1.3.3. SOS CHROMOTEST

For the detection of alkaline phosphatase activity P- nitro phenyl phosphate (PNPP)

was used as the standard SOS chromogen and the plates were incubated for 10 minutes

and it absorbance were measured at 405 nm. Alkaline Phosphatase activity reflects the

reduction factors which is represented as RF.

For β-galactosidase (b- gal) activity, 5- bromo-4- chloro-3-indolyl b- D-

galactopyranoside was used as the Standard SOS chromogen and the plates were

incubated for 60 minutes and its absorbance was measured at 620 nm. β- galactosidase

activity reflects the induction factors IF.

The ratio of the induction factor and reduction factor is the correction factor.




Page 122

Table No.17: The absorbance both at 405 nm and 620 nm

Sample

Concentration

Absorb

ance

(405 nm)

Sample

Concentration

Absorbance

(620nm)

Curcuma

aromatica

1000 0.676

Curcuma

aromatica

1000 0.317

500 0.671 500 0.287

250 0.609 250 0.269

Curcumin

1000 0.603

Curcumin

1000 0.365

500 0.567 500 0.354

250 0.514 250 0.280

Curcuma

zedoaria

1000 1.897

Curcuma

zedoaria

1000 0.371

500 0.880 500 0.358

250 0.652 250 0.350

Blank

0.897

Blank

0.408

The absorbance both at 405 nm and 620 nm was found to increase with the

concentration of hydro alcoholic extract of Curcuma aromatica, Curcuma zedoaria,

Curcumin.




Page 123

Table No.18: Determination of the Repression factor for the SOS Chromotest

Sample

Concentration

( µg/ml)

Absorbance

(405 nm)

RF values

Curcuma

aromatica

1000 0.676 0.7536

500 0.671 0.7480

250 0.609 0.6789

Curcumin

1000 0.603 0.6722

500 0.567 0.6321

250 0.514 0.5730

Curcuma

zedoaria

1000 1.897 2.1148

500 0.880 0.9810

250 0.652 0.7268

Blank

0.897

RF values were found to increase with the concentration hydro alcoholic extract of

Curcuma aromatica, Curcuma zedoaria and Curcumin.




Page 124

Table No.19: Determination of Induction Factor for the SOS Chromotest

Sample

Concentration

(µg/ml)

Absorbance

(620nm)

IF values

Curcuma

aromatica

1000 0.317 0.7769

500 0.287 0.7034

250 0.269 0.6593

Curcumin

1000 0.365 0.8946

500 0.354 0.8676

250 0.280 0.6862

Curcuma

zedoaria

1000 0.371 0.9093

500 0.358 0.8774

250 0.350 0.8578

Blank 0.408

Induction factor were also found to increase with the concentration of hydro alcoholic

extract of Curcuma aromatica, Curcuma zedoaria and Curcumin.




Page 125

Table No.20: Correction Induction Factor for the SOS Chromotest

Sample

Concentration

( µg/ml)

IF Value

RF Value

CIF= IF/RF

Curcuma

aromatica

1000 0.7769 0.7536 1.0309

500 0.7034 0.7480 0.9403

250 0.6593 0.6789 0.9711

Curcumin

1000 0.8946 0.6722 1.3308

500 0.8676 0.6321 1.3725

250 0.6862 0.5730 1.1975

Curcuma

zedoaria

1000 0.9093 2.1148 0.4299

500 0.8774 0.9810 0.8943

250 0.8578 0.7268 1.1802


For Curcuma aromatica the corrected induction factor produced at the concentration of

250 and 500 µg/ml was found to be more or less equal such as 0.9711 and 0.9403

where as the correction induction factor at the concentration of 1000 µg/ml was found

to be 1.0309. All these values were found to be less than 1.2 which is considered as an

index to represent genotoxicity. The hydro alcoholic extract of Curcuma aromatica

was considered to be non- genotoxic.




Page 126


For Curcuma zedoaria the corrected induction factor was found to be 0.4299, 0.8943

and 1.1975 for 1000, 500 and 250 µg/ml concentration respectively. The corrected

induction factor was found to be more at the concentration of 250 µg/ml. The corrected

induction factor found to decrease with the increase in the concentration of the hydro

alcoholic extract Curcuma zedoaria from 500 µg/ml to 1000 µg/ml. all these values

were found to be less than 1.2 which is considered as an index to represent

genotoxicity. From the values it was considered to be non- genotoxic.

6.1.3.3.3. Curcumin

For curcumin the corrected induction factor was found to be 1.3308, 1.3725 and 1.1975

for 1000, 500 and 250 µg/ml. Curcumin was found to be genotoxic even at the

concentration of 250 µg/ml onwards and corrected induction factor was found to

decrease with the increase in the concentration from 500 µg/ml to 1000 µg/ml.

Curcumin was considered to be genotoxic because all the values obtained were found

to be more than 1.2 which is considered as an index to represent genotoxicity.

In this model SOS Chromotest, curcumin alone was found to be genotoxic even at the

lowest concentration of 250 µg/ml where as the hydro alcoholic extract of Curcuma

aromatica and Curcuma zedoaria was found to be non- genotoxic even at the highest

concentration i.e 1000 µg/ml.

6.1.3.4. DNA Sugar Damage

Among the selected test compounds Curcuma aromatica, Curcumin, protected the

sugar moiety in DNA at the lowest concentration of 250µg/ml onwards where as




Page 127

Curcuma zedoaria protected the DNA only at 1000 µg/ml. Quercetin which is taken as

the control showed the protection at 250, 500 µg/ml. Curcumin showed the maximum

protection activity among all the tested material. The results are tabulated in Table

No.21 and represented in Graph No. 25.

Table No. 21: DNA sugar damage assay

6.1.3.5. Potato disc Assay

Positive control without extract

The potato disc which shows the mutation that is caused by the Agarobacterium

tumifaciens in the potato disc and it is shown in the Figure No.6 and it is considered as

positive control without extract.

Sample No

250ug/ml

500ug/ml

1000ug/ml

Blank 0.031 0.031 0.031

Curcuma zedoaria -0.063 0.003 0.025

Curcuma aromatica 0.037 0.079 0.223

Curcumin 0.110 0.124 0.284

Quercetin 0.028 0.082 0.090




Page 128

Negative control

Potato disc are kept as negative control. When treated with Iodine solution appearance

of blue colour indicates the presence of unmutated cells. This cells is considered as

negative control and it is shown in the Figure No.7.


The potato disc was treated with hydro alcoholic extract of Curcuma aromatica at the

concentration of 250 µg/ml. due to mutation the cells were not in a position to take the

iodine solution and turns blue in color. The cells were appearing white due to the

mutation that is caused by Agarobacterium tumifaciens and it is shown in Figure No.8.

When potato disc was treated with 500 µg of hydro alcoholic extract of Curcuma

aromatica. to some extend the extract protected the cells from mutation. The proportion

of blue color increased when compared to 250 µg/ml concentration and it is shown in

the Figure No.9.

When potato disc was treated with 1000 µg/ml of hydro alcoholic extract of Curcuma

aromatica, the cells were protected to a maximum extent at this concentration against

mutation which is indicated by the appearance of blue color in the cells and it is shown

in Figure No. 10.

6.1.3.5.2. Curcumin

When potato disc treated with was 250 µg/ml concentration of Curcumin, it showed

slight protection against the mutation that is caused by Agarobacterium tumifaciens and

it is shown in the Figure No. 11.




Page 129

When the potato disc treated with Curcumin at the concentration of 500 µg/ml, its

protection was good against the mutation that is caused by Agarobacterium

tumifaciens. When compared to 250 µg/ml and it is shown in Figure No.12.

When the potato disc treated with Curcumin at the concentration of 1000 µg/ml,

showed good protection against the mutation caused by Agarobacterium tumifaciens

and it is shown in Figure No.13.


Potato disc treated with hydro alcoholic extract of Curcuma zedoaria at the

concentration of 250 µg/ml showed slight protection against the mutation caused by

Agarobacterium tumifaciens and it is shown in the Figure No. 14.

When the potato disc treated with hydro alcoholic extract of Curcuma zedoaria at the

concentration of 500 µg/ml, protection was found to be better when compared to the

potato disc treated with 250 µg/ml concentrations and it is shown in the Figure No.15.

When the potato disc treated with hydro alcoholic extract of Curcuma zedoaria at the

concentration of 1000 µg/ml, protection was found to be good when compared to other

two concentrations of extract and it is shown in the Figure No.16.

These results suggested that the hydro alcoholic extract of Curcuma aromatica,

Curcuma zedoaria and Curcumin found to possess antimutagenic activity which was

found to increase with the concentration of the drug. Among the three tested

compounds curcumin showed good antimutagenic activity from the concentration 500

µg/ml to 1000 µg/ml.




Page 130

6.1.3.6. Comet assay

The lymphocytes were isolated from the blood sample which came to Drug testing

laboratory, JSS College of Pharmacy, Rockland’s, Udhagamandalam. The details of the

blood are as follows:

Agency which supplied the sample for sterility testing: Lions Club of Erode supreme

charitable trust blood bank, Erode.

Blood bag unit No: 1751

Group: “O” Positive

Quantity: 350 ml

Date of collection: 23.02.2012

Date of expiry: 27.03.2012

Date of arrival of the blood to the laboratory: 25.02.2012

Date on which the experiment is carried out: 25.02.2012.

The damage is in the human lymphocytes are classified as no damage, low damage,

medium damage, high damage and complete damage based on the ratio of tail to head

length. The extent of DNA damages was ascertained and scored as follows:

If the ratio of the tail and head length is less than 5.0 % the cells were considered as no

damage. If it is 5.1-20 % then it was considered as low damage. From 20.1-40.0% the

damage was considered to be medium and if the ratio is from 40.1- 95.0% the damage




Page 131

was considered to be high and if the ratio is more than 95% then the cells were

considered as completely damaged.

Among the slides 100 cells were scored and recorded.

In control samples 93±3 cells were found to have no damage in the presence of

metabolic activation factor where as in the absence of metabolic activation factor 97±3

cells were found to have no damage in the control sample.

At the concentration of 50 µg/ml

6.1.6.6.1. In the absence of metabolic activation factor

6.1.3.6.1.1. Curcuma aromatica salisb

No damage was found in 70±3 cells, low damage was found to be in 22±5 and medium

damage was found in 8±4 cells. In cells there was no high damage and complete

damage occurred in the cells.

6.1.3.6.1.2. Curcuma zedoaria (Christm.) Roscoe

The normal cells were found to be 79±3 cells. In 15±2 cells low damage was observed

and in 6±3 cells medium damage was observed.

6.1.3.6.1.3. Curcumin

Normal cells were found to be 62±4, 28±3 cells showed low damage and medium

damage had occurred in 10 ±4 cells.




Page 132

At the concentration of 50 µg/ml

6.1.3.6.2. In the presence of metabolic activation factor

6.1.3.6.2.1. Curcuma aromatica salisb

It caused high damage in 6±5 cells, medium damage was found in 12±3 cells, low

damage was found in 24±5 cells and normal cells were found to be 49±2 cells.

6.1.3.6.2.2. Curcuma zedoaria (Christm.) Roscoe

It caused high damage in 5±4 cells, medium damage in 11±5 cells and low damage in

30 ±1 cells and normal cells were found to be 54±3 cells.

6.1.3.6.2.3. Curcumin

It caused complete damage in 4±3, high damage in 8±3 cells. It caused medium

damage in 10±2 cells and low damage in 37±2 cells and normal cells were found to be

41±3 cells.

Among the three tested compounds, curcumin was found to be more genotoxic in

nature when compared by Curcuma aromatica, Curcuma zedoaria. These results

might either due to the presence of low concentration of curcumin in the extracts or due

to the presence of other phytoconsituents that are present in the extract. The damage of

the cells was found to be more in the presence of metabolic activation factor than the

absence of metabolic activation factor. This confirms that the phytoconstituents are not

genotoxic in nature but their metabolites that are formed in the presence of metabolic

activation factor is mainly responsible for their genotoxic effect. These results are




Page 133

tabulated in the Table No.22 and represented in Graph No. 26 and shown in the Figures

17-21.

Table No.22: Genotoxicity of Curcuma aromatica, Curcuma zedoaria, and

Curcumin in human lymphocytes in the comet assay.

Sample

Cell number

No damage Low

damage

Medium

damage

High

damage

Complete

damage

In the absence of metabolic activation factor

Control 93±3 3±3 0 0 0

Curcuma

aromatica

70±3 22±5 8±4 0 0

Curcuma

zedoaria

79±3 15±2 6±3 0 0

Curcumin 62±4 28±3 10±4 0 0

In the presence of metabolic activation factor

Control 97±3 5±2

Curcuma

aromatica

49±2 24±5 12±3 6±5 0

Curcuma

zedoaria

54±3 30±1 11±5 5±4 0

Curcumin 41±3 37±3 10±2 8±3 4±3




Page 134

6.1.3.7. Micronucleus test

Positive control- MMS (Methyl methane sulfonate at the concentration of 50 µg/ml)

was used.

The criteria that was followed for the identification of binucleated cells and micronuclei

as follows:

1. Shape should be round for both the nuclei and micronuclei

2. When compared to the main nuclei, the micronuclei should be 1/3 small.

3. The main nuclei should not touch the micronucleus

4. The color and intensity of the micronuclei must be same as the main nuclei.

6.1.3.7.1. Curcuma aromatica at the concentration of 50 µg/ml produced the

micronuclei at the rate of 12.333±2.223, 15.666±1.778, in the absence and presence of

metabolic activation factor respectively.

6.1.3.7.2. Curcuma zedoaria at the concentration of50 µg/ml produced the micronuclei

at the rate of 9.666±1.999, 14.666±2.666 in the absence and presence of metabolic

activation factor respectively.

6.1.3.7.3. Curcumin at the concentration of 50 µg/ml produced the micronuclei at the

rate of 14.666±1.555, 19.333±1.111 in the absence and presence of metabolic

activation factor respectively.

Positive control (Methyl methane sulfonate) produced the micronuclei at the rate of

25.666±1.555, 35.66±2.886 in the presence and absence of metabolic activation factor

respectively.




Page 135

When compared to the positive control (Methyl methane sulfonate at the concentration

of 50 µg/ml), all the test compounds such as hydro alcoholic extract of Curcuma

aromatica, Curcuma zedoaria, and Curcumin at the concentration of 50μg/ml showed

less toxicity to HEp-2 cells. The results are tabulated in Table No. 23, represented in

Graph No.27 and shown in the Figures 22 and 23.




Page 136

Table No.23: Mean Frequencies of Micronucleus observed in HEp-2 cells

Treatment

Repetition

Mean

±standard

deviation

1

2

3

Negative control 6 3 4 4.33±1.086

Positive control- in

the absence of S9

factor

40 32 35 35.66±2.886

Positive control- in

the presence of S9

factor

25 28 24 25.666±1.555

Curcuma aromatica salisb

In the absence of

S9 factor

13 15 09 12.333±2.223

In the presence of

S9 factor

18 16 13 15.666±1.778

Curcuma zedoaria (Christm.) Roscoe

In the absence of

S9 factor

8 9 12 9.666±1.999

In the presence of

S9 factor

12 17 15 14.666±2.666

Curcumin

In the absence of

S9 factor

14 17 13 14.666±1.555

In the presence of

S9 factor

21 18 19 19.333±1.111




Page 137

6.1.3.8. Plasmid nicking assay or DNA-damage protective activity

In this method plasmid DNA pBR322

was purchased from Chromos Biotech and it was

used to carry out the plasmid nicking assay or DNA-damage protective activity of the

test compounds

Quercetin was used as positive control

Plasmid DNA+ Fenton’s reagent + quercetin 250 µg/ml--- loaded in the lane 1 and 15

Plasmid DNA+ Fenton’s reagent + quercetin 500 µg/ml--- loaded in the lane 2 and 16

Plasmid DNA + Fenton’s reagent+ quercetin 1000 µg/ml—loaded in the lane 3 and17

Plasmid DNA+ Fenton’s reagent + Curcuma aromatica 250 µg/ml- loaded in the lane 4

and18

Plasmid DNA+ Fenton’s reagent+ Curcuma aromatica 500 µg/ml- loaded in the lane 5

and 19

Plasmid DNA+ Fenton’s reagent+ Curcuma aromatica 1000 µg/ml--- loaded in the

lane 6 and 20.

Plasmid DNA+ Fenton’s reagent+ Curcuma zedoaria 250 µg/ml---- loaded in the lane

7 and 21

Plasmid DNA+ Fenton’s reagent + Curcuma zedoaria 500 µg/ml------ loaded in the

lane 8 and 22

Plasmid DNA+ Fenton’s reagent+ Curcuma zedoaria 1000 µg/ml----- loaded in the

lane 9 and 23




Page 138

Plasmid DNA+ Fenton’s reagent + Curcumin 250 µg/ml---- loaded in the lane 10 and

24.

Plasmid DNA+ Fenton’s reagent +Curcumin 500 µg/ml------ loaded in the lane 11 and

25

Plasmid DNA + Fenton’s reagent+ Curcumin 1000 µg/ml------ loaded in the lane 12

and 26

Plasmid DNA- alone was used as control ---------- loaded in the lane 13 and 27.

Plasmid DNA treated with Fenton’s reagent was used as negative control--------- loaded

in the lane 14 and 28.

Quercetin at all the tested concentration such as 250, 500 and 1000 µg/ml showed the

damage to the plasmid and it might have occurred due to the incision in the DNA

Curcuma aromatica and Curcuma zedoaria protected the DNA from the concentration

of 250 µg/ml to 1000 µg/ml.

Curcumin caused slight damage when compared to quercetin as well as negative

control in all the tested concentration such as 250, 500 and 1000 µg/ml. The results are

shown in the Figure No. 24.

6.1.3.9. Sequencing method

In order the confirm the mutagenicity effect produced by the Fenton’s reagent and the

protection given by the tested compounds such as Curcuma aromatica, Curcuma




Page 139

zedoaria and Curcumin sequencing method was carried out and the samples were used

as follows:

Plasmid DNA+ Fenton’s reagent + Curcuma aromatica 50 µg/ml-

Plasmid DNA+ Fenton’s reagent+ Curcuma zedoaria 50 µg/ml

Plasmid DNA+ Fenton’s reagent + Curcumin 50 µg/ml

Plasmid DNA- alone was used as control

pBR322 specific primers used in the study:

807F: 5’-GCA GGA AAG AAC ATG TGA GCA AAA GGC CA-3’ (Forward primer)

1507R: 5’-CAA AAT CCC TTA ACG TGA GTT TTC GTT CC-3’ (reverse primer).

The results are tabulated in the Table NO. 24 and shown in the figures from 25-34.

Table No.24: Results for the sequencing

Name of the

sample

Primers

Sequence

Plasmid DNA

Forward primer

5’----GCA GGA AAG AAC ATG

TGA GCA AAA GGC CA -3’

CGT CCT TTC TTG TAC ACT CGT TTT

CCG GTC GTT TTC CGG TCC TTG GAT

TTT TCC GGC GCA ACG ACC GCA AAA

AGG TAT CCG AGG CGG GGG GAC TGC

TCG TAG TGT TTT TAG CTG CGT GTT

CAG TCT CCA CCG CTT TCG GCT GTC




Page 140

C-74 G-89T-70A-59

CTG ATA TTT CTA TGG TCC GCA AAG

GGG GAC CTT CGA GGG TGC ACG CGA

GAG GAC AAG GCT GGG ACG GCG AAT

GGC CTA TGG ACA GGC GGA AAG AGG

GAA GCC CTT CGC ACC GCG AAA GAG

TAT CGA GTG CGA CAT CCA TAG AGT

CAA GCC ACA

Reverse Primer

5’ CAA AAT CCC TTA ACG

TGA GTT TTG GTT CC3’

G-62 , T-63 , A-64, C- 53

GTT TTA GGG AAT TGC ACT CAA AGC

AAC CTG ACT CGC AGT CTG GGG CAT

CTT TTC TAG TTT CCT AGA AGA ACT

CTA GGA AAA AAA GAC GCG CAT TAG

ACG ACG AAC GTT TGT TTT TTT GGT

GGC GAT GGT CGC CCA CCA AAG GGG

CGG CCT AGT TCT CGA TGG TTG AGA

AAA AGG CTT CCA TTG ACC GAA GTC

GTC TCG CGT CTA TGG TTT ATG ACA

GGA AGA TCA CAT CGG CAT CAA TCC

GGT GGT GAA GT

Plasmid DNA

treated with

Curcuma

aromatica at the

concentration of

50 µg/ml

Forward primer



C-74 G-89T-70A-59














Page 141



CAA GCC ACA

Reverse Primer



G-62 , T-63 , A-64, C- 53











GGT GGT GAA GT

Plasmid DNA

treated with

Curcumin at the

concentration of

50 μg

Forward primer



C-74 G-89T-70A-59













CAA GCC ACA





Page 142

Reverse Primer



G-62 , T-63 , A-64, C- 53










GGT GGT GAA GT

Plasmid DNA

treated with

Curcuma

zedoaria at the

concentration of

50µg/ml

Forward primer



C-74 G-89T-70A-59













CAA GCC ACA

Reverse Primer










Page 143


G-62 , T-63 , A-64, C- 53






GGT GGT GAA GT

Plasmid DNA

treated with

Fenton’s reagent

Forward primer



Could not able to obtain any

information regarding the arrangement

of nitrogenous base

Reverse Primer



Could not able to obtain any

information regarding the arrangement

of nitrogenous base

A COMPARATIVE STUDY ON GENOTOXIC ACTIVITY OF CURCUMA AROMATICA Salisb AND CURCUMA ZEDOARIA(Christm.)Roscoe RHIZOMES

Discussion


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6.2. Discussion

Several substances in plants express cytotoxic and genotoxic activities and show

correlation with the incidence of tumors. Therefore, understanding the health benefits

and or potential toxicity of the plants is important (Yen et al., 2001). Although plant

extracts have been used in the treatment of diseases according to knowledge

accumulated over centuries, it is also known that many plants synthesize toxic

substances, which in nature acts as defense against infections, infects and herbivores.

Some substances present in some medicinal plants are potentially toxic and

carcinogenic and it has also been reported that some traditional medicines may have

genotoxic potentials. Assessment of the potential genotoxicity of traditional medicine

is indeed an important issue as damage to the genotoxic materials may lead to critical

mutation and therefore also to an increased risk of cancer and other disease. Major

bioactive phytochemicals that have been associated with many plants are different

types of saponins and flavonoid.

In over all safety evaluation of the botanicals, a modest trend towards increasing the

inclusion of information, a genotoxicity appeared in peak in the last four years because

of significant awareness of the impact of genotoxicity (Keui-Meng.Wu et al., 2010). In

in-vitro assay, recent investigations have revealed that many plants used as food or in

traditional medicine have mutagenic hazard (Esameldin et al., 2003). The isolated

compounds from the plants such as Quercetin, furoquinoline, alkaloids and

isothiocyanates were considered to be mutagens. It is very difficult to speculate the

compounds that are responsible for mutagenic response detected with plant extracts


Discussion


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because they are complex mixtures of organic compounds (Esameldin et al., 2003).

Short term in-vitro and in vivo studies as well as long term carcinogenicity studies, with

chemically treated animals confirmed that phytochemicals could also possess

antimutagenicity and anticarcinogenic effect. Epidemiological studies also supported

that the chemo preventive effect in which phytochemicals exhibit genotoxic/ mutagenic

effect by themselves or potential the effect of other xenobiotics (Volker Mersch

Sundermann et al., 2006).

6.2.1. Many literatures stressed the importance of carrying out the genotoxicity studies

for the medicinal plants. Botanicals contain multiple chemical constituents which may

be pharmacologically active with significant proportions of chemically undefined

constituents, the genotoxic information obtained from studies using a whole herbal o

multicomponent herb product is relatively lacking Based upon these literature, this

research work has been designed to carry out the genotoxicity studies for Curcuma

aromatica Salisb and Curcuma zedoaria (Christm.) Roscoe and the results which have

been obtained from these studies were compared with curcumin.

6.2.2. The presence of phytoconstituents including tannins, catechins, flavonones,

isoflavones are responsible for the possible genotoxic effects of plant extracts.

Flavonoids inhibit topoisomerase I and II enzyme which will interfere with the

replication and transcription process, inhibiting the relegation of DNA- double strand

breaks and enhancing the formation of cleavable DNA- enzyme complexes. Phenolic

rich extracts could lead to accumulated DNA breaks and mutation, thus contributing

significantly to genotoxicity. Because of these above statement, a systematic


Discussion


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phytoconstituents analysis has been carried out for Curcuma aromatica Salisb and

Curcuma zedoaria (Christm.) Roscoe. 50 % hydro alcoholic extract of Curcuma

aromatica salisb found to contain alkaloids, tannin and flavonoids where as Curcuma

zedoaria (Chrism.) Roscoe contain alkaloids and flavonoids. Total phenol and total

flavonol content was found to be more in Curcuma aromatica than Curcuma zedoaria.

Our studies showed the contrary results because even in the presence of high amount of

phenol and flavonol content Curcuma aromatica extract fails to produce genotoxicity

in many models. Curcuma zedoaria found to possess less phenol and flavonol content

but even then it produced the genotoxicity in the chromosomal aberration test in the

absence of S9 factor and it failed to protect the DNA sugar moiety at the concentration

of 250 µg/ml. From our studies it is understood, the presence of high phenol or flavonol

content is not having any role in producing the genotoxicity but circumstance in which

phytoconstituents are used decides the efficacy of the drug that are derived from the

plant source as far as these selected compounds are concerned.

The most important mechanism in antimutagenic and anticarcinogenic is the

scavenging of bioactive molecules. The extract which exhibit potent antioxidant and

free radical scavenging properties ascribed to its polyphenolic richness more

particularly to its flavonoid content (Volker Mersch sundermann et al., 2006). Free

radicals which are produced due to oxidative stress reported to produce damage in

DNA. Lipid peroxidation is also found to consider as a critical event in rat liver

mitochondria, microsome and spleenic lymphocytes due to the reaction of the hydroxyl

radicals generated with poly unsaturated fatty acids. Malondialdehyde which is the


Discussion


Page 147

product of lipid peroxidation forms adduct with cellular DNA (Maria Eurica Fracasso

et al., 2006). High content of flavonols, sulfur containing compounds and poly phenol

derivatives has been reported to exhibit antioxidative and free radical scavenging

abilities (Wonmekiat et al., 2008). Despite many pharmacological activities and

potential benefits to human health several flavonoids are described as mutagens (Santos

et al., 2006).

Many plant extracts had demonstrated potent cancer chemo preventive property and

most of them are known to exert their effects by antioxidant mechanism, either

quenching reactive oxygen species (ROS) or by inhibiting lipid peroxidation or by

stimulating cellular antioxidant defense (Hikinet Keles et al., 2010; Renato Mreira

Rosa et al., 2007) Genotoxicity might be related to hydrogen peroxide formation

arising from auto-oxidation of phenolic molecules (Volker Mersch Sundermann et al.,

2006). In-vitro antioxidant results showed that Curcumin as the potent antioxidant in

all the method employed. Among the plant extracts, 50 % hydro alcoholic extract of

Curcuma aromatica showed potent antioxidant activity than Curcuma zedoaria which

might be due to the presence of high phenolic and flavonol content. Involvement of

Na+/ H+ exchange and respiratory burst enzyme NADPH oxidases and Nitric oxide

synthase induce lipid peroxidation and DNA damage (Maria Eurica Fracasso et al.,

2006). At certain conditions, antioxidants such as vitamin C and E have been shown to

exert pro-oxidant effects in the presence of redox active elements (Wan-Ibrahim et al.,

2010). Curcumin which showed the potent antioxidant activity in our studies showed


Discussion


Page 148

the genotoxicity in chromosomal aberration test as well as SOS Chromotest.

Genotoxicity produced by curcumin might be due to its pro- oxidant activity.

6.2.3. By any single genotoxic test procedure the mutagenic potential of an agent can’t

be reliably determined because currently available mutagenicity assays have their own

strength and weakness ( Ann J. Dunnipace et al., 1992)

6.2.3.1. Ames reversion assay

Ames test is an in vitro mutagenicity assay which is useful for prescreening potential

carcinogens, as approximately 80% of carcinogens are proved to be mutagenic (Ann J.

Dunnipace et al., 1992). In Ames test where the target organism is unable to oxidize

chemicals, so necessary co factors must be supplied for an exogenous activation. The

major group of chemical carcinogen is activated by S9, generally comprises the hepatic

post- mitochondrial fraction from rats pre-treated with Aroclor 1254, a mixture of

polychlorinated bi phenyl which serve as a potent inducer of cytochrome P 450 families

in particular P 450 I and P 450 II. A clear mutagenic response is seen if the source of the

liver preparation is obtained from Arclor 1254- treated animals.

In the evaluation of genotoxicity of new chemicals, established mutagens are utilized

as positive control to ensure not only the responsiveness of the bacterial strains but also

the efficiency of the activating system during the routine employment of the Ames test.

To mutate all Salmonella typhimurium strain 2- amino anthracene is most widely used

mutagen which appears to be activated by hepatic system derived from all animal’s

species including man. N- hydroxylation catalyzes the activation of 2- amino


Discussion


Page 149

anthracene since Aroclor 1254 is an established inducer of P450- I family. The

mutagenicity of 2- aminoanthracene was markedly decreased when the animals are

treated with Arcoclor 1254. Microsome from the untreated animals could bioactivate

2- amino anthracene to mutagens (Ayrotones et al., 1992).

TA 98, TA 100 of Salmonella typhimurium are the histidine deficient (His-) mutant

tester strain. In many of the tester strain TA 98, TA 100 of Salmonella typhimurium

does not have intrinsic potential to induce mutation by increasing the number of His +

colonies. Exogenous metabolic activation is required for most of the chemicals to form

an ultimate mutagenic species to induce mutation in in- vitro assays including Ames

Salmonella test. Some electrophilic chemicals are mutagenic and directly act with

DNA.

For metabolic activation studies, in general 4-5 % Aroclor 1254- inducer S9 fraction in

the S9 mixture is used. In this study 1 % S9 factor was used. To induce the mutagenic

response, few chemicals require an elevated level of S9 fraction 10- 30 % in the S9

mixture (Meshram et al., 1992). Biological samples may cause problems in assays for

mutagenicity using Ames test/ Salmonella test because of the presence of autxotrophic

growth factors. In each of the colonies growing on the minimal agar plates, histidine

added by biological samples to the test system may extend the autxotrophic growth

phase of plated bacteria thereby increase the probability of spontaneous reversion to

prototrophy. Histidine- related growth factors added in the plate incorporation test may

give a false positive due to the consequence of extended autotrophic growth (Lars

Nylund and Pirkko Einisto, 1993). In this study, revertant colonies were found to


Discussion


Page 150

increase in the presence of metabolic activation factor at the concentration of 50 µg/ml.

In the absence of S9 factor, the revertant colonies were found to be 14±2 and in the

presence of S9 factor the number of revertant colonies were 18±3 for Curcuma

aromatica, for Curcuma zedoaria in the absence of S9 factor 10±1 and in the presence

of S9 factor the number of revertant colonies were 16±1. The number of revertant

colonies produced by Curcumin in TA 98 was found to be 13±1 and 16±1 in the

absence and presence of metabolic activation factor (S9 mix).

In TA 100 strains, in the absence of S9 factor, the number of revertant colonies was

found to be 16±3 for Curcuma aromatica, 18±1 for Curcuma zedoaria and 15±2 for

Curcumin. In the presence of S9 factor the revertant colonies were 64±10 for Curcuma

aromatica, 80±8 for Curcuma zedoaria and 76±7 for Curcumin. When compared to

positive control the number of revertant colonies was found to be more or less

equivalent to negative control which confirms the absence of genotoxic effect of test

compounds.

6.2.3.2. Chromosomal aberration test

Structural chromosome aberrations may be induced via DNA breaks by various types

of mutagens. Such DNA breaks may rejoin such that the chromosome is restored to its

original state, rejoin incorrectly or not rejoin at all. These last two cases may be

observable on microscopic preparations of metaphase cells. However, many of these

gross changes probably will not allow cell survival after division, but they serve as

indicators for the induction of smaller, not readily observable changes, which do allow


Discussion


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cell survival but may have deleterious consequences for the organism (Anderson et al.,

2001).

The chromosome aberration test is most often performed on human peripheral blood

lymphocytes. As peripheral lymphocytes are in the resting G0 stage of the cell cycle,

they have to be stimulated to divide by an aspecific antigen like phytohaemagglutinin.

After 46.5 hours just before fixation (at 48 hours) a spindle inhibitor like colchicine is

added to block the cells in the (pro) metaphase of the first mitosis (Anderson et al.,

2000). The results of this study, showed that hydro alcoholic extract of Curcuma

aromatica do not have chromosome aberration induction potential up to 312.5 µg/ml

both in the absence and presence of metabolic activation system, Curcumin have

chromosome aberration induction potential up to 40 µg/ml both in the absence and

presence of metabolic activation system, hydro alcoholic extract of Curcuma zedoaria

have chromosome aberration induction potential at the highest concentration tested

156.25 µg/ml in the absence of metabolic activation system under short-term treatment

duration, where no chromosome aberration induction potential up to 156.25 µg/ml in

the presence of metabolic activation system (1% v/v S9) in cultured human

lymphocytes.

6.2.3.3. SOS Chromotest

In the SOS Chromotest, it was ascertained that different concentrations of selected

compounds for this study such as hydro alcoholic extract of Curcuma aromatica,

Curcuma zedoaria and Curcumin added to the indicator bacteria were not genotoxic as

the induction factor induced by the tested doses was below 1.2. The compounds are


Discussion


Page 152

classified as non-genotoxic, if the induction factor (IF) remains <1.2 and genotoxic if

IF exceeds 1.2 (Manon Bombardier et al., 2001; Skandrani et al., 2010). For Curcuma

aromatica the corrected induction factor produced at the concentration of 250 and 500

µg/ml was found to be more or less equal such as 0.9711 and 0.9403 where as the

correction induction factor at the concentration of 1000 µg/ml was found to be 1.0309.

For Curcuma zedoaria the corrected induction factor was found to be 0.4299, 0.8943

and 1.1975 for 1000, 500 and 250 µg/ml concentration respectively. For Curcumin the

corrected induction factor was found to be 1.3308, 1.3725 and 1.1975 for 1000, 500

and 250 µg/ml. Curcumin alone was found to be genotoxic even at the concentration of

250 µg/ml where as the hydro alcoholic extract of Curcuma aromatica and Curcuma

zedoaria was found to be non- genotoxic even at the highest concentration i.e 1000

µg/ml.

6.2.3.4. DNA sugar damage

By normal cellular metabolism and by exogenous source such as genotoxic

compounds and ionizing radiations, reactive oxygen derived species including free

radical are formed in living cells which causes oxidative damage to DNA, resulting in

the formation of modified bases and sugars, DNA protein cross links, strand break,

base free site and tandem lesions such as 8,5’ cyclopurine- 2’ deoxyribonucleosides and

clustered damage sites. Among the free radical, the hydroxyl radical is highly reactive

and reacts with DNA by addition to double bond of heterocyclic DNA bases and by

abstraction of an H atom from the methyl group of thymine and from each of the C-H

bonds of 2’ deoxyribose (Winofred et al., 2006)


Discussion


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Concomitant damage to the sugar and base moieties of the same nucleotides is shown

by the formation of 8,5’ cyclopurine- 2’ deoxyribonucleoside that has been identified in

mammalian cells that are exposed to free radical- generating system

In base- excision repair (BER) of oxidative DNA sugar damage, numerous DNA

glycosylases which are substrate specificities are involved. Major products of oxidative

damage to DNA bases are substrates of known glycosylases. Some enzyme exhibit

cross reactivity specific for both pyrimidine- purine derived lesions and some enzymes

have more substrates than others. Eukaryotic counterparts possess narrow specificity

than prokaryotic enzyme (Miral Dizdaroglu, 2003) Malondialdehyde which is the


et al., 2006). This study showed that Curcuma aromatica, Curcumin which gave the

protection for the DNA sugar bone where as Curcuma zedoaria damaged the sugar

backbone at the concentration of 250, 500 µg/ml.

6.2.3.5. Potato disk assay

Potato disc tissue is an assay based on antimiotic activity which can detect a broad

range of known and novel antitumor effect by inhibition of Agarobacterium

tumifaciens induced tumors. Because of the tumerogenic mechanisms that are similar in

plants and animals, the validity of this assay is predicted. Initiation of crown gall tumor

inhibition on potato disc and subsequent growth showed good correlation with

compound and extracts active in 3PS leukemic mouse assay.

Agarobacterium tumifaciens, a gram negative bacterium, is the causative agent of

crown gall disease in which a mass of tissue bulging from stems and roots of woody


Discussion


Page 154

and herbaceous plant. On plant these tumors may be spongy or hard, and may be or

may not have a deleterious effect. A.tumifaciens produce tumors produced are

histologically similar to those found in humans and animals. A.tumifaciens contain a

tumor producing plasmid (Ti-Plasmid) during the infection of plant material, the

plasmid gets incorporated into plant’s chromosomal DNA. Phenols will be released

when the plant tissue is wounded, which activate the Ti- Plasmid causes the plant’s

cells to multiply rapidly without going through apoptosis resulting in tumor formation

similar in nucleic acid content similar to humans and animal cancer. Potato disc assay

is a fairly rapid, inexpensive and reliable method for screening antitumor activity. Plant

derived chemotherapeutic agent can be assessed with different modes of action in

potato disc tumor assay. FDA approved chemotherapeutic agent like Campothecin,

Paclitaxel, Podophyllin, vinblastin, vincristine originating from plants exhibit diverse

modes of action on the cell cycle (Coke et al., 2003; Carloalberto petti et al., 2009).

This study showed that hydro alcoholic extract of Curcuma aromatica, Curcuma

zedoaria and Curcumin found to possess antimutagenic activity which was found to

increase with the concentration of the drug. Among the three tested compounds

Curcumin showed good antimutagenic activity from the concentration 500 µg/ml to

1000 µg/ml.

6.2.3.6. Comet assay

Some 20 years ago, Ostling and Johnson developed the comet assay, which has its

origin in the micro gel electrophoresis technique which can be performed in different

ways and has been used for many different purposes during the last few years. By using


Discussion


Page 155

the alkaline version of the assay it is possible to detect not only DNA single strand

breaks and alkali labile sites but also DNA/DNA and DNA/Protein cross-links (Maria

Anderson et al., 2003). The assay which is capable of detecting the DNA damage in

individual cells is the comet assay. At the time of lysis, increased DNA migration

results from the induction of DNA- single strand breaks, alkali labile sites and

incomplete excision repair sites cell death arising from a non- DNA mediated process

or apoptosis is associated with increased DNA migration accompanied with DNA

fragmentation. In extreme cases like the apoptotic cells, the head and tail are well

separated and with an increasing number of breaks, DNA pieces migrate freely into the

tail of the comet. Information about the number of strand breaks is provided by the

intensity of fluorescence in the tail relative to the head. Over the wide range of

damage, tail length, percentage of total DNA in the tail and tail moment all reflect

DNA damage, though the percentage tail DNA generally seems to be most useful. A

measure of both the smallest detectable size of migrating DNA which is reflected in the

comet tail length and the number of relaxed/ broken pieces which is represented by the

intensity of DNA in the tail is incorporated in the tail moment. Inter individual

differences such as the age of blood donors and their physical activities, smoking habit

and cell cycle status are important and may reflect differences in the repair of an

induced DNA damage likely to add complexicity to the problem. During comet

formation in both alkaline and neutral assay system, the chromatin structure which is

fundamental to the replication and transcription activity affects the role of DNA

(Ruzica Rozgaj et al., 2002). Among the three tested compounds Curcumin was found


Discussion


Page 156

to be more genotoxic in nature when compared by Curcuma aromatica, Curcuma

zedoaria. These results might either due to the presence of low concentration of

Curcumin in the extracts or due to the presence of other phytoconsituents that are

present in the extract. The damage of the cells was found to be less in the presence of

metabolic activation factor than the absence of metabolic activation factor. This

confirms the presence of metabolic activation factor has an important role in preserving

the cells against the genotoxicant.

6.2.3.7. Micronucleus test

A micronucleus test is a test used in toxicological screening for potential genotoxic

compounds. The assay is now recognized as one of the most successful and reliable

assay for genotoxic carcinogens, i.e carcinogens that act by causing genetic damage and

is the OECD guidelines for the testing of chemicals. There are two versions of this test,

one is in vivo and other in vitro. A micronucleus is the erratic (third) nucleus that is

formed during the anaphase of mitosis or meiosis. Micronuclei are cytoplasmic bodies

having a portion of acentric chromosome or whole chromosome which was not carried

to the opposite poles during the anaphase. Their formation result in the daughter cell

lacking a part or all of a chromosome. These chromosome fragments or whole

chromosomes normally develop nuclear membrane and forms as micronuclei as a third

nucleus. After cytokinesis, one daughter cell ends up with one nucleus and the other

ends up with one large and one small nucleus, i.e., micronuclei. There is a chance of

more than one micronucleus forming when more genetic damage has happened. The

micronucleus test is used as a tool for genotoxicity assessment of various chemicals.


Discussion


Page 157

For the safety evaluation of new drugs or industrial chemicals in Japan as well as in

other countries, the micronucleus test has been widely used as a sensitive in vivo

genotoxicity test and has been recommended for use in regulatory guidelines. From

guidelines to guidelines, the experimental protocol of the test varies slightly in details

such as number of animals, sex, dosing and sampling time. Before the start of full scale

experimentation, a pilot preliminary experiment was to be carried out for each test

substances because the optimum response may vary according to the chemical

concerned (Hiroyasu Shimada et al., 1990). In this study, Curcuma aromatica at the

concentration of 50 µg/ml, produced the micronuclei at the rate of 12.333±2.223,

15.666±1.778, in the absence and presence of metabolic activation factor respectively

Curcuma zedoaria at the concentration of 50 µg/ml produced the micronuclei at the

rate of 9.666±1.999, 14.666±2.666 in the absence and presence of metabolic activation

factor respectively. Curcumin at the concentration of 50 µg/ml, produced the

micronuclei at the rate of 14.666±1.555, 19.333±1.111 in the absence and presence of

metabolic activation factor respectively

When compared to the positive control (Methyl methane sulfonate at the concentration

of 50 µg/ml), all the test compounds such as hydro alcoholic extract of Curcuma

aromatica, Curcuma zedoaria, and Curcumin at the concentration of 50μg/ml showed

less toxicity to HEp-2 cells. In the presence of S9 factor, micronucleus formation

found to increase when compared to its absence.


Discussion


Page 158

6.2.3.8. Plasmid nicking assay or DNA-damage protective activity

By damaging the cellular antioxidant defense mechanism, can induce oxidative damage

to vital cellular molecules including DNA, proteins and lipids. Genomic DNA is the

most important target in the living cells. Damage suffered by DNA includes strand

break and cross link of the intra and inter strand type. Malondialdehyde which is the


et al., 2006). Drugs can cause cellular damage through metabolic activation of these

compounds to highly reactive substances such as oxygen species which are derived

from the metabolism of oxygen that includes superoxide radicals, hydroxyl radicals,

hydrogen peroxide radicals which are often generated or obtained as byproducts of

biological reactions or from exogenous factors. Some of these reactive oxygen species

plays a positive role in cell physiology as well as cause great damage to the cell

membrane and DNA including oxidation that cause membrane lipid per oxidation,

decreased membrane fluidity and DNA mutation which leads to cancer and other

degenerative disease (Wan-Ibrahim et al., 2010). When singlet oxygen together with

small contribution of hydroxyl radical- mediated reactions through initially generated

superoxide radical produced/ induced the oxidative damage to DNA (Betina Kappal

Pereira et al., 2009). In this study Quercetin was used as a positive control. Quercetin at

all the tested concentration such as 250, 500 and 1000 µg/ml showed the damage to the

plasmid and it might have occurred due to the incision in the DNA. Curcuma

aromatica and Curcuma zedoaria protected the DNA from the concentration of 250

µg/ml to 1000 µg/ml. Curcumin caused slight damage when compared to quercetin as


Discussion


Page 159

well as negative control in all the tested concentration such as 250, 500 and 1000

µg/ml. The damage which is produced by the curcumin might be due to its biphasic

effect because of its pro- oxidant activity.

6.2.3.9. Sequential analysis

When the plasmid DNA treated with the Fenton’s reagent alone, we could not able to

obtain any information about the arrangement of nitrogenous base pairs both in case of

forward and reverse primers. This shows that the hydroxyl radical that has produced by

the Fenton’s reaction might have completely destroyed the plasmid. But at the same

time, in the presence of the test compounds such as hydro alcoholic extract of Curcuma

aromatica, Curcuma zedoaria and curcumin, even though we obtained differences in

the peaks height ( Some variations in the peak), we could able to get the information

about the arrangement of nitrogenous bases in the plasmid DNA. No mutation has

taken place based up the conditions under which we conducted this study. From these

studies we came to understood that all the tested compounds selected for this study

(Hydro alcoholic extract of Curcuma aromatica, Curcuma zedoaria and Curcumin)

was found to be non-genotoxic.

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