4. Results and Discussionshodhganga.inflibnet.ac.in › bitstream › 10603 › 2681 › 12 ›...

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4. Results and Discussion

Epidemiological evidences substantiate the health benefits of fruit and vegetable

consumption, which could be accounted for by nutrient content such as dietary fibers,

vitamins, minerals and phytochemicals. Medicinal and therapeutic properties of the

vegetables belonging to cruciferae family are well acknowledged through recorded

history. R. sativus is unique in its composition and a rich source of nutritive and non-

nutritive compounds including ascorbic acid, carotenoids, polyphenolics and GLs and

their hydrolysis products such as ITCs. Several studies established health and disease

fighting potential of these phytochemicals. However, a single phytochemical does not

offer the same benefit as those existing within chemically-related groups in whole food

sources possibly due to lack of synergistic or additive effects. This study was aimed at

understanding synergistic and additive effects of phytochemicals present in R. sativus

and to characterize components responsible for its biological activity.

4.1. Phytochemical analysis of R. sativus

4.1.1. Effect of solvents on yield of soluble substances

The yield of soluble substances, expressed as mg/g dry weight of root, stem and

leaves of R. sativus are closely dependent on the solvents, as shown in Table 4.1. The

highest yield was obtained, when extraction of root was performed with water, followed

by that obtained with methanol, acetone, ethyl acetate, chloroform and hexane. The

results were the same when stem and leaves were used, except that ethyl acetate gave a

higher yield than acetone.

4.1.2. Total isothiocyanate (ITC) content of R. sativus

Organic isothiocyanates are widely distributed especially in cruciferous plants

and responsible for a variety of beneficial effects. ITCs are quantified based on their

reaction with 1, 2-benzenedithiol to form a condensation product, 1, 3-benzodithiole-2-

thione, with absorption maxima at 365 nm. ITCs were detected in considerable amount

in root, stem and leaves of R. sativus (Table 4.2). However, root extracts contained the

highest level of ITCs, as compared to leaves and stem extracts. The amount of total ITCs

extracted with different solvents ranged from 0.42 – 3.81 mg/g for root, 0.08 – 0.16 mg/g

62

Table 4.1

Extraction yield (expressed as mg/g dry weight) of root, stem and leaves of R. sativus.

Parts Water Methanol Acetone Ethyl acetate Chloroform Hexane

Root 180.50 ± 12.95a 150.30 ± 19.60 96.70 ± 7.50 22.20 ± 2.87 27.30 ± 3.95 16.80 ± 2.14

Stem 106.10 ± 14.58 61.20 ± 5.26 20.80 ± 3.69 21.80 ± 1.84 14.40 ± 1.50 15.20 ± 1.26

Leaves 94.60 ± 8.24 61.20 ± 8.44 21.60 ± 4.06 18.50 ± 1.63 14.40 ± 0.98 21.60 ± 3.47

a Each value represents mean value ± standard deviation of three replicates.

Table 4.2

Total isothiocyanate (ITC) content (expressed as mg benzyl ITC/g dry weight) of root, stem and leaves of R. sativus.


Root 0.42 ± 0.04a 0.49 ± 0.01 1.82 ± 0.094 0.76 ± 0.02 1.18 ± 0.07 3.81 ± 0.182

Stem 0.08 ± 0.002 0.09 ± 0.00 0.16 ± 0.002 0.12 ± 0.007 0.14 ± 0.006 0.12 ± 0.004

Leaves 0.12 ± 0.008 0.13 ± 0.006 0.87 ± 0.024 0.16 ± 0.009 0.21 ± 0.004 0.24 ± 0.016


63

for stem and 0.12 – 0.87 mg/g for leaves of R. sativus. Root extract contained the highest

amount of ITCs, when hexane was used as an extraction solvent. However, higher

amounts of ITCs were recovered from stem and leaves, when acetone was used as an

extraction medium. Water and methanol seemed to extract similar amount of ITC from

R. sativus. While the amount of ITCs extracted in chloroform extracts was more than that

obtained with the ethyl acetate extracts.

Glucosinolates, precursors of ITCs are found in different proportions in different

parts of plants in response to different forms of synthesis pattern and environment stress

(Ciska et al, 2000). From this result, it can be deduced that ITCs were more concentrated

in root of thisvegetable and had approximately more than two-fold higher total ITC

content than Japanese species (Nakamura et al, 2001) and ten-fold higher amount than

Korean species (Suh et al, 2006), thus signifying its potential as a significant source of

dietary ITCs. These differences in ITC content could be attributed to genetic variability

that occurs among different varieties of R. sativus. Our findings are in line with Blazevic

and Mastelic (2009), who had reported significant amount of ITCs in roots of R. sativus,

as compared to its leaves, which possessed more of O-glycosidically-bound volatile

compounds (80.5 – 84.5%) and less of ITCs (0.3 – 5.7%).

4.1.3. Total polyphenolic content of R. sativus

Polyphenolics, on reaction with Folin–Ciocalteu reagent under basic conditions

dissociate to form a phenolate anion, which reduce molybdate in Folin–Ciocalteu

reagent forming a blue colored molybdenum oxide with maximum absorption near 700

nm. The intensity of blue colored complex is proportional to amount of polyphenolic

compounds present in the sample (Huang et al, 2005). However, this method is not

specific for phenolic compounds as other reducing compounds can interfere (Makkar,

1989) and its reactivity is different for different polyphenolics (Julkunen-Tiito, 1985).

Despite that, this method is generally preferred, since it is straightforward to obtain

comparative results with other plant materials accounted in the literature.

The total polyphenolics, expressed as catechin equivalents/g of dry weight is

shown in Table 4.3. The content of polyphenolics varied among different extracting

solvents and parts of plant used. Leaves were found to contain high amount of

64

polyphenolics, as compared to root and stem. Methanol was the solvent that could

extract most of the polyphenolics (86.16 mg/g) followed by acetone (78.77 mg/g). The

amount of total polyphenolics in water and ethyl acetate extract was in the range 34 – 37

mg/g, which was markedly less as compared to methanol and acetone extracts.

However, chloroform and hexane yielded least amount of polyphenolics which were

22.37 mg and 4.97 mg/g respectively. Stem contained lower amount of polyphenolics as

compared to leaves. It was found in the following order; methanol extract (56.69 mg/g),

water extract (23.55 mg/g), acetone extract (32.77 mg/g), ethyl acetate extract (30.43

mg/g), chloroform extract (16.78 mg/g) and hexane extract (1.92mg/g). In case of root

extracts, the highest amount was found in extract obtained with water (63.54 mg/g)

followed by that obtained with methanol (45.32 mg/g), acetone (32.77 mg/g), ethyl

acetate (30.02 mg/g), chloroform (20.73 mg/g) and hexane (13.18mg/g).

From these results, it becomes apparent that the recovery of polyphenolics was

dependent on extraction solvents and their polarity. The amount of polyphenolics

extracted into a solvent reduced as its polarity decreased. Several factors such as

extraction temperature, solvent type and solvent concentration can influence extraction

of polyphenolics (Li et al, 2006). Earlier study reported a similar trend, whereby most

typical polyphenolics were significantly extracted into polar solvents (Razali et al, 2008).

The polyphenolics extracted from R. sativus extracts were comparable to or higher than

that obtained for other cruciferous vegetables (Ahmed and Beigh, 2009; Ciska et al, 2005;

Koksal and Gulcin, 2008). The remarkable findings of this study are that aerial part (stem

and leaves) of this vegetable, usually discarded as waste, was found to contain higher

amount of polyphenolics than those reported for black kale leaves (Ayaz et al, 2008) and

ginger rhizomes (Bozin et al, 2008). In addition, polyphenolic content of leaves was

almost comparable to phenolic content of traditionally-rich sources such as black tea and

green tea respectively (Khokhar and Magnusdottir, 2002). Present findings suggest the

potential of leaves and stem to be exploited as novel source of nutritional polyphenolics

along with root of R. sativus.

65

Table 4.3

Total polyphenolic content (expressed as mg catechin/g dry weight) of root, stem and leaves of R. sativus.


Root 63.54 ± 3.62a 45.32 ± 1.63 32.77 ± 2.87 30.02 ± 0.85 20.73 ± 0.92 13.18 ± 2.66

Stem 23.55 ± 2.20 56.09 ± 0.00 56.84 ± 1.59 30.43 ± 2.31 16.78 ± 1.20 1.92 ± 0.08

Leaves 34.16 ± 3.44 86.16 ± 4.51 78.77 ± 5.32 36.81 ± 1.70 22.37 ± 1.51 4.97 ± 0.19


66

4.1.4. HPLC – DAD analysis of polyphenolics in R. sativus

Several polyphenolics such as catechin, protocatechuic acid, vanillic acid, syringic

acid, ferulic acid, sinapic acid, o-coumaric acid, myricetin and quercetin were identified in

root (Table 4.4.1), stem (Table 4.4.2) and leaves (Table 4.4.3) of R. sativus. Chromatographic

profile recorded at 280 nm for R. sativus extracts is presented as Figure 4.1 (standard),

Figure 4.2 (root), Figure 4.3 (stem) and Figure 4.4 (leaves) respectively. Significant amount

of catechin (10.54 mg/g) and sinapic acid (4.83 mg/g) were present in water extract of root.

The other phenolic acids were also present, but to different extent. Sinapic acid (5.29 mg/g)

and ferulic acid (2.65 mg/g) were identified as major phenolics in the methanolic extract of

root, while catechin (0.19 mg/g), protocatechuic acid (0.38 mg/g) and syringic acid (0.64

mg/g) were present in lesser amounts. Acetone extract did not yield considerable amount

of polyphenolics except ferulic acid (1.22 mg/g) from root. Sinapic acid (7.35 mg/g) was

the most abundant phenolics in ethyl acetate extract; syringic acid, vanillic acid and ferulic

acid were also present in variable amounts. The non-polar solvents such as chloroform and

hexane were poor in extracting polyphenolics from root. None of the polyphenolics

(standard phenolics used for analysis) were detected in chloroform extract. However, a

considerable amount of sinapic acid (2.66 mg/g) and small amount of protocatechuic acid

(0.27 mg/g), ferulic acid (0.14 mg/g) and o-coumaric acid (0.48 mg/g) were present in

hexane extract. Flavonols such as myricetin and quercetin were not detected in any of root

extracts.

HPLC profile of leaves and stem was almost similar, except that leaves contained

higher amounts of polyphenolics as compared to stem extract. Catechin (4.88 mg/g and

1.13 mg/g) was the most abundant phenolics in water extract of leaves and stem. Vanillic

acid, ferulic acid, sinapic acid and o-coumaric acid were predominant phenolic acids in

methanolic extract of leaves and stem. Catechin and vanillic acid were detected as major

phenolics in acetone extract. But, flavonols such as myricetin and quercetin were not

detected in any of these extracts. While myricetin (6.41 mg/g) was the main flavonol

identified in ethyl acetate extract of leaves. Chloroform extract contained moderate amount

of protocatechuic acid and quercetin. None of the polyphenolics (standards used for

analysis) were detected in hexane extracts of leaves and stem.

67

Figure 4.1

HPLC – DAD chromatogram of a mixture of standard polyphenolics. Detection at 280 nm.

Peaks: (1) catechin; (2) protocatechuic acid; (3) syringic acid; (4) vanillic acid; (5) ferulic acid;

(6) sinapic acid; (7) o-coumaric acid; (8) myricetin; (9) quercetin

0 10 20 30 40 50 60 min

0

100

200

300

400

500

600

mAU

6

2

3 1

4

5

9 8

7

68

Figure 4.2

HPLC-DAD polyphenolics profile of root of R. sativus. Detection at 280 nm.

(a) Water; (b) Methanol.

10 20 30 40 50 60 min

0

50

100

150

200

250

300

2 3

HBA

HCA

6

FLA HBA

1

HBA

HCA

b)

10 20 30 40 50 60 min

0

20

40

60

80

100

1

HBA

3

HCA

4

5 HCA

2

HCA

6

FL 7

HBA

a)

mAU

69

Figure 4.2

HPLC-DAD polyphenolics profile of root of R. sativus. Detection at 280 nm.

(c) Acetone; (d) Ethyl acetate.

10

20 30 40 50 60 min

0

50

100

150

200

250

300

HBA

HBA

FL

HCA

HCA 3

4 5

HBA

HCA

FL

6

FL

7

d)

10 20 30 40 50 60 min

0

50

100

150

200

mAU

HCA HBA

HBA

HBA

HBA

HCA

5

c)

70

Figure 4.2

HPLC-PDA polyphenolics profile of root of R. sativus. Detection at 280 nm.

(e) Chloroform; (f) Hexane extract.

10 20 30 40 50 60 min

0

20

40

60

80

100

120

HBA HBA

6

HBA

2 HBA

HBA HBA

5

f)

10 20 30 40 50 60 min

0

20

40

60

80

mAU

HBA

HBA HCA

HBA

HCA

FL

HBA

HBA

e)

71

Table 4.4.1

Polyphenolic content of root of R. sativus (mg/g dry weight)a

Extraction

solvent

Catechin

Protocate

chuic acid

Syringic acid

Vanillic acid

Ferulic acid

Sinapic acid

o-Coumaric

acid

Water 10.54 ± 1.20 0.30 ± 0.006 1.49 ± 0.040 1.44 ± 0.031 1.76 ± 0.025 4.83 ± 0.092 1.25 ± 0.037

Methanol 0.19 ± 0.004 0.38 ± 0.001 0.64 ± 0.002 NDb 2.65 ± 0.13 5.29 ± 0.24 ND

Acetone ND ND ND ND 1.22 ± 0.073 ND ND

Ethyl acetate ND ND 0.47 ± 0.001 0.64 ± 0.005 0.99 ± 0.013 7.35 ± 0.69 ND

Chloroform ND ND ND ND ND ND ND

Hexane ND 0.27 ± 0.001 ND ND 0.14 ± 0.002 2.66 ± 0.31 0.48 ± 0.006

a Values are means ± SD (n = 3).

b Not detected.

72

Figure 4.3

HPLC-DAD polyphenolics profile of stem of R. sativus. Detection at 280 nm.


10 20 30

40 50 60 min

0

20

40

60

80

100

120

140

mAU

HBA

1

4

5

HCA

HCA

HCA

6

6

HCA

FL

HCA

HCA

7

b)

10 20 30 40 50 60

min

0

20

40

60

80

100

120

4 HBA

1

3

HBA

7

a)

73

Figure 4.3


(c) Acetone; (d) Ethyl acetate.

10 20 30 40 50 60 min

0

20

40

60

80

)

FL

8

7

FL

3

HCA

2 HCA

HCA

6

HCA

HCA

HBA

d)

10 20 30 40 50 60 min

0

40

80

120

160

200

4 HCA

HCA

HCA

1

HBA

HCA

HCA 7

c)

74

Figure 4.3


(e) Chloroform; (f) Hexane.

10 20 30 40 50 60 min

0

10

20

30

40

mAU

f)

10 20 30 40 50 60 min

0

20

40

60

80

mAU

HBA

HCA

HCA

HCA

HBA

2

HCA

FL

e)

75

Table 4.4.2

Polyphenolic content of stem of R. sativus (mg/g dry weight)a

Extraction

solvent

Catechin

Protocate

chuicacid

Syringic

acid

Vanillic

acid

Ferulic

acid

Sinapic

acid

o-Couma

ric acid

Myricetin

Quercetin

Water 1.13 ± 0.034 NDb 0.70 ± 0.006 0.69 ± 0.004 ND ND 0.57 ± 0.006 ND ND

Methanol 0.22 ± 0.002 ND ND 1.76 ± 0.25 0.32 ± 0.003 1.32 ± 0.092 2.13 ± 0.049 ND ND

Acetone 1.03 ± 0.083 ND ND 1.64 ± 0.083 ND ND 0.86 ± 0.007 ND ND

Ethyl acetate ND 0.08 ± 0.000 0.19 ± 0.000 ND ND 0.75 ± 0.005 0.36 ± 0.003 0.41 ± 0.002 ND

Chloroform ND 0.33 ± 0.002 ND ND ND ND ND ND ND

Hexane ND ND ND ND ND ND ND ND ND


b Not detected.

76

Figure 4.4

HPLC-DAD polyphenolics profile of leaves of R. sativus. Detection at 280 nm.


10 20 30 40 50 60 min

0

50

100

150

200

HCA

FL

4

5

HCA FL

HCA

1

HBA

HCA

FL

6

HCA HCA

7

b)

mAU

10 \ 20 30 40 50 60 min

0

20

40

60

80

100

6 HBA HCA

1

HCA

HCA

7

a)

77

Figure 4.4


(a) Acetone; (b) Ethyl acetate.

10 20 30 40 50 60 min

0

40

80

120

160

HCA

3

HCA

HBA

HCA

HCA

4

6 FL

HBA

8

FL

9

d)

10 20 30 40 50 60 min

0

100

200

300

400

mAU

1

4 HBA

HCA

HCA

FL

HCA

FL

HCA

FL

7

c)

78

Figure 4.4


(a) Chloroform; (b) Hexane.

10 20 30 40 50 60 min

0

10

20

30

40

HCA HCA

HBA

f)

10 20 30 40 50 60 min

0

40

80

120

160

HBA

HBA HBA

HCA

HCA

HCA

FL

HCA

9

e)

mAU

79

Table 4.4.3

Polyphenolic content of leaves of R. sativus (mg/g dry weight)a

Extraction

solvent

Catechin

Protocatech

uic acid

Syringic

acid

Vanillic

acid

Ferulic

acid

Sinapic

acid

o-coumaric

acid

Myricetin

Quercetin

Water 4.88 ± 0.13 NDb ND ND ND 0.62 ±

0.008

1.05 ± 0.005 ND

ND

Methanol 0.36 ± 0.005 ND ND 4.13 ± 0.34 1.29 ±

0.046

3.21 ± 0.19 1.64 ± 0.061 ND

ND

Acetone 2.11 ± 0.097 ND ND 1.96 ±

0.096

ND ND 0.19 ± 0.001 ND

ND

Ethyl

acetate

ND ND

0.53 ±

0.003

1.39 ±

0.072

ND 1.09 ±

0.057

ND 6.41 ± 0.23 0.49 ± 0.000

Chloroform ND

ND

ND

ND

ND

ND

ND

ND

0.79 ± 0.004

Hexane ND ND ND ND ND ND

ND

ND

ND


b Not detected

80

Previous study reported the presence of sinapic acid esters and kaempferol as

major phenolics in Japanese R. sativus sprouts (Takaya et al, 2003). In the present study,

sinapic acid and catechin were found to be the most abundant phenolics in R. sativus.

Furthermore, assortment of phenolics was detected both in root and aerial part (leaves

and stem) of R. sativus. The significant findings of this study are that catechin content of

water extract of root was much higher than that reported for Lepidium meyenii (maca), a

plant belongs to crucifer family (Sandovala et al, 2002). Besides, catechin content of R.

sativus root was comparable to traditional sources of phenolics such as green tea and

black tea (Khokhar and Magnusdottir, 2002). Sinapic acid, abundant phenolic acid in R.

sativus, was found to be higher than those reported for cauliflower (Llorach et al, 2003)

and black cabbage (Ayaz et al, 2008). Similarly, ferulic acid content was found to be

higher than those reported for alfalfa, spinach, cabbage and bitter cumin (Ani et al, 2006;

Huang et al, 1986), but lesser than that reported for wheat bran (Huang et al, 1986).

Likewise, catechin content of water and acetone extract of leaves and stem were

comparable to traditional sources of phenolics such as green and black tea (Khokhar and

Magnusdottir, 2002). Sinapic acid was also found to be higher than those reported for

cauliflower (Llorach et al, 2003) and black cabbage (Ayaz et al, 2008). Similarly, ferulic

acid content of methanolic extract of leaves and stem were significantly higher than that

present in cabbage and bitter cumin (Ani et al, 2006; Ayaz et al, 2008; Huang et al, 1986).

Myricetin content of ethyl acetate extract of leaves was also considerably higher than

that present in red grapes' skin (Novek et al, 2008).

Apart from polyphenolics identified using standards, other peaks of HPLC

chromatogram could only be tentatively identified and quantified as derivatives of

hydroxybenzoic acid (HBA), hydroxy cinnamic acid (HCA), flavanol (FLA) and flavonol

(FL) based on their UV spectra. For this purpose, HBA, HCA, FLA and FL derivatives

were quantified as protocatechuic acid, ferulic acid, catechin and quercetin equivalents

and results are shown as Tables 4.5.1 – 4.5.3 for root, stem and leaves extract

respectively. The leaves had more polyphenolic content than root and stem. HBAs and

HCAs were detected in most of R. sativus extracts and HCAs seemed to be most

predominant polyphenolics. FLAs were detected only in water, methanol and acetone

extracts and found to be most significant polyphenolics in methanolic extract of root.

81

Table 4.5.1

Total polyphenolic index of root of R. sativus (mg/g dry weight) as determined by HPLC methods a.

Extraction

solvent

Total hydroxy

benzoic acids

Total hydroxy-

cinnamic acid

Total flavanols

Total flavonols

Total polyphenolic

index

Water 8.88 ± 1.36 14.79 ± 2.73 10.54 ± 1.40 NDb 34.21 ± 2.88

Methanol 8.08 ± 0.93 9.35 ± 1.45 12.44 ± 0.52 ND 29.86 ± 2.92

Acetone 3.25 ± 0.064 6.70 ± 0.94 ND ND 9.95 ± 0.97

Ethyl acetate 3.98 ± 0.12 10.36 ± 0.57 ND 2.45 ± 0.063 16.80 ± 0.83

Chloroform 1.22 ± 0.027 4.88 ± 0.36 ND 1.02 ± 0.009 7.11 ± 0.62

Hexane 2.33 ± 0.035 3.63 ± 0.28 ND ND 5.95 ± 0.26

a The concentrations of polyphenolic compounds were cumulative of individual compounds of same group quantified as equivalents

of representative standards. Values are means ± SD (n = 3). Total hydroxy benzoic acids include protocatechuic acid, syringic acid,

vanillic acid and their derivatives; total hydroxy cinnamic acids include ferulic acid, sinapic acid, o-coumaric acid and their

derivatives; total flavanols include catechin and their derivatives; total flavonols include myricetin, quercetin and their derivatives.

bND – not detected

82

Table 4.5.2

Total polyphenolic index of stem of R. sativus (mg/g dry weight) as determined by HPLC methods a.

Extraction

solvent

Total hydroxy

benzoic acids

Total hydroxy

cinnamic acid

Total flavanols

Total flavonols

Total polyphenolic

index

Water 4.98 ± 0.39 0.94 ± 0.018 1.13 ±0.006 NDb 7.04 ±0.73

Methanol 5.69 ± 0.62 27.42 ±3.66 0.22 ±0.007 1.60 ±0.022 34.93 ±3.15

Acetone 2.83 ± 0.037 19.08 ±1.08 1.03 ±0.012 ND 22.88 ±1.52

Ethyl acetate 2.31 ±0.075 12.11 ±0.69 ND 2.12 ± 0.038 16.55 ±0.72

Chloroform 1.99 ± 0.033 4.32 ±0.29 ND 0.72 ±0.010 7.03 ±0.39

Hexane 0.01 ± 0.000 0.06 ± 0.001 ND ND 0.07 ± 0.001






83

Table 4.5.3

Total polyphenolic index of leaves of R. sativus (mg/g dry weight) as determined by HPLC methods a.

Extraction

solvent

Total hydroxy

benzoic acids

Total hydroxy

cinnamic acid

Total flavanols

Total flavonols

Total polyphenolic

index

Water 1.02 ± 0.074 9.79 ±0.81 4.88 ±0.069 NDb 15.66 ±3.44

Methanol 9.31 ± 0.15 36.36 ±2.79 0.36 ±0.000 14.62 ±0.31 60.64 ±1.45

Acetone 3.06 ± 0.083 17.03 ±1.31 10.02 ±0.24 7.92 ±0.83 38.03 ±2.06

Ethyl acetate 3.18 ± 0.049 5.91 ±0.88 ND 7.85 ±0.31 16.93 ±0.97

Chloroform 2.56 ± 0.050 4.55 ±0.71 ND 1.85 ±0.014 8.97 ±0.68

Hexane 0.01 ± 0.000 0.09 ± 0.000 ND ND 0.10 ± 0.004






84

FLs were particularly detected in ethyl acetate and chloroform extracts of root, stem and

leaves of R. sativus. However, methanol and acetone extracts of stem and leaves

contained a considerable amount of FLs. Tentative identification of polyphenolics based

on comparison of their UV spectrum with that of standards enables to quantify related

compounds as derivatives of a particular group without actual identification of

individual compounds. This provides a quick overview of polyphenolics profile of

different parts of R. sativus. When chromatographic profiles obtained with root and

aerial part (stem and leaves) of this vegetable were compared, interestingly, profile of

polyphenolics was distinctly different; flavonols seemed to be concentrated in leaves

and stem extracts, but root of this vegetable contained significant amount of flavanols.

Further, total polyphenolic content as measured by HPLC was lower than that obtained

with Folin-Ciocaltau method. The observed difference could be due to minor

components that remained undetected by HPLC analysis or due to interference from

reducing substances present in plants.

4.2. Antioxidant properties of R. sativus

Several mechanisms have been proposed to be involved in antioxidant activity

such as hydrogen donation, termination of free radical mediated chain reaction,

prevention of hydrogen abstraction, chelation of catalytic ions and elimination of

peroxides (Gordon, 1990). Antioxidant activity is system- dependent and characteristic

of a particular system can influence outcome of analysis. Hence, a single assay would

not be representative of antioxidant potential of plant extracts. In this present study,

different models of antioxidant assays were employed, which could provide a more

consistent approach to assess antioxidant and radical scavenging potential of root, stem

and leaves of R. sativus.

4.2.1. Ferric reducing ability of R. sativus

FRAP assay is based on a redox-linked reaction, whereby antioxidants present in

plant extracts act as reductants while ferric ions in reagents serve as oxidants. Reduction

of ferric-tripyridyltriazine to ferrous complex forms an intense blue color with

maximum absorption at 593 nm, which is related to amount of antioxidants in the

85

sample. The ferric reducing ability of root, stem and leaves of R. sativus is shown in

Table 4.6. Water, methanol and acetone extract reduced ferric ions efficiently and had

reducing activity in the range of 0.82 – 2.83 mM/g, which was greater than or

comparable to synthetic antioxidant BHT (1.28 mM/g). Ethyl acetate and chloroform

extracts showed moderate reducing activity in the range of 0.43 – 0.89 mM/g. Hexane

extract of root displayed significant reducing ability and its activity was greater than

BHT. However, hexane extract of stem and leaves had negligible reducing activity. All

extracts were less effective, when compared with reducing activity of quercetin (15.61

mM/g).

Reduction of ferric to ferrous ion is frequently used as an indicator of electron-

donating activity, which is considered to be an important factor dictating antioxidant

and radical scavenging activity of plant. Figure 4.5 shows dose-response curves for

reducing power of different extracts from R. sativus root, stem and leaves. All root

extracts showed significant ability to reduce ferric ions in a dose-dependent manner.

Water and methanol extract showed highest reducing power, which was followed by

acetone, ethyl acetate, hexane and chloroform. Leaves and stem extract showed variable

reducing power with leaves displaying higher reducing power than stem extracts.

Reducing ability of methanolic extracts of leaves and stem were relatively more

pronounced than other extracts. Quercetin and BHT revealed potent reducing power,

which were distinctly higher than that of any of R. sativus extracts.

Antioxidant activity has been reported to be concomitant with reducing power of

plant extract (Gordon, 1990). Significant ferric reducing ability of R. sativus extracts

observed in this study suggest that polyphenolics present in the extracts have the ability

to donate electrons to free radicals by acting as reductones and thus could terminate free

radical-mediated oxidative reactions. Catechin, sinapic acid, ferulic acid, quercetin and

myricetin, which were identified in R. sativus have been shown to possess significant

ferric reducing ability in their pure form, suggesting that ferric reducing ability of R.

sativus could have been partly contributed by these phenolics (Pulido et al, 2000). Present

findings are in line with those of other investigators, who have also reported that

antioxidant properties are concomitant with development of reducing power (Chung et

al, 2005).

86

Table 4.6

Ferric Reducing Ability - FRAP (expressed as mM FeSO4/g dry weight) of root, stem and leaves of R. sativus.


Root 2.68 ± 0.052a 1.34 ± 0.015 0.82 ± 0.006 0.43 ± 0.002 0.43 ± 0.002 1.40 ± 0.000

Stem 1.68 ± 0.090 1.87 ± 0.034 1.44 ± 0.022 0.89 ± 0.001 0.48 ± 0.006 0.05 ± 0.000

Leaves 1.71 ± 0.031 2.83 ± 0.083 1.78 ± 0.012 0.59 ± 0.000 0.57 ± 0.005 0.06 ± 0.000


87

0

0.2

0.4

0.6

0.8

1

1.2

0 0.01 0.025 0.05 0.1 0.25

Concentration (mg/ml)

Absorbance (700 nm)

Quercetin

BHT

0

0.2

0.4

0.6

0.8

1

1.2

0 0.05 0.1 0.25 0.5 1


Absorbance (700 nm)

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

0.2

0.4

0.6

0.8

1

1.2

0 0.05 0.1 0.25 0.5 1


Absorbance (700 nm)

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

0.2

0.4

0.6

0.8

1

1.2

0 0.05 0.1 0.25 0.5 1


Absorbance (700 nm)

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

(a)

(c) (d)

(b)

Figure 4.5

Reducing power of R. sativus. Quercetin and BHT were used as reference antioxidant. (a) Standard; (b) Root; (c) Stem; (d) Leaves.

Values are means ± SD (n = 3).

88

4.2.2. Metal chelating activity of R. sativus

R. sativus extracts were evaluated for their ability to chelate ferrous ion by

competing with ferrozine in free solution. All extracts displayed an ability to chelate

ferrous ion in a dose-dependent manner (Figure 4.6). However, estimated IC50 was very

high (more than 2.0 mg/ml); particularly, in comparison with positive control EDTA

(7.75 µg/ml). Quercetin and BHT showed moderate metal chelating activity when

compared with EDTA with an IC50 of 134µg/ml and 86µg/ml respectively. Methanol,

water, ethyl acetate and hexane extract showed a chelating ability of 32.15, 28.54, 21.98

and 20.86% respectively at 1.0 mg/ml, whereas chelating ability of acetone (9.25%) and

chloroform (8.20%) were low and insignificant at 1.0 mg/ml. In case of leaves and stem

extract, metal chelating activity varied from 2.15% to 30.83%. Methanolic extracts were

the highest, followed by water, acetone and ethyl acetate extract. Chloroform and

hexane extract of stem and leaves displayed least activity and there was no significant

difference among them. EDTA, quercetin and BHT exhibited 99.23%, 60.54% and 71.36%

of chelating activity respectively, which were significantly higher than that of R. sativus

extracts.

Transition metal ions gain utmost significance in biological system due to their

ability to generate reactive free radicals. They can initiate Fenton type reaction with

production of hydroxyl radicals or Haber-Weiss reactions with superoxide radicals

(Kehrer, 2000; Wong and Kitts, 2001). They hasten peroxidation by decomposing lipid

hydroperoxides into peroxyl and alkoxyl radicals that can themselves abstract hydrogen

and perpetuate chain reaction of lipid peroxidation (Halliwell and Gutteridge, 1984;

Halliwell, 1991). Metal chelating capacity is imperative as it decreases concentration of

catalyzing transition metal ions in Fenton type reaction and protects system from

oxidative damage through inhibition of metal-dependent processes. Chelating agents

that form bonds with metals are effective as secondary antioxidants because they can

reduce redox potential by stabilizing oxidized form of metal ion (Gordon, 1990).

Regardless of reduced activity, R. sativus extracts did possess moderate iron binding

capacity, suggesting their protective action against lipid peroxidation-mediated

oxidative damage. This result is not surprising, as non-phenolic compounds are

supposed to be better chelators of metal ions than polyphenols (Chan et al, 2007).

89

0

20

40

60

80

100

0 0.01 0.025 0.05 0.1 0.25


Percent Inhibition

EDTA

Quercetin

BHT

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

(a)

(d)(c)

(b)

Figure 4.6

Metal chelating activity of R. sativus. EDTA was used as positive control. Quercetin and BHT were used as reference antioxidants.

(a) Standards; (b) Root; (c) Stem; (d) Leaves. Values are means ± SD (n = 3).

90

4.2.3. Inhibition of linoleic acid peroxidation

The ferric thiocyanate method evaluates the capacity of antioxidants to scavenge

peroxyl radicals, which cause oxidation of polyunsaturated fatty acids, through

hydrogen donation. The ferric ion generated from reaction of peroxide with ferrous ion,

form a red-colored complex with thiocyanate, absorbance of which is measured every 24

h until completion of reaction. The antioxidant activity was expressed by absorbance

value and lower the value, higher the antioxidant activity.

Inhibition of linoleic acid oxidation by R. sativus extracts at a concentration of

250µg/ml is shown in Figure 4.7. In the absence of extracts, linoleic acid was auto-

oxidized, which was followed by a rapid increase of peroxides started at 2nd day of

testing, reached maximum on 6th day and decreased on 7th day probably due to lack of

linoleic acid in reaction system. However, peroxidation of linoleic acid was significantly

delayed in the presence of R. sativus extracts, as compared to control. Of different

extracts tested, methanolic extracts were most effective in inhibiting peroxidation of

linoleic acid. The percentage inhibition of oxidation in linoleic acid system by 250µg/ml

of methanolic extract of root, stem and leaves at 6th day of analysis was found to be in

the range of 77 – 82%, which was greater than or comparable to reference antioxidants

such as quercetin (81.64%) and BHT (82.98%), as shown in Table 4.7. Water and acetone

extracts showed inhibitory activities in the range of 73 – 79%. The other extracts showed

inhibitory activity in the range of 34 – 67 %, which was comparatively lesser than that

obtained for standard antioxidants.

Lipid peroxidation is a free radical-mediated chain reaction and is recognized as

being involved in the pathogenesis of various chronic diseases, and its inhibition is

considered as one of the significant roles for antioxidants, because lipid peroxidation

causes disruption of membrane organization, induces changes in fluidity and

permeability, inhibits metabolic processes and alters ion transport (Halliwell, 1991).

Furthermore, peroxides and their secondary reaction products (aldehydes) are cytotoxic

and capable of modifying proteins and DNA bases (Halliwell, 1999). Data obtained in

this study demonstrate that R. sativus significantly inhibited peroxyl radical-induced

oxidation of linoleic acid. Polyphenolics identified in R. sativus are supposed to intercept

91

0

0.5

1

1.5

2

2.5

0 1 2 3 4 5 6 7

Days

Absorbance (500 nm)

Control

Quercetin

BHT

0

0.5

1

1.5

2

2.5

0 1 2 3 4 5 6 7

Days

Absorbance (500 nm)

Control

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

0.5

1

1.5

2

2.5

0 1 2 3 4 5 6 7

Days

Absorbance (500 nm)

Control

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

0.5

1

1.5

2

2.5

0 1 2 3 4 5 6 7

Days

Absorbance at 500 nm

Control

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

(a)

(c) (d)

(b)

Figure 4.7

Inhibition of linoleic acid oxidation by R. sativus at a concentration of 250µg/ml. Quercetin and BHT were used as reference

antioxidants. (a) Standards; (b) Root; (c) Stem; (d) Leaves. Results are of duplicate measurement.

92

Table 4.7

The percentage inhibition of oxidation in linoleic acid system by 250µg/ml of R. sativus extract and standards (quercetin and BHT) at

the 6th day of analysis


Root 76.64 ± 4.92a 80.48 ± 8.27 70.68 ± 3.38 57.63 ± 4.83 55.55 ± 3.52 62.40 ± 1.94

Stem 67.12 ± 6.33 76.55 ± 2.95 72.60 ± 6.71 62.54 ± 2.70 60.66 ± 2.77 57.01 ± 1.87

Leaves 79.68 ± 1.62 81.99 ± 4.50 73.32 ± 3.68 77.42 ± 6.81 67.12 ± 5.30 53.59 ± 4.59

Quercetin 81.64 ± 1.73

BHT 82.98 ± 1.05


93

peroxide-mediated reaction by donating hydrogen from their phenolic hydroxyl groups,

thereby forming a stable end product, which does not initiate or propagate further

peroxidation of lipids. Thus the presence of polyphenolics in R. sativus may partly

explain their significant effect on shielding linoleic acid from peroxidation, suggesting

their usefulness in protecting against oxidation-mediated diseases.

4.3. Free radical scavenging activity

Fundamental antioxidant property of plant extracts is their ability to scavenge

free-radicals, which are believed to contribute significantly to etiology and pathogenesis

of various chronic diseases. The free radical-mediated chain reaction is widely accepted

as a common mechanism of lipid peroxidation. The model of free radical scavenging is

used to assess chain-breaking activity in the propagation phase of lipid and protein

oxidation (Manzocco et al, 1998). Radical scavengers may directly react with and quench

reactive oxygen and nitrogen radicals to terminate peroxidation chain reaction, which is

thought to be due to their hydrogen donating ability (Gulcin et al, 2004). Polyphenolics

have been shown to exert antioxidant activity via this mechanism (Soobrattee et al, 2005).

4.3.1. DPPH radical scavenging activity

Basic information on efficacy of compounds in R. sativus extracts to quench free

radicals can be deduced from DPPH• assay. The DPPH• is a stable free radical, which is

recognized as a tool for evaluating radical scavenging ability of compounds and

antioxidant activity of foods (Sánchez-Moreno, 2002). It accepts an electron or hydrogen

radical to become a stable diamagnetic molecule. The reduction capacity of DPPH• is

determined by decrease in its absorbance at 517 nm, induced by antioxidants. It has also

been used to quantify antioxidants in complex biological systems, because of its ease and

convenience. Even though, DPPH radicals may not be biologically pertinent, it presents

an indication of hydrogen/ electron-donating capacity of plants and provides a useful

means to measure in vitro antioxidant activity.

All R. sativus extracts revealed a concentration-dependent scavenging of DPPH

radicals, with leaves presenting strongest effect followed by stem and root (Figure 4.8).

Of different extracts, methanolic extract showed strongest effect (IC50 at 31 µg/ml for

94

0

20

40

60

80

100

0 0.01 0.025 0.05 0.1 0.25


Percent Inhibition

Quercetin

BHT

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

(a) (b)

(c) (d)

Figure 4.8

DPPH radical scavenging activity of R. sativus. Quercetin and BHT were used as reference antioxidant.

(a) Standard; (b) Root; (c) Stem; (d) Leaves. Values are means ± SD (n = 3).

95

Table 4.8.1

Scavenging ability of root, stem and leaves of R. sativus and standard antioxidants on DPPH• as determined by their IC50, expressed

as mg/ml.


Root 0.335 ±0.014a 0.064 ±0.002 1.17 ±0.083 0.429 ±0.055 1.58 ±0.050 1.72 ±0.097

Stem 0.877 ±0.049 0.042 ±0.001 0.225 ±0.001 0.606 ±0.034 1.21 ±0.031 >2.0

Leaves 0.216 ±0.025 0.031 ±0.000 0.215 ±0.030 0.628 ±0.022 1.48 ±0.075 1.86 ±0.013

Quercetin 0.011 ± 0.002

BHT 0.493. ± 0.057

a All data were average (± SD) of three replicates.

96

leaves, 42 µg/ml for the stem and 64 µg/ml for the root), followed by water, acetone and

ethyl acetate extracts (Table 4.8.1). Chloroform and hexane extracts displayed weak

activity with IC50 of over 1.0 mg/ml. Comparison of DPPH radical scavenging activity

with standard antioxidants showed that the most potent R. sativus extracts had

scavenging ability higher than BHT (IC50 at 493 µg/ml), but lower than quercetin (IC50 at

11 µg/ml).

Effective DPPH• radical scavenging activity exhibited by R. sativus extracts

could be explained by the presence of polyphenolics in them, whose radical scavenging

properties were reported previously in various model systems (Fukumoto and Mazza,

2000). Radical scavenging ability of polyphenolics is attributed to their ability to donate

a hydrogen atom from a phenol to give DPPH-H and a phenoxyl radical. Methanolic

extracts contained more amounts of ferulic acid and sinapic acid, which could partially

explain higher ability to scavenge DPPH (Kim et al, 2008), in comparison with water,

acetone and ethyl acetate extracts. Catechin, the major component of water extracts was

found to be moderately active as an antioxidant in DPPH assay (Hwang et al, 2001). A

comparison between DPPH radical scavenging activity of R. sativus and common

cruciferous vegetables such as wasabi, cauliflower and broccoli showed that R. sativus

extracts were more potent in terms of radical scavenging activity whereby their IC50

values were comparatively much lower than these cruciferous vegetables (Lee et al, 2008;

Koksal and Gulcin, 2008; Borowski et al, 2007), thus further demonstrating effectiveness

of R. sativus root, stem and leaves as natural antioxidants.

4.3.2. Superoxide radical scavenging activity

Superoxide anion is a reduced form of molecular oxygen that is generated during

normal metabolic processes. It is known to be destructive to cellular components as a

precursor of other reactive oxygen species such as hydrogen peroxide, hydroxyl radical

or singlet oxygen (Stief, 2003), contributing to tissue damages and various chronic

diseases (Halliwall, 1991). The scavenging activity of R. sativus extracts on superoxide

radicals is shown in Figure 4.9. Extracts from different parts of R. sativus displayed

concentration dependent protective activity against superoxide radicals. Of which,

leaves were the most effective material followed by stem and root extracts (Table 4.8.2).

97

0

20

40

60

80

100

0 0.01 0.025 0.05 0.1 0.25


Percent Inhibition

Quercetin

BHT

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

(a)

(d)(c)

(b)

Figure 4.9

Superoxide radical scavenging activity of R. sativus. Quercetin and BHT were used as reference antioxidant.


98

Table 4.8.2

Scavenging ability of root, stem and leaves of R. sativus and standard antioxidants on superoxide radical (O2•) as determined by

their IC50, expressed as mg/ml.


Root 0.186 ±0.032a 0.060 ±0.004 0.841 ±0.052 0.738 ±0.049 1.52 ±0.083 1.48 ±0.066

Stem 0.418 ±0.020 0.052 ±0.002 0.189 ±0.023 0.131 ±0.013 0.965 ±0.051 >2.0

Leaves 0.328 ±0.008 0.023 ±0.002 0.046 ±0.007 0.061 ±0.002 0.806 ±0.034 1.85 ±0.059

Quercetin 0.010 ± 0.000

BHT 0.019. ± 0.002


99

Methanolic extracts of leaves (IC50 at 23 µg/ml), stem (IC50 at 52 µg/ml) and root (IC50 at

60 µg/ml) showed potent scavenging activity. Acetone and ethyl acetate extracts of

leaves also displayed significant activity with IC50 of 46 µg/ml and 61 µg/ml

respectively. Other extracts exhibited moderate activity with IC50 in the range of 131 –

841 µg/ml. While chloroform and hexane extracts showed low activity against

superoxide radical with IC50 of around 1.0 mg/ml. When radical scavenging activity of

R. sativus extracts compared to IC50 values calculated for reference antioxidants,

methanolic extract of leaves was as active as BHT (IC50 at 19 µg/ml), but less effective

than quercetin (IC50 at 10 µg/ml).

4.3.3. Hydrogen peroxide scavenging activity

Though hydrogen peroxide (H2O2) itself is not very reactive, it can occasionally

be toxic to cells, since it may give rise to potentially reactive hydroxyl radicals

(Halliwell, 1991). The scavenging activity of R. sativus extracts on H2O2 is shown in

Figure 4.10 and compared with quercetin and BHT as standard antioxidants. R. sativus

extracts were capable of scavenging H2O2 in a concentration-dependent manner. Of

different extracts, leaves showed strongest H2O2 scavenging activity, which was

followed by stem and root. The methanolic extract of leaves displayed the most potent

activity with IC50 at 67 µg/ml, which was comparable to quercetin (IC50 at 34 µg/ml) and

more effective than BHT (IC50 at 89 µg/ml). Other extracts displayed moderate activity

with IC50 in the range of 191 – 781 µg/ml, whilst chloroform and hexane extracts showed

low activity with IC50 of over 1.0 mg/ml (Table 4.8.3).

4.3.4. Nitric oxide scavenging activity

Nitric oxide (NO•) is an essential regulatory molecule, having multiple

physiological effects including blood pressure control, signal transduction, platelet

function, antimicrobial and antitumor activities, when present in nanomolar

concentration (Patel et al, 1999). However, when produced in higher concentrations, it

reacts with oxygen producing potentially deleterious reactive species called

peroxynitrite (Darley-Usmar et al, 1996). Recent studies demonstrate that NO• may act

by disrupting enzymatic activity of DNA repair proteins that play vital roles in the

100

0

20

40

60

80

100

0 0.01 0.025 0.05 0.1 0.25


Percent Inhibition

Quercetin

BHT

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

(a)

(d)(c)

(b)

Figure 4.10

Hydrogen peroxide scavenging activity of R. sativus. Quercetin and BHT were used as reference antioxidant.


101

Table 4.8.3

Scavenging ability of root, stem and leaves of R. sativus and standard antioxidants on hydrogen peroxide (H2O2) as determined by

their IC50, expressed as mg/ml.


Root 0.226 ±0.044a 0.259 ±0.016 0.781 ±0.033 0.528 ±0.008 1.55 ±0.067 1.54 ±0.073

Stem 0.652 ±0.028 0.197 ±0.032 0.680 ±0.009 0.644 ±0.013 1.97 ±0.019 >2.0

Leaves 0.628 ±0.037 0.067 ±0.003 0.457 ±0.041 0.488 ±0.029 1.86 ±0.022 >2.0

Quercetin 0.034 ±0.001

BHT 0.089 ±0.003


102

maintenance of genome integrity (Wink et al, 1991). NO• scavengers compete with

oxygen leading to reduced production of peroxynitrite. All R. sativus extracts revealed a

concentration-dependent scavenging of NO radicals (Figure 4.11). Of different extracts,

leaves showed significant activity, which was followed by stem and root (Table 4.8.4).

Overall, methanolic extract of leaves (IC50 at 56 µg/ml), stem (IC50 at 62 µg/ml)and root

(IC50 at 137 µg/ml) and acetone extract of leaves (IC50 at 91 µg/ml) showed highest NO•

scavenging activity as compared to other extracts. Water, acetone and ethyl acetate

extracts showed moderate NO• scavenging activity with IC50 in the range of 198 – 699

µg/ml. However, hexane extract of root alone was effective against NO• (IC50 at

626µg/ml), whereas hexane extract of stem and leaves did not display significant

scavenging activity against NO• generated in vitro. Comparison of NO• scavenging

activity with standard antioxidants showed that the most potent R. sativus extracts had

scavenging ability comparable to BHT (IC50 at 47 µg/ml), but lower than quercetin (IC50

at 36 µg/ml).

Findings from this study suggest that R. sativus extracts are able to neutralize

superoxide radicals, H2O2 and NO radicals by acting as chain-breaking antioxidants in a

dose-dependent manner. Even though, superoxide radicals, H2O2 and NO radicals are

weak oxidizing agents, they could generate potentially reactive oxygen and nitrogen

species such as singlet oxygen, hydroxyl radicals and peroxynitrite. These reactive

species are believed to act as inducers of cellular injury through initiation of lipid

peroxidation, oxidation of proteins and induction of DNA strand breaks (Halliwell,

1991). Several studies have reported relationship between polyphenolics structure and

antioxidant activity, demonstrating that polyphenolics possessing hydroxyl groups on

their phenyl rings effectively contribute to chain-breaking antioxidant activity by

stabilizing radical form in electron delocation (Rice-Evans, 1995). Among polyphenolics

detected in R. sativus, many have hydroxyl groups in their structure, which would make

it possible to inhibit free radical-induced chain reactions and thus, could contribute

significantly to antioxidant and radical scavenging activity of R. sativus. Furthermore,

antioxidant and radical scavenging activity are outcome of combination of diverse

polyphenolics having synergistic and/or additive effects.

103

0

20

40

60

80

100

0 0.01 0.025 0.05 0.1 0.25


Percent Inhibition

Quercetin

BHT

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1

Concentration (mg/mll)

Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

0

20

40

60

80

100

0 0.05 0.1 0.25 0.5 1


Percent Inhibition

Water

Methanol

Acetone

Ethyl acetate

Chloroform

Hexane

(a) (b)

(d)(c)

Figure 4.11

Nitric oxide radical scavenging activity of R. sativus. Quercetin and BHT were used as reference antioxidant.


104

Table 4.8.4

Scavenging ability of root, stem and leaves of R. sativus and standard antioxidants on nitric oxide (NO•) as determined by their IC50,

expressed as mg/ml.


Root 0.352 ±0.011a 0.137 ±0.046 0.411 ±0.007 0.334 ±0.010 0.397 ±0.005 0.626 ±0.046

Stem 0.682 ±0.009 0.062 ±0.007 0.198 ±0.002 0.424 ±0.035 0.551 ±0.015 1.69 ±0.009

Leaves 0.490 ±0.044 0.056 ±0.002 0.091 ±0.001 0.316 ±0.009 0.699 ±0.037 1.59 ±0.038

Quercetin 0.036 ±0.002

BHT 0.047 ±0.000


105

Polyphenolics of cruciferous vegetables are reported to have preventive and

curative properties against various chronic diseases. However, polyphenolics seemed to

be more concentrated in discarded parts of vegetables such as leaves and stem than in

their edible parts. Hollman and Arts (2000) have demonstrated higher content of

flavonoids in leaves of cauliflower than in their edible parts, where trace amount of

flavonoids is identified. Ayaz et al (2008) have likewise detected abundant phenolic acids

and excellent antioxidant property in leaves and seed of black cabbage. In line with

these findings, present results demonstrate that aerial parts (stem and leaves) of R.

sativus, often under-utilized parts of this vegetable, have high levels of polyphenolics

and show significant antioxidant and radical scavenging activity under in vitro

conditions, as compared to root extracts.

4.4. Antimicrobial activity of R. sativus

Ever increasing demands from consumers for use of natural agents as

additives/food preservatives as well as an increase in incidence of new and reemerging

infections has led to search for new and more effective antimicrobial compounds that

have diverse chemical structure and novel mechanism of action. Plants are an invaluable

source of pharmaceutical products as they have almost infinite ability to synthesize

compounds that reveal varying degree of antimicrobial activity against various

pathogenic and opportunistic microorganisms (Cowan, 1999).

4.4.1. Agar-well diffusion assay

Ten bacterial strains were utilized as taxonomical representatives such as Gram-

positive spore forming rods – Bacillus subtilis; Gram-positive cocci – Staphylococcus

aureus, Staphylococcus epidermidis and Enterococcus faecalis; Gram-negative enterobacteria

– Escherichia coli, Salmonella typhimurium, Enterobacter cloacae, Enterobacter aerogenes and

Klebsiella pneumoniae and Gram-negative non-enterobacteria – Pseudomonas aeruginosa; to

evaluate the effect of a candidate antimicrobial components against specific target

microbes. As shown in Table 4.9.1 – 4.9.3, extracts of root, stem and leaves of R. sativus

had exhibited antibacterial activity against different bacterial genre. Except for water

extracts, all extracts had significant antibacterial activity in agar well diffusion assay

106

against most of the bacteria tested. Antibacterial activity of root, stem and leaves of R.

sativus extracts and standard antibiotics such as penicillin, ampicillin, streptomycin,

ciprofloxacin and ofloxacin, depicting zone of inhibition on agar-well diffusion method

is also shown as Figures 4.12 – 4.17.

Ethyl acetate extract of root exhibited exceptionally large inhibition zones,

comparable with those obtained with standard antibiotics (Table 4.9.4), demonstrating

strong inhibitory activity towards all pathogenic bacteria tested. Acetone extract of root

also showed strong inhibitory activity comparable to ethyl acetate extract, especially

against S. epidermidis, S. typhimurium and E. aerogenes. However, acetone extract had no

effect on growth of K. pneumoniae, E. coli and P. aeruginosa. Methanol and chloroform

extracts had moderate to high antibacterial activity against all organisms tested. Hexane

extract of root showed a good activity only against E. faecalis and S. typhi and a weak

activity against other organisms. On the other hand, hexane extract was found to be

ineffective against E. coli and E. aerogenes.

Similar to root extracts, ethyl acetate extract of stem displayed considerable

inhibitory activity as compared to other extracts. However, it was significantly lesser

than that of ethyl acetate extract of root. Methanol and chloroform extracts showed

moderate activity against all organisms. Acetone extract of stem was moderately

effective against most of the organisms tested, but showed no inhibitory activity

towards S. aureus. Hexane extract of stem had exhibited a very weak inhibitory activity

and was found to be effective only against B. subtilis, S. epidermidis, S. aureus, S. typhi, E.

cloacae and P. aeruginosa.

The ethyl acetate extract of leaves also displayed significant inhibition zones,

equivalent to those obtained with ethyl acetate extract of root against all bacteria tested.

Methanol, acetone and chloroform extracts exhibited a strong to moderate inhibitory

activity. Whereas hexane extract of leaves showed a considerable activity only against S.

typhi (DIZ = 17.52 mm) and a weak activity against other bacteria. Besides, hexane

extract had no effect on growth of E. faecalis, E. aerogenes and P. aeruginosa.

107

Plate 1

108

Plate 2

109

Plate 3

110

Plate 4

111

Plate 5

112

Plate 6

113

Table 4.9.1

Antibacterial activity of R. sativus root extracts against pathogenic bacteria by agar well diffusion method.

Inhibition Zone (mm) Pathogenic

Organisms Water Methanol Acetone Ethyl acetate Chloroform Hexane

B. subtilis NIb 17.97 ± 0.42 a 23.43 ± 1.29 26.80 ± 0.36 17.93 ± 0.25 13.40 ± 0.60

S. aureus NI 19.83 ± 0.55 33.53 ± 1.36 23.83 ± 0.21 14.13 ± 0.49 14.33 ± 0.67

S. epidermidis NI 21.27 ± 0.40 29.57 ± 0.81 25.87 ± 0.41 24.73 ± 0.47 12.50 ± 1.26

E. faecalis NI 19.90 ± 0.36 35.53 ± 0.89 26.13 ± 0.15 16.57 ± 0.89 27.70 ± 2.50

S.typhimurium NI 24.37 ± 0.49 36.97 ± 0.15 26.37 ± 0.31 18.63 ± 0.59 18.50 ± 0.75

K.pneumoniae NI 17.43 ± 0.67 NI 24.53 ± 1.01 16.83 ± 0.49 10.00 ± 0.00

E.coli NI 18.07 ± 0.21 NI 19.67 ± 0.60 15.57 ± 0.83 NI

E.aerogenes NI 18.03 ± 0.55 34.17 ± 0.77 23.40 ± 1.04 18.43 ± 1.21 NI

E.cloacae NI 27.97 ± 0.57 20.50 ± 0.44 34.20 ± 0.66 23.40 ± 0.26 15.38 ± 0.17

P.aeruginosa NI 20.77 ± 0.38 NI 24.90 ± 0.60 19.43 ± 0.70 13.35 ± 0.51

Each value is mean ± standard deviation from three replicate. The concentration of extracts used was 1.0 mg/ml.

a Inhibitory zone in mm, including diameter of the well (8.0 mm)

b No inhibition or inhibition zone was less than 9.0 mm.

114

Table 4.9.2

Antibacterial activity of R. sativus stem extracts against pathogenic bacteria by agar well diffusion method.



B. subtilis NIb 16.47 ± 0.50 a 12.93 ± 0.12 18.30 ± 0.26 15.90 ± 0.79 10.66 ± 0.85

S. aureus NI 15.80 ± 0.20 NI 20.50 ± 0.50 14.83 ± 0.72 12.00 ± 0.00

S. epidermidis NI 16.97 ± 0.15 13.67 ± 0.49 18.30 ± 0.52 15.63 ± 0.41 11.95 ± 0.43

E. faecalis NI 12.03 ± 0.15 13.80 ± 0.35 18.17 ± 0.15 11.03 ± 0.15 NI

S.typhimurium NI 15.70 ± 0.36 12.60 ± 0.49 18.50 ± 0.56 11.93 ± 0.40 13.23 ± 1.33

K.pneumoniae NI 15.67 ± 0.58 10.73 ± 0.25 18.53 ± 0.51 14.87 ± 0.15 NI

E.coli NI 14.77 ± 0.49 10.80 ± 0.20 17.13 ± 0.32 12.10 ± 0.26 NI

E.aerogenes NI 16.67 ± 0.58 13.80 ± 0.53 20.73 ± 0.25 14.80 ± 0.20 NI

E.cloacae NI 19.90 ± 0.56 16.67 ± 0.31 21.67 ± 0.59 18.97 ± 0.25 10.50 ± 0.00

P.aeruginosa NI 14.37 ± 0.47 14.53 ± 0.50 19.53 ± 0.55 16.53 ± 0.42 13.30 ± 0.50

Each value is mean ± standard deviation from three replicate. The concentration of extracts used was 1.0 mg/ml.



115

Table 4.9.3

Antibacterial activity of R. sativus leaves extracts against pathogenic bacteria by agar well diffusion method.



B. subtilis NIb 17.97 ± 0.42 a 13.74 ± 1.58 26.80 ± 0.36 13.10 ± 0.10 12.35 ± 0.48

S. aureus NI 19.83 ± 0.55 20.19 ± 3.42 23.83 ± 0.21 16.20 ± 0.20 12.58 ± 0.91

S. epidermidis NI 21.27 ± 0.40 19.21 ± 1.53 25.87 ± 0.41 12.17 ± 0.21 10.20 ± 0.30

E. faecalis NI 19.90 ± 0.36 13.78 ± 0.89 26.13 ± 0.15 12.10 ± 0.10 NI

S.typhimurium NI 24.37 ± 0.49 16.10 ± 0.70 26.37 ± 0.31 15.97 ± 0.25 17.52 ± 1.14

K.pneumoniae NI 17.43 ± 0.67 19.00 ± 1.50 24.53 ± 1.01 14.17 ± 0.15 10.50 ± 0.20

E.coli NI 18.07 ± 0.21 16.48 ± 2.36 19.67 ± 0.60 18.40 ± 0.36 12.29 ± 0.39

E.aerogenes NI 18.03 ± 0.55 15.60 ± 0.55 23.40 ± 1.04 12.63 ± 0.32 NI

E.cloacae NI 27.97 ± 0.57 22.45 ± 0.69 34.20 ± 0.66 18.53 ± 0.21 11.80 ± 0.90

P.aeruginosa NI 20.77 ± 0.38 18.24 ± 1.80 24.90 ± 0.60 15.23 ± 0.25 NI

Each value is mean ± standard deviation from three replicates. The concentration of extracts used was 1.0 mg/ml.



116

Table 4.9.4

Antibacterial activity of standard antibiotics against pathogenic bacteria by agar well diffusion method.


Organisms Penicillin Ampicillin Streptomycin Ciprofloxacin Ofloxacin

B. subtilis 29.83 ± 0.29a 28.32 ± 0.28 24.33 ± 0.58 34.50 ± 0.50 34.97 ± 0.84

S. aureus 35.03 ± 0.06 39.55 ± 0.05 32.97 ± 0.06 27.57 ± 0.60 26.03 ± 0.65

S. epidermidis 31.67 ± 0.58 29.63 ± 0.47 19.67 ± 0.29 33.23 ± 0.25 34.33 ± 0.29

E. faecalis NIb NI NI 16.17 ± 0.29 18.13 ± 0.32

S.typhimurium 26.27 ± 0.46 34.82 ± 0.65 20.23 ± 0.40 30.30 ± 0.26 28.27 ± 0.31

K.pneumoniae 23.33 ± 0.58 18.70 ± 0.30 27.97 ± 0.05 29.33 ± 0.29 30.53 ± 0.42

E.coli 32.67 ± 0.57 25.46 ± 0.52 19.97 ± 0.06 25.43 ± 0.41 23.90 ± 0.10

E.aerogenes 26.30 ± 0.36 30.67 ± 0.32 14.17 ± 0.29 29.10 ± 0.10 27.33 ± 0.29

E.cloacae 22.83 ± 0.72 21.80 ± 0.25 24.97 ± 0.15 23.17 ± 0.21 21.17 ± 0.29

P.aeruginosa NI NI 21.33 ± 0.57 35.33 ± 0.35 32.33 ± 0.29

Each value is mean ± standard deviation from three replicates. The concentration of antibiotics used was 100µg/ml



117

The antibiotics penicillin, ampicillin, streptomycin, ciprofloxacin and ofloxacin

were effective against most of the organisms except that penicillin showed no activity

against E. faecalis and P. aeruginosa; streptomycin had no effect against E.

faecalis;ciprofloxacin and ofloxacin showed a lower activity against E. faecalis, S. typhi

and E. aerogenes. In contrast, inhibition zones of solvent control (methanol, acetone, ethyl

acetate, chloroform and hexane) were below 9.0 mm, indicating that they were inactive

against all microorganisms tested.

E. faecalis (resistant to penicillin and streptomycin) and P. aeruginosa (resistant to

penicillin) were significantly inhibited by acetone extract (DIZ = 35.53 mm) and hexane

extract of root (DIZ = 27.70 mm), and ethyl acetate extract of root, stem and leaves (DIZ

= 17.17 – 26.13 mm). Other extracts showed variable inhibitory activity towards these

resistant strains. E. cloacae were found to be a highly sensitive organism with DIZ in the

range of 15.83 – 34.20 mm. Selected food-borne pathogens used in this study were

susceptible to all extracts, but were highly sensitive to ethyl acetate extracts.

Further, both Gram-positive and Gram-negative bacteria were equally susceptible,

demonstrating broad spectrum inhibitory effect of R. sativus. Of different parts of R.

sativus used in this study, root extracts appeared to be more active than stem and leaves

extracts in inhibiting bacterial growth.

4.4.2. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal

Concentration (MBC) of R. sativus

The results obtained for MIC and MBC of R. sativus root, stem and leaves extracts

are presented in Table 4.10.1 – 4.10.3. Of the different root extracts, ethyl acetate extract

had lowest MIC and MBC, followed by acetone, methanol, chloroform and hexane. Over

half of MICs and MBCs for ethyl acetate extract were close to or equal to those of

positive controls (Table 4.10.4) and were in the ranges 0.016 – 0.064 and 0.016 – 0.512

mg/ml, respectively. MIC and MBC of acetone extract for E. aerogenes were 0.016 and

0.128 mg/ml respectively, which were relatively lesser than MICs and MBCs obtained

with standard antibiotics. Similarly, for other organisms, its MIC and MBC were found

to be in the ranges 0.032 – 0.256 and 0.064 – 0.512 mg/ml respectively. The methanol and

chloroform extracts also had a substantial antibacterial activity, with MICs in the range

118

0.064 – 0.512 mg/ml and MBCs in the range 0.256 – 4.10 mg/ml. Hexane extract showed

a lower inhibitory activity as compared to the other extracts with MICs in the range

0.512 – 1.02 mg/ml and MBCs in the range 2.05 – 4.10 mg/ml, except for E. faecalis where

MIC and MBC were 0.016 and 0.032 mg/ml respectively.

MICs and MBCs for ethyl acetate extracts of stem and leaves were in the ranges

0.064 – 0.256 and 0.128 – 2.05 mg/ml, which were, however, significantly higher than

MICs and MBCs of ethyl acetate extract of root. The methanol extract of stem and leaves

showed strong to moderate activity with MICs in the range 0.064 – 1.02 mg/ml and

MBCs in the range 0.256 – >4.10 mg/ml. For chloroform extracts of stem and leaves,

MICs were in the range 0.256 – 1.02 mg/ml and MBCs in the range 0.512 – >4.10 mg/ml.

Acetone extract of stem was weakly active against most of the pathogens with MICs in

the range 0.256 – 1.02 mg/ml and MBCs in the range 1.02 – >4.10 mg/ml. However,

acetone extract of leaves showed considerable antibacterial activity against S. aureus and

S. epidermidis with MICs of 0.064mg/ml and MBCs of 0.256 and 0.512 mg/ml

respectively. For remaining pathogens, MICs were in the range 0.128 – 1.02 mg/ml and

MBC in the range 1.02 – >4.10 mg/ml. Hexane extract of stem and leaves showed almost

identical activity with MICs in the range 0.512 – 1.02 mg/ml and MBCs in the range 2.05

– >4.10 mg/ml for most of the studied organisms.

Previous studies have indicated growth inhibitory activity of R. sativus root

extracts on few species of microorganisms (Abdou et al, 1972; Esaki and Onozaki, 1982;

Khan et al, 1985). However, this is the first time that antibacterial activity of different

parts of R. sativus has been demonstrated on a wide spectrum of bacteria. R. sativus root,

stem and leaves extracts had excellent bactericidal activity against both Gram-positive

and Gram-negative bacteria. Successful extraction of bioactive compounds from plant

material depends on solvent used in extraction procedure. In this study, it was observed

that extraction of plant with organic solvents resulted in extracts with considerable

antibacterial activity against all health damaging bacteria than extraction with water. In

particular, ethyl acetate extracts of R. sativus root, stem and leaves were very active

against all pathogens. These observations can be explained by different active

compounds being extracted with each solvent. These findings are in contrast with

results of Abdou et al (1972), who have described antibacterial activity of an aqueous

119

Table 4.10.1

MIC and MBC of R. sativus root extracts against health damaging bacteria.

MIC (MBC) mg/ml Pathogenic


B. subtilis NTa 0.256 (0.512) 0.064 (0.256) 0.032 (0.032) 0.256 (0.512) 1.02 (4.10)

S. aureus NT 0.064 (0.256) 0.064 (0.256) 0.032 (0.128) 0.512 (2.05) 0.512 (2.05)

S. epidermidis NT 0.128 (0.256) 0.032 (0.064) 0.032 (0.032) 0.128 (0.512) 1.02 (4.10)

E. faecalis NT 0.256 (2.05) 0.032 (0.128) 0.064 (0.128) 0.512 (4.10) 0.016 (0.032)

S.typhimurium NT 0.064 (0.256) 0.016 (0.128) 0.032 (0.128) 0.256 (1.02) 0.512 (4.10)

K.pneumoniae NT 0.256 (1.02) NT 0.064 (0.256) 0.256 (1.02) 1.02 (4.10)

E.coli NT 0.256 (1.02) NT 0.064 (0.512) 0.512 (4.10) NT

E.aerogenes NT 0.256 (1.02) 0.016 (0.128) 0.064 (0.512) 0.256 (2.05) NT

E.cloacae NT 0.064 (0.256) 0.256 (0.512) 0.016 (0.016) 0.256 (0.512) 0.512 (2.05)

P.aeruginosa NT 0.128 (1.02) NT 0.064 (0.512) 0.256 (4.10) 1.02 (4.10)

Results are shown as means of three measurements done on separate occasions.

a Not tested.

120

Table 4.10.2

MIC and MBC of R. sativus stem extracts against health damaging bacteria.



B. subtilis NTa 0.512 (2.05) 0.256 (1.02) 0.256 (0.512) 0.512 (2.05) 1.02 (4.10)

S. aureus NT 0.512 (2.05) NT 0.128 (0.256) 0.512 (2.05) 1.02 (2.05)

S. epidermidis NT 0.256 (1.02) 0.512 (1.02) 0.256 (1.02) 0.512 (2.05) 1.02 (>4.10)

E. faecalis NT 1.02 (>4.10) 1.02 (>4.10) 0.256 (2.05) 1.02 (>4.10) NT

S.typhimurium NT 0.256 (1.02) 1.02 (4.10) 0.256 (1.02) 1.02 (>4.10) 1.02 (>4.10)

K.pneumoniae NT 0.512 (2.05) 1.02 (4.10) 0.128 (0.512) 0.512 (2.05) NT

E.coli NT 0.512 (2.05) 1.02 (4.10) 0.256 (2.05) 1.02 (>4.10) NT

E.aerogenes NT 0.256 (1.02) 1.02 (4.10) 0.128 (0.512) 0.512 (2.05) NT

E.cloacae NT 0.128 (0.512) 0.512 (2.05) 0.128 (0.512) 0.256 (0.512) 1.02 (4.10)

P.aeruginosa NT 0.512 (2.05) 1.02 (>4.10) 0.128 (1.02) 0.256 (2.05) 1.02 (>4.10)


a Not tested.

121

Table 4.10.3

MIC and MBC of R. sativus leaves extracts against health damaging bacteria.



B. subtilis NTa 0.512 (2.05) 0.256 (1.02) 0.256 (1.02) 1.02 (4.10) 1.02 (4.10)

S. aureus NT 0.064 (0.256) 0.064 (0.256) 0.256 (1.02) 0.512 (2.05) 1.02 (4.10)

S. epidermidis NT 0.128 (0.512) 0.064 (0.512) 0.128 (0.512) 1.02 (>4.10) 1.02 (4.10)

E. faecalis NT 1.02 (4.10) 1.02 (>4.10) 0.256 (2.05) 1.02 (>4.10) NT

S.typhimurium NT 0.256 (1.02) 0.512 (2.05) 0.256 (1.02) 0.512 (2.05) 0.512 (4.10)

K.pneumoniae NT 0.256 (1.02) 0.256 (2.05) 0.128 (0.512) 0.512 (2.05) 1.02 (4.10)

E.coli NT 0.512 (4.10) 0.512 (4.10) 0.256 (1.02) 0.256 (1.02) 1.02 (4.10)

E.aerogenes NT 0.512 (4.10) 0.512 (2.05) 0.256 (1.02) 1.02 (4.10) NT

E.cloacae NT 0.128 (0.256) 0.128 (1.02) 0.064 (0.128) 0.256 (0.512) 1.02 (2.05)

P.aeruginosa NT 0.512 (2.05) 0.512 (4.10) 0.256 (2.05) 0.512 (>4.10) NT


a Not tested.

122

Table 4.10.4

MIC and MBC of standard antibiotics against health damaging bacteria.


Organisms Penicillin Ampicillin Streptomycin Ciprofloxacin Ofloxacin

B. subtilis 0.016 (0.032) 0.016 (0.032) 0.032 (0.032) 0.008 (0.016) 0.008 (0.016)

S. aureus 0.008 (0.016) 0.008 (0.016) 0.008 (0.008) 0.032 (0.032) 0.032 (0.032)

S. epidermidis 0.016 (0.016) 0.016 (0.032) 0.064 (0.064) 0.016 (0.016) 0.016 (0.016)

E. faecalis NTa NT NT 0.064 (0.128) 0.064 (0.128)

S. typhimurium 0.032 (0.128) 0.032 (0.064) 0.064 (0.128) 0.004 (0.008) 0.002 (0.008)

K. pneumoniae 0.064 (0.128) 0.064 (0.254) 0.032 (0.032) 0.008 (0.032) 0.008 (0.032)

E. coli 0.008 (0.008) 0.016 (0.016) 0.064 (0.128) 0.032 (0.064) 0.032 (0.064)

E. aerogenes 0.032 (0.064) 0.032 (0.032) 0.128 (0.512) 0.032 (0.064) 0.032 (0.064)

E. cloacae 0.064 (0.064) 0.064 (0.064) 0.032 (0.032) 0.032 (0.032) 0.032 (0.032)

P. aeruginosa NT NT 0.064 (0.256) 0.008 (0.032) 0.008 (0.032)


a Not tested.

123

extract of R. sativus tubercle against E. coli, P. pyocyaneus, S. typhimurium and B. subtilis.

Because no appreciable inhibitory activity was found for water extract of R. sativus at a

concentration of 1 mg/ml, it is supposed that aqueous extract used by Abdou et al (1972)

was of higher concentration than used in this study.

This study included E. faecalis resistant to penicillin and streptomycin and P.

aeruginosa resistant to penicillin, because these opportunistic bacteria can cause life-

threatening infections in humans, especially, in a nosocomial environment (Toye et al,

1997; Hancock, 1998). Interestingly, this study recorded a notable susceptibility of these

resistant strains, especially to root extracts, suggesting that components contained in

that particular extracts may provide an alternate strategy for combating these organisms

and could improve treatment of infections caused by these organisms. Further, different

parts of R. sativus appeared to have potent inhibitory activity towards food-borne

pathogens used in this study. Many previous studies reported the inability of natural

antimicrobial agents to inhibit growth of Gram-negative bacteria (Alzoreky and

Nakahara 2003; Weseler et al, 2002), perhaps, because of the presence of complex cell

wall structure that usually reduces penetration of bacterial cells by extracts. The

remarkable findings of this study are that R. sativus extracts are equally effective against

both Gram-positive and Gram-negative bacteria.

Isothiocyanates (ITCs) are regarded as the main constituents responsible for

antibacterial activity of cruciferous plants. This study detected the presence of different

amounts of ITCs in root, stem, and leaves of R. sativus. Root extracts seemed to contain

higher amounts of ITCs, as compared to leaves and stem. Further, it was noted that ITC

content was strongly dependent on solvent used, because hexane, chloroform, ethyl

acetate and acetone extracted significant amounts of ITCs than methanol and water.

Despite similar ranges of total ITC content of methanol and water extracts, all water

extracts were less effective at inhibiting growth of bacteria. Similarly, inhibitory activity

of ethyl acetate extracts was higher than that of hexane, chloroform and acetone extracts,

even though amount of ITCs was less than those extracts, thus excluding the possibility

that presence of ITCs in this plant was solely responsible for antibacterial activity

observed. Shin et al (2004) recently demonstrated that phenolic compounds, in addition

to isothiocyanates, could be responsible for antibacterial activity of wasabi.

124

However, hexane extract of root that contained considerable amount of ITCs

showed exceptionally potent and species-specific antibacterial activity especially against

E. faecalis. Similarly, acetone extract of root had a remarkable inhibitory effect against S.

epidermidis, S. typhimurium and E. aerogenes. Given the range of R side groups on parent

GLs, a wide spectrum of ITCs could possibly be present in R. sativus. Probable reasons

for these observed results could be due to influence of different R groups of ITCs on

antibacterial activity and possible synergism and/or antagonism among different ITCs.

Manici et al, (1997) have reported that various ITCs have different biocidal effect, which

depend both on species of microbes and chemical nature of ITC side chain.

4.4.3. Effect of pH and heat treatment on antibacterial activity of R. sativus

The ethyl acetate extracts of root, stem, and leaves of R. sativus, which had potent

inhibitory activity and more effective bactericidal activity than other extracts, were

further studied to determine effects of pH and temperature on their antibacterial

activity. The effects of pH and heat treatment on antibacterial activity of ethyl acetate

extracts of R. sativus are shown in Table 4.11.1 – 4.11.3 and 4.12.1 – 4.12.3 respectively. At

pH 3.0, inhibitory activity of ethyl acetate extracts was slightly higher than that of

control (pH 4.2). At pH 6.0, antibacterial activity seemed to be slightly lower than that of

control extract. At pH 9.0 inhibitory effects was significantly lower than that of control.

Thus, extracts studied had an excellent antibacterial activity when pH was maintained

around 3.0 – 6.0 and tended to lose their activity when pH was increased towards

alkaline side. Acid and alkali control solutions were not inhibitory to any of bacteria

tested. The inhibitory effect of heat-treated extracts was not significantly different from

that of untreated extracts, when extracts were incubated at or below 75ºC for 30 min.

However, boiling the extracts at 100ºC for 30 min significantly reduced, but did not

abolish, their antibacterial activity.

The acid tolerance and thermal stability of plant extracts are critical aspects of

their use in food-processing applications as natural preservatives to control bacterial

growth. In this study, it was observed that R. sativus had excellent antibacterial activity

at acidic pH, and that increasing pH of extracts towards alkaline side led to a significant

drop in their inhibitory action. It has been reported that antibacterial compounds

125

Table 4.11.1

Effect of pH of ethyl acetate extract of R. sativus root on inhibitory zone (mm) against pathogens.

pH Pathogenic Organisms

Control 3.0 6.0 9.0

B. subtilis 26.80 ± 1.36a 26.92 ± 1.54 25.15 ± 2.24 16.73 ± 0.64

S. aureus 23.83 ± 1.21 23.87 ± 2.64 22.31 ± 1.14 12.10 ± 0.08

S. epidermidis 25.87 ± 0.41 25.70 ± 1.58 23.96 ± 1.53 14.12 ± 0.35

E. faecalis 26.13 ± 0.15 28.50 ± 0.50 24.78 ± 0.42 15.21 ± 0.31

S. typhimurium 26.37 ± 2.31 27.54 ± 2.42 24.98 ± 0.63 14.13 ± 0.21

K. pneumoniae 24.53 ± 1.01 24.80 ± 2.50 23.55 ± 0.40 13.18 ± 0.29

E. coli 19.67 ± 0.60 20.51 ± 1.42 18.16 ± 0.20 11.64 ± 0.32

E. aerogenes 23.40 ± 1.04 24.89 ± 1.62 21.79 ± 2.43 10.33 ± 0.21

E. cloacae 34.20 ± 2.66 36.80 ± 1.48 32.58 ± 0.73 16.91 ± 0.30

P. aeruginosa 24.90 ± 0.60 25.66 ± 0.84 21.74 ± 0.23 13.55 ± 0.53

Each value is mean ± standard deviation from three replicates

The concentration of extracts used was 1.0 mg/ml


126

Table 4.11.2

Effect of pH of ethyl acetate extract of R. sativus stem on inhibitory zone (mm) against pathogens.


Control 3.0 6.0 9.0

B. subtilis 18.30 ± 0.26 a 19.66 ± 0.92 17.19 ± 0.31 10.72 ± 0.42

S. aureus 20.50 ± 0.50 21.82 ± 0.74 19.10 ± 0.06 12.30 ± 0.15

S. epidermidis 18.30 ± 0.52 18.50 ± 0.60 16.93 ± 0.57 11.58 ± 0.63

E. faecalis 18.17 ± 0.15 17.25 ± 1.08 16.75 ± 0.46 10.05 ± 0.16

S. typhimurium 18.50 ± 0.56 18.98 ± 1.87 16.81 ± 0.50 12.17 ± 0.36

K. pneumoniae 18.53 ± 0.51 19.00 ± 0.50 17.06 ± 0.08 18.53 ± 0.51

E. coli 17.13 ± 0.32 17.50 ± 1.20 16.10 ± 0.29 11.30 ± 0.47

E. aerogenes 20.73 ± 0.25 22.10 ± 0.24 18.75 ± 0.25 10.33 ± 0.21

E. cloacae 21.67 ± 0.59 22.43 ± 0.56 19.53 ± 0.41 21.67 ± 0.59

P. aeruginosa 19.53 ± 0.55 20.43 ± 0.92 17.47 ± 0.56 10.58 ± 0.33




127

Table 4.11.3

Effect of pH of ethyl acetate extract of R. sativus leaves on inhibitory zone (mm) against pathogens.


Control 3.0 6.0 9.0

B. subtilis 16.90 ± 0.36 a 17.30 ± 1.50 14.83 ± 0.31 9.57 ± 0.60

S. aureus 18.73 ± 0.25 19.10 ± 0.81 17.03 ± 0.25 12.10 ± 0.08

S. epidermidis 20.77 ± 1.25 20.87 ± 1.35 19.63 ± 1.60 12.13 ± 0.32

E. faecalis 17.17 ± 0.21 18.00 ± 0.00 16.27 ± 0.31 10.40 ± 0.85

S. typhimurium 18.67 ± 0.29 18.50 ± 0.82 16.13 ± 0.32 18.67 ± 0.29

K. pneumoniae 18.70 ± 0.30 18.92 ± 0.68 16.63 ± 0.60 11.03 ± 0.25

E. coli 18.30 ± 0.20 18.90 ± 0.71 16.80 ± 0.61 11.90 ± 0.56

E. aerogenes 18.33 ± 0.31 18.53 ± 0.15 16.23 ± 1.25 18.33 ± 0.31

E. cloacae 25.30 ± 0.10 26.41 ± 1.38 23.77 ± 0.93 14.33 ± 0.45

P. aeruginosa 18.33 ± 0.31 18.70 ± 1.20 16.10 ± 0.66 9.53 ± 0.35




128

Table 4.12.1

Effect of heat treatment on stability of ethyl acetate extract of R. sativus root.

Temperature (°C) Pathogenic

Organisms

Control 25 50 75 100

B. subtilis 26.80 ± 1.36a 26.30 ± 0.26 26.75 ± 1.30 26.15 ± 0.24 13.45 ± 0.62

S. aureus 23.83 ± 1.21 23.37 ± 0.35 23.80 ± 1.20 23.40 ± 0.40 12.65 ± 0.40

S. epidermidis 25.87 ± 0.41 24.93 ± 0.25 24.75 ± 1.97 24.60 ± 0.40 13.27 ± 0.62

E. faecalis 26.13 ± 0.15 26.36 ± 0.54 26.10 ± 0.00 26.12 ± 0.15 NDb

S. typhimurium 26.37 ± 2.31 25.60 ± 0.24 25.83 ± 2.61 25.87 ± 0.63 12.47 ± 0.33

K. pneumoniae 24.53 ± 1.01 24.58 ± 0.23 25.00 ± 0.50 24.00 ± 0.00 10.55 ± 0.45

E. coli 19.67 ± 0.60 18.69 ± 0.34 19.25 ± 2.50 19.24 ± 0.53 12.27 ± 1.67

E. aerogenes 23.40 ± 1.04 23.50 ± 0.75 23.15 ± 2.82 22.80 ± 0.42 10.82 ± 0.98

E. cloacae 34.20 ± 2.66 32.27 ± 0.35 32.73 ± 1.68 33.40 ± 0.20 12.50 ± 0.2

P. aeruginosa 24.90 ± 0.60 24.43 ± 0.58 25.30 ± 0.60 24.05 ± 0.50 12.00 ± 0.00

Each value is mean ± standard deviation from three replicates. The concentration of extracts used was 1.0 mg/ml.


b Not detected.

129

Table 4.12.2

Effect of heat treatment on stability of ethyl acetate extract of R. sativus stem.


Organisms

Control 25 50 75 100

B. subtilis 18.30 ± 0.26a 17.57 ± 0.40 18.54 ± 0.56 18.63 ± 0.32 10.26 ± 0.10

S. aureus 20.50 ± 0.50 19.80 ± 0.26 19.50 ± 0.84 20.10 ± 0.30 12.15 ± 0.33

S. epidermidis 18.30 ± 0.52 17.43 ± 0.51 18.49 ± 0.82 18.30 ± 0.20 11.20 ± 0.40

E. faecalis 18.17 ± 0.15 18.40 ± 0.20 18.24 ± 1.34 17.57 ± 0.68 NDb

S. typhimurium 18.50 ± 0.56 18.05 ± 0.4 18.50 ± 1.50 17.94 ± 0.55 10.89 ± 0.43

K. pneumoniae 18.53 ± 0.51 17.90 ± 0.36 18.43 ± 1.73 17.94 ± 0.72 10.20 ± 0.58

E. coli 17.13 ± 0.32 16.90 ± 0.20 17.24 ± 0.87 16.81 ± 0.68 10.35 ± 0.82

E. aerogenes 20.73 ± 0.25 19.80 ± 0.42 20.70 ± 0.69 20.15 ± 0.83 10.10 ± 0.22

E. cloacae 21.67 ± 0.59 20.94 ± 0.17 20.97 ± 0.48 20.05 ± 0.41 10.22 ± 0.42

P. aeruginosa 19.53 ± 0.55 19.30 ± 0.20 19.50 ± 1.46 18.97 ± 0.63 ND

Each value is mean ± standard deviation from three replicates. The concentration of extracts used was 1.0 mg/ml


b Not detected.

130

Table 4.12.3

Effect of heat treatment on stability of ethyl acetate extract of R. sativus leaves.


Organisms

Control 25 50 75 100

B. subtilis 16.90 ± 0.36a 16.70 ± 0.26 17.00 ± 0.10 16.54 ± 0.55 10.10 ± 0.21

S. aureus 18.73 ± 0.25 18.23 ± 0.25 18.55 ± 2.15 17.93 ± 0.12 10.50 ± 0.50

S. epidermidis 20.77 ± 1.25 20.07 ± 0.38 20.40 ± 2.35 20.00 ± 0.10 11.35 ± 0.94

E. faecalis 17.17 ± 0.21 17.13 ± 0.26 17.20 ± 0.60 17.02 ± 0.15 NDb

S. typhimurium 18.67 ± 0.29 18.60 ± 0.35 17.45 ± 0.67 17.68 ± 0.37 11.20 ± 0.30

K. pneumoniae 18.70 ± 0.30 17.85 ± 0.46 17.50 ± 1.20 18.20 ± 0.40 10.20 ± 0.74

E. coli 18.30 ± 0.20 18.67 ± 0.19 18.44 ± 0.68 18.50 ± 0.00 10.40 ± 0.20

E. aerogenes 18.33 ± 0.31 18.34 ± 0.76 18.30 ± 1.45 18.28 ± 0.17 9.80 ± 0.60

E. cloacae 25.30 ± 0.10 24.60 ± 0.25 25.44 ± 1.24 24.83 ± 0.72 10.64 ± 0.63

P. aeruginosa 18.33 ± 0.31 17.52 ± 0.62 18.27 ± 1.61 18.22 ± 0.14 ND

Each value is mean ± standard deviation from three replicates. The concentration of extracts used was 1.0 mg/ml


b Not detected.

131

seemed to be stabilized in cationic forms that may interact with and disrupt negatively

charged bacterial cells (Rhodes et al, 2006). Hence, dependence of antibacterial activity of

R. sativus on low pH suggests that molecular structure or charge of antibacterial species

maybe vital for its inhibitory effect. Heat treatment of R. sativus extracts at 100ºC for 30

min reduced their antibacterial activity, but these extracts still retained some of their

inhibitory effect. These results suggest that extracts have significant thermal stability,

which is regarded as an important property for compounds to be used in food

preservation.

4.4.4. GC – MS analysis of ethyl acetate extract of R. sativus root

Analysis of ethyl acetate extract of R. sativus by GC-MS revealed presence of 42

compounds (Figure 4.18). Of which, 21 compounds were identified, as shown in Table

4.13, by comparing their retention indices (RI) and mass spectra (MS) with the Wiley

library spectra database and literature data (Adams 1995; Vaughn and Berhow, 2005;

Blazevic and Mastelie, 2009). Fatty acids were found to be major components,

constituting almost 54% of total compounds present in it. Of which, 32% were

polyunsaturated fatty acids (PUFAs) and in which essential fatty such as 9, 12,-

octadecadienoic acid (linoleic acid) and 9, 12, 15-octadecatrienoic acid (α-linolenic acid –

ALA), were present in considerable amounts (3% and 22% respectively). The major ITCs

found in ethyl acetate extract were 4-(methylthio)-3-butenyl isothiocyanate (Z isomer)

(0.71%), 4-(methylthio) butyl isothiocyanate (1.83%), and 4-(methylthio)3-butenyl

isothiocyanate (E isomer) (9.04). Other components detected were alkanes, eugenol and

methyl cholesterol.

Essential fatty acids include both ω-6 fatty acids such as linoleic acid and ω-3

fatty acids such as α-linolenic acid (ALA). ALA and eicosapentaenoic acid (EPA) and

docosahexaenoic acid (DHA) (derived from ALA) are important in human nutrition as

it has been established to be beneficial in reduction of cardiovascular diseases. ALA is

abundantly available in edible plants, whereas EFA and DHA are particularly found in

marine vertebrates and fishes. EFA and DHA appear to be more beneficial than ALA.

Recent study demonstrated that ALA in diet was converted into more significant EPA

132

Figure 4.18

133

Table 4.13

Content and composition of compounds in ethyl acetate extract of R. sativus root, as

analyzed by GC-MS.

Sl.No Compounds RTa Homology

(%)

RCb

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

1,4-dimethyl tetrasulfide

Eugenol

Tridecane

Methylthio butenyl isothiocyanate

Methylthio butyl isothiocyanate

Methylthio butenyl isothiocyanate

Dodecanoic acid

Tetradecanoic acid

Pentadecanoic acid

n – Tetracosane

9,12,-octadecadienoic acid

7,10,13-hexadecatrienoic acid

n – Eicosane

n – Docosane

Methyl linolenate

Benzenepropanenitrile

n – Heptacosane

n – Docosane

n – Octacosane

n- Nonacosane

23-R-methyl cholesterol

10.97

12.90

13.36

13.69

13.89

13.95

15.84

18.07

19.80

20.09

21.40

21.48

21.64

21.94

24.89

25.78

26.09

27.04

28.20

29.59

34.42

95

97

95

97

97

97

95

95

95

95

95

96

97

97

95

95

97

97

97

97

99

0.86

0.18

1.89

0.71

1.83

9.04

2.98

1.14

17.78

1.68

2.32

8.29

4.91

1.09

21.52

2.15

1.44

1.21

1.08

0.54

1.50

a Retention time (min);

b Relative area percentage (peak area relative to the total peak area percentage)

134

and DHA in body, producing effects same as that of direct consumption of EPA and

DHA. Linoleic acid is also equally important for human nutrition and whose biological

effects are largely meditated by their conversion to eicosanoids. The conversion of

arachidonic acid to prostaglandins and leukotrienes provides many targets for

pharmaceutical drug development and treatment for many diseases, including tumor

proliferation.

Previous study showed that fatty acids function as important ingredients of food

additives due to their growth inhibitory effect on undesirable microorganisms (Freese et

al, 1973). Besides, PUFAs are bactericidal to significant pathogens, including methicillin-

resistant Staphylococcus aureus (Farrington et al, 1992; Knapp and Melly 1986), Helicobacter

pylori (Sun et al, 2003), and Mycobacteria (Seidel and Taylor, 2004). Further, Chen et al

(1994) displayed that ALA present in flaxseeds was stable at a temperature as high as

100ºC. Eugenol, a phenolic compound is present in essential oils of many plants. Several

studies showed antimicrobial activity of eugenol against various pathogenic bacteria

(Suresh et al, 1992), fungi (Bilgrami et al, 1992) and viruses (Pacheco et al, 1993). Besides,

eugenol was found to exhibit significant antimicrobial activity at an acidic pH (Ali et al,

2005). Significant antibacterial activity, acid tolerance and thermal stability of ethyl

acetate extract of R. sativus root could be attributed to complex mixture of

phytochemicals such as polyphenolics, PUFAs, ITCs and eugenol. Besides, this extract

may contain unknown components that might afford synergistic/additive inhibitory

effects.

4.5. Cytoprotective effect of R. sativus

Oxidative damage to biomolecules such as proteins, lipids and DNA by reactive

species may play a crucial role in the etiology of various degenerative diseases including

cancer (Halliwell 1994; Collins 1999). Diverse defense mechanisms exist in body to

alleviate potentially damaging free radicals. Regardless of these defense mechanisms,

oxidative damage still occurs within cells and accumulation of mutated DNA could very

well contribute to these chronic diseases. Numerous epidemiological studies highlight

the importance of consuming diets rich in phytochemicals, which could minimize

oxidative DNA damage and have a protective effect on health (Block et al, 1992).

135

4.5.1. Effect of R. sativus and H2O2 on viability of lymphocytes

The effect of R. sativus root, stem and leaves extracts on viability of normal

human lymphocytes was evaluated using MTT assay. The observed cell viability was

greater than 90%, when incubated with R. sativus ranging from 5 – 50µg/ml, indicating

that R. sativus did not display cytotoxicity to lymphocytes at the tested concentrations

(Table 4.14.1 – 4.14.3). However, there was a significant reduction in cell viability, when

concentration of R. sativus was increased to 100µg/ml. Based on data obtained,

concentration in the range of 5 – 50µg/ml, which related to greater than 90% cell

viability, was selected for use in consequent assays.

The concentration of H2O2 needed to induce significant oxidative damage to

lymphocytes was evaluated by incubating them with different concentration of H2O2 (0 –

500µM) for 10 min. Treatment with H2O2 reduced cell viability, but effect was not

significant at a lower concentration (10 – 50 µM). However, at a higher concentration

(100 – 500µM), there was a significant decrease in cell viability, as compared to untreated

control (Table 4.15).

Similarly, apparent DNA damage was detected at a concentration higher than

50µM and increasing the concentration to 500µM resulting in extensive DNA damage

with a majority of cells showing a tailed DNA. Based on the obtained data, 200 µM of

H2O2, which showed significant cell death and DNA damage was selected for use in all

subsequent assays.

4.5.2. Protective effect of R. sativus on H2O2 induced cytotoxicity in lymphocytes

Lymphocytes were pre-incubated for 3 h, with R. sativus (5 – 50 µg/ml) extracts

before exposure to 200 µM of H2O2 for 10 min. There was a significant reduction in cell

viability when lymphocytes were treated with 200 µM of H2O2 alone for 10 min, as

compared to untreated cells. However, pre-incubation of cells with R. sativus extracts led

to a significant reduction in cell mortality. Among R. sativus extracts used in this study,

hexane extract of root and methanolic extract of stem and leaves showed significant

protection against H2O2-induced cytotoxicity as shown in Table 4.16.1 – 4.16.3.

Interestingly, considerable degree of cytoprotection was obvious at a concentration as

low as 25µg/ml and no further significant increase in cytoprotection was observed as

136

Table 4.14.1

Effect of R. sativus root extracts on viability of lymphocytes

Concentration (µg/ml) Extraction solvent

5 10 25 50 100

Water 97.01 ± 1.31a 94.78 ± 2.15 94.03 ± 3.20 93.79 ± 2.95 52.81 ± 4.77

Methanol 96.54 ± 2.62 96.30 ± 1.75 94.33 ± 4.54 93.61 ± 3.83 53.12 ± 2.59

Acetone 95.52 ± 2.13 93.28 ± 2.43 92.73 ± 2.23 92.29 ± 3.73 41.58 ± 2.41

Ethyl acetate 94.39 ± 1.28 93.78 ± 2.43 92.86 ± 3.02 92.07 ± 2.43 50.40 ± 1.74

Chloroform 94.78 ± 1.21 93.81 ± 2.46 93.06 ± 2.59 91.32 ± 2.14 41.34 ± 3.43

Hexane 96.57 ± 1.66 92.83 ± 3.77 92.10 ± 1.75 91.62 ± 3.75 51.13 ± 3.39

a Data represent mean ± SD cell viability as a percentage of untreated control samples (n = 3).

137

Table 4.14.2

Effect of R. sativus stem extracts on viability of lymphocytes


5 10 25 50 100

Water 98.51 ± 0.95 a 96.84 ± 2.40 96.36 ± 1.36 95.39 ± 1.29 64.71 ± 3.61

Methanol 95.87 ± 1.35 94.23 ± 2.37 93.35 ± 3.84 93.19 ± 4.20 52.97 ± 3.47

Acetone 97.86 ± 1.75 97.62 ± 1.61 96.89 ± 2.59 95.92 ± 2.11 53.79 ± 3.39

Ethyl acetate 94.90 ± 1.97 94.32 ± 1.36 93.71 ± 2.60 91.10 ± 1.31 50.82 ± 1.92

Chloroform 98.79 ± 0.70 96.03 ± 1.72 93.29 ± 2.28 92.35 ± 1.83 51.47 ± 2.44

Hexane 96.66 ± 1.30 96.53 ± 2.81 95.81 ± 1.81 95.87 ± 1.43 64.21 ± 2.82


138

Table 4.14.3

Effect of R. sativus leaves extracts on viability of lymphocytes


5 10 25 50 100

Water 95.45 ± 2.65 a 94.66 ± 4.50 92.98 ± 3.94 92.29 ± 2.78 61.82 ± 4.82

Methanol 96.32 ± 1.84 96.20 ± 4.16 95.82 ± 2.47 95.43 ± 5.34 53.82 ± 2.59

Acetone 97.18 ± 1.13 97.10 ± 3.19 96.84 ± 3.31 96.27 ± 1.95 55.48 ± 1.52

Ethyl acetate 93.07 ± 0.83 93.05 ± 1.66 92.66 ± 2.93 92.28 ± 2.33 51.54 ± 3.26

Chloroform 94.92 ± 2.45 94.64 ± 1.35 94.23 ± 3.21 91.61 ± 3.16 51.57 ± 3.46

Hexane 93.19 ± 2.38 93.06 ± 2.92 92.27 ± 2.17 91.98 ± 2.37 61.43 ± 4.43


Table 4.15

Effect of H2O2 on viability of lymphocytes

Concentration (µM)

0 10 25 50 100 200 500

% Cell

Viability

100 ± 0.00a 90.25 ± 4.21 84.68 ± 2.57 71.43 ± 2.19 52.90 ± 1.20 37.32 ± 1.44 15.88 ± 0.53

139

Table 4.16.1

Protective effect of R. sativus root extracts on H2O2 induced cytotoxicity in lymphocytes

Concentration (µg/ml) + 200 µM of H2O2 Extraction solvent

0 5 10 25 50

Water 35.68 ± 2.81a 36.23 ± 1.35 49.57 ±.3.20 58.70 ± 2.93 65.22 ± 3.22

Methanol 36.23 ± 0.92 37.38 ± 2.57 39.16 ± 0.79 49.67 ± 2.81 52.39 ± 3.31

Acetone 35.15 ± 1.20 48.34 ± 1.51 62.83 ± 2.62 64.13 ± 2.99 65.57 ± 4.12

Ethyl acetate 37.34 ± 2.05 49.61 ± 2.92 52.48 ± 3.73 58.15 ± 4.15 60.04 ± 4.55

Chloroform 34.62 ± 1.44 53.55 ± 2.18 57.62 ± 1.46 64.46 ± 4.32 65.32 ± 5.38

Hexane 36.91 ± 1.73 71.93 ± 5.27 78.62 ± 4.23 87.61 ± 5.31 88.69 ± 3.29


140

Table 4.16.2

Protective effect of R. sativus stem extracts on H2O2 induced cytotoxicity in lymphocytes


0 5 10 25 50

Water 36.16 ± 1.63a 36.72 ± 2.37 37.38 ± 3.74 41.36 ± 3.36 42.59 ± 4.77

Methanol 35.31 ± 2.21 55.39 ± 3.47 68.33 ± 3.44 79.19 ± 4.49 79.72 ± 5.31

Acetone 35.88 ± 1.38 36.34 ± 2.19 38.14 ± 1.13 41.17 ± 1.80 45.43 ± 2.33

Ethyl acetate 37.44 ± 1.40 39.42 ± 3.20 41.39 ± 2.63 45.56 ± 3.11 45.96 ± 2.17

Chloroform 36.69 ± 0.82 36.77 ± 1.53 38.35 ± 1.52 39.51 ± 1.03 41.73 ± 1.99

Hexane 35.79 ± 1.80 36.27 ± 1.21 36.91 ± 0.90 37.65 ± 1.37 38.89 ± 1.60


141

Table 4.16.3

Protective effect of R. sativus leaves extracts on H2O2 induced cytotoxicity in lymphocytes


0 5 10 25 50

Water 35.24 ± 2.47a 36.88 ± 2.11 39.92 ± 3.12 40.74 ± 1.38 43.36 ± 2.50

Methanol 38.66 ± 1.52 58.73 ± 3.34 70.77 ± 3.19 82.86 ± 2.72 83.19 ± 3.85

Acetone 36.42 ± 2.31 46.10 ± 2.53 48.61 ± 2.58 51.11 ± 3.14 52.43 ± 1.18

Ethyl acetate 35.96 ± 1.26 44.72 ± 3.46 48.29 ± 3.71 51.23 ± 2.89 52.94 ± 2.89

Chloroform 38.17 ± 2.20 39.84 ± 0.84 42.88 ± 2.69 43.88 ± 2.47 46.17 ± 1.36

Hexane 34.70 ± 1.81 35.75 ± 1.53 36.65 ± 1.06 38.92 ± 0.94 39.17 ± 0.93


142

concentration increased to 50µg/ml. Other extracts did show a certain degree of

cytoprotection against H2O2-induced cell death, but effect was not significant as

compared to control. Based on these data, hexane extract of root and methanolic extract

of stem and leaves had chosen to study genoprotective ability of R. sativus.

4.5.3. Effect of R. sativus on H2O2 induced DNA damage in lymphocytes

Lymphocytes were incubated with hexane extract of root and methanolic extract

of stem and leaves (5 – 50µg/ml) for 3 h to determine their effect on DNA integrity. In

this study, we expressed DNA damage as assessed by the comet assay, in terms of

percentage tail DNA and OTM. None of the extracts investigated showed DNA damage

at the tested concentrations, as compared to untreated control (Figure 4.19). Exposure of

lymphocytes to 200µM H2O2 for 10 min resulted in significant DNA damage, both in

terms of % DNA in tail and OTM (Table 4.17 and Figure 4.19). Pretreatment of

lymphocytes with R. sativus extracts (5 – 50 µg/ml) for 3 h before exposure to 200µM

H2O2 for 10 min was found to diminish extent of DNA damage in a dose-dependent

manner (Table 4.17 and Figure 4.19). A significant decrease in % DNA inthe tail was

noticed from 32.71% in H2O2-treated cells to 7.18% in R. sativus treated cells. Similarly,

OTM was reduced from 9.36 to 2.94 in R. sativus extract-treated cells. In particular,

hexane extract of root showed the most potent genoprotective effect as compared to

methanolic extract of stem and leaves.

Single-cell gel electrophoresis, commonly known as the comet assay, is a simple,

sensitive and rapid method for detection and quantification of DNA damage by

oxidants. Of late, the comet assay has been frequently used to evaluate effects of diet on

DNA damage (Johnson and Loo 2000). Lymphocytes are considered to be an

appropriate cell system to study genoprotective effect of dietary phytochemicals, as they

are more susceptible to damaging effects of oxidants. Similarly, H2O2 is known to be an

appropriate oxidant for inducing artificial DNA damage because of its ability to

generate hydroxyl radicals (OH•) close to DNA molecules by Fenton reaction.

143

Comet pics

144

Table 4.17

Genoprotective effect of R. sativus on H2O2 induced oxidative DNA damage in lymphocytes

Concentration (µg/ml) + 200 µM of H2O2

R. sativus

DNA damage Untreated

Control 0 5 10 25 50

% tail DNA 4.85 ± 1.12a 32.71 ± 5.78 21.01 ± 2.92 11.90 ± 3.36 7.31 ±1.64 7.18 ± 2.08 Hexane

extract (R) OTM 1.59 ± 0.23 9.36 ± 2.19 5.85 ± 0.69 4.07 ± 1.49 3.21 ±0.32 2.94 ± 0.72

% tail DNA 4.85 ± 1.12 32.71 ± 5.78 26.49 ± 2.53 16.38 ± 1.76 10.58±2.34 10.26± 1.77 Methanolic

extract (S) OTM 1.59 ± 0.23 9.36 ± 2.19 7.34 ± 1.49 5.89 ± 1.71 4.16 ±0.95 3.97 ± 0.46

% tail DNA 4.85 ± 1.12 32.71 ± 5.78 25.86 ± 3.22 15.83 ± 2.60 10.45±2.92 10.20±2.07 Methanolic

extract (L) OTM 1.59 ± 0.23 9.36 ± 2.19 7.27 ± 1.55 5.27 ± 0.90 3.84±1.05 3.55 ± 0.61

a Values are expressed as means ± S.D [50 comets were scored for each concentration for their size and shape by computerized image

analysis (TriTek CometScoreTM)]. R – root; S – stem; L – leaves.

145

None of the extracts used in this study showed cytotoxic effect on lymphocytes,

when used at concentrations up to 50µg/ml. Among different extracts, hexane extract of

root and methanolic extract of stem and leaves showed significant cytoprotective effect.

Similarly, R. sativus extracts as such did not induce DNA damage at the tested

concentrations. Hexane extract of root exhibited the most potent genoprotective effect

followed by methanolic extract of leaves and stem.

Polyphenolics have been shown to ameliorate cell injuries and protect DNA from

lesions induced by oxidants, due to their ability to scavenge free radicals, thereby

reducing oxidative stress and impact of oxidant attack on DNA in living system

(Urquiaga and Leighton 2000). Methanolic extract of stem and leaves were found to be

rich in polyphenolics (Table 4.4.2 and 4.4.3), suggesting that potent cytoprotective and

genoprotective ability of methanolic extract of stem and leaves could be attributed to

those polyphenolics. Radical scavenging ability of polyphenolics has been reported

previously in various in vitro model systems (Fukumoto and Mazza, 2000). Previous

study reported relationship between polyphenolics structure and antioxidant activity,

demonstrating that polyphenolics possessing hydroxyl groups on their phenyl rings

effectively contribute to radical scavenging activity by stabilizing radical form in

electron delocation (Rice-Evans 1995). Polyphenolics found in stem and leaves contain

hydroxyl groups in their structure, which could make it possible to scavenge H2O2

effectively, thus, inhibiting H2O2-induced cytotoxicity and genotoxicity in lymphocytes.

In comparison with methanolic extract of stem and leaves, hexane extract of root

showed an exceptionally strong cytoprotective and genoprotective effect. This could be

explained by the presence of high content of isothiocyanates. ITCs are considered as

strong inducers of detoxification enzymes, which represent an essential part of cellular

defense against reactive oxidants through elimination of highly reactive intermediates as

water soluble products. Thus, these compounds are found to be strong antioxidants, by

virtue of their ability to activate detoxification enzyme system, rather than through

direct radical scavenging capability (Zhang et al, 1992). This property of ITCs is

considered to be one of the major contributors to its anti-cancer activity.

Several recent studies emphasized the importance of whole food extracts as rich

sources of phytochemicals and proposed that combination of phytochemicals in fruits

and vegetables is critical to powerful antioxidant and anticancer activity, as isolated

146

pure compound either loses its bioactivity or may not behave same way as found in

whole foods. Hence, polyphenolics and ITCs may act in synergistic or additive manner

and exert their protective effect through efficient removal of reactive oxidants by

enhancing cellular antioxidant enzymes and reduce impact of oxidant mediated cellular

injury and DNA damage.

4.6. Chemopreventive efficacy of R. sativus

In spite of all advances in cancer treatment and knowledge of processes

responsible for this disease, our understanding of identity of food components that

prevent cancer is not complete. The best way to ascertain chemopreventive potential of

dietary substances is to understand an additive or synergistic interaction, as diets

contain several components that may act on the same or different steps of

carcinogenesis.

4.6.1. Effects of R. sativus on growth inhibition of HeLa cells

HeLa cells were used as a model system to examine chemopreventive effect of R.

sativus. Cells were treated with root, stem and leaves of R. sativus (100 µg/ml) for 48 h.

As shown in Table 4.18, root exhibited substantial growth inhibition and percent

inhibition was in the range 40 – 95%. However, stem and leaves were ineffective in

reducing viability of cells and percent inhibition was in the range 10 – 40%. DMSO alone

at the concentration applied did not have any adverse effect on cellular proliferation.

Further, when cells were treated with root of R. sativus (0 – 100µg/ml) for 48 h, it was

observed that hexane extract of root showed the most potent growth inhibitory activity

against HeLa cells, as evident from Table 4.19. Consequently, hexane extract of root was

used in all further experiments for understanding molecular mechanism leading to

growth arrest and cell death.

147

Table 4.18

Effect of root, stem and leaves extracts of R. sativus on viability of human cervical carcinoma cell line (HeLa).

Cell viability (%)


Root 58.57 ± 3.75 a 57.37 ± 5.81 40.95 ± 4.82 46.29 ± 4.92 44.38 ± 4.28 6.49 ± 0.32

Stem 82.67 ± 4.22 64.80 ± 2.37 83.25 ± 6.53 90.45 ± 5.40 86.85 ± 4.50 88.97 ± 5.15

Leaves 80.43 ± 2.96 57.84 ± 3.44 69.37 ± 3.81 78.04 ± 6.12 83.14 ± 4.72 88.11 ± 3.64

Cells were incubated with R. sativus extracts at a concentration of 100 µg/ml for 48 hours.

Experiments were performed in triplicate. The cell viability was determined by MTT assay.

a Data represent mean ± SD cell viability as a percentage of untreated control samples.

148

Table 4.19

Effect of R. sativus root extracts on viability of human cervical carcinoma cell line (HeLa).

Concentration (µg/ml) Extraction

Solvent 5 10 25 50 100

IC50

Water 87.50 ± 5.74a 82.76 ± 5.21 77.06 ± 4.51 64.19 ± 2.22 59.22 ± 4.21 122.61 ± 3.57

Methanol 85.71 ± 6.32 81.78 ± 4.28 72.84 ± 4.28 63.81 ± 3.75 57.20 ± 3.48 118.82 ± 2.46

Acetone 76.50 ± 7.21 71.38 ± 5.69 59.72 ± 6.30 48.24 ± 4.82 41.91 ± 2.15 47.03 ± 1.88

Ethyl acetate 83.50 ± 4.33 78.21 ± 5.28 63.45 ± 4.41 54.55 ± 2.54 45.83 ± 3.72 71.32 ± 0.85

Chloroform 80.36 ± 5.74 76.80 ± 2.69 62.17 ± 4.38 52.76 ± 3.75 44.70 ± 1.91 60.42 ± 2.05

Hexane 60.56 ± 5.18 42.05 ± 4.88 19.77 ± 1.91 12.32 ± 1.11 7.15 ± 0.54 7.49 ± 0.44

Cells were incubated with R. sativus extracts for 48 hours. Experiments were performed in triplicate.

The cell viability was determined by MTT assay.


149

4.6.2. Effect of hexane extract of R. sativus root on viability of HeLa, A549,

MCF-7 and PC-3 cell lines

A set of four cancer cell lines of epithelial origins was used to evaluate growth

inhibitory potential of R. sativus extract. These cells were selected, as they represent

major organ sites including, cervix (HeLa), lung (A549), breast (MCF-7) and prostate

gland (PC-3). The effect of hexane extract of root on proliferation of HeLa, A549, MCF-7

and PC-3 cells are shown in Table 4.20.1 – 4.20.4. Cells were treated with different

concentration (0 – 25 µg/ml) of hexane extract and inhibition of cellcell

proliferation was evaluated after incubation for 24, 48 and 72 h. R. sativus extract

displayed a dose and time-dependent growth inhibitory effect on all human cancer cells

examined, with varying effect on different cell lines. In line with these profiles, IC50

values clearly indicated chemopreventive efficacy of R. sativus extract.

All cancer cell lines exhibited similar sensitivity to R. sativus extract.

Interestingly, significant growth inhibitory activity was seen within 24 h for HeLa, A549

and MCF-7, suggesting rapid inhibition of cell growth by R. sativus. However, in case of

PC-3 cells, effect was gradual and reached maximum at 72 h. Our findings indicate that

ability of R. sativus extracts to inhibit growth of cancer cells was related to cell types.

Furthermore, R. sativus extract at the concentration used for anti-proliferative activity

had a significantly negligible effect on viability of normal human lymphocytes (Table

4.14.1), signifying its selective activity towards cancer cells.

Etoposide was used as a positive control to determine sensitivity of cancer cell

lines to conventional anti-cancer drug as well as to compare potency of R. sativus extract

as chemopreventive agent. As shown in Table 4.21, all cancer cells were susceptible to

etoposide treatment with IC50 of 10.71 µg/ml for HeLa, 12.43 µg/ml for A549, 11.64

µg/ml for MCF-7 and 21.80 µg/ml for PC-3 cells respectively. Comparison of anti-

proliferative activity of R. sativus extract with standard anti-cancer drug showed that R.

sativus extract had growth arresting activity significantly higher than etoposide, thus,

demonstrating its effectiveness as a cancer preventing agent.

150

Table 4.20.1

Effect of hexane extract of R. sativus root on viability of human cervical cancer cell line (HeLa).

Concentration (µg/ml)

Time 1 2 5 10 25

IC50

24 h 91.25 ± 6.22a 74.89 ± 5.69 69.97 ± 3.72 45.79 ± 2.97 24.19 ± 1.64 8.78 ± 0.43

48 h 88.41 ± 4.27 71.83 ± 3.45 60.17 ± 3.23 41.30 ± 2.76 20.39 ± 1.52 7.40 ± 0.58

72 h 87.05 ± 3.85 70.74 ± 5.27 59.31 ± 4.67 38.70 ± 2.41 19.49 ± 0.97 7.15 ± 0.42



Table 4.20.2

Effect of hexane extract of R. sativus root on viability of human lung cancer cell line (A549)


Time 1 2 5 10 25

IC50

24 h 90.54 ± 4.27 a 87.58 ± 2.55 72.46 ± 4.73 56.15 ± 1.48 45.03 ± 2.16 10.24 ± 0.65

48 h 86.36 ± 3.85 74.85 ± 3.08 63.86 ± 3.48 45.30 ± 1.62 40.33 ± 1.80 8.03 ± 0.47

72 h 84.85 ± 3.26 73.67 ± 3.19 62.03 ± 2.92 43.37 ± 2.04 40.18 ± 2.33 7.71 ± 0.39



151

Table 4.20.3

Effect of hexane extract of R. sativus root on viability of human breast cancer cell line (MCF-7)


Time 1 2 5 10 25

IC50

24 h 94.09 ± 3.25 a 92.88 ± 4.52 67.81 ± 3.15 33.30 ± 1.64 13.48 ± 0.74 8.36 ± 0.21

48 h 85.50 ± 3.88 77.73 ± 4.75 45.63 ± 2.24 24.71 ± 1.50 11.40 ± 0.87 7.64 ± 0.57

72 h 84.36 ±4.21 76.50 ± 4.31 45.18 ± 2.53 23.23 ± 1.24 10.14 ± 0.97 7.51 ± 0.15

Experiments were performed in triplicate. The cell viability was then determined by MTT assay.


Table 4.20.4

Effect of hexane extract of R. sativus root on viability of human prostate cancer cell line (PC-3)


Time 1 2 5 10 25

IC50

24 h 97.52 ± 4.35 a 94.17 ± 3.56 89.98 ± 5.21 70.21 ± 4.23 57.07 ± 2.18 20.87 ± 0.77

48 h 92.88 ± 4.21 89.79 ± 2.25 85.53 ± 4.66 61.26 ± 3.54 36.51 ± 2.45 14.92 ± 0.42

72 h 90.78 ± 3.31 88.70 ± 3.08 82.03 ± 4.70 58.36 ± 3.20 31.01 ± 1.81 12.96 ± 0.34



152

Table 4.21

Effect of etoposide on viability of cancer cell lines


Cell lines 1 2 5 10 25

IC50

HeLa 88.18 ± 2.36a 84.74 ± 2.14 74.39 ± 3.38 57.07 ± 2.21 39.64 ± 2.57 10.71 ± 0.34

A549 96.54 ± 1.96 94.12 ± 2.20 82.01 ± 3.42 54.33 ± 2.16 49.48 ± 1.76 12.43 ± 0.09

MCF7 88.27 ± 2.35 83.08 ± 4.57 78.22 ± 3.41 57.63 ± 2.07 44.65 ± 2.34 11.64 ± 0.23

PC3 95.06 ± 1.65 93.46 ± 1.40 90.35 ± 2.25 70.02 ± 3.68 57.18 ± 3.22 21.30 ± 0.53

Cells were incubated with etoposide for 24 hours. Experiments were performed in triplicate.

The cell viability was determined by MTT assay.


153

4.6.3. Compositional analysis of hexane extract of R. sativus root

Analysis of hexane extract of R. sativus by GC-MS revealed the presence of 42

compounds (Figure 4.20). Of which, 18 compounds were identified, as shown in Table

4.22, by comparing their retention indices (RI) and mass spectra (MS) with the Wiley

library spectra database and literature data (Adams 1995; Vaughn and Berhow, 2005;

Blazevic and Mastelie, 2009). The major ITCs found in hexane extract were 4-

(methylthio)butenyl isothiocyanate (Z isomer) (33.92%), 4-(methylthio) butyl

isothiocyanate (15.09%), 4-(methylthio) butenyl isothiocyanate (E isomer) (5.82), 4-

methylpentyl isothiocyanate (1.61), 4-pentenyl isothiocyanate (0.86%) and sulforaphene

(0.49%). Other components detected were alkanes and fatty acids and their esters. In

addition, eugenol, phenylpropanoid with numerous biological activities was detected in

considerable amount (3.45%) in hexane extract.

A reduction in cell growth and an induction of cell death are considered to be

primary means for inhibition of tumor growth. Our findings demonstrate for the first

time that lipophilic root extract (hexane extract) of R. sativus exerted significant growth

inhibitory activity on various human cancer cell lines, at concentrations as low as 25

µg/ml. However, stem and leaves exhibited negligible growth inhibitory activity, which

could probably be due to their different phytochemical profile as compared to root of

this vegetable.

The main classes of compounds found in hexane extract of root were MTBITC

and erucin, whose anti-proliferative activity was proven against leukemia and colon

cancer cell lines (Barillari et al, 2008; Papi et al, 2008; Fimognari et al, 2004). Findings from

this present study extend anti-proliferative effects of these compounds to cervical, lung,

breast and prostate cancer cells. MTBITC was reported to be main volatile component of

R. sativus root responsible for its pungency (Coogan et al, 2001; Nakamura et al, 2001).

However, previous studies have demonstrated the presence of other ITCs such as 4-

methylpentyl ITC, hexyl ITC, 5-hexenyl ITC, 4-(methylthio) butyl ITC (erucin) and 5-

(methylthio) pentyl ITC (Visentin et al, 1992) in root of R. sativus. Recently, Blazevic and

Mastelic (2009) reported that erucin was the most abundant ITC in root of R. sativus.

Contrary to these findings, we found MTBITC to be the most predominant ITC in our

study. The observed differences on type and relative percentage of ITC could be

attributed to genetic variability that occurs among different varieties of R. sativus and

154

GCMS pics

155

Table 4.22

Content and composition of compounds in hexane extract of R. sativus root, as analyzed

by GC-MS.

Sl.No Compounds RTa Homology (%) RCb

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

4-pentenyl isothiocyanate

4-methyl pentyl isothiocyanate

4-methylthio pentenenitrile

Undecane

Dodecane

Eugenol

3-butenyl isothiocyanate

Tridecane

4-(methylthio)-3-butenyl isothiocyanate

4-(methylthio) butyl isothiocyanate

4-(methylthio)-3-butenyl isothiocyanate

Sulforaphene

Hexadecanoic acid

Octadecanoic acid

7-methyl linolenate

5-(methylthio) pentyl isothiocyanate

Pentacosane

Tetracosane

6.91

7.38

9.02

10.58

10.78

10.94

11.63

12.01

13.69

13.92

14.03

15.41

19.81

21.51

26.06

27.04

28.20

29.50

90

92

90

96

96

99

95

96

99

99

99

92

99

99

95

92

96

96

0.86

1.61

3.39

4.79

1.47

3.45

1.24

4.77

5.82

15.09

33.92

0.49

10.95

11.97

0.92

1.26

2.43

2.18

a Retention time (min)

b Relative area percentage (peak area relative to the total peak area percentage)

156

also due to contribution of different environmental factors. The marked

chemopreventive efficacy of R. sativus root could be attributable to synergism among

major and minor ITCs present in it. Stem and leaves of R. sativus contained significant

amount of polyphenolics, but low level of ITCs was detected as compared to root. This

could be the probable reason for low growth inhibitory activity of stem and leaves

extract.

Importantly, hexane extract was found to be capable of affecting cancer cells

selectively. Selective targeting and negligible toxicity to normal cells is basic

prerequisites for probable chemopreventive agents. The difference in R. sativus effect

towards cancer and normal cells could be due to a fact that it could be targeting a

particular molecular event exclusively present in cancer cells, but absent in normal cells.

4.6.4. Morphological changes following treatment with hexane extract of R.

sativus

The phenotypic characteristics of cancer cells treated with R. sativus extract were

evaluated by an inverted phase contrast microscope. Significant morphological changes

indicative of cell death and growth inhibition were observed in all cancer cell lines

treated with R. sativus extract for 24 h, as compared to untreated cells. Representative

control and treated cells are shown as Figures 4.21(HeLa), 4.22 (A549), 4.23 (MCF-7) and

4.24 (PC-3). Hexane extract of R. sativus severely affected spreading and elongation of

cells leading to a rounded morphology and eventual detachment from culture plates.

Detachment of cells is a common feature of apoptosis in a tissue culture that is thought

to be similar to separation of apoptotic cells that occurs in cancer tissues. To rule out

possibility of cell death due to necrosis, cells were examined with trypan blue and

greater than 90% of detached cells were found to exclude the dye.

Staining of cells with propidium iodide showed fragmentation and condensation

of chromatin and other morphological features characteristic of apoptotic cells in HeLa

cells treated with hexane extract for 24 h, as compared to untreated cells (Figure 4.25).

Untreated control cells exhibited a normal nuclear morphology characterized by

diffused chromatin structure. Similar results were obtained with other cancer cells such

as A549 (Figure 4.26), MCF-7 (Figure 4.27) and PC-3 (Figure 4.28). The observation of

157

Hela light

158

A549 light

159

Mcf light

160

Pc3 light

161

Hela flour

162

A549 flour

163

Mcf flour

164

Pc3 flour

165

apoptotic fragmentation of nuclei indicates that hexane extract of R. sativus induced

apoptosis in different cancer cells.

4.6.5. Quantification of DNA fragmentation by diphenylamine assay

DNA fragmentation is an index of oxidative DNA damage. Diphenylamine

reaction is a useful tool for quantification of fragmented DNA in cancer cells after

treatment with chemopreventive agents, which in turn enables to assess differential

cellular response to cancer therapy. Initial studies have employed this assay for

detection of apoptotic fragmentation of DNA in ovarian cancer cells induced by

conventional anti-cancer drugs, such as cisplatin and taxol (Bartlett, 2000). Our studies

also indicate that active components present in hexane extract induced DNA

fragmentation in a concentration-dependent manner (Table 4.23).

Apoptosis is a well-defined biological process responsible for the maintenance of

homeostasis of cell growth and proliferation. In cancer cells, this homeostasis is

disturbed, which leads to an uncontrolled proliferation and reduced apoptosis. Cancer

cells have an acquired ability to elude apoptosis through a variety of ways. Hence,

induction of apoptosis provides an important valuable strategy for the management of

cancer (Sun et al, 2004). Further, this could be applied as a useful marker for screening

active compounds for consequent development as potential chemopreventive agents.

In the present study, cancer cells treated with hexane extract showed

morphological features indicative of apoptosis such as rounded morphology,

detachment from substratum, cell shrinkage and DNA fragmentation. The overall data

demonstrate that hexane extract of R. sativus significantly inhibited cell growth and

proliferation through induction of apoptosis as the main death pathway in cancer cells.

Further, induction of apoptosis correlated inversely with decreased cell viability,

confirming that apoptosis was mostly accountable for cell death and growth inhibition.

An interesting finding in this present study is that hexane extract of R. sativus initiated

apoptotic cell death at a concentration range, which was well below the range reported

for other plant extracts (Li et al, 2009; Kilani, 2008; Srivastava and Gupta, 2007).

166

Table 4.23

Effect of hexane extract on DNA fragmentation as assessed by diphenylamine assay


Cell lines 0 1 2 5 10 25

HeLa 26.05 ± 3.24a 32.12 ± 2.40 38.36 ± 1.81 44.18 ± 2.88 57.27 ± 3.30 58.05 ± 2.15

A549 37.17 ± 1.58 44.61 ± 1.90 47.59 ± 1.47 49.65 ± 1.87 55.21 ± 2.74 55.98 ± 2.63

MCF7 40.18 ± 2.10 47.05 ± 2.33 49.90 ± 2.59 54.68 ± 2.14 62.92 ± 2.32 64.35 ± 1.84

PC3 37.19 ± 1.83 42.25 ± 2.15 42.69 ± 2.35 47.77 ± 2.61 49.01 ± 3.20 49.43 ± 1.57

a Data represent mean ± SD (expressed as % fragmented DNA)

167

4.6.6. Expression of genes related to apoptotic pathway

Effect of hexane extract of R. sativus on mRNA expression of genes related to

apoptotic pathway was analyzed by RT-PCR. Variable change in the expression of

apoptotic genes was noted in cancer cells treated with hexane extract, as shown in

Figure 4.29 (HeLa), 4.30 (A549), 4.31 (MCF-7) and 4.32 (PC-3).

The expression of Bax and caspases-3 was found to be augmented in all treated

cells, in comparison to untreated controls. Treatment with hexane extract increased the

expression of p53 in HeLa and A549 cells, but produced no significant change in MCF-7

cells. The expression of Bcl-2 and Bcl-XL was down-regulated in HeLa, MCF-7 and PC-3

cells. However, in case of A549 cells, effect of R. sativus on Bcl-2 and Bcl-XL gene

appeared to be less pronounced and relative gene expression level of R. sativus treated

cells was not significantly different from untreated control cells. In the control RT-PCR

assay, β-actin expression level remained unaltered following treatment with hexane

extract in all cell lines.

Our findings for the first time, demonstrate that R. sativus root extract induced

apoptosis both in p53-proficient human cancer cell lines such as HeLa, A549 and MCF-7

cells and p53-deficient cell line such as PC-3 cells, suggesting that a p53-independent

pathway may be operative in such a system and could act as a chemopreventive agent

regardless of p53 status of cancer cells. This induction of apoptosis occurred rapidly

within 12 h, implying that R. sativus extract could have induced apoptosis by activating

pre-existing apoptosis machinery. Earlier report indicated that p53 was not necessary for

the induction of apoptosis as p53-negative cells were equally sensitive to apoptotic

pathways (Hipp and Bauer, 1997). Shao et al (1995) have reported that a novel retinoid-

induced apoptotic pathway in human breast cancer cells via regulation of p21, Bcl-2 and

Bax in a p53-independent manner. It, thus, appears that in certain cells, apoptosis can be

induced in a p53 independent pathway either by inducing downstream genes of p53

such as p21, Bax, etc., or by an unknown mechanism.

Previous studies also reported that mutant p53 has a dominant negative effect on

a wild type p53 and presence of mutated p53 could render cancer cells resistant to

conventional chemotherapeutic agents or ionizing radiation (Scott et al, 1993). Moreover,

most of the chemotherapeutic drugs induce apoptosis via modulation of p53 expression,

168

Hela pcr

169

A549 pcr

170

Mcf pcr

171

Pc3 pcr

172

which could be one of the reasons for non-responsive nature of cancer cells to anti-

cancer drugs. Induction of apoptosis by R. sativus in both p53-proficient and p53-

deficient cells indicated that it has potential to be exploited as a novel chemopreventive

agent for cancer cells, which are resistant to conventional chemotherapeutic agents.

Bcl-2 family of homologous proteins signifies a crucial check point in most

apoptotic signaling pathways. They function either as pro-apoptotic (Bax, Bak, Bad) or

anti-apoptotic (Bcl2, Bcl-XL) regulators. In this study, we found that R. sativus extract

induced up-regulation of Bax mRNA expression in all cell lines studied. Bax plays a

significant role in promoting the activation of apoptotic signaling pathways. Bax act as

an apoptotic inducer by interacting with itself or with Bcl-2 or Bcl-XL, in a homo- and/or

hetero-dimeric state, in which relative amounts of each protein predetermine life or

death response of a cell to an apoptotic stimulus (Oltvai and Korsmeyer, 1994; Sedlak et

al, 1995). Previous report indicated that Bax gene is a direct transcriptional target of p53

(Miyashita and Reed, 1995). We found that Bax expression was up-regulated irrespective

of p53 status of the cell. In HeLa and A549 cells, upregulation of Bax expression was

associated with increased p53 expression, suggesting a p53-dependent apoptotic

pathway. However, in MCF-7 and PC-3 cells, R. sativus-induced cell death was not

accompanied by a change in p53 expression, but rather associated with an increased

expression of Bax.

Expression levels of anti-apoptotic genes such as Bcl-2 and Bcl-XL were found to

be variable. Bcl-2 and Bcl-XL was found to be down-regulated in HeLa, MCF-7 and PC-3

cells, which could probably be associated with increased apoptotic activity. However, in

A549 cells, there was no significant change in the expression level of Bcl-2 and Bcl-XL as

compared to untreated cells and apoptotic pathway seemed to be independent of Bcl-2

expression. Excess of Bax might counter death repressor activity of Bcl-2/Bcl-XL through

Bax: Bcl-2/Bcl-XL hetero-dimerization (Basu and Haldar, 1998). However, several

studies demonstrated that Bcl-2 family of proteins might function independently

without the formation of hetero-dimers (Cheng et al, 1996). A high level of apoptosis

even in the presence of Bcl-2 and Bcl-XL may imply the possibility of a treatment-

associated phosphorylation, which was reported to cause significant loss of their anti-

apoptotic function (Pratesi et al, 2000; Poruchynsky et al, 1998). The regulation of life and

death of a cell could probably be a multifaceted process and may be cell-type specific.

173

Our results suggest that sensitivity of cancer cells to R. sativus could be related to

interactions among Bcl-2 family proteins intrinsically modulated by R. sativus.

Caspase cascade is considered as a vital pathway in apoptotic signal

transduction. Caspases comprise of initiator caspases (caspase – 8 and 9), which are

involved in regulatory processes and effector caspases (caspase – 3 and 6), which are

involved in morphological changes associated with cell death. Activation and cleavage

of caspase-3 serve as a convergence point for apoptotic pathways (Porter and Janicke,

1999). Results from this study suggest that mechanism of R. sativus induced apoptosis in

cancer cells could entail caspase-3 activation and resultant cascade of reactions, since all

cancer cells overexpress caspases-3. It has been suggested that caspase-3 cleaves caspase-

activated DNase inhibitor and releases caspase-activated DNase (CAD) from the

complex. Once CAD is released, it enters into the nucleus and degrades chromatin into

smaller nucleosomal fragments, which consecutively promotes apoptosis (Degterev et al,

2003). Findings from this study, indicate that R. sativus activated caspase-3 and initiated

release of apoptotic factors, which in turn led to apoptosis.

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