61
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.
Parts Water Methanol Acetone Ethyl acetate Chloroform Hexane
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
a Each value represents mean value ± standard deviation of three replicates.
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.
Parts Water Methanol Acetone Ethyl acetate Chloroform Hexane
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
a Each value represents mean value ± standard deviation of three replicates.
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.
(a) Water; (b) Methanol.
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
HPLC-DAD polyphenolics profile of stem of R. sativus. Detection at 280 nm.
(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
HPLC-DAD polyphenolics profile of stem of R. sativus. Detection at 280 nm.
(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
a Values are means ± SD (n = 3).
b Not detected.
76
Figure 4.4
HPLC-DAD polyphenolics profile of leaves of R. sativus. Detection at 280 nm.
(a) Water; (b) Methanol.
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
HPLC-DAD polyphenolics profile of leaves of R. sativus. Detection at 280 nm.
(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
HPLC-DAD polyphenolics profile of leaves of R. sativus. Detection at 280 nm.
(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
a Values are means ± SD (n = 3).
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
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
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
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
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.
Parts Water Methanol Acetone Ethyl acetate Chloroform Hexane
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
a Each value represents mean value ± standard deviation of three replicates.
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
Concentration (mg/ml)
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
Concentration (mg/ml)
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
Concentration (mg/ml)
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
Concentration (mg/ml)
Percent Inhibition
EDTA
Quercetin
BHT
0
20
40
60
80
100
0 0.05 0.1 0.25 0.5 1
Concentration (mg/ml)
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/ml)
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/ml)
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
Parts Water Methanol Acetone Ethyl acetate Chloroform Hexane
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
a Each value represents mean value ± standard deviation of three replicates.
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
Concentration (mg/ml)
Percent Inhibition
Quercetin
BHT
0
20
40
60
80
100
0 0.05 0.1 0.25 0.5 1
Concentration (mg/ml)
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/ml)
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/ml)
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.
Parts Water Methanol Acetone Ethyl acetate Chloroform Hexane
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
Concentration (mg/ml)
Percent Inhibition
Quercetin
BHT
0
20
40
60
80
100
0 0.05 0.1 0.25 0.5 1
Concentration (mg/ml)
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/ml)
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/ml)
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.
(a) Standard; (b) Root; (c) Stem; (d) Leaves. Values are means ± SD (n = 3).
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.
Parts Water Methanol Acetone Ethyl acetate Chloroform Hexane
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
a All data were average (± SD) of three replicates.
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
Concentration (mg/ml)
Percent Inhibition
Quercetin
BHT
0
20
40
60
80
100
0 0.05 0.1 0.25 0.5 1
Concentration (mg/ml)
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/ml)
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/ml)
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.
(a) Standard; (b) Root; (c) Stem; (d) Leaves. Values are means ± SD (n = 3).
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.
Parts Water Methanol Acetone Ethyl acetate Chloroform Hexane
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
a All data were average (± SD) of three replicates.
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
Concentration (mg/ml)
Percent Inhibition
Quercetin
BHT
0
20
40
60
80
100
0 0.05 0.1 0.25 0.5 1
Concentration (mg/ml)
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
Concentration (mg/ml)
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.
(a) Standard; (b) Root; (c) Stem; (d) Leaves. Values are means ± SD (n = 3).
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.
Parts Water Methanol Acetone Ethyl acetate Chloroform Hexane
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
a All data were average (± SD) of three replicates.
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.
Inhibition Zone (mm) Pathogenic
Organisms Water Methanol Acetone Ethyl acetate Chloroform Hexane
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.
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.
115
Table 4.9.3
Antibacterial activity of R. sativus leaves 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 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.
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.
116
Table 4.9.4
Antibacterial activity of standard antibiotics against pathogenic bacteria by agar well diffusion method.
Inhibition Zone (mm) Pathogenic
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
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.
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
Organisms Water Methanol Acetone Ethyl acetate Chloroform Hexane
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.
MIC (MBC) mg/ml Pathogenic
Organisms Water Methanol Acetone Ethyl acetate Chloroform Hexane
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)
Results are shown as means of three measurements done on separate occasions.
a Not tested.
121
Table 4.10.3
MIC and MBC of R. sativus leaves extracts against health damaging bacteria.
MIC (MBC) mg/ml Pathogenic
Organisms Water Methanol Acetone Ethyl acetate Chloroform Hexane
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
Results are shown as means of three measurements done on separate occasions.
a Not tested.
122
Table 4.10.4
MIC and MBC of standard antibiotics against health damaging bacteria.
MIC (MBC) mg/ml Pathogenic
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)
Results are shown as means of three measurements done on separate occasions.
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
a Inhibitory zone in mm, including diameter of the well (8.0 mm)
126
Table 4.11.2
Effect of pH of ethyl acetate extract of R. sativus stem on inhibitory zone (mm) against pathogens.
pH Pathogenic Organisms
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
Each value is mean ± standard deviation from three replicates
The concentration of extracts used was 1.0 mg/ml
a Inhibitory zone in mm, including diameter of the well (8.0 mm)
127
Table 4.11.3
Effect of pH of ethyl acetate extract of R. sativus leaves on inhibitory zone (mm) against pathogens.
pH Pathogenic Organisms
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
Each value is mean ± standard deviation from three replicates
The concentration of extracts used was 1.0 mg/ml
a Inhibitory zone in mm, including diameter of the well (8.0 mm)
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.
a Inhibitory zone in mm, including diameter of the well (8.0 mm)
b Not detected.
129
Table 4.12.2
Effect of heat treatment on stability of ethyl acetate extract of R. sativus stem.
Temperature (°C) Pathogenic
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
a Inhibitory zone in mm, including diameter of the well (8.0 mm)
b Not detected.
130
Table 4.12.3
Effect of heat treatment on stability of ethyl acetate extract of R. sativus leaves.
Temperature (°C) Pathogenic
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
a Inhibitory zone in mm, including diameter of the well (8.0 mm)
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
Concentration (µg/ml) Extraction solvent
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
a Data represent mean ± SD cell viability as a percentage of untreated control samples (n = 3).
138
Table 4.14.3
Effect of R. sativus leaves extracts on viability of lymphocytes
Concentration (µg/ml) Extraction solvent
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
a Data represent mean ± SD cell viability as a percentage of untreated control samples (n = 3).
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
a Data represent mean ± SD cell viability as a percentage of untreated control samples (n = 3).
140
Table 4.16.2
Protective effect of R. sativus stem extracts on H2O2 induced cytotoxicity in lymphocytes
Concentration (µg/ml) + 200 µM of H2O2 Extraction solvent
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
a Data represent mean ± SD cell viability as a percentage of untreated control samples (n = 3).
141
Table 4.16.3
Protective effect of R. sativus leaves extracts on H2O2 induced cytotoxicity in lymphocytes
Concentration (µg/ml) + 200 µM of H2O2 Extraction solvent
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
a Data represent mean ± SD cell viability as a percentage of untreated control samples (n = 3).
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 (%)
Parts Water Methanol Acetone Ethyl acetate Chloroform Hexane
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.
a Data represent mean ± SD cell viability as a percentage of untreated control samples.
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
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.
Table 4.20.2
Effect of hexane extract of R. sativus root on viability of human lung cancer cell line (A549)
Concentration (µg/ml)
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
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.
151
Table 4.20.3
Effect of hexane extract of R. sativus root on viability of human breast cancer cell line (MCF-7)
Concentration (µg/ml)
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.
a Data represent mean ± SD cell viability as a percentage of untreated control samples.
Table 4.20.4
Effect of hexane extract of R. sativus root on viability of human prostate cancer cell line (PC-3)
Concentration (µg/ml)
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
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.
152
Table 4.21
Effect of etoposide on viability of cancer cell lines
Concentration (µg/ml)
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.
a Data represent mean ± SD cell viability as a percentage of untreated control samples.
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
Concentration (µg/ml)
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.