Journal of the Saudi Society of Agricultural Sciences (2012) 11, 135142

King Saud University

Journal of the Saudi Society of Agricultural Sciences

www.ksu.edu.sa
www.sciencedirect.com

FULL LENGTH ARTICLE

Enhancement of phenolics, avonoids and glucosinolates

of Broccoli (Brassica olaracea, var. Italica) as antioxidants
in response to organic and bio-organic fertilizers
Abd El-Moniem M. Naguib a, Farouk K. El-Baz b, Zeinab A. Salama
H. Abd El Baky Hanaa b, Hanaa F. Ali a, Alaa A. Gaafar b
a
b

b,*

Department of Biochemistry, Faculty of Agriculture, Cairo University, Egypt

Department of Plant Biochemistry, National Research Centre, Cairo, Egypt

Received 14 January 2012; accepted 6 March 2012

Available online 16 March 2012

KEYWORDS
Broccoli;
Antioxidant activity;
Organic fertilizers;
Bio-organic fertilizers;
Phenolics;
Flavonoids and
glucosinolates

Abstract A eld experiment was carried out to study the effect of organic and bioorganic fertilizers
on growth parameters, yield and the quality of two broccoli cultivars (Calabrese and Southern star).
Bio-organic fertilizers gave better results for all vegetative growth parameters for Southern star cultivar compared to Calabrese cultivar. Total phenolic content (TPC), total avonoid content (TFC)
and total glucosinolates content (TGsC) were almost higher in Calabrese cultivar as a result of
organic fertilizer treatment. The antioxidant activities of both cultivars were evaluated and Calabrese cultivar showed the higher1, 1-diphenyl-2-picrylhydrazyl DPPH. scavenging activity expressed
as IC50 (its concentrations for 50% inhibition) (16.56 lg/ml) compared to Southern star (19.42 lg/
ml). In addition, Calabrese cultivar showed the higher chelating power (75.36 lg/ml) than Southern
star (72.43 lg/ml) at (30 lg/ml) when the organic fertilizer was applied. The results indicated that
there is a good margin for enhancing antioxidant compounds of broccoli for economic production
using organic fertilization. This study indicated the potential application of broccoli as a potent natural source of antioxidants as nutraceuticals.
2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

* Corresponding author.
E-mail address: zeinabsalama70@hotmail.com (Z.A. Salama).
1658-077X 2012 King Saud University. Production and hosting by
Elsevier B.V. All rights reserved.
Peer review under responsibility of King Saud University.
http://dx.doi.org/10.1016/j.jssas.2012.03.001

Production and hosting by Elsevier

1. Introduction
Broccoli (Brassica olaracea, var. Italica) is mainly consumed
for its orets, has already been characterized in terms of glucosinolates (Rosa, 1997; Rosa and Heaney, 1996) and phenolics
(Ferreres et al., 2005; Sousa et al., 2005). Much attention has
been focused on broccoli as a source of bioactive compounds
such as phenolics, avonoids and glucosinolates, which possess
antioxidant and anticancer effects (Gundgaard et al., 2003;
Williamson et al., 1998; Zhang et al., 1994; Yoldas et al., 2008).

136
It is a signicant source of phenolics such as sinapic acid
(Vallejo et al., 2003a). Apart from other nutritional antioxidants, broccoli is also a signicant source of calcium, folic acid,
carotenoids, ascorbic acid and known to reduce risk of breast
and prostate cancer (Beecher, 1994; Williamson et al., 1998).
Nutrient and antioxidants may act together to reduce
reactive oxygen species level more effectively than single dietary antioxidant, because they can function as synergists
(Trombino et al., 2004).
The quality and quantity of phenolics in plant vary signicantly due to different factors, such as (1) genetic determinants
(Vallejo et al., 2003a,b); (2) environmental condition (temperature, light, water availability, nutrient availability) (Zhao
et al., 1994; Rosa et al., 1996); (3) agricultural practices (date
of harvest, etc.); and (4) post-harvest conditions (Rodriguez
and Rosa, 1999).
Fertilization is the most important and controllable factor
affecting the nutritional value of vegetables. The type and value of fertilizer and the level of application directly inuence
the level of nutrients available in plants and indirectly inuence
plant physiology and the biosynthesis of secondary compounds in plants. Secondary compounds in plants are known
as secondary metabolites or phytonutrients. General categories
of these compounds in broccoli are phenolics including avonoids, terpenes and nitrogen compounds including alkaloids
and sulfur compounds. These compounds are used in the treatment of human diseases. They can quench free radicals, act as
antiproliferative agents, promote detoxifying enzymes, induce
differentiation of cancer cells, stimulate the immune system
and inhibit tumor blood vessel formation (Heaton, 2001).
Organic food production is characterized by the absence isoavones, has been demonstrated to inhibit synthetic compound
(herbicides, pesticides) and readily soluble fertilizers and relies
more on natural mechanisms of controlling the growth, yield
and diseases. Application of compost to sandy soil increased
organic matter, cation exchange capacity, available nutrients
and biological properties (Lombardi-Boccia et al., 2004).
Manach et al. (2004) reported that nowadays, emphasis on
multi-strain biofertilizer has already been tidied. Biofertilizers
are biological preparations embodying, essentially, sufcient
densities of potent strains of microorganisms, having a tangible
benecial role in tting a proper rhizosphere for plant growth
(Saber, 2001) Some researchers suggest that, of all the measures
of nutrient quality, concentrations of phytochemicals have the
potential to differ the most between organic and conventional
foods (Woese et al., 1997). It has been claimed that due to their
higher amounts of secondary phenolic metabolites, organically
produced plant foods could be expected to be more healthpromoting than conventional foods Lundega and Rtensson,
2003). Organically grown cabbage, spinach, Welsh onion, and
green pepper generally had higher levels of avonoids and antioxidant activity (Young et al., 2000).
Organic manure can serve as alternative practice to mineral
fertilizers (Gupta et al., 1988; Wong et al., 1999; Naeem
et al., 2006) for improving soil structure (Bin, 1983; Dauda
et al., 2008) and microbial biomass (Suresh et al., 2004). Therefore, utilization of locally produced manures for vegetable production may increase crop yields with less use of chemical
fertilizers. In recent times, consumers are demanding higher
quality and safer food and highly interested in organic products. Organic fertilization has a stimulatory effect on accumulation of phenolics in broccoli orets. It is well known that, the

A.E.-M.M. Naguib et al.

higher concentrations of phenolics in orets can be explained
by the role of organic fertilizers in the biosynthesis which induces the acetate shikimate pathway, resulting in higher production of avonoids and phenolics (Sousa et al., 2008).
Also, because of the higher photo-pathogenic stress in organic
farming, which in turn may have abiotic stress, and causes an
increase of phenolics and avonoids and their antioxidant
activity grown organically (Young et al., 2000).
There is little information on the inuence of organic and
bio-organic fertilization on the potential activity of phenolics,
avonoides and glucosinolates as natural antioxidant from
broccoli. Therefore, the aim of this study was to investigate
the inuence of organic and bio-organic fertilizers on broccoli
yield and antioxidant content in two cultivars of broccoli.
2. Materials and methods
2.1. Plant materials
Two cultivars of broccoli (Brassica olaracea, var. Italica) were
used in the study. Calabrese was obtained from GSN semences
Co., France. While, Southern star was obtained from Takii
Co., Japan, family Brassicacea (Cruciferous).
2.2. 2- Field experiment
A eld experiment was carried out at El-Saff, Hellwan Governorate, Egypt, during winter season of 20072008, in newly reclaimed soil, to study the effect of organic and bio-organic
fertilizers compared with (NPK) as control treatment.
2.3. Agriculture conditions
Seeds of broccoli were sown in the foam trays lled with a mixture of peat moss and vermiculite (1:1 volume). Seedlings
45 days old were transplanted in the open eld on the center
of raw and spacing of 50 cm between plants. Ditches of
20 cm depth and 40 cm width were prepared in the sites of drip
irrigation lines; chicken manure, rocks phosphate, calcium
super phosphate and agricultural sulfur were mixed and were
added and covered with soil before transplanting. Plot area
was 3.5 lengths and 3.0 widths in three raw which equals
10.5 m2.
Note: one Feddan = 4200 m2.
2.4. Fertilizer treatments
2.4.1. Mineral NPK fertilizer (control)
Basic fertilizers were mixed with the soil in doses of ammonium sulfate (21.5% N) at a rate of 100 N2 kg/fed; calcium
super phosphate (15.5% P2O5), and rock phosphate (30% P)
at a rate of 60 P2O5 kg/fed and potassium sulfate (48%
K2O) at a rate of 40 K2O kg/fed. In addition, agricultural sulfur was used at a rate of (200 kg/fed).
2.4.2. Organic fertilizer
Complementary between 50% organic (chicken manure) at a
rate of 100 N2 unit/fed (5.850 m3/fed) and 50% mineral
(NPK) was used. NPK is formed from ammonium sulfate
(21.5% N) was used as a source of nitrogen, at a rate of 100
N2 kg/fed; calcium super phosphate (15.5% P2O5) and rock

Enhancement of phenolics, avonoids and glucosinolates of Broccoli (Brassica olaracea, var. Italica)

137

30
24.799
25

20.892

IC50 g/ml

19.423
20

16.561

19.699

17.694

15
10.066
10
5
0
C alabre se
NPK

C alabre se C alabre se South. Star South. Star South. Star

O rganic
Bioorganic
NPK
O rganic Bioorganic

BHT
Standard

Treatments

Figure 1 IC50 of DPPH. free radical of methanolic 80% extract of two fresh broccoli cultivars in presence of different types of fertilizers.
IC50: its concentrations for 50% inhibition All values are means of three replicates and are signicantly different at p P 0.05 standard
deviation.

phosphate (30% P) were used at a rate of 60 P2O5 kg/fed;

Potassium sulfate (48% K2O) was used at a rate of 40 K2O
kg/fed. In addition, agricultural sulfur was used at a rate of
(200 kg/fed) based on the recommended fertilizers requirements according to Ministry of Agriculture.
Ammonium sulfate and potassium sulfate were splinted
into three equal doses and applied as dressing (0, 30 and
60 day after transplanting) beside plants.
2.4.3. Bio-organic fertilizer
It was composed of organic treatment in addition to biofertilizers (50% Azotobacter chrococcum and 50% Bacillus
megaterium). Bio-organic fertilizer was applied once after 2,
5 and 8 weeks of transplanting at the level (10 ml/plant) at concentration 109 cell/ml. The mixture was injected near the root
plants.
The bio-organic fertilizer was kindly supported by the Agriculture Microbiology Department, National Research Center.
It was containing a mixture of N2-xing bacteria Azotobacter
chrococcum and phosphate dissolving bacteria Bacillus megaterium (1:1 v/v).
In all treatments, Drip irrigation line was spread over the
ditches and soil was irrigated continuously 3 days before
transplanting.

4. Determination of total phenolic content (TPC)

The total phenolic content (TPC) of fresh broccoli extracts was
spectrophotometrically determined by Folin Ciocalteu reagent
assay using gallic acid as a standard compound for the preparation of calibration curve (20120 mg/l) according to Singleton and Rossi (1965). Total phenolic content of samples was
measured at 670 nm and expressed as mg gallic acid equivalents (GAE)/g dry weight. All samples were analyzed in
triplicates.
5. Determination of total avonoid content (TFC)
Total avonoid content (TFC) of fresh broccoli extracts was
spectrophotometrically determined by the aluminum chloride
method using quercetin as standard (Zhishen et al., 1999).
After incubation at room temperature, samples were measured
at 512 nm and expressed as mg quercetin equivalents (QE)/g
fresh weight. Samples were analyzed in triplicates.
6. Determination of total glucosinolate content (TGsC)
Total glocosinolates was spectrophotometrically assayed
according to the method of Declercq and Daun (1989).

3. Preparation of plant samples for chemical analysis

7. Measurements of antioxidant capacity
The heads, harvested at fresh harvest maturity stage (uniformly green, compact) were put in polyethylene bags and
transported immediately to the laboratory, washed, chopped,
and the moisture content in the two cultivars was determined
using an oven at 105 C, for 24 h. Moisture was calculated
while expressing the data on fresh weight basis.
3.1. Preparation of fresh plant methanolic extract
The edible parts (broccoli orets) were extracted by liquid
nitrogen. 10 g of frozen tissues was soaked in 100 ml of 80%
methanol for (48 h).

7.1. DPPH. free radical scavenging assay

The method described by Blois (2002) was used to assess the
(1, 1-diphenyl-2-picrylhydrazyl) DPPH. radical scavenging
activity of fresh broccoli methanolic extract. 0.1 mM of DPPH.
in methyl alcohol was prepared and 1 ml of this solution was
added to 3 ml of broccoli methanolic extracts at different concentrations (5, 10, 20, 30 lg/ml). The mixture was shaken vigorously and allowed to stand at room temperature for 30 min.
The butylated hydroxy toluene (BHT) was used as positive
control; and negative control contained the entire reaction

138
Table 1

A.E.-M.M. Naguib et al.

Effect of organic and bioorganic fertilizers on yield of fresh orets and dry weight of two cultivars of broccoli.

Cultivars

Treatments

Calabrese

100% NPK (Control)

14.31a 0.37
50% NPK + 50% Organic
18.82bc 1.29
50% NPK + 50% Organic + Bio 19.77c 0.60

Southern star 100% NPK (Control)

50% NPK + 50% Organic
50% NPK + 50% Organic + Bio
L.S.D at 0.05

Florets dry weight (%) Yield of orets FW (ton/fed) Yield increases of orets FW (%)

14.24a 0.55
17.52b 1.22
17.32b 0.25
1.42

4.113a 0.16
6.53b 0.11
8.20d 0. 12

100
159
199

6.07b 0.20
7.40c 0.26
9.96e 0.02
0.36

100
122
164

All values are means of three replicates and are signicantly different at p P 0.05 standard deviation.
All values with the same letters are not signicantly different.

reagent except the extracts. Then the absorbance was measured at 517 nm against blank (methanol pure). Lower absorbance of the reaction mixture indicated higher free radical
scavenging activity.
The capacity to scavenge the DPPH. radical was calculated
using the following equation:
DPPH scavenging effectInhibition %
Ac  As =Ac  100
where Ac was the absorbance of the control reaction and As
the absorbance in the presence of the plant methanolic extract.
IC50: Its concentrations for 50% inhibition.
7.2. Ferrous chelating activity
The chelating of ferrous ions of broccoli was estimated according to Decker and Welch (1990). Five ml of 80% broccoli
methanolic extract, or EDTA solution as a positive control
at different concentrations (5, 10, 20, 30 lg/ml) was spiked
with 0.1 ml of 2 mM FeCl2 and 0.2 ml of 5 mM ferrozine solution. The reaction mixture was left for 10 min at room temperature then the absorbance was measured at 562 nm. The
percentage of ferrous ion chelating ability was calculated using
the following equation:
Iron chelating activity Inhibition % Ac  As =Ac  100
where Ac was the absorbance of the control reaction and As the
absorbance in the presence of the plant methanolic extract.
8. Statistical analysis
Data were statistically analyzed using Costat Statistical package (Anonymous, 1989).
9. Results and discussion
9.1. Inuence of organic and bio-organic fertilization on yield
orets fresh and dry weights of two broccoli cultivars
From the data presented in Table 1 it is clear that the yield of
orets in both cultivars of broccoli was signicantly increased
as a result of organic and bioorganic treatments. The yield was
increased by 59% and 99% in Calabrese cultivar and by 22%
and 64% in Southern star cultivar by the application of organic and bio-organic fertilizers respectively.

The highest orets yield of both cultivars of broccoli plants

was recorded by the combined effect of bio-organic fertilizer
and 50% organic +50% mineral treatment.
This superiority in orets yield might be referred to the increase in vegetative growth due to the reection of the high soil
fertility resulting by bio- organic fertilization and organic
manuring (Table 1).
These results are in agreement with those obtained in broccoli orets (Zaki et al., 2009) and head cabbage (Bimova and
Pokluda, 2009).
Organic and bio-organic fertilization has an important
inuence on slow release of nutrients which support root development leading to higher yield and better quality of broccoli
plant (Hameeda et al., 2007). Such promoting effect was maximal in response to combination of organic with mixed nitrogen xer and phosphate dissolving strains (Azotobacter
chrococcum + Bacillus megaterium) in both cultivars.
The benecial effect of organic and bio-organic fertilization
on increasing the yield may be due to the increase in the organic matter content as was reported by Wilkinson (1979). In
addition, the release of organic acids decreases the pH value
of the plant growth medium so leads to the improvement of
the soil conditions and in turn improves root growth and enhances nutrients uptake (Arisha et al., 2003).
9.2. Inuence of organic and bio-organic fertilization on total
phenolics, avonoid content and total glucosinolates content
(TPC, TFC and TGsC)
The application of organic and bio-organic fertilizers signicantly increased TPC in Calabrese cultivar from 9.15 mg/g
DW (control) to 13.83 mg/g DW (organic) and 11.56 mg/g
DW (bio-organic) respectively. These increases reached to
more than 51% and 26% increase respectively. However, in
Southern star cultivar it was increased from 7.68 mg/g (control) to 10.86 (41%) and 8.45 mg/g DW (10%) when organic
and bio-organic fertilizers were applied respectively compared
with control as shown in Table 2.
In addition, avonoid content of Calabrese cultivar showed
signicantly increases from 5.46 mg/g (control) to 9.27 mg/g
DW and 7.18 mg/g DW with organic and bio-organic fertilizer
respectively, (7032% increase) compared to those of control.
While, Southern star cultivar with organic and bio-organic fertilizer exhibited similar increases (5031% increases) compared
to those of control. Similar results of broccoli orets were recorded by Heimler et al. (2006).

Enhancement of phenolics, avonoids and glucosinolates of Broccoli (Brassica olaracea, var. Italica)

139

Table 2 Effect of organic and bio-organic fertilizers on total phenolics (TPCs), total avonoids (TFCs), total glucosinolates (TGs)
contents of broccoli cultivars.
Treatments

Calabrese

100% NPK (Control)

9.15c 0.35 100
50% NPK + 50% Organic
13.82f 0.42 151
50% NPK + 50% Organic + Bio 11.56e 0.19 126

Southern star 100% NPK (Control)

50% NPK + 50% Organic
50% NPK + 50% Organic + Bio
L.S.D at 0.05

TPC
(mg*/g DW)

TPC
TFC
TFC
TGs
TGs
increase (%) (mg**/g DW) increase (%) (l mol/g DW) increase (%)

Cultivars

7.68a 0.34
10.86d 0.50
8.45b 0.51
0.60

100
141
110

5.46b 0.21
9.27f 0.16
7.18e 0.15

100
170
132

48.72b 0.19
71.34f 0.14
57.35d 0.14

100
146
118

4.52a 0.17
6.76d 0.17
5.91c 0.27
0.29

100
150
131

44.04a 0.18
62.11e 0.14
53.01c 0.09
2.03

100
141
120

All values are the means of three replicates and are signicantly different at p P 0.05 standard deviation.
All values with the same letters are not signicantly different.
*
As gallic acid.
**
As quercetin.

It was also noticed that the higher values of TPC and TFC
were found in Calabrese cultivar. These results indicated that
organic fertilization has a stimulatory effect on accumulation
of phenolics in broccoli orets. It is well known that, the higher concentrations of phenolics in orets can be explained by
the role of organic fertilizers in the biosynthesis which induces
the acetate shikimate pathway, resulting in higher production
of avonoids and phenolics (Sousa et al., 2008). Also, because
of the higher photopathogenic stress in organic farming, this
in turn may have abiotic stress, and causes an increase of phenolics, grown organically (Young et al., 2000).
A similar trend was found in total glucosinolates content
(TGsC) hence, higher values (71.34 and 62.11 lmol/g DW)
were found when the organic fertilizer was applied followed
by bio-organic treatments (57.35 and 53.01 lmol/g DW) compared to control (48.72 and 44.04 lmol/g DW) in both cultivars (Table 2). These results supported the ndings of Rosa
and Rodrigues (2001) who reported that TGsC of different
broccoli cultivars were ranged from 15.20 to 59.30 lmol/g
DW. However, Miglio et al. (2008) found that TGsC of broccoli (cv. botrytis caput) was 71.40 lmol/g DW.
The results of Vallejo et al. (2003a) showed that the biosynthesis of sulforaphan is important for the biosynthesis of
glucosinolates.
Increase of the (TGsC) in response to sulfur availability has
also been documented in turnip rape (Brassica rapa L.) (Kim
et al., 2001) and Kale (Brassica oleracea L. Acephala Group)
(Kopsell and Kopsell, 2003).

It is also clear from the results given in Table 3 that the

yield of TPC was highly signicantly increased by organic fertilization application. It increased by more than 216% compared to control treatment. However, the response to
biofertilizer treatment was less effective. A similar trend was
obtained in case of TFC and TGsC.
10. Antioxidant activity
10.1. Scavenging activity on DPPH. radicals
DPPH. has been used widely for the determination of antioxidant activity of different plants, vegetables, fruits extracts
(Goupy et al., 1999; Yu and Zhou, 2004).
The main values of radical DPPH. scavenging activity of
Calabrese, Southern star cultivars and butylated hydroxy toluene (BHT, as synthetic antioxidant agent) are shown in
(Fig. 1). Plant extract of broccoli cultivars grown with organic
and bio-organic fertilizers exhibited a potential activity of
scavenging DPPH. free radical compared to control (NPK)
and synthetic antioxidant (BHT). The values of IC50 (the concentrations for 50% inhibition) Calabrese treated with organic
and bio-organic fertilizers (Fig. 1), were 16.56 lg/ml and
17.69 lg/ml, respectively compared to control (20.89 lg/ml).
On the other hand, the values of IC50 for Southern star cultivar
treated with organic and bio-organic fertilizers were 19.42 lg/
ml and 19.69 lg/ml respectively compared with control NPK

Table 3 Effect of organic and bioorganic fertilization on yield of total phenolics (TPCs), total avonoids (TFCs) and total
glucosinolates (TGsC) contents of orets in two cultivars of broccoli.
Cultivars

Treatments

Calabrese

100% NPK (control)

5.38a 0.19 100%
50% NPK + 50% Organic
17.01c 1.80 316
50% NPK + 50% Organic + Bio 18.72c 0.73 348

Southern star 100% NPK (Control)

50% NPK + 50% Organic
50% NPK + 50% Organic + Bio
L.S.D at 0.05

TPC yield
(kg/fed)

6.64a 0.46
14.09b 1.43
14.59b 1.12
1.91

TPC yield
TFC
increases (%) (kg/fed)

100%
212
220

TFC yield
TGsC yield
increases (%) (mol/fed)

TGsC yield
increases (%)

3.21a 0.10 100%

11.41d 1.15 356
11.63d 0.29 363

28.66a 1.171 100

87.73b 8.164 306
92.99b 5.66 324

3.91a 0.31
8.78 b 0.92
10.19c 0.49
1.06

38.08 2.60a
80.64b 8.16
91.48b 0.83
9.90

100%
225
261

TPC, TFC and TGsC (yields kg/fed) = {concentration (kg/Ton) yield of orets as DW (ton/fed)}/1000.
All values are means of three replicates and are signicantly different at p P 0.05 standard deviation.
All values with the same letters are not signicantly different.

100
212
240

140

A.E.-M.M. Naguib et al.

16
14
12
10
8
6
4
2
0
15

17

Figure 2

19

IC50 g/ml

21

(c)

y = -0.7033x + 20.034
2
R = 0.6078

12
10
8
6
4
2
0
15

23

17

19

21

23

Glucosinolates mol/g

(b)

y = -1.0488x + 30.412
2
R = 0.71

TFC mg/g dry Wt.

TPC mg/g dry wt.

(a)

y = -3.7409x + 127.99
2
R = 0.4878

80
60
40
20
0
15

17

IC50 mg/g

21
19
IC50 g/ml

23

Relationship between IC50 and Total phenolics (a), Flavonoids (b) and Glucosinolates content (c).

Table 4 Iron chelating activity by different concentrations of plant methanolic extracts for two cultivars of broccoli with different
treatments of fertilizer.
Cultivars

Treatments

Inhibition%
5 lg/ml

10 lg/ml

20 lg/ml

30 lg/ml

Calabrese

100% NPK (Control)

50% NPK + 50% Organic
50% NPK + 50% Organic + Bio

10.66 0.16
15.52 0.49
14.32 0.37

21.09 0.20
25.76 0.42
24.56 0.42

41.58 0.156
49.68 0.560
45.84 0.35

69.39 0.12
75.36 0.42
74.35 0.24

Southern star

100% NPK (Control)

50% NPK + 50% Organic
50% NPK + 50% Organic + Bio

8.63 0.16
13.20 0.42
12.08 0.45

18.49 0.23
23.04 0.37
21.68 0.29

39.26 0.20
43.12 0.21
42.16 0.29

65.91 0.08
72.43 0.26
71.28 0.49

19.02 0.06
0.488

44.35 0.11
0.449

82.32 0.17
0.440

96.49 0.06
0.409

EDTA as standard
L.S.D at 0.05

All values are means of three replicates and are signicantly different at p P 0.05 standard deviation.

mainly due to their redox properties, which allow them to

act as reducing agents, hydrogen donors, and singlet oxygen
quenchers (Rice-Evans et al., 1995).

(24.79 lg/ml). While, The IC50 value for BHT was 10.07 lg/ml.
The extracts of both cultivars were capable of directly reacting
and quenching DPPH radical. Calabrese cultivar exhibited the
highest activity when compared with Southern star cultivar.
Correlation coefcient of TPC, TFC and TGsC as well as
DPPH assay is given in Fig. 2. Results showed that TPC and
TFC are correlated with DPPH scavenging activity assay
(R2 = 0.710 and R2 = 0.6078). This means that TPC and
TFC (Fig. 2a and b) are contributed to the antioxidant capacities of broccoli methanolic extracts more than the correlation
coefcient with total glucosinolates (R2 = 0.4878), which
might partially be due to their lower antioxidant activity
(Fig. 2c).
In addition, organic treatment has a stimulating effect on
the biosynthesis of phenolics which possessed high potential
activity as antioxidant when compared with control and bioorganic treatments. The antioxidant activity of phenolics is

12
9
6
3
0
70

75

Iron chelating at 30 mg/ml

80

The effect of fertilization treatments on ferrous ion chelating

power of the methanolic extracts of broccoli cultivars (Calabrese and Southern star) is shown in Table 4.
Generally data show that the ferrous ion chelating activities
of all samples were increased by increasing the concentration
of plant methanolic extract. Calabrese and Southern star cultivars combined with organic fertilizer treatment showed the
highest chelating power followed by bio-organic treatment
(compared to control NPK) at all concentrations. In addition,
the highest chelating power was observed at concentrations of
30 lg/ml. The correlation between iron chelating activity, total
(c)

y = 0.4418x - 25.049
R2 = 0.8546

10
8
6
4
2
0
65

70

75

Iron chelating at 30 mg/ml

80

Glucosinolates mmol/g

15

65

Figure 3
(c).

(b)

y = 0.5899x - 31.9
R2 = 0.8005

TFC mg/g dry wt.

TPC mg/g dry wt.

(a)

10.2. Fe-chelating activity

y = 2.545x - 125.75
2
R = 0.8045

80
60
40
20
0
65

70

75

80

Iron chelating at 30 mg/ml

Relationship between iron chelating activity at 30 lg/ml and Total phenolics (a), Flavonoids (b) and Glucosinolates content

Enhancement of phenolics, avonoids and glucosinolates of Broccoli (Brassica olaracea, var. Italica)
phenolics, avonoids and glucosinolate contents of broccoli
cultivars is shown in Fig. 3.
From Fig. 3, there is a linear and direct correlation between
iron chelating activity and total phenolics of broccoli extracts
(R2 = 0.8005) Fig. 3a, (R2 = 0.8546) with avonoid content
(Fig. 3b) and (R2 = 0.8045) with glucosinolate content
Fig. 3c. The results indicated that the correlation of phenolics,
avonoids and glucosinolates was strong to moderate depending on the assay system used. Strong correlation of phenolics
with antioxidant scavenging activity of DPPH and Iron chelating power indicated that phenolics are key contributors to
antioxidant activity in broccoli. Flavonol compounds (quercetin and kaempferol), sinapic and caffoyl-quininc acids represent the major phenolic in broccoli which agreed with results
obtained by Vallejo et al. (2003a).
11. Conclusion
The response of broccoli cultivars to organic fertilizers in terms
of phenolic compounds, avonoids and glucosinolates and
their antioxidant activities suggested that, organic fertilizers
enhanced the yield of secondary metabolites. Therefore, this
study conrmed the potential application of broccoli as a potent natural source for antioxidants and other active substances. These metabolites have high potency as antioxidants
and intern can be applied as a good protective compounds.
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