Evaluation of antioxidant capacity and phenol content of five different vegetables from kolkata

EVALUATION OF ANTIOXIDANT CAPACITY
AND PHENOL CONTENT OF FIVE DIFFERENT
VEGETABLES FROM KOLKATA: A
COMPARATIVE STUDY
by Ipsita Bhattacharya,BRSN College M.Sc in Food & Nutrition 2012-06-15

Abstract:

The antioxidant capacity and phenol content of carrot (Daucus carota), cabbage (Brassica
oleracea) , broccoli (Brassica oleracea) , jack fruit seeds (Artocarpus heterophyllus) and
pumpkin (Cucurbita maxima) obtained from our local market in Kolkata, India was
detrmined by evaluating the scavenging activity and transition metal chelating activity using
1,1 –diphenyl-2-picrylhydrazyl (DPPH) and Ferrozine. They were also analyzed for total
phenolic content (TPC) and total flavonoids content (TFC). Both ethanol and water are the
best solvents for extracting phenols and flavonoids from these vegetables. Our results show
that the antioxidant activity of broccoli and jackfruit seeds extract co-relats with its total
phenol and flavonoid content. Although carrot and cabbage have high phenol and flavonoid
content but their antioxidant capacity is comparatively less in both ethanolic and aqueous
extracts. Interestingly both the extracts of pumpkin shows very high antioxidant activity but it
has comparatively less phenol and flavonoid content than other four vegetables. The results
indicated that pumpkin is one of the good sources of antioxidant compound along with the
broccoli and jackfruit seeds available in our region.

Keywords: DPPH-radical scavenging activity, Ferrozine- metal chelating activity, TPC- total
phenolic content, TFC- total flavonoid content

Introduction

Antioxidants are important in neutralizing free radicals. Free radicals are generated during
normal body metabolism as molecules with incomplete electron pairs which make them more
chemically unstable than those with complete electron pair (Fang et al. 2002). In present
times, it is believed that the regular consumption of dietary antioxidants may reduce the risk
of several serious diseases. Regular consumption of vegetables has always been associated
with health benefits, but their mechanism has become clear only in the recent decades.
Vegetables contain a wide variety of biologically active, non-nutritive compounds known as
phytochemicals. These phytochemicals impart health benefits beyond basic nutrition.
Recently, there have been great efforts to find safe and potent natural antioxidants from
various plant sources. As harmless sources of antioxidants, edible fruits have been
investigated for their antioxidant properties, the role of food constituents as essential nutrients
to one of preventing or delaying the premature onset of chronic disease late in life has now
been generally accepted. Reactive oxygen species (ROS), such as hydrogen peroxide (
and hypochlorous acid (HOCl), and free radicals, such as the hydroxyl radical (·OH) and
superoxide anion (O2−), are produced as normal products of cellular metabolism. Rapid
production of free radicals can lead to oxidative damage to biomolecules and may cause
disorders such as cancer, diabetes, inflammatorydisease, asthma, cardiovascular diseases,
neurodegenerative diseases, and premature aging(Young and Woodside, 2001). Many
vegetables contain a large amount of polyphenols, vitamin C, vitamin E, selenium, β-
carotene, lycopene, lutein, and other carotenoids, which play important roles in adsorbing and
neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides
(Djeridane et al., 2006). Several studies have demonstrated the antibacterial and/or
antioxidant properties of these plants, mainly using in vitro assays. Moreover, some
researchers reported that there is a relationship between the chemical structures of the most
abundant compounds in the plants and their above mentioned functional properties (Dean and
Svoboda, 1989; Farag et al., 1989).

Phenolic and polyphenolic compounds constitute the main class of natural antioxidants
present in plants, foods, and beverages and are usually quantified employing Folin’s reagent.
Vinson et al. (5) have measured phenolics in fruits and vegetables colorimetrically using the

Folin-Ciocalteu reagentand determined the fruit and vegetables’ antioxidant capacity by
inhibition of low density lipoprotein oxidation mediated by cupric ions. Vegetables cont a in
not only the above nutritional antioxidants but also a great quantity of non-nutritional
antioxidants, such as flavoniods, flavone, and polyphenol compounds (7-10) Many studies
have indicated that a frequent intake of cruciferous vegetables, such as broccoli, cauliflower,
leaf mustard, cabbage, Chinese broccoli, and turnip,could protect against cancer (11,12).
Numerous crude extracts and pure natural compounds from fruits were reported to have
antioxidant and radical-scavenging activities. Within the antioxidant compounds, flavonoids
and phenolics, with a large distribution in nature, have been studied (Li et al., 2009).
Phenolics or polyphenols, including flavonoids have received considerable attention because
of their physiological functions such as antioxidant, antimutagenic and antitumor activities
(Othman et al., 2007).
The growing interest in the antioxidant properties of the phenolics compounds in vegetables
and fruits derives from their strong activity and low toxicity compared with those of synthetic
phenolics and antioxidant such as BHT (butylated hydroxytoluene), BHA (butylated
hydroxyanisole), and propyl gallate (Cailet et al., 2006).The five vegetables which i have
taken for my study material, those are oftenly usea in our regular diet. Those vegetables are
carrot(Daucus carota), cabbage(Brassica oleracea) , broccoli(Brassica oleracea) , jack fruit
seeds(Artocarpus heterophyllus) and pumpkin (Cucurbita maxima). These have been grown
in their normal season time with normal and comfortable temperature. These vegetables are
obtained from West Bengal market.

The Nutritive Power of These Vegetables is below:
Health & Nutrition Benefits of Carrot (Daucus carota)

o Consuming Carrots are known to be good for the overall health and specially organs
like the skin, eyes, digestive system and teeth. Carrot is used in several Juice Therapy
Remedies for diseases. Given below are some benefits of this Vegetable.

o Carrots are rich in Beta carotene which is a powerful antioxidant which helps in
maintaining a healthy skin and also keep one away from many diseases.Carrots are
rich in alkaline elements which purify and revitalize the blood. They balance the acid
alkaline ratio in the body.

o Carrots have Potassium in it which helps to balance the high levels of sodium
associated with hypertension and keeps blood pressure under control. The high
soluble fibre content in carrot, it reduces cholesterol by binding LDL, the bad
cholesterol, and also increases the HDL which helps in reducing blood clots and heart
diseases.

o Carrots aid digestion by increasing saliva and supplying the minerals, vitamins and
enzymes required for it. Regular consumption of carrots helps in preventing gastric
ulcers and digestive disorders. Raw carrots are used as a home remedy for treating
worms in children. Raw or grated carrots can be used for wounds, cuts and
inflammation.

o Carrots are rich in Carotenoids which are beneficial to blood sugar regulation. Carrots
contain a phyto-nutrient called falcarinol which helps in promoting colon health and a
reducing the risk of cancers. Consuming carrots regularly are known to improve the
quality of breast milk in mothers.
Health & Nutrition Benefits of cabbage (Brassica oleracea)

o Cabbage is well-known for their anticancer properties. Cabbage is rich in
phytochemicals that help protect the body from free radical damage and help fight
against carcinogens. Research suggests those who regularly consume cabbage have a
lower risk of certain cancers, especially breast cancer.

One cup of cooked cabbage contains 91.7 %DV of vitamin K and 50.3 %DV of
vitamin C. Vitamin K promotes bone health by reducing bone loss, reducing the
changes of bone fractures. Vitamin C not only boosts immunity but offers protection
against premature aging and boosts skin health.

o The fibre in cabbage boosts digestive health and can lower cholesterol, reducing the
risk of cardiovascular disease and certain cancers.

o In particular, cabbage contains powerful anti-cancer compounds known as
glucosinolates. A 31/2-oz (100 g) serving of cooked cabbage provides 35 calories, 2.3
g of protein, no cholesterol, 0.4 g of fat, 7.2 g of carbohydrate, and 3.3 g of fibre.

o You can help cabbage retain its nutritional value by following a few simple
guidelines. Buy whole heads of cabbage rather than shredded cabbage, as shredded
cabbage may have lost its vitamin C. Store the cabbage in sealed plastic in the
refrigerator. Don't wash, cut or shred the cabbage until right before you're ready to use
it.

Health & Nutrition Benefits Brocoli (Brassica oleracea)

o The nutritional value of broccoli has garnered the spotlight in recent years. Broccoli,
after extensive scientific research, is now viewed as one of the top powerhouses when
it comes to nutrient density and benefits.

o Broccoli also gives a boost to enzymes which help to detoxify the body.
Detoxification leads to weight loss and helps prevent certain diseases. Just three
servings a month of raw broccoli or cabbage can reduce the risk of bladder cancer by
as much as 40 percent, researchers reported this week. These foods are rich in
compounds called isothiocyanates, which are known to lower cancer risk.

o Broccoli is considered a low-glycemic food which helps to normalize blood sugar.
One of the keys to weight loss is controlling the body's response to insulin. Broccoli
also gives a boost to enzymes which help to detoxify the body. Detoxification leads to
weight loss and helps prevent certain diseases.

o Exposure to UV sunlight over time may lead to an eye condition called macular
degeneration, which is the number one cause of blindness in US seniors. Researchers
at Johns Hopkins determined that broccoli sprouts can protect retinal cells from
ultraviolet light damage.

Health & Nutrition Benefits of Jack fruit seeds(Artocarpus heterophyllus)

o Phytonutrients of the jackfruit seeds is valuable for our body. They are particularly
super nutrient. The jackfruit seeds phytonutrients is benefited in the body to eliminate
the free radicals, increase the white blood cells count and help in preventing cancer.

o The patients suffering from asthma, interested in knowing that the jack fruit seeds are
very good for breathing free and easy. Being rich in potassium, jackfruit has been
found to be helpful in the lowering of blood pressure. The extract of Jackfruit root is
believed to help cure fever as well as diarrhoea.

o The fruit contains isoflavones, antioxidants, and phytonutrients, all of which are
credited for their cancer-fighting properties. Jackfruit is known to contain anti-ulcer
properties and is also good for those suffering from indigestion. Boasting of anti-
ageing properties, the fruit can help slow down the degeneration of cells and make the
skin look young and supple.

o Jackfruit serves as a good supply of proteins, carbohydrates and vitamins, for the
human body. It is believed that the fruit can help prevent and treat tension and
nervousness. Since it contains few calories and a very small amount of fat, jackfruit is
good for those trying to lose weight.

Health & Nutrition Benefits of Pumpkin (Cucurbita maxima)

o Pumpkin is incredibly rich in vital anti-oxidants and vitamins. This humble backyard
vegetable is very low in calories yet good source of vitamin A, flavonoid poly-
phenolic antioxidants like leutin, xanthins and carotenes.

o It is one of the vegetables which is very low calories; provides just 26 cal per 100 g
and contains no saturated fats or cholesterol; but is rich a source of dietary fiber, anti-
oxidants, minerals, vitamins. Recommended by dieticians in cholesterol controlling
and weight reduction programs.

o Zea-xanthin is a natural anti-oxidant which has UV (ultra-violet) rays filtering
actions in the macula lutea in reitina of the eyes. Thus, it helps protect from "age
related macular disease" (ARMD) in the elderly.
o
o They promote overall prostate health, apart from alleviating the problem of difficult
urination that is associated with an enlarged prostate.They comprise of L-tryptophan,
a compound that has been found to be effective against depression.They are believed
to serve as a natural protector against osteoporosis.

Review of Literature

 One of the study by Agnieszka Bartoszek etal (2007) shows that every genotype of
carrot has different antioxidant activity through phenolic content. This is also a
comparative study, which shows the free radical scavenging power is high in one
genotype when other genotype is not so high.

 Another study by Charanjit Kaur (2007) was showing aqueous and ethanolic extract
of cabbage were to be studied for free radical scavenging and metal chelating activity.
This is also a comperative study with this two extract where shows ethanolic has
greater potentiality from aqueous extract using different antioxidant tests.

 This study by María Elena Cartea etal (2001) shows that the storage period in low
temperature decreases the total antioxidant activity in Indian cabbage. The cabbages
were minimally processed after over than a week there was no detectable property of
antioxidant except vitamin c and citric acid.

 One of the study by Umesh B. Jagtap etal (2010)The potent antioxidative property of
the brassica family vegetables reduces the heart disease Depending on their structure
they can be classified into simple Phenolic content have received considerable
attention for being potentially protective factors. The total phenolic content was also
observed in those and they have very significant role also.

 Another study from Gary Williamson1 (2010) etal shows The cabbages are from
different seasons were obtained in different origin from Europe. Total phenolic
content was higher in one season as well as total free radical scavenging property is
higher different season at different origin this is also a comperative study.

 Another study of Abdul Mueed Bidchol etal 2007 Jackfruit pulp shows total flavonoid
and total phenol;ic content. The antioxidant activities of JFP extracts were correlated
with the total phenolic and flavonoids content. The ethanol and water are the best
solvents for the extracting phenols and flavonoids from the JFP In which the best
solvent was the either ethanolic extract. It was proved that the jackfruit pulp is the
natural source of antioxidant.

 This study from JIIN-TZONG GUO etal (2005) shows the edible portion and the seed
of avocado, jackfruit,longan, mango and tamarind were studied. were taken for
study. The seeds showed much higher antioxidant and phenolic activity than the
edible portion. Jack fruit seeds were also considered.

 Another study by Fei Que1,2 etal (2006) shows antioxidant activity of the major poly
phenolic compound was observed in broccoli. Total flavonoid and flavonol compound
were also observed in this syudy.

 One of this study from Azizah, M., 2009 in identical agricultural climatic condition,
the different type of individual variation of broccoli were taken for study. Total
phenolic content, the DPPH•, vitamin c and OH• radical-scavenging activities of
samples were determined.

 Another study by Wee, K. C. Etal 2009, was showing Broccoli and asparagus were
taken for antioxidant potentiality usind DPPH, ABTS. Asparagus and its juices
showed a better result from broccoli in all assays. Also showed a greater potentiality
in methanolic extract then aqueous.

 This study from Wang, H (2006) shown that flowers stem and leaves of broccoli were
taken in DPPH, reducing power and metal chelating activity. They were taken in
freeze dried condition. All of these part, The broccoli stem exhibited the highest

chelating ability among three parts of broccoli & the acetone extracts from stems
hardly showed any chelating ability as compared to alpha tocopherol and BHA. .

 Another study of Webb, D. (2008) was showing changing in boiling time of cooking
pumpkin, is compared with the stir frying showed a higher free radical scavenging
activity than the other assays. Boiling time reduces the power effectively.

 Another study by Fei Que1,2 etal 2006. Was shown that the antioxidant activities of
methanol extracts from pumpkin flours were studied in terms of total antioxidant
activity, reducing power, free radical scavenging, superoxide anion radical scavenging
and metal chelating activities. Different antioxidant activity assays were done with hot
dried and freeze dried condition of pumpkin. Hot dried pumpkin flowers have shoed a
stronger activity rather than the freeze dried flower.

 Another study by (Marja P etal 1999) have shown some plants including broccoli
and cabbage. In addition, potato peel and beetroot peel extracts showed strong
antioxidant effects. To utilize these significant sources of natural antioxidants, further
characterization of the phenolic composition is needed.

Aims and Objectives

This study has obvious focussed on some aims and objectives which are reliable and valid for
further extension and growth of this study. The objectives are summarized below

Characterization of different parts of five different vegetables in terms of their
antioxidant potency.

Determination of the comparative antioxidative power in the individual
extracts in terms of their free radical scavenging activity.

To compare the method of extraction and anti oxidative potency of some of the
plant products.

Comparison in the chelating power of the transition metal ion of these five
vegetables which are studied.

Materials and Methods

Chemicals and reagents
1. Ethanol
70% ethanol was required for the extract preparation and 99% ethanol was requires
for dpph assay. So the hydro alcoholic solution was prepared for extraction.

2. DPPH
2,2-diphenyl-1-picrylhydrazyl was prepared by 99% EtOH, 0.0008 gm. Of DPPH was
desolved in 10 ml ethanol, and the reagent was prepared with 2mM of DPPH. Each
170 μl of DPPH was required for each ml of extract.

3. Ferrozine
Ferrozine was prepared with 4 mg of sample, desolved in 1.5 ml of water, and the
reagent was made upto 6μl, 5mM for the ferrozine assay of each concentration of
sample,

4. Ferrous Chloride
The amount of 3.26 mg of sample was desolved in 10 ml of water and the reagent was
made for each sample assaying. The volume was made upto 2μl, 2mM. This sample
was prepared for the ferrozine assay.

5. Sodium Nitrite
For preparing this reagent the amount of 0.03ml was desolved in water, so now the
reagent was made by 5% of reagent, this was made for tolal flavonoid content assay.

6. Aluminium Chlorite
For the flavonoid content measurement this reagent was prepared with aqueous
solution. This was made by 0.1 gm of reagent was mixed with water, so the
concentration was achieved to 0.03 ml, 10% of reagent.

7. Sodium Hydroxide
This reagent was made for total flavonoid content measurement. The amount of 0.4
gm of NaOH was desolved in 20 ml of aqoueous solution.so thr concentration was
achieved to 0.2 ml, 1mM
,
8. Folin reagent
1 ml of this reagent was mixed with 1000 ml of distilled water as 1:1000 ratio. So in
this proportion, 750μl was prepared for each extraction, this reagent was prepared for
total phenolic content measurement.

9. Sodium Carbonate
This reagent was made for the the total phenolic content measurement. A 12 mg of
reagent was mixed with 15 ml of water solution, as to achieve the concentration of 1
ml, 0.06% of reagent

Plant materials or study materials

The edible portion of carrot (Daucus carota), the whole cabbage (Brassica oleracea) , the
flower of the broccoli (Brassica oleracea), the seeds of jackfruit(Artocarpus heterophyllus)
and the fruit pumpkin(Cucurbita maxima) were taken for our study materials.Sample

preparation and extraction
The five study materials were extracted in this way.

Ethanolic- aqueous extract:

At first the samples were dried at high temperature (50oC). 5 gms of dried sample was mixed
with 50 ml 70% ethanol. It was mixed in the cyclomixer for one hour using a magnetic
stirrer. After mixing, the sample was filtered and filtrate was separated out. The filtrate was
taken in a petridish & was allowed to evaporate, keeping the samples in a rotor evaporator at
about 50 °C for 24 hours. Next morning the residual was taken and dissolved in sterile double
distilled water (the double distilled water was taken according to the choice of the

concentrations.). It was filtered and the filtrate was taken as the clear solution. This extracts
was ready for assaying.

Aqueous-aqueous extract:

At first the samples were dried at high temperature (50oC). 5 gms of dried sample was mixed
with 50 ml boiled aqueous solution. It was mixed in the cyclomixer for one hour using a
magnetic stirrer and also regulating the high temperature. After mixing, the sample was
filtered and filtrate was separated out. The filtrate was taken in a petridish & was allowed to
evaporate, keeping the samples in a rotor evaporator at about 50 °C for 24 hours. Next
morning the residual was taken and dissolved in sterile double distilled water (the double
distilled water was taken according to the choice of the concentrations.). It was filtered and
the filtrate was taken as the clear solution. This extracts was ready for assaying.

Free Radical Scavenging Assay with DPPH

Principle
The disappearance of the DPPH radical absorption at a characteristic wavelength is
monitored by decrease in optical density. In this assay, the purple chromogen radical 2,2-
diphenyl- 1-picrylhydrazyl (DPPH•) is reduced by antioxidant/reducing compounds to the
corresponding pale yellow hydrazine .The scavenging capacity is generally evaluated in
organic media by monitoring the absorbance decrease at 515–528nm until the absorbance
remains constant or by electron spin resonance.
The scavenging reaction between (DPPH) and an antioxidant (H-A) can be written as:
(DPPH) + (H-A) DPPH-H + (A)
(Purple) (Yellow)

Procedure
The effect of different extractions at various concentrations (10, 20, 40 and 100 mg/ml) on
DPPH radical was estimated according to the method of Lee et al. (2007) adopting method of
Brand-william et al. (1995). According to the conc. the sample and solvent (ethanol) were
estimated and then the reagent (DPPH) was added to the samples. Next it was incubated for
30 minutes in room temperature. Spectrophotometric measurement then assays whether the

radical scavenging power was more or less with calculating the absorbance. The OD value
was taken in 517nm. The less absorbance (disintegrating violet colour) was determined more
scavenging activity. The assays were done in triplicate.

Calculating formula
Percent inhibition of DPPH radical
= [(controlOD- sampleOD)/controlOD)]×100

Metal Chelating Activity Assay with Ferrozine

Principle
A published method by Decker and Welch was adopted (2005). It has been determined by
spectrophotometric titration that ferrozine forms the expected tris complex with ferrous iron.
Most of these complexes are only weakly colored, are unstable under normal physical
conditions, orare formed over a very narrow pH range. A few of these compounds, however,
form stable, intensely colored species with the ferrous ion and are, therefore, suitable for the
quantitative determination of iron

Procedure
Five ml of the test solutions, including five sample materials extract and BHA solutions,
were spiked with 2.0μl of 2 mM FeCl2 and 6.0 μl of 5 mM ferrozine solutions. After reaction
for 10 min, the adduct violet colour was developed and the absorbance (at 562 nm) of
resulting solutions was recorded. A complex of Fe+2 /ferrozine has a strong absorbance at
562 nm. The higher the ferrous ion chelating ability of the test sample gives the lower
absorbance which disintegrates the violet colour. The percentage of ferrous ion chelating
ability is expressed by this given formula.

The calculating formula
Percentage of Ferrous ion chelating ability
= [(controlOD- sampleOD)/controlOD)]×100

Determination of TPC (Total Phenolic Content)

Principle
The total phenolic content of the extracts was determined using the Folin– Ciocalteu assay
(Singleton and Rossi 1965). The principle underlying that the reaction mechanism is that in
alkaline medium phenol reacts with folin reagent and results as producing of blue coloured
complex which have a maximum absorption of 770 nm.

Procedure
A known volume of the five different study materials with different concentrations of the
extract was mixed with 750 μl (10 times pre-diluted) of FC reagent. After standing for 5 min
at room temperature, 1 ml of (0.06% w/v) sodium carbonate solution was added. The
solutions were mixed and allowed to stand for 90 min at 22C temperature. The absorbance
was measured at 765 nm using a UV-visible spectrophotometer. A calibration curve was
prepared using a standard solution of gallic acid. Results were expressed as mg gallic acid
equivalents (GAE) per gram of sample.

Determination of TFC (Total Flavonoid Content)

Principle
The colorimetric method described by Sakanaka et al. (2005) was employed to determine the
total flavonoid content in the extracts.

Procedure
Briefly, a particular concentration (0.1ml) of the extract or (+)-quarcetin standard solution
was mixed with 0.3 ml of distilled water in a test tube, followed by addition of .03ml of a 5%
sodium nitrite solution. After 6 min incubation period, .03ml of a 10% aluminium chloride
solution was added and the mixture was allowed to stand for 5 min before 0.2 ml of 1 mM
sodium hydroxide was added. The mixture was brought to 1 ml with distilled water and
mixed well. The absorbance was measured immediately at 510 nm using a UV-visible
spectrophotometer.

Result

The result was determined after the study.

Type of DPPH assay % Ferrozine assay
samples of inhibition % of
metalchelation
E.E AE EE AE
Carrot 17.42 0.93 15.45 0.12 15.53 1.18 12.32 0.21
Cabbage 30.75 1.56 16.87 1.45 21.46 1.04 18.78 1.2
Broccoli 57.53 0.82 50.23 2.2 61.33 1.15 50.04 0.65
Jack fruit seeds 24.62 0.42 20.12 1.02 56.21 0.81 45.25 0.14
pumpkin 84.71 2.5 70.21 0.25 37.65 0.86 25.39 0.21
Table; 1 shows the radical scavenging activity in DPPH assay with compare to alcoholic and
aqueous extract. Also showing % of metal chelation with Ferrozine assay with comparing of
two extracts. The concentration have taken in 100 mg /gm and Values are represented as
mean ± SD (n=03);

EE – ethanolic extract, AE- aqueous extract

Type of **TPC (mg *TFC (mg
samples GAE/ g) QE/g)
EE AE EE AE
Carrot 49.83 0.23 55.23 0.33 15.5 0.66 17.65 0.42
Cabbage 38.5 1.42 45.46 .02 56.14 0.25 55.36 0.35
Broccoli 35.4 0.36 50.25 0.11 76.15 0.35 75.39 0.19
Jack fruit seeds 49.6 2.2 65.65 0.93 33.24 0.54 36.28 1.42
pumpkin 31.7 01.3 39.32 0.12 13.70 0.58 12.45 1.54
Table; 2 shows that TPC and TFC determination with comparing their ethanolic and aqueous
extract. Values are represented as mean ± SD (n=03); *Values are expressed as quercetine
equivalents in mg/g extract. The concentration have taken in 100 mg /gm and **Values are
expressed as gallic acid equivalent in mg/g extract

TPC- total phenolic content,

TFC- total flavonoid content,

EE – ethanolic extract, AE- aqueous extract

Sample extract Radical scavenging activity(in different
concentration)in DPPH
10 mg/gm 20 mg/gm 40 mg/gm 100 mg/gm
Carrot 13.6% 0.23 11.13% 0.66 15.05% 0.54 17.42% 0.19
cabbage 10.2% 0.93 11.6% 0.23 16.0% 1.54 30.75% 0.23
Broccoli 31.3% 0.66 44.5% 0.66 49.6% 0.54 57.53% 1.54
Jack fruit seeds 10.7% 0.93 16.7% 0.33 20.3% 1.54 24.62% 0.19
pumpkin 32.7% 0.33 43.22% 0.23 53.79% 0.23 84.71% 1.54

Table 3; shows the radical scavenging activity of the ethanolic extract at different
concentration, Values are represented as mean ± SD (n=03);

Sample extract Radical scavenging activity(in different
concentration)in DPPH
10 mg/gm 20 mg/gm 40 mg/gm 100 mg/gm
Carrot 12.6% 0.23 11.13% 0.66 15.05% 0.33 15.45% 0.19
cabbage 09.2% 0.93 10.6% 0.19 16.0% 0.93 16.87% 0.23
Broccoli 23.3% 0.19 44.5% 0.23 49.6% 0.66 50.23% 0.33
Jack fruit seeds 10.7% 0.66 16.7% 0.93 18.3% 0.19 20.12% 0.93
pumpkin 30.7% 0.33 42.22% 0.33 50.79% 0.23 70.21% 0.66
Table 4; shows the radical scavenging activity of the aqueous extract at different
concentration. Values are represented as mean ± SD (n=03);

Sample extract Metal chelating Activity(In Different
Concentration)with Ferrozine
10 20 40 100
Carrot 10.9% 0.93 12.1% 0.19 14.41% 0.93 15.34% 0.33
Cabbage 09.5% 0.33 10.8% 0.93 15.4% 0.19 21.6% 0.19
brocoli 16.24% 0.19 23.24% 0.33 27.0% 0.19 61.0% 0.33
Jackfruit seeds 25.57% 0.19 36.28% 0.19 50.71% 0.93 56.42% 0.93
pumpkin 20.0% 0.19 18.57% 0.19 25.14% 0.66 37.0% 0.19
Table 5; shows metal chelating activity of ethanolic extract (at Different Concentration) by
Ferrozine Assay. Values are represented as mean ± SD (n=03);

Sample extract Metal chelating Activity(In Different
Concentration)with Ferrozine
10 20 40 100
Carrot 10.9% 0.93 11.1% 0.19 12.01% 0.93 12.34% 0.33
Cabbage 09.5% 0.33 10.8% 0.93 15.4% 0.19 18.6% 0.19
brocoli 16.24% 0.19 23.24% 0.33 27.0% 0.19 50.0% 0.33
Jackfruit seeds 25.57% 0.19 36.28% 0.19 40.71% 0.93 45.42% 0.93
pumpkin 20.0% 0.19 22.57% 0.19 24.14% 0.66 25.0% 0.19
Table 6; shows metal chelating activity of aqueous extract (at Different Concentration) by
Ferrozine Assay. Values are represented as mean ± SD (n=03);

Statistical analysis
The experiment was done in triplicate, and there after their mean value was expressed in our
result. The standard deviation value was also obtained from mean to show the accuracy of the
mean value. Mean values have been obtained by summation of that triplicate value and
divided by 3. Then the SD value is obtained from squaring those triplicate value and
subtraction the value of mean square. Then the square root was done for obtaining the SD
value. Which was denoted as sign of accuracy.

The graphical representation of our results has been shown in the figures below.
100
90
80
70
carrot
% inhibition

60
50 cabbage
40 brocoli
30 jackfruit seeds
20
pumpkin
10
0
10 20 40 100
concentration (μl/100μl)

Figure 1; exhibits a comparison between five study samples according to their concentration.
In DPPH radical scavenging activity of ethanolic extract, there significant incretion of all
samples. Pumpkin and broccoli have the highest value in total but broccoli lower value in 40
conc. Values are represented as mean.

120

100

80
%inhibition

carrot
60
cabbage
40 brocoli
jackfruit seeds
20 pumpkin

0
1 2 3 4
concentration μg/ml

In DPPH radical scavenging activity of aqueous extract, there significant incretion of all

samples. Pumpkin and broccoli have the highest value in total but broccoli lower value in
100conc. other are more or less lower value. Values are represented as mean.

70

60

50

carrot
% chelation

40
cabbage
30 brocoli
jackfruit seed
20
pumpkin
10

0
10 20 40 100

In metal chelating activity with Ferrozine in ethanolic extract, there significant incretion of all
samples. Carrot and broccoli have the highest value in total but broccoli lower value in 40
conc. but it has a highest value in 100 conc. among all. Values are represented as mean.

160

140

120

100
% chelation

pumpkin
80 jackfruit seed
60 brocoli

40 cabbage
carrot
20

0
10 20 40 100

In metal chelating activity with Ferrozine in aqueous extract, there significant incretion of all

samples. Pumpkin and have the highest value in total but carrot have lowest value in 40 conc. but it
has a highest value in 100 conc. among all. Values are represented as mean.

100
90
80
70
% inhibition

60
50
40 EE

30 AE

20
10
0
carrot cabbage brocoli jackfruit seed pumpkin
samples extract

Figure 5 exhibits a comparison of antioxidant activity of five vegetables. Radical scavenging
activity using DPPH was done using ethanolic & aqueous extracts. Pumpkin has the highest
inhibition value than others, whereas carrot shows the lowest. Comparatively cabbage shows
relatively lower value in aqueous respect to other aqueous extract with respect to their
inhibition of free radicals.

70

60

50
% chelation

40

30 EE

20 AE

10

0
sample extract

Figure 6. exhibits a comparison of antioxidant activity of five vegetables. Metal chelating
activity using Ferrozine was done using ethanolic & aqueous extracts. Brocoli have the
highest chelation value than others, whereas carrot shows the lowest and also Comparatively

shows relatively lower value in aqueous respect to other aqueous extract with respect to their
chelation of transition metals.

80
70
total antioxidant activity

60
50
40
EE
30
AE
20
10
0
samples extract

Figure 7; exhibits a comparison of antioxidant activity of five vegetables. Total phenolic
content determination was done using ethanolic & aqueous extracts. Jackfruit seeds have the
highest value in aqueous than others, whereas pumpkin shows the lowest and also
Comparatively shows relatively lower value in aqueous respect to other aqueous extract with
respect to their total phenolic content.

90
80
70
total antioxidant activity

60
50
40 EE
30 AE
20
10
0
carrot cabbage brocoli jackfruit seeds pumpkin
samples extract

Figure 8; exhibits a comparison of antioxidant activity of five vegetables. Total flavonoid
content determination was done using ethanolic & aqueous extracts. brocoli have the highest
values than others, whereas pumpkin shows the lowest and also Comparatively shows

relatively lower value in aqueous respect to other aqueous extract with respect to their total flavonoid
content.

13.7
15.5 33.24
76.15 31.7
56.14
TFC

49.6 TPC
35.4 37.65
49.83

38.5 Ferrozine
61.33
56.21 DPPH
21.46
15.53 84.71

30.75 57.53
17.42 24.62

carrot cabbage brocoli jackfruit seeds pumpkin

Figure 9; shows a vast comparison of all study have done with all samples in ethanolic
extract. Pumpkin have highest value in DPPH assay, whereas carrot shows highest in TPC.
But jackfruit seeds have almost highest value in ferrozine assay. TFC value is more or less
same to all, but pumpkin have lower.

12.45
17.65 36.28
TFC
75.39
55.36 39.32
TPC

65.65 Ferrozine
55.23 50.25 25.39

45.46 DPPH
50.04
45.25 70.21
12.32 18.78
50.23
15.45 16.87 20.12

carrot cabbage brocoli jackfruit seeds pumkin

Figure 10; shows a vast comparison of all study have done with all samples in aqueous
extract. Pumpkin have highest value in DPPH assay, whereas carrot shows highest in TPC.
But jackfruit seeds have highest value in ferrozine assay. TFC value is more or less same to
all, but pumpkin have lower.

Discussion
Among all the samples, pumpkin have showing greater potentiality in radical scavenging
activity, whether in same concentration there is a reduced percentage of radical scavenging
activity in DPPH assay. Broccoli have also showing higher percentage than others. But there
is a sharp decrease of this activity in aqueous extract (Table 1). So we can say that the
scavenging activity is reduced in water (figure 5). As we consume our food in aqueous form
we get less antioxidant compared to its ethanolic extract. Antioxidant activities of pumpkin
and broccoli, along with jackfruit seeds in our study have shown higher activity. So we can
suggest consumption of these three vegetable in our area as a natural source of antioxidant
(Table 1). One of the study in India has also showed the antioxidant properties of both
extracts of Brassica oleracea and Daucus carota using different antioxidant tests, including
1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, in aqueous and ethanolic extracts
and also showed an reduced value in water compared to ethanol which has greater
potentiality of scavenging power (Charanjit Kaur ,2007). Our study showed comparatively
lower antioxidant activity in carrot in both ethanol and aqueous extract. Similar studies using
carrot from different genotype i.e. obtained from different origin of India, have showed
different values in scavenging property(give ref). Cultuvar bhavalpur have showed a low
scavenging power which is more or less same with our study (give ref), but the pusa kesar
cultivar carrot have showed a relatively high value (Agnieszka Bartoszek etal (2007) from our
study which was assayed in same concentration. But carrote of our region, there is a
significantly low scavenging value which is probably caused by the weather and for
cultivation technique.

Results of table 1 showed metal chelating activity with Ferrozine. Our study exhibited greater
scavenging activity for pumpkin However broccoli showed hifgest metal chelation power
followed by pumpkin and jackfruit seeds. At 100mg/ml concentration, carrot extract showed
lesser in metal chelation than scavengingo. But in other cultivar (pusa kesar cultivar), there is
a relatively higher value in metal chelating (Agnieszka Bartoszek etal (2007). Other study

showed relatively high amount of metal chelating activity by jackfruit pulp then the seeds of
jackfruit , and the seeds have almost no value for metal chelation (Umesh B. Jagtap, 2010),
but in our study there is a very significant value of metal chelation in seeds of jackfruit.
Another study assayed with several type of seeds and proved jackfruit seeds have more
higher value in metal chelation than the edible portion of that (JIIN-TZONG GUO etal (2005)
and this activity is more or less same with our study. In aqueous extract, there is a significant
reduction of these activity i.e. same as DPPH assay (figure 6). The stem of broccoli exihibits
a higher scavenging value than the flowers of broccoli, but the flower have showed relatively
high in metal chelating activity than the stem and also showed a greater value in ethanolic
and aqueous extract (Wang, H (2006). Our study also showed a greater value in flower of
broccoli in metal chelating activity (table 1), and also showed comparatively high value in
aqueous extract with respect to other samples (figure 6). So we can say that broccoli is not
good scavenger but a very good chelator.

Our study also support pumpkin as a very good scavenger but not so good in metal chelation.
So we can recommend this two vegetables in our regular diet along with jackfruit seeds
which will enhance antioxidant status.

Total phenolic content is also determined in all five samples (Table 2). Here we are surprised
with our result because carrots have been known for very good source of antioxidant. Our
variety of carrote showed very poor antioxidant activity by DPPH and Ferrozine assay. But
its TPC found to be good. Table 2 as well as Figure 9 showed lowest TPC in pumpkin
compared to broccoli, cabbage, jackfruit seeds. The vegetables from brassica family i.e.
cabbage and broccoli have been proved with very high in total phenolic content and they
have a significant role in reducing heart diseases ( Umesh B. Jagtap etal (2010). This study is
matched with our study as the broccoli and cabbage have showed significantly high TPC
(table 2). Here we observe a differentiation from above two assays. Aqueous extract have
showed relatively higher value i.e. happened with all my study samples in total phenolic
content than the ethanolic extract (figure 7). Jackfruit seeds have the highest value and carrots
have also the higher TPC in basically aqueous extract. Now we can say these are very much
beneficial for our diet, as the water act as a good solvent. This is basically caused by the
phenol group of poly phenol which is merged with the OH group of ethanol, and reduces the
polyphenol activity, but in water there is no such chance to happen. The pusa kesar cultivars

have showed higher TPC than the other cultivar in aqueous extract also (Agnieszka Bartoszek
etal (2007). This study support our results. As the carrot from Kolkata have also showed such
evidence in Total Phenolic Content.

Similar study was done for total flavonoid content these five samples in both ethanolic and
aqueous extract. As the flavonoid is the derivative compound of poly phenol so there should
not be such significance variance in TFC determination. But there is reduced the value of
TFC in carrot which one was high in TPC. Here broccoli have showed very significant
amount of flavonoid content and cabbage have also showed a significant amount of TFC after
the broccoli (Table 2). The vegetables from Brassica family have showed a very high amount
of flavonoid content which was conducted in both ethanolic and aqueous extract and also
have showed a significant role in heart diseases (Umesh B. Jagtap etal (2010). As our study
also have showed that the broccoli and cabbage (Brassica family) have very high amount of
flavonoid. In aqueous extract there is more or less same value of flavonoid as the ethanolic
extract . This exhibits.broccoli have the highest and carrots have the lowest amount of
flavonoid content (Figure 8). Only the jackfruit seeds have showing such higher amount of
flavonoid than the ethanolic in aqueous extract. The other vegetables as pumpkin and carrot
have showed no such significant amount of flavonoid in both ethanolic and aqueous extract.
Now we can say that in our diet the mixture of broccoli and cabbage can consumed. But as in
TPC we have seen that the carrot have significant amount of phenol and in TFC the amount is
reduced whether the broccoli have shown the highest amount of flavonoid. So a combination
of brassica family and carrot can be consumed together to enhance the total antioxidant
source in our food. Jackfruit pulps have showed a greater potentiality in TFC along with TPC
than the seeds portion of jackfruit (Abdul Mueed Bidchol etal 2007). This study is not
matched with our study where seeds portion have a high amount of flavonoid content in same
concentration of previous study. Whether another study have showed some different, various
fruit seeds are considered and among them jackfruit seeds have shown a greater potentiality
in flavonoid content than others in aqueous extract (JIIN-TZONG GUO etal (2005). This
value is more or less similar with my study, as this seeds have higher amount of flavonoid
content along with the poly phenol content in both aqueous and ethanolic extract. So the
seeds can be consumed in cooked form to encourage antioxidative status other than the edible
portion of jackfruit.

For the free radical scavenging assay and the metal chelating assay, four different
concentrations of extract were used for standardization of our study. Mostly all of the extracts
have shown a higher antioxidant activity with increase in concentration. In DPPH assay in
ethanolic extract, there is significant increase in all samples, except in 40 μg/ml
concentration, reduced the percentage inhibition value (figure 1). This may be due to some
ambiguity in sample preparation or handling of the samples. In aqueous extract, the inhibition
value is reduced surprisingly. Broccoli have shown increment upto 40μg/ml conc. but in
100μg/ml the values are reduced (figure 2). All other samples are reduced their value of
inhibition in all four conc. in aqueous than ethanolic extract (Table 5).

In metal chelating activity with Ferrozine assay, same four different concentrations are
observed. With increasing concentration, increase of the percentage value of chelating
power. Broccolis have shown higher value in 100μg/ ml conc. surprisingly in 10g/ml conc.
pumpkin have shown the high value which is not so high in 100 μg/ml. So, a small amount of
pumpkin can enhance antioxidant activity (Figure 3). But this is the ethanolic extract. In
aqueous extract the chelation percentage is reduced. But not as DPPH assay. Carrot have not
shown the activity in all four conc. but other samples have shown their activity in increasing
activity through conc. but not as much as ethanolic extract (figure 4).

Conclusion

Evaluating the two assays (DPPH and Ferrozine) for determination of antioxidant activity we
have observed that these five simple vegetables have greater antioxidant potency. Some of
them showed greater scavenging power while other vegetable showed high metal chelating
power. Interestingly vegetables are not showing not much activity as carrot. So a combination
of vegetables in diet is necessary. We can conclude that pumpkins have a greater source of
antioxidant along with broccoli and jackfruit seeds.

In total phenol content and total flavonoid content pumpkin showed very less value as
compared to their antioxidant activity as measurd by DPPH assay and ferrozine assay in
terms of ethanolic and aqueous extract. Broccoli, cabbage and jackfruit seeds have shown a
greater value in both in ethanol and water extract. We can conclude our study by
recommending pumpkin, cabbage and jackfruit seeds as our daily vegetables in our diet
which will be of low cost value. We can also consume carrots in alternative day as natural
source of antioxidant in our body.

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