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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 (H 2O 2¿ 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 Williamson 1 (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 anti- oxidative 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 samples
DPPH assay % of inhibition
Ferrozine assay % of metalchelation
E.E AE EE AECarrot 17.42±0.93 15.45±0.12 15.53±1.18 12.32±0.21Cabbage 30.75±1.56 16.87±1.45 21.46±1.04 18.78±1.2Broccoli 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±0.21Table; 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 samples
**TPC (mg GAE/ g)
*TFC (mg QE/g)
EE AE EE AECarrot 49.83±0.23 55.23±0.33 15.5±0.66 17.65±0.42Cabbage 38.5±1.42 45.46±.02 56.14±0.25 55.36±0.35Broccoli 35.4±0.36 50.25±0.11 76.15±0.35 75.39±0.19Jack 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±1.54Table; 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
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 DPPH10 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 Ferrozine10 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
Sample extract Radical scavenging activity(in different concentration)in DPPH10 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 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 Ferrozine10 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.
10 20 40 1000
10
20
30
40
50
60
70
80
90
carrotcabbagebrocolijackfruit seedspumpkin
concentration (μl/100μl)
% in
hibi
tion
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.
1 2 3 40
20
40
60
80
100
120
carrotcabbagebrocolijackfruit seedspumpkin
concentration μg/ml
%in
hibi
tion
Figure 2; exhibits a comparison between five study samples according to their concentration. 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.
10 20 40 1000
10
20
30
40
50
60
70
carrotcabbagebrocolijackfruit seedpumpkin
concentration μg/ml
% ch
elati
on
Figure 3; exhibits a comparison between five study samples according to their concentration. 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.
10 20 40 1000
20
40
60
80
100
120
140
160
pumpkinjackfruit seedbrocolicabbagecarrot
concentration μg/ml
% ch
elati
on
Figure 4; exhibits a comparison between five study samples according to their concentration. 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.
carrot cabbage brocoli jackfruit seed pumpkin0
10
20
30
40
50
60
70
80
90
EEAE
samples extract
% in
hibi
tion
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.
carrot cabbage brocoli jackfruit seed pumpkin0
10
20
30
40
50
60
EEAE
sample extract
% ch
elati
on
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.
carrot cabbage brocoli jackfruit seed pumpkin0
10
20
30
40
50
60
70
EEAE
samples extract
tota
l anti
oxid
ant a
ctivi
ty
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.
carrot cabbage brocoli jackfruit seeds
pumpkin0
10
20
30
40
50
60
70
80
EEAE
samples extract
tota
l anti
oxid
ant a
ctivi
ty
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.
carrot cabbage brocoli jackfruit seeds pumpkin
17.42 30.75 57.5324.62
84.7115.53 21.46
61.3356.21
37.6549.83
38.5
35.449.6
31.7
15.5
56.14 76.1533.24
13.7
TFC
TPC
Ferrozine
DPPH
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.
carrot cabbage brocoli jackfruit seeds pumkin
15.45 16.8750.23
20.12
70.2112.32 18.78
50.04
45.25
25.3955.23
45.46
50.2565.65
39.32
17.65
55.3675.39
36.28
12.45
TFC
TPC
Ferrozine
DPPH
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.
DiscussionAmong 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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