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

7/31/2019 Evaluation of Antioxidant Capacity and Phenol Content of Five Different Vegetables From Kolkata

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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
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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, -

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 Folins 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


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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.
http://en.wikipedia.org/wiki/Cucurbita_maximahttp://en.wikipedia.org/wiki/Cucurbita_maximahttp://en.wikipedia.org/wiki/Cucurbita_maximahttp://en.wikipedia.org/wiki/Cucurbita_maxima


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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


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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.


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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 backyardvegetable 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.


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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 Mara 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.


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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 ofAbdul 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 ofavocado,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.
http://www.springerlink.com/content/?Author=Umesh+B.+Jagtaphttp://informahealthcare.com/action/doSearch?action=runSearch&type=advanced&result=true&prevSearch=%2Bauthorsfield%3A%28Williamson%2C+Gary%29http://informahealthcare.com/action/doSearch?action=runSearch&type=advanced&result=true&prevSearch=%2Bauthorsfield%3A%28Williamson%2C+Gary%29http://informahealthcare.com/action/doSearch?action=runSearch&type=advanced&result=true&prevSearch=%2Bauthorsfield%3A%28Williamson%2C+Gary%29http://www.springerlink.com/content/?Author=Abdul+Mueed+Bidcholhttp://www.springerlink.com/content/?Author=Abdul+Mueed+Bidcholhttp://ukpmc.ac.uk/abstract/AGR/IND43644005/?whatizit_url_Species=http://www.ncbi.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=3435&lvl=0http://ukpmc.ac.uk/abstract/AGR/IND43644005/?whatizit_url_Species=http://www.ncbi.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=3489&lvl=0http://ukpmc.ac.uk/abstract/AGR/IND43644005/?whatizit_url_Species=http://www.ncbi.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=3489&lvl=0http://ukpmc.ac.uk/abstract/AGR/IND43644005/?whatizit_url_Species=http://www.ncbi.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=128017&lvl=0http://ukpmc.ac.uk/abstract/AGR/IND43644005/?whatizit_url_Species=http://www.ncbi.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=128017&lvl=0http://ukpmc.ac.uk/abstract/AGR/IND43644005/?whatizit_url_Species=http://www.ncbi.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=29780&lvl=0http://ukpmc.ac.uk/abstract/AGR/IND43644005/?whatizit_url_Species=http://www.ncbi.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=58860&lvl=0http://ukpmc.ac.uk/abstract/AGR/IND43644005/?whatizit_url_Species=http://www.ncbi.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=58860&lvl=0http://ukpmc.ac.uk/abstract/AGR/IND43644005/?whatizit_url_Species=http://www.ncbi.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=29780&lvl=0http://ukpmc.ac.uk/abstract/AGR/IND43644005/?whatizit_url_Species=http://www.ncbi.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=128017&lvl=0http://ukpmc.ac.uk/abstract/AGR/IND43644005/?whatizit_url_Species=http://www.ncbi.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=3489&lvl=0http://ukpmc.ac.uk/abstract/AGR/IND43644005/?whatizit_url_Species=http://www.ncbi.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=3435&lvl=0http://www.springerlink.com/content/?Author=Abdul+Mueed+Bidcholhttp://informahealthcare.com/action/doSearch?action=runSearch&type=advanced&result=true&prevSearch=%2Bauthorsfield%3A%28Williamson%2C+Gary%29http://www.springerlink.com/content/?Author=Umesh+B.+Jagtap


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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 scavengingand 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


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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.


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Comparison in the chelating power of the transition metal ion of these five

vegetables which are studied.


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Materials and Methods

Chemicals and reagents

1. Ethanol70% 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. DPPH2,2-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 l of DPPH was required for each ml of extract.

3. FerrozineFerrozine was prepared with 4 mg of sample, desolved in 1.5 ml of water, and the

reagent was made upto 6l, 5mM for the ferrozine assay of each concentration of

sample,

4. Ferrous ChlorideThe 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 2l, 2mM. This sample

was prepared for the ferrozine assay.

5. Sodium NitriteFor 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 ChloriteFor 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.


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7. Sodium HydroxideThis 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 reagent1 ml of this reagent was mixed with 1000 ml of distilled water as 1:1000 ratio. So in

this proportion, 750l was prepared for each extraction, this reagent was prepared for

total phenolic content measurement.

9. Sodium CarbonateThis 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


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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 515528nm 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


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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 2.0l of 2 mM FeCl2 and 6.0 l 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.


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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 FolinCiocalteu 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 l (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. Thesolutions 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.


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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.


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Result

The result was determined after the study.

Type ofsamples

DPPH assay %of inhibition

Ferrozineassay % of

metalchelation

E.E AE EE AE

Carrot 17.420.93 15.450.12 15.531.18 12.320.21

Cabbage 30.751.56 16.871.45 21.461.04 18.781.2

Broccoli 57.530.82 50.232.2 61.331.15 50.040.65

Jack fruit

seeds

24.620.42 20.121.02 56.210.81 45.250.14

pumpkin 84.712.5 70.210.25 37.650.86 25.390.21

Table; 1 shows the radical scavenging activity in DPPH assay with compare to alcoholic andaqueous 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);EEethanolic extract, AE- aqueous extract

Type of

samples

**TPC (mg

GAE/ g)*TFC (mg

QE/g)

EE AE EE AECarrot 49.830.23 55.230.33 15.50.66 17.650.42Cabbage 38.51.42 45.46.02 56.140.25 55.360.35

Broccoli 35.40.36 50.250.11 76.150.35 75.390.19Jack fruit

seeds

49.62.2 65.650.93 33.240.54 36.281.42

pumpkin 31.701.3 39.320.12 13.700.58 12.451.54

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 quercetineequivalents 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,

EEethanolic extract, AE- aqueous extract


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Table 3; shows the radical scavenging activity of the ethanolic extract at different

concentration, Values are represented as mean SD (n=03);

Table 4; shows the radical scavenging activity of the aqueous extract at differentconcentration. 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 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

Sample extract Radical scavenging activity(in differentconcentration)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


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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.


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The graphical representation of our results has been shown in the figures below.

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.


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.

0

10

20

30

40

50

60

70

8090

100

10 20 40 100

%i

nhibition

concentration (l/100l)

carrot

cabbage

brocoli

jackfruit seeds

pumpkin

0

20

40

60

80

100

120

1 2 3 4

%inhibition

concentration g/ml

carrot

cabbage

brocoli

jackfruit seeds

pumpkin


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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.


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.

0

10

20

30

40

50

60

70

10 20 40 100

%c

helation

concentration g/ml

carrot

cabbage

brocoli

jackfruit seed

pumpkin

0

20

40

60

80

100

120

140

160

10 20 40 100

%c

helation

concentration g/ml

pumpkin

jackfruit seed

brocoli

cabbage

carrot


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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.

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.

0

10

20

30

40

50

60

70

80

90

100

carrot cabbage brocoli jackfruit seed pumpkin

%i

nhibition

samples extract

EE

AE

0

10

20

30

40

50

60

70


%c

helation

sample extract

EE

AE


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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.

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.

0

10

20

30

40

50

60

70

80


totalantioxidan

tactivity

samples extract

EE

AE

0

10

20

30

40

50

60

70

80

90

carrot cabbage brocoli jackfruit seeds pumpkin

totalantioxidantactiv

ity

samples extract

EE

AE


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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.

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.

17.42 30.7557.53

24.62

84.7115.5321.46

61.33

56.21

37.6549.83

38.5

35.4

49.6

31.7

15.5

56.1476.15

33.24

13.7

carrot cabbage brocoli jackfruit seeds pumpkin

TFC

TPC

Ferrozine

DPPH

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

carrot cabbage brocoli jackfruit seeds pumkin

TFC

TPC

Ferrozine

DPPH


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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 ofBrassica oleracea and Daucus carotausing 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

cultivarcarrot 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
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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. Jagtapetal (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. Thepusa 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.
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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 familyhave 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. Jagtapetal (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 theseeds 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 g/ml
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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 40g/ml conc. but in

100g/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 100g/ ml conc. surprisingly in 10g/ml conc.

pumpkin have shown the high value which is not so high in 100 g/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).


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

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