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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