EVALUATION OF ANTIOXIDANT CAPACITY AND PHENOL CONTENT OF FIVE DIFFERENT VEGETABLES FROM KOLKATA: A COMPARATIVE STUDY

by Ipsita Bhattacharya,BRSN College M.Sc in Food & Nutrition 2012-06-15

Abstract:
The antioxidant capacity and phenol content of carrot (Daucus carota), cabbage (Brassica

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

Introduction
Antioxidants are important in neutralizing free radicals. Free radicals are generated during normal body metabolism as molecules with incomplete electron pairs which make them more chemically unstable than those with complete electron pair (Fang et al. 2002). In present times, it is believed that the regular consumption of dietary antioxidants may reduce the risk of several serious diseases. Regular consumption of vegetables has always been associated with health benefits, but their mechanism has become clear only in the recent decades. Vegetables contain a wide variety of biologically active, non-nutritive compounds known as phytochemicals. These phytochemicals impart health benefits beyond basic nutrition. Recently, there have been great efforts to find safe and potent natural antioxidants from various plant sources. As harmless sources of antioxidants, edible fruits have been investigated for their antioxidant properties, the role of food constituents as essential nutrients to one of preventing or delaying the premature onset of chronic disease late in life has now been generally accepted. Reactive oxygen species (ROS), such as hydrogen peroxide ( and hypochlorous acid (HOCl), and free radicals, such as the hydroxyl radical (OH) and superoxide anion (O2), are produced as normal products of cellular metabolism. Rapid production of free radicals can lead to oxidative damage to biomolecules and may cause disorders such as cancer, diabetes, inflammatorydisease, asthma, cardiovascular diseases, neurodegenerative diseases, and premature aging(Young and Woodside, 2001). Many vegetables contain a large amount of polyphenols, vitamin C, vitamin E, selenium, 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

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 antiageing 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 polyphenolic 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, antioxidants, 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 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.

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

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

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

This study from JIIN-TZONG GUO etal (2005) shows the edible portion and the seed of avocado, jackfruit,longan, mango and tamarind were studied. were taken for study. The seeds showed much higher antioxidant and phenolic activity than the edible portion. Jack fruit seeds were also considered. Another study by Fei Que1,2 etal (2006) shows antioxidant activity of the major poly phenolic compound was observed in broccoli. Total flavonoid and flavonol compound were also observed in this syudy.

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

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

This study from Wang, H (2006) shown that flowers stem and leaves of broccoli were taken in DPPH, reducing power and metal chelating activity. They were taken in freeze dried condition. All of these part, The broccoli stem exhibited the highest chelating ability among three parts of broccoli & the acetone extracts from stems hardly showed any chelating ability as compared to alpha tocopherol and BHA. .

Another study of

Webb, D. (2008) was showing

changing in boiling time of cooking

pumpkin, is compared with the stir frying showed a higher free radical scavenging activity than the other assays. Boiling time reduces the power effectively. Another study by Fei Que1,2 etal 2006. Was shown that the antioxidant activities of methanol extracts from pumpkin flours were studied in terms of total antioxidant activity, reducing power, free radical scavenging, superoxide anion radical scavenging and metal chelating activities. Different antioxidant activity assays were done with hot dried and freeze dried condition of pumpkin. Hot dried pumpkin flowers have shoed a stronger activity rather than the freeze dried flower. Another study by (Marja P etal 1999) have shown some plants including broccoli and cabbage. In addition, potato peel and beetroot peel extracts showed strong

antioxidant effects. To utilize these significant sources of natural antioxidants, further characterization of the phenolic composition is needed.

Aims and Objectives

This study has obvious focussed on some aims and objectives which are reliable and valid for further extension and growth of this study. The objectives are summarized below Characterization of different parts of five different vegetables in terms of their antioxidant potency. Determination of the comparative 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 l 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 6l, 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 2l, 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, 750l 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 2,2diphenyl- 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

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.

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 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. 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 E.E 17.42 30.75 57.53 24.62 AE 15.45 16.87 50.23 20.12 Ferrozine assay % of metalchelation EE 15.53 1.18 21.46 1.04 61.33 1.15 56.21 0.81

Carrot 0.93 0.12 0.21 Cabbage 1.56 1.45 1.2 Broccoli 0.82 2.2 0.65 Jack fruit 0.42 1.02 0.14 seeds pumpkin 84.71 2.5 70.21 0.25 37.65 0.86 25.39 0.21 Table; 1 shows the radical scavenging activity in DPPH assay with compare to alcoholic and aqueous extract. Also showing % of metal chelation with Ferrozine assay with comparing of two extracts. The concentration have taken in 100 mg /gm and Values are represented as mean SD (n=03); EE ethanolic extract, AE- aqueous extract

AE 12.32 18.78 50.04 45.25

Type of samples

Carrot 0.33 0.42 Cabbage .02 0.35 Broccoli 0.11 0.19 Jack fruit 0.93 1.42 seeds pumpkin 31.7 01.3 39.32 0.12 13.70 0.58 12.45 1.54 Table; 2 shows that TPC and TFC determination with comparing their ethanolic and aqueous extract. Values are represented as mean SD (n=03); *Values are expressed as quercetine equivalents in mg/g extract. The concentration have taken in 100 mg /gm and **Values are expressed as gallic acid equivalent in mg/g extract TPC- total phenolic content, TFC- total flavonoid content, EE ethanolic extract, AE- aqueous extract

**TPC (mg GAE/ g) EE 49.83 0.23 38.5 1.42 35.4 0.36 49.6 2.2

AE 55.23 45.46 50.25 65.65

*TFC (mg QE/g) EE 15.5 0.66 56.14 0.25 76.15 0.35 33.24 0.54

AE 17.65 55.36 75.39 36.28

Sample extract

Carrot cabbage Broccoli Jack fruit seeds

pumpkin

Radical scavenging activity(in different concentration)in DPPH 10 mg/gm 20 mg/gm 40 mg/gm 100 mg/gm 13.6% 0.23 11.13% 0.66 15.05% 0.54 17.42% 0.19 10.2% 0.93
31.3% 0.66 10.7% 0.93
32.7% 0.33

11.6% 0.23
44.5% 0.66 16.7% 0.33
43.22% 0.23

16.0% 1.54
49.6% 0.54 20.3% 1.54
53.79% 0.23

30.75% 0.23
57.53% 1.54 24.62% 0.19
84.71% 1.54

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

Sample extract

Carrot cabbage Broccoli Jack fruit seeds pumpkin

Radical scavenging activity(in different concentration)in DPPH 10 mg/gm 20 mg/gm 40 mg/gm 100 mg/gm 12.6% 0.23 11.13% 0.66 15.05% 0.33 15.45% 0.19 09.2% 0.93
23.3% 0.19 10.7% 0.66 30.7% 0.33

10.6% 0.19
44.5% 0.23 16.7% 0.93 42.22% 0.33

16.0% 0.93
49.6% 0.66 18.3% 0.19 50.79% 0.23

16.87% 0.23
50.23% 0.33 20.12% 0.93 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

Carrot Cabbage brocoli Jackfruit seeds pumpkin

Metal chelating Activity(In Different Concentration)with Ferrozine 10 20 40 10.9% 0.93 12.1% 0.19 14.41% 0.93 09.5% 0.33 16.24% 0.19 25.57% 0.19 20.0% 0.19 10.8% 0.93 23.24% 0.33 36.28% 0.19 18.57% 0.19 15.4% 0.19 27.0% 0.19 50.71% 0.93 25.14% 0.66

100 15.34% 0.33 21.6% 0.19 61.0% 0.33 56.42% 0.93 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

Carrot Cabbage brocoli Jackfruit seeds pumpkin

Metal chelating Activity(In Different Concentration)with Ferrozine 10 20 40 10.9% 0.93 11.1% 0.19 12.01% 0.93 09.5% 0.33 16.24% 0.19 25.57% 0.19 20.0% 0.19 10.8% 0.93 23.24% 0.33 36.28% 0.19 22.57% 0.19 15.4% 0.19 27.0% 0.19 40.71% 0.93 24.14% 0.66

100 12.34% 0.33 18.6% 0.19 50.0% 0.33 45.42% 0.93 25.0% 0.19

Table 6; shows metal chelating activity of aqueous extract (at Different Concentration) by Ferrozine Assay. Values are represented as mean SD (n=03);

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

The graphical representation of our results has been shown in the figures below.
100 90 80 70 60 50 40 30 20 10 0 10 20 40 100 concentration (l/100l)

% inhibition

carrot cabbage brocoli jackfruit seeds pumpkin

Figure 1; exhibits a comparison between five study samples according to their concentration. In DPPH radical scavenging activity of ethanolic extract, there significant incretion of all samples. Pumpkin and broccoli have the highest value in total but broccoli lower value in 40 conc. Values are represented as mean.
120 100 80 %inhibition 60 40 20 0 1 2 3 4 concentration g/ml carrot cabbage brocoli jackfruit seeds pumpkin

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.

70 60 50 % chelation 40 30 20 10 0 10 20 40 100 concentration g/ml carrot cabbage brocoli jackfruit seed pumpkin

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.
160 140 120 % chelation 100 80 60 40 20 0 10 20 40 100 concentration g/ml pumpkin jackfruit seed brocoli cabbage carrot

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.

100 90 80 70 % inhibition 60 50 40 30 20 10 0 carrot cabbage brocoli samples extract jackfruit seed pumpkin EE AE

Figure 5 exhibits a comparison of antioxidant activity of five vegetables. Radical scavenging activity using DPPH was done using ethanolic & aqueous extracts. Pumpkin has the highest inhibition value than others, whereas carrot shows the lowest. Comparatively cabbage shows relatively lower value in aqueous respect to other aqueous extract with respect to their inhibition of free radicals.
70 60 50 % chelation 40 30 20 10 0 carrot cabbage brocoli sample extract jackfruit seed pumpkin EE AE

Figure 6. exhibits a comparison of antioxidant activity of five vegetables. Metal chelating activity using Ferrozine was done using ethanolic & aqueous extracts. Brocoli have the highest chelation value than others, whereas carrot shows the lowest and also Comparatively shows relatively lower value in aqueous respect to other aqueous extract with respect to their
chelation of transition metals.

80 70 total antioxidant activity 60 50 40 30 20 10 0 carrot cabbage brocoli samples extract jackfruit seed pumpkin EE AE

Figure 7; exhibits a comparison of antioxidant activity of five vegetables. Total phenolic content determination was done using ethanolic & aqueous extracts. Jackfruit seeds have the highest value in aqueous than others, whereas pumpkin shows the lowest and also Comparatively shows relatively lower value in aqueous respect to other aqueous extract with
respect to their total phenolic content.
90 80 total antioxidant activity 70 60 50 40 30 20 10 0 carrot cabbage brocoli samples extract jackfruit seeds pumpkin EE AE

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.

15.5 56.14 76.15

13.7 33.24 31.7 TFC TPC Ferrozine 61.33 56.21 84.71 57.53 24.62 brocoli jackfruit seeds pumpkin DPPH

49.6 49.83 38.5 35.4

37.65

15.53 17.42 carrot

21.46

30.75 cabbage

Figure 9; shows a vast comparison of all study have done with all samples in ethanolic extract. Pumpkin have highest value in DPPH assay, whereas carrot shows highest in TPC. But jackfruit seeds have almost highest value in ferrozine assay. TFC value is more or less same to all, but pumpkin have lower.
12.45 17.65 55.36 75.39 39.32 TPC 65.65 Ferrozine DPPH 50.04 12.32 15.45 carrot 45.25 18.78 50.23 16.87 cabbage brocoli 20.12 jackfruit seeds pumkin 70.21 36.28 TFC

55.23 45.46

50.25

25.39

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

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

cultivar carrot have showed a relatively high value (Agnieszka Bartoszek etal (2007) from our
study which was assayed in same concentration. But carrote of our region, there is a significantly low scavenging value which is probably caused by the weather and for cultivation technique. Results of table 1 showed metal chelating activity with Ferrozine. Our study exhibited greater scavenging activity for pumpkin However broccoli showed hifgest metal chelation power followed by pumpkin and jackfruit seeds. At 100mg/ml concentration, carrot extract showed lesser in metal chelation than scavengingo. But in other cultivar (pusa kesar cultivar), there is a relatively higher value in metal chelating (Agnieszka Bartoszek etal (2007). Other study showed relatively high amount of metal chelating activity by jackfruit pulp then the seeds of jackfruit , and the seeds have almost no value for metal chelation (Umesh B. Jagtap, 2010), but in our study there is a very significant value of metal chelation in seeds of jackfruit. Another study assayed with several type of seeds and proved jackfruit seeds have more

higher value in metal chelation than the edible portion of that (JIIN-TZONG GUO etal (2005) and this activity is more or less same with our study. In aqueous extract, there is a significant reduction of these activity i.e. same as DPPH assay (figure 6). The stem of broccoli exihibits a higher scavenging value than the flowers of broccoli, but the flower have showed relatively high in metal chelating activity than the stem and also showed a greater value in ethanolic and aqueous extract (Wang, H (2006). Our study also showed a greater value in flower of broccoli in metal chelating activity (table 1), and also showed comparatively high value in aqueous extract with respect to other samples (figure 6). So we can say that broccoli is not good scavenger but a very good chelator. Our study also support pumpkin as a very good scavenger but not so good in metal chelation. So we can recommend this two vegetables in our regular diet along with jackfruit seeds which will enhance antioxidant status. Total phenolic content is also determined in all five samples (Table 2). Here we are surprised with our result because carrots have been known for very good source of antioxidant. Our variety of carrote showed very poor antioxidant activity by DPPH and Ferrozine assay. But its TPC found to be good. Table 2 as well as Figure 9 showed lowest TPC in pumpkin compared to broccoli, cabbage, jackfruit seeds. The vegetables from brassica family i.e. cabbage and broccoli have been proved with very high in total phenolic content and they have a significant role in reducing heart diseases ( Umesh B. Jagtap etal (2010). This study is matched with our study as the broccoli and cabbage have showed significantly high TPC (table 2). Here we observe a differentiation from above two assays. Aqueous extract have showed relatively higher value i.e. happened with all my study samples in total phenolic content than the ethanolic extract (figure 7). Jackfruit seeds have the highest value and carrots have also the higher TPC in basically aqueous extract. Now we can say these are very much beneficial for our diet, as the water act as a good solvent. This is basically caused by the phenol group of poly phenol which is merged with the OH group of ethanol, and reduces the polyphenol activity, but in water there is no such chance to happen. The pusa kesar cultivars have showed higher TPC than the other cultivar in aqueous extract also (Agnieszka Bartoszek etal (2007). This study support our results. As the carrot from Kolkata have also showed such evidence in Total Phenolic Content.

Similar study was done for total flavonoid content these five samples in both ethanolic and aqueous extract. As the flavonoid is the derivative compound of poly phenol so there should not be such significance variance in TFC determination. But there is reduced the value of TFC in carrot which one was high in TPC. Here broccoli have showed very significant amount of flavonoid content and cabbage have also showed a significant amount of TFC after the broccoli (Table 2). The vegetables from Brassica family have showed a very high amount of flavonoid content which was conducted in both ethanolic and aqueous extract and also have showed a significant role in heart diseases (Umesh B. Jagtap etal (2010). As our study also have showed that the broccoli and cabbage (Brassica family) have very high amount of flavonoid. In aqueous extract there is more or less same value of flavonoid as the ethanolic extract . This exhibits.broccoli have the highest and carrots have the lowest amount of flavonoid content (Figure 8). Only the jackfruit seeds have showing such higher amount of flavonoid than the ethanolic in aqueous extract. The other vegetables as pumpkin and carrot have showed no such significant amount of flavonoid in both ethanolic and aqueous extract. Now we can say that in our diet the mixture of broccoli and cabbage can consumed. But as in TPC we have seen that the carrot have significant amount of phenol and in TFC the amount is reduced whether the broccoli have shown the highest amount of flavonoid. So a combination of brassica family and carrot can be consumed together to enhance the total antioxidant source in our food. Jackfruit pulps have showed a greater potentiality in TFC along with TPC than the seeds portion of jackfruit (Abdul Mueed Bidchol etal 2007). This study is not matched with our study where seeds portion have a high amount of flavonoid content in same concentration of previous study. Whether another study have showed some different, various fruit seeds are considered and among them jackfruit seeds have shown a greater potentiality in flavonoid content than others in aqueous extract (JIIN-TZONG GUO etal (2005). This value is more or less similar with my study, as this seeds have higher amount of flavonoid content along with the poly phenol content in both aqueous and ethanolic extract. So the seeds can be consumed in cooked form to encourage antioxidative status other than the edible portion of jackfruit. For the free radical scavenging assay and the metal chelating assay, four different concentrations of extract were used for standardization of our study. Mostly all of the extracts have shown a higher antioxidant activity with increase in concentration. In DPPH assay in ethanolic extract, there is significant increase in all samples, except in 40 g/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 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).

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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