Evaluation and Comparison of Antioxidant Enzymes ...

Research & Reviews: A Journal of Food Science & Technology Volume 2, Issue 2, August 2012, Pages __________________________________________________________________________________________

Evaluation and Comparison of Antioxidant Enzymes from Different Local Varieties of Banana (Musa sp.) P. Poongodi*, S. Mohana Sundaram, J. Arun, S.Thirumalai, M. Pennnarasi, M.Prassana Department of Biotechnology, Karpaga Vinayaga College of Engineering and Technology, Maduranthagam, TamilNadu-603308, India ABSTRACT The present study mainly focused on quantification and comparison of enzymatic antioxidants from pulp extracts of nine varieties of banana, viz., Kadali, Karpooravalli, Monthan, Nenthiran, Pachainadan, Poovan, Rasthali, Robusta and Sevvazhai. The enzymatic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione s-transferase, glucose-6-phosphate dehydrogenase, polyphenol oxidase were quantified from banana pulp extract. The data obtained were subjected to statistical analysis and results were compared. All the banana varieties exhibited significant amount of antioxidant enzymes. Kadali, Monthan, Rasthali, Pachainadan, Poovan, Robusta showed highest amount of antioxidant enzymes compared to other varieties. Keywords: Free radicals, antioxidant enzymes, banana varieties, catalase, superoxide dismutase

*Author for Correspondence E-mail: [email protected], Tel: 91-44-27565486

system which includes various enzymes and

1. INTRODUCTION

high and low molecular weight antioxidants. Free radicals are a group of active molecules

Antioxidants

with unpaired electrons that react with

donating one of their own electrons, ending

biological systems, resulting in cell damage.

the electron stealing reaction. Foods rich in

Free radicals are involved in both the process

antioxidants have been shown to play an

of aging and the development of cancer [1].

essential

They attack many cellular targets including

cardiovascular

membranes, proteins and nucleic acids, and

neurodegenerative diseases, the most well-

cause structural damage to the cellular DNA.

known

These structural changes manifest as point

Alzheimer's

mutations and chromosomal alterations in

problems caused by cell and cutaneous aging

cancer-related

[4].

elderly

genes

people

are

[2].

Consequently,

predisposed

to

neutralize

role

of

in

free

the

radicals

prevention

diseases;

which

are

diseases,

by

of

cancers,

Parkinson’s

and

inflammation

and

the

development of cancer. Fortunately, certain

As with the chemical antioxidants, cells are

antioxidant supplements like vitamins C, E

protected against oxidative stress by an

and

much

interactive network of antioxidant enzymes

oxidative damage to DNA and thus reduce the

[5]. The antioxidant enzymes and free radical

ability of the oxidants to induce cancer [3]. To

scavengers

deal with the free radicals or so called ROS,

mechanism against the deleterious actions of

the body is equipped with an effective defense

ROS. Some of the antioxidant enzymes that

many

enzymes

can

prevent

may

© STM Journals 2012. All Rights Reserved Page 1

provide

a

defensive


are found to provide a protection against the

fruit cultivars are a man-made complex based

ROS are superoxide dismutase, catalase,

on two wild diploid species originating from

glutathione

glucose-6-

South-East Asia: Musa acuminata Colla (AA),

ascorbate

which is highly polymorphous, with spindly

oxidase [6]. Superoxide dismutase is an

plants that grow in clumps, and Musa

enzyme that repairs cells and reduces the

balbisiana Colla (BB), a homogeneous hardy

damage done to them by superoxide, the most

plant with a massive pseudo-trunk. There are

common free radical in the body. Catalase

diploid, triploid or tetraploid genome groups.

helps the body to convert hydrogen peroxide

The main genome groups are AA, AB, AAA,

into water and oxygen, thus preventing the

AAB and ABB [7]. The present study aimed to

formation of carbon dioxide bubbles in the

quantitate

blood. Glutathione peroxidase, the body’s

enzyme content in the pulp extracts of nine

primary antioxidant, which is in virtually

local varieties of banana may be useful to

every cell, is one of the most powerful free

combat free-radical-related diseases.

peroxidase,

phosphatedehydrogenase

and

and

compare

the

antioxidant

radical fighters that the body has in its arsenal. Glutathione s-transferase, family of enzymes

2. MATERIALS AND METHODS

utilize glutathione in reactions contributing to the transformation of a wide range of

2.1. Preparation of Banana Sample

compounds, including carcinogens, therapeutic

The

drugs, and products of oxidative stress. These

Karpooravalli,

enzymes play a key role in the detoxification

Pachainadan, Poovan, Rasthali, Robusta and

of such substances. Glutathione reductase is an

Sevvazhai, were collected locally from various

enzyme that reduces glutathione disulfide

places in Sathyamangalam (Tamil Nadu,

(GSSG) to the sulfyhydryl form GSH, which

India).

is an important cellular antioxidant. Bananas

authenticated by Dr. T. N. Balamohan,

are one of the most popular foods in the world

Professor and Head, Faculty of Horticulture,

and it will be known that fruits contain various

Tamil

antioxidant compounds such as gallocatechin

Coimbatore. A voucher specimen of the

and dopamine. Banana should be considered to

sample has been deposited in the herbarium of

be a good source of natural antioxidant for

the department. One gram of banana pulp was

foods and functional food source against

weighed and homogenized with 10 mL of

cancer

spp.,

0.2 M phosphate buffer pH 7.4. The sample

comprising banana and plantain are among the

was centrifuged at 4 ° C and the supernatant

world’s leading fruit crops and in terms of

was

economic value, it is the number five

antioxidants.

and

heart

disease.

Musa

banana

The

Nadu

used

for

agricultural crop in world trade. The edible


varieties,

viz.,

Monthan,

banana

Kadali, Nenthiran,

specimens

Agricultural

estimation

were

University,

of

enzymatic


2.1.1. Chemicals

after the addition of the acid reagent. The

All the solvents and other chemicals which

tubes were then heated for 10 min and the

were used during this study were purchased

color developed was read at 530 nm. The

from Merck and S. D. Fine-Chemicals,

activity of catalase was expressed as µmole of

Mumbai.

H2O2 decomposed/min/mg protein.

2.2. Methods

2.2.3. Estimation of Glutathione Peroxidase

2.2.1. Estimation of Superoxide Dismutase

Assay of glutathione peroxidase was done

The assay of superoxide dismutase was done

according to the procedure of Ellaman [10].

according to the procedure of Das [8]. In this

The reaction mixture contained 0.4 mL of

method, 1.4 mL of reaction mixture containing

buffer, 0.1 mL of sodium azide, 0.2 mL of

1.1 mL phosphate buffer, 75 µL methionine,

reduced glutathione, 0.1 mL of H2O2, 0.2 mL

40 µL triton X-100, 75 µL hydroxylamine

of enzyme and 1.0 mL of water were added to

hydrochloride, 100 µL EDTA and 100 µL of

a final incubation volume of 2.0 mL. The

sample was put into test tubes. The tubes were

tubes were incubated for 0, 30, 60, 90 s. The

pre-incubated at 37 ° C for 5 min. After

reaction was then terminated by the addition of

incubation, 80 µl of riboflavin was added. The

0.5 mL of TCA. To determine the glutathione

control tube contained buffer instead of

content, 2.0 mL of supernatant was removed

sample. The test and control tubes were

by centrifugation and added to 3.0 mL

exposed to UV light for 10 min. After the

disodium hydrogen phosphate solution and

exposure, 1 mL of Griess reagent was added to

1.0 mL of DTNB reagent. The color developed

all the tubes and absorbance of pink color

was read at 412 nm. Standards in the range of

developed was measured at 543 nm. One unit

200–1000 µg were taken and treated in the

of enzyme activity was defined as the amount

similar manner. The activity was expressed in

of SOD capable of inhibiting 50% of nitrite

terms of µg of glutathione utilized/min/mg

formation under assay condition.

protein.

2.2.2. Estimation of Catalase

2.2.4.

The assay of catalase was carried out

Transferase

according to the method of Sinha [9]. The

This enzyme was assayed by the procedure of

assay mixture contained 0.5 mL of H2O2,

Habig et al. [11]. To 1.0 mL of buffer, 0.1 mL

1.0 mL of buffer and 0.4 mL water; 0.2 mL of

of sample, 1.7 mL of water and 0.1 mL of

the enzyme was added to initiate the reaction.

CDNB were added and incubated at 37 ° C for

2.0 mL of the dichromate/acetic acid reagent

5 min. After incubation, 0.1 mL of reduced

was added after 0, 30, 60, 90 s of incubation.

glutathione was added. The increase in the

To the control tube, the enzyme was added

optical density of the enzyme for 1, 2, 3 min

Estimation


of

Glutathione

S-


was measured against blank at 340 nm. The

sample, 3.0 mL of buffer and 1.0 mL of

enzyme activity is calculated in terms of

catechol. The contents were mixed well and

µmoles of CDNB conjugate formed/min/mg

increased absorbance was measured at every

protein.

30 s up to 3 min. The enzyme activity was calculated

in

terms

of

0.001

OD

change/min/mg protein.

2.2.5. Estimation of Glutathione Reductase Assay of glutathione reductase was done by the method of Beutler [12]. 2 mL of sample,

3. RESULTS AND DISCUSSION

1.5 mL of buffer, 0.5 mL GSSG and 0.1 mL NADPH was added. The decrease in optical

3.1.

density of the enzyme was measured against

Metabolizing Enzymes

the blank at 340 nm. The enzyme activity is

The superoxide dismutase activity in pulp

calculated in terms of µmoles of NADPH

extracts of nine banana varieties varied from

oxidized/min/mg protein.

0.86 to 4.80 units mg−1 protein as depicted in

Activities

Hydrogen

Peroxide

Table I. The highest activity was found in 2.2.6. Estimation of Glucose-6-Phosphate

Rasthali and lowest activity was found in

Dehydrogenase

Karpooravalli banana. Therefore, Rasthali

This enzyme was assayed by the method of

banana has the highest potential to scavenge

Balinsky and Bernstein [13]. 0.4 mL of Tris

superoxide radicals. Superoxide scavenging

HCl buffer, 0.2 mL of NADP, 0.2 mL of

effect of alcoholic extracts of orange, tomato,

magnesium chloride, 1.0 mL of water and

grapes and gooseberries [15] were reported

0.2 mL of sample were taken in a test tube.

earlier.

The reaction was started by the addition of 0.2 mL

of

glucose-6-phosphate

and

the

The activity of catalase enzyme in pulp

increase in OD was measured at 340 nm. The

extracts of nine varieties banana varied from

activity was expressed in terms of units/mg of

6.19 to 26.17 units mg−1 protein as represented

protein, in which one unit is equal to the

in Table I. The highest hydrogen peroxide

amount of enzyme that brought about change

decomposing activity was recorded in Robusta

in OD of 0.01/min.

banana and least activity was found in Kadali banana. The presence of enzymes in various

2.2.7. Estimation of Polyphenol Oxidase

fruit varieties has already been reported [16].

The enzyme polyphenol oxidase was assayed

In plants, antioxidant enzymes namely catalase

by the procedure of Rocha and Morais [14].

[17] and peroxidase [18] have been shown to

The reaction mixture contained 2.0 mL of

increase when subjected to stress conditions.



Table I: Activities of Hydrogen Peroxide Metabolizing Enzymes in Pulp Extracts of Local Varieties of Banana. Catalase

Banana Varieties

Superoxide Dismutase

Kadali

1.73c ± .074

8.11b ± 0.105

Karpooravalli

0.86a ± 0.011

12.67c ± 0.065

Nendran

1.57c ± 0.264

13.69e ± 0.070

Monthan

3.83e ± 0.116

6.19a ± 0.085

Pachainadan

2.05d ± 0.132

16.59g ± 0.080

Poovan

1.38b ± 0.105

14.59f ± 0.055

Rasthali

4.80f ± 0.115

12.80d ± 0.095

Red

1.61c ± 0.068

19.74h ± 0.194

Robusta

1.94d ± 0.024

26.17i ± 2.217

Values represent mean ± SD of 3 replicates. Means followed by a common letter are not significantly different at the 5% level by DMRT. Units:

SOD – 50% inhibition of nitrate min−1 mg protein−1.

CAT – n moles of H2O2 decomposed min−1 mg protein−1. 3.2. Activities of Glutathione Utilizing

role in regulating the concentration of H2O2

Enzymes

and a wide variety of organic peroxides [19].

Glutathione peroxidase is the general name of

The activities of glutathione reductase enzyme

an enzyme family with peroxidase activity

in pulp extracts of nine varieties of banana

whose main biological role is to protect the

ranges from 1.29 to 9.4 units mg−1 protein. The

organism from oxidative damage. As depicted

Robusta and Kadali banana varieties exhibit

in Table II, the activity of glutathione

the highest activity and the lowest activity was

peroxidase enzyme in pulp extracts of nine

recorded in Poovan banana.

varieties of banana ranged from 0.012 to 0.083 units mg−1 protein.

The glutathione s-transferase activity in pulp extracts of nine varieties of banana ranges

The highest activity was found in Poovan

from 2.58 to 50.61 units mg−1 protein. The

banana and the least activity was found in

highest activity was shown in Pachainadan

Kadali banana. It has been well established

banana and the least activity was found in

that GPx a selenium enzyme, plays a major

Rasthali banana.



Table 2: Activities of Glutathione Utilizing Enzymes in Pulp Extract of Local Varieties of Banana. Banana Varieties

Glutathione Peroxidase

Glutathione

Glutathione S-

Reductase

Transferase

Kadali

0.012a ± .002

9.47f ± 0.113

22.16e ± 0.543

Karpooravalli

0.029b ± 0.002

5.60d ± 0.193

6.32c ± 0.190

Nendran

0.043c ± 0.004

2.27c ± 0.009

35.43f ± 0.292

Monthan

0.032b ± 0.002

2.17c ± 0.129

2.48a ± 0.405

Pachainadan

0.046c ± 0.003

1.74b ± 0.009

50.61g ± 0.207

Poovan

0.083e ± 0.004

1.29a ± 0.175

8.90d ± 0.166

Rasthali

0.057d ± 0.004

8.10e ± 0.159

2.58a ± 0.246

Red

0.044c ± 0.008

5.34d ± 0.345

8.54d ± 0.156

Robusta

0.055d ± 0.005

9.47f ± 0.374

3.23b ± 0.156

Values represent mean ± SD of 3 replicates. Means followed by a common letter are not significantly different at the 5% level by DMRT. Units: GPx – n moles of GSH consumed min−1 mg protein−1. GR – µmoles of GSH utilized min−1 mg protein−1. GST – µmoles of CDNB-GSH conjugate formed.

3.3.

Activities

of

Glucose-6-Phosphate

Dehydrogenase and Polyphenol Oxidase

and least activity was seen in Red banana. The

The

presence

activities

dehydrogenase

of and

glucose-6-phosphate polyphenol

oxidase

of

glucose-6-phosphate

dehydrogenase in various fruit varieties has

activities in pulp extracts of nine varieties of

already been reported [15]

banana are represented in Table III. The glucose-6-phosphate dehydrogenase activities

The polyphenol oxidase activity in pulp

in pulp extracts of nine banana varieties

extracts of nine banana varieties varied from

−1

protein.

0.08 to 16.53 units mg−1 protein. Monthan

The highest activity was recorded in Poovan

banana topped among other varieties of

banana.

banana and the least activity was recorded in

ranged from 0.75 to 9.70 units mg

Hence,

it

regeneration

of

maintaining

NADPH

helps

reduced

in

efficient

glutathione

by

Kadali banana as depicted in Table III.

Reduced

Occurrence and change of polyphenol oxidase

glutathione in turn forms conjugate with free

activity in some banana cultivars during fruit

radicals thereby combating several diseases

development was reported earlier [20].

levels.



Table III: Activities of Glucose-6-Phosphate Dehydrogenase and Polyphenol Oxidase in Pulp Extracts of Nine Varieties of Banana.

Banana varieties

Glucose-6-Phosphate Dehydrogenase

Polyphenol Oxidase

Kadali

0.99bc ± 0.083

0.08a ± 0.007

Karpooravalli

3.19e ± 0.180

0.09a ± 0.007

Nendran

2.49d ± 0.176

1.29c ± 0.1

Monthan

0.24a ± 0.017

16.53h ± 0.075

Pachainadan

2.26d ± 0.325

0.18b ± 0.009

Poovan

9.70f ± 0.275

13.31f ± 0.11

Rasthali

1.16c ± 0.529

13.93g ± 0.097

Red

0.75b ± 0.040

9.14d ± 0.097

Robusta

2.35d ± 0.105

12.84e ± 0.120

Values represent mean ± SD of 3 replicates. Means followed by a common letter are not significantly different at the 5% level by DMRT. Units:

G6PD, PPO – 0.01 OD min−1 mg protein−1.

Robusta. The pulp extracts were assayed for

4. CONCLUSIONS

different enzymatic antioxidant compounds The human body has been naturally blessed

like SOD, catalase, glutathione peroxidase,

with

glutathione

a

number

of

disease-combating

reductase,

glutathione

s-

compounds that are sensibly programmed to

transferase, etc. The results obtained were

act instantaneously. It is during the deficit of

subjected to two-way ANOVA and the

these substances that our body becomes

varieties were ranked according to their

afflicted with various ailments that may

enzymatic antioxidant content. All the nine

subsequently turn chronic. To prevent this, it is

banana

often recommended that people should intake

antioxidant enzyme content with Rasthali,

natural supplements of these substances and

Robusta,

antioxidants take the priority lead considering

Monthan topping the results.

varieties

Poovan,

showed

Kadali,

significant

Pachainadan,

their valuable functions in the body. The present work has been undertaken to evaluate

ACKNOWLEDGEMENT

and compare the antioxidant enzymes content of nine varieties of banana, viz., Kadali,

The authors are grateful to Mrs. Meenakshi

Karpooravalli,

Annamalai,

Monthan,

Nendran,

Pachainadan, Poovan, Rasthali, Red and

Director,

Karpaga

Vinayaga

College of Engineering and Technology, for



the complete support throughout the work with

13. Balinsky D., Bernstein, R. E. Biochemica

timely and valuable discussions.

et Biophysica Acta. 1963. 67. 13–15p. 14. Rocha A. M. C. N., Morais A. M. M. B. Food control. 2001. 12. 85–90p.

REFERENCES

15. Rani, P., Meena, K., Unni, K., et al. Indian 1. Halliwell,

B.

Acta

Neurologica

Journal of Clinical Biochemistry. 2004.

Scandinavica. 1989. 126. 23–33p.

19(2). 103–110p.

2. Cerutti, P., Ghosh, R., Oya, Y., et al.

16. Sivasankar, V., Moorthi, A., Sarathi

Environmental Health Perspectives. 1994.

Kannan, et al. Indian Journal of Clinical

102(suppl 10). 123–130p.

Biochemistry. 2004. 19(2). 103–110p.

3. Shigenga, K. K., Tory, M. H. and Bruce,

17. Hertwig, B. Steb, P. and Feierabend.

N. A. Proceedings of National Science

Journal

Academy. 1994. 91. 10771–10778p.

100(17). 1547–1553p.

4. Shahidi, F. and Wanasundara, P. K. J.

of

Plant

physiology.

1999.

18. Nouchi, I. Soil Science & Plant Nutrition.

Critical Reviews in Food Science and

1993. 39. 309–320p.

Nutrition. 1992. 8. 122p.

19. Sies, H. Planta Medica. 1991. 61. 515–

5. Sies, H. Experimental Physiology. 1997.

518p.

82. 291–295p.

20. Chang-Peng yang, Zhi-Rong nong, Jian-

6. Uday Bandyopadhyay, Dipak Das and

Lin lu, et al. Food Science and Technology

Ranajit Banerjee, K. Current Science.

Research. 2004. 10(1). 75–78p.

1990. 77. 658–666p. 7. Ramakrishnan Baskar, Selvaraj Shrisakthi, Babu Sathyapriya, et al. Food and Nutrition Sciences. 2011. 2. 1128–1133p. 8. Das, S., Vasisht, S., Snehlata, R., et al. Current Science. 2000. 78. 486–487p. 9. Sinha, A. K. Analytical Biochemistry. 1972. 47. 389p. 10. Ellaman, G. C. Archives of Biochemistry and Biophysics. 1959. 82. 70–77p. 11. Habig W. H., Pabst M. J., and Jakboy W. B. Journal of Biological Chemistry. 1973. 249. 7130–7139p. 12. Beutler E. A Manual of Biochemical Methods. Grune and Stratton, New York. 1984. 112–114p.
