International Journal of Current Research and Review (IJCRR)

Full Html


IJCRR - Vol 08 Issue 10, May

Pages: 01-04

Date of Publication: 22-May-2016


Print Article   Download XML  Download PDF

QUANTIFICATION OF ANTIOXIDANTS IN UNDERUTILIZED VEGETABLE LEAVES

Author: Mary Jenefer Sharmila P., Dorothy Jaganathan, Kumaravel S.

Category: Healthcare

Abstract:Introduction: Green leafy vegetables are an excellent source of bioactive compounds beyond human nutrition. There are many underutilized vegetable leaves which are equally a power house of bioactive compounds with reference to antioxidants. Much literature confirms that brassicca vegetables are high in antioxidants but the antioxidant studies these vegetable leaves are scarce as they are underutilized and neglected hence the present study was framed.
Aim: To analyze the total antioxidants, total phenols and total flavonoids in the leaves of cauliflower radish and beetroot leaves. Methodology: Total antioxidants were analysed using Dpph method and phenols by Folin-Ciocalteu method and Total flavonoids were determined spectrophotometrically. Results: The observations were read in triplicates. The data was analyzed statistically showing the presence of total antioxidants, total phenols and total flavonoids in leaves of cauliflower radish and beetroot which can a therapeutic role in the health of humans. Conclusion: Hence the present study reveals the benefits of these underutilized leaves which are packed with antioxidants that can prevent from all degenerative diseases and these antioxidant leaves can be also treated in formulation of functional foods and in food and health industry.

Keywords: Underutilized vegetable leaves, Total antioxidants, Total phenols, Total flavonoids, Degenerative diseases

Full Text:

INTRODUCTION
Humans have used different types of leaves as food since time immemorial. Plants possess many phytochemicals with various bioactivities including, carotenoids, ascorbic acid, tocopherol and polyphenols1 . A vegetable includes leaves, stems, roots, fl owers, seed, fruits, bulb and tubers2 . Among the food groups green leafy vegetables has a signifi cant place due to its abundant presence of vitamins, minerals bioactive health promoting compounds such as antioxidants and phytochemicals for health promotion and also in supply of, which provides benefi ts beyond basic nutrition. Though the availability of these leaves are cheap, they are highly perishable affected by post harvest losses, storage, handling and transport. Recently more attention is focused on importance of antioxidants for maintaining healthy life through many researches, books and media updates. Reactive Oxygen Species are a class of highly reactive molecules formed during aerobic life.3 A good source of natural antioxidants with reference to phenols and fl avonoids play a significant role in prevention against the formation of reactive oxygen species in maintaining a healthy body. Antioxidants are free-radical scavengers which can provide protection to living organisms from damage caused by uncontrolled production of reactive oxygen species4 .The most commonly used synthetic antioxidants are Butylated Hydroxyanisole (BHA), butylated hydroxytoluene (BHT), Propylgallate (PG) and butylated hydroquinone which have side effects such as liver damage and carcinogenesis5 .There is an emerging need for the application of natural antioxidant having less or no side effects, for use in foods or medicinal preparation in order to replace synthetic antioxidants. There is a growing interest in natural additives as potential antioxidants6 . Development of safer natural antioxidants from extracts of spices and other plant materials that can replace synthetic antioxidants is of interest7 . In this respect there are many varieties of green leafy vegetables in our locality, which are discarded and not used properly for human consumption that is antioxidant rich. Brassicas are known to possess antioxidant activity7 . Brassica vegetables such as caulifl ower and broccoli are popular and are among the most consumed in the world. Many researchers have proven the presence of potential antioxidants in caulifl ower on reviewing these perspectives an attempt was made to analyze the antioxidant activities in the leaves of caulifl ower, beetroot and radish belonging to similar plant family having high waste index.

MATERIALS AND MEHODS
The leaves of vegetables that are easily available, suitable for consumption, free of cost but neglected, discarded and are used as fodder for animals were the criteria’s considered in selection of leaves namely caulifl ower beetroot and radish. The nutritional knowledge about these leaves is also ignorant and hence avoided by majority of human population. The selected leaves were purchased fresh from the farm in Ooty Tamil Nadu. The leaves were separated from the stalks and washed twice under running tap water and blanched for 10- 15 sec. Later each variety of leaves was chopped and was spread on a fi lter paper at room temperature for two hours for the remaining water to drain the excess water from the leaves. It was then dried at 40oC over-night (12 hours) in hot air oven. The dried leaves of each variety were ground to a fi ne powder and packed in air tight containers for the analysis.

1. DETERMINATION OF TOTAL ANTIOXIDANTS
DPPH method is widely used to determine antiradical/ antioxidant activity of purifi ed phenolic compounds as well as natural plant extracts8 . Assays based on the use of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzthiazoline-6- sulfonic acid) (ABTS) radicals are among the most commonly used spectrophotometric methods for determination of the antioxidant capacity of foods, beverages, plant extracts and pure compounds due to the simple, rapid, sensitive, and reproducible procedures involved9 . The capacity of the leaf extracts to scavenge the stable 2,2’-diphenyl-2-picrylhydrazyl (DPPH) free radical was measured by extracting in 80% methanolic extract. To 0.1ml of freshly prepared caulifl ower leaf extraction, 6ml of DPPH solution was added. The test tubes were kept in dark room at 35o for one hour. The optimal density of the sample and the blank was read spectrophotometrically at 517 nm. The readings were read in triplicates. Butylated hydroxytoluene was used as standard. The same procedure was followed in beetroot and radish leaves.

2. DETERMINATION OF TOTAL PHENOLS
Polyphenols in plant extracts react with specifi c redox reagents (Folin-Ciocalteu reagent) to form a blue complex that can be quantifi ed by visible-light spectrophotometry10. The Folin-Ciocalteu method is described in several pharmacopoeias11.The leaf powders were treated with 80% ethanol for leaf extraction. To 0.5ml of freshly prepared samples each 8 ml of distilled water was added to all the test tubes. Later 0.5 ml of Follin’s Ciocalteau Reagent added to all the tubes and kept under incubation at 40ºC for 10 minutes. 1ml of Sodium Carbonate solution was added to all the test tubes. Then, the tubes were kept in the dark for incubation for one hour. The absorbance was read in triplicates using UV spectrophotometer at 660 nm using Gallic acid as standard. Readings were read in triplicates.

3. DETERMINATION OF TOTAL FLAVONOIDS
The spectrophotometric assay based on aluminium complex formation is one of the most commonly used procedure for the so-called total fl avonoid determination, as the content of these compounds is considered as an important parameter for evaluating food or medicinal plant samples12. One gram of each dried leaf sample was soaked in water over night. The contents were distilled in watt man fi lter paper. To 1.5mL of the leaf extract 3.5mL of distilled water and 0.3mL of 5% Sodium Nitrate was added to the tubes. After 5 minutes, 0.3mL of 10% Aluminum Chloride was added to all the tubes. At 6th minute, 2mL of 1M Sodium Hydroxide was added to mixture. Immediately, the contents of the reaction mixture were diluted with 2.4mL of distilled water and mixed thoroughly. Absorbance of the mixture was determined at 510n.m versus a prepared blank immediately. Quercetine was used as the standard for quantifi cation of total fl avonoids. The readings were read in triplicates.

RESULTS
The role of free radicals in many ailments has been well established. Accumulation of free radicals may lead to various disease conditions. Much research confi rms that foods or plants rich in antioxidants play an essential role in the prevention of free radical related diseases13.

Free radicals are inevitably produced in biological systems and also encountered exogenously, and are known to cause various degenerative disorders, like mutagenesis, carcinogenesis, cardiovascular disturbances and ageing14. Antioxidants are the compounds, which combat the free radicals by intervening at any one of the three major steps of the free radical mediated oxidative process, viz., initiation, propagation and termination15. The results statistically reveal that the three leaves have the scavenging capacity to fi ght against the free radicals (Dpph) was observed among the three leaf powders. Caulifl ower showed a good free radical scavenging capacity followed by beetroot and radish leaf powders.

Polyphenols (PPs) are a l a r g e and diverse class of compounds, many of which occur naturally in plants characterised by the presence of several phenolic groups. Naturally occurring polyphenols can be broadly divided into phenolic acids and fl avonoids16.The presence of higher total fl avonoid content in leaf suggests higher nutritional value of leaves, as fl avonoids possess strong antioxidant activity and inhibit oxidative stress17. Supporting the above study total fl avonoids was found to be higher in caulifl ower leaf powder. Statistically contribution of fl avonoids in caulifl ower leaf powder was double compared to beet root and radish leaf powders. Thus the fl avonoid content in these leaf powders confi rms the presence of polyphenols which is an essential bioactive compound for overall health.

DISCUSSION
The study reveals the signifi cant presence of total antioxidants total phenols and total fl avonoids in all the selected underutilized vegetable leaves. Statistically caulifl ower leaves scored high in total antioxidant capacity comparing radish and beetroot leaves. Total phenols were marginally high in radish leaves comparing caulifl ower leaves. Flavonoids were signifi cantly high in caulifl ower than radish and beetroot leaves. Hence the present study assures that these selected vegetable leaves that are easy to avail contain antioxidant activities.

CONCLUSION
The results of the study reveals the presence of bioactive compounds with reference to antioxidants, phenols and fl avonoids which have positive effects on the health even though their contribution is in smaller amounts, it can play a very signifi cant role in the health of humans. Caulifl ower, radish and beetroot are less utilized leaves in our locality which are in need to be focused for human consumption. Due to lack of nutritional knowledge, negligence and its identity as animal fodder, these leaves are not included in our daily diet inspite of their easy availability and low economic cost. Hence its prime time to realize the benefi ts of these underutilized leaves which are packed with antioxidants that can prevent from all degenerative diseases and these antioxidant leaves can be treated as a natural preservative and in formulation of functional foods.

ACKNOWLEDGEMENT
Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.

References:

1. Papas, A. M. In phytochemicals in Nutrition and Health, Meskin, M. S., Bidlack, W. R., Davies, A. J. and Omaye. S. T. New York: CRC Press, (2002) 61-78.

2. AchikanuC.E.et.al Determination of the vitamin and mineral composition of common leafy vegetables in south eastern Nigeri, Int.J.Curr.Microbiol.App.Sci (2013) 2(11): 347-353.

3. Gulcin,. M.E. Buyukokurolu, M. Oktay and O. Kufreviolu (2002), on the in vitro antioxidant properties of melatonin. J. Pineal Res.33: 167-171.

4. Ghosal.S. VK Tripathi; S Chaeruhann. Indian Journal o of Chemistry, 1996, 35, 941-948.

5. ER Sherwin; AL Brancn; PM Davidson; Salmincn S. Food Additives. Marvel Dekker Inc, New York, 1990, 139-193.

6. Gulcin. Antioxidant activity of caffeic acid (3,4dihydroxycinnamic acid).Toxicology217:213-220, 2006

7. Liyana-Pathirana, C.M. and F. Shahidi Antioxidant properties of commercial soft and hard winter wheats (Triticum aestivum L.) and their milling fractions. J. Sci. Food Agric (2006)86: 477-485.

8. Deng, G.-F.; Lin, X.; Xu, X.-R.; Gao, L.-L.; Xie, J.-F.; Li, H.-B. Antioxidant capacities and total phenolic contents of 56 vegetables. J. Funct. Foods (2012) 5, 260–266.

9. Li, X.L.; Zhou, A.G.; Han, Y. Anti-oxidation and antimicroorganism activities of purifi cation polysaccharide from Lygodium japonicum in vitro. Carbohydr. Polym. (2006) 66, 34–42.

10. Schofi eld, P.; Mbugua, D.M.; Pell, A.N. Analysis of condensed tannins: A review. Anim. Feed Sci. Tech. 2001, 91, 21–40.

11. Farmacopeia Brasileira, 5th ed.; Agência Nacional de Vigilância Sanitária: Brasília, Brazil, 2010

12. Anna Pkal and Krystyna Pyrzynska, Evaluation of Aluminium Complexation Reaction for Flavonoid Content Assay, Food Anal.methods 2014, 7:1776-1782 , DOI 10.1007/s12161-014-9814-x

13. Di Matteo, V. Esposito, E. Biochemical and therapeutic effects of antioxidants in the treatment of Alzheimer`s disease, Parkinson`s disease, and amyotrophic lateral sclerosis. Curr. Drug Targets CNS Neurol. Disord. 2003, 2, 95–107.

14. Singh S, Singh RP. In vitro methods of assay of antioxidants: An overview. Food Rev Int. 2008; 24(4):392–415. Doi: 10.1080/87559120802304269.

15. Cui K, Luo X, Murthy MRV. Role of oxidative stress in neurodegeneration: recent developments in assay methods for oxidative stress and nutraceutical antioxidants. Prog Neuropsychopharmacol Biol Psych. 2004; 28:771–799. Doi: 10.1016/j.pnpbp.2004.05.023. [PubMed] [Cross Ref]

16. Clifford, M.N. (2000). Chlorogenic acids and o t h e r cinnamates nature, occurrence, dietary b u r d e n , absorption and metabolism.Journal of the Science of Food and Agriculture, 80, 1033–1043.

17. Pourcel, L.; Routaboul, J.M.; Cheynier, V.; Lepiniec, L.; Debeaujon, I. Flavonoid oxidation in plants: From biochemical properties to physiological functions. Trends Plant Sci. 2007, 12, 29–36.