Evaluation of In-vitro Antioxidant Activity of Ethanolic Extract of Ugli Fruit
Chitra V, Tamilanban T*, Manasa K, Narayanan J
Department of Pharmacology, SRM College of Pharmacy, SRM Institute of Science and Technology,
Kattankulathur-603203, Tamilnadu, India.
*Corresponding Author E-mail: veera.srm@gmail.com
ABSTRACT:
Medicinal plants are rich sources of natural antioxidants which are used in the prevention and treatment of diseases like atherosclerosis, heart stroke, diabetes, cancer and to delay the process of aging. Antioxidants are the chemicals which scavenge the free radicals and help in preventing and treatment of several diseases. Human body produces oxygen free radicals and other reactive oxygen species (ROS) as by products through numerous physiological and biochemical processes. In the present study, antioxidant activity of rind extracts of Citrus reticulate x Citrus paradise (Ugli Fruit) was evaluated in vitro using DPPH, Nitric oxide, Hydrogen peroxide, Super oxide dismutase free radical scavenging methods. Different concentrations of test samples (ethanolic extracts) i.e. 50 μg/ml, 100 μg/ml, 250 μg/ml, 500 μg/ml and 1000 μg/ml were taken and each of the samples were analysed for their antioxidant status. Different concentrations of ethanolic extracts showed radical scavenging activity of which DPPH assay showed an IC50 value of about 18.9. The nitric oxide scavenging activity compared with Ascorbic acid and the IC50 values were found to be 46.59 µg/ml (Ascorbic acid) and 395.94 (Ugli fruit extract). The Ugli fruit extract was capable of scavenging hydrogenperoxide in a dose dependent manner. In superoxide dismutase assay, ascorbic acid is used as a standard and the ethanolic extract shows a better antioxidant property. Thus this study suggests that Citrus reticulate x Citrus paradise is an effective plant in terms of antioxidant potential that can be exploited for development of plant based antioxidant formulations.
KEYWORDS: Antioxidant, free radicals, Citrus reticulate x Citrus paradise, DPPH, nitric oxide, hydrogen peroxide, super oxide dismutase, scavenging ability.
INTRODUCTION:
Free radicals are defined as the atomic or molecular species having one or many unpaired electrons1. These hold a protective nature in biological systems, but when present in excess causes an extreme damage to biomolecules. It is defined as the protection against and repair of the oxidative damage due to free radicals. Antioxidants mimic the risk for chronic diseases like cancers, cardiovascular and neurodegenerative diseases, and immune dysfunction. Vitamin C inhibits the oxidation of LDL-cholesterol and reduces the risk of cardiovascular diseases2andalso has a significant antioxidant function in the lungs and protects the body from the free radical sources. Vitamin C is citrus fruit, such as lemons, oranges and grapefruit and carotene is rich in green leafy vegetables. Phytochemicals such as tea, garlic, oils, fruits and vegetables3 are proved to have a 10-fold greater antioxidant activity than of the vitamins found in animal diets. This potential nature of antioxidants helps in lowering the occurrence of chronic diseases by scavenging harmful free radicals in vivo, leads to the assessment of antioxidant activity of foods and nutraceuticals by using simple, accurate assays adaptable to the nutrition laboratory.
The present study provides the antioxidant activity of Citrus reticulate x Citrus paradise by certain in vitro techniques as no such pharmacological studies were reported in the literature. The objective was to quantify the antioxidant activity of selected fruit using a spectrophotometric assay4.
MATERIALS:
Collection and Authentication of the material:
The fresh fruits of Citrus reticulata × Citrus paradisi (Ugli fruit) were collected from Tambaram Fruit Market, Chennai, Tamil Nadu, in the month of January 2016. The fruits were authenticated by Prof. P. Jayaraman, Director; Plant Anatomy Research centre (PARC), Medicinal Plant Research Unit, West Tambaram, Chennai and a specimen was deposited.
Preparation of citrus reticulata × citrus paradisi extract:
After authentication the fruits were peeled off and their rind and shade dried ground and passed through sieve no.18 to get a coarse powder. Defatting was done with petroleum ether, filtered and dried. This defatted coarse powder was packed in a Soxhlet Extractor using Ethanol as solvent and the extraction was carried out for about 72hours. Later the extract was collected and evaporated in a Rotary Vacuum Evaporator until the thick paste of extract was obtained5.
Invitro Antioxidant Assay:
DPPH Assay 6-9:
Principle:
DPPH functions as a scavenger or trap for other free radicals. Addition of DPPH leads to reduction of compounds in a chemical reaction which is an indicator for the presence of free radicals. The DPPH radical shows a deep violet colour in solution due to its strong absorption band at 520 nm, and turns colourless or pale yellow when neutralized. This reaction can be monitored visually and the number of initial radicals can be counted from the change in the optical absorption at 520 nm or in the EPR signal of the DPPH.
METHOD:
0.3mM DPPH solution was prepared by dissolving 4.3mg of DPPH (2, 2-Diphenyl -1- picrylhydrazyl) in ethanol (6.6ml) and the test tubes were covered with an aluminium foil. Hundred and fifty microliter of DPPH was added to 3ml of ethanol and the absorbance was noted at 516nm for control reading. Concentrations of test samples i.e. 50 μg/ml, 100 μg/ml, 250 μg/ml, 500 μg/ml and 1000 μg/ml were taken and each of the samples was diluted with methanol up to 3ml, to it 150 μl DPPH was added. The samples were stored in a dark place for 15 min and the optical density was observed at 516nm using methanol as blank and ascorbic acid as standard. The free radical scavenging activity (%Antiradical activity) was calculated using the formula:
% Antioxidant activity=[(Control Absorbance X Sample Absorbance) / Control Absorbance] x100
Nitric oxide Scavenging Assay10-13:
Principle:
Nitric oxide radical scavenging activity was determined according to the method reported by Garrat. Aqueous solution of sodium nitroprusside spontaneously generates nitric oxide, reacts with oxygen and gives nitrite ions and assayed by Griess Illosvoy reaction. Nitric oxide (NO) is a strong pleiotropic mediator in neuronal signalling, smooth muscle relaxation, inhibition of platelet aggregation and regulates cell mediated toxicity. It also functions as an effector molecule in biological systems like neuronal messenger, vasodilatation and has antimicrobial and antitumor activities. In the pathological conditions, nitric oxide reacts with superoxide anion and forms cytotoxic peroxynitrite. Nitric oxide inhibitors have beneficial effects on inflammation and tissue damage in inflammatory diseases.
Method:
0.5 ml of the extract was mixed with 382 ml of 10 mM sodium nitroprusside in 0.5 mL phosphate buffer saline (pH 7.4) and incubated at 25oC for 150 min. 1.0 mL sulfanilic acid reagent was added to 0.5 ml of this mixture and incubated for 5 min at room temperature.1.0 mL naphthylethylenediamine dihydrochloride (0.1% w/v) was further added to the mixture and incubated for 30 min before measuring the absorbance at 540 nm .The nitric oxide radicals scavenging activity was calculated by the below formula:
S% = [(Acontrol – Asample)/Acontrol ×100
Acontrol = absorbance value of the blank control (containing all reagents except the extract solution) and Asample = absorbance value of the test.
Hydrogen peroxide Scavenging Capacity14,15:
Principle:
The Hydrogen Peroxide Colorimetric Detection assay is performed for the quantitative measure H2O2 in a variety of samples and should be read off the standard curve. Samples are mixed with the Colorimetric Substrate and the reaction initiated by addition of horse radish peroxidase (HRP) and is incubated for 15 min. In the presence of hydrogen peroxide, HRP reacts with the substrate to convert the colourless substrate into a coloured product (pink colour) was read at 560 nm. Rise in the levels of H2O2 cause a linear increase in colour.
Procedure:
Forty millimoles of hydrogen peroxide solution was prepared in phosphate buffer (pH 7.4). Extracts (50, 100, 250, 500, 1000 μg/mL) in distilled water were added to 0.6ml of 40mM hydrogen peroxide solution and the absorbance was determined 10 minutes against a blank solution containing the phosphate buffer at 560nm. The percentage of hydrogen peroxide scavenging of both ugli fruit extracts and standard ascorbic acid were calculated using the formula:
% Scavenged [H2O2] = [(AC – AS)/AC] x 100
Super oxide dismutase Assay16-19:
Principle:
The assay of SOD is based on the inhibition of the formation of NADH-phenazine methosulphate-nitroblue tetrazolium formazon. A bovine erythrocyte SOD standard generates a standard curve for the assay and all samples should be read off by the standard curve. The Substrate is added to xanthine oxidase reagent and incubated at room temperature for 20 minutes; which converts a colourless substrate in the detection reagent into a yellow coloured product read at 450 nm. Increasing levels of SOD in the samples causes a decrease in superoxide concentration and a reduction in yellow product. The results are expressed in terms of units of SOD activity per ml.
Method:
The assay mixture is 2.8 ml total volume in distilled water with1.2ml of sodium pyrophosphate buffer, 0.1ml of PMS, 0.3ml of NBT, 0.2ml of the enzyme preparation. NADH(0.2ml) was added to the mixture and incubated at 30°C for 90 seconds and 1.0ml of glacial acetic acid was added. N-butanol (4.0ml) was added to the reaction mixture, shaken and allowed to stand for 10 minutes and centrifuged. The chromogen intensity in the butanol layer was and measured at 560nm in a spectrophotometer.
% inhibition for the test sample(s): [(∆ A550n/minute) negative control - (∆ A550n/minute) test] x 100 = % Inhibition (∆ A550n/minute) negative control
RESULTS:
Table 1:
peel extract exhibited antioxidant activity at all the concentrations of test solutions. With the increase in concentration of the peel extract (50-1000µg/ml), the percentage of antioxidant activity also increased (78.6-91.3%). Among all maximum antioxidant activity (91.3%) was observed at 1000 μg/ml concentration with IC50 value of 18.9 and ascorbic acid showed 98% antioxidant activity.
Table 2:
The nitric oxide scavenging activity of ugli fruit peel extract was evaluated and compared with Ascorbic acid and the results are given in Table 2. The IC50 values are calculated from graph and were found to be 46.59 µg/ml (Ascorbic acid) and 395.94 (Ugli fruit extract).
Table 3:
The free radical scavenging ability of ethanolic extracts of ugli fruit on hydrogen peroxide is shown in Table3 and compared ascorbic acid as a standard. The ugli fruit extract was capable of scavenging hydrogenperoxide in an amount dependent manner. 1000, 500, 250, 100, 50μg/ml of the extracts exhibited following % scavenging activity on hydrogen peroxide.
Table 4:
In superoxide dismutase assay, ascorbic acid is used as a standard and the ethanolic extract shows a better antioxidant property. The % free radical scavenging activity of the extract is almost similar to that of the standard values.
Table 1: Results of DPPH Assay
|
S. No. |
Concentration (µg/ml) |
% Free radical scavenging activity of extract |
% Free radical scavenging activity of standard |
|
1 |
1000 |
91.3 |
98.0 |
|
2 |
500 |
88.2 |
92.1 |
|
3 |
250 |
85.3 |
87.3 |
|
4 |
100 |
82.9 |
84.6 |
|
5 |
50 |
78.6 |
80.2 |
Table 2: Nitric Oxide Assay
|
S. No. |
Concentration (µg/ml) |
% Free radical scavenging activity of extract |
% Free radical scavenging activity of standard |
|
1 |
1000 |
97.2 |
98.32 |
|
2 |
500 |
88.69 |
92.24 |
|
3 |
250 |
75.97 |
83.9 |
|
4 |
100 |
65.86 |
72.56 |
|
5 |
50 |
54.56 |
63.89 |
Table 3: Hydrogen Peroxide Assay
|
S. No. |
Concentration (µg/ml) |
% free radical scavenging activity of extract |
% free radical scavenging activity of standard |
|
1 |
1000 |
44.5 |
96.4 |
|
2 |
500 |
22.1 |
81.32 |
|
3 |
250 |
12.5 |
75.2 |
|
4 |
100 |
8.9 |
58.65 |
|
5 |
50 |
3.8 |
42.19 |
Table 4: Superoxide Dismutase Assay
|
S. |
Concentration (µg/ml) |
% free radical scavenging activity of extract |
% free radical scavenging activity of standard |
|
1 |
1000 |
89.23 |
99.7 |
|
2 |
500 |
76.9 |
86.6 |
|
3 |
250 |
57.32 |
75.78 |
|
4 |
100 |
25.76 |
64.32 |
|
5 |
50 |
13.61 |
59.24 |
DISCUSSION:
Oxidation leads to production of free radicals, leading to chain reactions and cause damage to the living cells; this in turn can be inhibited by an antioxidant5 which are either natural or artificial substances. However, its role in preventing diseases has not been justified. Vitamins C and E, selenium, beta-carotene, lycopene, lutein and zeaxanthin7 are some common antioxidants.
The unstable molecules formed in the body when the food gets converted to energy and in exercise are defined as “free radicals”20 which leads to “oxidative stress,” a process that can trigger cell damage. Oxidative stress is proved to play a role in a variety of diseases like cancer, diabetes, cardiovascular diseases neurodegenerative diseases, cataracts and age-related macular degeneration21.Antioxidant molecules counteract this oxidative stress in laboratory experiments (e.g., in cells or animal studies).
The common non-enzymatic antioxidants are vitamins A, C, and E, α-lipoic acid, mixed carotenoids, glutathione, coenzyme Q10 (CoQ10), several bioflavonoids, minerals (copper, zinc, manganese and selenium) and cofactors like albumin, uric acid, folic acid and vitamins B1, B2, B6, and B12 and the enzymatic antioxidants are catalase, superoxide dismutase, glutathione reductase, glutathione peroxidase22. Antioxidants has an effect on signal transduction and the immune response through normal physiological process, and thus shown a promising possibility in the prevention ,treatment and even decreased risks of certain cancers and cardiovascular and neuronal disorders23. In this study, the antioxidant activity of Citrus reticulate x Citrus paradise were evaluated and the results were documented with a markable antioxidant property.
In the present study, we have evaluated the ethanolic peel extracts of C. reticulata X C. paradisiaca for its antiradical activity by performing DPPH assay(Table1). The maximum antioxidant activity (91.3%) was observed at 1000 μg/ml concentration with IC50 value of 18.9. and ascorbic acid (standard) showed 98% antioxidant activity with IC50 value at 6.4μg/ml concentration. DPPH radical provides a rapid and convenient method to evaluate the antioxidants and radical scavengers9. The nitric oxide scavenging activity and Hydrogen peroxide assay of ugli fruit peel extract were also evaluated by comparing with Ascorbic acid and shows a successive activity14.Rise in the oxidative stress leads to over consumption of SOD and these levels were significantly improved on the treatment with the peel extracts18.
Phytochemical screening of C. reticulata X C. paradisiaca shows that ethanolic extract contains flavonoids and alkaloids24. Hence, we can assume that the flavonoids were responsible for the antioxidant activity which furthers needs an elaborated research to isolate and characterize the particular flavonoid.
ACKNOWLEDGEMENTS:
Authors are thankful to the Dean and Vice Principal, SRM College of Pharmacy and management of SRM University for providing necessary facilities to carry out this work.
REFERENCES:
1. Bjelakovic G, Nikolova D, Gluud C .Meta-regression analyses, meta-analyses, and trial sequential analyses of the effects of supplementation with beta-carotene, vitamin A, and vitamin E singly or in different combinations on all-cause mortality: do we have evidence for lack of harm?. Public Health Nutrition.2003; 7 (3): 407–22.
2. Abner EL, Schmitt FA, Mendiondo MS, Marcum JL, Kryscio RJ. "Vitamin E and all-cause mortality: a meta-analysis". Current Aging Science.2011; 4 (2): 158–70.
3. Cortes-Jofre M, Rueda JR, Corsini-Muñoz G, Fonseca-Cortés C, Caraballoso M, Bonfill Cosp X. Drugs for preventing lung cancer in healthy people. The Cochrane Database of Systematic Reviews.2012; 10.
4. Jiang L, Yang KH, Tian JH, Guan QL, Yao N, Cao N, Mi DH, Wu J, Ma B, Yang SH. Efficacy of antioxidant vitamins and selenium supplement in prostate cancer prevention: a meta-analysis of randomized controlled trials. Nutrition and Cancer.2010; 62 (6): 719–27.
5. Rees K, Hartley L, Day C, Flowers N, Clarke A, Stranges S. Selenium supplementation for the primary prevention of cardiovascular disease. The Cochrane Database of Systematic Reviews.2013;1
6. Gayatri S. Kamble, Rasika C. Torane, Kavita S.Mundhe, Nirmala R. Deshpande, Jyoti P. Salvekar. Evolution of Free Radical Potential of Embelia basalJournal of Chemical and Pharmaceutical Research.2011; 3(2):465-471.
7. Soler-Rivas C, Espin JC and Wichers HJ 2000. An easy and fast test to compare total free radical scavenger capacity of foodstuffs. Phytochemical Analysis.2000; 11:1-9.
8. Blois MS. Antioxidant determination by the use of stable free radicals. Nature.1958; 26:1199-1203.
9. Thamaraiselvi, P. Lalitha, P. Jayanthi Pelagia. Study of antioxidant activity of ethanolic extract of fresh Eichhornia crassipes (Mart.) Solms. Der Pharmacia Sinica. 2008; 3 (2):271-277.
10. Nagulendran K, Velavan S, Mahesh R, Begum VH. In Vitro Antioxidant Activity and Total Polyphenolic Content of Cyperus Rotundus Rhizomes. 2007; 4(3): 440-449.
11. Ruch RJ, Cheng SJ, Klaunig JF. Prevention of cytotoxicity and inhibition of intracellular communication by antioxidant catechins isolated from Chinese green tea, Carcinogenesis. 1989; 10: 10003-1008.
12. Keser, S., Celik, S., Turkoglu, S., Yilmaz, O., and Turkoglu, I. Determination of antioxidant capacities of ethanol and water extracts of Achillea millefolium L. (Yarrow). Asian Journal of Chemistry.2011; 23:3172-3176.
13. Mitsuda, H., Yuasumoto, K., and Iwami, K. Antioxidation action of indole compounds during the autoxidation of linoleic acid. Eiyoto Shokuryo. 1996; 19: 210-214.
16. Packer, L., Cadenas, E., and Davies, K.J.A. Free radicals and exercise: An introduction. Free Radical Biology and Medicine. 2008; 44: 123-125.
17. Ruch, R.J., Cheng, S.J., and Klaunig, J.E. Prevention of cytotoxicity and inhibition of intracellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis. Indian Journal of Pharmaceutical Sciences.1989; 10: 1003-1008.
18. Wells, P.G., McCallum, G.P., Chen, C.S., Henderson, J.T., Lee, C.J., Perstin, J., Preston, T.J., Wiley, M.J., and Wong A.W. Oxidative stress in developmental origins of disease: Teratogenesis, neurodevelopmental deficits, and cancer. Toxicological Sciences.2009; 108: 4-18.
19. Kussmaul L, Hirst J. The mechanism of superoxide production by NADH: ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Proceedings of the National Academy of Sciences. 2006; 103: 7607-7612.
20. Szeto HH. Mitochondria-targeted peptide antioxidants: novel neuroprotective agents. American Association of Pharmaceutical Scientists Journal.2006; 8: 521-31.
21. Robak J, Gryglewski RJ. Flavonoids are scavengers of superoxide anions. Biochemical Pharmacology. 1988; 37:837-41.
22. Nebot C, Moutet M, Huet P, Xu J-Z, Yadan J-C, Chaudiere J Spectrophotometric assay of superoxide dismutase activity based on the activated auto-oxidation of a tetracyclic catechol. Analytical Biochemistry.1993; 214:442-451.
23. Okado-Matsumoto A, Fridovich I. Subcellular distribution of superoxide dismutases (SOD) in rat liver. Journal of Biological Chemistry. 2001; 276:388-393.
24. Crichton GE, Bryan J, Murphy K J. Dietary antioxidants, cognitive function and dementia--a systematic review. Plant Foods for Human Nutrition. 2013; 68 (3): 279–92.
Received on 19.06.2017 Modified on 18.08.2017
Accepted on 17.09.2017 © RJPT All right reserved
Research J. Pharm. and Tech 2017; 10(11): 3826-3830.
DOI: 10.5958/0974-360X.2017.00694.1