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IN VITRO ANTIOXIDANT ACTIVITIES OF FERONIA LIMONIA LINN

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 Indo American Journal of Pharmaceutical Research, 2014 ISSN NO: 2231-6876

INDO AMERICAN
Journal home page: JOURNAL OF
http://www.iajpr.com PHARMACEUTICAL
RESEARCH

IN VITRO ANTIOXIDANT ACTIVITIES OF FERONIA LIMONIA LINN.

Jayashree V Hanchinalmath and Ramesh Londonkar*
Department of Biotechnology, Gulbarga University, Gulbarga, Karnataka, India-585106.

ARTICLE INFO ABSTRACT

Article history The antioxidant activities of Feronia limonia fruit pulp methanolic extract (MEFL) were
Received 15/06/2014 investigated by different in vitro assays. DPPH, ABTS radical scavenging activity and Total
Available online antioxidant activity were performed to evaluate the antioxidant capacities of the extract in
08/07/2014 vitro. MEFL exhibited good radical scavenging capacity in neutralization of DPPH, ABTS
radicals and phosphomolybdate in a concentration dependent manner. Total phenolic content
Keywords was evaluated by Folin Ciocalteau method and it was noted that MEFL had significant total
Feronia Limonia, phenolic content. The results of this study illustrate that the Feronia limonia methanol extract
Antioxidant Activity, can shield the oxidative stress, and the total phenolic content might be correlated with its
Phenolic Content, antioxidant and free radical scavenger effects. Hence, F limonia fruits are good source of
DPPH. natural antioxidants which can be taken as a dietary source.

Corresponding author
Prof. Ramesh Londonkar
Professor,
Dept of Biotechnology,
Gulbarga University Gulbarga,
Karnataka, India, 585106.
londonkarrmaesh53@gmail.com

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Please cite this article in press as Prof. Ramesh Londonkar et al. In Vitro Antioxidant Activities of Feronia Limonia Linn. Indo
American Journal of Pharm Research.2014:4(06).

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Vol 4, Issue 06, 2014. Prof. Ramesh Londonkar et. al. ISSN NO: 2231-6876

INTRODUCTION
Feronia limonia commonly known as wood apple is an important plant in Ayurveda, the traditional system of Indian
medicine. This fruit is recommended for the treatment of tumors, asthma, wounds, diarrhea, dysentery, cardiac dysfunction, hepatitis,
sore throat and is considered as tonic, astringent (when unripe), antiscorbutic, and alexiformic agent [1,2] . Feronia limonia fruit has
also been reported to possess antimicrobial,hypoglycemic and hypolipidaemic and wound healing properties [3,4,5].
Antioxidant activity is a fundamental property important for life. Many of the biological functions, such as antimutagenicity,
anticarcinogenicity, and antiaging, among others, originate from this property [6]. Antioxidants are compounds that can delay or inhibit
the oxidation of lipids or other molecules by inhibiting the initiation or propagation of oxidative chain reactions [7]. It has been
suggested that natural antioxidants like L-ascorbic acid are more safe and healthy than synthetic antioxidants such as butylated
hydroxytoluene (BHT) [8]. Due to this more safe, cost effective and less toxic antioxidants with high activity from natural sources are
required. Cancer is another deadly disease raising interest among scientists and food manufacturers towards the search of functional
food with specific health effects [9]. Potential sources of antioxidant compounds have been searched in several types of plant materials
such as fruits, leaves, seeds, barks, roots and crude plant drugs [10]. Crude extracts of plant materials rich in phenolics are increasingly
of interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional
value of food. The antioxidative effect is mainly due to flavonoids and phenolic acids present in the plants [11]. Many of the natural
antioxidants, especially phenolics, exhibit a wide range of biological effects including antibacterial, antiviral, anti-inflammatory,
antiallergic, antithrombotic, and vasodilatory actions [12]. There is need to search a potential plant source to combat the serious disease
from natural sources to overcome the negative impacts of synthetic drugs. Hence, present investigation was an attempt to explore the
total phenolic content, total antioxidant potentiality and capacity to scavenge the free radicals by Feronia limonia in different in vitro
test systems.

MATERIALS AND METHODS

Chemicals
2, 2’-Diphenyl-1-picrylhydrazyl (DPPH), 6-Hydroxy-2,5,7,8-tetramethylchroman-2 carboxylic acid (Trolox), Folin-Ciocalteu’s
phenol reagent, 2, 2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), Ascorbic acid, Butylated
hydroxytoluene (BHT), Gallic acid. All other chemicals and reagents were of analytical grade, and they were used as received.

Plant material and extraction

Fruits of Feronia limonia L were collected from local market Bellary, Karnataka, India during the month of March, 2012. It
was authenticated by the Department of P.G studies and Research in Botany, Gulbarga University, Gulbarga. Crude fruit pulp extract
was prepared by Soxhlet extraction method. About 50gm of powdered plant material was packed in a thimble and extracted
successively with 350ml of petroleum ether, chloroform, and methanol. The process of extraction is carried out until the solvent in
siphon tube of an extractor become colorless. The extract was taken in a petriplate and kept in hot air oven and heated at 30-40ºC till
the solvent got evaporated. Solvent free methanolic extract dissolved in 1% Dimethyl sulphoxide (DMSO) was used for the in vitro
studies.

Determination of total phenolic content (TPC)

The amount of total phenolics in MEFL was determined according to the Folin- Ciocalteu method [13]. 200 µL of samples
were introduced into test tubes; 1.0 ml of Folin-Ciocalteu’s reagent and 0.8 ml of sodium carbonate (7.5%) were added. The tubes
were mixed and allowed to stand for 30 min. Absorbance was measured at 765 nm (Perkin-Elmer UV-vis spectrophotometer,
Norwalk, CT). The total phenolic content was expressed as gallic acid equivalents (GAE) in milligrams per gram dry material.

DPPH radical scavenging activity

DPPH radical scavenging activity was performed according to the method described by [14]. A stock solution of DPPH (1.3
mg/ ml in methanol) was prepared so that 75 µL of it in 4ml methanol gave an initial absorbance of 1.93. Decrease in the absorbance
was measured at 517nm after 15 min at various concentrations. 1ml of DPPH (0.1 mM in methanol) was added to standard reference
BHT. The percentage of DPPH free radical scavenging activity was calculated using the following equation:
% Inhibition = [(A0 - At)/A0] x 100
where A0 is absorption of blank sample, At is absorption of test sample.

ABTS radical scavenging assay

ABTS radical scavenging activity was performed according to [15] with some modifications. The ABTS reagent was prepared
freshly by mixing 7mM ABTS and 2.45 mM potassium persulfate and incubated for 16 h at 37°C. The ABTS cations diluted with
ethanol to set O.D at 0.7 (±0.02). 3.9 ml of reagent was added to 0.1 ml of extract. The mixture was incubated at 37°C and absorbance
was measured at 734 nm after 2 min. Trolox was used as a standard reference. Results were expressed in μM Trolox Equivalents (TE)
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/g of plant extract.
% Inhibition = [(A0 - At)/A0] x 100
where A0 is absorption of blank sample, At is absorption of test sample.
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Vol 4, Issue 06, 2014. Prof. Ramesh Londonkar et. al. ISSN NO: 2231-6876

Phosphomolybdate assay (Total antioxidant capacity)

The total antioxidant capacity of MEFL was determined using ascorbic acid as standard following the phosphomolybdate
method of [16]. 0.1 ml of extract solution was mixed with 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and
4 mM ammonium molybdate). The tubes were incubated in a water bath at 95°C for 90 min. A typical blank contained 1 ml of reagent
solution and appropriate volume of solvent incubated under the same conditions. The tubes were cooled to room temperature and
absorbance was measured at 765 nm against a blank. The antioxidant capacity was estimated using following formula:
Antioxidant effect (%) =
[(control absorbance - sample absorbance) / (control absorbance)] × 100

Statistical analysis of data

Data were presented as means ± SD of three experiments. Analysis of variance was performed on the data obtained.
Significance of differences between means was determined by least significant differences (LSD) at P ≤ 0.05.

RESULT
Total phenolic content
Total phenolic compounds, as determined by Folin Ciocalteu method, are reported as Gallic acid equivalents (GAE) /g dry
material. It was noted that MEFL had significant total phenolic content of 76.77 to 351.45 mg GAE/g dry material at concentrations
ranging from 1.25 to 10 mg/ml (figure 1). The high amount of phenols in extracts may explain their high antioxidative activities.

Fig. 1 Total phenolic content of MEFL

DPPH radical scavenging activity

Concentration dependent DPPH radical scavenging activity was observed in both MEFL and standard. The decrease in
concentration of DPPH radical is due to the scavenging activity of MEFL and BHT which showed the significant free radical
scavenging inhibition activity of 52.74% -72.23 % and 67.15% -90.45 % respectively at 1.25-10 mg/ml (Figure 2).

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Fig. 2 DPPH free radical scavenging activity of MEFL

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Vol 4, Issue 06, 2014. Prof. Ramesh Londonkar et. al. ISSN NO: 2231-6876

ABTS radical scavenging activity of MEFL extract

ABTS radical scavenging activity of MEFL ranged from 43.13% to 76.21% and standard from 67.15% to 89.75% for the
concentrations from 1.25 to 10 mg/ml (Figure 3). The high phenolic content of MEFL extract might be responsible for the increased
ABTS radical scavenging activity.

Fig. 3 ABTS radical scavenging activity of MEFL

Phosphomolybdate assay
The phosphomolybdate method is quantitative, since the total antioxidant capacity (TAC) is expressed as ascorbic acid
equivalents. The results showed the antioxidant activity of MEFL in dose dependent manner at concentrations of 1.25 to 10 mg/ml
(Figure 4). Potent antioxidant activity of MEFL could be attributable to the presence of phenolic compounds.

Fig. 4 Total antioxidant capacity of MEFL

Relationship between phenolic content, radical scavenging and total antioxidant

Correlation analysis (Table 1) exhibited positive and strong correlation of TPC with DPPH (r = 0.98), ABTS (r = 0.99) and
TAC (r = 0.95). Correlation analysis also shows positive and strong correlation of DPPH with ABTS (r = 0.99) and TAC (r = 0.98);
ABTS with TAC (r = 0.97). The findings suggest strong involvement of phenolics in the antioxidant activity of MEFL. The high
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degree of correlation in the simple spectrophotometric assay showed that it would be a useful technique for rapid evaluation of
antioxidant activity in this plant.
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Vol 4, Issue 06, 2014. Prof. Ramesh Londonkar et. al. ISSN NO: 2231-6876

Table 1 :Relationship between phenolic content, radical scavenging and total antioxidant of MEFL

TPC DPPH ABTS TAC

TPC 1
DPPH 0.98 1
ABTS 0.99 0.99 1
TAC 0.95 0.98 0.97 1

DISCUSSION
Several techniques have been used to determine the antioxidant activity in vitro in order to allow rapid screening of
substances, since substances that have low antioxidant activity in vitro, will probably show little activity in vivo. Plant phenolics
constitute one of the major compounds as primary antioxidant or free radical terminator. Free radicals are known to play a definite role
in a wide variety of pathological manifestations. Antioxidants due to their scavenging activity are useful for the management of these
diseases. They exert their action either by scavenging the reactive oxygen species or protecting the antioxidant defense mechanisms
[17]
.Thus the total phenol content in various concentrations in MEFL was determined. The significant phenolic content in MEFL can
explain its high free radical scavenging activity due to its redox properties in accordance to [18]. This study reveals that the extract has
moderate to significant antioxidant and free radical scavenging activity. The present study suggests that Feronia limonia can be used
as a source of antioxidants for pharmacological preparations. Non-phenolic compounds of the plants such as trace elements may
decrease the antioxidant activity of the phenolic compounds [19]. Thus the measurement of phenolic content only could not be a good
indicator of the antioxidant capacity. Owing to the complexity of the antioxidant materials and their mechanism, it is obvious that no
single testing method is capable of providing a comprehensive picture of the antioxidant profile of a sample and a combination of
methods is essential. Thus DPPH and ABTS scavenging activity were carried out. Regardless of such limitations, DPPH assay is a
sensitive way for primary screening and finding of novel antioxidants [20]. A significant correlation was found between the total
phenolic content, DPPH, ABTS and TAC in methanol extract in our study.

CONCLUSION
Our investigations reveals that MEFL hold potent antioxidant activity, achieved by scavenging abilities observed against
DPPH, ABTS compared to standard antioxidants BHT and Trolox. These in vitro assays indicate that this plant extract is a significant
source of natural antioxidant, which might help prevent the progress of oxidative stress.. Efforts are in progress in our laboratory to
isolate and purify the active principle of this medicinal plant.

Authors’ Statements
Competing Interests
The authors declare no conflict of interest.

List of abbreviations
MEFL : Feronia limonia fruit pulp methanol extract
DPPH : 2, 2’-Diphenyl-1-picrylhydrazyl
ABTS : 2, 2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)
BHT : Butylated hydroxytoluene
FCR : Folin-Ciocalteu’s phenol reagent
DMSO : Dimethyl sulphoxide
GAE : Gallic acid equivalents
TE : Trolox Equivalents
TPC : Total phenolic content
TAC : Total antioxidant capacity
SD : Standard deviation.
LSD : Least significant differences

REFERENCES
1. Kirtikar KR, Basu BD, Indian Medicinal Plants, Lalit Mohan Basu Publications, Allahabad, India.1995; 1:2.
2. Morton JF, Fruits of warm climates, Indiana.USA: Centre for New crops & plant products. Purdue University. 1987,190-191.
3. Jayashree VH, Ramesh Londonkar, Comparative Phytochemical Studies And Antimicrobial Potential Of Fruit Extracts Of
Feronia Limonia Linn, Int J of Pharm Pharm Sci.2014,6(1),731-734.
2941

4. Kangralkar VA, Patil SD, Bandivadekar RM, Nandagaon VS, Burli SC, Hypoglycemic and hypolipidaemic effect of methanolic
extract of Feronia elephantum fruits in alloxan diabetic rats, Int J Pharm Sci Rev Res. 2010, 4(1),64-66.
5. Ilango K, Chitra V, Wound healing and anti- oxidant activities of the fruit pulp of Limonia acidissima Linn (Rutaceae) in rats,
Trop J Pharm Res. 2010,9(3),223-230.
6. Huang MT, Ho CT, Lee CY, Phenolic Compounds in Food and Their Effects on Health. II. Antioxidants and Cancer Prevention.
Page

ACS Symposium Series 507, American Chemical Society, Washington, DC 1992.

7. Sun, Chu, Wu, Liu, Antioxidant and antiproliferative activities of common fruits, J of Agri and Food Chem. 2002,50,7449-7454 .

www.iajpr.com
Vol 4, Issue 06, 2014. Prof. Ramesh Londonkar et. al. ISSN NO: 2231-6876

8. Galati EM, Mondello MR, Lauriano ER, Taviano MF, Galluzzo, Miceli NO, Fruit juice protects liver from carbon tetrachloride-
induced injury, Phytother Res. 2005,19(9),796-800.
9. Lo-liger J,The use of antioxidants in food, In Free Radicals and Food Additives; Taylor and Francis: London 1991, 129-150.
10. Ramarathnam N, Ochi H, Takeuchi M, Antioxidant defense system in vegetable extracts, In Shahidi F (ed) Natural Antioxidants:
Chemistry, Health Effects, and Applications. AOCS Press: Champaign 1997, 76-87.
11. Pietta PG. Flavonoids in medicinal plants. In CA Rice- Evans & L Packer (Eds) Flavonoids in health and disease. New York:
Dekker 1998, 61–110.
12. Divya Nambath et al. Free Radical Scavenging Activity of the Methanol Extract of Heracleam Candolleanum (Wight-Et-Arn),
Indo American Journal of Pharm Research.2014:4(05),2491-2495.
13. Kirankumar S, Ramesh L, Evaluation of total phenolic content and in vitro antioxidant activity of Ficus glomerata fruit extracts,
Int J of Pharmaceutical and clinical research. 2014,6(2),133-137.
14. Singleton VL, Rossi JA, Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents, Am. J. Enol. Viti.,
1965,(16)144-158.
15. Londonkar R, Kamble A, Evaluation of free radical scavenging activity of Pandanus odoratissimus, International journal of
Pharmacology, 2009,5(6,:377-380.
16. Re Pellegrino RN, Proteggente A, Pannala A, Yang M, Rice-Evans C, Antioxidant activity applying an improved ABTS radical
action decolonization assay, Free Radic Biol Med. 1999, 26,1231-1233.
17. Umamaheswari M, Chatterjee TK, In vitro antioxidant activities of the fractions of Coccinnia grandis L. leaf extract, Afr J Trad
Compl Altern Med. 2008,5,61–73.
18. Rice-Evans CA, Miller NJ, Paganga G, Antioxidant properties of phenolic compounds. Trend. Plant Sci. 1995,4,152-159.
19. Vinson JA, Hao Y, Su X, Zubik L, Phenol antioxidant quantity and quality in foods: vegetables, J Agric Food Chem.
1998,46,3630-3634.
20. Koleva II, Van Beek TA, Linssen JPH, De Groot A, Evstatieva LN, Screening of plant extracts for antioxidant activity: a
comparative study on three testing methods, Phytochem Anal. 2002,13,:8-17.

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