Determination of antioxidant properties of fig fruit extracts (Ficus carica L.)

Abstract

The fig tree (Ficus carica L.; Moraceae) is very common in the Mediterranean and in countries with dry and warm-temperate climates. Since ancient times figs have been used only for human consumption. Their nutritive and pharmacological properties have been investigated recently. Their consumption helps in the prevention of artery blockage, high fiber content has laxative effects, and the fig latex inhibits the growth of carcinoma cells. The aim of this study was to determine the content of some active components contained in extracts of five different fig cultivars ('Šaraguja', 'Termenjača', 'Crnica', 'Bjelica' and 'Bružetka bijela'). Fruit samples were freeze-dried. Extracts of total phenols and flavonoids were obtained by extraction in 70% methanol or 70% ethanol. For determination of antioxidant activity, scavenging capacity on DPPHß radicals and reducing power were performed. By high performance liquid chromatography (HPLC) some antioxidant compounds were detected and quantified. Total phenolics content in F. carica extracts was from 7.24 to 11.17 mg CAE/g of dry extract. All methanolic extracts showed higher content of total phenols. The DPPH radical scavenging capacity was found to exhibit IC50 value for the extract concentration lower than 0.40 mg/ml for extract cultivars 'Crnica', while for others this capacity was higher than 0.60 mg/ml. Using the reducing power antioxidant test, higher antioxidant activity was determined for 'Bjelica' than in all other extracts. Results obtained by the reducing power method were compared to activity of ascorbic acid, standard antioxidant compound.

Full text

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Determination of Fig Fruit Extracts (Ficus carica) Antioxidant Properties
I. Mujić and S. Dudas
Colegium Fluminense
Polytechnic of Rijeka
Carla Hugueas 6
Poreč
Croati
a
Z. Zeković,
Ž
. Lepojević, M. Radojković,
S. Vidović and S. Milošević
Faculty of Technology
University of Novi Sad
Bulevar cara Lazara 1
Serbi
a
Keywords: fig fruit, phenols, antioxidant activity
Abstract
The fig tree (Ficus carica L., Moraceae) is very common in the Mediterranean
and in countries with dry and warm-temperate climate. Since ancient times the figs
have been used for human consumption. Their nutritive and pharmacological
properties have been investigated recently. Fig consumption helps in the prevention of
vein blockage, high content in fibers has laxative effects, and the latex inhibits the
growth of carcinoma cells.
The aim of this study was to determine a content of some active components
found in extracts of five different figs cultivars (‘Šaraguja’, ‘Termenjača’, ‘Crnica’,
‘Bjelica’ and ‘Bružetka bijela’). Bioactive compounds were extracted from freeze-
dried fig fruits with 70% methanol or 70% ethanol. Analysis of fig extracts included
determination of total phenols and flavonoids, as it was determined that effects on
health derived from these compounds. For determination of antioxidant activity,
scavenging capacity of the DPPH* radicals and reducing power were determined.
Antioxidant compounds were detected and quantified by high performance liquid
chromatography (HPLC).
Total phenolics content in F. carica extracts varied from 7.24 to 11.17 mg
GAE/g of dry extract. All methanolic extracts showed higher content of total phenols.
The DPPH radical scavenging capacity was found to exhibit IC50 value for the extract
concentration lower than 0.40 mg/ml for extract cultivars ‘Crnica’, while for others
this capacity was higher than 0.60 mg/ml. Using reducing power antioxidant test,
higher antioxidant activity was measuered for ‘Bjelica’ than for all other extracts.
Results obtained by reducing power method were compared to activity of ascorbic
acid, a strong antioxidant compound.
INTRODUCTION
The fig tree (Ficus carica L., Moraceae) is very common in the Mediterranean
and in countries with dry and warm-temperate climate. Since ancient times the figs have
been used for human consumption. Their nutritive and pharmacological properties have
been investigated recently. Figs have been traditionally used for its medicinal benefits as
laxative, cardiovascular, respiratory, antispasmolitic nd anti-inflammatory remedies
(Guarrera, 2005; Oliviera et al., 2009).
Some studies have described the presence of several phenolic compounds in this
species (Oliviera et al., 2009; Texeira et al, 2006; Vaya and Mahmood, 2006; Guarrera,
2005), phytosterols and fatty acids in fruits and branches of fig trees (Jeong and
Lachance, 2001) and its antioxidant activity (Solomon et al., 2006; Veberic et al., 2008;
Oliviera et al., 2009). However, leaf, pulp and peels metabolite profile and biological
activity have not been compared.
Phenolic compounds are secondary metabolites that are widespread in nature.
These compounds play many physiological roles in plants and some of them are also
favourable to human health, since they are able to act as antioxidants by different ways: as
reducing agents, as hydrogen donors, as free radicals scavengers, and as singlet oxygen
quenchers (Kahkonen et al., 1999; Merken and Beecher, 2000; Espin et al., 2000;
Fattouch et al., 2007; Costa et al., 2009).
Proc. XXVIIIth IHC
–
IS on Emerging Health Topics
in Fruits and Vegetables
Ed.: Y. Desjardins
Acta Hort. 939, ISHS 2012

392
The aim of this study was to determine the content of some bioactive components
and evaluate antioxidant properties in extracts of five fig cultivars (‘Šaraguja’,
‘Termenjača’, ‘Crnica’, ‘Bjelica’ and ‘Bružetka bijela’), obtained with different extraction
protocols. Investigation included determination of total phenols and flavonoids, the
scavenging activity on DPPH radicals, reducing power and identification and
quantification of phenolic components using LC-MS.
MATERIALS AND METHODS
Chemicals
1,1-Diphenyl-2-picryl-hydrazyl-hydrate (DPPH), chlorogenic acid, catechin and
Folin-Ciocalteu reagent were purchased from Sigma (Sigma-Aldrich GmbH, Sternheim,
Germany). All other chemicals and reagents were of analytical reagent grade.
Plant Material and Sample Preparation
The aim of this study was to determine the content of some bioactive components
found in extracts of five fig cultivars (‘Šaraguja’, ‘Termenjača’, ‘Crnica’, ‘Bjelica’ and
‘Bružetka bijela’). Samples of Ficus carica L. were collected near the Adriatic coast
region, in Croatia, in July of 2008. Identification and classification of the fig cultivars was
carried out. Fig fruits were cleaned, freezed at -20°C and then freeze dried (LIO-10P,
Kambic d.o.o., Slovenia). Freeze dried fig fruit samples were stored in air-tight plastic
bags at room temperature. Samples were grounded to a fine powder with a blender before
extraction.
Fig samples (10.0 g) were extracted by 70% ethanol or 70% methanol (100.0 ml).
The extraction process was carried out using ultrasonic bath (Brason and Smith-Kline
Company, B-220) at the room temperature for 1 h. After filtration, 5 ml of liquid extract
was used to determine extraction yield. Solvent was evaporated by rotary evaporator
(Devarot, Elektromedicina, Ljubljana) under vacuum, and dried at 60C to a constant
mass. Dry extracts were stored in glass bottles at 4C to prevent oxidative damage until
analysis.
Determination of Total Phenols Content
The content of total phenolic (TP) compounds in investigated fig dry extracts, was
determined by Folin-Ciocalteu procedure (Kahkonen, 1999; Singleton and Rossi, 1965)
using chlorogenic acid as a standard. The absorbance measurements were performed
using a JANWAY 6300 VIS-spectrophotometer, at 750 nm. The standard diagram was
prepared with standard gallic acid solutions. The obtained equation is:
(1)
where: A is absorbance of sample solution; c is phenolics concentration, mg/ml.
The regression coefficient (r) was 0.9995. Total phenolic compounds content has
been expressed as mg of galic acid equivalent (GAE) per g of dry extract - mg GAE/g.
Determination of Total Flavonoids Content
Determination of flavonoids content was performed using a modified colorimetric
method (Jia, 1999). The absorbance measurements were performed using a JANWAY
6300 VIS-spectrophotometer, at 510 nm. The standard diagram was prepared with
standard catechin solutions and the obtained equation is:

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(2)
where: A is absorbance of sample solution; c is flavonoids concentration, mg/ml.
The regression coefficient (r) was 0.9997. The flavonoids contents were expressed
as mg catechin equivalent (CE) per g of dry extract - mg CE/ g.
DPPH Assay and Determination of Reducing Power
The free radical scavenging activity of fig fruits extracts was determined as
described by Espin (2000). Radical scavenging capacity (%RSC) was calculated by the
following equation:
(3)
where: Asample is absorbance of sample solution and Ablank is absorbance of blank sample.
This activity was also expressed as the inhibition concentration at 50% (IC50), the
concentration of test solution required to give 50% of decrease in absorbance compared to
the blank sample.
The reducing power of fig fruits extracts and ascorbic acid was determined by
Oyaizu method (1986). Absorbance of samples was measured at 700 nm. Higher
absorbance indicates a higher reducing power and higher antioxidant activity.
LC/MS Analysis
LC/MS analysis was performed on an Agilent MSD-TOF coupled to an Agilent
1200 series HPLC. Mobile phase A was 0.2% formic acid in water, and mobile phase B
was acetonitrile. The injection volume was 3 µl, and elution at 0.95 ml/min with gradient
program (0-20 min 5-16% B, 20-28 min 16-0% B, 28-32 min 40-70% B, 32-36 min 70-
99% B, 36-45 min 99% B, 45-46 min 99-5% B). A wavelength of 256 nm was selected
for detection. Mass spectra were acquired using an Agilent ESI-MSD-TOF. Drying gas
(N2) flow was 12 L/min; nebulizer pressure was 45 psig; drying gas temperature was
350°C. For ESI analysis, the parameters were: capillary voltage, 4000 V; fragmentor,
140 V; skimmer, 60 V; Oct RF V 250V, for negative modes. The mass range was from
100 to 2000 m/z.
RESULT AND DISCUSSION
It is believed that natural antioxidants have an important role in the prevention and
treatment of many diseases (Hollman and Katan, 1999). From the obtained results
presented in Table 1, total phenols content (mg GAE/g) and total flavonoids content (mg
CE/g) can be seen. All methanolic extracts had higher total phenols content. The highest
content of total phenols was found for ‘Bjelica’ extract obtained with methanol (11.17 mg
GAE/g). The highest content of total flavonoids was obtained by ‘Bjelica’ extracted with
methanol (5.68 mg CE7g), also.
Rutin was detected in all five samples, with LC/MS system (Fig. 1). Identification
of the compound was based on UV spectra and molecular formula obtained from accurate
mass measurement ([M-H]- and [2M-H]- ions, M=610.1629). Mass error was huge
(-15.6 ppm) due to interference with matrix. The identity of the compound was confirmed
by comparison of retention time and LC/MS data of standard compound.
The method of scavenging the stable DPPH* radical is a widely used method to
evaluate antioxidant activity in relatively short time. DPPH* is a stable free radical and it
accepts an electron or hydrogen radical to become a stable diamagnetic molecule

394
(Singleton and Rossi, 1965). The highest value of radical scavenging capacity was
achieved for ‘Crnica’ with 70% methanol extract, 18.75, 37.90, 60.17 and 75.26% for
extract concentrations of 0.1, 0.25, 0.5 and 1 mg/ml, respectively. Radical scavenging
activity was found to exhibit 50% of inhibition value (IC50 value) at the different
concentrations for different cultivar extracts (shown in Table 2). It can be seen that
different solvents affect the antioxidant activity of different kinds of figs extract. Also, by
examining the content of total phenols and IC50 values, we see that the antioxidant
activity comes from other compounds (non phenols).
For the measurements of the reductive ability of fig samples, Fe3+→Fe2+ trans-
formation in the presence of the fruit extracts was determined using the Oyiazu method
(Oyaizu, 1986). The reducing capacity of analyzed samples may serve as indicator of
their potential antioxidant activity. Figure 2 shows the reductive capabilities of the fig
extracts compared with reductive capabilities of ascorbic acid, a well know antioxidant
compound.
The reducing power increases with increases of concentration, for all extracts
(Table 3). It is clear that ascorbic acid has better reductive capability than all investigated
fig fruits extracts. At the same concentration of 0.5 mg/ml, the absorbance of the fig
samples was between 0.065 and 0.120 mg/ml.
CONCLUSION
Methanol proved to be a better solvent for the extraction of fig polyphenols. The
highest content of total phenols was measured in extracts of the cultivar ‘Bjelica’
(11.17 mg GAE/g). The highest content of flavonoids was found in ‘Bjelica’ (5.68 mg
CE/g), also. The highest radical scavenging capacity was measured in the cultivar
‘Crnica’. All methanolic extracts had higher antioxidant activity. The reducing power
increased with concentration of extracts. The main polyphenol found in all extracts was
rutin. Fig fruit extracts thus have high antioxidant activity. Because of its properties,
Ficus carica or their methanolic extracts can be used as natural source of antioxidants,
i.e., possible constituent in food or pharmaceutical products.
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Tables
Table 1. Total phenols (TP) and total flavonoids (TF) content.
Cultivar Solvent TP (mg GAE/g) TF (mg CE/g)
Šaraguja Methanol 10.54 5.12
Ethanol 9.11 4.77
Termenjača Methanol 10.82 5.53
Ethanol 9.42 4.97
Crnica Methanol 9.30 5.31
Ethanol 8.18 4.84
Bjelica Methanol 11.17 5.68
Ethanol 9.56 5.01
Bružetka bijela Methanol 10.24 5.23
Ethanol 7.25 4.43
Table 2. IC50 value of fig fruit extracts.
Cultivar Solvent IC50 (mg/ml)
Šaraguja Methanol 0.652
Ethanol 0.411
Termenjača Methanol 0.8188
Ethanol 0.4283
Crnica Methanol 0.3858
Ethanol 0.3947
Bjelica Methanol 0.8070
Ethanol 0.6504
Bružetka bijela Methanol 0.7061
Ethanol 0.7448

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Table 3. Reducing power of fig fruit extracts at different solvents.
Cultivar Solvent Concentration (mg/ml)
0.5 1 3 5
Šaraguja Methanol 0.078 0.150 0.307 0.789
Ethanol 0.077 0.148 0.301 0.780
Termenjača Methanol 0.077 0.159 0.327 0.910
Ethanol 0.065 0.138 0.305 0.807
Crnica Methanol 0.120 0.244 0.401 0.822
Ethanol 0.081 0.150 0.304 0.747
Bjelica Methanol 0.073 0.144 0.329 0.920
Ethanol 0.079 0.167 0.333 0.916
Bružetka bijela Methanol 0.077 0.148 0.305 0.784
Ethanol 0.075 0.141 0.299 0.742
Figurese
Fig. 1. Rutin, detected in all fig fruit extracts.
Fig. 2. Reducing power of ascorbic acid and fig fruits extract (cultivar ‘Bjelica’, using
methanol solvent) by spectrophotometric detection of Fe
3+
→Fe
2+
transformation.
Apsorbance
concentration [mg/ml]
vitamin C
Bijelica, methanol