banat s journal of biotechnology · 2017-01-05 · 2o 2), peroxyl radical (roo.), singlet oxygen...

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Banats Journal of Biotechnology

2013, IV(7),

47

ANALYSIS AND ANTIOXIDANT ACTIVITY OF PHENOLIC COMPOUNDS IN

OLIVE AND FIG LEAVES JUICE

DOI: 10.7904/2068–4738–IV(7)–47

Maliha, A. EL–MARZOUQ

Food Science & Nutrition Department, Faculty Agriculture Science & Foods, King Faisal University,

Saudi Arabia. e–mail: [email protected]

Abstract. The phenolic compounds of olive and fig leaves juice were extracted and

determination. The total phenolic contents of the juice were estimated and their ability to reduce the oxidation were tested using Rancimat and 1,1–diphenyl–2–picrylhydrozyl (DPPH) free radical scavenging. The phenolic compounds of olive and fig leaves juice were identified by high performance liquid chromatography (HPLC). The results indicated that the displayed potent antioxidant effect against the DPPH radical and high oxidative stability by Rancimat of phenolic compounds of olive and fig leaves juice. Generally, results obtained indicate that olive and fig leaves may become important as a cheap and noticeable natural source of antioxidant, which can be used in fatty foods.

Key words: phenolic compounds, olive leave, fig leaves, antioxidant.

Introduction The reactive oxygen species (ROC)

such as superoxide anion (O2–), hydroxyl

radical (HO.), hydrogen peroxide (H2O2), peroxyl radical (ROO.), singlet oxygen (1O2), and peroxynitrite (ONOO) can be generated from auto–oxidation and thermal oxidation of lipids and many cellular oxidative pathways and therefore are linked to many human diseases such as cancer, heart disease, and cerebrovascular disease [HUANG et al., 2005;

FANG et al., 2002; BRIANTE et al., 2003]. An imbalance between generation

and removal of ROC can cause an oxidative stress in which excess ROC attacks and damages virtually all biomolecules in the cells, leading to cell death and serious chronic disease [SCANDALIOS, 2005].

To minimize the physiological damages caused by excess ROS, a wide array of enzymatic and non–enzymatic endogenous antioxidant defense system have been evolved to compensate the generation of ROC [FRIDOVICH, 1997; SIES, 1993].

Recently, natural foods and food–derived antioxidant such as phenolic phytochemicals and vitamins have

received considerable attention, because they are known to function as chemopreventive agents oxidative damages [CARRASCO–PANCORBO et al., 2005; PEREZ–

BONILLA et al., 2006; VALAVANIDIS et al., 2004]. Oxidation could be also prevented

in foods by the addition of synthetic antioxidants such as BHT, BHA and TBHQ but more attention has recently been paid on natural antioxidants because of safety issues of synthetic forms.

Typical natural antioxidants include tocopherols, carotenoids, flavonoids, and polyphenolic compounds [AMRO et al., 2002;

PARK et al., 2005] that can potentially provide protection against the development of certain oxidation–linked chronic diseases [SKRGET et al., 2005, BUTNARIU et al., 2012].

Epidemiology studies have shown that the traditional Mediterranean diet is associated with low incidence of cardiovascular disease and certain cancers [ARTAJO et al., 2006; TUCK and HAYBALL, 2002].

These beneficial effects on human health have been attributed to the presence in the Mediterranean diet of antioxidants such as phenolic compounds, carotenoids, and tocopherols

Banat’s University of Agricultural Sciences and Veterinary Medicine from Timisoara,

Contact: web: http://www.bjbabe.ro, e-mail: bjb@usab–tm.ro

48

that play an important role in disease prevention [GIMENO et al., 2002].

The olive leaf (Oleaceae) has been widely used in folk medicine for several thousand years in European Mediterranean islands and countries.

Historically, olive leaf has been used as a remedy for fever and other diseases such as malaria [CIAFARDINI and

ZULLO, 2002; FERNANDEZ–ESCOBAR et al., 1999; GUCCI et

al., 1997 BUTNARIU et al., 2012]. Olive foods such as olive oil and

olive leaf in the Mediterranean diet are the primary source of phenolic compounds, which are also important markers for evaluating the quality of olive–based food products.

The major active components in olive leaf are known to be oleuropein and its derivatives such as hydroxytyrosol and tyrosol, as well as caffeic acid, p–coumaric acid, vanillic acid, vanillin, luteolin, diosmetin, rutin, luteolin–7–glucoside, apigenin–7–glucoside, and diometin–7–glucoside [BIANCO and UCCELLA,

2000; FARAG et al., 2003; RYAN et al., 2002; SAMUELSSON,

1951; TASIOULA–MARGARI and OLOGERI, 2001]. Also, Ficus carica Linn. is

commonly referred to as “fig”. It has many pharmacology effects,

including anti–tumour, antioxidant the ability to mediate body metabolism, hyperglycemia, hyperlipidemia and cholesterol levels, enhancement of oxidation resistance, antibiotic effects, antiviral properties, and the ability to mediate immunity and activate blood coagulation [ZHANG and JIANG, 2006; GILANI et al., 2008

PUTNOKY et al., 2012]. It has been traditionally used for its

medicinal benefits as laxative, cardiovascular, respiratory, antispasmodic and anti–inflammatory remedies [GUARRERA,

2005]. The present study was entailed on

the direct use of juice obtained by pressing olive and figure leaves without recourse to extraction and fractionation of the total polyphenols.

Total phenolic content, electron donating ability by DPPH and antioxidant activity, assay was Rancimat method.

As well, identification of the phenolic compounds in fresh juice (olive fruits and fig) by HPLC (high–pressure liquid chromatography) method.

Material and methods Source of olive and fig leaves:

The ripe olive and fig leaves were obtained during the season 2012 from Al–Hasa region, Saudi Arabia. Solvent and standard reagent:

All solvents used throughout the whole work were of analytical grade and were distilled before use. Caffeic acid (98%) was purchased from Aldrich Chemical Co. Ltd., England. Folin Cioculteau reagent and 1,1–diphenyl–1–pierylhyrazyl (DPPH) were obtained from Gerbsaure Chemical Co. Ltd., Germany. Source of sunflower oil:

Sunflower oil was obtained from supermarket, Al–Hasa, Saudi Arabia. Preparation of crude olive and fig

leave juice: Olive and fig leaves were cleaned

and remove seeds pressed by hydraulic laboratory press.

The resultant crude juice was concentrated using freeze dryer (Labconco Corporation, Kansas, City, MO, USA) and kept in a brown bottle at 5ºC until use. Determination of total polyphenolic:

The levels of total polyphenols of fresh leave juice were determined according to the method of Rancimat [GUTFINGER, 1981].

Caffeic acid was served as a standard compound for the preparation of the calibration curve. Phenolic fraction:

Phenolic fraction was isolated by solid phase extraction and analyzed by reversed–phase HPLC using a diode array UV detector [MATEOS et al., 2001].

A Hewlett–Packard series 1,100 liquid chromatographic system (Waldbronn, Germany) equipped with diode array detector and a lichrosorb RP18 column (4.00 mm, C250 mm, particle size 5mm, Merck, Darmstdt) used.

Elution was performed at a flow rate of 1.00 mL/min with mobile phase of



2013, IV(7),

49

water/acetic acid (98:2 v/v, solvent A) and methanol/ acetonitril (50:50, v/v, solvent B), starting with 5% B then increase to levels of 30 % at 25 min, 40% at 35 min., 52 % at 40 min.; 70% at 50 min., 100 % at 55 min., and kept at this stage for 5 min. quantification of phenolic compounds was carried out at wave length of 280 nm using p–hydroxybenzoic acid as an internal standard. DPPH free radical–scavenging

activity: The DPPH free radical scavenging

assay was cassied out, as previously reported by [LEE et al., 2009] with some modification.

The crude juices from olive and fig leaves at (200 ppm) were added to a 0.06 nm DPPH solution in ethanol and reaction mixture was shaken vigorously.

After incubation for 30 min at room temperature, the absorbance at 517 nm was recorded spectorphotometrically.

BHT at 200 ppm was used as a reference as the test compounds.

A control solution, without the tested compound, was prepared in the same manner as the assay mixture.

All the analysis was done in triplicate. The degree of disclorisation indicates the free radical scavenging efficiency of the substances.

The antioxidant activity was calculated as an inhibitory effect (IE %) of the DPPH radical formation as follows:

IE% = 100 X (A517cntrol–A517sample /A517cntrol)

Antioxidant activity by Rancimat: Different concentration of olive and

fig leaves juice (200, 400 and 800) and BHT (200 ppm) were individually added to sunflower oil to study their antioxidant behavior.

The designation of an induction period, measured by using a Rancimat instrument (679 Metrohom Ltd, CH–9100 Herisau, Switzerland), was taken as a tool to compare the effectiveness of the phenolic on sunflower oil stability [MENDEZ et

al., 1996]. Statistical analysis:

All data are expressed as mean as ± SD with the number of observations shown in parenthesis.

Statistical analysis was performed using the students test or one–way ANOVA followed by the post hoc Turkeys test. A value of P ≥ 0.05 was considered to be statistically significant.

Results and discussion

Total phenolic compounds content: Table 1 shows the polyphenolic

content of olive and fig leaves juice. The concentrations of total phenols

as determination by the Folin–Ciocalteu method varied from 524.00 to 624.50 µg/gm as caffeic acid.

Table 1.

The amount of total phenolics measured by Folin–Ciocalteu reagent Samples Plyphenols (µg/gm) Fig leaves 524.50±10.50 Olive leaves 623.00±5.80

Value are mean ± S.D. (N=3)

The highest phenolic content was presented in olive leave juice (623.00 µg/gm) followed by fig leave juice (523.50 µg/gm).

These results are in good agreement with other study by [SKEGET et al., 2005; VERBERIC et

al., 2008], in which total polyphenolic content of olive leaf extract were 444.00µg/ gm.

And fig leaf extract was 350.00 µg/ gm.

Furthermore, the profile of phenolic compounds identified in the extracts using HPLC was similar (data not shown) to the profile reported by [BENAVENTE–GARCIA et al., 2002]. The antioxidant activity by Rancimat

method: The antioxidant activities of olive

and fig leaves juice extracted from leaves (olive and fig) were assessed by the Rancimat method.



50

This method assigned the induction period for the onset of oxidative rancidity in sunflower oil at 100ºC.

In the present study, simple model systems comprising sunflower oil with various concentrations of olive and fig leaves juice were used to assess oxidation behavior.

An experiment was performed with sunflower oil and BHT (200ppm) to compare the antioxidant efficiency of the phenolic compounds from olive and fig leaves juice with the most commonly used synthetic antioxidant material.

Table 2 shows the effect of olive and fig leaves juice on the oxidative rancidity of sunflower oil.

Table 2. Oxidative stability method by Rancimat of olive and fig leaves juice and BHT

Samples Induction period (hrs) Control 7.00±1.00 BHT 200PPM 10.10±1.10 Fig leaves (200ppm) 9.50±1.01 Fig leaves (400ppm) 11.20±1.55 Fig leaves (800ppm) 15.30±2.00 Olive leaves (200ppm) 9.80±1.90 Olive leaves (400ppm) 11.70±1.77 Olive leaves (800ppm) 16.00±2.53

The results illustrate that all the

various concentrations of olive and fig leaves juice, exhibited antioxidant activity.

Statistical analysis showed that the olive leaves juice had no significant antioxidant effect on sunflower oil stability.

It is worth noting that olive and fig leaves juice at 400ppm level superior to that of BHT in retarding sunflower oil oxidative rancidity. DPPH radical–scavenging activity:

There are different methods for estimation of antioxidant activity but the most widely methods are those that involve generation of free radical species which are then neutralized by antioxidant compounds.

DPPH radical is commonly used as substrate to evaluate antioxidant activity; it is useful and stable free radical that can accept on electron or hydrogen radical to become a stable molecule.

The reduction of DPPH free radical was determined by the decrease in its absorbance at 517 nm induced by different antioxidants.

DPPH free radical reacts with antioxidant, consequentially, absorbance decreases and the DPPH free radical is converted into the DPPH form.

The degree of discoloration indicates the scavenging potential of antioxidant compounds of extracts in terms of hydrogen donating ability [JIAO et al.,

2012]. Table 3 three levels of the

concentrations of olive and fig leaves juice (200, 400 and 800 ppm) compared with BHT (200 ppm) were used with a very high scavenging capacity of 40.00 after only 10 min. in all cases the scavenging capacity did not increase after the first 10 min of incubation.

Table 3. Scavenging capacity of DPPH free radicals by olive and fig leaves juice and BHT

Samples Scavenging capacity (%) Control 15.00 BHT 200PPM 25.20 Fig leaves (200ppm) 20.50 Fig leaves (400ppm) 25.30 Fig leaves (800ppm) 40.00 Olive leaves (200ppm) 20.60 Olive leaves (400ppm) 26.00 Olive leaves (800ppm) 40.01



2013, IV(7),

51

The reactions of BHT with DPPH were similar to olive and fig leaves juice with DPPH; the scavenging capacities were similar.

These results were in agreement with the report by [AMRO et al., 2002; VEBERIC et al.,

2008], who showed that most of the olive sake and fig leaf extracts and fractions showed higher antioxidative activity than BHT.

The overall DPPH radical scavenging effect of the olive and fig leaves juice were to donate electron, which could then react with free radicals to convert them to more stable products, terminating radical chain reactions. Identification of phenolic

compounds:

Identification of phenolic compounds by HPLC technique was used to identify the major phenolic compounds in the olive and fig leaves juice.

The identification was based on comparisons of the chromatographic retention time and UV absorbance spectra of compounds in olive and fig leaves juice with of authentic standard.

Data of HPLC analysis of the olive and fig leaves juice were made up of 12 compounds.

The main phenolic compounds of olive and fig leaves juice were found p–hydroxyl benzoic acid, hydroxytyrosol, tyrosol and p–coumaric acid (Table 4).

Table 4. The amount of phenolic compounds by HPLC in olive and fig leave juice

Phenolic name Fig leaves Olive leaves p–Hydroxy benzoic acid 503.20±10.00 614.50±15.20 Vanilin 40.01±2.30 34.20±1.00 p–Coumaric acid 151.20±5.50 160.04±5.20 Gallic acid 50.20±3.40 14.04±0.52 Caffeic acid 42.30±1.40 12.30±0.71 Tyrosol 101.30±4.01 212.50±6.30 Ferulic acid 68.50±3.00 51.02±3.41 Hydroxy tyrosol 150.50±5.60 310.04±7.50 Ellagi acid 0.80±0.01 20.01±1.20 Syringic acid 50.90±5.50 11.30±0.21 Apigenin 90.02±4.30 48.50±0.90 Quercetin 0.09±0.001 0.01±0.001S

The high concentrations of these

compounds in olive leave juice. These results are in agreement with

report by [BENAVENTE–GARCIA, 2000; LEE et al., 2009]. It could be concluded from the

results of this study that the addition of olive and fig leave juice phenolic compounds to sunflower oil offers a good protection against oxidation especially are a level of (800 ppm).

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Received: February 3, 2013 Accepted: April 25, 2013

banat s journal of biotechnology · 2017-01-05 · 2o 2), peroxyl radical (roo.), singlet oxygen...

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