Oxidation is a common problem in olive oil due to its high content of unsaturated fatty acid, this leads to nutritional losses and quality degradation, especially under microwave heating. A number of studies have been done to add natural antioxidants to reduce oxidation. This project examines the possible application of olive leaves to reduce or inhibit olive oil oxidation under microwave heating. Olive leaves were able to increase the total polyphenol content as well as reducing the peroxide value in olive oil. However, the microwave heating caused severe quality degradation (increasing peroxide value and reducing total polyphenol) especially after 10 min of heating. Olive leaves prepared using a freeze dried method have an improved impact in olive oil above that of olive leaves prepared by an oven dried method in reducing peroxide values and increasing the total polyphenol content. Olive leaves serve as a natural antioxidant effectively reducing the oxidation in olive oil.
The outcome and the success of this project required a lot of guidance and assistance from lots of people to whom I am grateful I do not want to forget to thank.
All the thanks to my parents who gave me the chance to study overseas and provided me with unconditional emotional support from thousands of miles away.
I would like to gratefully acknowledge my scholarship (Saudi Arabian Cultural Mission) who supports me academically and financially and is helpful whenever I need them.
I also take this opportunity to express a deep sense of gratitude express and my hearty thanks to my supervisor Said Ajlouni for gave me the chance to do this project, answering all my questions throughout the project, being friendly and helpful. He gave me lots of his time, came with me in lab every time I needed him, coordinated me and advised me in all project areas.
I wish to remember the lab manager Michelle Rhee for her support and providing me with all the stuff I need & some guidelines.
I also want to thank Peter McSweeney for his great assistance in coordinating this project subject and providing useful lectures on how to start, write and present the project.
Oil Oxidation: Overview
Oil oxidation, according to Miller (2010) refers to the unwanted series of chemical reactions, which involve oxygen and lead to the degradation of the quality of an oil. Oxidation leads to the formation of rancidity in oil that is characterized by off smells and flavors. Miller (2010) and Emmanuel (1967) state that oil is always in a state if oxidation; it cannot be stopped, but various ways have been engineered to reduce the oxidation process. Miller (2010) recommends that attempts must be made to reduce oil oxidation at every stage of the manufacturing process. Miller (2010) and Emmanuel (1967) also state that oil oxidation is a multi step process; oil oxidation occurs in various stages and leads to formation of different products at each stage. The first stage leads to the formation of primary oxidation products such as free fatty acids, dienes, and peroxides; this is followed by the release of secondary oxidation products that include trienes, aldehydes and carbonyls (Emmanuel 1967). Oil oxidation culminates with the formation of tertiary products (Emmanuel 1967).
The process of oil oxidation varies and this is influenced by factors such as the presence of oxygen, UV light, temperature, moisture and metals such as iron (Emmanuel 1967; Ullah et al. 2003). Additionally, the type of oil also determines the rate of oxidation. For instance, those with high contents of polyunsaturated fatty acids (PUFAs) such as marine oil undergo oxidation at a faster rate (Ullah et al. 2003). Olive oil has monosaturated fats whose oxidation rates are slower than those of PUFAs. Miller (2010) and Ullah et al. (2003) contend that the high rate of oxidation in PUFAs is attributed to the high content of reactive double bonds. Conversely, saturated fatty acids lack double bonds hence oxidize rather slowly (Miller 2010; Ullah et al. 2003). Oil oxidation can be prevented by controlling various factors. Temperature is the leading cause of oxidation in oil. Thus, controlling temperature especially during the manufacturing and transportation process minimizes oil oxidation. However, in most households, microwaves are used to warm foods. As such, regulating temperature in order to minimize oxidation is unlikely to be achieved. Exposure to UV light is another factor that can be controlled in order to minimize oil oxidation (Emmanuel 1967). Additionally, reducing moisture content and exposure of metals can also reduce the rate of oxidation (Emmanuel 1967). The most efficient way to reduce oil oxidation is the use of antioxidants (Ullah et al. 2003). Natural antioxidants are preferred to synthetic ones; this study focuses on natural antioxidants present in olive leaves.
Previous studies on reducing oxidation in oil
A host of experiments have been conducted previously to determine the positive effect of antioxidants on the oxidative stability of vegetable oils used both domestically and in industrial processes (Yildirim et al 2000). Aluyor and Ori-Jesu (2008) contend that phenols are the most essential antioxidants. They have been found to be more effective than synthetic antioxidants such as Tert-butyl Hydroquinone (TBHQ), propyl gallate, butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA). The use of these phenols as antioxidants for vegetable oils meant for both domestic and industrial use is widespread. Aluyor and Ori-Jesu (2008) argue that vegetable oils in their natural forms have various constituents that act as natural antioxidants. Previous studies conducted to elucidate the effective of natural antioxidants present in red pepper oil showed that those constituents provide variable protection against auto-oxidation induced by light (Ullah et al. 2003). The researchers measured the peroxide values generated during the oxidation process and they realized that constituents from red pepper oil have an inhibitive effect on oxidation. Another study which assessed the inhibition action of tocopherols on the oxidation of palm kernel oil and rapeseed oil, by evaluating the amount of oxidation by product produced (monoaldehyde) showed that tocopherols have a positive antioxidant effect (Emmanuel et al. 1967).
Methanol extracts from Ocimum sanctum L. (holy basil) particularly from the stems, leaves, and inflorescence callas have been found to exhibit good antioxidant activity (Karem 2010; Yildirim et al 2000). This is due to the presence of phenolic compounds with one or more hydroxyl groups. In addition, methanol extracts derived from Piper betle and areca nut inflorescence had high levels of antioxidant polyphenols, which protect LDL from Ca2+ mediated oxidation. Satureja hortensis L., which is commonly known as summer savory has also been found to exhibit antioxidant activities (Karem 2010). Extracts derived from this plant have shown positive results in the elimination of reactive oxygen species and scientists believe that these extracts can be used in food industry as a preservative. Basil is another plant whose extracts have been exploited as potential natural antioxidants (Yildirim et al 2000). Water and ethanol extracts of this plant have the ability to scavenge free radicals as well as induce the reduction of ferrous ion (Karem 2010)
Furthermore, Yildirim et al (2000) warn that even though the reducing power of a natural antioxidant may be an indication of its antioxidant activity, the two properties may not be directly linked. Yildirim et al (2000) conducted a study involving water extracts from linden flowers as well as various black teas in an endeavor to evaluate their reducing and antioxidant properties. Surprisingly, water extracts from linden exhibited the lowest antioxidant activity, but was the most effective extract with respect to reducing power. Additionally, extracts from clove oil were found to have better antioxidant properties than BHT (Karem 2010).
Olive production, Use and Olive Oil oxidation
Olive oil is used widely across the globe especially for cooking purposes. Owing to the wide use of olive oil globally, the challenge of olive oil oxidation affects millions of users across the world. Olive oil is produced from olive dupes. As a result, oil leaves are given less consideration and yet they are a good source of antioxidants. Aluyor and Ori-Jesu (2008) define an oxidant as a molecule capable of preventing or slowing down the oxidation of other molecules. Antioxidants attain this goal via two methods: removing free radical intermediates or being oxidized themselves. In essence, antioxidants are reducing agents. Aluyor and Ori-Jesu (2008) posit that auto oxidation that occurs in various substances used domestically and in food systems is in the form of free radical chain reduction mechanism.
Assessing the Antioxidant Activity of plant Phenols
Methods that employed in the determination of the antioxidant activity of natural antioxidants can be categorized in two groups; SET reaction mechanisms and Hydrogen Atoms Transfer (HAT) reaction mechanisms.
Regimes under Hydrogen Atoms Transfer (HAT) reaction mechanisms
Oxygen Radical Absorbance Capacity (ORAC): This method assesses the antioxidant inhibition of peroxyl radical; as such, this method highlights radical chain breaking that involves the transfer of a hydrogen atom (Karem 2010). During this assay, there a reaction between the peroxyl radical and a florescent probe, this leads to the formation of a non-fluorescent product that can be assayed by fluorescence. Another technique is total radical-trapping antioxidant parameter. This method attempts to identify and quantify the ability of antioxidant compounds minimize the reaction between a probe and a peroxyl radical (Karem 2010). Chemiluminescence method on the other hand is best on a reaction between a marker compound and radical antioxidants, which produces excited state species that have the potential to emit chemically induced light (Karem 2010). Compounds that react with initiating radicals prevent the emission of light. Luminal is the most preferred marker. Additionally, photochemiluminescence assays rely on the generation of super oxide free radical photo-chemically as well as Chemiluminescence detection (Karem 2010). Furthermore, LDL oxidation can be analyzed to elucidate the anti-oxidizing activity of plant phenols. Regimes under SET reaction mechanisms; these reactions capitalize on the reeducation of ferric to a colored product (Karem 2010).
Olive Leaves Extracts: Anti oxidant activity
The medicinal properties of olive leaves, obtained from domesticated olive trees, have been documented since ancient times (Jemayi, Feki and Sayadi 2009). Olive leaves have the following constituents polyphenols (luteolin-7-glucoside, apegenin-7-glucoside and verbascoside), secoiridoids (oleuropein together with its derivatives), hydroxytyrosol, triterpenes and flavonoids (diosmin and rutin) (Thomson Research Inc., 2009). In 2007, a team of researchers from Australia studying the antioxidant properties of 55 medical herbs found out that antioxidants extracted from olive leaves had the highest free radical scavenging activity (Wojcikowski et al. 2007). Oleuropein has been found to minimize the oxidation of low-density lipoprotein (LDL) in vitro as well as in vivo (Visioli 1971; Coni et al. 2000).
A study conducted by Rafiee, Jafari, Alami and Khomeiri (2012) assessed the antioxidant properties of phenols found in olive oil. The researchers used Microwave Assisted Extraction (MAE) to extract the phenols. The researchers studied two varieties of olives: Roghani and Cronaiky. Methanol extract from the Cronaiky variety were found to exhibit the highest antioxidant effect. The researchers recommended that methanol extract from Cronaiky oil variety should be used instead of synthetic antioxidants in the establishment of oxidation stability of edible oils.
On the other hand, Papadopoulos and Boskou (1991) argue that phenols are the main constituents of the polar fraction of virgin olive oil. These phenols are usually extracted using methanol water mixtures (Wojcikowski et al. 2007). Papadopoulos and Boskou (1991) reports that the following compounds are present in the polar fraction of virgin olive oils: hydroxytyrosol, tyrosol, vanillic acid, p-coumaric acid, o-coumaric acid, 3, 4 hydroxyphenylacetic acid, syringic acid, 4-hydroxyphenylacetic acid and 4-hydroxybenzoic acid. Papadopoulos and Boskou (1991) and Wojcikowski et al. (2007) contend that there is a growing interest in the level of phenols in rape (a byproduct of the extraction process), olives and olive oil owing to the antioxidant properties of the polar fraction. However, Papadopoulos and Boskou (1991) regret that little is known about the effect of each component on the stability of the oil. Elucidation of such information is critical in the manufacturing process as well as quality evaluation. In their study, Papadopoulos and Boskou (1991) sought to measure protection factors of the chief phenols occurring in the polar fraction of virgin olive oils and to evaluate their contribution to the stability of the oil. The researchers noted that hydroxytyrosol and caffeic acid exhibited commendable antioxidant effect, which is greater than that of BHT. Conversely, vanillic acid, p-coumaric acid, o-coumaric acid, p-hydroxyphenylacetic acid, and 4-hydroxyphenylacetic acid had little or no effect. Additionally, Boskou (2006) argues that the polar phenolics compounds in olive are an essential class due to not only their biological role, but also the oil stabilization property. Boskou (2006) contends that various studies, which have assessed the anti oxidant properties of olive extracts have revealed that these extract, have a scavenging activity against hydrogen peroxide and superoxide anion and have the ability to stop the generation of free radical species. Table olives on the other hand have been found to possess a different quantitative and qualitative phenolic composition when compared to raw olive fruits. Boskou (2006) argues that an analysis of commercially available table olives has shown that they contain hydroxytyrosol as the leading phenolic compound. Bowden (2013) argues that the most the powerful constituent of olive leaf extract is the oleuropein. Bowden (2013) argues that oleuropein and its metabolite hydroxytyrosol have the most convincing antioxidant activity not only in vivo, but also in vitro.
In another study, Jemayi, Feki and Sayadi (2009) sought to assess the Alloxan-diabetic rats, which were derived from olive leaf extracts, in diabetic rats. Jemayi, Feki and Sayadi (2009) contend that although the mechanisms underlying diabetes mellitus remain unclear, most studies point out to oxidative stress as one of the mechanisms underlying this condition. Oxidative stress is believed to play a part in the development of various diabetic conditions (Wojcikowski et al. 2007). A host of defense mechanisms take part in the alleviation of diabetes induced complications and antioxidants are perfect examples. Antioxidants play a critical role in the elimination of free radicals (Wojcikowski et al. 2007). Herbal drugs are gaining popularity in the management of diabetes. Natural antioxidants are said to be safer regimes than synthetic ones (Wojcikowski et al. 2007). The olive tree is one of the leading sources of these natural antioxidants.
Jemayi, Feki and Sayadi (2009) attribute the medicinal role of byproducts from the olive tree to the presence of phenolic and antioxidant compounds, the most notable ones being tyrosol, hydroxytyrosol, oleuropein and oleuropein aglycone. Olive leaves are regarded as a cheap raw material and a crucial source of essential antioxidants. Jemayi, Feki and Sayadi (2009) argue that the leading antioxidants present in olive leaves include the glycosylated forms of ligstroside and oleuropein. However, upon purification, oleuropein is the single most abundant extract. Various studies have hypothesized and proven the antioxidant role of oleuropein (Bowden 2013). Other studies focused on hydroxytyrosol, which as been found to be effective in the elimination of hydroxyl radical and superoxide ion (Bowden 2013). Jemayi, Feki and Sayadi (2009) further argue that their previous studies involving hydroxytyrosol and oleuropein had registered positive results.
In addition, Lafka et al (2013) contend that olive mills as well as olive processing residual have become vital sources of natural antioxidants. Of all the sources, olive leaves are the single chief sources of antioxidants (Thomson Research Inc., 2009). Lafka et al (2013) argue pruning, which is a common stage in the cultivation of olive trees, can be a significant source of olive leaves for research instead of their present use as animal feed. For a long time olive leaves have been used as a traditional medicine. The phenolic properties of each plant material are unique and this calls for the establishment of optimal extraction conditions as well as extract evaluation with respect to composition and antioxidant properties (Thomson Research Inc., 2009). Solvent extraction has been devised for the extraction of soluble phenol from olive leaves extracts. The separation occurs by diffusion whereby the by-product diffuses from the olive matrix via a solvent. Several factors influence the solvent extraction process. These factors include the type of solvent employed, temperature, PH, the number of extraction steps, particle size of the solid matrix as well as solvent/solid ratio (Thomson Research Inc., 2009).
Lafka et al (2013) contends that supercritical fluid extraction (SFE) can be employed in the extraction of various bioactive substances without any substance residue as well as safety hazard. CO2-SFE has been found to be the most reliable method during the isolation of phenolic compounds because it leads to higher phenol recoveries as well than other regimes (Majid, Hasan and Pooran 2012; Lafka et al 2013). However, recent finding during the extraction of oleuropein from olive leaves, the application of CO2-SFE was found to be unsatisfactory and it required the addition of a polar modifier to enhance the selectivity of the process and the yield. Putting into consideration that bioactive materials in various plant materials vary and that extractability depends on method and solvent used, oleuropein Lafka et al (2013) sought to determine phenol extraction conditions from olive leaves. The researchers also evaluated and compared the phenolics contents as well as the antioxidant activity of SFE and solvent phenolic extracts as well as best extract application to oil protection. The study conducted by Lafka et al (2013) revealed that ethanol was the most suitable solvent for extraction under the following conditions: PH 2, Solvent to sample ratio 5:1 v/m at 180 minutes. The antioxidant potential of SFE extracts was however found to be higher than that of ethanol extracts. The study provided proof that leaf extracts are reliable protectors of sunflower and olive oils at the levels of 150 ppm.
Moreover, Silva et al (2006) argue that phenolic compounds are complex, but an essential group of naturally occurring plants. There is thus a growing interest in the phenolic compounds derived from the olive tree particularly their antioxidant role. Previous studies conducted on olive leaves extract have demonstrated the presence of a host of medical properties including antioxidant effect (Coni et al 2000). Fresh pulp of olive trees is believed to contain between 1 to 3 percent of phenolic compounds, which include flavonoids, phenolic alcohols, phenolic acids and secoiridoids (Coni et al 2000). Silva et al (2006) argue that hydroxytyrosol and tyrosol are the major alcoholic phenols founds found in olive leaves. Secoiridoids, unlike other phenols is has a limited occurrence; they are produced from secondary metabolism of terpenes and they are precursors for indole alkaloids. Secoiridoids have one unique property in that they contain elenolic acid or its derivatives (Coni et al 2000). On the other hand, hydroxytyrosol and oleuropein are abundant in olive fruits (Silva et al 2006). Oleuropein is abundant in unprocessed fruits while processed fruit and oil have high quantities of hydroxytyrosol (Coni et al 2000). Silva et al (2006) argue that it is essential to note that olives have complex phenols such as glycosides, which occur in a polar and hydrophilic form. The oil chiefly contains the aglyconic form, the lipid soluble part of the molecule (Silva et al 2006). The study conducted by Silva et al (2006) proposed that olive seeds are critical sources of natural antioxidants.
In another study, Majid, Hasan and Pooran (2012) conducted a study to elucidate the inhibitory effect of olive leaf extract on nephrotoxicity induced by gentamycin in rats. Such toxicity is attributed to the generation of free radical species. Majid, Hasan and Pooran (2012) found that the co-administration of gentamycin and of olive leaf extract led to a significant reduction the levels of serum and renal creatinine malondialdehyde and tubular necrosis. The researchers concluded that gentamycin-induced nephrotoxicity is eliminated by olive leaf extract through antioxidant activity. Majid, Hasan and Pooran (2012) and Silva et al (2006) argue that olive leaves are rich in biophenols particularly hydroxytyrosol, tyrosol, verbascoside, ligstroside and oleuropein. These compounds have exhibited biological activities such as anti-oxidation (Silva et al 2006).
In summary, this literature review has shown that oxidation leads to the formation of rancidity in oil that is characterized by off smells and flavors. It is essential to note that oil is always in a state if oxidation; this means that oxidation cannot be stopped, but various ways have been engineered to reduce the oxidation process. Oil oxidation occurs in various stages and leads to formation of different products at each stage. Olive oil is one of the commonly used cooking oils across the globe. As any other oils, olive oil undergoes oxidation. Owing to the wide use of olive oil across the globe, the challenge of olive oil oxidation affects millions of users. Olive oil is produced from olive dupes. As a result, oil leaves are given less consideration and yet they are a good source of antioxidants. Natural antioxidants are said to be safer regimes than synthetic ones. Olive leaves have for long time been used as a traditional medicine. The phenolic properties of each plant material are unique and this calls for the establishment of optimal extraction conditions as well as extract evaluation with respect to composition and antioxidant properties. The olive tree is one of the most ancient sources of these natural antioxidants. Olive leaves are an inexpensive source of natural antioxidants. The antioxidant capacity of natural antioxidants can be assessed using different regimes. SET reaction mechanisms and Hydrogen Atoms Transfer (HAT) reaction mechanisms. Regimes under SE reaction mechanisms capitalize on the reduction of ferric to a colored product.
Olive leaves have various constituents such as hydroxytyrosol, triterpenes and flavonoids (diosmin and rutin), polyphenols (luteolin-7-glucoside, apegenin-7-glucoside and verbascoside) and secoiridoids (oleuropein together with its derivatives) (Thomson Research Inc., 2009). In 2007, a team of researchers from Australia who were studying the antioxidant properties of 55 medical herbs found out that antioxidants extracted from olive leaves had the highest free radical scavenging activity (Wojcikowski et al. 2007). It is essential to note that olives have complex phenols such as glycosides, which occur in a polar and hydrophilic form. The oil mainly contains the aglyconic form, the lipid soluble part of the molecule (Silva et al 2006). Other experts argue leading antioxidants present in olive leaves include the glycosylated forms of ligstroside and oleuropein. However, upon purification, oleuropein is the single most abundant extract. Various studies have hypothesized and proven the antioxidant role of oleuropein. Solvent extraction is the most preferred method of extracting soluble phenol from olive leaves extracts. The separation occurs by diffusion in that the by-product diffuses from the olive matrix via a solvent. Several factors influence the solvent extraction process. The most prominent factors include the type of solvent employed, temperature, PH, the number of extraction steps, particle size of the solid matrix as well as solvent/solid ratio. Extracts from olive leaves derived from this plant have shown positive results in the elimination of reactive oxygen species and scientists believe that these extracts can be used in food industry as a preservative.
This study will assess total phenol content in the olive leaf extracts; thereafter, the free antioxidant activity of phenols extracted will be assayed using DPPH and the quantification of peroxide value. DPPH is commonly employed in research to monitor free radical activity. DPPH is a free radical trap; the rate of reduction of a given chemical reaction is assayed following the addition of DPPH. As such, it is critical in the evaluation of antioxidant activity of various plant extracts. In addition, measurement of peroxide value is another method used to assay the antioxidant activity of various substances. Peroxide is a primary product of oxidation; the quantification of its value can be used to estimate the antioxidant activity of a given substance. In this case, the peroxide level in olive oil will be assayed after microwave heating. This will be essential in the assessment of the antioxidant activity of olive leaves extracts. Findings from this study will assist people in the manufacturing sector to use proactive measures in the manufacture of olive oils; these measures should aim at reducing oxidation in olive oils.
Purpose of statement
Unsаturаted fаtty аcids аre the mаin tаrgets аnd substrаtes of oxidаtion, thаt in higher extent cаn form toxic аnd dаngerous compounds to the consumers heаlth (Lаguerre et аl., 2007, pp. 265), being therefore necessаry to find strаtegies to improve the stаbilizаtion аnd quаlity of olive oils. In the lаst yeаrs the аddition of аntioxidаnts of nаturаl sources hаs been а line of investigаtion with promising results. Severаl studies highlighted the beneficiаl effects of the аddition of nаturаl аntioxidаnts to olive oils (Lаlаs аnd Dourtoglou, 2003)
Olive leаves, а high cost, is considered а by-product of the olive oil extrаction industry. This sub-product, аccording to Bouаziz et аl. (2008) cаn reаch 10% of the totаl weight of processed olives, аnd аre аn increаsing cost for producers due to their removаl, storаge аnd eliminаtion. Therefore, wаys to vаlorize this sub-product аre needed аnd most welcome. Olive leаves аre а greаt source of nаturаl аntioxidаnt compounds with biologicаl properties (Briаnte et аl., 2002, pp. 132) аnd their extrаcts аlso reveаled to be effective stаbilizers in olive oils under oxidаtive induction (Bouаziz et аl., 2008, pp. 120). Our theory in this project is that olive leave extract will reduce or inhibit olive oil oxidation under microwave heating. We also assume that olive leave prepared using freeze dried method will have better effect in the oil than olive leave prepared by oven dried.
Aims / objectives
In this sense, with the present work we intend to study the effect of microwаve heаting in one of the most frequently consumed polyunsаturаted olive oil worldwide, the olive oil, аnd the cаpаcity to counterаct the oxidаtive process by the аddition of аqueous extrаcts of nаturаl sources, nаmely olive leаves. In other word, the project will also examine the possible application of olive leave extracts to inhibit or reduce olive oil oxidation and quality deterioration during microwave heating. A comparison will be made between using (a) olive oil and (b) olive oil with olive leaf extract (using freeze dried method) (c) olive oil with olive leaf extract (using oven dried method) to inhibit olive oil oxidation and deterioration during microwave heating (0, 5 & 10 min). The results are judged by measuring (Total phenolic content, DPPH & peroxide value).
Olive leaf preparation
Aqueous extrаcts was prepаred аs follows: lyophilized olive leаves was washed, dried and chopped, then divided into two parts: the first part was put in oven (50°C) for 7 hours, while the second part was put in freez dryer (-48°C) for 3 days. Both parts were then ground.
Three kinds of sample were prepared:
- Virgin Olive oil only
- Virgin Olive leaf (prepared using freeze dryer) with olive oil in a proportion 1g/100ml
- Virgin Olive leaf (prepared using oven) with olive oil in a proportion 1g/100ml
For each trial the prepared samples were microwaved under 1000w for 0, 5 and 10 min using the microwave available in the laboratory (household microwave)
In order to extract polyphenols, 10 ml were taken from each sample, mixed with 80% methanol, vortex, centrifuge for 10 minute, then collect supernatant to test total polyphenol content, Radical Scavenging Activity and peroxide value.
Total phenolic content determination
In order to measure the total polyphenol content, the method used by Singleton (1999). Breifely, 100 µl of each sample was mixed with 900 µl of distilled water and 500-µl Folin-Ciocalteu reagent. Then 4 ml of 7.5% Na2CO3 after 1 minute. Each of the mixtures was then incubated in a shaking incubator. The absorbance for each sample was determined at 756nm using spectrophotometer. A control was prepared using 100µl of distilled water instead of a sample extract. The total phenolic content was determined using a calibration curve prepeared using the same procedure from Epigallocatechin gallate (EGCG) as a standard (see appendix).
DPPH Radical Scavenging Activity
Samples were analyzed for their capacity to scavenge the stable DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical. each sample and a blank were prepared in dark room at room temperature and evaluated by mixing 1 ml of 0.1 mM DPPH (in methanol), vortex, then absorbance was read at 517 nm using spectrophotometer after 0, 5 & 10 minutes. Antioxidant capacity was calculated using the flowing equation:
% reduction= ADPPH (control) – Asample at the same time / ADPPH ( control) x 100
For measuring peroxide value, 9.4mL of the chloroform/methanol solvent was transferred into a 150/20mm test tube, 0.1ml of the liquefied sample was added, and 0.05mL of ammonium thiocyanate solution was added. 0.05mL of ferrous chloride solution was added. Then all are mixed thoroughly and allow standing for 5 minutes. Measure and record absorbance were Measured and recorded at 500nm against the chloroform/methanol solvent using spectrophotometer. 2 blank were prepared, first blank was the fat blank which prepared using the same procedure but no ferrous chloride were added, the second blank were regent blank that prepared with no sample adding (see appendix for more details). A calibration carve were prepared using ferric chloride in order to determine micrograms of iron corresponding to sample on the calibration curve (see appendix for more details). The flowing equations were applied to determine the Peroxide value:
Anet = A1 – (A2+A3)
Anet: net absorbance
A1: sample absorbance
A2: fat blank absorbance
A3 : reagent blank absorbance
PV (milligram-equivalents of oxygen per kilogram sample) = Fe (net)/ m*55.85
Fenet = micrograms of iron corresponding to Anet on the calibration curve.
M = sample weight (g)
Each experiment will be conducted in dolicates (two trials), and three measurments for each parameter will taken within each trial in order to determine the reliability of the method.
Data reported in this study were subjected to аnаlysis using:
- Exell software:
Was used to applied equations in the row data.
- Minitab software
Was used in order to apply an analysis of variance (ANOVA) and to make standard curve using general linear regression.
- Fisher’s LSD (Least Significant Difference) equation to show the significant diffrence between means in each test (see appendix)
All statistical tests were performed at a 5% signiﬁcance level.
Results and discussions
Any differences in 2 means equal or above LSD value will be significant difference
LSD (total polyphenol content) = 0.16
LSD (DPPH) = 4.34
LSD (Peroxide value) = 0.10
Total phenolic content determination:
The table present total polyphenol content (µg/ ml) in three samples with different heating time. As it can seen from the table, oil with freeze dried olive leaves gave the highest proportion of polyphenol, followed by oil with oven dried olive leaves and the oil only gave the lowest polyphenol content (4.22, 3.8 and2.29) with more heating time, the total polyphenol amount is decreased, and after 10 min the total poly phenol content become low and decreased almost 50% Thus, heating 10 min or over will reduce total polyphenol to half of the amount.
DPPH Radical Scavenging Activity
DPPH is unstable radical that can be reduced by antioxidant. As a result, the violet (purple colour) solution is changed to a pale yellow nonradical solution and the absorbance of DPPH is lowered at 517nm. Blank sample containing the same amount of DPPH and methanol and would show stable colour (no change to yellow). Percentage absorbance reduction of DPPH shows the antioxidant capacity in radical scavenging. The more antioxidants (polyphenols present), the larger the antioxidant activity, and the larger the %DPPH reduction (more yellow colour, and smaller OD values) ( Ajlouni, 2013).
However, in this experiment, results were opposite to was expected and what has been reported in literature, with more heat, the reduction is increased, and in the oil only the reduction was the highest. This means that a mistake was occurring in the experiment, as the spectrophotometer was giving changeable results. Therefore, it might be a problem in the spectrophotometer or the way of calculating the reduction. If the reduction percentage did not increase with olive leave, the total poly phenol will not be increased also. In addition, the heating time should give less percentage of reduction.
The peroxide value is measured to see the oxidative extension during microwave heating; the peroxide value is high in the primary stage of the oxidation, due to the formation of hyroperoxide product. It is clear from the table above that the peroxide value has been significantly decreased (Means difference > 0.10) with adding the leaves (both freeze dried and oven dried) compare to oil only. There was no significant differences between freeze dried leave and oven dried leave), but only after 5 min of microwaving, the differences has become significant as freeze dried olive leave gave significantly lower peroxide value. With more heating time, the peroxide value has increased in all the three samples and become very high. However, peroxide value could be decreased after 10 min as previous studies has shown (Malheiro, 2013), as the oxidation will moved to the secondary stage where hydroperoxide is degraded.
Based on the results of this study, we conclude that it is olive leaf can be used a natural antioxidant in olive oil to increase oxidative stability, as it shown positive effect on reducing peroxide value as well as increasing the total polyphenol content. The freeze dried method of drying olive live would be more effective than oven dried method. Using olive oil will help oxidative stability as well as reduce the cost for removing or get rid of the leave. Nevertheless, microwave heating has a negative impact in olive oil especially in 10 min or more, as it shown a significant reduction in polyphenol content as well as increasing the peroxide value, therefore, this study discourages using the microwave heating for more than 10 min.
In order to further evaluate the application of using olive leaves in olive oil, there are a number of areas that need to be studied,
- More heating time
This study used three different heating times (0,5 &10 min), and 10 min showed a high peroxide value and low total polyphenol content, there is a need to know whether a time before 10 min starts to cause this severe damage (eg, 10 min).
- Using different consentrations of olive oil
This study intend to study the impact 3 different consentration of oilve leaves, but due to time limitation, it only use one one consentration (1g/100 ml), so more consentration need to be studied to know what is the best consentration to be used
- Reexaminine the DPPH
DPPH results in this study shows opposite results to total polyphenol content, therefore, there is a need to study the impact of olive leaf in DPPH
- Test other paramenters
This study intends to also study the total tochopherol, as vitamin E is the majour antioxidant in the olive leave, but due to time limitation only three parameters were used (total polyphenol content, peroxide value & DPPH).