Olive (Olea europaea L.) leaf biophenols as nutraceuticals

Muhammad Kamran

    Research output: ThesisMasters Thesis

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    The use of nutraceuticals is increasing at rapid rate around the globe as, they are convenient and cheaper than expensive medical and surgical procedures. Moreover, nutraceuticals are surrounded by a halo of propaganda claiming a safer profile only due to their preparation from natural products. The way forward for giving these phytomolecules a legal ground to be applied in disease prevention and treatment will be through proper scientific research. Thus, there is a great need for comprehensive investigation to evaluate the efficacy of various nutraceuticals and to optimize their recovery from natural sources.
    Olive leaves were selected for this study as olive (Olea europaea L.) leaves are a significant source of biophenols all over the year. Though in the past olive leaves were mainly used for animal feeding, during the last two decades their health promoting properties have been recognized and many studies examined their potential applications. The superior health benefits of olive leaves are attributed to biophenols. These health promoting effects are further augmented because of high bioavailability of these biophenols. The biological activities of olive leaf biophenols have created a high demand for extensive research to develop the most effective method for the extraction and separation of olive biophenols, to identify these biophenols, and to determine their suitability as a nutraceutical or functional food.
    The first study investigated the best method for drying olive leaves so as to optimize the recovery of olive leaf biophenols especially oleuropein. The recovery of biophenolic compounds was compared between fresh, air-dried, freeze-dried and oven-dried (at 60 °C and 105 °C) olive leaves. The biophenol content and antioxidant activity were assessed by gross quantitative methods such as total phenol content (Folin-Ciocalteu’s method), total flavonoid content, total o-diphenol content and total antioxidant capacity using ABTS+◦ and DPPH◦ scavenging assays. In addition, the biophenolic composition of extracts was determined by high performance liquid chromatography (HPLC) equipped with diode array detection (DAD) with tandem mass spectrometery (MS/MS). The contribution of individual biophenols to the antioxidant activity of extracts was evaluted by an online ABTS scavenging assay. Extracts obtained from leaves oven-dried at 105 °C showed the highest phenol recoveries and antioxidant activities, whereas extracts obtained from oven-dried leaves at 60 °C had the lowest values. Oven drying of olive leaves at 105 °C for three hours increased oleuropein recovery up to 38 fold as compared with fresh olive leaves. Results of the study stress the paramount importance of sample pre-treatment in the preparation and analysis of herbal medicines. Futhermore, the limitations of sole dependence on gross assessment of total biophenolic composition and total antioxidant activity in studying plant samples were highlighted.
    Second study was a unique study that covered the complete olive tree cycle. This study was conducted to investigate the effect of climate, season and cultivar on the composition of olive leaf biophenols and antioxidant activity in order to determine the best harvest time for optimal recovery of biophenols from a particular cultivar. The biophenol content of an Australian cultivar was compared with cultivars of Italian origin, grown in Wagga Wagga. The correlation between biophenol content and the antioxidant activity was also determined. A new HPLC method developed for this study, resulted in rapid and clear separation of olive leaf biophenols without minimal co-elution. Olive leaf biophenols vary quantitatively during different stages of the biological cycle of the olive tree, however no qualitative differences were detected. Significant effects of cultivar/ variety as well as month of collection and season were observed for biophenol composition and antioxidant activity of olive leaves. Total flavonoids showed moderately positive correlation with oleuropein and antioxidant activity measured by ABTS and DPPH radical scavenging assays. Oleuropein showed a moderately positive correlation with ABTS activity, but a weak correlation with DPPH activity and total phenols. Total phenol content displayed a weak correlation with ABTS, but no correlation with DPPH activity. The time of collection is the most influential parameter with regards to olive leaves suitability for commercialization. None of the cultivars followed a specific pattern for accumulation of biophenols throughout the collection period. The summer season, was the best for the optimal antioxidant activity and for maximum recovery of biophenols, including oleuropein. For the better recoveries of total phenols, phenolic compunds, o-diphenols, hydrocinnamic acid derivatives and higher antioxidant activity the Leccino cultivar was most appropriate, whereas in order to get higher oleuropein and total flavonoid content, the Hardy’s Mammoth was the best cultivar.
    In the third study, in vitro antioxidant and antidiabetic activities of commercial olive leaf extracts were compared with laboratory prepared extract of Hardy’s Mammoth olive leaves. An array of antioxidant activity assays involving different mechanism such as ABTS and superoxide radical scavenging, hydrogen peroxide scavenging, copper chelation and ferric reducing antioxidant power assay were investigated. Crude and enriched extracts were prepared from leaves of the Hardy’s Mammoth cultivar and these extracts were compared with commercially available olive leaf extracts (OLEs). The oleuropein content of all extracts was determined by HPLC. Total phenols were also measured, as gallic acid and oleuropein equivalents, to find the contribution of total phenols towards antioxidant activity. Antidiabetic activity of OLEs, via α-amylase enzyme inhibition activity, was also investigated. OLEs demonstrated high potency as a source of antioxidant in all tested assays. They also confirmed their antidiabetic activity through inhibition of α-amylase in in vitro assay. Olive leaf biophenols as individual compounds and in a combined form of extract were excellent antioxidants. These results indicate that olive leaf biophenols are multi efficient bioactive molecules and have a great potential as nutraceuticals. Since olive leaf biophenols strongly inhibited the α-amylase enzyme, they could be beneficial in lowering blood glucose level and might prove to be an effective antidiabetic agent.
    In the fourth study, the potential of olive leaf extract as an antidiabetic agent was examined in a pilot clinical trial on pre-diabetic subjects. One in three people with prediabetes will develop type 2 diabetes but it is possible that prediabetics can return to normal with early pharmacological and intensive diet interventions. Therefore this study was planned to find out the suitability of OLE as a blood glucose lowering agent. Eighteen prediabetic adults were recruited, and randomly allocated to take either OLE or placebo capsules three times per day for 12 weeks. Reference blood and urine samples were taken at baseline and after 12 weeks to measure the levels of blood glucose, glycated haemoglobin (HbA1c) and lipid profile. Total phenols as well as the oxidative biomarkers STAT-8-isoprostane and 8-hydroxy-2'-deoxyguanosine were also measured. For all the studied parameters no significant differences were observed between the groups (p ˃ 0.05) and neither supplementation with the olive leaf extract or placebo produced any significant change compared to their baseline samples (p ˃ 0.05). Some individuals appeared to respond to the supplement but as a whole group there was no significant response, which indicated a highly individual response to the supplement. Though it was a pilot study and the number of participants too small enough to produce any significant effect but still it gives some valuable information about the baseline levels of blood parameters and effect of olive leaf extract on Australian prediabeteics.
    Olive leaf biophenols have a great potential as a nutraceutical. They demonstrated good antioxidant and antidiabetic activity in in vitro assays. However, in the pilot clinical study on prediabetic subjects, supplementation with olive leaf extract did not produce any significant change. It is suggested that the potential antidiabetic effect of olive leaf biophenols should be thoroughly investigated in controlled clinical trials with a larger sample size for the efficient utilization of these valuable nutraceuticals.
    Original languageEnglish
    QualificationMaster of Philosophy
    Awarding Institution
    • Charles Sturt University
    • Obied, Hassan, Principal Supervisor
    • Scott, Chris, Co-Supervisor
    • Hamlin, Adam, Co-Supervisor
    Award date01 Mar 2016
    Publication statusPublished - 2016


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