Alexandre Lorinia, Bianca Camargo, Aranhaa Bruna da Fonseca, Antunesb Deborah, Murowaniecki Otero, Andressa Carolina Jacquesb, Rui Carlos Zambiazia
https://doi.org/10.1016/j.foodchem.2020.128758
Abstract
Due to the appreciable amounts of bioactive compounds in olive leaves and the effect of abiotic stresses on their synthesis, this study evaluated the metabolic profile of leaves of olive cultivars (Arbequina, Manzanilla and Picual) collected in four periods of the year (autumn, winter, spring and summer). The determination of the profile of bioactive compounds (phenolic compounds, flavonoids, tannins, carotenoids and chlorophylls) by spectrophotometry and the individual compounds by liquid chromatography coupled to mass spectrum, as well as antioxidant potential tests were performed. As results it was possible to observe that the leaves of the cultivar Manzanilla presented the highest levels of phenolic compounds and that the leaves collected in the summer presented a number of compounds much more relevant than the others. Thus, it was possible to conclude that the cultivar and the collection season significantly affect the bioactive content and the antioxidant potential of olive leaves.
1. Introduction
One of the oldest plants cultivated in the world is the olive tree (Olea europaea L.), which is found in several regions of the globe, with the two main products from this plant, olive oil and table olives. However, the fruit of the olive tree (olives) is not the only part of the plant that can be used; the leaves, which are considered a co-product from the production of olives, can be found in large quantities in the olive oil industries and in the plantations during the pruning period of the trees (Guo et al., 2018, Lama-Muñoz et al., 2020).
Olive leaves are considered a material rich in bioactive compounds and because of this they become a source with great biological potential. Some products are already made from leaves, which are being marketed in various forms (Özcan et al., 2019), in addition leaf extracts can be incorporated into foods, used as nutraceuticals and/or used by the pharmaceutical industry (Guinda et al., 2015). The main compounds identified in olive leaves are in the class of simple phenolics, flavonoids and secoiridoids (Talhaoui et al., 2015). All of these compounds originate from the specialized metabolism of the plant and have a protective function for the olive tree and for other plants in which they are synthesized (Rahmanian, Jafari & Wani, 2015).
The studies about the composition of bioactive compounds allows the addition of value to the raw material, reutilization of by-products, and generation of jobs throughout its productive cycle, making it a profitable and promising alternative to the academy, industry and farmers. In addition, increasing consumer concern on the use of synthetic substances in the industry has attracted a research interest in the field of biomaterial processing and pollution control for the development of clean technologies (Rosa et al., 2019).
These various metabolite compounds present in plants are a source of study for the field of metabolomics, and have become a very important tool for several areas in food science, such as regulatory compliance, processing, quality, safety and microbiology (Cevallos-Cevallos et al., 2009). With the advance of liquid chromatography coupled with mass spectrometry it is possible to identify a very large range of compounds in a single analysis, quickly and accurately.
Therefore, it is believed that the temperature variation resulting from the change of seasons as well as the planted cultivar affect the metabolic profile of the leaves. Thus, in this study, the influence on the bioactive content of leaf metabolism from three olive cultivars (Arbequina, Manzanilla and Picual) was evaluated during the changes of the Pampa gaucho season, aiming to find the best cultivar and time of year for collection of leaves for possible use by the population and / or industry.
2. Material and methods
2.1. Sample collection and preparation
Olive leaves (Olea europaea L.) were collected at the end of the autumn (June), winter (September), spring (December) and summer (March) seasons, which constitute the seasons of the year. Daily temperatures (°C) and solar radiation (MegaJoule m2) were monitored through a weather station (Fig. 1). Three cultivars were chosen for this research, two with the production of olives for the extraction of oil (Arbequina and Picual) and one with the production of table olives (Manzanilla). All cultivars were grown under the same agronomic and environmental conditions, in the same olive groves located on a private property in Pinheiro Machado (RS) (31° 29′59.4″ S and 53° 30′32.7 ″ O).
The leaves were collected from the external regions of adult trees (≥5 years), planted 7 × 5 m apart, and cultivated in order to grow with a single trunk. Leaves were collected from 50 trees of each cultivar at each time of collection, totaling approximately 5 kg in leaves. After collected, the leaves were gathered in a single reservoir and taken to the Chromatography Laboratory of the Federal University of Pelotas (UFPel/Pelotas/RS). The material was crushed and ground in a mill with the aid of liquid nitrogen, stored in polyethylene packaging and kept at −80 °C.
2.2. Determination of the metabolomic profile
2.2.1. Phenolic compounds
The quantification of phenolic compounds was performed using the methodology of Singleton, Orthofer and Lamuela-Raventós (1974), with adaptations. For the extraction, methanol P.A was used, and the samples kept under stirring for 24 h at 25 °C in the dark. For the reaction, Folin-Ciocalteu and 20% NaCO3 were used, with a 2-hour reaction and reading at 765 nm on a UV–VIS spectrophotometer (model JENWAY 6705). For the quantification, a calibration curve with a gallic acid standard (5 to 140 mg L−1) was used, and the results were expressed in milligrams equivalent of gallic acid per gram of dry sample (mg EAG g−1).
2.2.2. Flavonoids
To determine the total flavonoids, the methodology of Funari and Ferro (2006) was used, in which an ethanolic extract was obtained, following the same parameters as the phenolic compounds extract. The reaction was performed using AlCl3 with a 40-minute time and reading at 415 nm in a UV–VIS spectrophotometer (model JENWAY 6705). For quantification, a calibration curve was obtained using quercetin as a standard (5 to 100 mg L−1). The results were expressed in milligrams equivalent of quercetin per gram of dry sample (mg EQ g−1).
2.2.3. Hydrolyzable tannins
The quantification of hydrolyzable tannins followed the methodology described by Brune, Hallberg, and Skanberg (1991), using the same extract obtained for the analysis of phenolic compounds. The analysis was done through a reaction of the extract with a FAZ solution (urea: acetate, gum arabic and ferric ammonium sulfate) for 15 min, and final reading at 680 nm in UV–VIS spectrophotometer (model JENWAY 6705). For quantification, a calibration curve was obtained using gallic acid as a standard (0 to 400 mg L−1), and the results were expressed in milligrams equivalent of gallic acid per gram of dry sample (mg EAG g−1).
2.2.4. Condensed tannins
The condensed tannin content was measured by the method of Price, Scoyoc, and Butler (1978), using methanol acidified with 1% HCl for extraction of the compounds, the extraction solution being kept under constant agitation for 2 h, and afterwards centrifuged for 10 min at 5700xg. For the reaction the supernatant was collected and added with vanillin, and for each sample a blank using methanol acidified with 4% HCl was prepared. The reading was performed at 500 nm in a UV–VIS spectrophotometer (model JENWAY 6705), after 20 min of reaction in a thermostatic bath at 30 °C. For quantification, a calibration curve was made using catechin as a standard (0–400 mg L−1), and the results were expressed in milligrams equivalent of catechin per gram of dry sample (mg EC g−1).
2.2.5. Totals carotenoids
To quantify the total carotenoids, the Rodriguez-Amaya (2001) methodology was used. The samples were added with cold acetone for 10 min, then petroleum ether was added in a separating funnel, washed three times with water and the nonpolar portion was collected, which was read in a UV–VIS spectrophotometer (model JENWAY 6705) at 433 nm. A calibration curve using β-carotene standard was constructed (10 to 50 mg L−1), and the results were expressed in milligrams equivalent of β-carotene per gram of dry sample (mg Eβ g−1).
2.2.6. Chlorophylls
For the determination of total chlorophylls and chlorophylls “a” and “b”, the methodology of Lichtenthaler (1987) was used, the extraction was performed using 80% acetone and centrifugation at 1050xg for 15 min. The supernatant was separated and reading performed at 647 and 663 nm in a UV–VIS spectrophotometer (model JENWAY 6705). For quantification, Eqs. (1)–(3) were used, and the results were expressed in milligrams per gram of dry sample (mg g−1):
2.2.7. Individual phenolics by HPLC-MS-ESI-QTOF
The extraction was carried out according to the methodology of De Vos et al. (2007), with adaptations, where 100 mg of olive leaves were added with 990 µL of methanol grade HPLC 90% acidified with 0.1% formic acid and 10 µL reserpine (1 mg mL−1) as an internal standard. The mixture was placed in an ultrasonic bath for 15 min at 25 °C, and centrifuged at 14500xg for 10 min at 4 °C. The supernatant was separated and added with an additional 1 mL of 90% methanol HPLC acidified with 0.1% formic acid, repeating the extraction steps, added to the previous supernatant. The extract was filtered through nylon filters with 0.22 µm porosity and injected in a high-performance liquid chromatography system (UFLC, Shimadzu, Japan) coupled to a high-resolution quadrupole-time-of-flight mass spectrometer (Maxis Impact, Bruker Daltonics, Bremen, Germany) for the separation and identification of phenols.
For the chromatographic separation, the Luna C18 column (75 × 2 mm) was used, where the mobile phases used were: acidified water with 0.1% formic acid (eluent A) and acetonitrile (eluent B) with a flow of 0.2 mL min−1 and the column temperature was maintained at 40 °C, with the gradient used (min:% B): (0:10); (2:10); (10:75); (15:75); (18:90); (21:90); (23:10) and (30:10).
The mass spectrometer was operated in negative ESI mode, with spectra acquired over a mass range from m/z 50 to 1200. The acquisition parameters were: capillary voltage at 4 kV, nebulization gas pressure (N2) bar, drying gas in 8 mL min−1, source temperature 180° C, RF collision of 150 Vpp; 70 mS transfer and 5 mS pre-pulse storage. The equipment was calibrated with 10 mM sodium formeate, covering the entire acquisition range (from m/z 50 to 1200). In addition, automatic MS/MS experiments were carried out by adjusting the collision energy values as follows: m/z 100, 15 eV; m/z 500, 35 eV; m/z 1000, 50 eV, nitrogen being used as collision gas.
The phenolic compounds were quantified based on standard curves (39–2500 ng mL−1) of apigenin, hydroxybenzoic acid, campferol, luteolin, oleuropein, quercetin, rutin and tyrosol. And the results expressed in micrograms equivalent of the standards per gram of dry sample (μg g−1).
2.3. Antioxidant potential
The antioxidant potential of the samples was determined using the methanolic extract using three different methods. For all, calibration curves (0 to 300 mg L−1) were made using the Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) as the reference standard, and the results were expressed in millimol of Trolox per gram of dry sample (mmol Trolox g−1).
2.3.1. Sequestration of the DPPH radical
The sequestration of the DPPH radical (2,2-diphenyl-1-picrilhidrazil) was determined using Brand-Williams, Cuvelier, and Berset (1995) methodology, the extract being mixed with a solution of DPPH with absorbance adjusted to 1800, and after 100 min of reaction it was the reading was performed at 517 nm in a UV–VIS spectrophotometer (model JENWAY 6705).
2.3.2. Ferric reducing antioxidant power (FRAP)
The iron reducing capacity (FRAP) was determined by the reaction of the extract with the FRAP solution, which was obtained with 10 mL of acetate buffer, 1 mL of TPTZ (2,4,6-Tris (2-pyridyl) - s-triazine) and 1 mL of ferric chloride. The reaction took place in a thermostatic bath at 30 °C for 30 min, and the reading was performed at 595 nm on a UV–VIS spectrophotometer (model JENWAY 6705) (da Silva, Muniz & Nunomura, 2013).
2.3.3. ABTS radical scavenging assay
The capture of the ABTS radical (2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)) was determined by the reaction of the extract with the ABTS solution, which was obtained with 5 mL of the radical (7 mM) and 88 mL of potassium persulfate (140 mM), left to stand for 16 h before analysis. The reaction duration was 6 min and the reading were performed at 734 nm on a UV–VIS spectrophotometer (model JENWAY 6705) (Gülçin et al., 2010).
2.4. Statistical analysis
The design used was completely randomized in a factorial scheme of 3 cultivars and 4 times of collection. For data analysis, means and respective standard deviations were performed, as well as factorial analysis of variance. For the variables that presented significance, Scott-Knott test was performed considering 5% of significance. Simple correlation analysis between variables was also performed (p < 0.05).
3. Results and discussion
Bioactive compounds are influenced by several factors intrinsic and extrinsic to the leaves, each region and each cultivar will present different peculiarities and therefore the need for this study. Through the obtained results it was observed that, for the total of phenolic compounds, flavonoids and hydrolyzable tannins in the leaves, there is no interaction between the cultivars and the seasons. Thus, the statistics of the separate variables showed that leaves of the cultivar Manzanilla have higher concentrations of phenolic compounds and hydrolyzable tannins (Table 1).
Table 1. Content of polar compounds and antioxidant potential of olive leaves (Olea europeae) in different cultivars and season.
| Cultivars (annual means) | |||||||
|---|---|---|---|---|---|---|---|
| Phenolic Compounds (mg EGA g−1) | Flavonoids (mg EQ g−1) | Hydrolyzable Tannins (mg EAG g−1) | Condensed Tannins | DPPH (mmol Trolox g−1) | FRAP (mmol Trolox g−1) | ABTS (mmol Trolox g−1) | |
| Arbequina | 8.25B* | 5.25A | 29.04B | ND** | 65.17C | 59.84C | 65.50C |
| Manzanilla | 10.05A | 5.56A | 30.59A | ND | 93.53A | 80.86A | 84.79A |
| Picual | 8.45B | 3.94B | 26.09C | ND | 72.04B | 66.48B | 71.99B |
| Seasons (cultivar means) | |||||||
| Autumn | 8.59B | 5.13A | 26.60B | ND | 71.88C | 68.28B | 72.20B |
| Winter | 7.61C | 4.19B | 25.23B | ND | 59.82D | 57.80C | 67.01C |
| Spring | 9.08B | 4.42B | 29.15A | ND | 84.29B | 68.42B | 73.11B |
| Summer | 10.40A | 5.94A | 33.33A | ND | 91.66A | 81.75A | 84.06A |
| CV (%): | 6.71 | 17.43 | 5.75 | – | 8.1 | 6.05 | 5.09 |
*Equal capital letters between means in the column do not differ statistically by the Scott-Knott test considering 5% of significance; **Not detected; CV: coefficient of variation.
Phenolic compounds are derived from the specialized metabolism of plants and are related to its defense against adverse conditions during life (Le Gall et al., 2015). Many variables can affect the synthesis and content of phenolic compounds of the olive tree, both in the leaves, in the fruits and in the oil, variables that include the position in the tree, minerals present in the soil, sun exposure and geographic location and cultivar (Otero et al., 2020). Studies have shown that different cultivars present differentiated production of bioactive compounds, including phenolic and flavonoid compounds (Lama-Muñoz et al., 2020, Martín-García et al., 2019, Nicolì et al., 2019).
The interest in the potential health benefits of olive leaves has in-creased among scientists in various fields such as pharmaceutical, cosmetic, and food industries (Medina, Romero, García & Brenes, 2019). The primary reason for this interest in olive tree leaves is their beneficial effects on metabolism when used as a traditional herbal drug which is attributed to the bioactive compounds, such as phenolic compounds, they contain (Contreras et al., 2020, Hannachi et al., 2019, Lama-Muñoz et al., 2020, Rosa et al., 2019).
When evaluating only the seasons and their contribution to the increase of certain bioactive compounds, it was noticed that winter was the season of the year where the lowest synthesis of phenolic compounds occurred, in the summer it was possible to observe a greater synthesis of these compounds, the which indicates that the temperature has a direct influence on the mechanism, where, high temperatures can result in the activation of protection mechanisms of the olive tree, and as a consequence induce greater synthesis of these compounds. In a study by Martinez et al. (2016) the influence of saline and thermal stress on tomato leaves was noted, and it is observed that for quercetin and campferol, thermal stress has a great influence, causing the content of some of these compounds to be elevated in more than 100% with increasing temperature.
Flavonoids are one of the most common and widely distributed group of olive leaves polyphenols (Özcan et al., 2019). In the present study, the total levels of flavonoids showed equal averages between autumn/summer (5.13 and 5.94 mg EQ g−1) and winter/spring (4.19 and 4.42 mg EQ g−1), while the levels of hydrolysable tannins showed equal averages between autumn/winter (26.20 and 25.23 mg EAQ g−1) and spring/summer (29.15 and 33.33 mg EAQ g−1); thus suggesting that changes between seasons and consequently in daily temperatures (Fig. 1), influence plant metabolism in different ways for each class of compounds present in the leaves. As noted by Martinez et al. (2016), the thermal stress applied to tomato leaves increased the flavonoid content, however a reduction in the content of caffeoylquinic acid was observed with the application of heat treatment and an increase in content with the application of salinity.
It was observed that in the three tested methods, the extract of the leaves that showed greater antioxidant activity was from the cultivar Manzanilla (95.53 mmol Trolox.g−1), behavior also observed for the content of bioactive compounds. Authors report the antioxidant potential of leaves related to bioactive compounds found in these matrices (Fernández-Agulló et al., 2020, Khanum et al., 2020). There was also no significant interaction for the potential antioxidants of olive leaves collected in different seasons and different cultivars in the analysis of variance (Table 1).
Antioxidant compounds, such as phenolic and flavonoids, have the function of regulating the balance of free radicals in the plant's organism and preventing its death (Le Gall et al., 2015); therefore, they are responsible for the antioxidant potential of vegetables, acting as regulators of the stresses that vegetables go through during life. The literature reports the antioxidant capacity of olive leaves (Guglielmotti et al., 2020, Khanum et al., 2020), which are evaluated by different methodologies in order to provide the most representative results possible, since the methods react through different mechanisms with the samples (Antunes et al., 2020).
In general, the content of the most polar compounds showed no interaction, as well as the potential antioxidants, since these potentials were tested with the polar extracts. However, the contents of more nonpolar compounds, such as chlorophylls and carotenoids (Table 2), were analyzed variables that showed significant interaction between cultivar and season, indicating that these behave differently between each season of the year for each grow crops.
Table 2. Content of nonpolar compounds of olive leaves (Olea europeae) in different cultivars and season.
| Chlorophyll A (mg g−1) | ||||
|---|---|---|---|---|
| Autumn | Winter | Spring | Summer | |
| Arbequina | 1.03aA* | 0.78bA | 0.80bA | 0.78bB |
| Manzanilla | 0.81bB | 0.61cB | 0.85bA | 1.09aA |
| Picual | 0.64bC | 0.49cB | 0.64bB | 0.83aB |
| Chlorophyll B (mg g−1) | ||||
| Autumn | Winter | Spring | Summer | |
| Arbequina | 0.50aA | 0.36bA | 0.34bA | 0.29bB |
| Manzanilla | 0.34bB | 0.27cB | 0.36bA | 0.45aA |
| Picual | 0.25bC | 0.21bB | 0.25bB | 0.31aB |
| Total Chlorophyll (mg g−1) | ||||
| Autumn | Winter | Spring | Summer | |
| Arbequina | 2.09aA | 1.65bA | 1.79bA | 1.64bB |
| Manzanilla | 1.78bB | 1.41cB | 1.73bA | 2.23aA |
| Picual | 1.44bC | 1.19bB | 1.34bB | 1.81aB |
| Total carotenoids (mg Eβ g−1) | ||||
| Autumn | Winter | Spring | Summer | |
| Arbequina | 14.20aA | 13.31aA | 12.24aA | 12.94aA |
| Manzanilla | 11.82bB | 9.59cB | 11.98bA | 14.18aA |
| Picual | 10.43bB | 8.19cB | 9.70bB | 12.22aA |
*Same lowercase letters between means in the line and uppercase letters in the column do not differ statistically by the Scott-Knott test considering 5% of significance; CV (%) for chlorophyll A, B and Total of 10.16; 12.52 and 8.12 and carotenoids of 9.00.
The literature reports that olive leaves are an excellent source of pigments, mainly chlorophylls, which can be extracted or mixed with oils to provide more intense color, in addition, in the dark, this pigment is also endowed with antioxidant activity, even if under light conditions it can act as a prooxidant, reacting with triplet oxygen to form the excited-state singlet oxygen (Malheiro et al., 2013). The leaves of the cultivar Arbequina showed higher levels of total chlorophylls (2.09 mg g−1), chlorophyll “a” (1.03 mg g−1) and “b” (0.5 mg g−1) in autumn, while the leaves cultivars Manzanilla and Picual had the highest concentrations in summer. The leaves of cultivar Manzanilla showed high rates only in summer, while in autumn, spring and winter, the content in cultivar Arbequina was statistically higher than other cultivars.
Arbequina leaves showed no difference in carotenoid content between seasons, since this cultivar is the most adapted for the growing region. This demonstrates that fluctuations in temperature and solar radiation (Fig. 1) did not influence these compounds in the leaves of this cultivar. In the colder seasons, the leaves of the cultivar Arbequina stood out in terms of the total carotenoid content, while in the warmer seasons there was no difference between the content in the leaves of the three cultivars. Thus, it is observed that cultivars less adapted to the heat of the region needed to direct their metabolism towards the production of protective compounds against UV radiation, which contrasts with the higher levels of polar compounds, described in this research.
The cultivar Arbequina had an average content throughout the year of 13.17 mg g−1 for total carotenoids, while Picual was the cultivar with the lowest concentration (10.13 mg g−1), these values being higher than those described by Magdich et al., 2016, Benjeddou et al., 2019 who report the total carotenoid concentration of 3.26 and 3.0 mg g−1 fresh weight in olive leaves. Carotenoids are important organic compounds, which give color to plant leaves, foods and agriculture products, these pigments have a significant role in light harvesting and photoprotection in photosynthesis in plants (Teramukai et al., 2020). Because of the high functionalities and importance as natural colorants, much attention has been paid to the effective method to separate carotenoids from natural resources, with olive leaves being an excellent source for the recovery of these compounds.
The analysis of individual phenolics showed significant interaction between cultivars and seasons (Table 3 and Fig. 2), showing that the seasons have a strong influence on the content of bioactive compounds, with higher contents being observed for most compounds in the leaves (apigenin, hydroxybenzoic acid, campferol, luteolin, quercetin and tyrosol) in the summer. Researchers observed that the different seasons of the year (Ben Mansour-Gueddes et al., 2020, Özcan et al., 2019, Wang et al., 2019) as well as the different cultivars (Guo et al., 2018, Medfai et al., 2020, Özcan et al., 2019) had an influence on the metabolomic profile of the leaves.
Table 3. Individual bioactive compounds quantified in olive leaves (Olea europeae) in different cultivars and season (µg g−1).
| Autumn | Winter | Spring | Summer | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Arbequina | Manzanilla | Picual | Arbequina | Manzanilla | Picual | Arbequina | Manzanilla | Picual | Arbequina | Manzanilla | Picual | |
| Apigenin | 83.75bB* | 156.44aA | 87.34bB | 111.99bA | 168.89aA | 71.57cC | 71.62bC | 110.68aB | 38.04cD | 87.46cB | 163.91aA | 98.63bA |
| Hydro. acid | 1.81cD | 17.42aA | 13.73bA | 4.26cC | 11.4aC | 6.18bC | 6.18cB | 13.15aB | 10.91bB | 8.16bA | 3.92cD | 12.54aA |
| Kaempferol | 2181.40aB | 774.93cC | 1500.32bB | 1864.88aB | 769.7cC | 1462.92bB | 2032.38aB | 1343.29bB | 859.91cC | 4566.06bA | 4889.17aA | 3597.35cA |
| Luteolin | 48.94aB | 16.54cC | 25.86bB | 43.41aB | 15.09cC | 31.97bB | 44.35aB | 30.17bB | 15.20cC | 92.01bA | 117.79aA | 90.14bA |
| Oleuropein | 0.0cA | 0.09bA | 0.11aA | 0.03bA | 0.07aB | 0.07aB | 0.03bA | 0.03bD | 0.06aC | 0.04bA | 0.04bC | 0.05aC |
| Quercetin | 3.51bB | 3.68bB | 4.11aB | 3.57aB | 2.79bC | 2.53bC | 3.18aB | 3.41aB | 2.09bD | 4.21bA | 4.39bA | 5.40aA |
| Rutin | 1804.03bB | 2053.64bA | 2898.33aB | 1205.23cC | 1930.32bA | 3707.94aA | 2716.96bA | 847.2cC | 3384.25aA | 1427.29aC | 1217.29aB | 1646.23aC |
| Tyrosol | 6.43cC | 13.11aA | 10.82bA | 7.40bB | 9.94aC | 7.36bC | 9.20bA | 12.20aB | 9.70bB | 4.55cD | 7.99bD | 11.29aA |
*Means followed by equal lower case letters between cultivars in the same collection period and upper case letters between collection periods in the same cultivar do not differ statistically from each other by the Scott-Knott test considering 5% significance. CV (%) for apigenin, acid. hydrox., campferol, luteolin, oleuropein, quercetin, rutin and tyrosol, respectively of 6.12; 9.91; 7.54; 9.50; 13.26; 9.62; 9.76 and 3.84.
Summer was the season that most caused changes in plants, and that these changes, mainly the increase in the content of bioactive compounds, may be related to a higher incidence of light / UV-B and to an increase in temperature. This effect has already been evidenced by authors who evaluated the leaf bioactive profile (Brahmi, Mechri, Dhibi, & Hammami, 2013), in addition, higher phenol content is likely associated with lower rainfall levels (Arslan, Karabekir, & Schreiner, 2013).
As for the contents of the investigated phenolic compounds, it was noticed that the leaves of the cultivar Manzanilla, in practically all seasons of the year (autumn, winter and spring), presented higher amounts of apigenin, hydroxybenzoic acid and tyrosol. The same was observed for the leaves of the cultivar Arbequina regarding the contents of campferol and luteolin. The highest oleuropein and rutin contents were observed in all seasons in the leaves of the cultivar Picual. The literature reports different compositions and concentrations of phenolic compounds present in the leaves of olive trees grown in different countries. Oleuropein, luteolin and apigenin glucosides are referred in many studies as the main components (more abundant) in leaves, however in this study the phenolic compounds mentioned above were not found in abundance (Ben Mansour-Gueddes et al., 2020, Guo et al., 2018, Medfai et al., 2020, Özcan et al., 2019, Wang et al., 2019). It is believed that the results may have been influenced by the type of extraction used, since the purpose of this analysis was to perform a scan of the phenolic compounds and not to optimize the concentration from the extraction. Besides that, these differences are related to intrinsic and extrinsic factors of plants, studies have already shown that the metabolic profile varies between species and geographic location (Hoffmann et al., 2017), as well as in different cultivars (Brahmi, Mechri, Dhibi, & Hammami, 2013).
The correlation analysis (Table 4) showed that there was a high correlation between the potential antioxidants and the content of phenolic compounds (r = 0.8989; 0.907 and 0.8638). According to Talhaoui et al. (2014), when they assessed the correlation between the content of phenolic compounds in olive leaves and the antioxidant po- tential measured by the DPPH radical scavenging method, found a high degree of correlation between radical elimination and the content of phenolic compounds (r = -0.9525).
Table 4. Simple correlation between variables.
| Phenolics | Flavonoids | Chlorophyll A | Chlorophyll B | Total Chlorophyll | Total Carotenoids | DPPH | FRAP | ABTS | Hydrolyzable Tannins | |
|---|---|---|---|---|---|---|---|---|---|---|
| Phenolics | – | 0.5626 | 0.5109 | 0.2973 | 0.554 | 0.324 | 0.8989 | 0.907 | 0.8638 | 0.769 |
| Flavonoids | * | – | 0.5858 | 0.4839 | 0.6303 | 0.4831 | 0.3994 | 0.5042 | 0.4612 | 0.5494 |
| Chlorophyll A | * | * | – | 0.9509 | 0.9818 | 0.7853 | 0.4236 | 0.4306 | 0.3306 | 0.6209 |
| Chlorophyll B | NS | * | * | – | 0.9211 | 0.749 | 0.2226 | 0.233 | 0.134 | 0.4509 |
| Total Chlorophyll | * | * | * | * | – | 0.7808 | 0.4124 | 0.4346 | 0.3454 | 0.6175 |
| Total Carotenoids | NS | * | * | * | * | – | 0.1889 | 0.2006 | 0.1141 | 0.5791 |
| DPPH | * | * | * | NS | * | NS | – | 0.9297 | 0.9021 | 0.7662 |
| FRAP | * | * | * | NS | * | NS | * | – | 0.9191 | 0.7276 |
| ABTS | * | * | * | NS | * | NS | * | * | – | 0.6858 |
| Hydrolyzable Tannins | * | * | * | * | * | * | * | * | * | – |
*Significant correlation by the Tukey test considering 5% significance. NS – Not significant.
Finally, the hypothesis tested was proven, revealing that the cultivars chosen for planting, as well as the existing climatic changes with the season changes, affect the metabolic profile of olive leaves (Olea europeae L.)
4. Conclusions
High added-value ingredients can be obtained by recovering bioactive compounds from olive by-products (leaves) which are of interest to the pharmaceutical, cosmetic and food industries. Each olive cultivar presented a different response to climatic factors, in addition to its own metabolism, results that are relevant for the industries, since they are important information to optimize their processes. In Arbequina, the highest levels of carotenoids were found in the cold seasons (winter and autumn), Manzanilla presented higher levels of phenolic compounds and hydrolyzable tannins in the summer and Picual intermediate values of the evaluated compounds.
The leaves of the cultivar Manzanilla showed greater antioxidant potential and higher content of phenolic compounds compared to the other cultivars, and the phenolic compounds present in olive leaves demonstrated to be responsible for their antioxidant capacities. The different seasons of the year have an influence on the metabolism of olive trees, which, depending on the cultivar, led to increases or decreases in the synthesis of phenolic compounds, carotenoids and flavonoids. In general, in summer, the highest concentrations were found for all bioactive compounds evaluated.
Funding sources
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) – Finance Code 001.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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