Olive trees (Olea europaea
L.), belonging to the family Oleaceae, are one of the important economic crops all over the world [1
]. In 1960, olive trees were introduced from the Mediterranean region into China as a commercial crop [2
]. According to statistical analysis, almost 80,000 hectares of olive trees were cultivated by the end of 2017, generating approximately 600,000 tons/year of abandoned leaves in China [4
]. Olive leaves, an agricultural by-product obtained after the pruning and harvesting of olive trees, are thrown away, burned, or scattered in the field, potentially causing environmental damage and increasing waste disposal cost for farmers [5
]. Olive leaves were highly valued in Mediterranean folk medicine for the treatment of influenza, common cold, malaria, dengue, diarrhea, and surgical infections [3
]. Because of their health promoting properties, olive leaves have recently gained increasing interest and have been used as an inexpensive raw material for various technological, scientific, and commercial applications [1
Recent studies have demonstrated that olive leaves are mainly composed of moisture, proteins, lipids, minerals, and carbohydrates [10
]. Although olive leaves contain large quantities of nutrients, the phenolic content is of major interest because of its health benefits [11
]. Olive leaves contain an abundance of high-quality polyphenols. These compounds are mostly classified into secoiridoids, acids, and flavonoids, and they exhibit strong preventive effects against oxidation [12
]. Based on the potential health benefits, several studies have evaluated the effect of phenolic extracts (PEs) derived from olive leaves in the treatment of various diseases, such as cardiovascular diseases, cancer, myocardial oxidative damage, and atherosclerosis [13
]. In particular, oleuropein, the main phenol in olive leaves, exhibits remarkable biological and pharmacological activities, especially antioxidant, antimicrobial, and anticancer effects [16
Seasonal variation in chemical compositions is a well-known phenomenon in plants, and it is associated with the biosynthesis, stability, and degradation of secondary metabolites in olives [19
]. In addition, quantitative and qualitative changes in the biochemical composition of olive leaves also depends on the plant variety, climatic conditions, sampling time, genetics, and geographical origin [19
]. Recent studies have typically focused on olives grown in a few countries in the Mediterranean region. In China, the Liangshan variety is produced on the largest scale under climate conditions characterized by four different seasons and variations in “weather within 10 km” [23
]. The climatic conditions in Liangshan strongly affect the genetic quality of olive cultivars, olive fruit, and oil [24
]. For example, Chen et al. [24
] reported that olives from China, which possess a higher moisture content in the fruit, show unique characteristics compared with olives grown in the Mediterranean region. However, data related to the bioactive ingredients of olive leaves at different times under the climate of Liangshan are available. Determination of the seasonal effects on the bioactive constituents in Liangshan olive leaves is essential for understanding the impact of harvesting time on olive leaves and ensuring optimal concentrations of active ingredients.
The aim of this study was to investigate the seasonal variations in the chemical compositions of Liangshan olive leaves from January to December, including total phenolic content (TPC), total flavonoid content (TFC), free amino acid content (FAAC), soluble sugar content (SSC), and soluble protein content (SPC), as well as the contents of seven major phenolic compounds. In addition, the in vitro antioxidant capacities of PE as scavengers of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide anions, as well as the reducing power, were evaluated. The potential anticancer effects of PE on human embryo kidney cells (HEK293), human cervical cancer cells (Hela), and ascites tumor cells (S180) were also assessed.
2. Material and Methods
2.1. Materials and Chemicals
Trees of O. europaea cultivar “Manzanillo” are major olive cultivars grown in Liangshan, Sichuan (China). The leaves were sampled on the 15th of each month from January to December in 2018. After collection, the leaves were dried and ground. Then, the dried powder was sieved and stored at −20 °C.
The following standards were purchased from Acros Organics (Geel, Belgium) and Chengdu Kelong Chemical Factory (Chengdu, China): methanol and acetonitrile, 2,2-diphenyl-picrylhydrazyl (DPPH), and ethanol. All reagents were of analytical grade, while six phenolic compounds were high-performance liquid chromatography (HPLC) grade (Chengdu Must-biotechnology, Chengdu, China).
2.2. Sample Preparation
Powdered olive leaves (10 g) were mixed in ethanol–water (1:1) solution in a sample-to-extract ratio of 1:25 (v/w). The beaker containing the mixture was placed in an ultrasonic device (KQ300DV, 40 kHz, Kunshan Ultrasonic Instrument Co. Jiangsu, China) at 60 °C, and the extraction was performed at 270 W for 40 min. The crude extracts were centrifuged at 4500 rpm for 10 min. Subsequently, the supernatants were filtered using 0.45 µm syringe filters and were used directly to estimate TPC, TFC, and FAAC, as well as to perform HPLC analyses of the phenolic compounds.
The sample solution obtained in January was concentrated with a rotary evaporator and dried in a vacuum drying oven to obtain PE. The PE samples were stored at −20 °C in the dark for further analysis.
2.3. Determination of Chemical Composition
2.3.1. Total Phenolic Content (TPC)
The amount of TPC in the olive leaves was estimated according to the Folin–Ciocalteu method [27
]. Briefly, the extract (0.1 mL) was reacted with 2.5 mL of Folin–Ciocalteu reagent for 2 min, followed by the addition of 1 mL sodium carbonate (7.5% w
), after which it was diluted to 10 mL with distilled water. The tube containing the reaction solution was incubated at 25 °C for 90 min without light. The absorbance was measured by a microplate reader (Spectramax M2, USA) at 760 nm. The TPC was expressed as milligrams of gallic acid equivalent (GAE) per gram dry weight of the sample (mg GAE/g).
2.3.2. Total Flavonoid Content (TFC)
Total flavonoid content (TFC) of the extract was estimated by the AlCl3
(aluminum chloride) method with some modifications [28
]. Briefly, a 2 mL sample was added to 4 mL AlCl3
), and the solution was adjusted to 25 mL with distilled water. The absorbance was measured at 415 nm. The TFC was expressed as milligrams of routine equivalent (RE) per gram dry weight of the sample (mg RE/g).
2.3.3. Free Amino Acid Content (FAAC)
The FAAC of the olive leaves was determined using ninhydrin colorimetry with slight modifications [29
]. Briefly, the sample supernatant (0.5 mL) was mixed with 1.5 mL ninhydrin (2% w
), 0.5 mL distilled water, and followed by the addition of 0.05 mL ascorbic acid (0.1% w
). Finally, the mixture was incubated at 100 °C for 10 min and cooled immediately in an ice-water bath for 20 min. The absorbance was measured at 570 nm. The FAAC was expressed in terms of glutamate standard.
2.3.4. Soluble Sugar Content (SSC)
The SSC of the olive leaves was determined based on the method of Dubois et al. [30
]. The method entailed mixing 0.1 g of dried powder in 5 mL of distilled water. The sample tube was immersed in an 80 °C water bath. After 30 min, 0.2 mL solution was reacted with a mixture of 0.2 mL sample solution (6% w
) and 0.5 mL sulfuric acid. The absorbance at 490 nm was recorded, with glucose solution as the standard.
2.3.5. Soluble Protein Content (SPC)
The SPC of the olive leaves was determined based on the method of Meda et al. [31
]. A lysis buffer consisting of 10 mM NaCO3
and 10 mM NaHCO3
was prepared. The Coomassie Brilliant Blue (CBB) color reagent was obtained by mixing 5 mL ethanol (95%) and phosphoric acid (85%) and diluting to 100 mL. The olive dried powder (0.25 g) was extracted using lysis buffer (5 mL) in an ultrasonic device (240 W) at 20 °C for 50 min. The extracts were centrifuged (8000 rpm, 5 min), and 0.1 mL supernatant was distilled to 0.3 mL with lysis buffer. Finally, 0.5 mL CBB color reagent was added. After 2 min, the absorbance was read at 595 nm. SPC was used to quantify the standard curve with bovine serum albumin (BSA) as the reference.
2.4. HPLC Analysis of Phenolic Compounds
Analyses of phenolic compounds were identified and quantified on an Agilent 1260 high-performance liquid chromatography (HPLC) chromatogram (Agilent Technologies Agilent Technologies Singapore International Pte., Ltd.Singapore). The sample was separated by a Agilent 1260 HPLC system equipped with a UV–vis DAD detector at 350 nm, and detected on a Zorbax Eclipe Plus-C18 column (5.0 µm, 150 × 4.6 mm). The injection volume was 10 µL, and the temperature was maintained at 30 °C. The mobile phase comprised solvent A (0.2% aqueous phosphoric acid) and solvent B (acetonitrile). The flow rate was 0.8 mL/min, and the gradient profile was 84% A to 16% B at 0–3 min, 70% A to 30% B at 3–20 min, 60% A to 40% B at 20–25 min, 84% A to 16% B at 25–30 min, and 84% A to 16% B at 30–33 min. The amounts of the phenolic compounds were calculated using the calibration curve (C), and the equation was as follows: extraction yield (mg/g) = [(C × V)/W]/1000, where V is the volume of the extract (mL), and W is the dried weight of sample (g).
The oleuropein was determined at 254 nm during 10 min. The mobile phase A was water (75%), and mobile phase B was acetonitrile (25%).
2.5. Antioxidant Activity Assays
2.5.1. Superoxide Radical-Scavenging Activity
The superoxide radical-scavenging activity of PE was determined according to the method of Jia et al. [32
]. Briefly, PE was dissolved in deionized water and diluted with different concentrations of PE (0.2–2.0 mg/mL) to obtain sample solution. Then, the sample solution was mixed 0.2 mL nitroblue tetrazolium (NBT, 0.08 mM), 0.4 mL nicotinamide adenine dinucleotide (NADH, 0.25 mM), and 0.2 mL phenazine methyl sulfate (PMS, 0.06 mM) to obtain reaction solution. The reaction mixture was incubated at 25 °C for 15 min. The deionized water and ascorbic acid (Vc) were used as the blank and positive control, respectively. The absorbance at 560 was measured. Inhibition of scavenging effect was calculated with the following equation: scavenging activity (%) = (1 − (As1
) / As0
) × 100, where As0
, and As2
are the absorbance of the control, the reaction solution, and the sample solution, respectively.
The IC50 (50% effective concentration) values were calculated from the dose–response curve using SPSS 19, where the abscissa represented the concentration of tested PE as the average inhibition percentage.
2.5.2. DPPH Radical-Scavenging Activity (DPPH)
The DPPH radical-scavenging activity was assayed based on the method of Brand-Williams et al. [33
]. Briefly, different solutions of PE (0.2–1.2 mg/mL) were reacted with DPPH solution (0.4 mM) in ethanol. The reaction solution was incubated in the dark at 37 °C for 10 min, and the absorbance was measured at 517 nm. Ethanol served as the blank control, and Vc served as the positive control. Inhibition of scavenging activity was calculated by the equation: scavenging activity (%) = (1 − (As1
) × 100, where As0
, and As2
are the absorbance of the control, the reaction solution, and the sample solution, respectively.
2.5.3. Reducing Power
The reducing power was evaluated as described in the methods of Moein et al. [34
]. Briefly, 0.5 mL phosphate buffer (0.2 M, pH 6.6) was mixed various concentrations of PE (0.2–1.2 mg/mL) and 0.5 mL potassium ferricyanide (1% w
) for 20 min at 50 °C. A total of 0.5 mL trichloroacetic acid (10% w
) was supplemented and centrifuged (3000 rpm, 10 min). The 0.5 mL supernatant was made up with 0.5 mL distilled water. The mixture reacted with 0.1 mL ferric chloride (0.1% w
). Distilled water served as the blank control, and Vc served as the positive control. Then, the absorbance at 700 nm was read.
2.6. Anticancer Activity Assays
2.6.1. Cell Culture
HEK293, HeLa, and S180 cells were kindly provided by the cell storeroom of the Chinese Academy of Sciences (Shanghai, China). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Culture plates were placed in an incubator maintained at 37 °C with 5% CO2.
2.6.2. Cell Viability
Cytotoxicity and cell viability assays were analyzed by the CCK-8 assay, as detailed by the manufacturing company (Dojindo Molecular Technologies, Tokyo, Japan). HEK293, HeLa, and S180 cells were plated in 96-well plates at an initial density of 2 × 104 cells/mL. Cells were added with different concentrations of PE and were cultured for different times. After different times, the old medium of DMEM was completely removed, and 100 µL fresh growth medium was supplemented. After 2 h of incubation (37 °C, 5% CO2), the density of each well was recorded at 450 nm. Inhibition rate was determined using the following formula: (Ac − As)/(Ac − Ab) × 100%, where As, Ac, and Ab are the absorbance of a well with reaction solution (cells, CCK-8 solution, and sample solution), cells and CCK-8 solution (without a sample solution), and medium and CCK-8 solution (without cells and sample). Cell viability was determined in triplicate in independent experiments using 6 wells per concentration. IC50 values were calculated using SPSS 19 from CCK-8 assay data.
2.6.3. Mitochondrial Membrane Potential (∆Ψm)
The mitochondrial membrane potential of S180 cells was assessed using a JC-10 assay, following instructions described by the manufacturer (Solarbio Science and Technology, Beijing, China). The mitochondrial membrane potential assay kit is used to specifically evaluate the ability of a compound to rapidly disrupt the polarity of the mitochondrial membrane. S180 cells were plated in six-well microplates at a concentration of 106 particle/mL and cultured for 24 h. Different concentrations of PE (0, 160, and 200 μg/mL) were added, respectively. At 24 h, the treated cells were incubated with JC-10 at 37 °C and then washed twice with PBS.
2.6.4. Caspase-3 and Caspase-9 Activity
The caspase-3/9 activity of the S180 cell lysates was determined by using a caspase-3/9 solarbio colorimetric assay kit according to instructions described by the manufacturer (Solarbio Science and Technology, Beijing, China). S180 cells were treated with different concentrations of PE (0, 160, and 200 μg/mL) for 24 h. The old medium was completely replaced. Cells were lysed with lysis buffer for 15 min after being washed with cold PBS, and they were resuspended in trypsin. Caspase-3/9 activity was quantified, and the absorbance was measured at 405 nm using a microplate spectrophotometer. Compared with the untreated cells, the activity was expressed as the fold of enzyme activity.
2.7. Statistical Analysis
All assays were expressed as the mean value ± standard deviation (SD), and all experiments were repeated three independent times in triplicate form. Statistical analysis was conducted using SPSS 19 software (SPSS, Inc., Chicago, IL, USA). Results were tested for statistical significance by one-way analysis of variance (ANOVA). Statistically significant differences among treatments (p < 0.001) were assessed using Duncan’s honestly significant difference (HSD) and multiple range tests (DMRT).
Olive trees (Olea europaea L.) have been cultivated in China for decades, but their variations in chemical composition have not been extensively explored. The objectives of this study were to determine the seasonal variations in the chemical compositions of Liangshan olive leaves from January to December and to evaluate their antioxidant and anticancer activities based on polyphenol content. The results showed that the TPC, TFC, SPC, SSC, and FAAC in Liangshan olive leaves decreased in April and September but increased in spring and winter. HPLC analysis revealed that OE and luteolin-4’-O-glucoside were the primary components, followed by apigenin-7-O-glucoside, quercetin, and rutin, and lower amounts of luteolin and apigenin were detected. These compounds were highest in spring and winter and lowest in summer and autumn as a whole. In addition, excellent antioxidant activity of PE was exhibited when DPPH radical and superoxide radical scavenging were tested, which were 91.29% and 62.26%, respectively, with IC50 values of 0.14 and 0.93 mg/mL. The reducing power of PE was also tested. Furthermore, the potent toxicity against HEK293 cells showed that the IC50 value of PE was found as 841.48 μg/mL, and a low inhibitory effect was observed when HeLa cells were treated with 0–800 μg/mL of PE for 24 h, but S180 cells exhibited a higher sensitivity to PE concentrations. PE induced S180 cell mortality via increased mitochondrial membrane potential depolarization and up-regulated caspase-3 and caspase-9 activity. Taken together, the potential antioxidant and anticancer activities of phenolic extracts, especially that of Liangshan olive leaves in January and December, can be a potential and alternative source applied in the food, cosmetic, and pharmaceutical fields.