In Vivo and In Vitro Antioxidant Activities of Methanol Extracts from Olive Leaves on Caenorhabditis elegans

The aim of this study was to evaluate the antioxidant activities of extracts from olive leaves (EOL). The main contents of EOL were determined by colorimetric methods. The antioxidant activities were assessed by measuring the scavenging free radicals in vitro. To investigate the antioxidant activity in vivo, we detected the survival of Caenorhabditis elegans, under thermal stress. Subsequently the reactive oxygen species (ROS) level, activities of antioxidant enzymes, the expression of HSP-16.2 and the translocation of daf-16 were measured. The results showed that, polyphenols was the main component. EOL could well scavenge DPPH and superoxide anion radicals in vitro. Compared to the control group, the survival rate of C. elegans treated with EOL was extended by 10.43%, under heat stress. The ROS level was reduced, while the expression of hsp-16.2 was increased to protect the organism against the increasing ROS. The level of malondialdehyde (MDA) also decreased sharply. The activities of inner antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) were potentiated, which might have had a correlation with the DAF-16 transcription factor that was induced-turned into the nuclear. Therefore, EOL showed a strong antioxidant ability in vitro and in vivo. Hence, it could be a potential candidate when it came to medicinal and edible plants.


Introduction
Olive (Olea europare L.) deriving from the Mediterranean was introduced to China in the 1960s. The oil from olive fruits had been historically used as a kind of cooking oil in the Mediterranean. Additionally, olive oil had rich monounsaturated fatty acids and a wide array of bioactive compounds, such as polyphenols, flavonoids, and squalence, which were also renowned as liquid gold [1]. Studies have showed that olive oil has a wide range of pharmacological applications, such as reduction in blood pressure and anti-bacterial, anti-aging, and anti-tumor activity; these effects are more prominently seen is Europe [2][3][4]. Olive fruit extracts are also widely used as nutrition supplements, food additives, and medicinal and ceremonial ingredients. Consequently, olive has attracted the attention of the world, due to its highly beneficial effect on humans [5]. By 2012, the cultivation area of olive trees, in China, had reached over three million hectares. According to statistics, approximately 25 kg olive leaves are produced from one olive tree, every year, after the pruning of branches. The majorities of olive leaves are abandoned or incinerated, which leads to environmental pollution and resource waste [6]. Studies have shown that the main compounds in olive leaves are the total polyphenol and the total

Components of EOL
As showed in Table 1, polyphenols and flavonoids were the main contents of EOL, accounting for 41.77 ± 2.38% and 30.01 ± 0.76%, respectively. A similar outcome was obtained by Le et al., who showed extracts obtained from olive leaves contained about 45% polyphenol compounds [24]. The content of proteins in this study was found to reach to 20.40 ± 0.29%. However, free amino acids was only about 0.09 ± 0.02% and soluble sugars was only 14.14 ± 0.29%.

In Vitro Antioxidant Activities
A single antioxidant assay could not evaluate the antioxidant capacity of extracts, comprehensively. Several testing methods should be carried out to value the antioxidant abilities of plant extracts [25,26]. Therefore, in this study, the antioxidant activity of EOL was estimated by determining its scavenging effect on DPPH, superoxide anion radicals, and its reducing power.
DPPH is widely used as a tool to estimate the free radical scavenging activities of antioxidants. As shown in Figure 1a, the maximum scavenging ability of EOL on the DPPH radical was 91.03%, at 1.2 mg/mL. After 0.8 mg/mL, there was no significant difference between the DPPH scavenging ability of the EOL and ascorbic acid (Vc) (p > 0.05). The result was in agreement with previous studies. Brahmi et al. found that olive leaves extracts could well scavenge DPPH, by 90% [27]. The IC 50 of EOL was found to be 0.38 mg/mL (IC 50 refers to the concentrations at which 50% of the DPPH radical was scavenged). The result was well in accordance with the research that the DPPH IC 50 values of olive leaves extracts varied from 0.17 to 0.97 mg/mL [28].

In Vivo Biological Activities
Worms could reproduce a number of offspring that quickly consumed the limited OP50 supply. Therefore, the OD value of the wells without EOL, significantly descended ( Figure 2). The addition of 0.4 mg/mL to the mediums, showed no notable effect on food clearance, compared to the control, As shown in Figure 1b, the EOL could scavenge the superoxide anion free radical, with a maximum clearance of 73.82% at 1.2 mg/mL and an IC 50 = 0.33 mg/mL. Compared with Vc, the absorbance of EOL was comparatively low, but the tendency of the curve ascended, meaning the reducing power of EOL conformed to the dose-dependent effect (Figure 1c).
A high reducing activity at a lower concentration indicated a potential higher antioxidant ability. Studies have demonstrated that the reducing power of bioactive compounds is related to its antioxidant activity [29,30]. Other experiments have showed that a higher content of the total polyphenol and total flavonoid could contribute to a higher biological activity. Falleh found that the polyphenol from Cynara cardunculus L. leaves displayed a good scavenging radical ability [31]. Xian et al. also discovered that the flavonoid extracts from Ferns could scavenge about 95% of the superoxide anion radical [32]. In this study, polyphenols were the main compounds in EOL (Table 1). Therefore, the polyphenols and the flavonoids could be the main reason for a high antioxidant activity in vitro. Antioxidant activity assays in vitro were consistent with previous work [33].

Determination of a Suitable Concentration
Worms could reproduce a number of offspring that quickly consumed the limited OP50 supply. Therefore, the OD value of the wells without EOL, significantly descended ( Figure 2). The addition of 0.4 mg/mL to the mediums, showed no notable effect on food clearance, compared to the control, while worms treated with 0.3 mg/mL, 0.5 mg/mL, and 0.6 mg/mL had a negative effect on food clearance. Based on the above analysis, 0.4 mg/mL was found to be the most suitable concentration.

Determination of a Suitable Concentration
Worms could reproduce a number of offspring that quickly consumed the limited OP50 supply. Therefore, the OD value of the wells without EOL, significantly descended ( Figure 2). The addition of 0.4 mg/mL to the mediums, showed no notable effect on food clearance, compared to the control, while worms treated with 0.3 mg/mL, 0.5 mg/mL, and 0.6 mg/mL had a negative effect on food clearance. Based on the above analysis, 0.4 mg/mL was found to be the most suitable concentration.

Verification of the Non-Toxic Concentration
To find out the suitable concentration of treatment for worms, the fertilization in C. elegans was investigated. After treatment by EOL, for 48 h, the experimental group (laid 21 ± 1) showed a 36.96% increase in progeny, compared to the control group (laid 15.33 ± 1.53) (Figure 3). The result did not

Verification of the Non-Toxic Concentration
To find out the suitable concentration of treatment for worms, the fertilization in C. elegans was investigated. After treatment by EOL, for 48 h, the experimental group (laid 21 ± 1) showed a 36.96% increase in progeny, compared to the control group (laid 15.33 ± 1.53) (Figure 3). The result did not indicate that this level of concentration had a toxic effect on C. elegans. Therefore, this concentration level was considered to be suitable for the following experiment. indicate that this level of concentration had a toxic effect on C. elegans. Therefore, this concentration level was considered to be suitable for the following experiment.

Survival under Thermal Stress
Extracts from plants could improve the resistance in C. elegans under thermal stress. Extra virgin olive oil could extend the survivals of worms, under heat stress [23]. Vayndorf and co-workers found extracts from apple could strength resistance to heat, UV radiation, and oxidative stress [34]. The polyphenol from the Zalema grape pomace could also increase the resistance in C. elegans, under

Survival under Thermal Stress
Extracts from plants could improve the resistance in C. elegans under thermal stress. Extra virgin olive oil could extend the survivals of worms, under heat stress [23]. Vayndorf and co-workers found extracts from apple could strength resistance to heat, UV radiation, and oxidative stress [34]. The polyphenol from the Zalema grape pomace could also increase the resistance in C. elegans, under thermal stress [35]. Hence to investigate the effect of EOL on heat resistance in C. elegans, treated worms were exposed to 35 • C, for 5 h. The result showed that the EOL heightened the resistibility of C. elegans, by 10.43% (Figure 4).

Survival under Thermal Stress
Extracts from plants could improve the resistance in C. elegans under thermal stress. Extra virgin olive oil could extend the survivals of worms, under heat stress [23]. Vayndorf and co-workers found extracts from apple could strength resistance to heat, UV radiation, and oxidative stress [34]. The polyphenol from the Zalema grape pomace could also increase the resistance in C. elegans, under thermal stress [35]. Hence to investigate the effect of EOL on heat resistance in C. elegans, treated worms were exposed to 35 °C, for 5 h. The result showed that the EOL heightened the resistibility of C. elegans, by 10.43% ( Figure 4).
Plant extracts of acai palm, apple, asparagus, blueberry, cinnamon, and cocoa, have been proved to have active antioxidant defense mechanisms to increase survival rates of organisms under environmental stress [36]. The thermal stress resistance effect of polysaccharides from Panax notoginseng on C. elegans was augmented, as it raised the antioxidant enzyme activities and sharply decreased the ROS levels [37,38]. In this study, we assessed the ROS levels to make a further exploration of how EOL increases the resistance in C. elegans under acute stress.

The ROS Level
Under the thermal condition, the ROS levels in the C. elegans rose sharply, producing more free radicals, which were the main reason of aging and destruction of cellular structure [39]. Some reports showed that the survival rate was lower with a high ROS level in C. elegans. However, extracts from natural plant could reduce the increasing ROS levels. José et al. found extracts from the Zalema grape pomace could increase the survival rate of worms and reduce the ROS level [35]. A similar outcome was obtained by Liao, using extracts from curcumin [19]. Hence, to investigate whether the EOL could reduce the ROS levels, worms were treated with EOL sample. Compared to the control group, the ROS level was significantly decreased by 54.15% ( Figure 5). Therefore, the EOL might increase resistance to thermal stress by reducing the increasing ROS in C. elegans. Plant extracts of acai palm, apple, asparagus, blueberry, cinnamon, and cocoa, have been proved to have active antioxidant defense mechanisms to increase survival rates of organisms under environmental stress [36]. The thermal stress resistance effect of polysaccharides from Panax notoginseng on C. elegans was augmented, as it raised the antioxidant enzyme activities and sharply decreased the ROS levels [37,38]. In this study, we assessed the ROS levels to make a further exploration of how EOL increases the resistance in C. elegans under acute stress.

The ROS Level
Under the thermal condition, the ROS levels in the C. elegans rose sharply, producing more free radicals, which were the main reason of aging and destruction of cellular structure [39]. Some reports showed that the survival rate was lower with a high ROS level in C. elegans. However, extracts from natural plant could reduce the increasing ROS levels. José et al. found extracts from the Zalema grape pomace could increase the survival rate of worms and reduce the ROS level [35]. A similar outcome was obtained by Liao, using extracts from curcumin [19]. Hence, to investigate whether the EOL could reduce the ROS levels, worms were treated with EOL sample. Compared to the control group, the ROS level was significantly decreased by 54.15% ( Figure 5). Therefore, the EOL might increase resistance to thermal stress by reducing the increasing ROS in C. elegans. When an organism is placed in harsh environments like thermal stress, heat shock proteins (HSPs) are produced to prevent protein interaction which has a bad effect on the health of cells. Thus, every cell would maintain normal physiological activities with the help of HSPs. Moreover, HSPs  When an organism is placed in harsh environments like thermal stress, heat shock proteins (HSPs) are produced to prevent protein interaction which has a bad effect on the health of cells. Thus, every cell would maintain normal physiological activities with the help of HSPs. Moreover, HSPs have an influence on the resistance of C. elegans to stress [40]. Therefore, to value the affection of EOL on the expression of HSP-16.2 in C. elegans, a test was conducted by using the transgenic strain. Compared to the control group, the expression of HSP-16.2 was increased ( Figure 6). Therefore, EOL could enhance the expression of HSP-16.2, to improve the resistance of C. elegans to thermal stress (Figure 7). Patrícia found Guarana extract could reduce intracellular ROS and increased the expression HSP-16.2 [41]. Therefore, we could assume that the EOL could not only lower the ROS level but could also increase the expression of HSP-16.2, to protect the whole organism against severe conditions. Figure 5. The effect of EOL on reducing ROS in C. elegans. Differences were considered to be significant at p < 0.05 (*).

The Expression of HSP-16.2
When an organism is placed in harsh environments like thermal stress, heat shock proteins (HSPs) are produced to prevent protein interaction which has a bad effect on the health of cells. Thus, every cell would maintain normal physiological activities with the help of HSPs. Moreover, HSPs have an influence on the resistance of C. elegans to stress [40]. Therefore, to value the affection of EOL on the expression of HSP-16.2 in C. elegans, a test was conducted by using the transgenic strain. Compared to the control group, the expression of HSP-16.2 was increased ( Figure 6). Therefore, EOL could enhance the expression of HSP-16.2, to improve the resistance of C. elegans to thermal stress ( Figure 7). Patrícia found Guarana extract could reduce intracellular ROS and increased the expression HSP-16.2 [41]. Therefore, we could assume that the EOL could not only lower the ROS level but could also increase the expression of HSP-16.2, to protect the whole organism against severe conditions.

The Antioxidant Enzymes Activities and MDA Levels
ROS quickly accumulates in cells as a result of thermal stress, therefore, leading to oxidative damage of tissue, and consequently, accelerated death of organisms [42,43]. In vivo, antioxidant enzymes including CAT, SOD, GSH-Px play the main role in defending the organ system against over-production of ROS [44]. SOD has been documented to play a crucial role in turning free radicals into water and H2O2 [45]. CAT is regarded as the principal H2O2 scavenging enzyme. GSH-Px is also b a Figure 6. The effect of EOL on expression of HSP-16.2 in C. elegans. Differences were considered significant at p < 0.001 (***). Figure 5. The effect of EOL on reducing ROS in C. elegans. Differences were considered to be significant at p < 0.05 (*).

The Expression of HSP-16.2
When an organism is placed in harsh environments like thermal stress, heat shock proteins (HSPs) are produced to prevent protein interaction which has a bad effect on the health of cells. Thus, every cell would maintain normal physiological activities with the help of HSPs. Moreover, HSPs have an influence on the resistance of C. elegans to stress [40]. Therefore, to value the affection of EOL on the expression of HSP-16.2 in C. elegans, a test was conducted by using the transgenic strain. Compared to the control group, the expression of HSP-16.2 was increased ( Figure 6). Therefore, EOL could enhance the expression of HSP-16.2, to improve the resistance of C. elegans to thermal stress (Figure 7). Patrícia found Guarana extract could reduce intracellular ROS and increased the expression HSP-16.2 [41]. Therefore, we could assume that the EOL could not only lower the ROS level but could also increase the expression of HSP-16.2, to protect the whole organism against severe conditions.  2.3.6. The Antioxidant Enzymes Activities and MDA Levels ROS quickly accumulates in cells as a result of thermal stress, therefore, leading to oxidative damage of tissue, and consequently, accelerated death of organisms [42,43]. In vivo, antioxidant enzymes including CAT, SOD, GSH-Px play the main role in defending the organ system against over-production of ROS [44]. SOD has been documented to play a crucial role in turning free radicals into water and H2O2 [45]. CAT is regarded as the principal H2O2 scavenging enzyme. GSH-Px is also b a 2.3.6. The Antioxidant Enzymes Activities and MDA Levels ROS quickly accumulates in cells as a result of thermal stress, therefore, leading to oxidative damage of tissue, and consequently, accelerated death of organisms [42,43]. In vivo, antioxidant enzymes including CAT, SOD, GSH-Px play the main role in defending the organ system against over-production of ROS [44]. SOD has been documented to play a crucial role in turning free radicals into water and H 2 O 2 [45]. CAT is regarded as the principal H 2 O 2 scavenging enzyme. GSH-Px is also considered to be a part of the enzymatic scavenger systems to protect organisms from environmental stress [46]. After treatment with EOL, for 48 h, the CAT, GSH-Px, and SOD activities were found to have increased by 10.81%, 52.23%, and 30.97%, respectively, compared to the control group (Figure 8).
Polyunsaturated fatty acid components of phospholipids in cellular membranes, were sensitive to the free radical attack. Therefore, when ROS attacked the cellular membranes, MDA was produced by free radical-initiated oxidative damage of polyunsaturated fatty acids. The MDA was a consequence of lipid peroxidation possessing cytotoxicity [47]. In this study, the MDA content decreased by 72.96%, in comparison to the control group (Figure 8d).
Previous studies have found that the antioxidant enzymes promoted the protection of worms under harsh conditions, after C. elegans was treated with extracts from natural plants. Feng et al. found the polysaccharides from P. notoginseng could increase the activities of CAT and SOD [37]. Therefore, the hypothesis could be delivered that the EOL cleared the increasing free radicals produced by ROS by promoting the activities of inner antioxidant enzymes, such as CAT, SOD, and GSH-Px.

8).
Polyunsaturated fatty acid components of phospholipids in cellular membranes, were sensitive to the free radical attack. Therefore, when ROS attacked the cellular membranes, MDA was produced by free radical-initiated oxidative damage of polyunsaturated fatty acids. The MDA was a consequence of lipid peroxidation possessing cytotoxicity [47]. In this study, the MDA content decreased by 72.96%, in comparison to the control group (Figure 8d).
Previous studies have found that the antioxidant enzymes promoted the protection of worms under harsh conditions, after C. elegans was treated with extracts from natural plants. Feng et al. found the polysaccharides from P. notoginseng could increase the activities of CAT and SOD [37]. Therefore, the hypothesis could be delivered that the EOL cleared the increasing free radicals produced by ROS by promoting the activities of inner antioxidant enzymes, such as CAT, SOD, and GSH-Px.

Nuclear Translocation of daf-16::GFP
In C. elegans, the daf-16 transcription factor could reinforce the resistance to stress via certain pathways [48]. With pretreatment of EOL for 24 h, the daf-16 nuclear localization was significantly increased, by 18.33% (p < 0.001) (Figures 9 and 10), the cytosolic localization was reduced to about 23.34% (p < 0.001) (Figure 9a). After the daf-16 transcription factor turns to the nucleus, the expression of relevant genes would be activated, such as the genes of antioxidant enzymes. Studies have showed that extracts from plants could accelerate the daf-16 nuclear localization. Extracts from Cistanche deserticola could well increase daf-16 nuclear localization [49]. Feng et al. found that the polysaccharide extracts from P. notoginseng accelerated the daf-16 translocation factor nuclear localization, while the activities of antioxidant enzymes, such as CAT and SOD, were enhanced [37,38]. Therefore, in this study, when treated with EOL, the daf-16 nuclear localization had increased, which was associated with the increasing activities of antioxidant enzymes. Hence, the EOL might enhance the activities of the antioxidant enzymes via activation of the daf-16 translocation factor.

Nuclear Translocation of daf-16::GFP
In C. elegans, the daf-16 transcription factor could reinforce the resistance to stress via certain pathways [48]. With pretreatment of EOL for 24 h, the daf-16 nuclear localization was significantly increased, by 18.33% (p < 0.001) (Figures 9 and 10), the cytosolic localization was reduced to about 23.34% (p < 0.001) (Figure 9a). After the daf-16 transcription factor turns to the nucleus, the expression of relevant genes would be activated, such as the genes of antioxidant enzymes. Studies have showed that extracts from plants could accelerate the daf-16 nuclear localization. Extracts from Cistanche deserticola could well increase daf-16 nuclear localization [49]. Feng et al. found that the polysaccharide extracts from P. notoginseng accelerated the daf-16 translocation factor nuclear localization, while the activities of antioxidant enzymes, such as CAT and SOD, were enhanced [37,38]. Therefore, in this study, when treated with EOL, the daf-16 nuclear localization had increased, which was associated with the increasing activities of antioxidant enzymes. Hence, the EOL might enhance the activities of the antioxidant enzymes via activation of the daf-16 translocation factor.

Materials and Chemicals
Olive leaves of Pendolino were collected in July 2017 from the Olive planting base in Beihe, Xichang, Sichuan (China). The olive leaves were washed and dried at 45 °C. All dried samples were ground to fine powder screened by a 60 mesh sieve.

Methanol Extraction from Olive Leaves
Extraction of olive leaves was carried out according to the method stated in [50], with some modifications. Briefly, 20 g powder was mixed with 1 L methanol at 50 °C for 46 min, through ultrasound-assisted extraction with a power of 270 W by ultrasonic device (KQ300DV, 40 kHz, Kunshan Ultrasonic Instrument Co., Jiangsu, China). After being concentrated by a rotary evaporator, the concentration was dried in a vacuum drying oven, and then was kept at 4 °C.

Polyphenols Content
Polyphenols of EOL was determined by the Folin-Ciocalteu reagent method [51]. Briefly, after being resolved in methanol, 0.1 mL EOL sample was added to 0.25 mL Folin-Ciocalteu reagent and 1 mL sodium carbonate solution (7.5% w/v), then the solution was diluted with distilled water, to 10 mL. The reaction mixture was incubated at room temperature, for 2 h, in dark. Absorbance of the b c a

Materials and Chemicals
Olive leaves of Pendolino were collected in July 2017 from the Olive planting base in Beihe, Xichang, Sichuan (China). The olive leaves were washed and dried at 45 • C. All dried samples were ground to fine powder screened by a 60 mesh sieve.

Methanol Extraction from Olive Leaves
Extraction of olive leaves was carried out according to the method stated in [50], with some modifications. Briefly, 20 g powder was mixed with 1 L methanol at 50 • C for 46 min, through ultrasound-assisted extraction with a power of 270 W by ultrasonic device (KQ300DV, 40 kHz, Kunshan Ultrasonic Instrument Co., Jiangsu, China). After being concentrated by a rotary evaporator, the concentration was dried in a vacuum drying oven, and then was kept at 4 • C.

Polyphenols Content
Polyphenols of EOL was determined by the Folin-Ciocalteu reagent method [51]. Briefly, after being resolved in methanol, 0.1 mL EOL sample was added to 0.25 mL Folin-Ciocalteu reagent and 1 mL sodium carbonate solution (7.5% w/v), then the solution was diluted with distilled water, to 10 mL. The reaction mixture was incubated at room temperature, for 2 h, in dark. Absorbance of the solution was measured at 760 nm by microplate reader (Spectramax M2, USA). The total polyphenol was calculated with a standard curve made with the gallic acid standard as a reference.

Flavonoids Content
Total flavonoids of EOL was determined by the aluminum chloride coloration method [52]. Briefly, 0.2 mL EOL sample was added to 0.4 mL aluminum chloride (1% w/v), then the solution was diluted with distilled water, to 2.5 mL. The reaction mixture was incubated at room temperature, for 10 min, in dark. Absorbance was measured at 415 nm. The total flavonoids was calculated with a standard curve made with the rutoside standard as a reference.

Soluble Proteins Content
Soluble proteins of EOL was determined using the Coomassie Brilliant Blue (CBB) method with some slight modifications [53]. Ten grams of CBBG-250 was mixed with 5 mL 95% ethanol and 10 mL 85% phosphoric acid, then the mixture was diluted to 100 mL to obtain the CBBG-250 color reagent. About 0.1 mL of the EOL sample diluent was added to 0.5 mL CBBG-250 color reagent, then the solution was diluted to 1.5 mL. Absorbance of the reaction mixture was measured at 595 nm. The soluble proteins was calculated with a standard curve, with BSA as a reference.

Soluble Sugars Content
Soluble sugars of EOL was determined by the phenol-sulfuric acid method [54]. Briefly, 0.1 mL EOL sample was mixed with 0.1 mL 6% phenol solution and 0.5 mL sulphuric acid, then absorbance was measured at 490 nm. The soluble sugars was calculated with a standard curve made with glucose as a reference.

Free Amino Acids Content
Free amino acids of EOL was determined by the ninhydrin colorimetry method with slight modifications [55]. Basically, 1 mL EOL sample was mixed with 0.5 mL 2% ninhydrin and 0.5 mL phosphate buffered saline (pH = 8.0), then the mixture was incubated at 99 • C for 15 min. The absorbance of the reaction solution was measured at 570 nm, after being diluted to 25 mL. Free amino acids was calculated with a standard curve made with glutamate standard as a reference.

DPPH Radical Scavenging Assay
EOL was resolved in methanol to different concentrations. The DPPH radical scavenging abilities of EOL were determined by the method described in [56], with some slight modifications. In brief, 35 µL ascorbic acid (Vc)-used as positive control-and the EOL sample (0-1.2 mg/mL), were added to 165 µL of DPPH ethanol solution (0.4 mM), respectively. The reaction solutions were incubated at 37 • C, for 10 min, in the dark, and then absorbance was measured at 517 nm by microplate reader (Spectramax M2, USA). The DPPH radical scavenging ability was calculated by the following formula: DPPH radical scavenging activity (%) = (1 − A 1 /A 0 ) × 100 (1) where A 1 is the absorbance of the reaction solution with the sample and A 0 is the control group replaced with distilled water.

Superoxide Radical Scavenging Assay
The superoxide radical scavenging ability of the EOL was determined by the method described in [57], with some slight modifications. In bried, 0.2 mL Vc-used as a positive control-and the EOL sample (0-1.2 mg/mL), were mixed with 0.2 mL NBT (0.08 mM), 0.4 mL NADH (0.25 mM), and 0.2 mL PMS (0.06 mM), respectively. Then the reaction solution was measured at 560 nm after being incubated at room temperature, for 10 min, in dark. The superoxide radical scavenging ability was calculated by the following formula: Superoxide radical scavenging ability (%) = (1 − A 1 /A 0 ) × 100 (2) where A 1 is the absorbance of the reaction solution with the sample and A 0 is the control group replaced with distilled water.

Reducing Power Assay
The reducing power of EOL was determined by the method described in [58], with some slight modifications. About 0.2 mL of the EOL sample (0-1.2 mg/mL) was added to 0.5 mL phosphate buffer (0.2 M) and 0.5 mL K3[Fe(CN 6 )] (1% w/v). The mixed solution was incubated at 50 • C, for 20 min, and subsequently cooled at 0 • C, for 5 min, after adding 0.5 mL TCA (10% w/v). About 0.5 mL aliquot of supernatant fluid was added to 0.5 mL distilled water and 0.1 mL FeCI 3 (0.1% w/v). Then, the absorbance of the reaction solution was measured at 700 nm, after being incubated at room temperature, for 10 min. Increasing absorbance was directly associated with an increasing reducing power. C. elegans and Escherichia coli OP50 strain were obtained from the Caenorhabditis Genetics Center (CGC). The C. elegans were maintained on nematode growth medium (NGM) agar plates, with a layer of E. coli OP50 as food sources, at 20 • C. Synchronized worms were obtained by the sodium hypochlorite method [59].

Food Clearance Assay
The optimal concentration of the EOL sample was determined by a food clearance assay, described in previous study [60]. Approximately 30 synchronized L4 nematodes grew in an S medium, containing EOL and OP50 in 96-well plates at 20 • C. The absorbance of the S medium was measured at 600 nm for 6 days.

Fertility Assay
Synchronized L4 larva worms were transferred to a fresh NGM, coated with 0.4 mg/mL EOL sample or 1% DMSO, and were subsequently trained for 48 h. Four worms were transferred to a new NGM incubated for 1.5 h, then the number of eggs that had incubated to L2 or L3 stage, were counted [61]. All experiments were conducted for three times, individually.

Thermal Stress Assay
The thermal stress assay was carried out according to the method with some slight modifications [62]. Briefly, after the sodium hypochlorite treatment, the eggs were transferred onto the NGM containing the E. coli OP50, until the L4 larvae stage. Subsequently, the L4 larva worms were transferred to a new fresh NGM, containing 0.4 mg/mL of the EOL sample and 25 µM 5-FUDR, for 48 h, while the control group was coated with 1% DMSO. 5-FUDR could prevent worms from reproduction. To determine the effect of EOL on the heat shock tolerance of the worms, the control and treated worms were incubated at 35 • C for 5 h, then scored for viability.

Determination of the ROS Level
ROS level determination was conducted by the method described in [19]. Briefly, the synchronized L1 worms were transferred to a fresh NGM plate containing the EOL sample or 1% DMSO. After 24 h exposure, the plates were placed at 35 • C for 1 h. Then the worms were washed three times by M9, to remove the OP50. Subsequently, the worms were incubated in the M9-contained 50 µM DCFH-DA, for 0.5 h, then the fluorescence was measured by microplate reader (Spectramax M2, USA).
3.5.6. Visualization of the HSP-16.2::GFP Carrying a hsp-16.2::GFP, the TJ375 strain was used to test the expression of HSP-16.2. Briefly, synchronized L1 worms were transferred to a fresh NGM plate containing the EOL sample or 1% DMSO. After 24 h exposure, the worms were fixed on a slide with 15 mM sodium azide, after being placed at 35 • C for 1h. HSP-16.2::GFP fluorescence was examined by a fluorescence microscopy (Olympas-BX51TRF, Japan).

Measurement of Antioxidant Enzymes and the MDA Level
To assess the SOD, CAT, and GSH-PX activities and the MDA level of C. elegans, under thermal stress, the control and treated worms were incubated at 35 • C for 3 h, then harvested by an M9 buffer. The worms were crushed by an ultrasonic wave to obtain a suspension. Then, the suspension was centrifuged at 2500 rpm/min, for 10 min. The supernatant liquid was determined for the protein content, the antioxidant enzyme activities, as well as the MDA level, according to the instructions of the assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Nuclear Localization of daf-16
Carrying a daf-16::GFP, the TJ356 stain was used to test the translization of daf-16, in the nucleus. Briefly, synchronized L1 worms were transferred to a fresh NGM plate, with the absence or presence of the EOL sample. After 24 h treatment, the worms were fixed on a microscopy slide with 15 mM sodium azide, then the subcellular DAF-distribution was observed under a fluorescence microscope (Olympas-BX51TRF, Japan). The location of daf-16 was categorized as cytosolic, intermediate, and nuclear.

Statistical Analyses
The data were analyzed statistically by Student's t-test (t-test) (GraphPad Prism 6) (GraphPad Software, Inc., La Jolla, CA, USA). The fluorescence intensity was quantified by the Image-J software (National Institutes of Health, Bethesda, MD, USA). All values were expressed as mean ± standard deviation (SD), n = 3. All experiments were performed in triplicates. Difference at the p < 0.05 level was considered to be significantly different.

Conclusions
In this study polyphenols was found to be the main compound in the EOL. In the investigations, it was obvious that the EOL expressed a good antioxidant ability. The most effectively scavenging ability of the DPPH radical was 91.03%, with an IC 50 of 0.38 mg/mL. Under heat stress, the EOL could strength the resistance in the C. elegans, by conspicuously reducing the ROS level and increasing the expression of the HSP-16.2. The daf-16 transcription factor turned into the nucleus, and then activated the downstream target genes. Hence, the activities of antioxidant enzymes, such as SOD, CAT, and GSH-P X , were enhanced and the MDA content was sharply lowered. This study provided a definite evidence that extracts from olive leaves could efficiently scavenge free radicals in vitro and significantly increase expression of antioxidant enzymes in C. elegans, under acute stress. Therefore, olive leaves could be a good choice when it comes to antioxidants. The results in this study also laid a theoretical foundation for further development and utilization of olive leaves, which possess a potentially economic as well as medical values.