Comprehensive Metabolite Profiling of Chemlali Olive Tree Root Extracts Using LC-ESI-QTOF-MS/MS, Their Cytotoxicity, and Antiviral Assessment

The large quantity of olive roots resulting from a large number of old and unfruitful trees encouraged us to look for ways of adding value to these roots. For this reason, the current research work is devoted to the valorization of olive roots by identifying active phytochemicals and assessing their biological activities, including the cytotoxicity and antiviral potential of different extracts from the Olea europaea Chemlali cultivar. The extract, obtained by ultrasonic extraction, was analyzed using the liquid chromatography-mass spectrometry technique (LC-MS). The cytotoxicity was evaluated through the use of the microculture tetrazolium assay (MTT) against VERO cells. Subsequently, the antiviral activity was determined for HHV-1 (Human Herpesvirus type 1) and CVB3 (Coxsackievirus B3) replication in the infected VERO cells. LC-MS analysis allowed the identification of 40 compounds, classified as secoiridoids (53%), organic acids (13%), iridoids (10%), lignans (8%), caffeoylphenylethanoid (5%), phenylethanoids (5%),sugars and derivatives (2%), phenolic acids (2%), and flavonoids (2%). It was found that extracts were not toxic to the VERO cells. Moreover, the extracts did not influence the appearance of HHV-1 or CVB3 cytopathic effects in the infected VERO cells and failed to decrease the viral infectious titer.


Introduction
Tunisia is regarded as the most important olive-producing country in the southern Mediterranean region. More than 30% of the agricultural land proved to be devoted to cultivating olive trees. These trees are distributed from north to south, with very different bioclimatic conditions and a wide range of varieties. Today, Tunisia ranks the fourth country in the world for the number of cultivated olive trees, estimated at more than 86 million trees covering an area of 1,800,000 hectares [1]. In addition to oil as the basic product, the olive oil industry produces a large number of by-products, including pruning products, pomace, and margines. The valorization of these residues has become crucial in order to avoid increased contamination. Prunings display multiple uses, including animal feed [2] and compost production [3].
These residues are known for their beneficial properties for human health, referring to their richness in phenolic compounds, especially oleuropein [4]. These compounds exhibit, among others, antioxidant, anticancer, and antimicrobial potentials, which makes them very significant for the health and food industries [5,6]. The growing interest in phenolic

The Quantitative Study of the Different Extracts
The extraction yield of dry residue rich in phenolic compounds from the external part of the olive roots was 31.16 g/100 g, the internal part was 10.23 g/100 g, and the root sum was 15.72 g/100 g. The percentages of extraction yields are illustrated in Table 1. Table 1. The extraction yield of the dry residue rich in phenolic compounds from olive tree roots.

Organs Yield (%)
External part 31.61 Internal part 10.23 Root sum 15.72 Our results show that extract yields for all three samples are important, ranging on average from 10.23% to 31.61%. The highest yield was registered for the outer part of the olive root, with a value of 31.61%.
The quantitative analysis of the different phenolic compounds was undertaken by highperformance liquid chromatography (HPLC). Several compounds from different families (flavonoids, secoiridoids, lignans, iridoids, organic acids, and sugars) were quantified (Table 2), such as oleuropein, which is the major compound, whose concentration varies between 5.892 mg/g for the root bark and 10.362 mg/g for the inner part of the root; elenolic acid, with concentrations varying from 4.54 mg/g to 0.116 mg/g; oleuropein hexoside content ranges from 3.484 mg/g to 0.141 mg/g;and ligstroside, which varied from 2.112 mg/g to 5.241 mg/g. Figure 1 displays the structure of the detected main compounds. Figure 2 illustrates a comparison between the content of selected compounds in roots and dry leaves studied by Talhaoui et al. [13]. The oleuropein content in olive roots varies from 5.892 to 10.362 mg/g, while that in leaves is around 18.01 mg/g. The verbascoside concentration in dry leaves is around 4 mg/g, while in the inner part of olive roots it amounts to 3.207 mg/g. Moreover, the ligesroside content of olive roots (5.241 mg/g) is higher than that of olive leaves (3.845 mg/g).    Figure 2 illustrates a comparison between the content of selected compounds in roots and dry leaves studied by Talhaoui et al. [13]. The oleuropein content in olive roots varies from 5.892 to 10.362 mg/g, while that in leaves is around 18.01 mg/g. The verbascoside concentration in dry leaves is around 4 mg/g, while in the inner part of olive roots it amounts to 3.207 mg/g. Moreover, the ligesroside content of olive roots (5.241 mg/g) is higher than that of olive leaves (3.845 mg/g).

Qualitative Profiling via HPLC-DAD and LC-ESIMS/ MS and Their Relative MS/MS Data
The identified phenolic compounds marked by HPLC-DAD and LC-ESI-MS/MS in negative ionization mode are exhibited in Table 3. Each compound is related to its retention time (RT), its molecular monoisotopic neutral mass, its experimental m/z, its molecular formula, its mass error (in ppm), and its main MS/MS fragments.  Figure 2. Comparison of the content of selected compounds in roots and leaves.

Qualitative Profiling via HPLC-DAD and LC-ESIMS/MS and Their Relative MS/MS Data
The identified phenolic compounds marked by HPLC-DAD and LC-ESI-MS/MS in negative ionization mode are exhibited in Table 3. Each compound is related to its retention time (RT), its molecular monoisotopic neutral mass, its experimental m/z, its molecular formula, its mass error (in ppm), and its main MS/MS fragments.   To fully assess the phenolic composition of olive roots, we have chosen to study each part on its own in order to compare them with each other. The first part corresponds to the outer part or bark as represented by the chromatogram (A); the second part corresponds to the inner part of the root as represented by the chromatogram (B); and the third part corresponds to the sum of the roots as represented by the chromatogram (C). The basepeak chromatograms (BPC) of olive roots are plotted in Figure 3, indicating their richness in phenolic compounds, as 40 phenolic compounds were identified for the roots. Olive metabolites were classified into eight structural classes: sugars and derivatives, organic acids, phenolic acids (simple), flavonoids, caffeoyl phenylethanoid derivatives, iridoids and derivatives, secoiridoids and derivatives, and lignans. sponds to the inner part of the root as represented by the chromatogram (B); and the third part corresponds to the sum of the roots as represented by the chromatogram (C). The base-peak chromatograms (BPC) of olive roots are plotted in Figure 3, indicating their richness in phenolic compounds, as 40 phenolic compounds were identified for the roots. Olive metabolites were classified into eight structural classes: sugars and derivatives, organic acids, phenolic acids (simple), flavonoids, caffeoyl phenylethanoid derivatives, iridoids and derivatives, secoiridoids and derivatives, and lignans.

Sugars and Derivatives
Low-molecular-weight sugars share multiple chemical properties. They are optically active aliphatic polyhydroxy compounds that are generally highly soluble in water. The presence of sugars and their derivatives was denoted with a peak at 1.49 min, representing the monosaccharide D-mannitol (C6H14O6) (181.07 m/z) with its fragment (163.06 m/z) (C6H12O5), corresponding to the loss of water molecules (-H2O), (89.0244 m/z), (71.0141 m/z), and (119.0343 m/z). This is in good agreement with previous data addressing olive wood from Spanish cultivars [14,15].

Organic Acids
Organic acids are ubiquitous in nature, as they are found in animal, vegetable, and microbial sources. They contain one or more carboxylic acid groups that can be covalently bonded with such groups as amides, esters, and peptides. Five compounds were

Sugars and Derivatives
Low-molecular-weight sugars share multiple chemical properties. They are optically active aliphatic polyhydroxy compounds that are generally highly soluble in water. The presence of sugars and their derivatives was denoted with a peak at 1.49 min, representing the monosaccharide D-mannitol (C 6 H 14 O 6 ) (181.07 m/z) with its fragment (163.06 m/z) (C 6 H 12 O 5 ), corresponding to the loss of water molecules (-H 2 O), (89.0244 m/z), (71.0141 m/z), and (119.0343 m/z). This is in good agreement with previous data addressing olive wood from Spanish cultivars [14,15].

Organic Acids
Organic acids are ubiquitous in nature, as they are found in animal, vegetable, and microbial sources. They contain one or more carboxylic acid groups that can be covalently bonded with such groups as amides, esters, and peptides. Five compounds were observed in olive root extracts, including: gluconic acid (C 6

Iridoids and Derivatives
Iridoids are known to offer a wide range of health benefits, including resisting a wide assortment of physical, biological, and chemical stressors. Multiple scientific studies have highlighted that iridoids possess antioxidant, antibacterial, anti-cancer, and anti-viral properties. Iridoids have been identified in olive tree root extracts. Among the most important ones, we state loganic acid (C 16  This type of monoterpene is widely distributed in the Oleaceae (Obied et al., 2008) [20]. It is to be noted that their fragmentation patterns are consistent with previous works [10,14,21]. This type of monoterpene is widely distributed in the Oleaceae (Obied et al., 2008) [20]. It is to be noted that their fragmentation patterns are consistent with previous works [10,14,21].

Secoiridoids and Derivatives
Secoiridoids are the major class of secondary metabolites identified in olives. Among them, oleuropein corresponds to the major compound in leaf, stem, and root extracts. Several secoiridoids were detected in the extracts, and all were identified by comparing their ECs and fragmentation pathways to those found in the literature and databases [10,16].

Secoiridoids and Derivatives
Secoiridoids are the major class of secondary metabolites identified in olives. Among them, oleuropein corresponds to the major compound in leaf, stem, and root extracts. Several secoiridoids were detected in the extracts, and all were identified by comparing their ECs and fragmentation pathways to those found in the literature and databases [10,16].
A total of 22 known secoiridoids have been tentatively identified in olive roots. They notably correspond to oleoside (C 16    All aforementioned lignans were previously reported in olive wood [14,15].  Figure 7 depicts a proposed mechanism for the fragmentation of ligstroside. The fragmentation model was checked through comparison with previous studies [22].    [14,15]. Figure 8 illustrates the extracted ion chromatogram (EIC) profiles and MS-MS spectra of selected phenolic compounds identified in methanolic extracts from Chemlali olive roots.

Cytotoxicity and Antiviral Activity
The dose-response effect of the extracts (external part M-EX, internal part M-IN, and all the roots M-T) on the viability of VERO cells is portrayed in Figure 9. The exact CC 50 values (concentrations of the tested extract decreasing the cellular viability by 50%) could not be assessed but were above 500 µg/mL for all tested extracts. According to a previously published work [24], no significant cytotoxicity of plant extract is recorded when the CC 50 is above 500 µg/mL.

Cytotoxicity and Antiviral Activity
The dose-response effect of the extracts (external part M-EX, internal part M-IN, and all the roots M-T) on the viability of VERO cells is portrayed in Figure 9. The exact CC50 values (concentrations of the tested extract decreasing the cellular viability by 50%) could not be assessed but were above 500 µ g/mL for all tested extracts. According to a previously published work [24], no significant cytotoxicity of plant extract is recorded when the CC50 is above 500 µ g/mL. None of the tested extracts displayed any noticeable effect on the formation of virus-induced cytopathic effects(CPEs) in the infected VERO cells. Subsequent end-point dilution assays revealed that the infectious titers of both viruses in the extract-treated samples were comparable to the titers in the appropriate virus control samples.
At this stage of analysis, it is noteworthy that the extracts obtained from the underground parts of the olive tree Chemlalai cultivar exhibit no significant cytotoxicity towards non-cancerous VERO cells. Interestingly, methanolic extracts from the twigs of Chemlali and Chétoui cultivars presented moderate cytotoxicity towards VERO cells with CC50 values of 155 and 151 μg/mL [25]. Aqueous extracts from the leaves of two olive tree varieties (Meski and Chemlali) proved to increase the viability of murine oligodendrocytes (158N) in the concentration range of 5-200 μg/mL, while at 400 μg/mL, the viability of 158N was reduced [26]. The olive leaf ethanolic extracts also suppressed cytotoxicity induced by oxidative stress on the mouse embryonic fibroblast (MEF) [27]. Hydroethanolic extract from the leaves of the Chemlali cultivar proved to inhibit the proliferation of a cell line derived from human chronic myeloid leukemia (K562) and to exert Luteolin-7-O-glucoside

Cytotoxicity and Antiviral Activity
The dose-response effect of the extracts (external part M-EX, internal part M-IN, and all the roots M-T) on the viability of VERO cells is portrayed in Figure 9. The exact CC50 values (concentrations of the tested extract decreasing the cellular viability by 50%) could not be assessed but were above 500 µ g/mL for all tested extracts. According to a previously published work [24], no significant cytotoxicity of plant extract is recorded when the CC50 is above 500 µ g/mL. None of the tested extracts displayed any noticeable effect on the formation of virus-induced cytopathic effects(CPEs) in the infected VERO cells. Subsequent end-point dilution assays revealed that the infectious titers of both viruses in the extract-treated samples were comparable to the titers in the appropriate virus control samples.
At this stage of analysis, it is noteworthy that the extracts obtained from the underground parts of the olive tree Chemlalai cultivar exhibit no significant cytotoxicity towards non-cancerous VERO cells. Interestingly, methanolic extracts from the twigs of Chemlali and Chétoui cultivars presented moderate cytotoxicity towards VERO cells with CC50 values of 155 and 151 μg/mL [25]. Aqueous extracts from the leaves of two olive tree varieties (Meski and Chemlali) proved to increase the viability of murine oligodendrocytes (158N) in the concentration range of 5-200 μg/mL, while at 400 μg/mL, the viability of 158N was reduced [26]. The olive leaf ethanolic extracts also suppressed cytotoxicity induced by oxidative stress on the mouse embryonic fibroblast (MEF) [27]. Hydroethanolic extract from the leaves of the Chemlali cultivar proved to inhibit the proliferation of a cell line derived from human chronic myeloid leukemia (K562) and to exert Luteolin-7-O-glucoside At this stage of analysis, it is noteworthy that the extracts obtained from the underground parts of the olive tree Chemlalai cultivar exhibit no significant cytotoxicity towards non-cancerous VERO cells. Interestingly, methanolic extracts from the twigs of Chemlali and Chétoui cultivars presented moderate cytotoxicity towards VERO cells with CC 50 values of 155 and 151 µg/mL [25]. Aqueous extracts from the leaves of two olive tree varieties (Meski and Chemlali) proved to increase the viability of murine oligodendrocytes (158N) in the concentration range of 5-200 µg/mL, while at 400 µg/mL, the viability of 158N was reduced [26]. The olive leaf ethanolic extracts also suppressed cytotoxicity induced by oxidative stress on the mouse embryonic fibroblast (MEF) [27]. Hydroethanolic extract from the leaves of the Chemlali cultivar proved to inhibit the proliferation of a cell line derived from human chronic myeloid leukemia (K562) and to exert G0/G1 cell cycle arrest on the 1st and 2nd days of incubation, and then also at G2/M phase (3rd and 4th days). Additionally, the extract induced apoptosis and differentiation of K562 cells towards the monocyte lineage [28]. Essafi Rhouma et al. [29] extracted the flowers of the Chemlali cultivar with 80% (v/v) ethanol and tested their cytotoxicity towards breast cancer cells (MCF-7) and human mammary epithelial cells (MCF-10A). The extracts in the concentration range between 31.25 and 250 µg/mL decreased the viability of MCF-7 cells without displaying a detrimental effect on MCF-10A. Moreover, the western blot analysis revealed the potential pro-apoptotic activity of flower extracts in MCF-7 cells [29].
None of the tested extracts demonstrated antiviral activity against the CVB3 or HHV-1 viruses replicating in the VERO cells. Leila et al. [25] investigated the cytotoxicity and antiviral activity against CVB3 or HHV-2 (Human Herpesvirus type 2) of twig extracts obtained using hexane, dichloromethane, ethyl acetate, and methanol from two Tunisian olive cultivars (Chemlali and Chétoui). Extracts from both cultivars displayed no effect on the replication of HHV-2. However, hexane extracts from Chemlali and Chétoui cultivars exhibited antiviral effects towards CVB-3 with IC 50 (50% inhibitory concentration) of 20.41 and 23.28 µg/mL, respectively, and a selectivity index (SI) > 5 [25]. The hydroethanolic (water:ethanol; 50:50 (v/v)) extracts from the leaves of O. europaea var. sativa and O. europaea var. sylvestris were found to inhibit the replication of HHV-1 in the HeLa cells, reducing the infectious titer (plaque-forming assay) as well as the viral load (real-time PCR) [30]. Despite the literature data describing olive twigs or leaves as potential sources of antiviral compounds, we have not observed any antiviral activity for the extracts from underground parts of the olive tree (Chemlalai cultivar). In the case of hexane extracts from twigs of Chemlali and Chétoui cultivars, antiviral activity against CVB-3 was observed, but no detailed phytochemical analysis was performed. It was suggested that 2,4-di-tertbutylphenol was the bioactive compound responsible for this activity, but this was only shown through bio-guided assays using thin-layer chromatography [25]. This compound was not present in the root extracts we studied. The O. europaea var. sativa and O. europaea var. sylvestris extracts showed potent anti-HHV-1 activity, and this activity was probably due to the presence of oleuropein [30]. We have also identified oleuropein in the extracts from underground parts. Still, the content of this compound is the highest in leaves, and this may be the reason for the lack of antiviral activity of root extracts. Unfortunately, the cytotoxicity of root extracts did not allow us to test the antiviral activity using higher concentrations than those presented in Figure 10.

Sample Extraction
The olive root (2 kg) was collected from Chemlali olive trees grown in the Sfax region (Tunisia). The olive trees were about 45 years old. Olive root samples were collected in early January 2021. The samples were dried for 90 days at room temperature in a dark and airy room, and then the root was scraped at a local sawmill to separate the bark from

Sample Extraction
The olive root (2 kg) was collected from Chemlali olive trees grown in the Sfax region (Tunisia). The olive trees were about 45 years old. Olive root samples were collected in early January 2021. The samples were dried for 90 days at room temperature in a dark and airy room, and then the root was scraped at a local sawmill to separate the bark from the root wood. All samples were then ground to a fine powder using a domestic mill before being extracted. The roots of the olive tree have been separated into three parts, namely the external part, the internal part, and the total roots. Each part of the olive roots was placed separately in amber glass bottles and homogenized in a solution of methanol/water 80:20 (v/v) using an ultrasonic bath for 30 min. After filtration with Whatman filter paper No. 42 (125 mm), the extracts were put in a rotary evaporator under vacuum at 40 • C. Eventually, the extracts were kept at −20 • C until future analysis. After extraction and obtaining dry residues, the extraction yield was computed.

HPLC Analysis
In order to characterize the phenolic compounds by HPLC (Agilent 1200 series), the mobile phases relied on a combination of solvent A (water) and solvent B (methanol). The following multi-step gradient was applied: 0 min, 50% B; 5 min, 60% B; 20 min, 80% B; 25 min, 100% B; 26 min, 50% B; 35 min, 50% B. The flow rate was set at 1 mL/min throughout the gradient. The separation was undertaken with a Gemini 5 mcm NX-C18 110A column (250 × 4.6 mm) at room temperature. UV spectra were recorded from 190 to 600 nm. The injection volume was 10 uL. The identification of each compound was based on a comparison of their retention times with those of the reference compounds and recording the UV spectra in the 190-400 nm range. The quantitative determination was performed using calibration curves for phenolic standards. The content of the phenolic compounds was expressed in mg/g of dry olive roots.

MS-MS Analysis
The system was coupled to a 6540 Agilent Ultra-High-Definition Accurate-Mass QTOF, equipped with an ESI source (Agilent Dual Jet Stream) (Agilent Technologies, Palo Alto, CA, USA). The operating conditions in negative ionization mode were as follows: gas temperature, 350 • C; drying gas, nitrogen at 12 L/min; nebulizer pressure, 40 psig; sheath gas temperature, 400 • C; sheath gas flow, nitrogen at 12 L/min; capillary voltage, 4000 V; skimmer, 645 V; octopol radiofrequency voltage, 750 V, with the corresponding polarity automatically set. The spectra were acquired over a mass range from 100 to 1000 m/z, and for MS/MS experiments, from 100 to 1000 m/z. The reference mass correction of each sample was carried out with a continuous infusion of Agilent API TOF reference mixture (61,969-85,001). The data analysis was conducted on a Mass Hunter Qualitative Analysis B.10.00 (Agilent Technologies).

Cytotoxicity Testing
Cytotoxicity was tested according to the previously described methodology [31]. The monolayer of VERO cells in 96-well plates was treated with serial dilutions of extracts in cell media for 72 h. Afterwards, the media were removed. After washing with PBS, the MTT solution in DMEM was added, and the incubation continued for 4 h. Eventually, the formazan crystals were dissolved using SDS/DMF/PBS solvent. Following the overnight incubation, the absorbance (540 and 620 nm) was determined using the Synergy H1 Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). Collected data were exported from Gen5 software (ver. 3.09.07; BioTek Instruments, Inc., Winooski, VT, USA) to GraphPad Prism (v7.0.4) for further analysis.

Antiviral Activity
The antiviral activity was tested, as previously described [31]. The VERO cells seeded in 48-well plates were infected with HHV-1 or CVB3 at a 100-fold CCID 50 infectious titer. After 1 h of pre-incubation allowing virus attachment, the cell monolayer was washed with PBS and treated with extracts in non-toxic concentrations. The non-toxic doses were selected based on dose-response curves obtained during cytotoxicity studies. They were defined as concentrations of selected extracts that did not reduce cellular viability by more than 10%. Incubation continued until a typical CPE was observed in the virus control (infected, non-treated cells). Next, the sample-treated infected cells were also observed for the occurrence of CPE using an inverted microscope (CKX41, Olympus Corporation, Tokyo, Japan) equipped with a camera (Moticam 3+, Motic, Hong Kong), and the results were recorded (Motic Images Plus 2.0, Motic, Hong Kong). Afterwards, the plates were thrice frozen (−72 • C) and thawed, and the samples were collected and kept frozen at −72 • C until the assessment of the viral infectious titer was carried out.
Virus titrations were conducted using an end-point dilution assay [31]. Briefly, monolayers of VERO in 96-well plates were treated with tenfold dilutions of samples from the above-described antiviral assays. After 72 h, the viral titers were calculated using an MTT assay. The antiviral activity was specified, referring to the difference in viral titer between the extract-treated samples and the virus control. A significant antiviral activity needs at least a 3 log reduction of the viral infectious titer.

Conclusions
Overall, olive roots may be considered a source of polyphenolic compounds. Spectrometric analysis applied through the use of the liquid chromatography-mass spectrometry technique proved to be a powerful tool for the characterization of several compounds in olive roots. 41 molecules of different families were identified, including secoiridoids, iridoids, lignans, and organic acids. Moreover, according to the quantification by UHPLC-DAD, oleuropein is the major compound detected in olive roots, with a concentration of 10.36 mg/g. Ligestroside has also been detected, and its amount is higher than that in leaves. These results provide a deeper and better insight into the bioactive compounds in the less-considered organs of the olive tree. Several compounds remained unidentified, revealing the chemical diversity of Olea. The extracts proved to be non-toxic to VERO cells and did not exert any noticeable antiviral effect against HHV-1 or CVB3 replicating in VERO cells. Indeed, in future works, we may tackle the identified compounds, attempt to isolate and investigate their characteristics, and fully understand how significant they are.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.