Chemical Profile, Cytotoxic Activity And Oxidative Stress Reduction Of Different Syringa Vulgaris L. Extracts Part 2

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2.5. Antioxidant Activity Assays

The in vitro antioxidant capacity of S.wulgaris extracts was evaluated by three different methods:2,2-diphenyl-picrylhydrazil(DPPH●)scavenging assay, ferric-reducing antioxidant power(FRAP), and cupric ion reducing antioxidant capacity(CUPRAC)(Table 9). The three used methods were chosen in order to bring arguments that prove the antioxidant capacity of tested samples by three different mechanisms [31-33].

Antioxidant activity for the flowers and fruit of S.vulgaris is reported by Toth et al. using the DPPH bleaching assay and revealed an effective antioxidant activity of these methanolic extracts with IC50 =65.25 ug/mL and IC50 =67.39 ug/mL, respectively [10]. The same assay is used by Varga et al. and showed a superior antioxidant activity of leaves and bark extracts （ICs0=25.25μg/mL， ICs0 =40.61 ug/mL respectively）【11】. Our study revealed that the flowers extract showed significant (p <0.001)higher antioxidant activity （IC50 = 36.83 μg/mL） compared to the other analyzed extracts in the following order: flowers >leaves >bark > fruit (Table 9). Similar results for flower extract were obtained by CUPRAC and FRAP assays (p <0.001). Moreover, the present study brings originality by offering, for the first time, proof of the antioxidant capacity of the tested samples by different other mechanisms that are highlighted using the two other assays, FRAP, and CUPRAC. At the same time, the study offers for the first time a comparative view on flowers, fruit, leaves, and bark, bringing evidence on the fact that flowers represent the most important antioxidant potential.

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Moreover, the originality that is brought by the performed assays, the study of the antioxidant capacity of the tested samples becomes even more important, as it represents the basis of the cytotoxic activity. The antioxidant capacity is highly related to the phenolic composition of the tested samples [10,11], as it is largely known and accepted that phenylpropanoids, flavonoids, and also secoiridoids exhibit antioxidant and antiproliferative capacity [34-36], but moreover, the antioxidant capacity can be, at the same time, highly related to the cytotoxic activity of these samples, as the antioxidant activity may represent one of the most important mechanisms at the basis of the cytotoxicity [37].

2.6. Cytotoxicity Assays

To investigate the in vitro antiproliferative potential of the four S.vulgaris ethanolic extracts on two human cancer cell lines, HeLa and B16F10, the MTT assay was conducted. Results are presented in Figures 3 and 4.

Figure 4. Inhibitory effects on B16H10 cell line of S. Vulgaris leaves, fruit, bark, and flowers extracts at five different concentrations C1-C5 calculated according to the TPC(umol GAE/mL) determined for each extract: leaves（11.34-56.7 mmol GAE/mL）， fruit（6.66-33.2 μmol GAE/mL）， bark（9.875-49.37 μmol GAE/mL） and flowers （11.69-58.475 μmol GAE/mL）; Negative control—untreated cells， Internal control—Ethanol， Positive control—Cisplatin. Values represent the mean ± SD of three determinations.*p<0.05;**p<0.001;**p<0.0001(Differences between extract-treated cells and the negative control).

The flowers ethanolic extract displayed significant cytotoxic activity on both B16F10 and HeLa cells (Figures 3 and 4). The viability of both melanoma and carcinoma cells was significantly decreased (p <0.0001) compared to the untreated control for concentrations of 23.39-58.475 μmol GAE/mL and 11.69-58.475 μmol GAE/mL， respectively. No, statistically significant (p>0.05) differences between the viability values determined by these concentrations were noticed, indicating that the cytotoxic activity is not dose-dependent. Similar to the flower extract, the leaves extract showed a significant cytotoxic effect on both cell lines (p<0.001). Furthermore, when tested against the HeLa cell line, these two extracts' inhibitory activity was comparable to the positive control—Cisplatin treated cells (Figure 3).On the B16F10 cells, similar efficacy was displayed only by the flower extract, while the leaf and bark extracts possessed a lower cytotoxic effect compared to Cisplatin. The fruit extract proved the lowest cytotoxic effect (Figure 4).

Thus, our results demonstrate the in vitro cytotoxic properties of S.vulgaris extracts on two tumoral cell lines. HeLa cell line proved to be more susceptible than B16F10 cells; varying in vitro and in vivo sensitivity between differing tumor cell types is documented by literature [38,39].

A non-linear regression analysis of the dose-response curve determined half-maximal inhibitory concentration (ICs0)values for cytotoxic activity and the results are described in Table 10.

These results indicate that the lowest ICso values in the case of S.vulgaris ethanolic extracts were obtained for fruit and bark, respectively. However, these values are calculated

according to the TPC (umol GAE/mL) determined for each extract. Thus, considering the complex chemical composition established for all four extracts, correlations between the in vitro antiproliferative activity and identified groups and/compounds were determined. No correlation was found between cell viability with TPC（r²=-0.4）. A strong correlation was noticed between the inhibitory effect on cell viability and certain identified and quantified compounds that are described in Table 1. The highest quantity of the following compounds 1, 2, 5, 7, 9, 11, 13, and 14 was attributed to the flowers extract chemical composition, while the rest of the compounds were the most abundant in the bark extract (Table 1). Among these compounds, acetonide and echinacoside quantities appeared to strongly correlate with the cytotoxicity displayed by both flower（r²= 0.84-0.97 and 0.87-0.99, respectively) and bark (r2=0.87-0.94 and 0.85-0.99, respectively)extracts toward the B16H10 cell line. In the case of the HeLa cell line, the correlation was observed for ligstroside （r-=0.89-0.99）， syringalactone A （r²=0.90-0.99）, and oleuropein-aglycone （r²=0.95-0.98）. Although all these three secoiridoids compounds were found at the highest quantity in the case of the bark extract, correlation coefficients between their contents in the bark extracts and antiproliferative activity were relatively low（r²=-0.80-0.49）.

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These findings underline phenylpropanoids (acteoside and echinacoside) and sec-iridoids (ligstroside, syringalactone A, oleuropein-aglycone) among the compounds responsible for the S. Vulgaris ethanolic flower extract antiproliferative efficacy toward B16H10 and HeLa cell line, respectively. These results are in agreement with previous reports demonstrating in vitro antiproliferative efficacy of secoiridoids [4].

Previous studies pointed out the antioxidant and antitumor potential in the case of several Syringa species, suggesting these properties might be related to certain major identified compounds, namely iridoids and lignans 4,5]. Aqueous extracts obtained from flowers and leaves of S. pubescens were in vitro cytotoxic when tested against L2215 cell line, while S. patula floral buds extract and two isolated compounds,syringaresinol and oleoside 11-methyl ester, were able to inhibit HepG2 cells proliferation [4,5]. Similarly, oleuropein and 3,4-dihydroxy phenyl ethyl alcohol 8-O-β-D-glucopyranoside, two compounds isolated from S.pubescens subsp. patula, expressed cytotoxicity against P-388, L-1210, SNU-5, and HL-60 cells [5].

Several compounds isolated from S.vulgaris leaves were reported to exhibit anti-tumoral potential [4,40]. Relatively weak cytotoxicity was highlighted in the case of isooleoacteoside and syringopicroside B when tested against LOX-IMVI melanoma cell line and NCI-H522 lung cancer cell line, respectively [4,18]. Additionally, the hydrolysis product of is oleuropein displayed moderate cytotoxic activity against lung cancer cell lines DMS273 and DMS114 [4,40].

With all these in view and corroborating the results obtained in the phytochemical analysis, in the cytotoxicity assays, and in the antioxidant assays, the link between all these becomes obvious and it can be concluded that the antiproliferative activity of the tested samples is significant and may be due to an antioxidant mechanism. To the best of our knowledge, this is the first study aimed to evaluate and compare the chemical profile, antioxidative and antiproliferative properties of ethanolic extracts obtained from flowers, leaves, bark, and fruit of S. Vulgaris.

3. Materials and Methods 3.1. Chemicals and Reagents

Acetonitrile for the HPLC-DAD-MS analysis was purchased by Merck (Darmstadt, Germany), while water was purified with a Direct-QUV system by Millipore (Darmstadt, Germany). Chlorogenic acid, rutin, and oleuropein (analytical purity)were purchased from Sigma Aldrich (Darmstadt, Germany). All other chemicals used were purchased from Alfa-Aesar, Karlsruhe, Germany. The cytotoxicity was tested on two tumoral cell lines: murine melanoma cells (B16F10 cells) and human cancer cell lines (HeLa). The selected cell lines were obtained from the American Type Cell Collection(ATCC)(Manassas, VA, USA). The B16F10 cells were maintained in RPMI 1640 medium (Sigma Aldrich, Darmstadt, Germany)

supplemented with 10% fetal bovine serum(EuroClone, MI, Pero, Italy) and 1% Antibiotic-Antimvcotic 100×(Sigma Aldrich, Darmstadt, Germany). Hela cells were maintained in DMEM/F-12 medium (Gibco Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum (EuroClone, MI, Pero, Italy) and 1%Antibiotic-Antimycotic 100×(Sigma Aldrich, Darmstadt, Germany). The two cell lines were cultured in a 5%CO, incubator (Advantage-Lab, Schilde, Belgium) at 37°Cin a humidified atmosphere. Cisplatin (Ebewe Pharma Ges. m.b. H.Nfg. KG, Unterach am Attersee, Austria) was included as a standard positive control for cytotoxicity assay.

3.2.Plant Material and Preparation of Extracts

The vegetal material was harvested from Cluj county, North-Western Romania. Flow-ers and leaves were harvested in April-May 2020, during the flowering period of the species, while fruit and bark were harvested in September 2020. Voucher specimens for the har-vested species are deposited in the herbarium of the Pharmacognosy Department of the Faculty of Pharmacy Cluj-Napoca (Voucher no83).

The harvested samples were air-dried. For the obtention of extracts, the ground vegetal material of each sample was cold macerated with 70% o/v ethanol, in a ratio of 1:10. The solution was then subjected to percolation, filtered, and used for the phytochemical analysis and biological activity testing of antioxidant capacity and cytotoxic activity [41].

3.3. HPLC-DAD-ESI+ Analysis of Polyphenolic Compounds

Evaluation of polyphenolic compounds was performed on HP-1200 liquid chromatography, which was equipped with a quaternary pump, autosampler, DAD detector, and MS-6110 single quadrupole API-electrospray detector(Agilent-Technologies, Santa Clara, CA, USA). Detection of phenolic compounds was carried out in positive ionization mode. Different fragment, in the range 50-100 V, was applied. Separation of compounds was performed on an Eclipse XDB-C18（5 μm;4.5×150 mm i.d.）column（Agilent）， using as a mobile phase 0.1%acetic acid in water(A)and 0.1%acetic acid in acetonitrile (B).a multistep linear gradient was employed for elution, with the following composition:5% B for 2 min; from 5%to 90% of B in 20 min, hold for 4 min at 90%B, then 6 min to arrive at 5% B. Flow rate was maintained at 0.5 mL/min and temperature at 25±0.5°C. The phenolic compounds in the extract were analyzed by comparing the retentions times, UV visible and mass spectra of each separated compound with three reference standards, as follows: for the flavonoids, compounds were quantified using the calibration curve of rutin obtained using five different concentrations, varying from 10 to 80ug/mL and expressed as equivalents of rutin (mg rutin/g plant material (R2=0.9973)), for phenylpropanoids, the compounds were quantified using the calibration curve of chlorogenic acid obtained using five different concentrations, varying from 10 to 50 ug/mL and expressed as equivalents of chlorogenic acid/plant material (R2=0.9937), while for iridoids the compounds were quantified using the calibration curve of oleuropein obtained using five different concentrations， varying from 10 to 100 μg/mL and expressed as equivalents of chlorogenic acid/g plant material (R-=0.9966). Positively charged ions were detected by mass spectrometry, using the Scan mode. The following conditions for mass spectrometry were used: gas temperature 3500 C, nitrogen flow 7 L/min, nebulizer pressure 35 psi, capillary voltage 3000 V， fragment 100 V, and m/z 120-1200. Chromatograms were recorded at λ=280 and 340 nm. Data acquisition was performed using the Agilent ChemStation software [42,43].

3.4. FT-IR SpectroScopy

The Fourier Infrared transform spectroscopy was made at the Spectroscopy laboratory from Life Sciences Institute"King Michael I of Romania" from Cluj-Napoca. Prior to FT-IR analysis, the S. Vulgaris leaf, flower, bark, and fruit extracts were dried on a clean microscope slide for 12 h at room temperature. The dried extracts were then removed from the microscope slide and mixed with the KBr powder in a proportion of 1:100 and placed in a spectral pellet press chamber steel kit. To form the translucent KBr pellet we applied pressure of 10tfor2min. The FT-IR spectra were collected with a Jasco FT-IR4100 spectrometer (Jasco, Germany), in the 4000-400 cm' spectral range, using 256 scans/sample at 4 cm-resolution. Furthermore, the obtained Ft-IR spectra were corrected for CO, and H2O using the Spectra Manager program of the same used software. Finally, the FT-IR data analysis was carried out using OriginPro Version 8.5.1 software (OriginLab Corporation, Northampton, MA, USA).

3.5.Quantification of Total Polyphenols, Flavonoids, and Phenolic Acids Content

Total phenolic content (TPC) was assessed by a spectrophotometric method based on the color reaction of polyphenols with the Folin-Ciocalteu reagent, according to the Euro-pean Pharmacopoeia, using a calibration curve of gallic acid (R2= 0.9928). Results were expressed as mg gallic acid equivalents (GAE)/g dried vegetal material. Determination of total flavonoids (TFC) was also performed by a spectrophotometric, using the aluminum chloride method, based on a calibration curve of rutin (R-=0.9981)and expressing results as mg of rutoside equivalents (RE)/g dried vegetal material. Total phenolic acids(TPA)was assessed by a spectrophotometrical method, using Arnow's reagent, similar to the one existing in the 10th Edition of the Romanian Pharmacopoeia(Cynarae folium mono-graph). Results of the TPA determination were expressed as mg caffeic acid equivalents (CAE)/g dried vegetal material and calculated using a caffeic acid calibration curve graph (R2= 0.9956). All these experiments were performed in triplicate [31,33,44].

3.6. GC-MS Analysis

The GC-MS analysis was carried out on a Dani Master GC-MS System. A SH-Rxi-5 ms column with 30 m ×0.25 mm×0.25 μm was used for the separation of compounds. Nitrogen was used as a carrier gas, with a 10 mL/min flow rate. The temperature of the system followed the gradient found in Table 11.

Five μL of each sample， diluted 1 to 10 with absolute ethanol， were injected. The EIS-MS detector identified compounds with molecular weights from 50 to 600 daltons. The ion source was operated at 200°C. The compounds were tentatively identified based on the matching factor, using the NIST MS 2.2 spectra database. A matching score higher than 80% was considered acceptable for the identification of the compounds. Quantitative analysis was performed by area normalization method and the results were expressed as area percentage (%) [31,45]. 3.7.Antioxidant Activity Assays

3.7.1.DPPH Radical Scavenging Activity

For assessing the antioxidant capacity of S. Vulgaris extracts, the DPPH bleaching assay was used. It is a spectrophotometric method, based on the reaction of the DPPHe reagent and antioxidants that are present in tested extracts mL of each extract of different concentrations were added to 2 mL0.1 g/L DPPH·methanolic solution and maintained at 40 °C in a thermostated bath, for half an hour. Absorbance and their variation were measured at 517 nm. Inhibition of the DPPHe radical was calculated using the formula: DPPH scavenging ability%=(A control- A sample/A control)× 100, where A control is an absorbance of the control, composed of the DPPH· radical solution + methanol(a mixture containing all reagents except the tincture) and A sample is the absorbance of DPPH radical+samples. The percentage of DPPHdecrease was expressed in Trolox equivalents (TE, R2=0.987).

DPPHradical scavenging activity of the tincture was expressed as IC50(ug/mL). Assays were performed in triplicate [32,33,46].

3.7.2. Ferric-Reducing Antioxidant Power Assay (FRAP)

The FRAP method is a spectrophotometric method based on the color change of a 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ) radical complex with Fe3+. This color change is assessed by the reduction of the ferric ion (Fe3) to the ferrous ion (Fe2+) in this complex[47]. The FRAP reagent consists of a mixture of 2.5 mL of a 10 mM TPTZ solution in 40 mM HCl, mixed with 2.5 mL20 mM ferric chloride solution and 25 mL of acetate buffer at pH=3.6. Of each tested sample,4 mL was diluted to 1.8 mL with water and mixed with 6 mL of this reagent. A blank solution was prepared in the same manner, but replacing extracts with water Antioxidant capacity was evaluated in correlation with the color change, by measuring absorbances at 450 nm, using Trolox as a reference and a calibration curve (R2=0.992). Results were expressed as uM Trolox equivalents/g dry weight vegetal product and the assays were performed in triplicate [48].

3.7.3. Cupric long Reducing Antioxidant Capacity (CUPRAC)

The CUPRAC method is a spectrophotometric method based on the reduction of the copper ion (II) to the copper iron (I) in the neoprene (2,9-dimethyl-1,10-phenantroline)complex. This reduction determines a color change from light green to red-orange. The change of color was correlated with the antioxidant capacity by measuring the absorbance at 450 nm. The calibration curve was plotted using concentrations of the Trolox standard and results were expressed as mM Trolox equivalent/g dry weight vegetal product [32,49,50]. 3.8. Cytotoxicity Assays Cytotoxicity study on tumoral cell lines was performed using the MTT assay (3-(45-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma Aldrich) [51,52]. For both cell lines, the cells were plated (1 × 10°cells/well) in 96-well plates for 24 h in normal propagation media (200 uL cell suspension in each well). The extracts were added to the complete medium in five distinct volums （5 μL，10 μL， 15 μL，20 μL，25 μL）， with the result-ing concentrations (C1, C2, C3, C4, and C5) calculated according to the TPC determined for each extract and expressed as umol GAE/mL as follows: flowers(C1—11.69 umol GAE/mL， C2—23.39 μmol GAE/mL， C3—35.08 umol GAE/mL， C4—46.78 μmol GAE/mL， C5—58.475 μmol GAE/mL）， leaves（C1-11.34 μmol GAE/mL， C2—22.68 umol GAE/mL， C3—34.02 μmol GAE/mL， C4—45.36 μmol GAE/mL， C5—56.7 μmol GAE/mL）， bark （C1—9.875 μmol GAE/mL， C2—19.75 umol GAE/mL， C3—29.625 umol GAE/mL， C4—39.5 umol GAE/mL， C5—49.37 μmol GAE/mL） and fruit （C1—6.66 umol GAE/mL， C2—13.32 μmol GAE/mL， C3—19.98 umol GAE/mL， C4—26.64 umol GAE/mL， C5—33.2 umol GAE/mL). The negative control was represented by cells lines cultured in a normal expansion medium (untreated cells), while 70% o/v ethanol and Cisplatin (0.2 uM) were included as the internal control and the positive control, respectively.

The cells viability following 24 h incubation at 37°C in a humidified atmosphere with 5% CO, was evaluated using the MTT assay according to a previously published protocol [51,52]. The formazan particles formed by adding 0.5 mg MTT to each well were dissolved with dimethyl sulfoxide (DMSO) (Sigma Aldrich, St.Louis, MO, USA), and the absorbance was read at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). The cell viability percentages(%)were calculated based on the absorbance ratio between cell cultures treated with extracts and the negative controls (untreated cells) multiplied by l00. For each extract, the cytotoxic activity expressed as IC5so values representing the extract concentration required to inhibit 50% of cell proliferation were calculated from the dose-response curve obtained using non-linear regression. All experiments were performed in triplicates.

3.9. Statistical Analysis

All statistical analyses were conducted using ANOVA GraphPad Prism software, version 6.0(GraphPad, San Diego, CA, USA). The results were expressed as the mean standard deviation (SD). One-way analysis of variance (ANOVA)was used, followed by Tukey's post hoc test, to determine statistical significance. The Pearson correlation analysis was performed to determine the correlation between extracts cytotoxic activity, total phenolic content, and identified compounds, respectively. A p-value lower than 0.05 was considered statistically significant.

4. Conclusions

To the best of our knowledge, this is the first study aimed at evaluating and comparing the chemical profile, antioxidative and antiproliferative properties of ethanolic extracts obtained from flowers, leaves, bark, and fruit of S. Vulgaris. The performed methods highlighted that S.vulgaris extracts, in particular the ones obtained from flowers and leaves, are valuable sources of compounds with significant antioxidant and cytotoxic potential.

This article is extracted from Molecules 2021, 26, 3104. https://doi.org/10.3390/molecules26113104 https://www.mdpi.com/journal/molecules