HESI-MS/MS Analysis of Phenolic Compounds from Calendula aegyptiaca Fruits Extracts and Evaluation of Their Antioxidant Activities

Considering medicinal plants as an inexhaustible source of active ingredients that may be easily isolated using simple and inexpensive techniques, phytotherapy is becoming increasingly popular. Various experimental approaches and analytical methods have been used to demonstrate that the genus Calendula (Asteraceae) has a particular richness in active ingredients, especially phenolic compounds, which justifies the growing interest in scientific studies on this genus’ species. From a chemical and biological viewpoint, Calendula aegyptiaca is a little-studied plant. For the first time, high-performance liquid chromatography combined with negative electrospray ionization mass spectrometry (HPLC-HESI-MS) was used to analyze methanolic extracts of Calendula aegyptiaca (C. aegyptiaca) fruits. Thirty-five molecules were identified. Flavonoids (47.87%), phenolic acids (5.18%), and saponins (6.47%) formed the majority of these chemicals. Rutin, caffeic acid hexoside, and Soyasaponin βg’ were the most abundant molecules in the fruit methanolic extract, accounting for 17.49% of total flavonoids, 2.32 % of total phenolic acids, and 0.95% of total saponins, respectively. The antioxidant activity of the fruit extracts of C. aegyptiaca was investigated using FRAP, TAC, and DPPH as well as flavonoids and total phenols content. Because the phenolic components were more extractable using polar solvents, the antioxidant activity of the methanolic extract was found to be higher than that of the dichloromethane and hexane extracts. The IC50 value for DPPH of methanolic extract was found to be 0.041 mg·mL−1. Our findings showed that C. aegyptiaca is an important source of physiologically active compounds.


Introduction
Medicinal plants have a variety of biological and therapeutic properties that are helpful to one's health and effective in the treatment of a variety of disorders [1]. In both a therapeutic and preventive context, these natural resources have the potential to be a viable alternative to synthetic medications. Tunisia has the most diverse flora in North Africa. This wealth is due to the geographic and climatic changes observed from north to south of the country. Nonetheless, from a phytochemical and biological point of view, this floristic fortune has only briefly been examined. The Asteraceae are angiosperms' most important family, with around 25,000 species grouped into 1600 genus [2]. Calendula is the most well known of the Asteraceae family, with roughly 25 species (C. officinalis and C. arvensis . . . ). From an economic and medicinal standpoint, this genus is extremely

Total Phenolics and Flavonoids Contents of Various Extracts from C. aegyptiaca Fruits
Two families of chemicals were detected in all extracts: phenolic acids and flavonoids. Total phenolics and flavonoids contents of C. aegyptiaca fruits extracts, expressed in mg of gallic acid equivalent per g of dried extract (mg GAE/g DE) and mg of quercetin equivalent per g of dried extract (mg QE/g DE), respectively, are summarized in Table 2. The MeOH extract had a higher concentration of phenolic acids (275.38 mg GAE/g DE) and flavonoids (204.57 mg QE/g DE) than the DCM and hexane extracts (p 0.05). There was no information on the total phenol and flavonoid contents of Calendula species fruits in previous studies. In comparison to C. arvensis flowers, total phenols and flavonoids contents of dried MeOH extract did not exceed 118.18 mg GAE/g and 74.14 mg QE/g, respectively [5]. This result indicates the richness of C. aegyptiaca fruits in phenols, mainly flavonoids.

Phytochemical Constituents
LC-MS/MS was used to describe and characterize the major metabolites found in C. aegyptiaca's methanolic extract of fruits. Figure 1 depicts the total ion mass chromatogram profile of this extract. Table 3 shows the MS/MS data of the substances which were tentatively identified. QE/g, respectively [5]. This result indicates the richness of C. aegyptiaca fruits in phenols, mainly flavonoids.

Phytochemical Constituents
LC-MS/MS was used to describe and characterize the major metabolites found in C. aegyptiaca's methanolic extract of fruits. Figure 1 depicts the total ion mass chromatogram profile of this extract. Table 3 shows the MS/MS data of the substances which were tentatively identified.

Antioxidant Activity of Fruit Extracts In Vitro
The total antioxidant capacity (TAC) of the extracts (Table 4) was calculated using the phosphomolybdenum method. By forming a green phosphomolybdenum complex (V) with a maximum absorbance at 695 nm, the antioxidant compounds converted Mo(VI) to Mo(V). MeOH extract had the highest antioxidant capacity (253.394 mg gallic acid equivalents (GAE/g extract), followed by DCM (181.414 mg GAE/g extract) and n-hexane (123.771 mg GAE/g extract) extracts, which could be explained by its high levels of total phenolic acids and flavonoids contents ( Table 2).
The DPPH scavenging activity of phenols and flavonoids was also investigated ( Table 4). When compared to DCM and n-hexane extracts (IC 50 = 0.050 mg·mL −1 and IC 50 = 0.054 mg·mL −1 ), which presented a moderate and low significance, respectively, the MeOH extract of C. aegyptiaca had significantly higher DPPH scavenging activity (IC 50 = 0.041 mg·mL −1 ). Our findings suggest that hydroxyl groups could intervene as electron donors, transforming free radicals into much more stable substances by scavenging radicals. According to the literature [34], the methanolic extract of this plant had higher DPPH scavenging activity (IC 50 = 0.041 mg·mL −1 ) than the hydro-methanol extract of C. officinalis leaves (0.57 mg·mL −1 ) and lower than that of flowers (0.35 mg·mL −1 ).
The Ferric reducing activity power (FRAP) method is based on electron-donating antioxidants reducing the Fe 3+ tripyridyltriazine complex (colorless complex) to Fe 2+tripyridyltriazine (blue complex) at low pH. The reducing power of extracts and vitamin C was determined (Figure 3). The FRAP test revealed an increase in absorbance with increasing doses of the tested extracts, which corresponded to an increase in reducing power. The obtained results revealed that the extracts' reducing power increased in direct proportion to their concentration. Because of its highest levels of phenolic and flavonoid content (Table 2), MeOH extract had the highest reducing power (p < 0.05), followed by DCM and n-hexane extracts.

Plant Material
C. aegyptiaca fruits were collected from Sfax south Tunisia in March 2020, placed in the shade in a well-ventilated area with low humidity (22-25%) at a temperature range of 18-25 °C for 21 days, and then crushed. The plant was recognized by Pr. Mohamed Chaieb [35], Biology Department Faculty of Sciences of Sfax, and a voucher specimen (LCSN150) Figure 3. Ferric reducing antioxidant power (FRAP) assays (n = 3) compared to vitamin C as standard; the differences were analyzed using Duncan and Tukey's post hoc test for multiple comparisons with p < 0.05.

Correlations
To evaluate the influence of phytochemical constituents on antioxidant capacity, the correlations between the phenolics and flavonoids contents and antioxidant activity of extracts were measured. Table 5

Plant Material
C. aegyptiaca fruits were collected from Sfax south Tunisia in March 2020, placed in the shade in a well-ventilated area with low humidity (22-25%) at a temperature range of 18-25 • C for 21 days, and then crushed. The plant was recognized by Pr. Mohamed Chaieb [35], Biology Department Faculty of Sciences of Sfax, and a voucher specimen (LCSN150) was stored at the herbarium of the Laboratory of Organic Chemistry (LR17-ES08), Faculty of Sciences, University of Sfax, Tunisia.

Extraction
The dried fruits were crushed in a grinder from Fritsch Company (reference 14.3000.00) in order to obtain much finer particles (2, 3 mm) and then stored in airtight jars away from humidity at room temperature. The moisture content of fruits was evaluated to be 19.71%. The obtained powder was extracted successively with organic solvents of increasing polarities (n-hexane, dichloromethane and methanol) with mechanical stirring (plant material/solvent ratio 1:8 (w/v)). Each extraction was carried out three times at room temperature and for 24 h each time. The macerates were then filtered and evaporated under vacuum to concentrate the extracts. The evaporation process resulted in crude extracts that had no moisture content.

Determination of Phenolic Content
The spectrophotometric method was used to determine the total phenol content (TPC) [36]. A total of 0.5 mL of Folin-Ciocalteu reagent was added to a solution containing 1 mL of a known concentration extract (1 mg·mL −1 ) and 3 mL of distilled water. After 5 min, 0.5 mL of 2% aqueous sodium carbonate (Na 2 CO 3 ) was added. After 90 min of incubation at 25 • C, the absorbance at 760 nm was measured. The test was carried out three times. A standard gallic acid graph was used to calculate TPC, which was expressed in milligrams of gallic acid equivalent per gram of dry weight of extract.

Determination of Flavonoid Content
The method established by Heimler et al. [37] was used to determine total flavonoid content (TFC). The approach is based on the creation of a very stable combination between aluminum chloride and the oxygen atoms found on the flavonoids' carbons 4 and 5, with a maximum absorbance of 430 nm. The calibration curve was generated using quercetin (commercial, Sigma-Aldrich, St. Louis, MO, USA). An amount of 1 mL of 2% aluminum trichloride (AlCl 3 ) was blended with 1 mL of sample (1 mg·mL −1 ). The absorbance of the mixture was measured at 430 nm with a spectrophotometer after 15 min of incubation at room temperature. TFC was measured in milligrams of quercetin equivalent (QE) per gram of extract. The experiment was repeated three times.

Free Radical Scavenging Activity
The DPPH test was used to assess the extracts' capacity to scavenge free radicals, as described earlier [38]. DPPH radicals were absorbed at 517 nm; however, absorbance dropped when they were reduced by an antioxidant agent. The decrease in absorbance at 515 nm was measured using UV spectrometry. For concentrations of 0.063, 0.125, 0.25, 0.5, and 1 mg·mL −1 of plant extract, vitamin C was employed as a positive control, and all tests were carried out three times. For the assay, different concentrations were used. A total of 2 mL of the DPPH solution and 2 mL of the sample were mixed and left to react in the dark at 37 • C for 30 min as well as a blank test. The results of radical scavenging tests were expressed as 50% inhibition concentration (IC 50 ).

Total Antioxidant Capacity
Total antioxidant capacity of the extracts was assessed using the method of phosphomolybdenum complex formation [39]. The reduction of ammonium molybdate and the transmission of electrons are the basis of this approach. A green ammonium phosphate/molybdate complex formed during the process. In total, 1 mL of the reagent solution (sodium phosphate, sulfuric acid, and ammonium molybdate) was combined with 0.1 mL of the sample. The mixes were then incubated for 1 h 30 min in boiling water (95 • C). After the samples cooled, the absorbance was determined at 695 nm. The total antioxidant capacity was expressed as mg of gallic acid equivalents per g of extract. The test was performed in triplicate.

Reducing Power Assay
The procedure used was that of Barros et al. [40]. At various concentrations, 1 mL of each sample was treated with a mixture of potassium ferricyanide (1%) and sodium phosphate (0.2 M). The mixtures were incubated at 50 • C for 20 min. The trichloroacetic acid was then added, and the mixture was placed in the centrifuge for 10 min. After recovery, the supernatant of each mixture was mixed with the ferric chloride solution 0.1% in 2.5 mL of distilled water. Every test was performed three times.

LC-HESI-MS
Fruit methanolic extract of C. aegyptiaca was investigated using a Thermo Scientific LTQ XL Mass Spectrometer fitted with a hot electrospray ionization source in the negative mode. Thermo Xcalibur software was used to record ion spectra. A C 18 reversed phase Luna column at 30 • C (5 µm, 150 mm × 2.1 mm) was delivered to Vanquish HPLC (Thermo Scientific Inc., Waltham, MA, USA) for analysis. A: 0.1% formic acid in water (5% ACN), v/v and B: 0.1% formic acid in acetonitrile, v/v, were the selected solvents. The elution gradient was set from 0 to 40% of B during 40 min, 100% B after 50 min, and the column was re-equilibrated between individual runs. The mobile phase had a flow rate of 0.2 mL·min −1 , and the injection volume was 20 µL. The ion spray voltage was fixed at 3.5 V, the ESI source and the capillary temperature was calibrated at 300 • C, and the sheath and auxiliary gas pressures were set to 50 and 5 psi, respectively. The spectral range was from m/z 50 to 1200. The approach combined full scans and MS/MS experiments using a collision energy ranging from 10 to 35 eV, depending on the molecular mass of compounds.

Statistical Analysis
A one-way ANOVA was used to assess statistical significance followed by Tukey's post hoc test for multiple comparisons with p = 0.05 and correlation coefficients (r). The Statistical Product and Service Solutions application (SPSS) version 20 was used to conduct these analyses.

Conclusions
The HPLC-HESI-MS n method was effectively established in this study for the quick separation and identification of various chemicals in the methanol extract of C. aegyptiaca fruits. Thirty-five chemicals were identified: six phenolic acids (compounds 1-4, 14, and 16), then flavonoids including apigenin derivatives (compounds 6 and 18), quercetin derivatives (compounds 7-9, 11, and 19) and isorhamnetin derivatives (compounds 10, 12, and 17), four fatty acids (compounds 24, 41, 46, and 50), and fifteen saponins. Oleanolic acid derivatives and hedragenin derivatives were the most commonly reported saponins. As far as we know, compounds 34-36, 44, and 45 were described for the first time for this species in this paper. Oleanolic acid saponins are known to have anti-inflammatory, anticancer, antihepatotoxic, antidiabetic, and cytotoxic properties. MeOH extract had the highest total phenolic content, as well as the highest total flavonoid contents (275.38 ± 0.39mg GAE/g DE and 204.57 ± 4.101 mg QE/g DE, respectively). These findings imply that phenolic acids (particularly caffeic acid, which accounts for 2.32%) and flavonoids (rutin 17.57%, quercetin-3,4 -di-O-glucoside 8.8%, quercetin-3-O-glucoside 7.57%) could be responsible for this plant's antioxidant properties. As a result, fruits of C. aegyptiaca should be thought of as a novel source of bioactive compounds with potential applications in a variety of fields. However, more research is required to investigate additional biological activities.