Phytochemical Constituents and Biological Activities of Jasonia glutinosa L.: The First Report for the Plant Growing in North Africa

Jasonia glutinosa (rock tea), also known as Chiliadenus glutinosa Cass., is a medicinal plant growing in the Mediterranean Basin. It is used for the treatment of depression, gastrointestinal complaints, infammations, appendicitis, colds


Introduction
Production of reactive oxygen radicals is a physiological process in the human body and is essential for several normal cellular activities, including signal transduction, immunity functions, and normal aging of the cells [1,2].Overproduction of reactive oxygen species (ROS) is considered a pathological pathway associated with the development of several diseases, including cancer, atherosclerosis, neurodegenerative disorders, and liver and kidney malfunctions [3,4].Te environmental pollution, infections, crowdedness, and exhausts of various industries, in addition to certain human lifestyles and diets, are also stress factors afecting the body's soft tissues, including the liver, kidney, and brain [5][6][7].All these diseases are linked to the serious degenerative efect of ROS on the body's macromolecules, such as DNA, nucleic acids, proteins, lipids, and carbohydrates [3,8].Cancers and infectious diseases are serious medical conditions that afect human life and impede the continuous improvement of people's living standards around the world [9,10], and their complications have been linked to oxidative stress and higher production of ROS [11].
Te primary role of vegetables and fruits in the prevention of oxidative stress-related diseases is well understood by the public and the scientifc community.Te role of these natural ingredients in the treatment of liver dysfunction, cardiovascular diseases, central nervous system degeneration, and other soft tissue illnesses, which are initiated, progressed, and exaggerated by ROS, has been reported [12,13].
Te worldwide-spreading family, is one of the largest families of fowering plants.Te family comprises about 1600 genera and 25,000 species, which approximates 10% of the global fora [14].Chiliadenus Cass.has been described in the nineteenth century as a small genus that belongs to the family Asteraceae, and includes only ten species distributed in the Mediterranean and adjacent areas [15][16][17].In Libya, only two species of the genus Chiliadenus have been identifed [18].Tese species were previously classifed as members of the genera Jasonia and Varthemia, but Brullo, classifed them as members of the genus Chiliadenus based on taxonomic characteristics [19].Jasonia glutinosa or Chiliadenus glutinosa Cass.commonly known as rock tea, is a medicinal plant present in the Mediterranean Basin.It is considered an aromatic plant that grows in rocky crevices and limestone landings with an altitudinal range of 250 to 1800 m.Tey bloom in the summer, from July to September [20].
Te phytochemical examination of Jasonia has led to the isolation and identifcation of numerous bioactive phytoconstituents of diverse chemical classes, e.g., sesquiterpenes, favonoids, monoterpenes, and diterpenes [15].Te simultaneous estimation of C. glutinosa revealed the presence of sesquiterpene, lucinone and glutinone [21,22], and sesquiterpene alcohol (eudesmane alcohol) [23].Several phenolics and favonoids have also been detected in the aerial parts of the plant [24,25].Other research showed that the most abundant phenolic acid was dicafeoylquinic acid [26].In addition, favonoid methyl derivatives, e.g., patuletin, quercetin glucopyranosides, and kaempferol glucuronopyranosides, were also identifed in the plan [24,25].In addition, C. glutinosa is an aromatic plant, and its essential constituents have been identifed from the plant species growing in diferent areas [27,28].Te overall analysis of the essential oil constituents of C. glutinosa indicated that camphor and borneol were the major constituents in the plant [27,28].
In both folk and modern traditional medicine, Jasonia glutinosa is an important species and a popular stomachic herb.Te plant is also used as a component in preparations showing gastrointestinal benefcial efects, e.g., antispasmodic and digestant, and as an anti-infammatory drug [29].Other traditional uses include treating appendicitis, cold, and respiratory diseases, as well as treating depression [20].Several studies on the biological properties of C. glutinosa have been previously published and showed antioxidant [30], anti-infammatory [22], and antimicrobial activities [31].Moreover, the acetone extracts obtained from aerial parts of the plant exerted an antiparasitic efect [32].
Furthermore, ethnobotanical studies have revealed that this plant is not only used where it grows but can also be found in some traditional taverns, restaurants, pharmacies, and herbal remedy shops [33].
Te current study investigated the phytochemical constituents of Jasonia glutinosa by spectrophotometric analysis and the spectroscopic technique, LC-MS.Te biological activities, i.e., antioxidant, cytotoxic, and antimicrobial activities, were also investigated for the plant extract.Te growing area where Jasonia glutinosa was collected is part of what makes our work novel, as there have been no previous studies for the plant species growing in Libya.Te study also emphasized the biological activity and phenolic components of Jasonia glutinosa, the plant that is immensely popular in the North African and Mediterranean countries and has a long history of usage in traditional medicine.

Plant Materials, Collection, Identifcation, and Extraction
Procedure.Te plant was collected in February 2022 from El-Jabal Al Akhdar area in Libya.Te identifcation of the plant sample was precisely confrmed by the herbarium of Benghazi University's Faculty of Science, Botany department, Benghazi, Libya.250 gm of the plant was chipped into small pieces, homogenized in a mixer, and extracted with methanol 70% using Soxhlet apparatus until complete exhaustion.Te obtained extract was concentrated by removing the solvent under vacuum using a rotary evaporator.Te resulting residues were weighed and kept in desiccators.

Total Phenolic and Total Flavonoid Contents.
Te total contents of phenols (TPC) were estimated using Folin-Ciocalteu reagent and by using a UV-vis spectrophotometer, according to the method of Attard 2013 [34].Te technique involved mixing 10 μL of the sample/standard with 100 μL of the Folin-Ciocalteu reagent previously diluted 1 : 10 with distilled water and 80 μL of 1M Na 2 CO 3 in a 96-well microplate.Te plate was incubated at room temperature in the dark for 20 minutes.Te intensity of the resulted blue color was measured at 630 nm against a blank consists of all ingredients except the sample.Te standard calibration curve was plotted using the absorbance of various concentrations of gallic acid.Te results were calculated from triplicate measurements and expressed as mg gallic acid equivalents/g of the weight of the dry extract (mg GAE/ g) [35].
Te total favonoids content was evaluated in microplates by conducting the aluminum chloride reagent method as described by Kiranmai et al. [36].Te analysis was conducted on a Beckman DU-650 spectrophotometer in a 96-well microplate, whereas 15 μL of the sample/standard was mixed with 175 μL of methanol, 30 μL of 1.25% AlCl 3 , and 30 μL of 0.125 M C 2 H 3 NaO 2 and incubated for 5 minutes.Te color was measured at 420 nm against a blank methanol solution.Te average of absorbance values of rutin at diferent concentrations (5,10,20,40,80,100,120, and 160 μg/mL in methanol) were employed to plot the calibration curve.Te TFC in the extract was expressed as milligrams of the standard rutin equivalent per gram of the dried extract (mg RE/g).

LC-MS Analysis.
All solvent used in the LC-MS analysis were of analytical grade.Shimadzu ExionLC (Shimadzu, Kyoto, Japan) equipped with a TurboIonSpray, SCIEX X500 R QTOF (SCIEX, Framingham, MA, USA) was used for the extract scanning.Accurately, 1 mg of the extract was dissolved in 2 ml of DMSO and centrifuged at 5000 rpm for 2.0 min.Accurately, 1.0 ml of the clear solution was transferred to the autosampler, and the injection volume was adjusted to 3.0 μl.Te instrument was operated using ion source gas 1 (psi): 50 and ion source gas 2 (psi): 50, and ion funnel electrospray source.Te instrument parameters were adjusted as follows: capillary voltage (negative, −4000 V), nebulizer gas (2.0 bar), nitrogen fow (8 L/min), and dry temperature (200 °C).Te mass accuracy was <1 ppm, the mass resolution was 50,000 FSR (full sensitivity resolution), and the TOF repetition rate was up to 20 kHz.Te chromatographic separation was performed on C18 reversephase (RP) column, 100 × 2.1 mm, 3.0 μm from GL-Science (Japan) at 50 °C, autosampler temperature 8.0 °C, with a fow rate of 0.35 mL/min, and total run time of 40 min using the gradient elution.Te eluents consisted of as follows: mobile phase A: 0.1% formic acid in water and mobile phase B: 100% acetonitrile.

ABTS.
Te ABTS radical cation decolorization analysis was employed to investigate the free radical scavenging capacity of the extracts.Te analysis was performed in microplates following the technique outlined by Arnao et al [37], with little modifcations.A 192 mg of ABTS were dissolved in deionized water and transferred to a 50 mL volumetric fask, and the fnal volume was made with the addition of distilled water.Accurately, 1 mL of the prior solution was mixed with 17 μL of 140 mM potassium persulphate and stored for 24 hours in a dark place.Te reaction mixture was then diluted to 50 mL using methanol to attain the fnal ABTS dilution utilized in the experiment.In a 96well plate (n = 6), 190 μL of freshly produced ABTS reagent was combined with 10 μL of the sample/standard, and the reaction was placed in an incubator for 30 minutes in a dark chamber with temperature control set at room temperature (22 °C).At the end of the incubation time, the drop in the intensity of ABTS color was measured at 734 nm.Te results were recorded using FluoStar Omega microplate reader (BMG Labtech, Ortenberg, Germany).

ORAC.
Te oxygen radical absorbance capacity (ORAC) assay was performed using the technique of Liang et al. [38], with little modifcations; 10 μL of the prepared sample was incubated with 30 μL fuorescein (100 nM) for 10 minutes at 37 °C.For background detection, the measurements of fuorescence (485 EX, 520 EM, nm) were conducted for three rounds (90 sec each).Following that, 70 μL of newly prepared 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) (300 mM) was immediately added to each well.Te temperatures must be set at some higher point to ensure that azide decomposition temperature in the wells is reached.Te measurements of forescent (485 EX, 520 EM, nm) was proceeded for 60 min (40 cycles, each 90 sec) by using FluoStar Omega microplate reader (BMG Labtech, Ortenberg, Germany).

FRAP.
Te test was carried out in microplates according to the technique described by Benzie and Strain with little modifcations [39].A newly produced TPTZ reagent (300 mM acetate bufer (PH 3.6), 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl3, in a ratio of 10 : 1:1 v/v/v, respectively).In a 96-well plate, 190 μL of freshly made TPTZ reagent were combined with 10 μL of sample, and the reactions were kept at ambient temperature for 30 minutes in a dark chamber.At the end of the incubation time, the resultant blue color was detected at 593 nm by FluoStar Omega microplate reader equipment (BMG Labtech, Ortenberg, Germany), and the activity of the extract was calculated as mg Trolox equivalent per gram of the plant's dried extract using the FRAP-Trolox calibration curve.

Antiproliferative Assay. Te sulforhodamine B (SRB)
assay was applied to assess cell viability in the presence of J. glutinosa.Tree cell lines were chosen for the cytotoxic evaluation of the plant extract, i.e., breast adenocarcinoma (MCF-7), hepatocellular carcinoma (HepG2), and pancreatic cancer (PANC-1) cell lines.Aliquots of 100 μL suspension of the cells (5 × 10 3 cells) were seeded into 96-well plates and kept incubated for 24 hours.Another aliquot of 100 μL media contaning J. glutinosa extract at various concentrations (from 0.001 to 100 μg/mL) was delivered to the cells.Following 72 h of cells exposure to the J. glutinosa extract, cells were maintained by substituting the media with 150 μL of 10% trichloroacetic acid (TCA) and kept in an incubator at 4 °C for one hour.After discarding the TCA solution, the cells were rinsed fve times with deionized water.Aliquots of 70 μL SRB solution (0.4% w/v) were inserted and allowed to stand at ambient temperature for 10 min in a dark chamber.Ten, 1% acetic acid was used to wash the plates.After that, 150 μL of TRIS (10 mM) was applied to dissolve the protein-bound SRB stain.BMG LABTECH ® -FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany) was used to measure the absorbance at 540 nm [40,41].
2.6.Antimicrobial Assay.Te antibacterial activity was assessed using a modifed Kirby-Bauer disk difusion susceptibility test procedure [42].Paper disks containing the methanolic plant extract (50 μl of J. glutinosa at 100 mg/mL) were prepared and attached on the surface of agar plates inoculated with the bacteria (Escherichia coli ATCC 8739, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 29213, Salmonella typhimurium ATCC 14028, Candida albicans ATCC 10231, and Saccharomyces cerevisiae ATCC 9763).Te negative control was made of the same volume of DMSO, while the positive control was standard disks of antibacterial agents.Te plates were kept inverted in the incubator for one day at 37 °C.After incubation, the plates were inspected to measure the zones of inhibition.Diameters less than 5 mm were considered as no efect.Te experiment was carried out three times.

Statistical Analysis.
Results were presented as means ± standard deviation (SD).Statistical analysis was conducted by applying Student's t-test and P values less than 0.05 was recorded as signifcant.Correlations between acquired data were analyzed using the correlation coefcient statistical tool in the MS Excel software.

Spectrophotometric Analysis of Plant Constituents.
Te total contents of phenolics and favonoids were measured quantitatively as gallic acid (GAE) and rutin (RE) equivalents per gram of the plant extract.Te results (Table 1) revealed the presence of 14.67 ± 0.67 GAE and 46.42 ± 3.12 RE of the phenolics and favonoids, respectively, in mg/g of the extract.Environmental conditions and soil content have been reported as intrinsic factors afecting plant constituents [43,44].Te total phenolics and favonoids of J. glutinosa growing in other areas have been reported.For instance, the phenolic contents have been measured for the commercial plant samples collected from local markets in Spain [26].Te methanol and water extracts of the plant have been shown to contain signifcantly higher levels of the phenolics compared to the current result, with 155.2 and 163.3 mg GAE/g of the extracts, respectively [26].Te large diference in phenolic content between Libyan and Spanish growing species could be attributed to the efects of environmental conditions and soil contents on the levels of the plant's phenolic constituents.However, the higher signifcant diferences in phenolic contents between Libyan and Spanish species of the plant could be attributed in part to variations and conditions of the phenolic determination protocols, as well as the presence of other constituents other than phenolics capable of reducing the Folin-Ciocalteu reagent, such as reducing sugars and ascorbic acid, as previously reported [45].

Spectroscopic Analysis of Plant Constituents.
Te individual phenolics and favonoids in the Jasonia glutinosa extract were tentatively identifed by the LC-MS spectroscopic analysis.Te molecular ions and related mass fragments of the identifed compounds were detected in the negative and positive mass ion modes of analysis.Out of dozens of peaks in both positive and negative chromatograms, only thirty compounds related to the phenolic acid and favonoid classes were tentatively identifed (Table 2).Te m/z of the molecular ions in the negative modes along with the m/z for the mass fragments and molecular formula of the identifed compounds were all represented in Table 2 and arranged according to their retention times.Te total relative percentages of the identifed compounds were calculated at 18.69% based on individual percentage of each identifed peak in relation to the total peaks in the negative mode chromatogram.Te identifcation of the compounds was established based on the mass fragments and molecular ion peaks of each compound in relation to the literature and also on the available literature of the phenolic and favonoid constituents of the plant.Te mass fragments of the sugar moieties and aglycones have also been used in the confrmation of the compound's tentative identity (Figure 1).For example, the presence of the aglycone fragment after the subtraction of 162 atomic mass unit (amu) indicated the hexoside nature of the sugar [60].However, the presence of the fragment at m/z 191 amu indicates the removal of the quinic acid from its glycosidal forms.Te quinic acid as a cyclitois sugar moiety has been identifed in four compounds (3-cafeoylquinic acid (1), 1-cafeoylquinic acid (8), 1,3-dicafeoylquinic acid (9), and 1,5-O-dicafeoylquinic acid ( 10)) due to the presence of its m/ z mass fragment unit at 191 in the negative mode spectra of these compounds.Furthermore, the aglycone part in these four compounds, 1, 8, 9, and 10, was identifed due to the presence of a fragment at 179 amu assigned for the cafeic acid in the negative ion mode fragment spectra.Te sugar moiety, glucuronide, was detected in two of the identifed compounds, i.e., quercetin-3-O-glucuronide (5) and mearnsetin-O-glucuronide (7), by the presence of the aglycones mass unites at m/z 301 and 331, indicating the loss of 176 amu from the molecular masses [M-H] -at m/z 477 and 507, respectively.In addition, a loss of 162 amu was assigned for the removal of hexose units, which are mainly considered as glucosides.Losses of 162 amu were found in the mass fragmentation spectra of quercetin-3-glucoside ( 4 ) were marked by the removal of 204 amu from both compounds, which were assigned for the removal of hexosyl and acetyl residues.
Te overall analysis of the LC-MS spectral results confrmed the presence of phenolics and favonoids in considerable amounts in the plant extract and revealed the 4 Journal of Chemistry presence of 12.83% of the phenolic acid derivatives, including 3.82% of the phenolic glycosides.Te analysis also revealed the presence of 5.86% favonoids, of which 1.67% were glycosylated favonoids, 4.19% were favonoid aglycones, 3.11% were methoxylated favonoids, and 0.44% were isofavonoids.Te analysis also revealed the dominance of the cinnamic acid derivatives, including the cafeoylquinic acids and the methoxylated favonoids, which is consistent with the reported LC-MS analysis for the plant species growing in Spain [26].6 Journal of Chemistry 3.3.Antioxidant Activity of J. glutinosa.Te antioxidant activity of J. glutinosa has been reported for plant species growing in diferent locations other than Libya [20,26].Te current results also revealed the remarked antioxidant activity for the plant, which was assessed by three diferent assays, i.e., ABTS, ORAC, and FRAP (Table 1).Tese methods are common in vitro assays used to measure the free radical scavenging (ABTS and ORAC) and reducing power (FRAP) of plant extracts and biological samples.Te current results are consistent with the fndings of the spectrophotometric (TPC and TFC) and spectroscopical analysis (LC-MS) of the plant ethanolic extract, which revealed the presence of a considerable amount of the phenolic constituents measured at 18.69% of the total plant constituents that appeared in the LC-MS chromatogram.In addition, the antioxidant efect of polyphenols specially, favonoids are well-known in literature [61,62].Te results showed 265.55, 513.32, and 27.10 μM Trolox eq/mg of extract in the ABTS, ORAC, and FRAP assays, respectively (Table 1).Compared to the reported antioxidant activity of the plant, our results demonstrated signifcantly higher activity compared to the plant species growing in Spain, which has been shown to have an ABTS power of 15.3 μM Trolox eq/100 g of the plant extract [26].Furthermore, the same species inhibited the DPPH free radical with 17.4 μM Trolox eq/mg extract [26].Te ORAC and FRAP activities of the plant extract have been reported for the species growing in Spain and showed 2.72 μM Trolox eq/mg extract and 52.09 μmol Fe 2+ /g of extract, respectively [63].Tese reported results also revealed the higher antioxidant content of the Libyan species, which exhibited 188x activity in the ORAC compared to the result reported for the Spanish species [63].To the best of our knowledge, the current report is the frst to discuss the antioxidant activity of the plant, J. glutinosa, from the African northern species.Te results, when compared to the available reported of  antioxidant activity, also revealed the efect of environmental conditions on the plant constituents and their biological activities.In addition, the antioxidant activity of the plant is refecting its beneft and safety for the human use as part of the traditional medicine system in the north Africa.
3.4.Antiproliferative Activity of J. glutinosa.Table 3 shows the antiproliferative activity of J. glutinosa extract, which demonstrated weak cytotoxic activity, with cell growth inhibition of 9.23, 11.42, and 34.01%at a concentration of 100 g/mL against tested cancer cells, namely MCF-7, HepG2, and PANC-1, when compared to the standard anticancer agent, DOX.
Te current study's fndings for J. glutinosa extract could be a sign of the safety of the plant for mammalian cells and be consistent with previous fndings for the plant, which showed no cytotoxicity against the mouse embryonic fbroblast cell line, 3T3-L1 cells, until the concentration of 200 μg/mL; however, the plant extract showed benefcial efects in the prevention of obesity, diabetes, and related metabolic complications [63].A confict between the current results of the week's cytotoxic efect and the presence of several constituents known for their cytotoxic and anticancer activities is a point of contention that needs to be explained.For instance, quercetin, taxifolin, luteolin, chrysin, kaempferol, and their glycosides, which were detected in the plant extract by the LC-MS analysis, have been reported for their cytotoxic activities [64][65][66].In addition, cafeoylquinic acid derivatives, which were represented in the plant extract at ≈ 3.6%, are also responsible for the remarkable antiproliferative activity in plants [67].Te presence of favonoids and phenolic mixture might be the reason for the weak cytotoxic activity of the plant due to some mechanistic interference, which needs further studies to be clearly demonstrated.Te results also refect the safety of the plant, as it is consumed as food and in traditional medicine.
3.5.Antimicrobial Activity of J. glutinosa.Te antimicrobial test of J. glutinosa has been conducted against six microorganisms using the common assay, the agar well difusion method.In the test, two Gram negative bacteria, E. coli, Salmonella typhimurium; two Gram positive bacteria, Bacillus subtilis, Staphylococcus aureus; and two yeasts, Candida albicans and Saccharomyces cerevisiae, were used to provide ideas about the antimicrobial activity of J. glutinosa.Te results clearly demonstrated no antimicrobial activity for the plant extract against all mentioned microorganisms at the tested dose of 100 mg/mL (Figure 2).Te plant, J. glutinosa, has been listed as one of the plants employed in the Mediterranean area as an antimicrobial agent, particularly for intestinal infection [68].However, other report has indicated very weak antimicrobial activity for J. glutinosa.For instance, diferent types of extracts obtained from the plant exhibited very weak antifungal activity against Rhizopus stolonifer, whereas dichloromethane extract showed a minimum inhibitory concentration above 1000 μg/mL.However, ethyl acetate and methanol extracts have been shown to have no activity against Rhizopus stolonifer [30].Te current result for the antimicrobial activity of the plant did not support the claim of traditional use of the plant as antimicrobial agent [69].

Conclusion
Te phytochemical and biological investigations of J. glutinosa growing in Libya were reported here for the frst time.Te fndings support the former reports, which confrm the presence and abundance of phenolics and favonoids in the plant.Te results also demonstrated considerable antioxidant activity and weak cytotoxic efect of the plant extract, which might refect the safety of the plant, as it is consumed as food and is used in traditional medicine for curing several ailments.Te results also supported the previous fndings for the safety of the plant towards normal cells and its benefcial efects in the prevention of obesity, diabetes, and related metabolic complications.According to current results, the plant could be potential source for the phenolics and favonoids with antioxidant activity.Te fndings of the antimicrobial assay for the plant extract showed no action, contradicting the assertion that it has historically been used as an antibacterial agent.However, the antimicrobial assay of the plant should be repeated against a wide variety of microorganisms and at a higher concentration of the plant extract than that used in the current study.Te plant's nonpolar constituents may be more efective as antibacterial agents; hence, nonpolar extracts of the plant should also be investigated, which is part of our future plan.

Table 1 :
Quantitative measurements of the phenolics, favonoids, and antioxidant activity of Jasonia glutinosa.