Journal of Essential Oil Bearing

ISSN: 0972-060X (Print) 0976-5026 (Online) Journal homepage: https://www.tandfonline.com/loi/teop20

Chemical Composition and Biological Activity of Capetula and Shoots Essential Oils of glaucus L.

Taha Ramadan, Ahmed Zaher, Ahmed Amro & Raoof Sultan

To cite this article: Taha Ramadan, Ahmed Zaher, Ahmed Amro & Raoof Sultan (2020) Chemical Composition and Biological Activity of Capetula and Shoots Essential Oils of Senecio￿glaucus L., Journal of Essential Oil Bearing Plants, 23:1, 168-183, DOI: 10.1080/0972060X.2020.1742797 To link to this article: https://doi.org/10.1080/0972060X.2020.1742797

Published online: 26 Mar 2020.

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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=teop20 TEOP 23 (1) 2020 pp 168 - 183 168 ISSN Print: 0972-060X ISSN Online: 0976-5026

Chemical Composition and Biological Activity of Capetula and Shoots Essential Oils of L.

Taha Ramadan 1*, Ahmed Zaher 2, Ahmed Amro 1, Raoof Sultan 1,3

1 Department of Botany and Microbiology, Faculty of Science, Assiut University, Assiut 71516, Egypt 2 Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71516, Egypt 3 Biology Department, Faculty of Applied Science, Taiz University, Taiz, Yemen Received 01 February 2020; accepted in revised form 07 March 2020

Abstract: The chemical composition, antimicrobial, anti-inflammatory, antioxidant and cytotoxic activities of the extracted essential oils (EOs) of capetula and shoots of Senecio glaucus L. were determined. The main constituents are m-mentha-1(7),8-diene (31.4 % and 25.6 % of total capetula and shoot oils, respectively), cis-m-mentha-2,8-diene (22.9 % and 8.2 %), dehydrofukinone (17.2 % and 19.9 %), α-terpinolene (3.9 % and 3.1 %) and 2,5-cyclohexadiene-1,4-dione,2-(1,1- dimethylethyl)-5-(2-methyl-2-propen-1-yl)- (3.9 % and 3.4 %). Sabinene constitutes about 2.7 % of the capetula EO, but not detected in shoot oil. α-Fenchene and 1,3,8-p-menthatriene represented by 5.6 % and 5.3 % respectively, in the shoot EO, but not detected in capetula oil. More than half of the oils was monoterpenes, followed by sesquiterpenes. Both EOs differentially exerted antimicrobial effects on Gram-positive and Gram-negative bacteria, yeasts and molds. Capetula oil showed anti-inflammation effect higher than the shoots oil. The EOs showed strong antioxidant effect with μ EC50 values for scavenging DPPH radical of 1.6 and 1.9 l/ ml for capetula and shoot EOs, respectively, compared to 16 μg/ ml for ascorbic acid. So, the capetula EO was more potent against human breast cancer cells than shoots EO.

Key words: Anti-inflammation; antimicrobial; antioxidant; cytotoxicity; essential oils; Senecio glaucus.

Introduction tries due to their antimicrobial, antioxidant and The number of described structures of second- other biological properties 2,3. The genus Senecio ary metabolites exceeds 100,000 from phy- (family ) is widely distributed and com- tochemistry investigation of only 20-30 % of prises nearly 3000 species 4 of which about six plants 1. By more investigation of plants and us- occur in Egypt including S. flavus, S. glaucus, S. ing more advanced methods and analytical instru- vulgaris, S. aegyptius, S. belbeysius and S. ments, the number is going to be much higher. hoggariensis 5,6. The importance of this genus is Essential, or volatile, oils as secondary metabo- based on its botanical, pharmacological and toxi- lites from plants have been widely used in phar- cological properties 4,7. maceutical, agricultural, cosmetic and food indus- The genus Senecio have been used in folk medi-

*Corresponding author (Taha Ramadan) E-mail: < [email protected]; [email protected] > © 2020, Har Krishan Bhalla & Sons Taha Ramadan et al., / TEOP 23 (1) 2020 168 - 183 169 cine for the treatment of various ailments particu- but also provide a way for plants to call for de- larly treatment of dysentery, conjunctivitis, infec- fensive help from other organisms. tions, rheumatism, cancer, cough suppressant, Analysis of essential oils of some Senecio spe- asthma, bronchitis, eczema, inflammation and cies showed the presence of α-thujone, β-caryo- muscular pain 8,9. In Sinai, species of Senecio that phyllene, germacrene D, α-phellandrene, spathu- inhabit desert wadies and sandy plains are used lenol, ocimene, α-pinene, farnesol, p-cymene, as sedative of central nervous system, emetic and myrcene, dehydrofukinone and sabinene as ma- diuretic 10. Some species, that are common in jor constituents along with some other mono and many regions, have been used for the treatment sesquiterpenoides 19,51-56. Furthermore, biological of wounds and as antiemetic, anti-inflammatory activities such as antibacterial or antimicrobial and vasodilatory preparations 11. 18,19,23 and cytotoxic activities 47 have been reported Previous works have been done on the chemi- for few species. The essential oil of S. graveolens cal composition of the essential oils of some Sene- has antibacterial effect on Micrococcus luteus, and cio species such as: S. trapezuntinus 12, S. platy- Staphylococcus aureus, as well as antifungal ef- phyllus var. platyphyllus 13, S. vernalis 4,13,14, S. fects on Candida albicans 19. The essential oil of glaucus subsp. coronopifolius 15, S. leucostachys Senecio longipenicillatus has a strong antibacte- 16, S. squalidus 17, S. aegyptius var.discoideus 18, rial activity against Staphylococcus aureus and S. graveolens 19,20, S. farfarifolius 21, S. nutans and Enterococcus faecalis 23. S. viridis 22, S. longipenicillatus 23, S. graciliflorus Despite Senecio is one of the largest genera in 24,25, S. pogonias and S. oreophyton 26, S. family Asteraceae, the studied species for essen- anteuphorbium 27, S. mikanioides 28, S. scandens tial oils constituents are still few and not more 29, S. vulgaris 30, S. laetus 31, S. pterophorus 32, S. than 10 % of the species belonging to this genus. jacobaea 33, S. mustersiiy, and S. subpanduratus However, the aim of this study is to investigate 34, S. othonnae, S. racemosus, and S. nemorensis the differences in composition, antimicrobial, anti- 35, S. pandurifolius 36, S. pedunculatus 37, S. inflammatory, antioxidant and cytotoxic activi- rufinervis 38, S. coincyi 39, S. scandens 29,40, S. ties between the EOs extracted from capetula and polyanthemoides 41, S. giganteus 42,43, S. bombay- shoots of S. glaucus. ensis 44, S. belgaumensis 45 and S. flammeus 46. Volatile constituents of Senecio species mainly Materials and methods contain monoterpene and sesquiterpene hydrocar- material bons and their oxygenated derivatives. Senecio Shoots and flowers (whole capitula) of Senecio crude extracts are known to possess various bio- glaucus L. were freshly collected (one kg from logical activities such as antibacterial, antifungal, each) during the flowering stage on April 2018 antitubercular, molluscicidal and cytotoxic activi- from a wild population inhabiting the newly re- ties 47. On the other hand, essential oils have in- claimed area of the eastern desert at Assiut, Egypt, sect repellent properties and the plant became for latitude 27° 15' 8" N and longitude 31° 18' 45" E. some extend not palatable for livestock and re- The plant was identified by taxonomists, accord- pelling the potential herbivores even before they ing to Boulos 5,6 and a voucher specimens have take a trial bite 48. Interesting studies by Turlings been deposited at the Assiut University Herbarium et al.49 and Kessler et al.50 showed that certain (ASTU). monoterpenes and sesquiterpenes are produced and emitted from many plant species only after Extraction of essential oils insect feeding has already begun. These sub- Immediately, one kg of fresh capitula or shoots stances repel ovipositing herbivores and attract were separately hydrodistilled in a Clevenger natural enemies, including predatory and parasitic apparatus. The plant material was cut into small insects, that kill plant-feeding insects and so help pieces, about 100 g fresh weight were added to minimize further damage. However, volatile ter- 500 ml distilled water in a one liter flask and sub- penes are not only defenses in their own right, jected to hydrodistillation for 3 h 14,41 (4 appara- Taha Ramadan et al., / TEOP 23 (1) 2020 168 - 183 170 tus were separately used at the same time). The Fungi Science Center, Faculty of Science, Assiut final condensed oil together with water was col- University. Shoots or capetula essential oils were lected and the upper phase (essential oil) was sepa- dissolved in dimethyl formamide (DMF) to pre- rated from the lower one (water), dried over an- pare stock solutions of 100 μl/ml. hydrous sodium sulphate and kept at 4°C in sealed The antimicrobial activity was determined by brown vials for analysis. Yield was calculated as the agar well diffusion method 58,59. Briefly, 1 ml average of three distillations (the collected oil of a suspension of the test microorganism 0.5 from the 4 apparatus was considered as yield of McFerland (1.5*108 cells/ml) was spread on Nu- one hydrodistillation) and related to the fresh trient agar plates for bacteria and Sabouraud Dex- weight. trose Agar (SDA) for the fungi. Six mm diameter wells were cut from the agar using a sterile cork- GC/MS analysis of essential oils borer, and 100 μl of the test substances were de- The essential oils were analyzed by GC/MS livered into the wells. The plates were incubated (Shimadzu GCMS-QP2010; Tokyo, Japan) for 24-48 hrs at 35°C. Antimicrobial activity was equipped with Rtx 5MS fused bonded column (30 evaluated by measuring the inhibition zone pro- m x 0.25 mm i.d. x 0.25 μm film thickness; Restek, duced against the test organism. Chlorampheni- USA) and a split-splitless injector. The initial col- col 1 mg/ml (for bacteria) and fluconazole 10 μl/ umn temperature was kept at 45°C for 2 min (iso- disk (for fungi) were used as positive controls and thermal) and programmed to 300°C at a rate of DMF as negative control. 5°C/min, and kept constant at 300°C for 5 min (isothermal). Injector temperature was 250°C. Determination of the minimal inhibitory con- Helium carrier gas flow rate was 1.41 ml/min. All centration (MIC) the mass spectra were recorded applying the fol- The minimum concentration of S. glaucus es- lowing condition: (equipment current) filament sential oils at which no growth occurred (MIC) emission current, 60 mA; ionization voltage, 70 were determined against S. aureus, B. subtilis, E. eV; ion source, 200°C. Diluted samples (1 % v/v) coli, P. aeruginosa, C. albicans, C. tropicalis were injected with split mode (split ratio 1: 15). strains using broth dilution method according to The identification of compounds was achieved the guidelines of the Clinical and Laboratory Stan- by library search on a Wiley 275 L GC/MS data dards Institute 60. Sterile 96-well rounded bottom base (Thermo Fisher Technology, Waltham, Mas- microwell plates were used. Samples were sachusetts, United States), observed Kovats in- vortexed before the experiment to ensure homog- dex and confirmed by comparing the retention enous distribution of the particles. Nutrient broth indices and mass fragmentation patterns to those and Sabouraud Dextrose broth (100 μl) was dis- of the reported data 57. Quantification was done pensed into all wells of the microwell plate. Each by external standard method using calibration tested extract was added to the first well followed curves generated by running GC analysis of rep- by 10 serial two-fold dilutions. Bacterial and yeast resentative authentic compounds. suspensions were added into all wells except for control wells. Plates were incubated at 37°C for Antimicrobial activity 24-48 hrs and evaluated for the minimum inhibi- The antimicrobial activities of the essential oils tory concentration by visual examination of the of S. glaucus were evaluated against two Gram culture turbidity. positive bacteria (Staphylococcus aureus and Bacillus subtilis), two Gram negative bacteria Anti-inflammatory assay (Escherichia coli and Pseudomonus aeruginosa), Anti-inflammatory activity was evaluated us- two yeasts (Candida albicans and Candida ing carrageenan induced paw edema method 61. tropicalis) and two molds (Aspergillus flavus and The experiment was performed on adult mail al- Fusarium oxysporum). All these organisms were bino rats, weighing 150-200 g, housed in a room obtained from the Prof. Dr. Abdel Aal Mubasher’ temperature at 23±2°C with a 12 h light/dark Taha Ramadan et al., / TEOP 23 (1) 2020 168 - 183 171 cycle. Food and water were provided ad libitum, equation: but the rates were fasted for 24 h before the ex- Potency = Percentage edema inhibition of periment with free access to water. The rats were essential oil treated group / Percentage edema divided into four groups, 4 rats each (control, ref- inhibition of indomethacin treated group x 100 erence and the tested capetula or shoot oils). The tested oils and the standard drug were adminis- Evaluation of the antioxidant activity tered intraperitoneally in the form of a suspen- Quantification of antioxidant activity was de- sion (using 1 % carboxymethyl cellulose) in dis- termined spectrophotometrically using DPPH free tilled water. Carrageenan solution (0.1 % in ster- radical scavenging method 62. In this method, 2 ile 0.9 % NaCl solution) in a volume of 0.1 ml ml of varying concentrations of oils (1, 2, 3, 4 was injected subcutaneously into the sub-plantar and 5 μl/ml methanol) were mixed with 2 ml region of the right hind paw of each rat, except methanolic solution of 0.1 mM DPPH. The mix- the first control group. Care and treatment of ani- ture was allowed to react at room temperature in mals were conducted according to approval of the dark for 30 minutes. The negative control was ethics regulation at Assiut University. DPPH solution, while L-ascorbic acid (2.5, 5.0, At the beginning of the experiment, the thick- 7.5, 10.0 and 12.5 μg/ ml methanol) was used as nesses of the paws were measured in mm using standard reference. The decrease in absorbance Vernier Caliper. The first group was kept as nega- (A) was measured at 518 nm using spectropho- tive control (non-treated) and injected by saline tometer. solution, while the second group received in- Inhibition percentage (I %) of free radical DPPH domethacin (positive control) in a dose of 8 mg/ or the DPPH free radical scavenging activity (%) kg (intraperitoneal). The other groups were sepa- was calculated from the absorption according to rately, intraperitoneally administered the oil of the following equation: μ capetula or shoots in doses of 200 l/kg. Acontrol - Asample After 30 minutes from the administration, the DPPH scavenged % = x 100 inflammation was induced. The thicknesses of the Acontrol paw in all groups were measured using Vernier The antioxidant activity is expressed as effec-

Caliber after 1, 2, 3, 4 and 5 hours. The differ- tive concentration (EC50) values. The lower the ence between the thickness of the right and the EC50 value, the more effective antioxidant acti- left paws was taken as a measure of edema. The vity is. The EC50 value, defined as the concentra- anti-inflammatory efficacy of the tested fractions tion of the sample leading to 50 % reduction of was estimated by comparing the magnitude of paw the initial DPPH concentration, was calculated swelling in the pretreated animals with that in- from the linear regression of plots of concentra- duced in control animals. tion of the test extracts (μl/ml) against the mean The percentage of edema and the percent edema percentage (n=3) of the scavenged DPPH. The inhibition was calculated from the mean effect of results were also compared to EC50 obtained by the control and treated animals according to the ascorbic acid. following equation: % Variation edema = (Right paw thickness-Left Cytotoxicity assays paw thickness) / Right paw thickness x 100 The effect of essential oils on the proliferation rate was analyzed by the MTT assay using Cell ® Percent edema inhibition = (1- Tt/Tc) x 100 Titer 96 Non-Radioactive Cell Proliferation As-

Where Tt and Tc are the mean increase in paw say kit (Promega, USA) following the thickness of oil treated and control groups of rats, manufacturer’s instructions. Briefly, 1 x 104 MCF- respectively. 7 cells were plated in wells of 96-well plate in Potency of the tested essential oils was calcu- triplicates at a final volume of 100 μl. Cells were lated relative to indomethacin ‘‘reference stan- maintained in DMEM medium supplemented with dard’’ treated group according to the following 10 % fetal bovine serum (Gibco, USA), penicil- Taha Ramadan et al., / TEOP 23 (1) 2020 168 - 183 172 lin antibiotic and streptomycin as antimycotic. % of the total oil. The major constituents of this Then two-fold serial dilutions of the capetula or oil were m-mentha-1(7),8-diene (31.4 %), cis-m- shoot EOs, in DMSO, were added to cells fol- mentha-2,8-diene (22.9 %), dehydrofukinone α lowed by incubation at 37°C under 5 % CO2 in- (17.2 %), -terpinolene (3.9 %), 2,5-cyclohexa- cubator (Thermo Fisher Scientific, USA) for 24 diene-1,4-dione,2-(1,1- dimethylethyl)-5-(2-me- hours. Cells treated with the solvent were consid- thyl-2-propen-1-yl)- (3.9 %) and sabinene (2.7 %). ered as controls. After incubation, 10 μl of MTT Five another constituents in the capetula essen- reagent was added to cells and incubated at 37°C tial oil, α-pinene, β-curcumene, germacrene D, for a further 4 hours. Finally, the stop solution humulene and euparin each represented by 1.0 % was added and incubated, and the absorbance was - 1.7 % of the oil (= 5.0 - 8.5 mg per 100 g fresh measured at a wavelength of 570 nm and a refer- weight). In the shoots oil, 33 compounds were ence wavelength of 630 nm using Epoch Micro- identified representing 96.0 % of the total oil. The plate Spectrophotometer (BioTek, USA). Percent- major constituents were m-mentha-1(7),8-diene age of cell viability was calculated by dividing (25.6 %), cis-m-mentha-2,8-diene (8.2 %), the OD values of the drug-treated sample by that dehydrofukinone (19.9 %), α-fenchene (5.6 %), of control samples. 1,3,8-p-menthatriene (5.3 %), β-ocimene (3.4 %), 2,5-cyclohexadiene-1,4-dione, 2-(1,1-dimethyl- Results ethyl)-5-(2-methyl-2-propen-1-yl)- (3.4 %) and Components of essential oils α-terpinolene (3.1 %). From these major constitu- Hydrodistillation of the fresh capetula and ents, α-fenchene and 1,3,8-p-menthatriene did not shoots of S. glaucus gave pale yellow essential detected in capetula oil. As shown in Table 1, there oils in yields of 0.5 % and 0.25 % of fresh weight, are 9 compounds each represented by 1.0 % - 1.8 respectively. A quantitative and qualitative varia- % of the shoot oil (=2.5 - 4.5 mg per 100 g fresh tion between both oils were detected, and the com- weight). pounds with concentrations more than 0.1 % of Figure 1 shows the distribution of chemical the oil are represented in Table 1. The capetula classes in the volatile oils of S. glaucus. The com- oil contained 33 components, representing 97.1 pounds were separated into terpenoids (monoter- Essential oil of shoots Essential oil of capetula

Figure 1. The chemical class distribution in the essential oils of S. glaucus L. represented as mg (100 g)-1 fresh weight and as a percentage from the total oil Table 1. Chemical composition of capetula and shoots essential oils of Senecio glaucus L. Only compounds with concentrations more than 0.1% of the oil are represented

Capetula oil Shoots oil No. Compounds Rta RIb RIc Concentration mg/100 g Concentration mg/ 100 g %d FW %d FW

1 1-Nonene VII 6.2 906 889 0.3 1.7 0.9 2.3 2 α-Pinene I 7.4 948 939 1.0 5.0 1.7 4.2 Taha Ramadan 3 α-Fenchene I 7.8 953 945 - 5.6 14.0 4 Sabinene I 8.6 961 969 2.7 13.7 - 5 m-Mentha-4,8-diene I 9.3 990 987 22.9 114.5 8.2 20.4 6 m-Mentha-1(7),8-diene I 10.2 1013 1000 31.4 157.2 25.6 63.9 7 1,3,8-p-Menthatriene I 10.3 1029 1110 - 5.3 13.1 et al β I 8 (Z)- -Ocimene 10.6 1030 1032 1.0 5.0 3.4 8.5 ., / TEOP 23(1) 9 (E)-β-Ocimene I 10.9 1040 1044 0.4 2.2 1.3 3.4 10 α-Terpinolene I 12.1 1052 1087 3.9 19.7 3.1 7.6 11 p-Mentha-2,4(8)-diene I 12.2 1080 1088 0.3 1.4 0.5 1.3 12 Estragole II 15.6 1191 1196 0.1 0.6 0.7 1.7 13 1-Octenyl acetate VI 15.8 1199 1198 0.4 2.0 0.4 1.1 14 Methyl eugenol VII 21.5 1398 1403 - 0.2 173 0.4 2020 168-183 15 9,10-Dehydro-isolongifolene III 21.9 1398 1390 0.1 0.6 0.2 0.5 16 Caryophyllene III 22.8 1412 1408 1.1 5.3 1.2 2.9 17 α-Cubenene III 23.4 1440 1495 0.2 1.1 0.5 1.2 18 Dehydro-β-ionone VII 23.5 1440 1477 0.6 3.0 - 19 Humulene III 23.7 1448 1454 1.3 6.3 1.8 4.5 20 ValenceneIII 23.9 1471 1496 0.2 1.0 0.5 1.1 21 Germacrene D III 24.6 1476 1481 1.3 6.5 1.7 4.3 22 γ-Curcumene III 24.8 1480 1482 1.7 8.3 1.7 4.1 23 α-Selinene III 26.6 1483 1493 0.7 3.3 0.8 2.1 24 γ-Amorphene III 27 1489 1495 0.5 2.7 1.3 3.2 25 β-Vetispirene III 27.2 1489 1493 0.4 1.9 1.7 4.2 26 δ-Cadinene III 27.6 1517 1523 0.4 0.6 0.7 0.9 table 1. (continued).

Capetula oil Shoots oil No. Compounds Rta RIb RIc Concentration mg/100 g Concentration mg/ 100 g %d FW %d FW Taha Ramadan 27 cis-Sesquisabinene hydrate IV 28.4 1523 1520 0.1 0.7 0.3 0.8 28 Dihydro-β-agarofuran IV 28.6 1527 1522 0.3 1.7 1.1 2.6 29 Geranyl isovalerate IV 29.8 1586 1599 0.4 2.0 0.9 2.3 30 Dehydrofukinone IV 31.2 1678 1688 17.2 86.0 19.9 49.6 31 Mint sulfideVII 31.5 1736 1740 0.2 0.8 0.1 0.2 et al 32 2,5-Cyclohexadiene-1,4-dione, 32.5 1858 - 3.9 19.5 3.4 8.4

2-(1,1- dimethylethyl)-5-(2-methyl- ., / TEOP 23(1) 2-propen-1-yl)- VII 33 Euparin IV 33.2 1888 1889 1.5 7.5 0.9 2.4 34 Pseudo phytolVII 37.3 2045 2018 0.1 0.4 0.3 0.7 35 HeneicosaneVII 43.9 2109 2100 0.2 1.0 - 36 Octacosane VII 49.9 2804 2800 0.3 1.6 0.1 0.1 0018-13174 2020 168-183 Total identified 97.1 484.8 96.0 238.0 a Rt: Retention time (in min) b RI: Retention indeces on Rtx5MS fused bonded column (30 m x 0.25 mm i.d. x 0.25 μm film thickness; Restek, USA) in reference to n-alkanes c RI: Literature retention indices on DB-5 column (0.25 mm i.d. x 30 m, 0.25 micron coating thickness, fused silica capillary) according to Adam57 d Quantification was done by external standard method using calibration curves generated by running GC analysis of representative authentic components I Monoterpene hydrocarbons II Oxygenated monoterpenes III Sesquiterpene hydrocarbons IV Oxygenated sesquiterpenes, V Diterpenes VI Terpene related compounds and VII Others Taha Ramadan et al., / TEOP 23 (1) 2020 168 - 183 175 pene hydrocarbons, oxygenated monoterpenes, Anti-inflammatory activity sesquiterpene hydrocarbons, oxygenated sesquit- Data in Table 3 show that both of capetula and erpene, diterpen and terpene related compounds), shoots essential oils of S. glaucus exerted the high- hydrocarbons, and others. The major constituents est anti-inflammatory effect after 3 and 4 hours were monoterpene hydrocarbons (63.3 % and 54.2 from carrageenan injection then the activity de- % in the oils of capetula and shoots, respectively). creased again after 5 hours. The essential oils In the oil of capetula and shoots of S. glaucus, 12 showed the highest anti-inflammatory activities and 15 terpenoid compounds were identified, re- and the same potency as indomethacin after 4 spectively. hours of treatment. As shown in Table 3, both volatile oils did not differentiate significantly from Antimicrobial activity indomethacin especially after 2, 3, and 4 hours The oils of S. glaucus were tested for antibac- with potencies between 0.8 and 0.9 compared with terial activity against two Gram-positive and two the reference standard drug. Gram-negative bacteria, and were found to inhibit the growth of the four organisms S. aureus, B. Antioxidant activity subtilis, P. aeruginosa and E. coli. The inhibition The results of the antioxidant activity of zone of capetula and shoots oils was ranging be- capetula and shoots essential oils of S. glaucus tween 10 and 13 mm, with a higher activity for are shown in Table 4. The DPPH scavenging abil- capetula oil than of shoot oil by about 12 % (Table ity of capetula and shoots essential oils showed a 2). For S. aureus and P. aeruginosa, the minimum concentration-dependent activity profile with a inhibitory concentration (MIC) of both oils was maximum inhibition of 63.0 %, 55.9 % and 44.8 6.3 μl, but B. subtilis was more sensitive and the % at 2.5 μl/ml concentration, respectively, com- MIC was 3.1 μl. It was observed that at low con- pared to 89.6 % at 25 μg/ml of ascorbic acid. The centrations, the EOs inhibited selectively B. EC50 value calculated for the capetula and shoots subtilis growth while at high concentrations it essential oils was found to be 1.6 and 1.9 μl/ml, inhibited also E. coli. whereas for standard ascorbic acid it was found The results of the antifungal activity of oils to be 16 μg/ml. against C. albicans, C. tropicalis, A. flavus, and F. oxisporum are shown in Table 2. Both oils exhi- Cytotoxicity bited antifungal activity with zones of inhibition The results of the cytotoxic effect of the essen- ranging from 9-17 mm. Moderate activity was tial oil, which was evaluated by the MTT assay observed against F. oxisporum with inhibition using the breast cancer cell lines, are shown in zone about 9 mm, but both oils were more active Table 4. The oils that exhibited >50 % inhibition against A. flavus. against the cell line were assayed further to de- Table 2. Antimicrobial activity of capetula and shoots essential oils of S. glaucus

Gram-positive Gram-negative Yeasts Molds Treatment S. B. P. E. C. C. A. F. aureus subtilis aeruginosa coli tropicalis albicans flavus oxysporum

Capetula oil 12(6.3) 13(3.1) 12(6.3) 13(6.3) 13(3.1) 12(6.3) 17 10 Shoots oil 10(6.3) 13(3.1) 11(6.3) 12(12.5) 10(3.1) 11(6.3) 15 9 Chloramphenicol 19 20 18 20 - - - - Fluconazole 10 - - - - 20 19 23 20

Values of inhibition zones are in mm including the well diameters. Values of minimum inhibitory concentration MIC, in parentheses, are expressed in μl ml-1. No inhibition zones ware observed in negative control Table 3: Effect of capitula and shoots essential oils of S. glaucus on carrageenan - right hind paw edema

Time after administration (hours)

Group (dose) 12345 Taha Ramadan

Negative control (3 ml/kg) Edema thickness (mm) 4.5c±0.0 4.8b±0.1 4.5c±0.3 4.6b±0.2 4.5c±0.3 %Variation edema 60.0 61.5 60.0 61.5 60.0 Indomethacin (8 mg/kg) Edema thickness (mm) 1.8a±0.1 1.1a±0.1 0.8a±0.1 0.9a±0.2 2.0a±0.5

% Variation edema 37.5 26.8 21.1 23.1 40.0 et al % Inhibition 36.7 46.8 50.0 49.2 33.3 Capetula oil (200 μl/kg) Edema thickness (mm) 0.3b±0.2 1.8a±0.3 1.3ab±0.1 1.4a±0.2 3.3b±0.1 ., / TEOP 23(1) % Variation edema 50.0 37.2 30.2 31.8 52.4 % Inhibition 20.0 38.7 43.3 42.6 16.7 Shoots oil (200 μl/kg) Edema thickness (mm) 3.3b±0.3 2.0a±0.4 1.5b±0.3 1.3a±0.3 3.0b±0.2 % variation edema 52.4 40.0 33.3 30.2 50.0

% Inhibition 16.7 35.5 40.0 44.320.0 176 2020 168-183 F-value 30.5*** 33.5*** 55.2*** 51.8*** 11.9** Potency capetula oil 0.55 0.83 0.87 0.87 0.50 Potency shoots oil 0.45 0.76 0.80 0.90 0.60

Values of edema thickness (means ± S.E, n= 4) with different letters are significantly differ according to Duncan’s t-tes F-values of One Way ANOVA with ** or *** are significant at P< 0.01 or P< 0.001, respectively The potency of capetula and shoots EOs are calculated as relative to indomethacin Taha Ramadan et al., / TEOP 23 (1) 2020 168 - 183 177 Table 4. Antioxidant (or the percentage of DPPH scavenged) and cytotoxic activities of capetula and shoots essential oils of S. glaucus

μ -1 Extracts oil ( l ml )EC50 Cytotoxic activity μ -1 0.5 1.0 1.5 2.0 2.5 l ml IC50 on MCF7 cell line

Capetula oil 27.6 34.4 46.2 54.3 63.0 1.6 1.5 % Shoots oil 17.7 29.7 40.3 48.4 55.9 1.9 2.1 % Ascorbic acid μg ml-1 5 10 15 20 25 16 μg ml-1 % DPPH scavenged 15.1 27.8 44.3 66.5 89.6

termine IC50 value and a dose-dependent cytotoxi- essential oils as well as other active agents in city relation was observed. The capetula flower plants 69,70. essential oil of Senecio glaucus was found to be Comparing the data of this study with those pre- most potent against human breast cancer cells IC50 viously reported in literature, the studied essen- = 1.5 % followed by shoots oil against the same tial oils displayed different chemical profiles, al- cell line with IC50 of 2.1 %. though monoterpene hydrocarbons have been re- ported as the main constituents of the essential Discussion oils of several species of the genus Senecio In this study, the yields of capetula (0.5 %) or 16,24,47,71-74. However, some species of this genus shoots (0.25 %) essential oils on the bases of fresh such as , Senecio aegyptius var. weight are considered high compared to other discoideus and Senecio chrysanthemoides are species of the genus Senecio. The yield of essen- characterized by high percentage of oxygenated tial oils from S. jacobaea 33 and S. giganteus 43 in compounds 4,18,75. Sesquiterpene hydrocarbon, that was found to be only 0.02 % on the air found to be a major component in the studied dry samples. The yield in case of S. graveolens Senecio glaucus, has been reported as the main was 0.5 % 19, S. aegyptius was 0.4 % 18, in fresh constituent in S. aegyptius var. discoideus 75. flowers, leaves and stems of S. polyanthemoides The essential oil of S. glaucus capetula have in South Africa was 0.07- 0.23 % 41 and in S. more antimicrobial activity, as indicated by the perralderianus was 0.1 % 63. These variations in inhibition zone of similar concentrations, than that the yield of essential oil may be attributed to spe- of the shoots. This may be attributed to relatively cies, origin, habitat conditions, harvest period and the high percentage of monoterpenes that found the used extraction technique. However, the stud- in capetula oil compared to the shoots. The anti- ied S. glaucus was collected from a very dry habi- bacterial and antifungal activities of an essential tats in the eastern desert at Assiut province, Egypt. oil are linked to its chemical composition, to the A total 33 compounds were identified accounting functional groups of the major compounds for 97.1 % and 96.0 % of the composition of the (alcohols, phenols, terpene compounds and ke- essential oil of capetula and shoots of S. glaucus, tones) and to its synergistic effects 76. Pinene-type respectively. To the best of our knowledge, this is monoterpene hydrocarbons (like α-pinene) are the first report on the presence of m-mentha- well known chemicals having antimicrobial po- 1(7),8-diene (31.4 % and 25.6 %) and cis-m- tentials 77. Also, it is known, amongst several mentha-2,8-diene (22.9 % and 8.2 % in the es- monoterpenes, to be toxic to numerous insects and sential oil of capetula and shoots, respectively) herbivores repellent. The activity of the oil varies in S. glaucus. Chemical variation of essential oils with its concentration and kind of bacteria. These has been attributed to differences in environmen- variations in the susceptibility of the test organ- tal and genetic factors 64-68. Furthermore, ecologi- isms to essential oil could be attributed to varia- cal factors, particularly, light and temperature tion in the rate of monoterpenes penetration have been reported to influence the production of through cell wall and cell membrane structures. Taha Ramadan et al., / TEOP 23 (1) 2020 168 - 183 178 The ability of essential oil to disrupt the perme- the presence of compounds having hydrogen do- ability barrier of cell membrane structures and the nating ability 86,87. accompanying loss of chemiosmotic control is the The pharmacological effect of these essential most likely source of its lethal action 78. oils depict is believed to be the outcome of the The antimicrobial activity of essential oils of synergistic effect of the major constituents. Lone several Senecio spp., against Gram negative and et al.24 attributed the potent antioxidant and cyto- Gram positive bacteria and fungi, including S. toxic effect of Senecio graciliflorus essential oil graveolens, S. aegyptius, S. subpanduratus S. to synergistic effect of the major constituent mustersii, S. pandurifolius, S. atacamensis, Sene- monoterpenes. The value of IC50 of capetula es- cio nutans, Senecio viridis and S. graciliflorus sential oil of Senecio glaucus against human 18,19,22,25,34,36,79 were reported . Components such as breast cancer cells, and also the EC50 value (= the α-pinene, 1,8-cineole, γ-terpinene, linalool and concentration of the oil leading to 50 % reduc- α-terpineol have been found to have relatively tion of the initial DPPH concentration) were found strong antimicrobial properties 80. In addition, the to be less than that of the shoots oil. However, essential oils of many species of family Asteraceae the essential oil of capetula is richer with monot- (e.g. A. trifurcata, S. oreophyton, and S. pogonias erpenes than that of the shoots. In addition to showed a good antibacterial activity 26. Galvez et major constituents, some minor constituents may al.22 found that the essential oils from aerial parts also be contributing to the observed bioactivity of S. nutans and S. viridis, have a moderate anti- 88. It is very difficult to attribute the antioxidant fungal activity on Fusarium and were mostly in- effect of the essential oil to one or a few active active on Aspergillus. The oil of S. viridis principles, because an essential oil always con- synergized the effect of fungicides and food pre- tains a mixture of different chemical compounds, servatives on F. verticillioides which could be due and the minor compounds may make a signifi- to the presence of 9,10-dehydrofukinone. How- cant contribution to the oil’s activity. ever, these differences in the antimicrobial acti- vity of essential oils between the species of the Conclusions same genus or between populations of the same The chemical composition of capetula and species depend on the dissimilarity in the chemi- shoots essential oils of Senecio glaucus showed, cal composition of oils collected from different for some extent, a qualitative and quantitative environments or geographical regions. variations. The five compounds that identified at The development of edema has been described higher percentages (m-mentha-1(7),8-diene; cis- as biphasic, the initial phase is due to the release m-mentha-2,8-diene; dehydrofukinone; α- of mediators of inflammation like histamine, terpinolene and 2,5-cyclohexadiene-1,4-dione,2- serotonins, dopamine and kinins in the first hour. (1,1-dimethylethyl)-5-(2-methyl-2-propen-1-yl)) The more pronounced second phase is related to collectively represented by about 79.4 % and 59.9 the release of prostaglandins in 2-3h. Hence, the % of the total oil of capetula and shoots, respec- significant anti-inflammatory effect of both es- tively. The capetula EO contained 22 compounds sential oils may be due to inhibitory effect ex- did not identified in the shoots EO, while the erted predominantly on the mediators of inflam- shoots EO contained 16 compounds did not iden- mation 81-83. The anti-inflammatory activity of S. tified in the capetula EO. Monoterpene hydrocar- glaucus essential oils may be attributed not only bons were the main constituents of the oils, but to their antioxidant activities, that documented in with higher percentage in capetula oil than in this study, but also as suggested by Miguel et al. shoots oil. The shoots oil contained oxygenated 84 to their interactions with signaling cascades in- terpenes more than capetula oil. The capetula volving cytokines and regulatory transcription essential oil were dominated by m-mentha-1(7),8- factors, and on the expression of pro-inflamma- diene and cis-m-mentha-2,8-diene (54.3 %) which tory genes. Such activity was suggested to be due can explain its high antioxidant and antimicro- to the high amounts of monoterpenes 85 or due to bial activities compared with shoots oil. Taha Ramadan et al., / TEOP 23 (1) 2020 168 - 183 179 Acknowledgment Disclosure statement Authors are grateful to Assiut University for The authors declare no conflicts of interest. financial support.

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