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South African Journal of Botany 91 (2014) 63–70

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Phytochemicals, antioxidant and antifungal activities of roseum var. grandiflorum subvar. typicum Regel.

Lamia Sakka Rouis-Soussi a, Naima Boughelleb-M'Hamdi b, Asma El Ayeb-Zakhama a, Guido Flamini c, Hichem Ben Jannet d, Fethia Harzallah-Skhiri a,⁎

a Laboratory of Genetic Biodiversity and Valorisation of Bioresources (LR11ES41), High Institute of Biotechnology, Rue Tahar Haddad, Monastir 5000, b Department of Biological Sciences and Protection, High Institute of Agronomy of Chott Meriem, University of Sousse, Chott Meriem 4042, Tunisia c Dipartimento di Farmacia, Faculty of Farmaci, Via Bonanno 33, 56126 Pisa, Italy d Laboratory of Heterocyclic Chemistry, Natural Products and Reactivity, Team: Medicinal Chemistry and Natural Products, Faculty of Sciences, Monastir, Tunisia

article info abstract

Article history: The chemical composition of essential oil hydrodistillized from Allium roseum var. grandiflorum subvar. typicum Received 15 August 2013 Regel. leaves was analyzed by GC and GC/MS. Nine extracts obtained from flowers, stems and leaves and Received in revised form 2 December 2013 and bulblets of A. roseum var. grandiflorum were tested for their total phenol, total flavonoid and total flavonol Accepted 22 December 2013 content. All these extracts and the essential oils from fresh stems, leaves and flowers were screened for their pos- Available online 20 January 2014 sible antioxidant and antifungal properties. The results showed that the hexadecanoic acid was detected as the Edited by V Steenkamp major component of the leaf essential oil (75.9%). The ethyl acetate extract of stems and leaves had the highest • antioxidant activity with a 50% inhibition concentration (IC50) of 0.35 ± 0.01 mg/mL of DPPH and •+ Keywords: 0.71 ± 0.01 mg/mL of ABTS . All the extracts appeared to be able to inhibit most of the tested fungi. The essen- Allium roseum var. grandiflorum subvar. typicum tial oil of the leaves had an antifungal growth effect on Fusarium solani f. sp. cucurbitae and Botrytis cinerea (39.13 Regel. and 52.50%, respectively). This could be attributed to the presence of hexadecanoic acid, known for its strong Essential oil antifungal activity. In conclusion, in addition to the health benefits of A. roseum, it can be used as an alternative Chemical composition pesticide in the control of plant disease and in the protection of agriculture products. Total polyphenol content © 2013 SAAB. Published by Elsevier B.V. All rights reserved. Antioxidant activity Antifungal activity

1. Introduction interest (Rivlin, 2001; Griffiths et al., 2002). This genus is also one of the major sources of polyphenol compounds (Shon et al., 2004; Singh Much research has been performed to investigate the et al., 2009). However, polyphenols are bioactive molecules widely phytotherapeutic properties of the Allium genus. Recent studies have distributed in many plant species, with a great variety of structures, confirmed the antibacterial, antifungal, antiviral, immuno-stimulating, ranging from simple compounds to very complex polymeric substances and antioxidant properties and cholesterol lowering effects of Allium (Pârvu and Pârvu, 2011). Among polyphenols, flavonoids are the best species (Pittler and Ernst, 2007; Stajner et al., 2008). Due to their health known and best characterized group. This group is further subdivided beneficial effects, extensive scientific investigations have been mainly into classes which include flavones, flavonols, isoflavonoids and conducted on the phytochemistry and biological properties of Allium proanthocyanidins (Ferguson Lynnette, 2001). The different polyphenol sativum L. and Allium cepa L. (Corzo-Martinez et al., 2007). classes share the ability to act as chain-breaking antioxidants, Allium and their extracts contain different chemical com- which confers protection against the damage caused by free radicals pounds; an abundance of bioactive constituents namely organo-sulfur to DNA, membrane and cell components (Halliwell, 1996; Madhujith compounds, volatile sulfur compounds and proteins. Prostaglandins, and Shahidi, 2009). The total polyphenol content is a good indicator of fructan, vitamins, polyphenols, fatty acids and essential oils have also the antioxidant capacity and various studies have reported a high been identified (Corzo-Martinez et al., 2007). Because of its secondary correlation between antioxidant capacity and this value (Simonetti metabolite production, in particular because of its sulfur and other nu- et al., 1997; Pellegrini et al., 2000). Moreover, polyphenols are also merous phenolic compounds’ content, Allium species are of great shown to exhibit antibacterial, anti-inflammatory, antiallergenic, anti- arthrogenic and antithrombotic effects (Ajila et al., 2010). Different fla- vonoids have showed distinct antioxidant and antibacterial activities (Akroum et al., 2010). ⁎ Corresponding author at: High Institute of Biotechnology of Monastir, University of Although antioxidant activity of some Allium species has already Monastir, Rue Tahar Haddad, 5000 Monastir, Tunisia. Tel.: +216 73405405; fax: +216 73405404. been reported elsewhere (Yin and Cheng, 1998), little attention was E-mail address: [email protected] (F. Harzallah-Skhiri). paid to their antifungal potential (Yin and Tsao, 1999). extracts

0254-6299/$ – see front matter © 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sajb.2013.12.005 64 L. Sakka Rouis-Soussi et al. / South African Journal of Botany 91 (2014) 63–70 have been demonstrated to have antifungal properties against soil dried over sodium sulfate, weighed and stored in sealed glass vials in borne fungal pathogens (Sealy et al., 2007). a refrigerator at 4 °C until use. Allium roseum L. is a bulbous perennial plant native from the Mediterranean. A. roseum var. grandiflorum Briq. has big flowers with 2.4. Phytochemical analysis large obtuse . Leaves are flattened, 5–10 mm wide, papillose on the edges; it grows on undergrowth and grassy slopes. The subvariety 2.4.1. Analytical GC typicum Regel. is characterized by inflorescences that do not have - Gas chromatograph: HP 5890-series II instrument equipped with a lets with well developed flowers. In Tunisia, this subvariety is wide- flame ionization detector (FID), HP-5 (30 m × 0.25 mm ID, 0.25 μm spread in the North East and the North West of the country and on film thickness) fused silica capillary column, carrier gas nitrogen mountainous regions located in the Center and has also reached the (1.2 mL/min). The temperature oven was programmed from 50 °C South (Cuénod et al., 1954). Le Floc'h (1983) reported that A. roseum (1 min) to 280 °C at 5 °C/min (1 min). Injector and detector tempera- was used since ancient times as a vegetable, spice or herbal remedy to tures were 250 °C and 280 °C, respectively. Volume injected: 0.1 μLof treat headache and rheumatism. 1% hexane solution. The identification of the components was Investigations dealing with A. roseum growing wild in Tunisia are performed by comparison of their retention times with those of pure scarce. Recently, some studies have described the chemical composition authentic samples and by means of their linear retention indices of the essential oil and the antimicrobial and antioxidant activities of (LRIs) relative to the series of n-hydrocarbons. A. roseum var. odoratissimum (Desf.) Coss. collected in certain regions of Tunisia (Najjaa et al., 2007, 2011; Dziri et al., 2012; Zouari et al., 2.4.2. Analytical GC–MS 2012, 2013). Nevertheless, no information concerning the A. roseum GC–EIMS analyses were performed with a Varian CP-3800 var. grandiflorum growing wild in Tunisia has been published, except gas-chromatograph equipped with a HP-5 capillary column the work of BenJannetetal.(2007)who studied the chemical compo- (30 m × 0.25 mm; coating thickness 0.25 μm) and a Varian Saturn sition of essential oils of flowers and stems of the same species as 2000 ion trap mass detector. Analytical conditions: injector and transfer reported upon in this study. line temperatures 220 and 240 °C respectively; oven temperature pro- Therefore, the aim of the present work was to study the phytochem- grammed from 60 °C to 240 °C at 3 °C/min; carrier gas helium at ical content of the essential oil and organic extracts of A. roseum var. 1 mL/min; injection of 0.2 μL (10% hexane solution); split ratio 1:30. grandiflorum subvar. typicum Regel. and their antioxidant and antifungal Identification of the constituents was based on comparison of the properties used in the Tunisian traditional medicine or generally as retention times with those of authentic samples, comparing their linear food. retention indices relative to the series of n-hydrocarbons and on computer matching against commercial (NIST 98 and ADAMS) and 2. Materials and methods home-made library mass spectra built up from pure substances and components of known oils and MS literature data. Moreover, the 2.1. Plant material molecular weights of all the identified substances were confirmed by GC–CIMS, using MeOH as CI ionizing. A. roseum var. grandiflorum subvar. typicum was collected in the re- gion of Sousse (coastal region, in the Center-East of Tunisia, geographi- 2.4.3. Total phenolic content cal coordinates are 35°49′32″ North, 10°38′28″ East), during their The content of total phenolic compounds in organic extracts was blooming stage in March 2007 (for the extraction of organic extract) measured with a spectrophotometry method based on a colorimetric and in March 2011 (for the extraction of essentials oils). Identification oxidation/reduction reaction. The oxidizing agent was the Folin– was performed according to the “Flora of Tunisia” (Cuénod et al., Ciocalteu's phenol reagent (Merck) (Singleton et al., 1965; AOAC, 1954), by the botanist Pr. Fethia Harzallah-Skhiri and a voucher speci- 1984). 50 μL of each diluted organic extract was added to 750 μL of dis- men (Al.104) has been deposited in the laboratory of Genetic Biodiver- tilled water/Folin–Ciocalteu solution (28:2 v:v). After 3 min, 200 μLof sity and Valorisation of Bioresources, High Institute of Biotechnology of sodium carbonate solution (20%) was added. The reaction mixture Monastir, Tunisia. was kept in a boiling water bath for 1 min. For the control, 50 μLof methanol was used. The absorbance was measured at 765 nm. Tests 2.2. Preparation of A. roseum var. grandiflorum organic extracts were carried out in triplicate. Quantification was obtained by reporting the absorbance in the calibration curve prepared with gallic acid, results Plant samples were separated in three parts: (flowers), (both stems are expressed as mg of gallic acid equivalents (GAE) per 100 g Dry and leaves) and (both bulbs and bulblets). Fresh flowers and both stems Weight (DW). and leaves of A. roseum var. grandiflorum were air-dried for five weeks then ground into fine powder, whereas the bulbs and bulblets were 2.4.4. Total flavonoid content used fresh. One hundred grams of each plant part was extracted sepa- Total flavonoid content in each organic extract was determined rately at room temperature with 200 mL of acetone–H2O mixture (8:2 using a spectrophotometric method (Lamaison and Carnat, 1991) with v:v). Extraction was performed twice for 5 days at room temperature. slight modifications, based on the formation of the complex flavo- The resulting extract was filtered and the solution was evaporated to re- noid–aluminum. To 0.5 mL diluted organic extract, 0.5 mL of 2% alumi- move acetone under reduced pressure in a rotary evaporator (Büchi num chloride (AlCl3) dissolved in methanol was added. The sample was Rotavapor R-200, Büchi Heating Bath B-490). The remaining aqueous incubated for 15 min at room temperature and the absorbance of the solution was extracted sequentially with the following solvents: chloro- reaction mixtures was measured at 430 nm. Rutin was used as standard form, ethyl acetate and butanol. The extracts were separately concen- flavonoid. The total flavonoid content was expressed as mg of rutin trated with a rotary evaporator under reduced pressure and stored at equivalents per 100 g DW, by using a standard graph and the values 4 °C until tested. were presented as means of triplicate analyses.

2.3. Preparation of A. roseum essential oils 2.4.5. Total flavonol content Total flavonol content in each organic extract was determined ac- The fresh stems, leaves and flowers (100 g of each sample in 300 mL cording to Miliauskas et al. (2004) with slight modifications. One milli- of distilled water) were separately submitted to hydrodistillation in a gram of each the organic extract was dissolved in 1 mL of methanol.

Clevenger-type apparatus for 4 h. The essential oils were collected, One milliliter of this solution was added to 1 mL of 2% AlCl3 dissolved L. Sakka Rouis-Soussi et al. / South African Journal of Botany 91 (2014) 63–70 65 in methanol and 3 mL of 5% sodium acetate in methanol. After incuba- (TEAC). The TEAC value of a sample represents the concentration of a tion at room temperature for 2 h 30 min, the absorbance was measured Trolox solution that has the same antioxidant capacity as this sample. at 440 nm. Rutin was used as standard flavonol. The flavonol content was expressed as mg of rutin equivalents per 100 g DW. The experi- 2.6. Antifungal activity ments were always performed in triplicate. 2.6.1. Fungal isolates Fungi were collected from various localities in Tunisia. Table 1 lists 2.5. Antioxidant activity each fungus, the host, plant part from which it was isolated, locality and date of collection. All fungal isolates were identified and samples The antioxidant potential of A. roseum var. grandiflorum organic ex- of each fungus were deposited in the collection bank at the Plant Pathol- tracts and of essential oils was determined by two complementary rad- • ogy Laboratory (High Institute of Agronomy of Chott-Meriem, Tunisia). ical scavenging assays: the 2,2-diphenylpicrylhydrazyl radical (DPPH ) Fungal isolates were maintained on Potato Dextrose Agar (PDA, Difco and the 2,2′-azinobis-(3-ethylbenzothiazoline-6 sulfonic acid radical •+ Laboratories Inc., Detroit, MI, USA), stored at room temperature and (ABTS ). A. roseum var. grandiflorum samples were weighed and dis- sub-cultured once a month. The isolates were dispensed in sterile solved in absolute ethanol at concentration ranges of 0.5–5 mg/mL. Petri dishes and grown for 7–10 days before use.

• 2.5.1. Scavenging activity of DPPH free radical 2.6.2. Extracts and essential oils dissolution The hydrogen atoms or electron-donation ability of A. roseum var. The organic extracts and the essential oils of A. roseum var. fl grandi orum organic extracts and essential oils was measured by the grandiflorum were dissolved in methanol and dimethyl sulfoxide • bleaching of a purple-colored methanol solution of DPPH .Thisspectro- (DMSO 10%), respectively, in order to have an initial concentration of ′ photometric assay uses the stable 2,2 -diphenyl-1-picrylhydrazyl 10 mg/mL. (DPPH•) radical as a reagent and was adapted from Ramadan et al. (2003) with slight modifications. Thus, 950 μLof10−4 MDPPH• meth- 2.6.3. Antifungal screening anol solution was added to 50 μLofA. roseum var. grandiflorum diluted The disk qualitative and quantitative antifungal assays of A. roseum samples. Each mixture was homogenized and allowed to stand at room var. grandiflorum organic extracts and essential oils were carried out temperature in the dark for 30 min. The absorbance was read against a using the disk diffusion method (Boughalleb et al., 2005). A conidial sus- blank (absolute ethanol) at 515 nm. The decrease in absorbance in- pension (1 mL) of each fungus was added into each Petri dish. Thereaf- duced by the tested samples (change of color from deep-violet to ter, 15 mL of Potato Dextrose Agar (PDA) medium supplemented with light-yellow) was compared to that of the positive control Trolox. The streptomycin sulfate (100 mg/L) was added to the dish containing the antioxidant activities of samples were expressed by IC values which 50 spore suspension. Once the substrate solidified a disk of Whatman indicate the concentration of samples (mg extract/mL ethanol) required paper no. 3 (5 mm in diameter) was soaked with 20 μLofeach to scavenge 50% of the DPPH• radical. All the tests were carried out in A. roseum var. grandiflorum sample, allowed to dry and placed on the in- triplicate. oculated Petri dishes. In the case of the control, the disk was moistened with methanol for the organic extract and with DMSO (10%) for the 2.5.2. Scavenging activity of ABTS•+ free radical essential oil. Then the plates were incubated at 25 °C for 8 days. The an- The scavenging activity of the ABTS•+ (2,2′-azino-bis(3- tifungal activity was evaluated by calculating the percentage of myceli- ethylbenzothiazoline-6-sulfonic acid) radical was measured as de- um growth inhibition %I = ((C − T) / C) ∗ 100, according to the scribed by Re et al. (1999).ABTS•+ radical cation was generated by method of Singh et al. (1993), where C is the mean of mycelium growth the interaction of ABTS (7 mM) and potassium persulfate (K2S2O8; (mm) of controls and T is the mean of mycelium growth (mm) of those 2.45 mM). The mixture was allowed to stand at room temperature in treated with organic extracts or essential oil. All the tests were per- dark for 12–16 h to give a dark green solution. Oxidation of ABTS•+ be- formed in three replicates. gins immediately, but the absorbance was not maximal and stable until more than 6 h had elapsed. The radical cation was stable in this form for 2.7. Statistical analysis more than 2 days when stored in the dark at room temperature. Prior to assay, the dark green solution was diluted in ethanol (1:80 v:v) to give Simple regression analysis was performed to calculate the dose–re- an absorbance at 734 nm of 0.70 ± 0.03 in a 1 cm cuvette. One millili- sponse relationship of standard solutions used for calibration as well ter of the resulting solution was mixed with 10 μLofA. roseum var. as samples tested. Linear regression analysis was performed. All the re- grandiflorum diluted samples. The absorbance was measured exactly sults are expressed as mean ± standard deviation of three parallel mea- 20 min after initial mixing. Trolox was used as an antioxidant standard. surements. The data were processed using Microsoft Excel 2003 then All tests were performed in triplicate. The antioxidant activities of sam- were subjected to one way analysis of variance (ANOVA) and the signif- ples were expressed by IC50 values, which indicated the concentrations icance of differences between means was calculated by the Duncan of samples (mg extract/mL ethanol) required to scavenge 50% of ABTS•+ multiple range test using SPSS for Windows (Standard Version 14.0 radical, expressed also as Trolox Equivalent Antioxidant Capacity SPSS Inc., Chicago. IL.), p values b0.05 were regarded as significant.

Table 1 Fungal isolates used to test the inhibitory activity of Allium roseum var. grandiflorum subvar. typicum Regel.

Collection date Collection locality Plant part sampled Plant source Fungi

11/05/2000 Bou Argoub (North) Fruits Vine Botrytis cinerea 23/08/2004 Chott-Meriem (North East coastal region) Leaves Tomato Alternaria solani 23/08/2004 Sbiba (North) Roots Apple tree Pythium ultimum 05/07/2001 Jebeniena (Center) Roots and stems Watermelon Rhizoctonia solani 23/05/2001 Skhira (South) Roots Watermelon F. oxysporum f. sp. niveum 23/05/2001 Skhira (South) Roots Watermelon F. solani f. sp. cucurbitae 66 L. Sakka Rouis-Soussi et al. / South African Journal of Botany 91 (2014) 63–70

Table 2 carotenoid-derived compounds, hexahydrofarnesyl acetone (10, 1.9%) Abbreviations and yields of organic extracts and essential oils from different parts of Allium and (E)-β-ionone (4, 1.2%), were identified. Four sulfur compounds roseum var. grandiflorum subvar. typicum Regel. were identified namely dimethyl tetrasulfide (1), dipropyl trisulfide (2), Abbreviation Plant parts Extract/essential oil Yield (%) di-2-propenyl trisulfide (3) and di-2-propenyl tetrasulfide (6), which FlCh Flowers Chloroformic 1.109 accounted for 3.8% of the totality of the oil. FlEa Flowers Ethyl acetate 0.099 Hexadecanoic acid (syn. palmitic acid) was found to be the most FlB Flowers Butanolic 0.895 abundant component in some essential oils such as those from Oryza SLCh Stems and leaves Chloroformic 0.829 sativa L. (74.57%) (Sayaka and Mitsuo, 2006)andfromArdisia brevicaulis SLEa Stems and leaves Ethyl acetate 0.154 SLB Stems and leaves Butanolic 0.534 Diels leaves (43.32%) (Pu et al., 2009). In the case of Pyrenacantha BbCh Bulbs and bulblets Chloroformic 0.158 staudtii (Engl.) Engl., hexadecanoic (28.32%) and tetradecanoic BbEa Bulbs and bulblets Ethyl acetate 0.074 (20.60%) acids were the major components in the leaf essential oil BbB Bulbs and bulblets Butanolic 0.151 (Falodun et al., 2009). Hexadecanoic acid is also present in some Allium FlEO Flowers Essential oil 0.026 species, where it represented 19.03% and 16.75% of the totality of the es- LEO Leaves Essential oil 0.010 SEO Stems Essential oil 0.008 sential oil in Allium jesdianum Boiss aerial parts (Amiri, 2007)andAllium ursinum L. leaves, respectively (Blazewicz-Wozniak and Michowska, 2011). In contrast, only smaller percentages of hexadecanoic acid 3. Results and discussion (3.74%) and tetradecanoic acid (0.25%) were present in the essential oil of A. roseum var. odoratissimum (Desf.) Coss. flowers (Zouari et al., 3.1. Extract and essential oil yields 2013). Although, in the previous study by Ben Jannet et al. (2007), hexadecanoic acid was absent in essential oils from flowers and stems Table 2 gives abbreviation and the yield for each organic extract and of A. roseum var. grandiflorum. Hexahydrofarnesyl acetone was detected fl essential oil from A. roseum var. grandi orum. Extract yields varied from in very small amounts (0.6%) in the essential oil of Allium fl 0.074% (bulbs and bulblets' ethyl acetate extract) to 1.109% ( owers' sphaerocephalon L. inflorescences (Lazarevic Jelena et al., 2011) com- chloroform extract). The yield of the essential oil from stems, leaves pared to the results in this study. Methyl tetradecanoate (7) was also fl fl and owers of A. roseum var. grandi orum was 0.008, 0.010 and present in lower percentages in flower and stem essential oils (0.94 0.026% (w/w), respectively. and 2.47%, respectively) in the same species A. roseum var. grandiflorum studied by Ben Jannet et al. (2007). The latter authors also noted the 3.2. Chemical composition of A. roseum var. grandiflorum leaves' essential presence of methyl pentadecanoate (9) and methyl hexadecanoate oil (12) in stem oils (1.54 and 2.56%, respectively). Sulfur components were also detected in oils of some Allium species. The chemical composition of essential oils of flowers and stems of Dimethyl tetrasulfide (1) was present in small amounts in the essential A. roseum has been studied recently by Ben Jannet et al. (2007) while oils of A. roseum var. odoratissimum flowers (0.34%) (Zouari et al., 2013). that from the leaves has never been investigated. Di-2-propenyl trisulfide (3) was previously detected present in small The essential oil of A. roseum var. grandiflorum leaves was light yel- amounts in the leaves' essential oil of three A. ursinum ecotypes (0.19, low, liquid at room temperature and its odor was piquant. The compo- 1.82 and 2.83%, respectively) (Blazewicz-Wozniak and Michowska, sition of this oil was analyzed and reported for the first time in this 2011). Finally, di-2-propenyl tetrasulfide (6) was present in A. ursinum study. The identified components, their percentages, identification leaves' essential oil (1.97%) (Godevac et al., 2008). methods, calculated linear retention indices (LRIs) and their compari- son according to the LRI-values previously published in the literature are listed in Table 3. A total of 13 constituents accounting for 96.8% of 3.3. Polyphenol content in A. roseum var. grandiflorum extracts the whole oil were identified (Table 3). The leaf essential oil was rich in carboxylic acids. Hexadecanoic acid As shown in Table 4, the total phenol content in the nine organic ex- (13) was detected as the major component, with 75.9% of the totality of tracts ranged from 31.83 ± 0.04 to 193.27 ± 0.44 mg GAE/100 g DW. the oil. It was identified for the first time in the essential oil of this species. FlB extract contained the highest total phenol content, followed by Furthermore, tetradecanoic acid (8) and pentadecanoic acid (11)consti- BbEa extract. BbB, SLCh and SLB extracts have moderate total phenol tuted 2.9% of this oil. The three carboxylic acid esters (7, 9 and 12)were content compared to the lowest amount in FIEa extract. particularly abundant with 10% of the totality of this oil, which only meth- The highest level of total flavonoid content was measured in the FlB yl hexadecanoate (12) detected in appreciable amounts (8.5%). Two extract (Table 4), followed by the SLCh extract. Significant amounts

Table 3 Chemical composition of Allium roseum var. grandiflorum subvar. typicum Regel. leaves' essential oil as determined by GC, MS, RI.

Peak number Constituent Retention index (RI) Percentage

Calculated Literature (references)

1 Dimethyl tetrasulfide 1215 1215 (Bonaïti et al., 2005)0.4 2 Dipropyl trisulfide 1299 1302 (Mochizuki et al., 1998)0.7 3 Di-2-propenyl trisulfide 1376 1374 (Kim et al., 1995)1.7 4 (E)-β-Ionone 1487 1485 (Khanavi et al., 2004)1.2 5 1-Pentadecene 1490 1492 (Radulovic et al., 2011)0.7 6 Di-2-propenyl tetrasulfide 1540 1540 (Zoghbi et al., 2002)1.4 7 Methyl tetradecanoate 1727 1726 (Píno et al., 2004)0.7 8 Tetradecanoic acid 1770 1769 (Conforti et al., 2009)2.0 9 Methyl pentadecanoate 1825 1824 (Dickschat et al., 2005)0.8 10 Hexahydrofarnesyl acetone 1844 1843 (Lazarevic Jelena et al., 2011)1.9 11 Pentadecanoic acid 1868 1865 (Radulovic et al., 2011)0.9 12 Methyl hexadecanoate 1928 1919 (Quijano et al., 2007)8.5 13 Hexadecanoic acid 1963 1963 (Zeng et al., 2007)75.9

Percentage calculated by GC-FID on apolar capillary column HP-5. L. Sakka Rouis-Soussi et al. / South African Journal of Botany 91 (2014) 63–70 67

• were also obtained in SLB and FlCh extracts. Thus, the flower part and As shown in Table 5,IC50 values of the DPPH radical scavenging both stem and leaf parts have the highest content of total flavonoid in activity ranged from 0.35 ± 0.01 to 4.58 ± 0.06 mg/mL for the nine or- chloroform and butanol extracts, while a moderate content in ethyl ac- ganic extracts. Essential oils have an IC50 values N 5 mg/mL. Interesting etate extracts. In contrast, all extracts of bulbs and bulblets contained results were shown by the SLEa extract. This extract can be considered quite low amounts of these chemicals. to have a high percentage of inhibition of DPPH•, because after complet- The total flavonoid content increased from the ethyl acetate extract ing the reaction, the final solution always possessed some yellowish to the butanol extract then the chloroform extract for all the plant parts, color. FlEa, SLB and SLCh extracts also gave good results with IC50 values except in the case of the flowers where the total flavonoid content was of 0.54, 0.65, and 0.96 mg/mL, respectively. The different extracts of higher in butanol than in chloroform extract. A. roseum var. grandiflorum bulbs and bulblets have the lowest DPPH•

The total flavonol content ranged from 35.36 ± 0.19 to radical scavenging activity, with IC50 values ranging from 3.57 to 179.72 ± 0.69 mg rutin/100 g DW (Table 4). The highest level was 4.58 mg/mL. The DPPH• radical scavenging capacity of the organic ex- measured in FlB extract. Interesting results were also observed for chlo- tracts and essential oils has not been previously reported. roform and butanol extracts of stems and leaves (125.17 ± 0.85 and In earlier studies, the IC50 values for the Italian A. roseum were deter- 118.46 ± 0.94 mg/100 g DW, respectively). The lowest amount was mined for the flowers and leaves' aqueous extracts (1.01 ± 0.032 to detected in ethyl acetate extract for all plant parts. 3.08 ± 0.108 mg/mL, respectively) (Nencini et al., 2007) and in the The most significant of the observations was in the butanol flower leaves' hydroethanolic (aqueous ethanol) extract (6.59 ± 0.26 mg/mL) extract of A. roseum var. grandiflorum which were similar to the results (Nencini et al., 2011).Thesameauthorsreportedthatthebulbsandbulb- of Dziri et al. (2012), where the highest total phenol content and flavo- lets from the same species exhibited a lower effectiveness (Nencini et al., noid content were detected in methanol flower extract of A. roseum var. 2007, 2011), in good agreement with results in this study, where the odoratissimum (Table 4). bulbs are the part of the plant presenting with the weakest antioxidant Concerning the total flavonol content, interesting results were also activity. achieved in butanol and chloroform bulb extracts of A. roseum var. In the DPPH• assay, the free radical scavenging activities decreased grandiflorum. Previous studies reported high values for the total flavonol from ethyl acetate extracts to butanol and chloroform extracts for content in bulbs of A. cepa (Rhodes and Price, 1996; Rodrigues et al., each of the plant parts of A. roseum var. grandiflorum,evidencinga 2011). clear secondary metabolite content due to the solvent–solvent Analysis of the polyphenol content in A. roseum var. grandiflorum partitioning processes (Lixiang et al., 2009). indicated that the flowers' butanol extract contained the highest concentration of total phenol, flavonoid and flavonol. This organ- 3.4.2. ABTS•+ assay •+ dependant distribution of total polyphenols has recently been reported As shown in Table 5,theIC50 values of the ABTS radical scavenging for A. roseum var. odoratissimum and for other Allium species with the activity ranged from 0.71 ± 0.01 to 1.49 ± 0.02 mg/mL (Trolox fl owers and leaves being the richest organs in terms of total polyphenols IC50 = 0.273 ± 0.17 mg/mL). However, no 50% of inhibition was (Najjaa et al., 2011; Nencini et al., 2011). From a biological perspective, achieved for essential oils with concentrations up to 5 mg/mL, which the observed trend in polyphenol accumulation in some organs seems mimicked that observed in the DPPH• assay. to pinpoint that they may have a protective role (due to their antioxi- The best results were observed with the SLEa (correlating to DPPH• dant properties) against some abiotic factors, such as UV-B radiations assay also) and the SLB extracts followed by BbEa, SLCh, FlEa and FlB (Zouari et al., 2012). extracts. In contrast, FlCh and BbCh extracts had the lowest ABTS•+ rad- On the other hand, the obtained results with A. roseum var. ical scavenging activity. grandiflorum extracts showed that total polyphenol content was depen- The TEAC value is defined as the molar concentration of Trolox solu- dent on the solvent nature, because of the different degrees of polarity tion having an antioxidant capacity equivalent to the sample solution among different compounds (Jayaprakasha et al., 2001). being tested (Miraliakbari and Shahidi, 2008). Table 5 illustrates the TEAC values of A. roseum var. grandiflorum organic extracts, where BbB extract and essential oils did not show any activity. 3.4. Antioxidant activity of A. roseum var. grandiflorum organic extracts The best values of TEAC were obtained with the SLEa and SLB ex- and essential oils tracts (0.33 ± 0.00 and 0.32 ± 0.00 mg/mL, respectively). Statistically, all extracts were more active than Trolox, except the flowers and bulbs • 3.4.1. DPPH assay and bulblets' chloroform extracts (FlCh and BbCh) with the lowest TEAC The lower the IC50 value, the greater the free radical scavenging values (0.24 ± 0.00 and 0.22 ± 0.00 mg/mL, respectively). activity observed (Lixiang et al., 2009). All the organic extracts were If each organ (the flowers, or the leaves and stems) is examined sep- • able to reduce the stable, purple-colored radical, DPPH into the arately, it is remarkable to note that the best antioxidant activity was yellow-colored DPPH-H reaching 50% reduction. Table 5 • •+ Table 4 Antioxidant activity assessed by DPPH and ABTS radicals of Allium roseum var. grandiflorum subvar. typicum Regel. organic extracts. Total phenol, flavonoid and flavonol content of Allium roseum var. grandiflorum Regel. organic extracts. Samples DPPH• ABTS•+

Samples Total phenol Total flavonoid Total flavonol IC50 (mg/mL) IC50 (mg/mL) TEAC (mg/mL) (mgacidgallic/100gDW) (mgrutin/100gDW) (mgrutin/100gDW) FlCh 2.38 ± 0.09f 1.28 ± 0.02g 0.24 ± 0.00b FlCh 67.83 ± 1.84c 101.14 ± 0.41f 70.73 ± 1.52c SLCh 0.96 ± 0.01d 0.90 ± 0.01d 0.29 ± 0.00f SLCh 80.48 ± 1.98e 145.77 ± 0.67h 125.17 ± 0.85g BbCh 4.58 ± 0.06i 1.49 ± 0.02h 0.22 ± 0.00a BbCh 39.50 ± 1.69b 23.22 ± 0.14c 109.41 ± 1.48e FlEa 0.54 ± 0.00c 1.04 ± 0.02e 0.27 ± 0.00e FlEa 31.83 ± 0.04a 44.03 ± 0.06d 35.36 ± 0.19a SLEa 0.35 ± 0.01b 0.71 ± 0.01b 0.33 ± 0.00i SLEa 39.13 ± 0.19b 70.13 ± 0.27e 50.09 ± 0.19b BbEa 3.57 ± 0.01g 0.84 ± 0.00c 0.30 ± 0.00g BbEa 126.19 ± 0.38f 3.10 ± 0.02a 36.21 ± 0.12a FlB 1.47 ± 0.01e 1.08 ± 0.02f 0.26 ± 0.00d FlB 193.27 ± 0.44g 171.77 ± 1.33i 179.72 ± 0.69h SLB 0.65 ± 0.02c 0.72 ± 0.01b 0.32 ± 0.00h SLB 75.09 ± 0.82d 115.57 ± 0.80g 118.46 ± 0.94f BbB 3.97 ± 0.18h N5.00 b0.15 BbB 81.54 ± 1.66e 5.11 ± 0.15b 79.51 ± 0.76d Trolox 0.078 ± 0.00a 0.273 ± 0.00a 0.25 ± 0.00c

Means with the same letter in a column are not significantly different at p N 0.05 (ANOVA Means with the same letter in a column are not significantly different at p N 0.05 (ANOVA test). test). 68 L. Sakka Rouis-Soussi et al. / South African Journal of Botany 91 (2014) 63–70 generally found in the ethyl acetate extracts followed by butanol and An interesting inhibition of Alternaria solani was observed for the chloroform extracts in the order. TEAC values of the extracts decreased stems and leaf extracts, where SLB was as potent as benomyl on myce- in the following order: SLEa N SLB N SLCh; FlEa N FlB N FlCh. Ethyl lium growth, followed by SLCh and SLEa. While a moderate inhibition acetate seems to be the solvent that best concentrates antioxidant sub- was reported for FlCh and BbCh. All the other extracts and essential stances of intermediate polarity; this is in accordance with the findings oils did not inhibit this fungus. Pythium ultimum was inhibited only by of Anagnostopoulou et al. (2006). the butanol extracts of bulbs and bulblets (BbB: 42.31%) and of stems In this study, both leaves' and stems' extracts of A. roseum var. and leaves (SLB: 40.38%). The hyphal growth of Rhizoctonia solani and grandiflorum possessed the higher TEAC values and best antioxidant ac- Fusarium oxysporum f. sp. niveum was only reduced by the two extracts, tivity. In the literature, the highest antioxidant activity was observed in SLEa and BbCh (19.23% for each sample). the leaves of Allium schoenoprasum L., Allium giganteum L. and A. roseum In general, organic extracts and essential oils of A. roseum var. (Stajner et al., 2004, 2006, 2008). grandiflorum showed strong antifungal activity against F. solani f. sp. cucurbitae. This is supported by previous studies on the antifungal activ- ity of A. sativum against F. solani (Mart.) Sacc. and other fungi, where the 3.5. Antifungal activity A. sativum extract completely inhibited the mycelium growth of F. solani (Shrestha and Tiwari, 2009). In another study, the mycelial develop- The antifungal activity of the tested essential oils and the different ment of the phytopathogenic fungi including F. solani and R. solani organic extracts from A. roseum var. grandiflorum on some fungi varied Kühn was strongly inhibited by A. sativum extract (Bianchi et al., according to the type of solvent used, the essential oil and the fungus 1997). In addition, the growth of P. ultimum var. ultimum was entirely tested (Table 6). blocked by aqueous extract of A. sativum (Bianchi et al., 1997). Singh Interesting results were obtained against Fusarium solani f. sp. and Singh (1980) also found that suppressed sclerotial forma- cucurbitae. In fact, all organic extracts and all essential oils have antifun- tion in R. solani and killed the microorganism when hyphal disks were gal activity, except the FlB extract. The percentage of mycelium growth exposed to the oil in the Petri plate assay. It was concluded that the in- inhibition ranged from 28 to 56% compared to the positive control ben- hibition of sclerotia was in part due to the result of the inhibition of hy- omyl (69.80%). SLEa and BbCh extracts displayed a highly significant phal growth (Singh and Singh, 1980). growth inhibition of this fungus. Pârvu et al. (2009, 2010, 2011) and Pârvu and Pârvu (2011) investi- BbB, FlEa and FlCh extracts revealed potential to inhibit the myceli- gated the antifungal activities of some Allium sp. In fact, Allium obliquum um growth of F. solani f. sp. cucurbitae with a value of the inhibition of L. hydroethanolic extracts inhibited B. cinerea and F. oxysporum (Pârvu 36% for each extract. SLCh extract has the lowest inhibition against et al., 2009)andAllium fistulosum extract had antifungal effects against F. solani f. sp. cucurbitae. As for the essential oils, LEO exhibited the F. oxysporum (Pârvu et al., 2010). Furthermore, Pârvu et al. (2011) men- highest inhibition with 39.13%, followed by FlEO then SEO. tioned that A. ursinum flower and leaf hydroethanolic extracts had anti- The antifungal growth inhibition varied from 30.43 to 52.17% for fungal effects against B. cinerea and F. oxysporum. In another study, the organic extract against Botrytis cinerea, with the exception of butanol ex- inhibitory effect of the A. ursinum flower extract was stronger than tracts (FlB. SLB and BbB) which showed no effect against this fungus. In that of the leaf extract for all concentrations and for all the tested contrast, the most active extracts were the chloroform and ethyl acetate fungi (Pârvu and Pârvu, 2011). samples of the different parts of A. roseum var. grandiflorum. B. cinerea In our study, we noted that the inhibition growth of B. cinerea by the was also significantly inhibited by the leaf essential oil (52.50%). flower chloroform extract was higher than that by the stems and leaves'

Table 6 Effect of different organic extracts and essential oils of Allium roseum var. grandiflorum subvar. typicum Regel. on mycelium growth (mm) and percentage of inhibition (%) of phytopathogenic fungi.

Extracts/essential oils Fusarium solani f. sp. cucurbitae Botrytis cinerea Alternaria solani Pythium ultimum Rhizoctonia solani Fusarium oxysporum f. sp. niveum

FlCh 32.00 ± 1.00de 28.00 ± 1.00e 37.00 ± 1.00cd 52.00 ± 0.00d 52.00 ± 0.00c 52.00 ± 0.00c (36%) (39.13%) (22.92%) (0%) (0%) (0%) SLCh 38.00 ± 2.00g 32.00 ± 1.73f 33.00 ± 1.73b 52.00 ± 0.00d 52.00 ± 0.00c 52.00 ± 0.00c (24%) (30.43%) (31.25%) (0%) (0%) (0%) BbCh 28.00 ± 1.32c 26.00 ± 2.65de 38.00 ± 2.00d 52.00 ± 0.00d 52.00 ± 0.00c 42.00 ± 1.00b (44%) (43.48%) (20.83%) (0%) (0%) (19.23%) FlEa 32.00 ± 1.80de 24.00 ± 2.65cd 48.00 ± 0.00f 52.00 ± 0.00d 52.00 ± 0.00c 52.00 ± 0.00c (36%) (47.83%) (0%) (0%) (0%) (0%) SLEa 22.00 ± 1.00b 22.10 ± 1.00c 36.00 ± 1.00c 52.00 ± 0.00d 42.00 ± 1.00b 52.00 ± 0.00c (56%) (52.17%) (25%) (0%) (19.23%) (0%) BbEa 36.00 ± 0.00fg 28.00 ± 0.00e 48.00 ± 0.00f 52.00 ± 0.00d 52.00 ± 0.00c 52.00 ± 0.00c (28%) (39.13%) (0%) (0%) (0%) (0%) FlB 50.00 ± 0.00h 46.00 ± 0.00h 48.00 ± 0.00f 52.00 ± 0.00d 52.00 ± 0.00c 52.00 ± 0.00c (0%) (0%) (0%) (0%) (0%) (0%) SLB 34.00 ± 0.50ef 46.00 ± 0.00h 30.00 ± 2.65a 31.00 ± 1.50c 52.00 ± 0.00c 52.00 ± 0.00c (32%) (0%) (37.50%) (40.38%) (0%) (0%) BbB 32.00 ± 1.00de 46.00 ± 0.00h 48.00 ± 0.00f 30.00 ± 1.00b 52.00 ± 0.00c 52.00 ± 0.00c (36%) (0%) (0%) (42.31%) (0%) (0%) LEO 28.00 ± 2.18c 19.00 ± 1.73b 42.00 ± 0.00e 52.00 ± 0.00d 52.00 ± 0.00c 52.00 ± 0.00c (39.13%) (52.50%) (0%) (0%) (0%) (0%) SEO 32.00 ± 2.00de 40.00 ± 0.00g 42.00 ± 0.00e 52.00 ± 0.00d 52.00 ± 0.00c 52.00 ± 0.00c (30.43%) (0%) (0%) (0%) (0%) (0%) FlEO 30.00 ± 1.73cd 40.00 ± 0.00g 42.00 ± 0.00e 52.00 ± 0.00d 52.00 ± 0.00c 52.00 ± 0.00c (34.78%) (0%) (0%) (0%) (0%) (0%) Benomyl 10.10 ± 1.00a 5.00 ± 0.00a 28.50 ± 2.00a 6.30 ± 1.50a 5.90 ± 0.50a 5.00 ± 1.00a (69.80%) (92%) (25.5%) (76.10%) (90.30%) (86.70%)

Benomyl as positive control (1 mg/mL by a disk). Means with the same letter in a column are not significantly different at p N 0.05 (ANOVA test). L. Sakka Rouis-Soussi et al. / South African Journal of Botany 91 (2014) 63–70 69 chloroform extract (Table 6). In contrast, the inhibition of growth by the Bianchi, A., Zambonelli, A., D'Aulerio, Z.A., Bellesia, F., 1997. Ultrastructural studies of the fl effects of Allium sativum on phytopathogenic fungi in vitro. Plant Disease 81, ower ethyl acetate extract was lower than stems and leaves' ethyl 1241–1246. acetate extract against B. cinerea. Concerning Alternaria sp., Zarei Blazewicz-Wozniak, M., Michowska, A., 2011. The growth, flowering and chemical com- Mahmoudabadi and Gharib Nasery (2009) reported a remarkable position of leaves of three ecotypes of L. Acta Agrobotanica 64, 171–180. activity of Allium ascalonicum L. bulbs aqueous extract. Bonaïti, C., Irlinger, F., Spinnler, H.E., Engel, E., 2005. 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