Phenolic content, antioxidant and allelopathic activities of various extracts of Thymus numidicus Poir. organs

Industrial Crops and Products 62 (2014) 188–195

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Phenolic content, antioxidant and allelopathic activities of various extracts of Thymus numidicus Poir. organs Imen Ben El Hadj Ali a,∗ , Radhia Bahri b , Maher Chaouachi b , Mohamed Boussaïd a , Fethia Harzallah-Skhiri b a

Laboratory of Plant Biotechnology, National Institute of Applied Sciences and Technology, Carthage University, B.P. 676, 1080 Tunis, Tunisia Laboratory of Genetic Biodiversity and Valorisation of Bioresources, Higher Institute of Biotechnology of Monastir, University of Monastir, Monastir, Tunisia b

a r t i c l e

i n f o

Article history: Received 12 April 2014 Received in revised form 12 August 2014 Accepted 17 August 2014 Keywords: Thymus numidicus Organ Polyphenols Antioxidant capacity Phytotoxic activity

a b s t r a c t Total phenol content, antioxidant and allelopathic properties of roots, steems and leaves extracts of Tunisian endemic T. numidicus were studied. Three solvent systems with varying polarities (petroleum ether, ethyl acetate and methanol) were used. The highest amounts of polyphenols (98.66 ± 3.17 mg EAG/g DW), flavonoids (54.28 ± 1.6 mg RE/g DW), flavonols (27.23 ± 1.71 mg RE/g DW) and proanthocyanidins (5.12 ± 0.8 ␮g cyanidin chloride/g DW) were shown by the polar subfraction of the leaf methanolic extracts. The efficiency of the solvents used to extract phenols from the three organs varied considerably. The level of antioxidant activity estimated by DPPH and ABTS test systems was high for leaf (IC50 = 11.06 ± 0.33 ␮g/ml; 235.46 ± 2.14 ␮g TE/mg DW) and stems (IC50 = 15.29 ± 0.9 ␮g/ml; 233.75 ± 1.2 ␮g TE/mg DW) methanolic extracts. A significant correlation between radical-scavenging capacities of extracts with total phenolic compound content was observed. The results also indicate that the root extracts inhibited the shoot and root growth of Medicago sativa and Triticum æstivum seedlings. Thus, the T. numidicus extracts may be used as a natural herbicide. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Tunisian flora includes more than 350 spontaneous species considered as medicinal and aromatic plants and used in traditional phytotherapy (Le Floc’h, 1983). Among the aromatic plants, the Thymus genus, belonging to the Lamiaceae family, is noteworthy for the numerous species and varieties of wild-growing plants. This genus comprises about 400 species of perennial aromatic plants with many subspecies, varieties, subvarieties and forms. The plants are extensively used, fresh and dried, as a culinary herb (Dob et al., 2006). In recent years, several reports have been published concerning the composition and the biological properties of the essential oils and extracts of this genus. In deed, Thymus species are widely used in pharmaceutical, cosmetic and perfume industry, and for flavoring and preservation of several food products (Hazzit et al., 2009). Also, they are employed in popular medicinal for its expectorant, antitussive, analgesic, antibroncholitic, antispasmodic,

∗ Corresponding author. Tel.: +216 71703829/929. E-mail address: [email protected] (I. Ben El Hadj Ali). http://dx.doi.org/10.1016/j.indcrop.2014.08.021 0926-6690/© 2014 Elsevier B.V. All rights reserved.

carminative and diuretic effects (Dob et al., 2006; Hazzit et al., 2009). Others findings suggested that Thymus essential oils have been widely used mainly due to their antibacterial, antifungal, antioxidant and anti-inflammatory properties (Giordani et al., 2008; Hazzit et al., 2009; Mahmoudi et al., 2008). Thymus numidicus Poir. is an endemic species of Tunisian North-West and Algeria (Pottier-Alapetite, 1981). It is a short lived and outcrossing shrub predominantly bee-pollinated. It is a hermaphrodite species and possess a capacity for asexual reproduction by either vegetative propagation (Pottier-Alapetite, 1981). It can reach 10–15 cm in height. Leaves are opposite and linear/lanceolate (4–15 mm). Flowers are hermaphrodite, large (15 mm) and grouped in dense terminal heads with an uneven calyx (3 mm) and a pink corolla (6 mm). Flowering takes place between April and June (Pottier-Alapetite, 1981). In Tunisia, T. numidicus populations are distributed from the lower humid to the upper semi-arid bioclimates at altitudes ranging from 450 to 1100 m (Nabli, 1995). The species grows on poor fertile calcareous soils and occurs in scattered small populations. Previous work on Algerian T. numidicus showed that essential oils showed strongly antibacterial activity, particularly against

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Bacillus subtilus, Staphylococcus aureus and Enterobacter aerogenes (Kabouche et al., 2005). Moreover, thymol (68.2%), carvacrol (16.92%) and linalool (11.5%) were the main identified compounds (Kabouche et al., 2005). The flavonoids of T. numidicus was also investigated (Benkiniouar et al., 2010). Plant phenols exhibit significant antioxidant, antitumoral, antiviral, anti-inflammatory and antibiotic properties (Apak et al., 2007; Zhang et al., 2011). Phenolics are antioxidants with redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers. In recent years, there is a wide interest in finding phytochemicals from natural sources that could replace synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) which are commonly used in food industry because of their toxicity (Canadanovic-Brunet et al., 2006). Antioxidants such as flavonoids, phenolics, terpenoids, flavonols, proanthocyanidins and tannins are found in various plant products (Jeong et al., 2004). For this reason, there is a growing interest in separating these plant antioxidants and using them as natural antioxidants. However, there are several methods established for the extraction of polyphenols from plant materials. Those methods vary in solvents and conditions used. Therefore, the extraction method is essential for the accurate quantification of antioxidant content and capacity (Alothman et al., 2009; Santas et al., 2008). The aims of this study were: (i) to assess the polyphenols, flavonoids, flavonols and proanthocyanidins contents of various extracts from roots, stems and leaves of Tunisian T. numidicus; and (ii) to investigate their antioxidant and allelopathic activities. 2. Materials and methods 2.1. Plant material The starting material consists of mature plants, at the vegetative stage, growing wild in the northwest area of Tunisia, to be precise from the Jendouba region. From each plant branches with leaves and roots were collected and transported to the laboratory. The fresh plants were separated into roots, leaves and stems. Before analyses, specimens were air-dried at room temperature for 16 days. The moisture content of samples was 3.5%. The studied species was identified at the Department of Botany, Higher Institute of Biotechnology of Monastir (ISBM, Tunisia) and voucher specimens (number: ThN. 1) are kept at the herbarium of the of the Department of Botany in the cited institute. 2.2. Chemical and reagents DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid)), Trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid), gallic acid, Na2 CO3 , AlCl3 6H2 O, C2 H3 NaO2 , rutin, cyanidin chloride, nBuOH–HCl, NH4 Fe(SO4 )2 and Folin–Ciocalteu reagent, were purchased from Sigma–Aldrich (St. Louis, MO). All solvents and reagents used were of the highest purity. 2.3. Preparation of the plant extracts Plant materials were ground and macerated for extraction. Eighty grams of each organ were weighed into 1 l Erlenmeyer flasks, and then 400 ml of different solvents of increasing polarity (petroleum ether, ethyl acetate and methanol) were added to the plant organs. After filtration through filter paper (Whatman No. 4), the residue was re-extracted twice, and then the combined extracts of every organ were evaporated at room temperature. For water extraction, a 20 g of each organ was mixed with 100 ml of boiling water and filtered through Watman No. 1 paper. Extraction

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was carried out by shaking at room temperature for 3 days. Each extract was stored in a brown bottle at 4 ◦ C prior to further analysis. 2.4. Phenolic compounds content 2.4.1. Total phenolic content The total phenolic content of organs was assessed using the Folin–Ciocalteu reagent, following Singleton and Rosi’s (1965) method, based on the reduction of a phosphowolframate–phosphomolybdate complex by phenolics to blue reaction products and slightly modified by Dewanto et al. (2002). An aliquot of each diluted sample extract was mixed with 2.5 ml Folin–Ciocalteu reagent. 2 ml of sodium carbonate solution (7.5%) was added to the mixture after 3 min. After incubation (90 min) in dark, the absorbance at 760 nm was read versus the prepared blank. The standard curve was prepared by solutions of gallic acid in methanol. The concentration of total phenolic compounds in the extracts was determined as ␮g of gallic acid equivalent using an equation obtained from the standard gallic acid graph, and expressed as mg gallic acid/g dry weight of the plant material (mg EGA/g DW). The data were presented as the average of triplicate analyses. 2.4.2. Total flavonoid content The total flavonoid contents of plant samples were determined according to aluminum chloride colorimetric method (Djeridane et al., 2006). Each extract was dissolved in 1 ml of the appropriate solvent. 1 ml of this solution was mixed with 1 ml of 2% AlCl3 methanolic solution. After incubation at room temperature for 15 min, the absorbance was measured at 430 nm. Rutin was chosen as a standard. Using a standard curve, the levels of total flavonoid contents in the sample extracts were determined in triplicate, respectively. Total flavonoids were expressed as mg rutin equivalent/g DW (mg RE/g DW). Analyses were done in triplicate. 2.4.3. Total flavonol content The content of flavonols was determined according to aluminum chloride colorimetric method by Miliauskas et al. (2004). Each extract was dissolved in 1 ml of the appropriate solvent. 1 ml of this solution was mixed with 1 ml of AlCl3 methanolic and 3 ml of sodium acetate solutions. The absorption at 440 nm was read after 2h 30 min at 20 ◦ C. Rutin was chosen as a standard. Using a standard curve, the levels of total flavonol contents in the sample extracts were determined in triplicate, respectively. Total flavonols were expressed as mg rutin equivalent/g DW (mg RE/g DW). All determinations were carried out in triplicate. 2.4.4. Total proanthocyanidin content The total amount of proanthocyanidins was measured using the HCl/butan-1-ol assay described by Bahorun et al. (2003). To each stoppered tube, 0.5 ml of the extracts was added, flowed by 6 ml of an n-BuOH–HCl solution (95:5, v/v) and 0.2 ml of NH4 Fe–(SO4 )2 12H2 O in 2 M HCl. The tubes were thoroughly vortexed and incubated for 40 min at 100 ◦ C. After cooling in the dark, the red coloration was read at 550 nm against a blank standard. The amount of total proanthocyanidins was expressed in ␮g cyanidin chloride/g of dry weight (DW). All samples were analyzed in three replications. 2.5. Antioxidant activity evaluation The antioxidant activity of T. numidicus extracts from all organs was assessed using free radical-scavenging activity (RSA) with DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2 -azinobis-(3ethylbenzothiazoline-6-sulphonic acid)) radical assay.

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2.5.1. DPPH radical scavenging activity The evaluation of the free radical-scavenging activity of extracts was based on the measurement of the reducing ability of antioxidants toward the DPPH radical. The method described by Brand-Williams et al. (1995) was used with slight modifications. 20 ␮l of different extracts (at different concentrations) was added to 980 ␮l of the methanolic DPPH solution (90 ␮M). The reaction was allowed to stand at room temperature in the dark for 30 min and the absorbance was recorded at 517 nm against a blank (methanol solution) using a UV–vis spectrophotometer. The measurements were performed in triplicate. The radical-scavenging activity was calculated using the following equation: I% = [(AB − AA)/AB] × 100, where I is the DPPH• inhibition, %; AB and AA are the absorbance values of the control and of the test sample, respectively. DPPH• scavenging activity is presented by IC50 value, defined as the concentration of the antioxidant needed to scavenge 50% of DPPH• present in the test solution. All tests were carried out in triplicate and IC50 values were reported as means ± SD of triplicates. 2.5.2. ABTS radical cation decolourisation assay The determination of ABTS•+ radical scavenging was carried out as reported by Dorman and Hiltunen (2004). This assay assesses the total radical-scavenging capacity based on the ability of a compound to scavenge the stable ABTS radical (ABTS•+ ). The ABTS•+ radical cation was produced by reacting ABTS with potassium persulfate (K2 S2 O8 ) (Re et al., 1999). The ABTS•+ was produced by the reaction between 7 mM ABTS in water and 2.45 mM potassium persulfate, stored in the dark at room temperature for 12 h. Before usage, the ABTS•+ solution was diluted to get an absorbance of 0.703 ± 0.025 at 734 nm. 20 ␮l of different extracts was added to 980 ␮l ABTS•+ solution and the absorbance at 734 nm was measured. Sample absorbance was compared to a blank where 20 ␮l of the solvent were added to 980 ␮l of the ABTS•+ solution. The absorbance was read at ambient temperature at 6 min after addition of the antioxidant. The standard curve was linear between 0 and 250 ␮g/ml Trolox. Additional dilution was needed if the ABTS value measured was over the linear range of the standard curve. All determinations were performed in triplicate. Results were expressed in inhibition percentage versus samples concentrations (mg/ml). The percentage decrease of the absorbance at 734 nm was calculated by formula: %inhibition = [(AB − AA)/AB] × 100, where I is the ABTS•+ inhibition %; AB and AA are the absorbance values of the control and of the test sample, respectively. Thereafter, results are expressed in ␮g of Trolox equivalents (TE) per milligrams of dry weight (␮g TE/mg DW). 2.6. Phytotoxic assay The inhibitory potential of the T. numidicus extracts obtained from roots, stems and leaves on the seed germination, the hypocotyl and root lengths and the seedling dry weight of Medicago sativa L. and Triticum æstivum L. seeds was investigated. Different concentrations (0.2, 0.4, 0.6, 0.8 and 1 mg/ml) of extracts were dispersed in sterile Petri dishes (9 cm diameter) lined with double-sterile filter paper (Whatman No. 2). M. sativa and T. æstivum seeds were surface sterilized for 20 min in 1% NaClO before use. Dishes prepared without solvent were used as a negative control. Then, 4 ml of distilled water was added to each Petri dishes, those were sealed with Parafilm to prevent water loss and stored in the dark at 25 ◦ C for 7 days. A seed was considered germinated, when the protrusion of the radical became evident. Seeds that did not germinate were considered to have a radical length of 0 mm. All phytotoxic assays were conducted in triplicate, and in total 60 seedlings were measured for each target species. After 7 days, the germination percentage was determined. Then, the seedlings of M. sativa and T. æstivum seeds were collected,

the hypocotyls and radical lengths were measured, and the fresh and dry weights per Petri dish were determined to evaluate the allelopathic activity of the T. numidicus extracts. The inhibitory or stimulatory effects were calculated using the following equation, with slight modifications from Chung et al. (2001): Inhibition (−)/stimulation (+) % = ((EXe − Ce)/Ce) × 100; where EXe (extract effect) is the parameter measured in the presence of T. numidicus extracts and Ce (control effect) the parameter measured in the presence of distilled water. 2.7. Statistical analysis Each assay was done three times from the same extract in order to determine their reproducibility. Data were performed using the software using the SPSS version 13.0 for Windows. Quantitative differences were assessed by ANOVA procedure followed by Duncan’s multiple range test. Values were expressed as means ± standard deviations (SD). Differences were considered significant at p < 0.05. Correlations among data obtained were calculated using Spearman coefficient (r) using Spearman–Kendall’s rank test (Saez, 1995). The significance of the correlation was tested after 1000 permutations. 3. Results and discussion 3.1. Extract yields and total phenolic contents The yield extracted from the roots, stems and leaves of Tunisian endemic T. numidicus, collected at the vegetative stage, by three different solvents with varying polarities (petroleum ether, ethyl acetate and methanol) is reported in Table 1. It is apparent from this current work that the highest extract yield was obtained by methanol extraction of all organs, followed by ethyl acetate and by petroleum ether. In different organs, the yields of methanolic extracts were significantly high: 12.92% for roots, 22.77% for stems and 28.58% for leaves. However, the petroleum ether efficiency as a solvent was lower than the efficiencies of all other solvents. Thus, the variation in the yields of various extracts can be attributed to the polarities of the different compounds in the different organs. Such differences have been reported in the literature (Khlifi et al., 2011). There is no other study in the literature which investigated the yield organs extracted with different solvents in this endemic species. The total phenolic, flavonoid, flavonol and proanthocyanidin contents varied significantly among the studied parts in the different extracts of the studied species (Table 1). Leaves, determined in the methanolic extract, had the highest contents of polyphenols, flavonoids, flavonols and proanthocyanidins: 98.66 ± 3.17 mg EAG/g DW, 54.28 ± 1.6 mg RE/g DW, 27.23 ± 1.71 mg RE/g DW and 5.12 ± 0.8 ␮g cyanidin chloride/g DW, respectively. However, roots present lower level of all contents: 23.76 ± 1.29 mg EAG/g DW of phenols, 13.67 ± 1.1 mg RE/g DW of flavonoids, 12.96 ± 1.9 mg RE/g DW of flavonols and 0.35 ± 0.2 ␮g cyanidin chloride/g DW of proanthocyanidins. This level of total phenols were found to be lower than the values reported in the literature for other Thymus species such as T. caramanicus (124.30 ± 2.62 ␮g/mg) reported by Safaei-Ghomi et al. (2009), T. spathulifolius (141 ␮g/mg of the polar subfraction of a methanol extract) reported by Sokmen et al. (2004) and T. serpyllum (113 ␮g/mg of an ethanol extract) reported by Mata et al. (2007). The variance analysis performed on averages of these compounds analyzed for roots, stems and leaves showed significant organ and solvent effects (Table 1). Results revealed that methanol was better solvents than the others in extracting polyphenol, flavonoid, flavonol and proanthocyanidin compounds due to their polarity and good solubility for phenolic components from plant materials. Therefore, the recovery of polyphenols from plant

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Table 1 Average of total polyphenol, flavonoid, flavonol and proanthocyanidin contents according to organs and solvent extraction systems. Polyphenols (mg EAG/g DW)

Flavonoids (mg RE/g DW)

Flavonols (mg RE/g DW)

Petroleum ether extracts 1.4 Roots Stems 2.4 Leaves 4.39

8.72 ± 0.83a 14.29 ± 2.16a 26.31 ± 1.65b

7.59 ± 2.77a 11.87 ± 0.99a 13.48 ± 0.73a

1.08 ± 0.22a 3.9 ± 0.99a 5.36 ± 1.04a

0.27 ± 0.11a 0.44 ± 0.21a 0.83 ± 0.12ab

Ethyl acetate extracts Roots Stems Leaves

6.14 8.94 10.5

16.73 ± 1.9a 38.04 ± 2.4c 80.63 ± 1.62d

11.52 ± 0.91a 20.16 ± 2.27b 41.49 ± 2.07c

5.91 ± 1.12a 8.08 ± 1.4ab 15.15 ± 1.9b

0.64 ± 0.12ab 1.12 ± 0.35b 1.91 ± 0.9b

Methanol extracts Roots Stems Leaves

12.92 22.77 28.58

23.76 ± 1.29b 81.85 ± 2.75d 98.66 ± 3.17d

13.67 ± 1.1a 38.11 ± 0.98c 54.28 ± 1.6d

12.96 ± 1.9b 19.09 ± 1.5c 27.23 ± 1.71c

0.35 ± 0.2a 1.71 ± 0.25b 5.12 ± 0.8c

Assay

Yields (%)

Proanthocyanidins (␮g CC/g DW)

Means in each column followed by different letters are significantly different (p < 0.05).

materials is influenced by the solubility of the phenolic compounds in the solvent used for the extraction process (Kequan and Liangli, 2006; Roby et al., 2013). Furthermore, solvent polarity will play a key role in increasing phenolic solubility (Naczk and Shahidi, 2006). So, it could be concluded from the results that polar fractions had more phenolics in them than had non-polar fractions. Hence, this could be used as an important descriptor to characterize the extracts of T. numidicus. These results are almost similar to those reported by Hernandez-Hernandez et al. (2009). The same finding is reported for other medicinal plants such as T. vulgaris, Salvia officinalis and Origanum majorana (Roby et al., 2013), Rosmarinus officinalis and Origanum vulgare (Hernandez-Hernandez et al., 2009) and Globularia alypum (Khlifi et al., 2011). On the other hand, our results revealed also that leaves of T. numidicus at vegetative stage are characterized by high amounts of phenols, flavonoids, flavonols and proanthocyanidins. Thus, the high rate of these compounds should be explained by the beginning of senescence of organs accompanied by biochemical, physiological and molecular changes evolving phenol synthesis changes. Then, the differential accumulation of these compounds between organs should be related to their specific tissues and cells (i.e. mesophyll, epiderm, thickness cuticle, chloroplasts, trichomes). Previous reports have attributed the high presence in leaves of these compounds and their low content in roots to the close interaction between organs and to the different processes of biosynthesis and/or degradation, to the transport involved in the distribution of these polyphenols at the plant level and to the phenological organ growth (Fico et al., 2000; Hudaib et al., 2002).

extracts for roots. Also, these activities are significantly higher than that of the synthetic antioxidant BHT (butylated hydroxytoluene) with IC50 = 25 ␮g/ml and the trolox with IC50 = 32.0 ␮g/ml, as a standard reference product. We can deduce, that the extracts obtained using high polarity solvents were considerably more effective radical-scavengers than were those using low polarity solvents. These extracts exhibited also the highest ABTS value (235.46 ± 2.14 ␮g TE/mg DW and 233.75 ± 1.2 ␮g TE/mg DW in methanol extracts for leaves and stems, respectively). This result confirms the agreement between the solvent polarity and antioxidant activity where the methanol extract presented higher total phenolic quantity. Change in solvent polarity alters its ability to dissolve a selected group of antioxidant compounds and influences activity estimation. Our results are comparable to previous reports which attested an important in vitro antioxidant activity of various extracts of Thymus genus such T. praecox subsp. skorpilii var. skorpilii (Ozen et al., 2011) and T. vulgaris (Roby et al., 2013). While, the Tunisian T. numidicus radical-scavenging activity can be considered high compared to the results reported on the other members of Thymus family such as T. caramanicus methanol extract (IC50 = 43 ␮g/ml) reported by Safaei-Ghomi et al. (2009), highlighting the considerable potential of this plant as an antioxidant food additive. In this study, the high free radical-scavenging inhibition activity evaluated by the DPPH• and the ABTS•+ radical scavenging test may be related to the presence of flavonoid-type compounds and other phenolics. So, the high contents of polyphenol compounds in the studied species contribute to their important antiradical and antioxidative activities. Others findings reports the presence of different phenolic compounds in the Thymus genus such as rosmarinic acid, caffeic acid, ferulic acid, carnosic acid, quinic acid, p-coumaric acid, caffeoylquinic acid derivative, quercetin7-o-glucoside, cinnamic acid, methyl rosmarenate, nareingenin, luteolin-7-o-rutinose and ferulic acid derivative, and flavonoids such as 5,7,4 -trihydroxyflavone (apigenin); 4 -dihydroxy-6,7,8trimethoxyflavone (xanthomicrol); 5,7,3 ,4 -tetrahydroxyflavone 5,3 ,4 -trihydroxy-6,7,8-trimethoxyflavone (sider(luteolin); itoflavone) and 5-hydroxy-6,7,3 ,4 -tetramethoxyflavone

3.2. Antioxidant activity The different extracts of T. numidicus exhibited remarkable reduction activities (Table 2). The radical-scavenging activity, expressed as inhibition concentration (IC50 ), varied according to solvent and organ extracts (Table 2). The best activity was observed in methanol extracts for leaves (IC50 = 11.06 ± 0.33 ␮g/ml) and stems (IC50 = 15.29 ± 0.9 ␮g/ml), and the lowest (IC50 = 66.6 ± 1.22 ␮g/ml) in petroleum ether

Table 2 Total antioxidant capacity determined by DPPH and ABTS test systems of Thymus numidicus extracts according to organs obtained from different solvent extraction systems. Assay

Petroleum ether extracts Ethyl acetate extracts Methanol extracts

DPPH IC50 (␮g/ml)

ABTS (␮g TE/mg DW)

Roots

Stems

Leaves

Roots

Stems

Leaves

66.6 ± 1.22b 39.1 ± 1.03ab 22.51 ± 0.99a

58.1 ± 1.0b 29.06 ± 0.95a 15.29 ± 0.9a

47.31 ± 0.72b 23.51 ± 1.15a 11.06 ± 0.33a

62.12 ± 2.04a 173.14 ± 2.41b 217.32 ± 6.5b

68.26 ± 2.66a 206.83 ± 1.34b 233.75 ± 1.2b

78.97 ± 1.92a 220.41 ± 1.69b 235.46 ± 2.14b

Means in each column followed by different letters are significantly different (p < 0.05).

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(5-desmethylsinensetin) (Benkiniouar et al., 2010; Jordan et al., 2009; Loziene et al., 2007; Roby et al., 2013). However, flavonoids interrupt the propagation of the free radical autoxidation chain by contributing a hydrogen atom from a phenolic hydroxyl group, with the formation of a relatively stable free radical that does not initiate or propagate further oxidation processes (Bahramikia et al., 2009). The presence of these compounds in the subfraction of T. numidicus extracts from different organs may also be the main cause of its high radical-scavenging activity and high total phenolic contents. Furthermore, flavonoids are a group of polyphenolic components with known various properties such as inhibition of hydrolytic and oxidative enzymes, anti-inflammatory action, to enhance human immunity, reduction blood-lipid and glucose, and free radical scavenging (Atoui et al., 2005). Those findings justify the use of different plant organs of the studied species by ancient and actual local human populations. According to Zhang et al. (2011), the antioxidant activity is generally attributed to phenolic and flavonoid compounds in plant extracts. In deed, a significant negative correlation between the polyphenol contents and IC50 antioxidant activity values were observed indicating that extracts with highest polyphenol contents show lower IC50 values (Khlifi et al., 2011; Liu

et al., 2007; Sengul et al., 2009; Verzelloni et al., 2007). In the present study, a negative significant correlations (−0.99 < r < −0.7; 7.68 × 10−6 < p < 0.05), estimated by the Spearman’s coefficient, were observed between the total phenol, flavonoids, flavonols or proanthocyanidins contents in different organ extracts and the free radical-scavenging values IC50 (Table 3). Moreover, significant correlations were obtained between ABTS and phenols contents except for proanthocyanidins contents in petroleum ether extracts of all organs and for phenols contents in ethyl acetate extracts of roots (Table 3). Thus, the high antioxidant activity revealed mainly for leaf extracts could be attributed to the high phenolic content (98.66 mg EAG/g DW) probably due to compounds such as such as 4 -dihydroxy-6,7,8-trimethoxyflavone (xanthomicrol), 5,3 ,4 -trihydroxy-6,7,8-trimethoxyflavone (sideritoflavone), 5-hydroxy-6,7,3 ,4 -tetramethoxyflavone (5desmethylsinensetin), 5,7,4 -trihydroxyflavone (apigenin) and 5,7,3 ,4 -tetrahydroxyflavone (luteolin) (Benkiniouar et al., 2010). This is the first correlation obtained for different extracts of T. numidicus between polyphenols, flavonoids, flavonols or proanthocyanidins and antioxidant activity, so this correlation can guide the search for molecules responsible for the antioxidant activity. Therefore, our results confirm that phenolic compounds contribute to the

Table 3 Spearman correlation coefficient between total polyphenols, flavonoids, flavonols and proanthocyanidins with antioxidant activity. Assay

DPPH IC50 (␮g/ml)

ABTS (␮g TE/mg DW)

r

p

r

p

Petroleum ether extracts Roots

Polyphenols Flavonoids Flavonols Proanthocyanidins

−0.91 −0.71 −0.85 −0.49

0.013 0.046 0.033 0.073

0.91 0.74 0.84 0.45

0.016 0.049 0.029 0.076

Stems

Polyphenols Flavonoids Flavonols Proanthocyanidins

−0.71 −0.89 −0.90 −0.88

0.046 0.024 0.020 0.028

0.95 0.98 0.95 0.43

0.013 0.005 0.012 0.081

Leaves

Polyphenols Flavonoids Flavonols Proanthocyanidins

−0.91 −0.98 −0.88 −0.34

0.016 0.00043 0.029 0.126

0.71 0.48 0.85 0.49

0.046 0.079 0.033 0.064

Roots

Polyphenols Flavonoids Flavonols Proanthocyanidins

−0.71 −0.79 −0.82 −0.94

0.046 0.040 0.038 0.011

0.89 0.39 0.41 0.37

0.026 0.112 0.110 0.133

Stems

Polyphenols Flavonoids Flavonols Proanthocyanidins

−0.76 −0.75 −0.95 −0.82

0.043 0.044 0.014 0.038

0.72 0.71 0.92 0.84

0.041 0.046 0.016 0.036

Leaves

Polyphenols Flavonoides Flavonols Proanthocyanidins

−0.89 −0.76 −0.98 −0.87

0.022 0.043 0.002 0.030

0.99 0.94 0.86 0.98

0.003 0.016 0.028 0.004

Roots

Polyphenols Flavonoids Flavonols Proanthocyanidins

−0.89 −0.99 −0.99 −0.99

0.024 7.68 × 10−6 0.001 5.56 × 10−6

0.87 0.7 0.46 0.7

0.027 0.048 0.070 0.049

Stems

Polyphenols Flavonoids Flavonols Proanthocyanidins

−0.7 −0.99 −0.89 −0.74

0.049 0.0002 0.021 0.041

0.82 0.44 0.48 0.48

0.039 0.109 0.080 0.083

Leaves

Polyphenols Flavonoids Flavonols Proanthocyanidins

−0.85 −0.73 −0.98 −0.85

0.034 0.042 0.006 0.033

0.99 0.7 0.98 0.71

0.021 0.049 0.006 0.046

Ethyl acetate extracts

Methanol extracts

−79.2e −53.1c −59.3c

−46b

−60d −42.3c −47c

−34.1b

−23c −19b

−74.5d −53.7b −60.5c −63.1d −45.4b −53c −52.2d

−31.3ab −40c

−32 c −24ab −31c −20.2c −10.2a −13.2ab −79.5d

−15.6b −56.7d −39.4c −30.8d −16.3d Leaves

Means in each column followed by different letters are significantly different (p < 0.05).

−67.6d −42d −74d

−39.4e −27.9c −19.2e −13.5cd Roots

Stems

−48.1d −33.7b

−59.6d −50.9c

−65.4c −71.2d

−21.1c −15b

−25.6b −30c

−32.8b −35.9b

−72.8d −41.1b −56c

−55.8b −63.1c

−13.1ab −55.9b −33.8b −27.6b −11.5ab −54.1b −38.8ab −29.6b −20.4b −11.2c Methanol extracts

Leaves

−21.2c

−32.8c −23.1ab −25.8b

−47.1d −34.1b −39.9c

−29.3ab −19.8a −14ab −52.2b −46b

−23.1ab −17.9a −18.1b −11a −60.9c −49.2a −51.2c −34a −42.2c −27.6a

−17.6a −27.1b

−37c

−15.8b −12.5ab −63.5c −54.7b

−43.4b −50c

−55c

−41.3c −30.6ab −31.8c −19ab −17.9b −10.8ab −60.2c −51b −41.8b −38.8ab −32.7b −26.5ab −32.5d −17.3ab −14.3d −8.2c Stems

Leaves

Ethyl acetate extracts

Roots

−27ab −18.9a −23ab −14.9ab

−18.9ab −10.8a

−6.8b −2.7a −6.8b Roots Stems

−32.2ab

−33.2b −23.2a

−40.3c −32b

−30.2b −23 a −25 a −23a −19.9a

−21a −18.8a

−20.2ab −15 a

−15ab −10 a −12.5a −57.5b −44.6a −48.8a −47b −33.1a −38a −29a

−37.6b −26a

−28b −16.8a −24ab −12ab −7.9 a −9.8 a −55.1b −40.2a

−48ab −34.2a

−43.2b −32.5a

−32b −24.3a −29.5ab −24b −14a −21b −11.7ab −6.8a

−8.7a −51.4b

−55.4b −43.2a −39.2ab −31a

−35.1ab

−13.6ab −25c Leaves

−56.1c −40ab

−47b −31.4 a −36.3 a

−46.3b −35.1b −29.9ab −22.8ab −11.2a −51.1b −20.9ab −10a −47ab −38.2ab

−27.6a −30ab −19ab −21.3b −45a −55b −35ab −45b −20a −30b −5ab −10c Stems

Roots

−44.9b

−29.7a −31.1ab

−38b

−28.2b −15.9a −17.3b −9.3a −56.4b −43.4a −48.3b −34.6a −34.2b −14.3a

−16.6b −12.1ab −65c −50c −35b

−30d −10a −15d Water extract

193

Petroleum ether extracts

0.8

−34.8b −23.7a

0.6

−26.8b −19a

0.4

−13ab −9.5 a

0.2

−56.2b −48.6a −47.6b −36.5a

1 0.8 0.6 0.4 0.2

Hypocotyl length

1 0.8 0.6 0.4 0.2

Radicle length

1 0.8 0.6 0.2

0.4

Seed germination

Concentration (mg/ml)

Inhibitory effect compared to control (%)

Parameter controlled

Table 4 Allelopathic effects of different tissues extracts obtained from different solvent extraction systems of Tunisian T. numidicus at the vegetative stage on Medicago sativa L. seedlings.

Seedling dry weight

3.3. Phytotoxic potential The phytotoxic effects of the roots, stems and leaves extracts of T. numidicus by three different solvents with varying polarities (petroleum ether, ethyl acetate and methanol) and by the water are summarized in Tables 4 and 5. The allelopathic influence on M. sativa L. and T. æstivum L. germination and seedling growth varied significantly according to the solvent and plant parts. A significant inhibitory effect on the germination of M. sativa seeds was found (Table 4). In deed, the allelopathic effects were significantly different (p < 0.05) (Table 4). The germination percentages varied between 57.5 (of stems petroleum ether extract at 0.2 mg/ml) and 22.5% (of leaf methanol extract of at 1 mg/ml) at the seventh day of germination. The T. numidicus extracts at different concentrations showed very high phytotoxic effects against M. sativa, with an inhibition of seed germination of 79.2% at 1 mg/ml. Therefore, water, petroleum ether, ethyl acetate and methanol extracts marked germination and seedling growth inhibition that was concentration-dependent. Significant allelopathic effect was also observed at the lowest concentration tested, 0.2 mg/ml. The highest allelopathic effects were obtained by methanol extraction of all organs, followed by ethyl acetate then water extract and by petroleum ether. From the results shown in Table 4, it is evident that the recovery of inhibitory effect was dependent on the solvent used and its polarity (for all organs). For roots, methanol extract gave the highest inhibition of the seed germination, the hypocotyl and radicle lengths and the seedling dry weight of M. sativa with significant differences between extract concentrations. The inhibition of the radicle growth varied from 6.8 (stems petroleum ether extract at 0.2 mg/ml) to 79.5% (roots methanol extract at 1 mg/ml) and that of the hypocotyl from 7.9 to 74.5%. The biomass production was slightly inhibited in the presence of different extracts at 0.2 mg/ml concentration, and the dry weight of the seedlings treated with 1 mg/ml was highly reduced by 79.2% with root methanol extract. Table 5 showed that the phytotoxic effects of T. numidicus plant parts extracted with different solvents on T. æstivum germination and seedling growth was solvent and dose-dependent. In deed, the allelopathic effects of extracts were significantly different (p < 0.05). The best activities were also observed at methanol extracts. The lowest activities were observed for the stems petroleum ether extracts at different concentration. The root extracts with methanol solvent had the highest allelopathic activity, which the inhibition percentage of the germination was 74% (at 1 mg/ml). The inhibition of the radicle and the hypocotyl growth varied from 6.7 (stems petroleum ether extract at 0.2 mg/ml) to 79.5% (at 1 mg/ml of roots methanol extract), and from 7 (stems water extract at 0.2 mg/ml) to 74.5% (at 1 mg/ml of roots methanol extract), respectively. The dry weight of the seedlings treated with roots methanol extract was highly reduced by 73.8% at 1 mg/ml. Our results demonstrated that the volatile compounds isolated from the Tunisian endemic T. numidicus tissues with various solvents significantly delayed the seed germination and the growth of M. sativa and T. æstivum seedlings. This inhibitory activity can be mainly due to toxic compounds present in the roots. In deed, the reduction in seed germination and shoot length may be attributed to the reduced rate of cell division and cell elongation due to

−42.6c −26.6a

1

radical scavenging activity of Tunisian T. numidicus extracts. Moreover, several studies have focused on the relationship between the antioxidant activity of phenolic compounds as hydrogen donating free radical-scavengers and their chemical structure. Phenolic antioxidants are products of secondary metabolism in plants, and the antioxidant activity is mainly due to their redox properties and chemical structure, which can play an important role in inhibiting lipoxygenase and scavenging free radicals (Evans et al., 1996).

−67.2d −42ab

I. Ben El Hadj Ali et al. / Industrial Crops and Products 62 (2014) 188–195

I. Ben El Hadj Ali et al. / Industrial Crops and Products 62 (2014) 188–195 −73.8e −59.1c −58.3c −39 c −43.3d −59.6d −22.9 ab −34.5bc −45c −29.9b −35.8bc −42.4c −32.1d −19.4b −21.6b

the presence of the allelochemicals (Javaid and Anjum, 2006). In line with our findings, few studies reported that some essential oils containing a toxic compounds (e.g. caryophyllene oxide, limonene, spathulenol, etc.) also have interesting phytotoxic potentials (Chung et al., 2001; Mabrouk et al., 2013). The Tunisian T. numidicus phytotoxic potential can be considered high compared to the results reported by Shao et al. (2013). These results confirm that T. numidicus could be used as a potential allelopathic substance and should be tested as a potential natural herbicide resource. 4. Conclusion This study is the first to investigate the global chemical composition of various extracts from roots, steems and leaves of T. numidicus, endemic species from Tunisia, and their antioxidant and allelopathic activities. Our results clearly showed that the leaves seems the organ which give the highest level in polyphenol, flavonoid, flavonol and proanthocyanidin contents which favor them in industrial use for extraction of polyphenol compounds. Also, we can conclude that leaf methanolic extracts with higher antioxidant capacity (IC50 = 11.06 ± 0.33 ␮g/ml by DPPH assay and 235.46 ± 2.14 ␮g TE/mg DW by ABTS assay) could be considered a potential natural antioxidant alternative for use as a natural additive in food, cosmetic and pharmaceutical industries for the prevention or treatment caused by microorganisms and free radicals. Furthermore, extracts with higher antioxidant capacity also had higher polyphenol contents. Moreover, root extracts showed high allelopathic activity. All those results emphasize the importance of the chemical composition of this Tunisian endemic plant.

−40e −22.2c −28.3c −20.2c −10.2a −14.2ab −79.5e −55.8c −63.1d −67.6d −32.8b −35.9b −32.9c −21.6b −25.5b

−52.2d −31.3c −39.4cd

−62.9e −41.4c −51.6d

−74.5e −53.7d −60.5d

−59.3c −49.1b −50b −44.6c −36.1b −38.7b −31.4b −38.5c −19.9ab −25.7a −22.3 ab −30b −28.1c −15.4a −17.5ab −33.6d −16.3b −29.6c −16ab −12.5ab −13.6ab −63.5d −51.7c −55.7c

Acknowledgments

−70.8d −42.9b −52.5c

This research was supported by a grant of the Ministry of Scientific Research and Technology, the National Institute of Applied Science and Technology (research grant 99/UR/09-10) and the Higher Institute of Biotechnology of Monastir (Laboratory of Genetic Biodiversity and Valorisation of Bioresources).

Means in each column followed by different letters are significantly different (p < 0.05).

−23.1c −15 ab −15.6ab −74d −65.4c −71.2d −60e −51d −55.7d −48.1d −33.7c −39.4c −26c −12bc −16.3bc Methanol extracts Roots Stems Leaves

−39.4d −25.9b −30.8c

−14.3bc −8.2b −11.2bc Ethyl acetate extracts Roots Stems Leaves

−23.5b −32.7c −17.3ab −26.5b −20.4b −29.6b

−31.8a −38.8b −38.8b

−60.2c −51b −54.1b

−18b −10.8ab −11.5ab

−52.7c −41.5b −48.7c −41.3c −30.6b −33.8b −32.3c −21.5b −30c

−42.2d −27.6b −34.2c

−53.3d −34.2b −45.4c

−61d −49.2c −52.2d

−39.6b −28a −32.4a −28.5b −30b −15.3a −20.5a −20.9 ab −24.8a −21.2b −11.1a −13.6a −6.8b −2.7a −6.8b Petroleum ether extracts Roots Stems Leaves

−18.9ab −27b −10.8a −18.9a −14.9a −23ab

−39.2b −55.4b −31.1a −43.2a −35.1ab −51.4b

−11.7ab −6.7a −8.7a

−37.6c −26b −29b −29c −11.7a −22c −41.5b −55.1c −11.8ab −30.5ab −40.2ab −7.9a −38.5b −47.7b −9.8b −32b −24.3a −29.5ab −24.5b −12.8a −20.2b

−44.7c −30.7b −37.7b

−57.5d −44.6c −48.8c

−42ab −34.2a −37.5a

−36.4b −29.1a −31.8a −32.6b −21.7a −26.9a −29.2b −16.5a −25.8b −21.2b −14.6a −17ab −32.8b −27.7a −28.8a −21.7a −17a −18.9a −13.1ab −16.3a −9.7a −11.6a −11.1a −13 a −31.3ab −46b −10.1a −26.8a −37.8a −7 a −29a −40.6ab −8a −18.3ab −27.7ab −16.9ab −20 a −17.6ab −21.4a −14.2ab −10.4ab −13.9ab −63c −48.2a −55.6b −44.4c −33.3a −42.7c −21.2c −11.1bc −18.5bc

−30c −37c −18.5ab −22.2ab −22.2b −29.6b

0.8

1 0.6 0.4 0.2

Water extract Roots Stems Leaves

0.4 0.2 Concentration (mg/ml)

0.6

0.8

1

0.2

0.4

0.6

0.8

1

0.2

0.4

0.6

0.8

1

Seedling dry weight Hypocotyl length Radicle length Seed germination Parameter controlled

Inhibitory effect compared to control (%)

Table 5 Allelopathic effects of different tissues extracts obtained from different solvent extraction systems of Tunisian T. numidicus at the vegetative stage on Triticum æstivum L. seedlings.

−64.8d −49.3b −52.6b

194

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