Vol. 11(4), pp. 73-86, 25 January, 2017 DOI: 10.5897/JMPR2016.6278 Article Number: 22FCD8E62531 ISSN 1996-0875 Journal of Medicinal Research Copyright © 2017 Author(s) retain the copyright of this article http://www.academicjournals.org/JMPR

Full Length Research Paper

New insights of Nettle ( urens): Antioxidant and antimicrobial activities

Lobna Maaroufi1,2, M. Sazzad Hossain2,3*, Wiem Tahri1 and Ahmed Landoulsi1

1Laboratory of Biochemistry and Molecular Biology, Faculty of Science of Bizerte, University of Carthage, Zarzouna 7021, Bizerte, Tunisia. 2Department of Biochemistry and Physiology of Plants, University of Bielefeld, 33501 Bielefeld, Germany. 3Department of Agronomy and Haor Agriculture, Sylhet Agricultural University, Sylhet 3100, Bangladesh.

Received 21 October, 2016; Accepted 14 December, 2016

Urtica urens is classified into the sub family of , belonging to the family. Their well-established beneficial properties to human health w e r e mainly related to their phenolic content. Nettle extracts showed that an antioxidant and antimicrobial potentials. Among the different nettle extracts methanol extract (EMO) has the highest phenolic content (86.5±1.36 mg EGA/g DM) and lowest value was observed in the water extract (EAO) (43.915 ± 19.95 mg EAG/g DM). The stronger inhibitory effect on 1,1-diphenyl-2-picryl-hydrazil radicals (DPPH•) corresponds to those obtained from methanol extract. A comparative analysis between two fungi Penicelleum notatum and Fusarium oxysporum, showed a decrease growth rate and was monitored at the range of 1 to 2.5 mg/ml. Complete growth inhibition of these two fungi was obtained at the concentrations of 5, 10, and 15 mg/ml of water extract (EAO). The antibacterial activity was also significant while addition of the nettle extracts and depends on the strain tested. Gram positive bacterial strains (Enterococcus faecalis and Enterecoccus faecium) showed higher sensitivity to the extracts, in comparison with Gram negative strains Escherichia coli and Salmonella typhimurium. The antioxidant activities were correlated (r2> 0.9) with the phenolic content, suggesting that the phenolic content are playing major role in scavenging free radicals. Our total findings suggest that the Nettle (Urtica urens) extract has important antioxidant and antimicrobial activities which is positively correlated with its phenolic content.

Key words: Urtica urens, polyphenol, antioxidant, antimicrobial and 1,1-diphenyl-2-picryl-hydrazil radicals (DPPH•).

INTRODUCTION

All living beings have a primary metabolism which subsequent chemical reactions. There is no doubt that provides the basic molecules (carbohydrates, amino plants are a good source of biologically active natural acids, nucleic acids, proteins, lipids etc.) from where products. To investigate bioactive natural compounds, it secondary metabolites are produced through the is essential to have access to simple biological tests to

*Corresponding author. E-mail: [email protected].

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 74 J. Med. Plants Res.

locate required activities (Beart et al., 1985). The effect of the solvent and the effect of the nettle itself, the preservative effect of many species and herbs antibacterial assay was conducted with the water extract (EAO) of suggests the presence of antibacterial and antioxidant nettle. To prepare the water extract plant powder was mixed with the boiled water and centrifuged at 4000 g for 20 min and finally constituents in their tissues (Bobis et al., 2015; filtered on filter paper. The filtrate was then stored at 4°C for Chew et al., 2015; Kukrić et al., 2012; Singh further use. et al., 2012; Hirasa and Takemasa., 1998). Plants contain phenolic compounds and nitrogen compounds having essential alkaloids and glycosides. Determination of total phenolic contents

These metabolites often play a role in plant defense The assay for the determination of total phenolic content in U. urens where they are produced. Besides the antioxidant was performed according to the method of Namjooyan et al. and antimicrobial activities, polyphenols have several (2007). Amounts of 1.85 ml of distilled water, 0.125 ml of Folin- other beneficial effects on human health (Najjaa et al., Ciocalteu reagent and 0.5 ml of a 20% sodium carbonate (Na2CO3) 2007). Faced with the therapeutic limit of chemical solution were added to 25 µl of liquid extract sample in a test tube, drugs, the developmental research on medicinal making a final volume of 2.5 ml. The solution was homogenized and left to stand for 30 min, and the absorbance was determined plants has been directed towards obtaining herbal from at 750 nm. The total phenols were calculated as milligrams of folk medicines (Škrovánková et al., 2012). This gallic acid equivalents per gram of dry matter (DM). development is an essential step for promoting the entire sector linked to the need not only for therapeutic purposes but also cosmetics and food industries Determination of total carotenoid (Fattouch et al., 2007). Determination of total carotenoid was performed according to the Urticaceae is one of the important sources of method of Wellburn (1994) with few modifications. 50 mg of fresh polyphenols. The beneficial effect of nettle on human plant material was ground in 10 ml of acetone (80%). The obtained health is already very well known (Heoa et al., 2015; extract was centrifuged and filtered. The reading of the Fattahi et al., 2014; Mansoub, 2011; Gulcin et al., absorbance is carried out at three different wavelengths of 2004). The most abundant and endemic species of 470, 663 and 647 nm and the concentration is determined as follows: Carotenoid = 5 × DO + 2.846 × DO -14.87 × DO nettle in Tunisia are and Urtica urens. 470 663 647 The result is expressed in mg per 100 g dry matter (DM). They are spontaneous grass and their aerial parts are used as salad particularly in North-Africa and Mediterranean countries. There is some research work Determination of total flavonoids done about the antioxidant activity of Urtica dioica (Gulcin et al., 2004) but no more information is available The principle is based on the formation of the complex about Urtica urens in this context. The main aim of this flavonoids-aluminum chloride (Djeridane et al., 2006; Chang et al., 2002). The color intensity is proportional to the study is to evaluate the potentials of U. urens as a new concentration of the flavonoids. A volume of 250μL of the extract source of natural phenolic compounds and its is diluted 5 times, supplemented with 75 μL of a solution of Na antioxidant and antimicrobial activities. NO2 (5%), followed by a rest of 6 min before adding 150 μL of aluminum chloride (AlCl3, 6H2O, 10%) after 5min of incubation, 500 μL of NaOH (1 M) are added. The final volume of the MATERIALS AND METHODS solution is adjusted to 2500 μL with distilled water. The absorbance of the mixture was read at 510 nm. The reference range is Plant materials prepared with the catechin. The content of total flavonoids expressed as mg catechins per gram of dry matter (DM). Aerial parts of U. urens were collected from the province of Bizerte in Noth-Africa during the spring 2010 which were the main plant material of the study. Determination of condensed tannins

In the presence of concentrated sulfuric acid, condensed Preparation of the plant extracts tannins depolymerize and transformed in the presence of vanillin anthocyanidols red color whose intensity is measured Aerial parts of U. urens were washed and dried in the open air at spectrophotometrically at 500 nm. A volume of 3 ml of vanillin room temperature, frozen and grounded in liquid nitrogen. The (4%) was added to 50 µl of the extract and 1500 µl of resulting powder was used for the preparation of methanol (EMO) concentrated HCl. The resulting mixture was incubated for 15 and acetone (ECO) extracts. The powder obtained was min at room temperature and the absorbance is measured at 500 macerated in the appropriate solvent (like methanol and acetone) nm. The standard is like catechin flavonoids, condensed tannins under stirring for 30 min at room temperature. The mixture was content in mg catechin equivalents per gram of dry matter (mg CE left at 4°C for 24 h to be subsequently filtered through a / mg DM). ® Whatman paper (No.4 Sigma Aldrich ). The filtrate is collected and stored in dark at 4°C for next use. Determination of total anthocyanins

Preparation of the aqueous extract Plant powder of 1 g was mixed with 12 M HCl (1%). The mixture is incubated at a temperature of 4°C for 48 h with In order to avoid any controversial contradiction between the continuous stirring followed by centrifugation and filtration of Maaroufi et al. 75

supernatant. The absorbance is read at two different wavelengths diameter of the mycelium formed every 24 h. (530 nm and 675 nm). The concentration of anthocyanins was expressed in g per 100 g of dry matter (DM). Statistical tests

Total antioxidant capacity The ANOVA was used to verify the difference between the control and the different doses and for the four strains bacterial Total antioxidant capacity of the extract was evaluated by the and two fungi. Student’s T-test was performed in other cases. method of phosohomolybdenum described by Prieto et al. (1999). A volume of 1ml of a solution (0.6 M sulfuric acid, 28mM sodium phosphate and 4 mM ammonium molybdate) is added to 100 µL of the 10 times diluted extract. The mixture obtained is RESULTS AND DISCUSSION incubated at 95°C in the dark for 90 min. The absorbance reading was taken at 695 nm against a blank containing the Determination of antioxidants of different extracts of solvent and the reagent. A standard range is prepared and nettle the results are expressed in mg gallic acid equivalent per range of dry matter (DM). The environmental pollution, chemical products, food additives, physical stress and normal usage of oxygen Free radical scavenging activity by our body all contribute to the production of harmful radicals that damage healthy cells. Antioxidants act by The free radical scavenging activity of the nettle extract was trapping free radicals to isolate their single electrons measured by 1,1-diphenyl- 2picryl-hydrazil (DPPH) was evaluated using colorimetric method of Shimada et al. (1992) and improved and transforming later in stable molecules or ions. Most by Islam et al (2013). At room temperature and in the presence of the antioxidants produced by the plants are then of an antioxidant, the DPPH radical has an intense purple color. processed by humans as humans are unable to The transition to non-radical form DPPH (after saturation of its synthesize them (Fattouch et al., 2007). electronic layers) was accompanied by the disappearance of the The total polyphenols content varies depending on violet color (Soler-Rivas et al., 2000). The decrease in the intensity the type of solvent used as summarized in (Figure 1). of the color is monitored by spectrophotometry at 517 nm. The estimation of this activity was measured. The free radical In fact, methanol seems to be the most suitable solvent scavenging activity of each sample is expressed as percent for extraction of polyphenols (with a grade of inhibition of DPPH using the following formula: 88.75±12.25 mg EAG / g DM) followed by acetone (with an average of 56.5±13,66mg EAG / g DM). The DPPH = [(A0-A)/A0] × 100 lowest value was observed in the aqueous extract (with a grade of 43.915 ± 19.95mg / g DM). The study of the Where, A0 is absorbance at 517 nm DPPH without extract and A is sample absorbance. variation of the total polyphenol content varies significantly depending on the type of solvent used (p <0.05). Polyphenols are fundamental constituents of the Bacterial strains plant thanks to their purifying capacity provided by their hydroxyl group (Hatano et al., 1989). Four reference bacterial strains were used to evaluate antibacterial activity: two Gram negative (Escherichia coli JW1772 According to recent works, a strong correlation exists and Salmonella typhimurium ATCC14028) and two Gram-positive between polyphenols and antioxidant power and this is (Enterococcus faecium and Enterococcus faecalis) provided by the demonstrated in many plants (Vinson et al., 1998; microbiology laboratory at the Faculty of Sciences of Tunis. Velioglu et al., 1998; and Oktay et al., 2003). Phenolic compounds could contribute in a direct way to the

Bacterial growth antioxidant activity (Duh et al., 1999). It is suggested that polyphenolic compounds may have anti-mutagenic Turbidity is measured by spectrophotometry (Thermofisher). and anti-carcinogenic in humans. When they are Samples were taken in sterile conditions and Optical Density ingested with a daily dose of 1 g for a diet composed (OD) is measured every 1 h. A growth curve is established, expressing the OD as a function of time: OD = f (t). only of fruits and vegetables (Tanaka et al., 1998). In addition, it has been reported that the phenolic compounds have been associated with antioxidant Fungi and its processing by the nettle extract activity and play an important role in the stabilization of

Two reference fungi Penicillium notatum and Fusarium lipid peroxidation (Yen and Duh, 1994). oxysporum, provided by the microbiology laboratory at the The aerial parts of nettle contain carotenoids with a Faculty of Sciences of Tunis, were used to evaluate antifungal content of about 3.33 μg and anthocyanins in particular activity. The PDA (Potato dextrose agar) medium was poured in the smaller quantities of approximately 2 μg (Figure 2). presence of the extract of specific volume, to obtain different Proanthocyanin also called "condensed tannins" are desired concentrations of 1, 2.5, 5, 10 and 15mg / ml of the substrates of enzymatic browning. The predominant aqueous extract of nettle. After the extract and the medium were well mixed and solidified, then appropriate fungal spores were physicochemical property of tannins is their ability to placed on the agar. Incubate for 3 days at an optimal temperature bind by hydrophobic interactions and/or exchange of of 20°C. The growth kinetic was measured by calculating the hydrogen bonds with proteins and polysaccharides 76 J. Med. Plants Res.

Figure 1. total polyphenol content contained in the three extracts of nettle (EMO extraction of nettles in methanol, ECO extraction in acetone and EAO extraction in water) expressed as gallic acid equivalent per gram DM (p <0.05).

Figure 2. Content of total carotenoids and anthocyanin with nettle expressed in 100 g of dry matter.

(Jeantet et al., 2007). They form salivary protein on the type of solvent used as shown in Figure 4. The complexes with condensed tannins, which give the contents are as follows the methanol extract with a astringency of fruit (grapes, peaches, persimmons, content of 67.5±13.5 µg CE / g DM, the extracted with apples, pears, etc.), beverages (wines, ciders, tea, beer, acetone (with a content of 66.1±6.5 µg CE / g DM), and etc.) and the bitterness of chocolate (Santos-Buelga and the aqueous extract (with a content of only 46±5.9 µg CE Scalbert, 2000). The condensed tannins content varies / g DM). The polarity of the solvent doesn’t seem to have depending on the solvent used as shown in Figure 3. an impact on the extraction since the most polar solvent In fact, acetone seem to be the most suitable (water) showed the lowest extraction capacity for the all solvent for extraction of condensed tannins (with a investigated polyphenols. content of 115.33±2.6 µg CE / g DM) followed by methanol (with a content of 52.33±15.22 µg CE / g DM). The lowest value was observed in the aqueous extract Antioxidant power and anti-radical activity of U. urens (with a content of only 39.28 ± 2.28 µg CE / g DM). The study of the variation of the condensed tannin content The antioxidant activity was attributed to several varies significantly depending on the type of solvent mechanisms among which we mention the peroxide used (p <0.05). decomposition and reducing free radicals. Many methods Flavonoids are antioxidants belonging to the family of have been proposed to evaluate the antioxidant power. polyphenols (Fernandez-Pachon et al., 2004; Zhishen et Among these techniques, we adopted total antioxidant al., 1999). Flavonoid content also tends to vary depending activity and anti-radical activity by the free radical M a a r o u f i e t al. 77

Figure 3. Tannins content contained in the three extracts of nettle (EMO extraction nettle in methanol, ECO extraction in acetone and EAO extraction in water) expressed as catechin equivalents per gram of DM (P <0.05). Salivary protein complexes with condensed tannins are responsible for the astringency of fruit (grapes, peaches, persimmons, apples, pears, etc.), beverages (wines, ciders, tea, beer, etc.) and the bitterness of chocolate (Buelga and Scalbert 2000).

Figure 4. flavonoids content contained in the three extracts of nettle (EMO extraction nettle in methanol, ECO extraction in acetone and EAO extraction in water) expressed as catechin equivalents per gram of DM (P > 0.05). The study of the variation in the content of flavonoids shows a non-significant variation. The extraction of flavonoids is independent of the type of solvent used.

DPPH. 5). The antioxidant activity is also dependent on the The extract showed antioxidant activity depending on dose used in nettle extract. The effect of antioxidants the dose or the solvent used (Kataki et al., 2012). on anti-radical activity of the DPPH is due to their ability However, a strong antioxidant activity was observed to donate a proton. The DPPH is a stable free radical with the methanol extract and acetone extract higher that accepts an electron or hydrogen radical to become a than gallic acid activity used as a positive control (Figure stable diamagnetic molecule. The anti-radical method of 7 8 J. Med. Plants Res.

e

0.35 EMO 0.25 d c c

0.15 b OD A695 OD b b 0.05 a

-0.05 0 50 100 200 500 (A) (B)

d 0.35 ECO 0.25 c c 0.15 b b OD A695 OD a 0.05 a

-0.05 0 50 100 200 500

Concentration [µg / ml] (C)

Figure 5. Antioxidant activity of three extracts of nettle (EAO): aqueous extract of nettle; EMO: methanol extract of nettle, ECO: ketone extract of nettle) and comparison with a positive control antioxidant gallic acid.

DPPH is a technique used to evaluate the antioxidant activity and total polyphenol content with an r 2 > 0.94 activity in a short time compared to other methods as shown in Figure 7. Similar results were found by (Soares et al., 1997). other authors who showed that there is good The reduction of the DPPH• radical is determined by correlation between total phenolic profile and the absorbance at 517nm induced by antioxidants. antioxidant activity of plant extracts emphasizing the Figure 6 shows a significant decrease (p<0.05) on the role of phenolic compounds in antioxidant activity concentration of DPPH due to the anti-radical power of (Tunalier et al., 2002; Duh et al., 1999). the nettle except for the concentration of 10 µg / ml. The choice of solvent does not appear to have an effect on anti-radical activity. Based on this data, we Correlation between the total polyphenol content can conclude that the nettle appears to have an and anti-radical activity of nettle effective antioxidant power that reacts with free There is a strong correlation between the antioxidant radicals. This finding is interesting knowing the capacity 2 activity and total polyphenol content with an r > 0.85 all of polypehnols to limit the damage caused by these extract confused as shown in Figure 8. The strong radicals in the body. The comparison between the correlation observed between the antioxidant potential antioxidant capacity of the extracts (IC obtained in 50%) and the total polyphenol content suggests that our laboratory with different extracts of nettle (extract polyphenols are the main compounds that contribute to with water, extracted with acetone and methanol this activity. Indeed, rats poisoned by CCl xenobiotic extract) and IC of Urtica dioica obtained can be 4 50% and treated with nettle extracts showed significant considered as comparable. increases in liver antioxidant enzymes (Yen and Duh, Indeed, it is 31.6 for EAO against 44.77±2.12 for 1994). the roots with a different extraction protocol (Gulcin et In addition, the purification of H O has been al., 2004). 2 2 attributed to phenols thanks to their ability to donate

electrons to H2O2, and neutralizing it in water form by Correlation between the content of total the called process water-water cycle which take place in polyphenols and total antioxidant activity plants (Asada et al., 2000). The capacity of the EAO to purify the H2O2 can be There is a strong correlation between the antioxidant attributed to its structural characteristics of its active

Maaroufi et al. 79

0.8 a b EMO

0.6 c 0.4 0.2 d OD A517 OD 0 1 2.5 5 10 (A) (B)

1 a

0.8 b

0.6 ECO 0.4 c c A517 OD 0.2

0

1 2.5 5 10

Concentration [µg / ml] (C)

Figure 6. Comparison of the anti-radical activity of the nettle with the radical DPPH (EAO: aqueous extract of nettle; EMO: methanol extract of nettle, ECO: acetone nettle extract).

77 154

153 76

152 R² = 0.9829 75 activity 151 R² = 1 150 74

149 activity

148 73 [mg EAG / g DM] 147 Total antioxidant 72 146 antioxidant 71 [mg EAG [mgEAG /DM] g 0 50 100 150 0 20 40 60 80

Total Total polyphenol content [mg / g DM] Total polyphenol content [mg / g DM] (A) (B)

64 62 R² = 0.9474 60 58

activity activity 56

54

[mg EAG [mgEAG /DM] g Total antioxidant 52 0 20 40 60 Total polyphenol content [mg / g DM] (C)

2 Figure 7. Curve correlation (r ) between the total polyphenol content and antioxidant activity in Urtica urens and determination of the correlation of coefficient. 80 J. Med. Plants Res.

31 30.5 42

41.5 %

30 41 29.5 R² = 0.9829 % IC IC 50 40.5 R² = 1

29 IC 50 40 39.5 28.5 39 0 50 100 150 0 20 40 60 80

Total polyphenol content [ mg / g DM] (A) Total polyphenol content [mg / g DM] (B)

33 32.5

32 % 31.5

IC 50 R² = 0.8848 31 30.5 30 0 20 40 60 Total polyphenol content [mg / g DM] (C)

Figure 8. Curve correlation (r2) between the content of total polyphenols and anti- radical activity in Urtica urens and determination of the correlation of coefficient.

components that determine their ability to donate 12). electrons. The scavenging capacity of EAO on H2O2 was compared to that of BHA, BHT and α-tocopherol as a standard. The concentrations used have shown Escherichia coli gram negative activity by 23% H2O2. In comparison, the same concentration of BHA, BHT and α-tocopherol show The E. coli growth (Figure 9) shows that there is inhibition 38.86 and 57% respectively. These results show that of cell growth while addition of the aqueous extract of nettle in the bacterial culture medium. Indeed, the the scavenging effect of EAO on H2O2 is lower but still interesting in comparison with BHA, BHT and α- concentration of 5 mg / ml resulted in a tendency to decrease the growth rate is of 0.32 h-1 lower than the tocopherol (Halliwell, 1991). -1 control (0.44 h ), this growth rate is even lower for the extract concentration of 10 mg / ml and 15 mg / ml, the μmax (the maximum specific growth rate of the The effect of aqueous nettle extract on the growth of -1 various microorganisms microorganisms)decreases from 0.29 to 0.25 h . In addition, the bacterial culture treated with aqueous nettle The effect of EAO on bacterial growth was extract between the stationary phase at a cell density demonstrated with by the effect of the extract on significantly lower than that of the cultivation of the control kinetic growth inhibition. A starting OD600 nm at 0.1 nm bacteria. was used in all the assays with an appropriate volume of aqueous extract of nettle to obtain the following Salmonella typhimurium gram negative concentrations: 5, 10 and 15 mg/ml. For monitoring of the biomass, measurements of OD at 600 nm were As shown in Figure 10, the addition of aqueous nettle carried out on samples taken every hour for 9 hours. extract at different concentrations of 5 to 15 mg / ml at Measurements are plotted on curves expressing the OD starting 0.1 resulted in a lag phase longer than the OD600 nm versus time. A reference inoculum is phase of the control. It lasts approximately 2 h. The treated the same way but in the absence of aqueous application of statistical tests showed that the observed extract of nettle establish the control curve of the changes are significant (p < 0.05). Indeed during the bacterial growth. Analyses were replicated three times exponential phase, the calculated bacterial control -1 -1 and reproducibility of the results was verified (Figure 9 to growth is of 0.41 h while it decreases to 0.36 h for Escherichia coli Enterococcus faecium 1.6 0.5 1.4 0.45 1.2 0.4 0.35 Cont 1 Cont 5g/l 0.3 5g/l 0.8 0.25

10g/l 0.2 10g/l A 600nm A

0.6 A600nm 15g/l 0.15 15g/L 0.4 0.1 0.2 0.05 0 0 0 2 4 6 8 10 0 2 4 6 8 10 Time [h] Time [h] Maaroufi et al. 81

Escherichia coli Enterococcus faecium Escherichia coli Enterococcus faecium 1.6 Salmonella typhymurium Enterococcus faecalis 1.6 0.5 1.4 0.5 0.45 0.45 0.5

1.4 1.4 1.2 0.4 0.45 0.4 1.2 Cont 1.2 0.35 Cont 1 0.35 0.4 Cont 0.3 Cont 1 5g/l 0.3 1 Cont5g/l 0.35 Cont 0.8 5g/l 0.25 5g/l 0.3 0.8 10g/l0.25 0.2 5g/l10g/l 5g/l A 600nm A 0.8

0.6 A600nm 10g/l 0.2 10g/l 0.25

A 600nm A 15g/l 0.15 10g/l15g/L 10g/l A600nm 0.6 600nm A 0.2 0.4 15g/l 0.150.6 15g/L A600nm 0.4 0.1 15g/l 0.15 15g/L 0.2 0.10.4 0.05 0.2 0.1 0 0.050.2 0 0.05 0 0 2 4 6 8 10 0 0 0 2 4 6 8 10 0 0 2 4 6 8 10 0 2 4 Time6 [h] 8 10 0 1 2 3 4 Time5 [h] 6 7 8 9 10 0 2 4 6 8 10 Time [h] (A) Time [h] Time [h] (B) Time [h]

Escherichia coli Enterococcus faecium 1.6 0.5 Salmonella typhymurium 0.45 Enterococcus faecalis 1.4 Salmonella typhymurium Enterococcus faecalis 1.2 0.4 1.4 0.5 0.45 Cont 1.40.35 Cont 0.5

1 0.3 1.2 0.45 0.4

5g/l 5g/l 0.8 1.20.25 1 Cont 0.4 0.35 Cont 10g/l 10.2 10g/l5g/l 0.35 0.3 Cont 5g/l A 600nm A 0.8 Cont

0.6 A600nm 0.25 15g/l 15g/L10g/l 0.3 10g/l 0.80.15 600nm A 5g/l 0.2 5g/l 0.4 0.6 0.25 A600nm 0.1 10g/l 15g/l 10g/l 15g/L A 600nm A 0.4 0.2 0.15

0.6 A600nm 0.2 15g/l 0.1 0.40.05 0.2 0.15 15g/L 0 0 0.1 0.05 0.2 0 0 0 2 4 6 8 10 0 2 4 6 8 10 0.05 0 0 1 2 3 4 5 6 7 8 9 10 0 0 2 4 6 8 10 Time [h] 0 1 2 3 4 Time5 [h] 6Time 7 [h] 8 9 10 (C) 0 2 4 Time6 [h] 8 10 (D) Time [h] Time [h]

Figure 9. Effect of aqueous extract of nettle on the growth of E. coli cell growth was monitored during the treatment of cells with the aqueous extract of nettle (control inoculum, C1 = 5 mg / ml (0.85 mg / ml eqv DM), C2 = 10 mg / ml (1, 7 mg / ml eqv DM), C3 = 15 mg / ml (2.55 mg / ml eqv DM) (p <0.05)). Salmonella typhymurium Enterococcus faecalis 1.4 0.5 0.45

1.2 0.4 1 Cont 0.35 Cont 5g/l 0.3 5g/l 0.8 EAO concentration0.25 of 5mg / ml and even a lower µ of Comparative analysis of the curves corresponding to 10g/l max10g/l A 600nm A 0.2

0.6 A600nm 15g/l -1 -1 0.4 about 0.350.15 h for EAO 10 mg/ ml and a µx of 0.27 h 15g/Lfor the treated bacteria and bacteria that corresponding to 0.1 0.2 a concentration0.05 of extract of 15 mg / ml. the controls shows that the addition of aqueous extract 0 0 0 1 2 3 4 5 6 7 8 9 10 0 2 4 6 8 10 significantly affect the growth of E. faecalis (Figure 12). Time [h] Time [h] It seems that the gram positive strains are more Enterococcus faecalis gram positive sensitive to our extract than Gram negatives strains tested. This can be explained by the fact that gram The application of statistical tests showed that the positive bacteria are devoided of the extra outer observed variations are significant (p<0.05). Comparative membrane that enhance their vulnerability and allow an analysis of the curves of the treated bacteria and control easier penetration of the active substances contained in shows that the addition of aqueous extract significantly the EAO. affects the growth of E. faecium especially in the first three hours. Indeed, the growth rate is relatively low -1 (0.041, 0.04 and 0.02 h , respectively for concentrations Effect of the nettle on fungal growth EAO 5mg / ml to 10 mg / ml and 15 mg/ ml) against a -1 μmax of 0.2 h of the control strain during the growth Nettle extract was added in the presence of the culture phase. During the stationary phase, the gap is smaller medium of Fusarium oxysporum at different concern. between different concentrations. It was 0.16 h-1 for the The Figures 10 and 11 shows that the growth of untreated strain and 0.11 h-1 for strain treated with a rate Penicillium notatum is inversely proportional to the dose of EAO 5 mg/ml and 0.1 h-1for treated strains with EAO EAO added. Indeed, the higher is the dose administered rate of 10 and 15 mg / ml. It seems that the treated in the medium, the stronger is the effect of the mycelium strain resume growth with the exponential phase, one inhibition. which is noticeable from Figure 10 (a,b and could even speak of a phase shift that can be c) for the dose of 1 mg / ml as well as a dose of 2.5 mg / explained by an adaptation of the bacteria to the new ml up to complete disappearance of mycelium starting at environment for pursuing of growth (Figure 11). a dose of 5 mg / ml and for the dose 10 mg / ml and the dose of 15 mg / ml. The application of statistical tests showed that the Enterococcus faecalis gram positive variations between control and the different doses observed are significant (p < 0.05). In fact, the higher The application of statistical tests showed that the dose administered in the medium a higher is the effect observed variations are significant (p < 0.05). on the mycelium growth. It dramatically decreases (from 82 J. Med. Plants Res.

Te 1 mg / ml 2.5 mg / ml

5 mg / ml 10 mg / ml 15 mg / ml

Figure 10. Effect of aqueous nettle extract on the growth of Salmonella typhimurium Cell growth was monitored during the treatment of cells with the aqueous extract of nettle (control inoculum, C1 = 5 mg / ml (0.85 mg / ml eqv DM), C2 = 10 mg / ml (1, 7 mg / ml eqv DM), C3 = 15 mg / ml (2.55 mg / ml eqv DM) (p <0.05)).

5

d 4

3 c

2 b

Relative growth Relative 1 a a a 0 Te 1 mg / ml 2.5 mg / ml 5 mg / ml 10 mg / ml 15 mg / ml

Figure 11. Effect of aqueous nettle extract on the growth of Enterococcus faecium. Cell growth was monitored during the treatment of cells with the aqueous extract of nettle (control inoculum, C1 = 5 mg / ml (0.85 mg / ml eqv DM), C2 = 10 mg / ml (1, 7 mg / ml eqv DM), C3 = 15 mg / ml (2.55 mg / ml eqv DM) (p <0.05)).

1 to 2.5 mg / ml) or completely disappears (no growth is nettle is used by European farmers, as a natural seen) while adding 5, 10 and 15 mg/ml (Figure 13). The fertilizer called compost or manure nettle to fight against growth of Fusarium oxysporum is strongly conditioned by plant pests such as F. oxysporum. the EAO added dose. This is even more likely that the Organic farming has only a very limited range of Maaroufi et al. 83

6 d 5

4

3

2 c Relative growth Relative 1 b a a a 0

Te 1 mg /ml 2.5 mg / ml 5 mg / ml 10 mg / ml 15 mg / ml

Figure 12. Effect of aqueous nettle extract on the growth of Enterococcus faecalis. Cell growth was monitored during the treatment of cells with the aqueous extract of nettle (control inoculum, C1 = 5 mg / ml (0.85 mg / ml eqv DM), C2 = 10 mg / ml (1, 7 mg / ml eqv DM), C3 = 15 mg / ml (2.55 mg / ml eqv DM)) (p <0.05)).

Te 1mg/ml 2.5 mg / ml

10 mg / ml 5 mg / ml 15 mg / ml

Figure 13. Effect of OCT on the growth of F. oxysporum at different doses ((Te: control without extract, 1 mg / ml, 2mg / ml, 5mg / ml 10mg / ml and 15mg / ml of EAO).

fertilizers and pesticides approved. In Tunisia, most of effects of a product imported from marine algae these products are imported, hence the interest to Algadul®. These treatments were applied on a culture of seek new local natural products able to substitute it. early vegetables pepper greenhouse shed, on which Fertilizer effects of nettle were compared to fertilizer several parameters were evaluated and have yielded 84 J. Med. Plants Res.

the following results: nettle and Algadul® had similar been shown recently, inhibition of the synthesis of effects on fruit quality. endothelin, a vasoconstrictor peptide directly Thus, extracts of nettle (dry or fresh) prove able to responsible for vascular disease and atherosclerosis. In replace imported liquid organic fertilizer (Algadul) fact, polyphenols would act primarily by inhibiting (Hattab et al., 2006). Hence the confirmation of our phosphorylation enzymes, tyrosine kinases that are results that show strong inhibition of the fungi treated involved in the synthesis of endothelin (Parr and with EAO. Interestingly, Bhalodia and Shukla (2011) Bolwell, 2000). demonstrated the antifungal activities of different title of We also have shown a strong correlation (r > 0.85) the extracts from Cassia fistula against three fungal between the antioxidant activity and anti-radical on one strains-Aspergillus niger, Aspergillus clavatus, Candida hand and the total polyphenol content on the other albicans and observed a growth inhibition of the treated hand. U. urens, also showed an interesting antibacterial fungi. activity of various extracts. This result corroborates the Plants produce a large number of compounds that are work of Gulcin et al. (2004) who noted antibacterial not directly produced by photosynthesis, but resulting activity extracts from U. dioica, with bacteria Gram from subsequent chemical reactions (Stavrianakou et positive and Gram negative strains. That would explain at al., 2005). These compounds are referred to as least in part the efficiency of the plant in traditional secondary metabolites. These metabolites often play a medicine for the treatment of skin infections and defensive role of the plant that manufactures them. In gastrointestinal (Yesilada et al., 1993). addition to their antioxidant; anticancer (Buk-Gu et al, Furthermore, our results show that the antibacterial 2015) and antimicrobial activities (Lan et al., 2015), activities depend on the strain tested. Gram positive these metabolites including polyphenols have many bacterial strains (E. faecalis and E. faecium) showed other beneficial effects for human health. Hence the higher sensitivity to the extracts, than the Gram renewed interest in plants. negative strains Escherichia coli and Salmonella U. urens the subject of this study is sorted in the typhimurium. The differential action of the nettle extract order of Rosales, belonging to the family Urticaceae where Gram positive strains seem to be more sensitive better known as nettle. Its beneficial properties for than Gram negative strains could be explained by the human health were established and found mainly extra outer membrane characteristic of gram negative. correlelated to its phenolic content. It is known in Several mechanisms may be involved in the inhibitory traditional therapy that Urticaceae have a hypertensive effect of aqueous extracts of nettle on the membrane free effect (Gulcin et al, 2004). Nettle came into interest strains (gram positive) including adsorption secondary when it was found that this plant rich in polyphenolic metabolites in bacterial membranes and interactions compounds such as tannins, mono and tri-terpenes, with cellular enzymes preventing them from functioning vitamins, acids, polysaccharides, choline, xanthophylls, normally (Fallah et al., 2008; Scalbert et al., 1991). The phenolic acids and other phenolic. Our research has most marked sensitivity was observed with fungi shown that nettle contains carotenoids and anthocyanins Penicillium notatum and Fusarium oxysporium with with rates of 3.33 μg / 100 g DM and 2 μg / 100 g significant variation confirmed by statical tests. DM. It has also demonstrated a strong flavonoid This antifungal activity is consistent with research content of 120±13.5 μg CE / g DM. It is reported that Gulcin et al. (2004) in ethnopharmacology where the flavonoids are beneficial to health since many flavonoids use of nettle infusion for treatment of foot fungus is are involved in the inhibition of blood platelet widespread in Turkey and the Mediterranean countries. aggregation and adhesion on the vessel wall, The U. urens also has a fairly significant antifungal phenomena which can be the source of clots that cause activity since the mycelium growth is completely inhibited thrombosis (Parr and Bolwell, 2000; Knekt et al., 2002). at a concentration of EAO of about 5 mg / ml. This On one hand, high polyphenol content was also activity is confirmed for two fungal strains as P. notatum revealed in our study, exceeding 80 mg/g DM and F. oxysporum. This is all the more plausible that regardless of the extraction solvent used. On the other the use of nettle compost is widespread in Europe and hand, our results also show that the antioxidant particularly in France where it substitutes synthetic activities are related to the nature of the solvent. fertilizers. Its antifungal activity has been the work of Indeed, the aqueous extract seems to be the less Hattab et al. (2006) where the nettle is revealed to be a rich in antioxidants (flavonoids, total polyphenols and natural fertilizer as effective as imported liquid organic condensed tannins). The methanol extract is fertilizer (Algadul®) both for the preservation of the quality particularly rich in flavonoids and total polyphenol of plants with a better efficiency. closely followed by the acetone extract. The study finding put together, demonstrate the strong Thus, it was possible to demonstrate the existence of a activity of plants as antioxidant, antifungal which are in positive correlation between the protective effect of agreement with those obtained by Bobis et al. (2015). certain foods and their content of polyphenolic So far antibiotics are largely as powerful weapon for compounds. Polyphenols are main player in several fighting against infection with the well-known side processes, like decreasing blood pressure and, as has effects that they can engender and emergence of Maaroufi et al. 85

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