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

Urtica urens is classified into the sub family of Rosales, belonging to the Urticaceae 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 (r 2 > 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.


INTRODUCTION
All living beings have a primary metabolism which provides the basic molecules (carbohydrates, amino acids, nucleic acids, proteins, lipids etc.) from where secondary metabolites are produced through the subsequent chemical reactions.There is no doubt that plants are a good source of biologically active natural products.To investigate bioactive natural compounds, it is essential to have access to simple biological tests to *Corresponding author.E-mail: hossain.sazzad@uni-bielefeld.de.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License locate required activities (Beart et al., 1985).The preservative effect of many plant species and herbs suggests the presence of antibacterial and antioxidant constituents in their tissues (Bobis et al., 2015; C h e w e t a l ., 2 0 1 5 ; Kukrić et al., 2012;Singh et al., 2012;Hirasa and Takemasa., 1998).
Plants contain phenolic compounds and nitrogen compounds having essential alkaloids and glycosides.These metabolites often play a role in plant defense where they are produced.Besides the antioxidant and antimicrobial activities, polyphenols have several other beneficial effects on human health (Najjaa et al., 2007).Faced with the therapeutic limit of chemical drugs, the developmental research on medicinal plants has been directed towards obtaining herbal from folk medicines (Škrovánková et al., 2012).This 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 (Fattouch et al., 2007).
Urticaceae is one of the important sources of polyphenols.The beneficial effect of nettle on human health is already very well known (Heoa et al., 2015;Fattahi et al., 2014;Mansoub, 2011;Gulcin et al., 2004).The most abundant and endemic species of nettle in Tunisia are Urtica dioica and Urtica urens.They are spontaneous grass and their aerial parts are used as salad particularly in North-Africa and Mediterranean countries.There is some research work done about the antioxidant activity of Urtica dioica (Gulcin et al., 2004) but no more information is available about Urtica urens in this context.The main aim of this study is to evaluate the potentials of U. urens as a new source of natural phenolic compounds and its antioxidant and antimicrobial activities.

Plant materials
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.

Preparation of the plant extracts
Aerial parts of U. urens were washed and dried in the open air at room temperature, frozen and grounded in liquid nitrogen.The resulting powder was used for the preparation of methanol (EMO) and acetone (ECO) extracts.The powder obtained was macerated in the appropriate solvent (like methanol and acetone) under stirring for 30 min at room temperature.The mixture was left at 4°C for 24 h to be subsequently filtered through a Whatman paper (No.4 Sigma Aldrich ® ).The filtrate is collected and stored in dark at 4°C for next use.

Preparation of the aqueous extract
In order to avoid any c o n t r o v e r s i a l contradiction between the effect of the solvent and the effect of the nettle itself, the antibacterial assay was conducted with the water extract (EAO) of nettle.To prepare the water extract plant powder was mixed with the boiled water and centrifuged at 4000 g for 20 min and finally filtered on filter paper.The filtrate was then stored at 4°C for further use.

Determination of total phenolic contents
The assay for the determination of total phenolic content in U. urens was performed according to the method of Namjooyan et al. (2007).Amounts of 1.85 ml of distilled water, 0.125 ml of Folin-Ciocalteu reagent and 0.5 ml of a 20% sodium carbonate (Na 2 CO 3 ) solution were added to 25 µl of liquid extract sample in a test tube, making a final volume of 2.5 ml.The solution was homogenized and left to stand for 30 min, and the absorbance was determined at 750 nm.The total phenols were calculated as milligrams of gallic acid equivalents per gram of dry matter (DM).

Determination of total carotenoid
Determination of total carotenoid was performed according to the method of Wellburn (1994) with few modifications.50 mg of fresh plant material was ground in 10 ml of acetone (80%).The obtained extract was centrifuged and filtered.The reading of the absorbance is carried out at three different wavelengths of 470, 663 and 647 nm and the concentration is determined as follows: Carotenoid = 5 × DO470 + 2.846 × DO663 -14.87 × DO647 The result is expressed in mg per 100 g dry matter (DM).

Determination of total flavonoids
The principle is based on the formation of the complex flavonoids-aluminum chloride (Djeridane et al., 2006;Chang et al., 2002).The color intensity is proportional to the concentration of the flavonoids.A volume of 250μL of the extract is diluted 5 times, supplemented with 75 μL of a solution of Na NO 2 (5%), followed by a rest of 6 min before adding 150 μL of aluminum chloride (AlCl 3 , 6H 2 O, 10%) after 5min of incubation, 500 μL of NaOH (1 M) are added.The final volume of the solution is adjusted to 2500 μL with distilled water.The absorbance of the mixture was read at 510 nm.The reference range is prepared with the catechin.The content of total flavonoids expressed as mg catechins per gram of dry matter (DM).

Determination of condensed tannins
In the presence of concentrated sulfuric acid, condensed tannins depolymerize and transformed in the presence of vanillin anthocyanidols red color whose intensity is measured spectrophotometrically at 500 nm.A volume of 3 ml of vanillin (4%) was added to 50 µl of the extract and 1500 µl of concentrated HCl.The resulting mixture was incubated for 15 min at room temperature and the absorbance is measured at 500 nm.The standard is like catechin flavonoids, condensed tannins content in mg catechin equivalents per gram of dry matter (mg CE / mg DM).

Determination of total anthocyanins
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 continuous stirring followed by centrifugation and filtration of supernatant.The absorbance is read at two different wavelengths (530 nm and 675 nm).The concentration of anthocyanins was expressed in g per 100 g of dry matter (DM).

Total antioxidant capacity
Total antioxidant capacity of the extract was evaluated by the 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 incubated at 95°C in the dark for 90 min.The absorbance reading was taken at 695 nm against a blank containing the solvent and the reagent.A standard range is prepared and the results are expressed in mg gallic acid equivalent per range of dry matter (DM).

Free radical scavenging activity
The free radical scavenging activity of the nettle extract was measured by 1,1-diphenyl-2picryl-hydrazil (DPPH) was evaluated using colorimetric method of Shimada et al. (1992) and improved by Islam et al (2013).At room temperature and in the presence of an antioxidant, the DPPH radical has an intense purple color.The transition to non-radical form DPPH (after saturation of its electronic layers) was accompanied by the disappearance of the violet color (Soler-Rivas et al., 2000).The decrease in the intensity of the color is monitored by spectrophotometry at 517 nm.The estimation of this activity was measured.The free radical scavenging activity of each sample is expressed as percent inhibition of DPPH using the following formula: Where, A0 is absorbance at 517 nm DPPH without extract and A is sample absorbance.

Bacterial strains
Four reference bacterial strains were used to evaluate antibacterial activity: two Gram negative (Escherichia coli JW1772 and Salmonella typhimurium ATCC14028) and two Gram-positive (Enterococcus faecium and Enterococcus faecalis) provided by the microbiology laboratory at the Faculty of Sciences of Tunis.

Bacterial growth
Turbidity is measured by spectrophotometry (Thermofisher).Samples were taken in sterile conditions and Optical Density (OD) is measured every 1 h.A growth curve is established, expressing the OD as a function of time: OD = f (t).

Fungi and its processing by the nettle extract
Two reference fungi Penicillium notatum and Fusarium oxysporum, provided by the microbiology laboratory at the Faculty of Sciences of Tunis, were used to evaluate antifungal activity.The PDA (Potato dextrose agar) medium was poured in the presence of the extract of specific volume, to obtain different desired concentrations of 1, 2.5, 5, 10 and 15mg / ml of the aqueous extract of nettle.After the extract and the medium were well mixed and solidified, then appropriate fungal spores were placed on the agar.Incubate for 3 days at an optimal temperature of 20°C.The growth kinetic was measured by calculating the diameter of the mycelium formed every 24 h.

Statistical tests
The ANOVA was used to verify the difference between the control and the different doses and for the four strains bacterial and two fungi.Student's T-test was performed in other cases.

Determination of antioxidants of different extracts of nettle
The environmental pollution, chemical products, food additives, physical stress and normal usage of oxygen by our body all contribute to the production of harmful radicals that damage healthy cells.Antioxidants act by trapping free radicals to isolate their single electrons and transforming later in stable molecules or ions.Most of the antioxidants produced by the plants are then processed by humans as humans are unable to synthesize them (Fattouch et al., 2007).
The total polyphenols content varies depending on the type of solvent used as summarized in (Figure 1).In fact, methanol seems to be the most suitable solvent for extraction of polyphenols (with a grade of 88.75±12.25 mg EAG / g DM) followed by acetone (with an average of 56.5±13,66mg EAG / g DM).The lowest value was observed in the aqueous extract (with a grade of 43.915 ± 19.95mg / g DM).The study of the variation of the total polyphenol content varies significantly depending on the type of solvent used (p <0.05).Polyphenols are fundamental constituents of the plant thanks to their purifying capacity provided by their hydroxyl group (Hatano et al., 1989).
According to recent works, a strong correlation exists between polyphenols and antioxidant power and this is demonstrated in many plants (Vinson et al., 1998;Velioglu et al., 1998;and Oktay et al., 2003).Phenolic compounds could contribute in a direct way to the antioxidant activity (Duh et al., 1999).It is suggested that polyphenolic compounds may have anti-mutagenic and anti-carcinogenic in humans.When they are ingested with a daily dose of 1 g for a diet composed only of fruits and vegetables (Tanaka et al., 1998).In addition, it has been reported that the phenolic compounds have been associated with antioxidant activity and play an important role in the stabilization of lipid peroxidation (Yen and Duh, 1994).
The aerial parts of nettle contain carotenoids with a content of about 3.33 μg and anthocyanins in particular smaller quantities of approximately 2 μg (Figure 2).Proanthocyanin also called "condensed tannins" are substrates of enzymatic browning.The predominant physicochemical property of tannins is their ability to bind by hydrophobic interactions and/or exchange of hydrogen bonds with proteins and polysaccharides   (Jeantet et al., 2007).They form salivary protein complexes with condensed tannins, which give the astringency of fruit (grapes, peaches, persimmons, apples, pears, etc.), beverages (wines, ciders, tea, beer, etc.) and the bitterness of chocolate (Santos-Buelga and Scalbert, 2000).The condensed tannins content varies depending on the solvent used as shown in Figure 3.
In fact, acetone seem to be the most suitable solvent for extraction of condensed tannins (with a content of 115.33±2.6 µg CE / g DM) followed by methanol (with a content of 52.33±15.22µg CE / g DM).The lowest value was observed in the aqueous extract (with a content of only 39.28 ± 2.28 µg CE / g DM).The study of the variation of the condensed tannin content varies significantly depending on the type of solvent used (p <0.05).
Flavonoids are antioxidants belonging to the family of polyphenols (Fernandez-Pachon et al., 2004;Zhishen et al., 1999).Flavonoid content also tends to vary depending on the type of solvent used as shown in Figure 4.The contents are as follows the methanol extract with a content of 67.5±13.5 µg CE / g DM, the extracted with acetone (with a content of 66.1±6.5 µg CE / g DM), and the aqueous extract (with a content of only 46±5.9 µg CE / g DM).The polarity of the solvent doesn't seem to have an impact on the extraction since the most polar solvent (water) showed the lowest extraction capacity for the all investigated polyphenols.

Antioxidant power and anti-radical activity of U. urens
The antioxidant activity was attributed to several mechanisms among which we mention the peroxide decomposition and reducing free radicals.Many methods have been proposed to evaluate the antioxidant power.Among these techniques, we adopted total antioxidant activity and anti-radical activity by the free radical

DPPH.
The extract showed antioxidant activity depending on the dose or the solvent used (Kataki et al., 2012).However, a strong antioxidant activity was observed with the methanol extract and acetone extract higher than gallic acid activity used as a positive control (Figure 5).The antioxidant activity is also dependent on the dose used in nettle extract.The effect of antioxidants on anti-radical activity of the DPPH is due to their ability to donate a proton.The DPPH is a stable free radical that accepts an electron or hydrogen radical to become a stable diamagnetic molecule.The anti-radical method of DPPH is a technique used to evaluate the antioxidant activity in a short time compared to other methods (Soares et al., 1997).
The reduction of the DPPH• radical is determined by the absorbance at 517nm induced by antioxidants.Figure 6 shows a significant decrease (p<0.05) on the concentration of DPPH due to the anti-radical power of 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 can conclude that the nettle appears to have an effective antioxidant power that reacts with free radicals.This finding is interesting knowing the capacity of polypehnols to limit the damage caused by these radicals in the body.The comparison between the antioxidant capacity of the extracts (IC50%) obtained in our laboratory with different extracts of nettle (extract with water, extracted with acetone and methanol extract) and IC50% of Urtica dioica obtained can be considered as comparable.
Indeed, it is 31.6 for EAO against 44.77±2.12 for the roots with a different extraction protocol (Gulcin et al., 2004).

Correlation between the content of total polyphenols and total antioxidant activity
There is a strong correlation between the antioxidant activity and total polyphenol content with an r 2 > 0.94 as shown in Figure 7. Similar results were found by other authors who showed that there is good correlation between total phenolic profile and antioxidant activity of plant extracts emphasizing the role of phenolic compounds in antioxidant activity (Tunalier et al., 2002;Duh et al., 1999).

Correlation between the total polyphenol content and anti-radical activity of nettle
There is a strong correlation between the antioxidant activity and total polyphenol content with an r 2 > 0.85 all extract confused as shown in Figure 8.The strong correlation observed between the antioxidant potential and the total polyphenol content suggests that polyphenols are the main compounds that contribute to this activity.Indeed, rats poisoned by CCl 4 xenobiotic and treated with nettle extracts showed significant increases in liver antioxidant enzymes (Yen and Duh, 1994).
In addition, the purification of H 2 O 2 has been attributed to phenols thanks to their ability to donate electrons to H 2 O 2 , and neutralizing it in water form by the called process water-water cycle which take place in plants (Asada et al., 2000).
The capacity of the EAO to purify the H 2 O 2 can be attributed to its structural characteristics of its active   components that determine their ability to donate electrons.The scavenging capacity of EAO on H 2 O 2 was compared to that of BHA, BHT and α-tocopherol as a standard.The concentrations used have shown activity by 23% H 2 O 2 .In comparison, the same concentration of BHA, BHT and α-tocopherol show 38.86 and 57% respectively.These results show that the scavenging effect of EAO on H 2 O 2 is lower but still interesting in comparison with BHA, BHT and αtocopherol (Halliwell, 1991).

The effect of aqueous nettle extract on the growth of various microorganisms
The effect of EAO on bacterial growth was demonstrated with by the effect of the extract on kinetic growth inhibition.A starting OD600 nm at 0.1 nm was used in all the assays with an appropriate volume of aqueous extract of nettle to obtain the following concentrations: 5, 10 and 15 mg/ml.For monitoring of the biomass, measurements of OD at 600 nm were carried out on samples taken every hour for 9 hours.Measurements are plotted on curves expressing the OD600 nm versus time.A reference inoculum is treated the same way but in the absence of aqueous extract of nettle establish the control curve of the bacterial growth.Analyses were replicated three times and reproducibility of the results was verified (Figure 9 to 12).

Escherichia coli gram negative
The E. coli growth (Figure 9) shows that there is inhibition of cell growth while addition of the aqueous extract of nettle in the bacterial culture medium.Indeed, the concentration of 5 mg / ml resulted in a tendency to decrease the growth rate is of 0.32 h -1 lower than the control (0.44 h -1 ), 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 microorganisms)decreases from 0.29 to 0.25 h -1 .In addition, the bacterial culture treated with aqueous nettle extract between the stationary phase at a cell density significantly lower than that of the cultivation of the control bacteria.

Salmonella typhimurium gram negative
As shown in Figure 10, the addition of aqueous nettle extract at different concentrations of 5 to 15 mg / ml at OD starting 0.1 resulted in a lag phase longer than the phase of the control.It lasts approximately 2 h.The application of statistical tests showed that the observed changes are significant (p < 0.05).Indeed during the exponential phase, the calculated bacterial control growth is of 0.41 h -1 while it decreases to 0.36 h -1 for EAO concentration of 5mg / ml and even a lower µmax of about 0.35 h -1 for EAO 10 mg/ ml and a µx of 0.27 h -1 for a concentration of extract of 15 mg / ml.

Enterococcus faecalis gram positive
The application of statistical tests showed that the observed variations are significant (p<0.05).Comparative analysis of the curves of the treated bacteria and control shows that the addition of aqueous extract significantly affects the growth of E. faecium especially in first three hours.Indeed, the growth rate is relatively low (0.041, 0.04 and 0.02 h -1 , respectively for concentrations EAO 5mg / ml to 10 mg / ml and 15 mg/ ml) against a μmax of 0.2 h -1 of the control strain during the growth phase.During the stationary phase, the gap is smaller between different concentrations.It was 0.16 h -1 for the untreated strain and 0.11 h -1 for strain treated with a rate of EAO 5 mg/ml and 0.1 h -1 for treated strains with EAO rate of 10 and 15 mg / ml.It seems that the treated strain resume growth with the exponential phase, one could even speak of a phase shift that can be explained by an adaptation of the bacteria to the new environment for pursuing of growth (Figure 11).

Enterococcus faecalis gram positive
The application of statistical tests showed that the observed variations are significant (p < 0.05).
Comparative analysis of the curves corresponding to the treated bacteria and bacteria that corresponding to the controls shows that the addition of aqueous extract significantly affect the growth of E. faecalis (Figure 12).It seems that the gram positive strains are more sensitive to our extract than Gram negatives strains tested.This can be explained by the fact that gram positive bacteria are devoided of the extra outer membrane that enhance their vulnerability and allow an easier penetration of the active substances contained in the EAO.

Effect of the nettle on fungal growth
Nettle extract was added in the presence of the culture medium of Fusarium oxysporum at different concern.The Figures 10 and 11 shows that the growth of Penicillium notatum is inversely proportional to the dose EAO added.Indeed, the higher is the dose administered in the medium, the stronger is the effect of the mycelium inhibition.which is noticeable from Figure 10 (a,b and  c) for the dose of 1 mg / ml as well as a dose of 2.5 mg / ml up to complete disappearance of mycelium starting at 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 variations between control and the different doses observed are significant (p < 0.05).In fact, the higher dose administered in the medium a higher is the effect on the mycelium growth.It dramatically decreases (from  1 to 2.5 mg / ml) or completely disappears (no growth is seen) while adding 5, 10 and 15 mg/ml (Figure 13).The growth of Fusarium oxysporum is strongly conditioned by the EAO added dose.This is even more likely that the nettle is used by European farmers, as a natural fertilizer called compost or manure nettle to fight against plant pests such as F. oxysporum.Organic farming has only a very limited range of 1 mg / ml 2.5 mg / ml 5 mg / ml 10 mg / ml 15 mg / ml  1 mg /ml 2.5 mg / ml 5 mg / ml 10 mg / ml 15 mg / ml Te 1mg/ml 2.5 mg / ml 5 mg / ml 10 mg / ml 15 mg / ml the following results: nettle and Algadul® had similar effects on fruit quality.Thus, extracts of nettle (dry or fresh) prove able to replace imported liquid organic fertilizer (Algadul) (Hattab et al., 2006).Hence the confirmation of our results that show strong inhibition of the fungi treated with EAO.Interestingly, Bhalodia and Shukla ( 2011) demonstrated the antifungal activities of different title of the extracts from Cassia fistula against three fungal strains-Aspergillus niger, Aspergillus clavatus, Candida albicans and observed a growth inhibition of the treated fungi.
Plants produce a large number of compounds that are not directly produced by photosynthesis, but resulting from subsequent chemical reactions (Stavrianakou et al., 2005).These compounds are referred to as secondary metabolites.These metabolites often play a defensive role of the plant that manufactures them.In addition to their antioxidant; anticancer (Buk-Gu et al, 2015) and antimicrobial activities (Lan et al., 2015), these metabolites including polyphenols have many other beneficial effects for human health.Hence the renewed interest in plants.
U. urens the subject of this study is sorted in the order of Rosales, belonging to the family Urticaceae better known as nettle.Its beneficial properties for human health were established and found mainly correlelated to its phenolic content.It is known in traditional therapy that Urticaceae have a hypertensive effect (Gulcin et al, 2004).Nettle came into interest when it was found that this plant rich in polyphenolic compounds such as tannins, mono and tri-terpenes, vitamins, acids, polysaccharides, choline, xanthophylls, phenolic acids and other phenolic.Our research has shown that nettle contains carotenoids and anthocyanins with rates of 3.33 μg / 100 g DM and 2 μg / 100 g DM.It has also demonstrated a strong flavonoid content of 120±13.5 μg CE / g DM.It is reported that flavonoids are beneficial to health since many flavonoids are involved in the inhibition of blood platelet aggregation and adhesion on the vessel wall, phenomena which can be the source of clots that cause thrombosis (Parr and Bolwell, 2000;Knekt et al., 2002).
On one hand, high polyphenol content was also revealed in our study, exceeding 80 mg/g DM regardless of the extraction solvent used.On the other hand, our results also show that the antioxidant activities are related to the nature of the solvent.Indeed, the aqueous extract seems to be the less rich in antioxidants (flavonoids, total polyphenols and condensed tannins).The methanol extract is particularly rich in flavonoids and total polyphenol closely followed by the acetone extract.
Thus, it was possible to demonstrate the existence of a positive correlation between the protective effect of certain foods and their content of polyphenolic compounds.Polyphenols are main player in several processes, like decreasing blood pressure and, as has been shown recently, inhibition of the synthesis of endothelin, a vasoconstrictor peptide directly responsible for vascular disease and atherosclerosis.In fact, polyphenols would act primarily by inhibiting phosphorylation enzymes, tyrosine kinases that are involved in the synthesis of endothelin (Parr and Bolwell, 2000).
We also have shown a strong correlation (r > 0.85) between the antioxidant activity and anti-radical on one hand and the total polyphenol content on the other hand.U. urens, also showed an interesting antibacterial activity of various extracts.This result corroborates the work of Gulcin et al. (2004) who noted antibacterial activity extracts from U. dioica, with bacteria Gram positive and Gram negative strains.That would explain at least in part the efficiency of the plant in traditional medicine for the treatment of skin infections and gastrointestinal (Yesilada et al., 1993).
Furthermore, our results show that the antibacterial activities depend on the strain tested.Gram positive bacterial strains (E.faecalis and E. faecium) showed higher sensitivity to the extracts, than the Gram negative strains Escherichia coli and Salmonella typhimurium.The differential action of the nettle extract where Gram positive strains seem to be more sensitive than Gram negative strains could be explained by the extra outer membrane characteristic of gram negative.Several mechanisms may be involved in the inhibitory effect of aqueous extracts of nettle on the membrane free strains (gram positive) including adsorption secondary metabolites in bacterial membranes and interactions with cellular enzymes preventing them from functioning normally (Fallah et al., 2008;Scalbert et al., 1991).The most marked sensitivity was observed with fungi Penicillium notatum and Fusarium oxysporium with significant variation confirmed by statical tests.
This antifungal activity is consistent with research Gulcin et al. (2004) in ethnopharmacology where the use of nettle infusion for treatment of foot fungus is widespread in Turkey and the Mediterranean countries.The U. urens also has a fairly significant antifungal activity since the mycelium growth is completely inhibited at a concentration of EAO of about 5 mg / ml.This activity is confirmed for two fungal strains as P. notatum and F. oxysporum.This is all the more plausible that the use of nettle compost is widespread in Europe and particularly in France where it substitutes synthetic fertilizers.Its antifungal activity has been the work of Hattab et al. (2006) where the nettle is revealed to be a natural fertilizer as effective as imported liquid organic fertilizer (Algadul®) both for the preservation of the quality of plants with a better efficiency.
The study finding put together, demonstrate the strong activity of plants as antioxidant, antifungal which are in agreement with those obtained by Bobis et al. (2015).So far antibiotics are largely as powerful weapon for fighting against infection with the well-known side effects that they can engender and emergence of bacterial resisitance lead to search and develop new tools.Plants can be used as a substitute, and thisopen up for new alternatives (Guyue et al., 2014).

Conclusion
The U. urens r e v e a l i t s potentials as a new source of natural phenolic compounds and its antioxidant and antimicrobial activities can be used as a substitute of new alternatives for fighting against infection and harmful effects of human.

Figure 1 .
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 .
Figure 2. Content of total carotenoids and anthocyanin with nettle expressed in 100 g of dry matter.

Figure 3 .
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 .
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.

Figure 5 .
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.

Figure 6 .
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).

Figure 7 .Figure 8 .
Figure 7. Curve correlation (r 2 ) between the total polyphenol content and antioxidant activity in Urtica urens and determination of the correlation of coefficient.