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Iron, zinc and copper chelation activity of Phragmites australis leaves extracts.


Water is something of a rare commodity in Algeriadue to the impact of climate change. Renewable natural water resources are estimated at approximately 15 billion [m.sup.3] per year that is approximately 404 [m.sup.3] per individual per year, near the threshold of 500 [m.sup.3] per capita per year, which is widely recognized as the deficiency threshold that indicates developing of crises [4].

In Algeria sustainable water management is one of the main areas of sustainable development, since the water must meet the needs of present and future generations meet. Agriculture is the largest consumer of water resources, given the reduced water inflows recorded for several decades. Farmers, especially those in inland areas, are interested in the use of wastewater wadis. The future development of water resources depends on solutions characterized by high energy consumption, for example sea water desalination, the reuse of wastewater and the introduction of drip irrigation. Development of the water sector will therefore be closely tied to the development of the energy sector, but the cost of treatment is still a problem, it is minimized by system of phytoremediation [5].

A large number of plants, have very interesting biological properties, which find their application in various fields especially in environment. The evaluation of phytoremediation properties, such as accumulation and chelation of heavy metals is considered very important and very useful, especially for plants, which are economics [14].

Heavy metal pollution caused by a combination of natural leaching and anthropogenic activity is becoming a significant environmental problem. Thousands of hectares of arable land have been contaminated, representing a significant health hazard to the population. Phytoremediation is seen as a favorable strategy to remove the contamination. The optimal phytoremediating plant needs to be highly productive in terms of biomass and efficient in terms of accumulation of heavy metals. One such species is the Phragmites australis, a most effective accumulator of Cd, Pb and Zn, and it has been widely exploited as a sewage treatment wetland species. However, the most efficient accumulators appear to be poor in terms of biomass production [15].

Phragmites australis is a macrophyte plant of economic interest frequently found in wetlands throughout temperate and tropical regions of the world. In addition, it can accumulate several metals, including Fe, Zn and Cu [11].

This plant can withstand extreme environmental conditions. Phragmites ecotypes exist with their own stable morphological, physiological and ecological characteristics, which are adapted to specific environments, including the presence of toxic heavy metal contaminants, such as zinc, iron and copper [6]. Consequently, P. australis is considered a plant with high detoxification and phytoremediation potential and has been widely used to engineer wetlands for the treatment of industrial wastewater containing heavy metals. Therefore, much attention has recently been focused on the response of this plant to heavy metal stresses [9].

The objective of this study is to explore the chelating activity of the leaves of the common reed (P. australis) using different solvents (hexane, chloroform, ethyl acetate and methanol)


2.1. Materials:

2.1.1. Chemicals:

All chemicals products were purchased from sigma-aldrich or fluka.

2.1.2. Collection and Extraction preparation of plant materials:

Ariel part (leaves) of P. australis were collected from Elhamadia region situated in south of Bordj Bou Arreridj (east of Algeria) in the month of March 2013. The collected plant materials were washed with running tap water and dried in shade for four weeks. After drying the plant leaves were ground in moulinex mill until a fine powder of clear green color. The powder is prepared just prior to extraction.

The air dried leaf powders (21 g) were successively extracted by soxhlet extraction using solvents (190 ml) of increasing polarity Hexane, chloroform, ethyl acetate and methanol for six cycles for each one. The extracts were then concentrated to dryness at 45 [degrees]C in a rotary vacuum evaporator (Buchi). Finely, the extracts were dried and stored at 4[degrees]C in a sterile container for further use [10].

2.2. Methods:

2.2.1. Ferrous ion chelating activity:

The chelation of iron (II) ions by the P. australis extracts was determined as described by Gulzar et al. (2013) with some modification. A 500 [micro]l of hexane, chloroform, ethyl acetate and methanol extracts solutions (1 mg/ml) in DMSO or EDTA (1 mg/ml)in distilled water was mixed with 50 [micro]l FeS[O.sub.4] (2 mM) in distilled water and 450 [micro]l methanol. Five minutes later, 50 [micro]l of ferrozine (5mM in distilled water) was added to the mixture. The controls contained all the reaction reagents except the extract. After a 10 min equilibrium period, the absorbance of the solution was measured spectrophotometrically at 562 nm. The percentage of inhibition of ferrozine-[Fe.sup.2+] complex formation was given by the formula: % inhibition = [([A.sub.C] - [A.sub.S])/[A.sub.C]] x 100

Where [A.sub.C] was the absorbance of the control and [A.sub.S] was the absorbance in the presence of the sample of P. australis extracts and standard.

2.2.2. Zinc and copper chelating assay:

The chelating activity of zinc ions for P australis leaf extracts solution 1mg/ml or 10mg/ml in DMSO for Zn and Cu chelating activity respectively was measured by the method of murexide described by Watak and patil, (2012) with slight modification. A volume of 0,5 ml of DMSO containing a different volumes (25, 50, 75, 100, 125 and 175 [micro]l) of HLE, CLE, EALE, MLE or EDTA (1mg/ml in deionized water as a standard) from Zn chelation assay or 0,5ml of different concentrations (1, 2, 3, 4, 5, 7mg/ml) and (10, 20, 30, 40, 50, 70 [micro]g/ml) for EDTA from copper chelation was mixed with 1,6 ml deionized water and 0,1 ml of Zn[Cl.sub.2] or CuS[O.sub.4] 8mM in HCl buffer (0,1M prepared by addition of KCl 0,1M at pH 5). After 30 second 0,1 ml of murexide (5mM in the same buffer) was added. Murexide reacted with the divalent zinc or copper to form stable magenta complex species that where very soluble in water. The mixture was then kept at room temperature in the dark for 10 min, and the absorbance was measured at 462 nm. The control containing deionized water without sample. A lower absorbance of the reaction mixture indicated a higher [Zn.sup.2+] or [Cu.sup.2+] chelating activity. The test was carried out in triplicate. The percentage of inhibition of murexide-[Zn.sup.2+] or [Cu.sup.2+] complex formation was given by the formula: % inhibition = [([A.sub.C] - [A.sub.S])/[A.sub.C]] x 100.

Where [A.sub.C] was the absorbance of the control and [A.sub.S] was the absorbance in the presence of the sample of plant extracts and standard.

Statistical Analysis:

Results are expressed as mean [+ or -] SD of three parallel replicates. Student's t test used to compare all products (extracts and standard) against control using Excel software. Parametric one-way analysis of variance (ANOVA) followed by LSD comparisons to compare the mean values among the groups was performed using CoStat software. P values < 0.01 were regarded as significant and P values < 0.001 very significant.


The iron-chelating activity is based on absorbance measurement of iron (Il)-ferrozine complex. This complex produced a red chromophore with a maximum absorbance at 562 nm. The chelators agents are able to capture ferrous ion beforferrozine. Figure 1 demonstrate that the addition of increased volumes of hexane leaf extract and EDTA as a standard induces a significant (p [less than or equal to] 0,01) decrease dose-dependent in the optical density at 562 nm compared to the control suggesting that the hexane extract possesses a high capacity to iron chelate where the absorbance obtained decreased to a lesser extent 0,2 [+ or -] 0,011 compared to the EDTA (standard chelator) 0,04 [+ or -] 0,013. The addition of the same volumes of the rest leaf extracts (chloroform, ethyl acetate and methanol) showed a high stable absorbances which is still higher than 1, this prove that those extracts have a less capacity to chelate ferrous ions compared to the EDTA (standard chelator) and hexane leaf extract.

The difference between hexane extract and the control was statistically significant with maximum inhibition that exceeds 85% from 100 pi. The EDTA (standard chelator) produced to a significant (p [less than or equal to] 0.01) effect (97 %) compared to the control. The comparison of variances between our products used (extracts and EDTA) showed a difference very significant in the following order EDTA > HLE > CLE > MLE > EALE. This analysis showed also that EDTA and HLE regroup in the same range.

In the zinc or copper chelating assay, the chelator were able to capture the ions and inhibit the formation of [Zn.sup.2+] or [Cu.sup.2+-] murexide complex which has an absorption maximum at 462 nm where the low absorbance indicate a high chelating activity. The addition of different volumes (25, 50, 75, 100, 125 and 175 [micro]l) of HLE, CLE, EALE and MLE or EDTA as positive control showed that HLE and EDTA have highest significant (p [less than or equal to] 0.01) capacity dose dependent among them with increased absorbance's 0,41 [+ or -] 0,01 and 0,42 [+ or -] 0,02 (figure 2) and highest percentage of inhibition (56% and 55%) at 100 [micro]l respectively.

In the copper chelation assay, the uses of different concentrations (1, 2, 3, 4, 5, 7mg/ml) of the same extracts or (10, 20, 30, 40, 50, 70 [micro]g/ml) of the EDTA showed a significant (p [less than or equal to] 0.01) dose dependent ability to chelate copper ions with 0,43 [+ or -] 0,03 at 7mg/ml compared to the control and the rest extracts (CLE, EALE and MLE) which presented a high stable absorbance, but this capacity still low compared to the standard chelator which gave a very significant (p [less than or equal to] 0.001) capacity with 0,13 [+ or -] 0,005 at only 70 [micro]g/ml.

The statistical analysis between all products showed a difference very significant where the HLE and EDTA present always greater.

According to [8] Phragmites australis has a high capacity to absorb and accumulate iron and Zinc by roots and their transfer to the aerial parts.

Some studies confirm the results obtained by Phragmites australis. Indeed, in vivo work proves that this plant has a large bioaccumulate heavy metals these latter is transferred by the adsorption process.

Roots of Phragmites australis can absorb and accumulate a great quantity of heavy metals in order Zn and Cu because of the cortex parenchyma with a large intercellular air spaces. Then the heavy metals transferred to aerial parts where accumulated in leaf vacuoles [1].

Some studies have also developed that the concentration of zinc increases by order root> stem> leaves with exogenous increase of zinc [7].

Practically the chelation of heavy metals by the leaves of P. australis gives the best results with extracts of hexane. According To [12] this results due to the presence of propionate and iso-butyrate (fatty acids with short aliphatic chain).where propionic acid and isobutyric acid cheeks the role of electron donors and transforms the ferrous or ferric iron is therefore inhibits the formation of the complex ferrous-ferrozine [2], as these fatty acids have the ability to form complex with copper and zinc faster than murexide.


In this study, the chelation effect of hexane, chloroform, ethyl acetate and methanol extracts from plant Phragmites australis against the iron, zinc and copper ions was evaluated in vitro. The results obtained showed that the hexane extract is a good chelator. This activity was evaluated by the chelation tests of iron, zinc and copper prove that the extract of the hexane Phragmites australis possess very advantageous chelating properties compared to standard chelator. The chelating activity of heavy metals from the extract of the obtained hexane Phragmites australis is probably due to the presence of fatty acid short aliphatic chain which cheeks the role of an electron donor in the case of iron, and making complex with zinc and copper.

Received 28 December 2015; Accepted 28 January 2016; Available online 24 February 2016


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(1) Sellal A., (1) Melloul D., (1) Benghedfa N., (2) Belattar R and (1) Bouzidi A

(1) Faculty of Natural Sciences and Life, Department of Biochemistry, University. Setif 1, Algeria

(2) Department of Ecology and Plant Biology University of Constantine. Algeria

Address For Correspondence:

Sellal A., Laboratory of Biochemistry, Faculty of Natural Sciences and Life, Department of Biochemistry, University.Setif 1, Algeria. Email:
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Author:Sellal, A.; Melloul, D.; Benghedfa, N.; Belattar, R.; Bouzidi, A.
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:6ALGE
Date:Jan 1, 2016
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