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Study of the Chelating Ability of Hexane, Chloroform, Ethyl acetate and Methanolic Root Extracts from Algerian Phragmites australis Species

Introduction: Heavy metals that enter organisms can cause illness and disease. Chelation therapy is a solution that uses chelating agents to protect these organisms by high accumulation. Materials and methods: The objective of this study is to evaluate the capability of different roots extracts from reed or EDTA (as standard) to complexate iron using, ferrozine-ferrous reducing assay or Zn and Cu using the murexide-Zn or Cu reducing assay in vitro. Results: the results proved that the organic phase of the first extract and EDTA (standard chelator), have the strongest chelation activities with respective rates of 70% and 97% for iron, 55% and 56% for zinc and 47% and 88% for copper compared to the control groups considered to be 100% of the complexation. Finally, the HPLC analysis showed the presence of six organic acids, which are probably responsible for this effect. Conclusion: The hexane extract and EDTA (standard), have the strongest chelation activities.


INTRODUCTION
Pollution is an unfavorable change in the environment caused totally or partially by humans either directly or indirectly, as it can occur naturally under the influence of different physical, chemical and biological agents and therefore resulting in deterioration of the quality of water, air or soil. It affects the distribution of energy flows, radiation levels and the physico-chemical properties of the ecosystem. This change can affect humans directly or through agricultural, water or biological resources. The degradation of the quality of the environment as a result of pollution is due to the loss of vegetation, biological diversity, excessive amounts of harmful chemicals in the atmosphere and in cereals intended for food (Zhao et al. 2019) 1 .
Heavy metals are considered a major source of environmental contamination. They are toxic, persistent and can negatively affect all life forms. In humans they are absorbed directly by ingestion or inhalation and concentrated in different parts of the human body resulting in chronic or acute effects on human health, including physiological disorders of the respiratory system, renal and gastrointestinal (Sellal et Chelation is a treatment that uses chelating agents to treat disorders caused by an abnormally high accumulation of metal ions in organisms. Although heavy metals are toxic to humans and plants and can cause pollution of water, soil and air, they need to be removed before they are released to the environment. (Wu, 2016) 3 .
Chelating agents are organic or inorganic compounds that have the ability to bind to metal ions to result in the formation of a "chelate" which is a complex ring-like structure (Flora and Pachauri, 2010) 4 .
The short-chain aliphatic fatty acids of the plant Phragmites australis represent a group of monocarboxylic organic acids, which are also classified among the natural chelators of heavy metals (Cu, Zn and Fe) thanks to the presence of carboxylic group (Kumar et al. 2015) 5 .
The aim of this research is to study the chelating power of different extracts from the root part of the Phragmites australis plant and to identify the active substances in the effective extract.

Extracts preparation
The roots of reed plant were collected in the southern of Bordj Bou Arreridj (eastern Algeria). The material was rinsed and dried. The material is then ground up to obtaining a fine powder. Then, the 25 g of the last preparation were extracted by 190 ml of different solvents with increased polarity hexane, chloroform, ethyl acetate and methanol successively using soxhlet, and the obtained were then evaporated at 45 ° C in a rotary vacuum evaporator (Buchi), dried and stored at 4 °C (Sanjeevkumar et al. 2014) 6 .

Ferrozine-ferrous reducing assay
The ferrous chelating ability of roots P. australis extracts was evaluated by the method described by Le et al. (2007) with some modifiations 7 . The five hundred µl of Ethylene Diamine Tetra acetic Acid (1 mg/ml as standard) or different roots extracts (1 mg/ml as sample) are added to fifty µl FeSO4 (2 mM) and 450 µl methanol. 5 minutes later, the complexing reaction is stimulated by the addition of fifty µl ferrozine (5mM) and the absorbance of the solution was measured spectrophotometrically at 562 nm after 10 min. The control group contained all product except the extract.

Murexide-zinc or copper reducing assay
The zinc and copper chelating ability of roots P. australis extracts was determined by the method described by Watak and Patil, (2012) 8 . practically, the volume of 0.5 ml containing 25, 50, 75, 100, 125 and 175 µl for each extract taken from a stock solution (1 mg/ml) or Ethylene Diamine Tetra acetic Acid (1 mg/ml) was prepared for Zn chelation assay. For copper chelation the same volume contains a different concentrations (1, 2, 3, 4, 5, 7 mg/ml of extracts) or (10, 20, 30, 40, 50, 70 µg/ml of Ethylene Diamine Tetra acetic Acid) is also prepared. Preparations are then added to 1.6 ml deionized water and 0.1 ml of ZnCl2 or CuSO4 (8 mM). 30 seconds later, the complexing reaction is stimulated by the addition of 0.1 ml of murexide (5 mM) and the mixture is incubated in the dark for 10 min. After, the absorbance was measured spectrophotometrically at 562 nm against a blank, which contains all the solutions used except the extracts.
The percentage of inhibition of ferrozine-Fe 2+ or murexide-Zn or murexide-Cu complexes formations was given by the formula: % inhibition= [(Ac -As)/ Ac] ×100 Where: AC was the absorbance of the control and As was the absorbance in the presence of the sample of P. australis extracts and standard.

HPLC
The analysis and identification of organic acids (fumaric, formic, malic, propionic, isobutyric, acetic, oxalic, citric and succinic acid) of the root hexane extract are carried out by the chromatographic method UV-Vis at 206 nm according to the method described by Sa et al. (2011) 9 .
Standard solutions (1000 mmol/l) are prepared separately in ultra-pure distilled water (HPLC grade) and stored in the freezer until 'in use.
The chromatographic chain (HPLC) used for the analytical control is brand waters 2695 and includes the following elements: a degasser (waters), two pumps (waters) of an isocratic system, an auto sampler and a waters 2996 detector connected with computer software (Empower). The analytical column used is an ion exchange column of the brand Aminex HPX-87H used for the analysis of carbohydrates, volatile fatty acids and fatty acids with a short aliphatic chain (length 300 mm, diameter 7.8 mm and the size of the 9 µm particles).
The mobile phase is a sulfuric acid solution at 0.005 mol/l introduced at 55 ° C with a flow rate of 0.9 ml/min and injection volume of 20µl in isocratic mode after filtration (on a membrane omnipore TM merck millipore Ltd with a porosity of 0.45 µm) and degassing by ultrasound.

Statistical analysis
The results are presented as mean ± SD of three replicates. All products (extracts and standard) are compared against the control using Student's t test. The ANOVA was used to compare the means between groups using CoStat software. P < 0.01 considered as significant.

Ferrozine-ferrous reducing assay
Ferrozine forms a red complex with ferrous ions. the chelation effect is therefore inversely proportional with the red color. In general, hexane extract and EDTA showed significantly (p ≤ 0.01) higher ferrous ion-chelating capacity than the other extracts, where the obtained absorbance dose-dependent decreased to a lesser extent of 0,54  0,01 and 0,046  0,01 respectively ( Figure 1). In contrary, the same volumes of the other roots extracts (chloroform, ethyl acetate and methanol) showed a high stable absorbance which is still higher than 1. The difference between hexane extract or EDTA and the witness was statistically significant with maximum inhibition of 70% and 97% respectively.

Murexide-zinc or copper reducing assay
For zinc chelation, the hexane extract of root part and EDTA at a volume of 175 μl taken from a stock solution of 1 mg/ml gives a lower highly significant absorbance's (p≤0.01) of 0.41  0,005 and 0.40  0.005 respectively than that of the negative control 0.93  0.02 considered as 100% of the complexation and to the other extracts which are given high and stable absorbances despite the increase in concentration (Figure 2).
The hexane roots extract from the reed is generated therefore an effect very close to that of EDTA where the zinc ions are chelated with an approximate percentage of 55% and 56% respectively compared to control group.

Figure 2: The murexide-Zn reducing ability of different root reed extracts and EDTA (as a standard) at 562 nm. The absorbance's presented as mean ± SD of three replicates and P < 0.01 taken as significant.
For copper chelation, the addition of hexane extract to the mixture induces a marked reduction in absorbance compared to the control (1.20 ± 0.01, p≤0.01). Indeed, the hexane extract of the leaf reveals an absorbance of 0,63 ± 0.01 with a concentration of 7 mg/ml (Figure 3 A). The copper complexation with murexide was thus reduced by approximately 47% compared to the negative control considered as 100% of the complexation. These results remain lower than those obtained with EDTA, where the minimum absorbance obtained with only a volume of 70 µl is 0,13 ± 0.005 (Figure 3 B) which corresponds to a maximum inhibition of 88% compared to the control. Finally, the ANOVA comparison between all products used (extracts and EDTA) for the assays showed a significant difference in the following order EDTA ˃ HRE ˃ MRE ˃ CRE ˃ EARE.

High performance liquid chromatography analysis of hexane root extract
The qualitative analysis based on the comparison of retention times between the sample (hexane root extract) and the standards proved the presence of six organic acids (figure 4 and 5) and the quantities presented in table 1 also calculated using the chromatograms areas comparison between the sample and standards.

DISCUSSION
Chelation means the insertion of a mineral ion into a complex cyclic structure by an organic or inorganic ligand which represents the chelating agent. Generally, sulfur, nitrogen and / or oxygen are the electron donor atoms that are found in the chelating agent because they contain pairs of solitary electrons. The strength of the chemical bonds within the complexes formed between the chelators and the metal ions rests on the elements involved and their stereochemistry (Sears, 2013) 10 .
Chelation is considered a favorable solution to reduce the toxicity of metals. Complex structures are formed (chelates) after binding of chelating agents with toxic metal ions, these structures are easily removed from inside or outside of cells (Flora and Pachauri, 2010   The roots of reed plant can absorb and accumulate high quantities of toxic metals. this capacity is mainly due to the parenchyma cortex which has large intercellular air spaces. Then the toxic elements were transferred in vacuoles to the leaf parts (Sellal et al. 2016) 12 .
Our results also, confirm that the hexane extracts from the leaves and stems of the Phragmites australis plant have the strongest chelation activities with respective rates of 89% and 86% for iron, 56% and 54% for zinc and finally 64% and 44% for copper (

CONCLUSION
The chelating power of different extracts from the root part of the reed plant against Fe, Zn and Cu was evaluated in vitro.
The obtained results, showed that the hexane extract and EDTA (standard), have the strongest chelation activities with respective rates of 70% and 97% for iron, 55% and 56% for zinc and 47% and 88% for copper compared to the control groups. These effects are probably due to the presence of oxalic, citric, malic, fumaric, formic and propionic acids confirmed by an HPLC analysis.