Application of Cyanidin in Quantitative Estimation of Metals in Fish Samples

The use of cyanidin as a metallochromic agent in analyses of heavy metal is reported. Cyanidin is a ligand that was extracted from Gmelina arborea fruit and characterized. The cyanidin was used to form complexes with metals in five fish samples for the quantitative determination of Cu, Zn, Ca and Mg. The optimum pH for absorbances of the cyanidin-metal complexes was observed at 5. Experimental results obtained using cyanidin were compared with analyses results obtained by Atomic absorption spectrophotometry (AAS) and both methods were evaluated using paired T-test to ascertain the suitability of cyanidin as metallochromic agent for the quantitative determination of heavy metals in fish samples. A null hypothesis that cyanidin method is a good alternative to AAS was accepted for the analyses of Cu and Zn (p > 0.05). The paired T-test, however rejected the null hypothesis for the determination of Ca and Mg (p < 0.05). This study has provided a cheap, sensitive, rapid, simple and easy method for metal determination in analytical samples.


Introduction
Anthocyanins belong to plants whose pigments are water soluble [1] [2]. They are widely distributed in nature and their pigment shades range from red, purple and blue. They are found in flowers, fruits, vegetables and other parts of higher plants. Anthocyanins belong to the class of flavonoids and have a general structural formula of C 6 C 3 C 6 [1] [3]. Anthocyanins are hydroxyl B-ring substituted is a derivative of anthocyanin and is a chromic indicator that has been isolated from flowers of Hibiscus sabdariffa [7], red beet [1] and many others [3]. Cyanidin is a sensitive metallochromic indicator for detecting changes in UV-Vis absorption parameters as a result of its interaction with a plethora of metal ions. Ukwueze [11].
An important feature of anthocyanins is their pH dependence and their structure in aqueous solutions is distinguished from other forms with variations in pH [12] [13]. This also favours stability of their complexes with metals [14] [15] [16] [17]. The oxidation state of metal ions is another important factor that plays a crucial role in the formation of anthocyanin-metal complexes [18]. The most common metals in anthocyanin complexes are Sn, Cu, Fe, Al, Mg, and K [19]. The metal binding ability of anthocyanins presents a simple, sensitive, cheap, rapid and environmentally friendly approach/analytical tool for metal determination in environmental samples [8] [9] [10] [11]. Metal binding activity of anthocyanins is investigated via a variety of analytical techniques and to date UV-Vis spectroscopy is considered the most informative tool for the detection of anthocyanins [14]. Jangantakumar and Shukla [20] and Okoye et al. [10] have registered that spectral characteristics in visible wave bands as well as additional maximum absorption in the UV region are the most potent ways to monitor metal binding activities of anthocyanins. On the other hand AAS is known for its versatility, reliability, sensitivity and accuracy. It still has limitation such as the use of reagents that are not eco-friendly [21]. Considering the increase in metal concentration in the environment which may affect life of fish and other aquatic organisms and man through food chain, there is the need for metal monitoring of fish as a source of food to man. Therefore this work is aimed at developing cyanidin as an indicator from Gmelina arborea whose fruit is non-edible in Nigeria for the purpose analytical importance and further validating its suitability by comparing its performance with AAS.

Experiments
All reagents and fish samples used were purchased in local market and the reagents were analar grade. Fresh stock and standard solutions were used through-out the experiment. The metal ions were determined in fish samples using Uv-Vis spectrophotometer (Jenway 6405 model), AAS (210CGP Bulk scientific) and Elico pH meter.

Pigment Extraction and Purification
500 g of Gmelina arborea fruits (see Figure 1) were washed under tap and rinsed with distilled H 2 O. The fruits were separated from its seeds; ground and 400 cm 3 of distilled H 2 O were added to the extract. The ground fruits were filtered using Whatman filter paper No. 1. The supernatant was concentrated to 300 cm 3 and diluted with 100 cm 3 conc. HCl and refluxed for 2 hours. The solution was allowed to cool until crystals began settling out. The solution was later put in the refrigerator to allow for more crystal formation. The crystals were filtered, recrystallized from hot methanol and air dried in the laboratory at room temperature. The crystals were stored in a dark sample bottle to avoid auto oxidation.

Spectral Characterization of Crystals
5% solution of the purified crystals were prepared by dissolving 5 g of the crystals in methanol containing 0.01% conc. HCl and made up to 100 cm 3 in a standard flask. 1 cm 3 of this solution was diluted to 10 cm 3 and its λmax was determined spectrophotometrically in a 1 cm 3 cuvette by scanning from 200 -700 nm.

Determination of Wavelength of Maximum Absorption (λmax) of Crystal-Metal Complexes
Fresh standard solutions of Cu 2+ , Zn 2+ , Ca 2+ , and Mg 2+ were prepared at various concentrations ranging from 1.0 to 3.0 ppm. 5 cm 3 of each standard solution were introduced into five sample bottles containing 5 cm 3 of crystal solution and kept at pH5. After equilibration of the solutions, the absorbances of the crystal-metal complexes were recorded at maximum wavelength after scanning from 200 to 700 nm.

Sample Preparation and Analysis
Five tilapia fish samples were dried at 105˚C until constant weight was maintained. The dried fish samples were pulverized and weighed into porcelain crucibles. 1 cm 3 conc. HNO 3 was added to each crucible and ashed at 450˚C. The ashes were dissolved in 5 cm 3 1:1 HCl and samples transferred into 250 cm 3 flask containing 1 cm 3 5% crystal solution at optimum pH. The crystal-sample solutions were made up to mark with distilled water and analysed using UV-Vis spectrophotometer and AAS (210CGP bulk scientific).

Statistical Analysis
Paired T-test was used to determine significant variation between the developed method using the Gmelina arborea crystals and AAS.

Spectra Characterization of Purified Crystals from Gmelina arborea Extract
The spectral data obtained from the analysis of the extract in comparison with literature values are shown in

Selection of Optimum pH at λmax of Metal Ions
The effect of pH on the formation of cyanidin-metal complexes was observed from a calibration curve. At various pH values, these cyanidin-metal complexes showed reproducible results. However, all the metal complexes showed prominent absorbances at pH = 5. In consideration of selectivity and sensitivity of the method, pH value of 5 was chosen as the optimum pH for the simultaneous determination of these metals complexes with cyanidin in aqueous media. American Journal of Analytical Chemistry

Analyses of Fish Samples Using the Cyaniding Complex
The analyses results of metals from fish samples analysed with AAS and UV-Vis is shown in Table 2. The metal distribution was Ca > Mg > Cu > Zn for AAs and Cu > Mg > Zn > Ca when cyaniding method was used. These distribution patterns in the fish samples suggest different levels of the metals in the different environments the fishes habited. Table 3 shows the paired T-test of the significant differences in the methods and how they affect the H o .
H 0 was accepted as summarised in Table 3. Therefore Zn can be analysed using both AAS and cyaniding method. This may be said for Cu since there mean differences supported the H 0 (p > 0.05, −2.5706 ≤ t ≥ 2.5706) as shown in Table 3.
This can be attributed to the stable complexes they formed with cyanidin extracted from Gmelina arborea.

Conclusion
From the results obtained and the statistical evaluation of the data, cyanidin method and AAS were both good alternatives to analysing Zn and Cu. Conversely, cyanidin method seems not a very good alternative to AAS in the quantitative determination of Ca and Mg. The method has shown good sensitivity, reliability and easy preparation of the reagent compared with other existing extractive spectrophotometric determination methods.

Recommendations
This work recommends the following: firstly the authors have added Gmelina arborea as a source of cyanidin to published list of plants that contain cyanidin.
There is plethora of plant sources yet to be identified and analysed for cyanidin.
Such works should be done on plants that are yet to be explored to determine their analytical importance. This work has demonstrated the usefulness this method has on aquatic samples like fish. It is recommended that this method be used for metal analyses on other environmental samples such as soil, water, dust and plant tissues. As a work in progress, the authors are isolating other ligands contained in Gmelina arborea to determine their suitability in metal analyses.