# Anthocyanins, Phenolic Compounds, and Antioxidants from Extractions of Six _Eucalyptus_ Species
## 2 Materials and Methods
### Chemical Characterisation  
Based on the best performing extraction method, the following analyses were performed:  
The total phenol content of extracts was determined by the Folin-Ciocalteu method [32; 33]. An Agilent spectrophotometer, the Cary-60 VIS-UV (Agilent Technologies, Santa Clara, CA, USA), with double internal beam and pulsing xenon lamp was used, and absorbance was read at 760 nm. A gallic calibration curve at five gallic acid concentrations (0, 4, 10, 25, 50 mg/L) was obtained. The procedure described for the standard gallic acid was followed and the absorbance was recorded for each extract. Samples were prepared in triplicate for each analysis and the average absorbance value was used to plot the calibration curve to determine the level of phenols in the extracts. The total phenolic content of the extracts  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Solvent**} & \multirow{2}{*}{**Type of Solvent**} & **Dielectric Constant F/m** & \multirow{2}{*}{**High**} & **Polarity** & \multirow{2}{*}{**Low**} & **Boiling Temperature (\({}^{\circ}\)C)** & **Density (g/mL)** \\ \hline Acetone (CH\({}_{3}\)COCH\({}_{3}\)) & dipolar aprotic & 20.7 & & X & & 56 & 0.786 (25 \({}^{\circ}\)C) \\ \hline Dichloromethane CH\({}_{2}\)Cl\({}_{2}\) & Non-polar & 9.1 & & & X & 40 & 1.326 (20 \({}^{\circ}\)C) \\ \hline Ethanol (CH\({}_{3}\)CH\({}_{2}\)OH) & polar proctic & 24.3 & X & & & 79 & 0.789 (20 \({}^{\circ}\)C) \\ \hline Hexane & \multirow{2}{*}{non-polar} & \multirow{2}{*}{2.02} & \multirow{2}{*}{X} & \multirow{2}{*}{69} & \multirow{2}{*}{0.655 (25 \({}^{\circ}\)C)} \\ (CH\({}_{3}\)CH\({}_{2}\)CH\({}_{2}\)CH\({}_{2}\)CH\({}_{3}\)) & & & & & \\ \hline Methanol (CH\({}_{3}\)OH) & polar proctic & 33 & X & & 65 & 0.791 (20 \({}^{\circ}\)C) \\ \hline Water (H\({}_{2}\)O) & polar proctic & 80 & X & & 100 & 1.000 (20 \({}^{\circ}\)C) \\ \hline \hline \end{tabular}
\end{table}
Table 2: Characteristics of the solvents used.  
was expressed as mg gallic acid equivalents (GAE) per gram of sample on a dry weight basis (mg/g).  
Anthocyanins measurements were done by the acid hydrolysis in butanol method [34]. The absorbance was recorder at 530 nm, absorption maximum observed to anthocyanin [30], and the results were expressed as based on cyanine equivalent. A cyanine calibration curve at five concentrations (0, 5, 10, 15, 30 mg/L) was obtained. A standard calibration method was followed, the commercial cyanine chloride standard, 1 mg, was dissolved in 1 mL of methanol and the 5 mL flask was made up to the mark with distilled water. From this solution, which is 200 ppm, the necessary dilutions are prepared to make the standard line. The results were expressed as mg cyanine chloride /g extract.  
Antioxidant activity was assessed by two methodologies: (1) ABTS- + method; 3 mL of ABTS- + solution was mixed with 10 \(\mu\)L of extracts. A decrease in absorbance was measured at \(\lambda\) = 734 nm [30]; (2) CUPRAC assay: the CUPRAC solution included copper (II) chloride (0.01 M in water), ammonium acetate buffer (0.001 M, pH = 7), and neocuproine (0.0075 M in ethanol) (ratio 1:1:1:1). Then, 3 mL of CUPRAC reagent was mixed with 10 \(\mu\)L of extracts. An increase in absorbance was recorded at \(\lambda\) = 450 nm [31].