2.3.7. Antioxidant Properties

The antioxidant properties of LOMW were evaluated by using specific tests (inhibition capacity towards the lipophilic (DPPH) and hydrophilic (ABTS) radicals and to measure the total antioxidant activity (TCA)).

The scavenging activity of LOMW in an organic environment was evaluated in terms of decrease of the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•) concentration [23]. A total of 1.0 mL of hydro-alcoholic solutions (50:50 *v*/*v*) of LOMW were added to 4.0 mL of hydro-alcoholic mixture (50:50 *v*/*v*) and 5.0 mL of DPPH• ethanolic solution (200 μM). The mixture was kept at 25 ◦C for 30 min and the residual concentration of the DPPH•

radical was spectrophotometrically evaluated at 517 nm. The percentage of inhibition of the DPPH• radical was calculated according to the following equation:

$$\text{Inhibition } \text{(\%)}= (\text{A}\_0 - \text{A}\_1)/\text{A}\_0 \times 100\tag{3}$$

where A0 is the absorbance of the control solution prepared under the same conditions but without sample, while A1 is the absorbance recorded analyzing LOMW sample. The scavenging activity of LOMW against the lipophilic radical DPPH was expressed in terms of IC50. Each measure was performed in triplicate and data expressed as means ( ±SD).

The scavenging activity in the aqueous environment of LOMW was determined in terms of reduction of the radical 2,2-azino-bis(3-ethylbenzothiazolin-6-sulphonic) (ABTS•) [24]. Aqueous solutions of LOMW were prepared, at different concentrations and 2.0 mL of the aqueous solution of the ABTS radical was added to 500 μL of each. The solutions were then kept in the dark for 6 min and the residual concentration of the ABTS radical was spectrophotometrically evaluated at 734 nm. The percentage of inhibition of the ABTS• was calculated according to the Equation (3), while LOMW scavenging activity was expressed in terms of IC50. Each measure was performed in triplicate and data expressed as means ( ±SD).

Total antioxidant capacity (TAC) of LOMW was evaluated by mixing 300 μL of LOMW hydro-alcoholic solutions (50:50 *v*/*v*) with 1.2 mL of the reagen<sup>t</sup> solution (28.0 mmol L−<sup>1</sup> Na3PO4, 4.0 mmol L−<sup>1</sup> (NH4)2MoO4, 0.6 mol L−<sup>1</sup> H2SO4) [25]. The solutions were kept at 95 ◦C in the dark for 150 min and then by measuring the adsorbance at 695 nm. A calibration curve was constructed by the method of least square, using 8.0, 16.0, 24.0, 32.0 and 40.0 μM hydro-alcoholic solutions (50:50 *v*/*v*) of CT. The TAC was expressed in milligrams of CT per gram of LOMW (mg CT/g LOMW). Each measure was performed in triplicate and data expressed as means ( ±SD).

### *2.4. Synthesis of the Tara Gum Conjugate by Grafting Procedure*

The synthesis of polymeric conjugate was carried out according to the methods reported in literature, with some modifications [26]. In a 100 mL glass flask, 0.250 g of tara gum was dissolved in 37.5 mL of purified water, then 12.5 mL of H2O2 (120 vol) and 0.3 g of ascorbic acid were added at 25 ◦C. After 2 h, LOMW (equivalent to 0.035 g of CT) was dissolved into the reaction flask. After 24 h, the mixture was introduced into dialysis tubes (MWCO: 12,000–14,000 Dalton) and dipped into a glass vessel containing distilled water for 48 h. The conjugate (PLOMW) was checked to be free of unreacted antioxidant and any other compounds by LC analysis after purification step. LC analysis was performed on a Knauer (Asi Advanced Scientific Instruments, Berlin, Germany) system equipped with two pumps Smartiline Pump 1000, a Rheodyne injection valve (20 μL) and a photodiode array detector equipped with a semi-microcell. The resulting solution was frozen and dried with "freezing-drying apparatus" to provide a vaporous solid. A control polymer, blank tara gum (BTG), was also prepared under the same conditions but without LOMW.

### *2.5. Characterization of Tara Gum Conjugate*

### 2.5.1. H-NMR Analysis of Tara Gum Conjugate

The samples of PLOMW and BTG (5.8 mg) were dissolved in 0.6 μL of D2O (99.9% D). 1H-NMR spectra were performed at 25 ◦C using a Brucker Advance 200 spectrometer of 300 MHz equipped with a 13C/1H dual probe. The NMR experiments were recorded with a spectral width of 6983.240 Hz, an acquisition time of 10.20 s, a number of 64 scans, a relaxation time of 2 s and, a pulse width of 7 s. The spectra were processed by XWIN-NMR.

### 2.5.2. Differential Scansion Calorimetry (DSC)

The DSC studies were performed using a SETARAM 131 instrument. The amount of each sample was around 3–10 mg. Analyses were performed from 25 to 650 ◦C at a temperature scan rate of 10 ◦C min−1, under nitrogen flux.

The method used is set out in more detail below:


### 2.5.3. Antioxidant Properties of the Tara Gum Conjugate

PLOMW conjugate was characterized in terms of available phenolic groups, phenolic acids and flavonoids content by the methodologies previously described, expressing the results as mg of CT per gram of PLOMW. Similarly, antioxidant performances were recorded by evaluation of TAC (expressed as mg of CT per gram of PLOMW) and scavenging activity if both aqueous (against ABTS radical) and organic (against DPPH radical) environments.

2.5.4. Toxicity of the Tara Gum Conjugate
