fruits: A new source of bioactive anthocyanins

The extraction conditions and chromatographic analysis from seeds of Inga edulis were optimized and provided one anthocyanin from aqueous fraction and a mixture of three anthocyanins from methanolic fraction. The pure anthocyanin (500 mg) obtained was subjected to structural modifications and the products obtained were subjected to chemical and pharmacological assays, as well as quantum chemical calculations based on DFT and TD-DFT methods. Hence, the anthocyanin fractions were evaluated for their chemical-pharmacological potential through chemical and biological assays: antioxidant activity by the DPPH free radical inhibition test, determination of the Solar Protection Factor (SPF) and cytotoxic activity (hepatocellular carcinoma infected with hepatitis C virus). The results indicated that even the anthocyanin and derivatized compounds having high antioxidant potential showed an SPF lower than six, which is lower than the minimum accepted by current Brazilian legislation. In addition, none of compounds presented significant cytotoxic activity against the tumour cell line studied.


Plant material
Fresh fruits of I. edulis were collected in Teófilo Otoni city (Minas Gerais state, Brazil) on may 10 th , 2017 taxonomically identified and deposited in the collection from UFVJM herbarium in Diamantina city (Exsicates -HED 2555). After collection, the fruits were then separated into the bark, seed, and pulp. The seeds, where are concentrated the anthocyanins, were then dried at room temperature since high temperatures degrade the pigments responsible for the coloring of seed coatings.

Preparation of extracts and obtaining derivatized anthocyanin compounds
Anthocyanins are highly soluble in water and alcohol solutions. In water, they are generally more stable at low pH values and at high pH values tend to be easily degraded, and as result produce phenolic acid and aldehydes according Pascual-Teresa and Sanchez-Ballesta (2008).
The anthocyanins were extracted with the use of water (purified with Millipore Milli-Q system) acidified with 1% HCl and/or methanol (methanol PA from Tedia) acidified with 1% HCl, providing the aqueous and methanolic extracts. After extraction, the samples were then filtered and concentrated in a rotary evaporator (54-Rotavapor R-220, brand: Büchi, with vacuum pump Vacuum Contoller V-805, brand: Büchi and water circulator). After TLC analysis, the extracts were purified on Amberlite® XAD16N 20-60 mesh (Sigma-Aldrich) chromatographic column. The two fractions obtained rich in anthocyanins were subjected to purity was investigated by means of the HPLC analysis, then evaluated by ultraviolet/visible scanning spectroscopy, which used the Perkin Elmer® double beam spectrophotometer (model Lambda 20), whose tests were also performed in triplicate. Further, the methanolic fraction clearly showed to be composed of three anthocyanins, while the aqueous fraction provided only one anthocyanin (500 mg). In this case, the anthocyanin isolated from the aqueous fraction was characterized by 1 H and 13 C NMR analysis and submitted to structural modification according to the methodology described by Bordignon Jr et al. 2009.

RP-HPLC-PDA and 1 H NMR analysis
As a preliminary analysis, the obtained fractions were applied on reverse phase silica (C18) (20 x 20 cm X 0.2 mm) chromatoplates of the Sorbent brand.
The Shimadzu Chromatograph equipped with two Shimadzu LC-10AD pumps, Shimadzu SIL 10A auto-injector, UV-VIS array detector model Shimadzu SPD MX AVP. Data acquisition and processing were performed using a Pentium II 500 MHz computer using Shimadzu CLASSLC10 software (version 1.64A). This analysis was performed on both chromatographs from the analytical model in the condition for exploratory gradient in methanol (HPLC grade of the JT Baker® brands) in a Phenomenex C18-Hydro chromatographic column (250 x 4.6mm, 4μm) at room temperature, flow 1 mL.min -1 , injection volume was 30 μL.
The mono-and bidimensional NMR spectra from isolated anthocyanins were obtained on the Varian Inova 500® Spectrometer.

Evaluation of chemical and pharmacological potential from anthocyanins and derivatized compounds.
Antioxidant potential: To evaluate the antioxidant potential, in vitro photocolorimetric method of free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) described by Mensor et al. (2001). Briefly, 20 μg/mL of the methanolic solution of the diluted samples was added to 220 μg/mL of a free radical methanolic DPPH solution. After 30 minutes of reaction, the reading was carried out at 517 nm using a UV-Vis spectrophotometer (Shimadzu UV 1601). All readings were performed in triplicate and the average of the obtained data was then calculated. After that, the percentage of the antioxidant activity of the extracts was estimated, by the following formula: Where AA = percentage of antioxidant activity; Aa = absorbance of the sample; Ab = white absorbance; Ac = control absorbance.
Determination of the Sun Protection Factor (SPF) from anthocyanins: For the determination of SPF, the spectrophotometric methodology of Mansur et al. (1986). In Absorbance.

Computational details
All of the DFT and TDDFT calculations were performed in the gas phase using the Gaussian 09 software. The molecular geometries of all anthocyanin and derivatized compounds, are shown in Figure S5, and were theoretically evaluated at the B3LYP/6-31+G level. This theoretical level was also used for the frequency calculations and electronic excitations via a single point TD-DFT.      Table S1. DPPH Inhibition potential and cytotoxic potential from anthocyanin and derivatized compounds.