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  • br Extraction of the chelate soluble pectin fraction


    2.2. Extraction of the chelate-soluble pectin fraction (CSF)
    The frozen papaya pulp was ground in N2 and incubated three times in boiling chloroform:methanol (1:1; 15 min). The solid residue was further washed in boiling 80% ethanol and acetone and dried at ambient temperature to obtain the papaya's total cell wall. The Puromycin was ex-tracted three times with deionized water (20 min). Then, the remaining insoluble material was extracted three times with 0.08 M ammonium oxalate (pH 4.5; 30 min) accordingly to Prado et al. [21]. The superna-tants from the extraction in ammonium oxalate of each ripening-time point were combined to give the chelate-soluble pectic fraction, which was dialyzed (MWCO 3.5 kDa, Millipore, Billerica, MA, USA) against cold water for 72 h and freeze-dried. The CSF obtained on the first until the fifth days after harvest (DAH) were named 1CSF, 2CSF, 3CSF, 4CSF, and 5CSF, respectively. Analysis for ash, starch, proteins and phe-nolic compounds were done in order to test the purity of CSF extracted from papayas.
    2.3. Interaction between CSF and galectin-3
    The interaction between 1-5CSF and galectin-3 was measured by the ability of pectin fractions to inhibit galectin-3-mediated agglutination, similar to what was described previously [24,25]. Firstly, recombinant galectin-3 was produced (Nangia-Makker et al. [26]). Then, the mini-mum amount of recombinant galectin-3 that induces the hemaggluti-nation of a 4% rabbit erythrocytes solution (determined as 4 μg galectin-3/mL) was determined. Next, a solution of 4% rabbit erythro-cytes was added to V-bottom 96 well-plates and mixed with distinct concentrations of 1-5CSF added or not to 4 μg/mL galectin-3. After incu-bation (90 min) at room temperature, the minimum concentration of each pectin fraction that inhibits galectin-3-mediated hemagglutination was determined. PBS or 1% BSA was used instead of galectin-3 as con-trols. Erythrocytes mixed only with CSF were used to observe distinct effects rather than galectin-3-mediated hemagglutination (results equal to controls). Lactose was used as a positive control to inhibit the galectin-3 hemagglutination (minimum concentration assay). The assay was repeated at least three times to confirm the results.
    2.4. Effects of CSF on colon cancer cells
    Colon cancer cell lines HCT116 and HT29 were purchased from American Type Culture Collection guidelines (ATCC) or Rio de Janeiro Cell Bank (BCRJ, Rio de Janeiro, Brazil). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing penicillin (100 μL/mL) and streptomycin (100 μg/mL) added to 10% fetal bovine serum. Cells 
    were maintained under standard conditions and subcultured when they reached a confluence of 70 to 90%. Cells (1 × 104 cells/well; 96-well plate) were plated overnight for the MTT assay. Then the medium was replaced, and the cells were treated or not with 2-4CSF (0.05, 0.1 or 0.2%) or lactose (33 or 100 mM) for 24, 48, and 72 h. The higher concentration was also tested for sucrose (100 mM) and for another type of pectin (citrus pectin by sigma − 1%) to attest that these concentrations did not affect cell prolif-eration by osmotic effects (Supplemental Fig. 1). After incubation, 0.5 mg/mL MTT was added (3 h, 37 °C). Next, the supernatant was re-moved, and the formazan crystals were solubilized with DMSO. Absor-bance at 490 nm was measured in a microplate reader (Bio-Rad, Hercules, CA). The cells' viability at each incubation time was expressed in relation to the untreated cells (control).
    2.5. Structural characterization of CSF
    2.5.1. Homogeneity and molecular weight
    Chelate-soluble fractions were analyzed by high-performance size-exclusion chromatography coupled with a refractive index detector (HPSEC-RID). A 1250 Infinity system (Agilent, Santa Clara, CA) was used, equipped with four PL aquagel-OH columns (60, 50, 40, and 30; 429,300 × 7.5 mm) connected in tandem. The eluent was 0.2 M NaNO3/0.02% NaN3 (0.6 mL/min), and the RID temperature was set at 30 °C. Average molecular weight was calculated using a standard curve of dextrans (MW 5–1800 kDa).