Phloroglucinol

Cranberry fruit (Vaccinium macrocarpon Ait.) was collected at the Marucci Center for Blueberry and Cranberry Research, Chatsworth, NJ. Purified C-PAC extract was isolated from cranberries of the ‘Early Black’ cultivar utilizing solid-phase chromatography according to well established methodology [9 (link)–12 (link)]. In brief, the fruit was homogenized in 70% aqueous acetone, filtered and the pulp discarded. Collected cranberry-derived proanthocyanidins were concentrated under reduced pressure and purified extract isolated using bioassay-directed fractionation. The absence of absorption at 360 nm and 450 nm confirm all but proanthocyanidins are removed. Additional methods including ¹³C NMR, electrospray mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and acid catalyzed degradation with phloroglucinol were utilized to verify the presence of A-type linkages as well as to determine the concentration of proanthocyanidins in the purified extract. C-PAC is comprised of five main proanthocyanidins as previously characterized by Dr. Howell and colleagues [11 (link)]. The proanthocyanidin molecules largely consist of epicatechin units with degrees of polymerization of 4 or 5, as well as epigallocatechin and catechin. C-PAC contains three types of linkages, two common B-type linkages (C4→C6 and C4→C8) and at least one unique A-type ether linkage (C2→O→C7) found only in cranberry, chokeberry, plums and avocado [14 (link), 15 (link)]. Purified C-PAC was freeze-dried and stored at −80°C. C-PAC concentrations chosen for study were informed by our earlier research which determined the LD50 to be in the 50 to 100 μg/ml range in various cancer cell lines [16 (link)–18 (link)]. Consideration was also given to earlier evaluations by Howell and colleagues showing 50 μg/ml of C-PAC inhibits adhesion of p-fimbriated uropathogenic E. coli bacteria in vitro and that 36 mg/day of C-PAC delivered in 10 ounces of juice inhibits bacterial adhesion in the urinary tract wall of humans [9 (link)–13 (link)]. Importantly, the concentrations of C-PAC under evaluation in this series of preclinical investigations are readily achievable in humans and are already under evaluation for oral and urinary tract health benefits.

Fucoidan with an average Mw of 735 kDa was provided by MMBI KSC RAS, Murmansk, Russia. Fucoidan was extracted from F. vesiculosus by ultrasound-assisted extraction [72 (link)] according to the procedure described previously [18 (link)]. The yield of fucoidan was 4.9% by mass. Fucoidan contained 79.5% of neutral carbohydrates, 27.0% of sulfate residues, and 0.7% of uronic acid. Carbohydrates were represented by fucose (73.5 mol%), glucose (11.8 mol%), galactose (3.7 mol%), xylose (6.6 mol%), mannose (0.2 mol%), and arabinose (0.2 mol%). The molar ratio of fucose, glucose, galactose, xylose, mannose, and arabinose was 1.0:0.16:0.05:0.09:0.03:0.03, respectively, as evidenced by high performance liquid chromatography (HPLC) [18 (link)]. According to the literature data, fucoidans from Fucus spp. are represented mainly by (1→3)- and (1→4)-linked α-l-fucopyranose residues [3 (link),36 (link)]. Electrospray ionization mass spectrometry evidenced that sulfate groups are attached mostly at C-2 and sometimes at C-4 of sugar residues in fucoidans from brown seaweeds [73 (link)].
The concentration of total polyphenols was determined with Folin–Ciocalteu reagent as described previously [74 (link)]. Phloroglucinol (Sigma, St. Louis, MO, USA) was used as reference. The total polyphenol content was 4.7 ± 0.5 mg PGEq g⁻¹ (milligram of Phloroglucinol equivalent PGEq per 1 g fucoidan).

The assessment of lignin deposition on vascular tissue (xylem) was performed using the method described by Saxena et al. [26 ]. The control and pre-treated chilli plants with bioagents were used for the observation of lignin deposition on cell-wall. After 30 and 60 days of treatment with bioagents, the second internodal region of the stem was selected. The transverse section of the stem of treated and untreated samples was made with hand-cut approx. 0.5 mm thickness with the help of a steel blade. The sections were fixed in 1% (w/v) phloroglucinol solution containing 50% HCl in the ratio of 3:1 and kept for 5 min. After that, the sections were mounted with glycerin on a glass side and covered with a coverslip. The stained tissue was observed under Olympus binocular microscope. The deposition of lignin in stem tissues was examined as red-pink color. The experiment was repeated thrice.