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. 2015 Apr 29:6:359.
doi: 10.3389/fmicb.2015.00359. eCollection 2015.

Comparison of α-glucosyl hesperidin of citrus fruits and epigallocatechin gallate of green tea on the Loss of Rotavirus Infectivity in Cell Culture

Steven M Lipson  1 , Fatma S Ozen  1 , Samantha Louis  1 , Laina Karthikeyan  2
Affiliations

Comparison of α-glucosyl hesperidin of citrus fruits and epigallocatechin gallate of green tea on the Loss of Rotavirus Infectivity in Cell Culture

Steven M Lipson et al. Front Microbiol. .

Abstract

A number of secondary plant metabolites (e.g., flavonoids) possess antiviral/antimicrobial activity. Most flavonoids, however, are difficult to study, as they are immiscible in water-based systems. The relatively new semisynthetic α-glucosyl hesperitin (GH), and the natural plant product epigallocatechin gallate (EGCG) are unique among most flavonoids, as these flavonoids are highly soluble. The antiviral activity of these plant metabolites were investigated using the rotavirus as a model enteric virus system. Direct loss of virus structural integrity in cell-free suspension and titration of amplified RTV in host cell cultures was measured by a quantitative enzyme-linked immunosorbent assay (qEIA). After 30 min. 100 × 10(3) μg/ml GH reduced RTV antigen levels by ca. 90%. The same compound reduced infectivity (replication in cell culture) by a similar order of magnitude 3 to 4 days post inoculation. After 3 days in culture, EGCG concentrations of 80, 160, and 320 μg/ml reduced RTV infectivity titer levels to ca. 50, 20, and 15% of the control, respectively. Loss of RTV infectivity titers occurred following viral treatment by parallel testing of both GH and EGCG, with the latter, markedly more effective. Cytotoxicity testing showed no adverse effects by the phenolic concentrations used in this study. The unique chemical structure of each flavonoid rather than each phenolic's inherent solubility may be ascribed to those marked differences between each molecule's antiviral (anti-RTV) effects. The solubility of EGCG and GH obviated our need to use potentially confounding or obfuscating carrier molecules (e.g., methanol, ethanol, DMSO) denoting our use of a pure system environ. Our work further denotes the need to address the unique chemical nature of secondary plant metabolites before any broad generalizations in flavonoid (antiviral) activity may be proposed.

Keywords: ELISA; a-glucosyl hesperidin; epigallocatechin gallate; infectivity titers; rotavirus.

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Figures

FIGURE 1
FIGURE 1
Rapid in vitro determination of rotavirus infectivity. Rotavirus/flavonoid preparations (and controls) were added in triplicate to MA-104 grown in 96-well cluster plates. After 5 days, the viral capsid protein (i.e., VP6) was quantitatively measured by antigen capture enzyme-linked immunosorbent assay using spectrophotometric readings as the objective signal standard. Spectrophotometric readings were reported as the mean ± SE of the means. P-values <0.05 were considered statistically significant.
FIGURE 2
FIGURE 2
Effect of glucosyl hesperidin (GH) on the loss of rotavirus (RTV) antigen in cell-free suspension. RTV/GH complexes were maintained for 5 and 30 min. at room temperature (23°C) followed by the quantitative measurement (using qEIA) of the VP6 capsid antigen. Data points from spectrophotometric readings represent the mean ± SE of the means. P < 0.05 is assumed statistically significant.
FIGURE 3
FIGURE 3
Effect of GH (100 × 103 μg/ml) on the isolation [antigen detection-cell culture amplification (Ag-CCA)] of RTV in MA-104 cell culture monolayers. Input titers: 3.1 × 103 to 3.1 × 104 TCID50/ml. Day 2 experimental (3.1 × 104) vs. Positive control (3.1 × 104 TCID50/ml): p = 0.08 (n = 3). Spectrophotometric readings performed at 450 nm. Data points represent the mean ± SE of the mean. P < 0.05 was assumed statistically significant.
FIGURE 4
FIGURE 4
Loss of rotavirus (RTV) infectivity following treatment with glucosyl hesperidin and epigallocatechin gallate (EGCG). A markedly reduced concentration of EGCG was effective in reducing RTV infectivity by 1 log10. Virus treatment with glucosyl hesperidin failed to approach that of EGCG, regardless of using the inordinately high flavanone concentration of 5000 μg/ml. Mean ± SE of the mean. P < 0.05 was assumed statistically significant.
FIGURE 5
FIGURE 5
Effect of EGCG on RTV infectivity in MA-104 host cell cultures. RTV was treated with increasing concentrations of EGCG, incubated in suspension for 60 min., followed by inoculation of 25 μl of the virus-catechin complex into microtiter plates (96-well cluster plates) containing MA-104 cells in monolayer culture. After 5 days in culture, viral capsid antigen levels were quantitatively determined. The appearance (or absence) of the cytopathic effect (CPE) was recorded as well. Replicate cultures were monitored for a period of 8 days, showing no differences in the appearance of the RTV CPE. Data points based on spectrophotometric readings represent the mean ± SE of the means. P < 0.05 was assumed statistically significant. 160 vs. 320 μg/ml: P = 0.24. Insert A Photomicrograph of the RTV CPE shows a breakage of cell monolayer with the appearance of cellular rounding and clumping (see Lipson, 1992). Mock infected cell cultures remained intact for the duration of the experiment (not shown).
FIGURE 6
FIGURE 6
Cytotoxicity testing of glucosyl hesperidin and epigallogatechin gallate MA-104 cell cultures. Cytotoxicity testing employed the “ToxilightR Non-Destructive Cytotoxicity Assay”. Readings were performed using a Modulus luminometer (Turner Biosystems/Promega Corp., Sunnyvale, CA, USA) with data presented as relative light units (RLUs). Data points represent the mean ± SE of the means. P < 0.05 were assumed statistically significant.
FIGURE 7
FIGURE 7
Selected flavanones reported to possess antiviral activity. (A) Hesperidin, a flavanone glycoside; (B) hesperitin, an aglycone of hesperidin; (C) α-glucosyl hesperidin (A semi-synthetic derivative of hesperidin. U.S. Patent number: 5,652,124); (D) EGCG.
FIGURE 8
FIGURE 8
Effect of EGCG on MA-104 cell cultures. (A) Three hundred microgram EGCG induces a precipitation effect in microtitier wells containing MA-104 cell cultures maintaned in minimal essential medium plus supplements. (B) Untreated MA-104 monolayer culture. Amino acids and proteins are known to bond onto EGCG in a stacking pattern to which the above effect may be ascribed in (see text for details).
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