Aqueous Extracts of Hibiscus sabdariffa Calyces Decrease Hepatitis A Virus and Human Norovirus Surrogate Titers

Abstract

Hibiscus sabdariffa extract is known to have antioxidant, anti-diabetic, and antimicrobial properties. However, their effects against foodborne viruses are currently unknown. The objective of this study was to determine the antiviral effects of aqueous extracts of H. sabdariffa against human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)) and hepatitis A virus (HAV) at 37 °C over 24 h. Individual viruses (~5 log PFU/ml) were incubated with 40 or 100 mg/ml of aqueous hibiscus extract (HE; pH 3.6), protocatechuic acid (PCA; 3 or 6 mg/ml, pH 3.6), ferulic acid (FA; 0.5 or 1 mg/ml; pH 4.0), malic acid (10 mM; pH 3.0), or phosphate buffered saline (pH 7.2 as control) at 37 °C over 24 h. Each treatment was replicated thrice and plaque assayed in duplicate. FCV-F9 titers were reduced to undetectable levels after 15 min with both 40 and 100 mg/ml HE. MNV-1 was reduced by 1.77 ± 0.10 and 1.88 ± 0.12 log PFU/ml after 6 h with 40 and 100 mg/ml HE, respectively, and to undetectable levels after 24 h by both concentrations. HAV was reduced to undetectable levels by both HE concentrations after 24 h. PCA at 3 mg/ml reduced FCV-F9 titers to undetectable levels after 6 h, MNV-1 by 0.53 ± 0.01 log PFU/ml after 6 h, and caused no significant change in HAV titers. FA reduced FCV-F9 to undetectable levels after 3 h and MNV-1 and HAV after 24 h. Transmission electron microscopy showed no conclusive results. The findings suggest that H. sabdariffa extracts have potential to prevent foodborne viral transmission.

Reduction of Enteric Viruses by Blueberry Juice and Blueberry Proanthocyanidins

“…Statistical analysis was carried out using ANOVA with SAS software (version 9.3, SAS Institute, Cary, NC, USA) and Tukey's test with data obtained from the three replications for each individual treated virus and compared to control untreated virus assayed in duplicate as described in previous studies (Su and D'Souza, 2011;Su and D'Souza 2013;Su et al, 2010a;Su, Howell, and D'Souza 2010b;Joshi et al 2015).…”

“…Hepatitis A virus (HAV; strain HM175) and fetal rhesus monkey kidney (FRhK4) cells were provided by our collaborator, Dr. Kalmia Kniel (University of Delaware). As described before, all three host cell lines were maintained using Dulbecco's Modified Eagle's Medium/Ham's F-12 (DMEM-F12; HyClone Laboratories, Logan, UT) containing 10 % heat-inactivated fetal bovine serum (FBS, HyClone Laboratories) and 19 Anti-Anti (Antibiotic-Antimycotic, Invitrogen, Grand Island, NY) at 37°C in an atmosphere containing 5 % CO 2 (Su and D'Souza 2011;Joshi et al 2015).…”

“…Briefly, the viruses were harvested by 1 to 3 freeze thaw cycles followed by centrifugation at 5,0009g for 10 min. The resulting supernatant was filtered through a 0.2 lm filter, aliquoted, stored at -80°C until further use and titers of the recovered viral stocks were determined using standard reported plaque assays as described earlier (Su and D'Souza 2011;Joshi et al 2015).…”

“…Equal volumes of each virus at a titer of *5 log PFU/ml was individually mixed with BJ (pH 2.8), BB-PAC (1, 2, 4, and 10 mg/ml), malic acid (10 mM, pH 3.0), neutralized BJ (pH 7 using 4 M sodium hydroxide, NaOH), 10 % ethanol (control; as 10 mg/ml BB-PAC stock was prepared in 10 % ethanol and then subsequently diluted in sterile water to get lower working stock solutions) or phosphate-buffered saline (PBS; 7.2 as control), and incubated at 37°C for 5 (0.08 h), 15 (0.25 h), 30 (0.5 h), 60 (1 h), 120 (2 h), 180 (3 h), 360 min (6 h), or 24 h. Treatments were stopped using initial serial dilutions of the treated virus with cellculture media containing 10 % heat-inactivated FBS, followed by dilutions in cell-culture media containing 2 % FBS. The infectivity of viruses was evaluated using standard plaque assays as described before (D'Souza and Su 2010; Su and D'Souza 2011;Joshi et al 2015). Each experiment was replicated thrice and assayed in duplicate.…”

Selected Plant Extracts Show Antiviral Effects against Murine Norovirus Surrogate

“…The powdered dried calyx extracts are used as diet nourishment [12]. It has been demonstrated that aqueous extract of the plant possesses antiviral effect on human norovirus surrogates feline calicivirus-9 (FCV-F9) and murine norovirus (MNV-1) as well as in hepatitis A virus [13]. Hibiscus sabdariffa possesses antioxidant, anti-diabetic and antimicrobial properties [13].…”