key: cord-0725597-axrzrgua
authors: Ferrer, Gustavo; Betancourt, Arian; Go, Camille Celeste; Vazquez, Hector; Westover, Jonna B.; Cagno, Valeria; Tapparel, Caroline; Sanchez-Gonzalez, Marcos A.
title: A Nasal Spray Solution of Grapefruit Seed Extract plus Xylitol Displays Virucidal Activity Against SARS-Cov-2 In Vitro
date: 2020-11-23
journal: bioRxiv
DOI: 10.1101/2020.11.23.394114
sha: 8a55428e3bb9fbd1975ee210bc9dfb8004042e07
doc_id: 725597
cord_uid: axrzrgua

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the ongoing pandemic coronavirus disease 2019 (COVID-19) has triggered worldwide concerted efforts in an attempt to identify effective therapies. In the present study, we have identified two candidate agents with potential activity against SARS-CoV-2 which can be administered intranasally, namely, xylitol and grape seed fruit extract (GSE). A commercially available nasal spray (Xlear) combining xylitol and GSE has been available for years, but the antiviral effects of this solution have not been documented. This in vitro study examined the virucidal effect of Xlear against SARS-CoV-2. To this end, two independent sets of experiments were carried out to test the hypothesis that Xlear is an effective (Experiment I) and replicable (Experiment II) means to deactivate SARS-CoV-2. When tested against SARS-CoV-2, the test compound GSE 0.2% was the only compound effective at reducing >3 log10 CCID50 infectious virus from, 3.67 log10 CCID50/0.1 mL to an undetectable amount of infectious virus. The present results validated by two independent sets of experiments, performed by different labs, on different viral strains, provide early evidence to encourage further pilot and clinical studies aimed at investigating the use of Xlear as a potential treatment for COVID-19

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the 33 ongoing pandemic coronavirus disease 2019 (COVID-19) has triggered worldwide concerted 34 efforts in an attempt to identify effective therapies. In the present study, we have identified two 35 candidate agents with potential activity against SARS-CoV-2 which can be administered 36 intranasally, namely, xylitol and grape seed fruit extract (GSE). A commercially available nasal 37 spray (Xlear) combining xylitol and GSE has been available for years, but the antiviral effects of 38 this solution have not been documented. This in vitro study examined the virucidal effect of 39

Xlear against SARS-CoV-2. To this end, two independent sets of experiments were carried out to 40 test the hypothesis that Xlear is an effective (Experiment I) and replicable (Experiment II) means 41 to deactivate SARS-CoV-2. When tested against SARS-CoV-2, the test compound GSE 0.2% 42 was the only compound effective at reducing >3 log10 CCID50 infectious virus from, 3.67 log10 43 The initial global outbreak of the severe acute respiratory syndrome coronavirus 2 55 (SARS-CoV-2), responsible for the ongoing pandemic coronavirus disease 2019 (COVID-19) , 56 was initially identified in Wuhan, China in December 2019. As of July 2020, there were more 57 than 13.3 million confirmed cases worldwide, with total deaths exceeding 573,000 (Dong et al., 58 2020) . Worldwide concerted efforts have been made in an attempt to characterize the disease and 59 identify effective therapies targeting SARS-CoV-2 including lines of studies focusing on the 60 route of infection, the potential routes of administration of therapeutic agents as well as the 61 potential efficacy of antiseptics (Meister et al., 2020) . In this vein, a landmark study found that 62 the coronavirus infects the nasal cavity via the angiotensin-converting enzyme 2 (ACE2) protein 63 which appears to be the host-cell receptor for SARS-CoV-2 (Hoffmann et al., 2020). Since the 64 nasal epithelium cells have the highest percentage of ACE2 expressing ciliate cells in the 65 proximal airways, it is plausible to suggest that pharmacological agents such as sprays that are 66 used via the intranasal route of administration might be optimal candidates for providing 67 effective therapies against COVID-19 (Jia et al., 2005) . symptoms. Taken together the aforementioned studies support our rationale that therapeutic strategies should be aimed at reducing the viral load in the nose by targeting this mild-moderate 77 phase of the disease process, and hence the use of a nasal spray might be an effective means to 78 accomplish this therapeutic strategy. 79

In the present study, we have identified two candidate agents with potential activity 80 against SARS-CoV-2 which can be administered intranasally, namely, xylitol and grape seed 81 fruit extract (GSE). Xylitol, a sweetener with antimicrobial and anti-inflammatory properties, 82 has been shown effective in decreasing the incidence of dental caries and improving chronic 83 Ethanol (90%) was tested in parallel as a positive control and water only as a virus control. 119

The test solutions were incubated at room temperature (22 ± 2ºC) for 15 minutes with 120 SARS-CoV-2 or Rhinovirus-16. The solutions were then neutralized by a 1/10 dilution in 121 the test media of each specific virus. The virucidal assays were performed in triplicate, then after neutralization, the triplicate samples were pooled, serially diluted, and assayed for 123 infectious virus. 124

The surviving virus from each sample was quantified by standard end-point 126 dilution assay. Briefly, the neutralized samples were pooled and serially diluted using 127 eight log dilutions in test medium. Then 100 µL of each dilution was plated into 128 quadruplicate wells of 96-well plates containing 80-90% confluent Vero 76 (SARS-CoV-129

2) or HeLa cells (Rhino-16). The toxicity controls were added to an additional 4 wells of 130

Vero 76 or HeLa cells and 2 of those wells at each dilution were infected with virus to 131 serve as neutralization controls, ensuring that the residual sample in the titer assay plate 132 did not inhibit growth and detection of the surviving virus. Plates were incubated at 37 ± 133 2ºC with 5% CO 2 for 5 days and at 33 ± 2ºC with 5% CO2 for 4 days for the SARS-CoV-2 134 assay and the Rhinovirus-16 assay, respectively. Each well was then scored for the 135 presence or absence of an infectious virus. The titers were measured using a standard 136 endpoint dilution 50% cell culture infectious dose (CCID50) assay calculated using the 137 were further confirmed with toxicity assays (Figure 1b) . 188

In the virucidal assays (Figure 2) , Xlear showed virucidal activity at the different 189 concentrations tested. Complete inhibition of viral infectivity was observed for the 90%, 80%, 190 60% condition, and a reduction of 2.17 log of viral titer in the 20% condition. In this assay, the 191 mixture of virus and Xlear was neutralized by a 1/10 dilution before addition on cells, therefore 192 diluting the compound below the toxic doses determined in the toxicity assay (Figure 1b) . The precise mechanism of action of GSE is poorly understood. However, according to 215 the present virucidal tests, the active component of the spray is the GSE, which is in line with 216 previous reports demonstrating that the extract was is effective to inactivate different enveloped 217 and non-enveloped viruses (Su and D'Souza, 2011) . 218 219 Moreover, it seems that the mechanism of action of GSE targets the viral adsorption (or viral 220 binding) to a greater extent than viral replication. It is worth mentioning that studies of the 221 precise mechanism of action of GSE are beyond the scope of this work. 222

As with any research study, the present experimental design is not free from some 

This study demonstrates the strong virucidal effects against SARS-CoV-2 of the Xlear 231 nasal spray compound with xylitol and GSE. Using a virucidal nasal spray could become a 232 cutting-edge element in the prevention and treatment of COVID-19 disease. To further ascertain 233 the impact of this nasal spray in SARS-CoV-2, we propose to perform further a randomized 234 placebo-controlled study of intranasally delivered Xlear in patients with mild to moderate SARS-235

CoV-2 and randomized placebo-controlled preventive trial in healthcare workers. 236 1h at 37 C. At the end of the incubation, mixtures were serially diluted and added on Vero-E6 320 cells. Cells were fixed 48hpi and scored for presence or absence of cytopathic effect and 321 

Effects of short-term xylitol 248 chewing gum on pro-inflammatory cytokines and Streptococcus mutans: a randomized, 249 placebo-controlled trial

Grape-252 seed procyanidins modulate inflammation on human differentiated adipocytes in vitro

An interactive web-based dashboard to track COVID-19 in 255 real time

Acid production from sugars and sugar alcohols 257 by probiotic lactobacilli and bifidobacteria in vitro

Intranasal Antiviral Drug Delivery and Coronavirus Disease 2019 260 (COVID-19): A State of the Art Review

The 262 novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the 263 cellular protease TMPRSS2 for entry into target cells

ACE2 receptor expression and severe acute 266 respiratory syndrome coronavirus infection depend on differentiation of human airway 267 epithelia

Inhibitory effect of grapefruit seed extract (GSE) on 270 avian pathogens

Virucidal 273 Efficacy of Different Oral Rinses Against Severe Acute Respiratory Syndrome 274 Coronavirus 2

Xylitol's Health Benefits beyond 276 Dental Health: A Comprehensive Review

Coronavirus envelope protein: current knowledge

COVID-19 illness in native and immunosuppressed states: A 280 clinical-therapeutic staging proposal

Gastrointestinal tolerance of erythritol and xylitol 282 ingested in a liquid

Grape seed extract for control of human enteric viruses

Effects of Consuming Xylitol on Gut Microbiota and Lipid Metabolism in Mice

Xylitol: a review on 289 bioproduction, application, health benefits, and related safety issues. Critical reviews in 290 food science and nutrition

Xylitol nasal irrigation in the management of 292 chronic rhinosinusitis: a pilot study

Ameliorating Effect of Dietary Xylitol on Human 294 Respiratory Syncytial Virus (hRSV) Infection

The authors are grateful to Mr. Nathan Jones for donating the reagents and testing solutions for 239 this study.

Funding 242The study was funded thanks to the financial support of the "Fondation privée des HUG" and the 243Carigest Foundation to CT. 244 245 246