A Nasal Spray Solution of Grapefruit Seed Extract plus Xylitol Displays Virucidal Activity Against SARS-Cov-2 In Vitro

Abstract

ABSTARCT
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

Full text

Journal: Draft 1
Type: Original Research (Basic Science) 2
Abstract text: 199 3
Text: 2272 4
5
6
A Nasal Spray Solution of Grapefruit Seed Extract plus Xylitol Displays 7
Virucidal Activity Against SARS-Cov-2 In Vitro 8
9
Gustavo Ferrer, M.D.1,2 Arian Betancourt, M.D. 2; Camille Celeste Go, M.D. 2; Hector Vazquez, 10
MD 2; Jonna B. Westover, Ph.D.3; Valeria Cagno, Ph.D. 4; Caroline Tapparel, Ph.D. 4; Marcos A. 11
Sanchez-Gonzalez, MD, Ph.D.2,5*
12
1 Medical Innovations, Nova Southeastern University, Davie, FL, USA 13
2 Research & Development, Aventura Pulmonary Institute, Miami, FL, USA 14
3 Institute for Antiviral Research, Utah State University, Logan, Utah, USA 15
4Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of 16
Geneva, Geneva, Switzerland 17
5 Lake Erie College of Osteopathic Medicine, Bradenton, FL, USA 18
19
20
21
*Correspondence: 22
Marcos A. Sanchez-Gonzalez, MD, PhD 23
5000 Lakewood Ranch Blvd 24
Bradenton, FL, 34211 25
(850) 559-47676 26
msanchez-gonzalez@LECOM.edu 27
28
Declarations of interest: none 29
30
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

ABSTARCT 31
32
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
CCID50/0.1 mL to an undetectable amount of infectious virus. The present results validated by 44
two independent sets of experiments, performed by different labs, on different viral strains, 45
provide early evidence to encourage further pilot and clinical studies aimed at investigating the 46
use of Xlear as a potential treatment for COVID-19 47
48
KEYWORDS: severe acute respiratory syndrome coronavirus, xylitol, grapefruit seed extract, 49
intranasal spray 50
51
52
53
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

1 Introduction 54
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).
68
In a recent literature review conducted by Higgins et al. it is highlighted that intranasal 69
drug delivery represents an important area of research for viral diseases and COVID-19 (Higgins 70
et al., 2020). They concluded that the intranasal method of drug delivery has potential relevance 71
for future clinical trials in the setting of disease prevention and treatment of SARS-CoV-2 in 72
addition to other viral diseases (Higgins et al., 2020). Subsequently, Siddiqi et.al (2020), in a 73
diagram of COVID-19 disease progression, illustrated that the viral response phase is highest 74
during the early infection of the disease process, of which patients manifest mild constitutional 75
symptoms. Taken together the aforementioned studies support our rationale that therapeutic 76
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

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
rhinitis as well as important microbiota and immunological modulatory effects (Akgül et al., 84
2020; Haukioja et al., 2008; Weissman et al., 2011; Xu et al., 2016). Xylitol has been reported to 85
have multiple health benefits as well as is generally safe and well-tolerated for most adults in 86
doses up to 35 grams per day and up to 20 grams per day in children (Salli et al., 2019; Storey et 87
al., 2007; Ur-Rehman et al., 2015). A derivative of grapefruit seeds, GSE, is associated with 88
abundant health benefits due to the presence of antioxidants and proanthocyanidin complexes 89
(Chacón et al., 2009). Also, GSE has been documented to have inhibitory effects against the 90
avian influenza virus, Newcastle disease virus, infections bursal disease virus, as well as other 91
pathogenic enteric viruses (Komura et al., 2019; Su and D'Souza, 2011). A commercially 92
available nasal spray combining xylitol and GSE, marketed as Xlear (American Fork, UT, USA), 93
has been widely used in the United States for several decades, but the antiviral effects of this 94
solution have not been documented. Accordingly, the aim of the present in vitro study was to 95
examine the virucidal effect of Xlear against SARS-CoV-2. To this end, two independent sets of 96
experiments were carried out to test the hypothesis that Xlear is an effective (Experiment I) and 97
replicable (Experiment II) means to deactivate SARS-CoV-2 the causative microorganism of 98
COVID-19. 99
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

100
2 MATERIALS AND METHODS 101
2.1 Experiment I: Xlear Virucidal Activity Efficacy 102
2.1.1 Procedure 103
SARS-CoV-2, USA-WA1/2020 strain, virus stock was prepared before testing by 104
growing 2 passages in Vero 76 cells. Culture media for prepared stock (test media) was 105
MEM with 2% fetal bovine serum (FBS) and 50 µg/mL gentamicin. Human rhinovirus 16, 106
strain 11757 purchased from ATCC (Gaithersburg, Maryland, USA), was grown in 3 107
passages of HeLa cells in MEM with 2% fetal bovine serum (FBS), 25 mM MgCl2, and 50
108
µg/mL gentamicin. Test media is the growth media with 5% FBS. 109
2.1.2 Virucidal Assay 110
Test compounds including commercially available Xlear containing purified water, 111
11% Pure Xylitol (Shandon Lujian, Shandong, China), 0.6%NaCL (Saline), and 0.015% 112
GSE (Chemie Research & Manufacturing Co., Casselberry, FL, USA) were obtained from 113
the manufacturer in liquid form and stored at room temperature. The test compound 11% 114
xylitol in saline was diluted 1:2 with water before testing. Each solution was mixed directly 115
with virus stock so that the final concentration was 90% of each test compound and 10% 116
virus stock. A single concentration was tested in triplicate. Test media without virus was 117
added to duplicate tubes of the compounds to serve as toxicity and neutralization controls. 118
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 122
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

after neutralization, the triplicate samples were pooled, serially diluted, and assayed for 123
infectious virus. 124
2.1.3 Virus Quantification 125
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% CO2 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
Reed-Muench (1948) equation and the log reduction value (LRV) of each compound 138
compared to the negative (water) control was calculated. 139
2.2 Experiment II: Xlear Virucidal Activity Replication 140
2.2.1 Procedure 141
SARS-CoV2/Switzerland/GE9586/2020 virus stock was amplified and titrated in 142
Vero E6 cells by plaque assay cultured in DMEM HG with 5% fetal bovine serum (FBS) 143
and 1% penicillin/streptomycin. 144
Dose-response Assay 145
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

Xlear nasal spray was serially diluted in DMEM HG and incubated with SARS-CoV2 146
(MOI 0.003 corresponding to 200 pfu/well) for 1 hour at 37°C and subsequently added on Vero 147
E6 cells for 1 hour at 37°C. The inoculum was then removed, cells were washed and overlaid 148
with DMEM HG with 5% FBS and Avicel 0.8%. 48hpi cells were fixed with PFA 4% and 149
stained with crystal violet. Plaques were counted and percent of infection calculated in 150
comparison with untreated wells. The experiments were performed twice independently, and 151
each was performed in duplicate. 152
Virucidal Assay 153
Xlear spray was mixed in different concentrations with SARS-CoV2 stock (105pfu). The 154
compound was mixed directly with the virus solution with a final concentration of respectively 155
90%, 80%, 60%, or 20%. PBS was used as control. The solution and virus were incubated at 37 156
°C for 1 hour. The solution was then neutralized by a 1/10 dilution in test media. A 60% 157
condition was repeated in two independent experiments while the other dilutions were performed 158
in a single experiment in duplicate. 159
The infectious virus from each sample was quantified by standard end-point dilution 160
assay. 100 µL of each dilution were plated into quadruplicate wells of 96-well plates containing 161
80-90% confluent Vero 76 cells. Plates were incubated at 37ºC with 5% CO2 for three days. 162
Each well was then scored for the presence or absence of the virus. The end-point titers 163
(TCID50) values were calculated using the Reed- Muench (1948) equation. 164
2.2.2 Toxicity assay 165
Vero-E6 (13000 cells per well) were seeded in 96-well plate. Xlear was serially diluted in 166
DMEM supplemented with 5% FBS and added on cells for 1h, followed by a washout, addition 167
of DMEM supplemented with 5% FBS for additional 48h hours. MTT reagent (Sigma Aldrich) 168
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

was added on cells for 3h at 37°C according to manufacturer instructions, subsequently cells 169
were lysed with pure DMSO and absorbance read at 570 nm. Percentages of viability were 170
calculated by comparing the absorbance in treated wells and untreated. 171
3 RESULTS 172
3.1 Experiment I 173
Virus titers and LRV of Rhinovirus-16 and SARS-CoV-2 when incubated with a 174
single concentration of the Xlear solutions are shown in Table 1. After a 15-minute contact 175
time, the Xlear nasal spray was not effective at reducing the infectious Rhino-16 virus. 176
When tested against SARS-CoV-2, the test compound GSE 0.2% was the only compound 177
effective at reducing >3 log10 CCID50 infectious virus from, 3.67 log10 CCID50/0.1 mL
178
to an undetectable amount of infectious virus (Table 1). The Xlear nasal spray and the 179
GSE 0.2% had some toxicity in the top rows (1/10 dilution of the test sample) which may 180
have contributed to the virucidal effect of the GSE. The 11% xylitol and 11% erythritol 181
had no cytotoxicity. The positive control and neutralization control performed as expected. 182
3.2 Experiment II 183
SARS-Cov2 is inhibited in the dose-response assay (Figure 1) by different concentrations of 184
Xlear spray. However, the dilution 1:2 in medium evidenced damage to the cells with almost 185
complete loss of the cells, while with the dilution 1:6 a partial damage to the cell was evidenced, 186
while no morphologic changes in cells were visible from dilution 1:12 onwards. These results 187
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
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

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). 193
194
4 DISCUSSION 195
The present study sought to evaluate the in vitro virucidal effects of a solution combining 196
xylitol and GSE in a nasal spray formulation known as Xlear. The novel results of this study 197
support our hypothesis that Xlear displays virucidal activity against SARS-CoV-2. The present 198
results validated by two independent sets of experiments, performed by different labs, on 199
different viral strains, provide early evidence to encourage further pilot and clinical studies 200
aimed at investigating the use of Xlear as a potential treatment for COVID-19. 201
Xlear is a solution of xylitol and GSE, in line with previous reports, the latter displayed 202
antiviral activity. Komura et al. demonstrated the efficacy of GSE as an antimicrobial agent on 203
avian pathogens including avian influenza virus, Newcastle disease virus, infectious bursal 204
disease virus, Salmonella Infantis, and Escherichia coli (Komura et al., 2019). Also, GSE has 205
shown similar antiviral activities against human enteric pathogens including Hepatitis A virus in 206
a dose-dependent manner (Su and D'Souza, 2011). Interestingly, GSE antiviral activity seems to 207
be particularly effective on enveloped viruses. Since SARS-CoV-2 is an enveloped virus the 208
GSE characteristics to induced or target the viral envelope should not be overlooked as candidate 209
therapies for COVID-19 emerge (Schoeman and Fielding, 2019). On the other hand, xylitol did 210
not show in vitro virucidal properties in the present study. However, it seems that the viral 211
protective effects of xylitol are evident in vivo a suggested by studies demonstrating ameliorating 212
effects against human respiratory syncytial virus and changes in the microbiota when consumed 213
orally (Uebanso et al., 2017; Xu et al., 2016). 214
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

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 223
limitations. The minimum time required for the Xlear solution to exert the virucidal effect was 224
not investigated. Furthermore, to assess the relevance of the time-dependent effect of Xylitol 225
effect in vivo, it will be important to verify if the addition of the spray-on cells previously 226
infected at nontoxic doses would exert a reduction of the viral titer. Also, whether pre-treating 227
the cells with the spray and subsequently adding the virus would decrease the rate of infection 228
would be needed to assess the possible preventive use of the nasal spray. 229
CONCLUSIONS 230
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
237
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

Acknowledgments 238
The authors are grateful to Mr. Nathan Jones for donating the reagents and testing solutions for 239
this study. 240
241
Funding 242
The study was funded thanks to the financial support of the “Fondation privée des HUG” and the 243
Carigest Foundation to CT. 244
245
246
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

5 REFERENCES 247
Akgül, Ö., Ak, A., Zorlu, S., Özdaş, D., Uslu, M., Çayirgan, D., 2020. Effects of short-term xylitol 248
chewing gum on pro-inflammatory cytokines and Streptococcus mutans: a randomized, 249
placebo-controlled trial. Int J Clin Pract, e13623. 250
Chacón, M.R., Ceperuelo-Mallafré, V., Maymó-Masip, E., Mateo-Sanz, J.M., Arola, L., 251
Guitiérrez, C., Fernandez-Real, J.M., Ardèvol, A., Simón, I., Vendrell, J., 2009. Grape-252
seed procyanidins modulate inflammation on human differentiated adipocytes in vitro. 253
Cytokine 47(2), 137-142. 254
Dong, E., Du, H., Gardner, L., 2020. An interactive web-based dashboard to track COVID-19 in 255
real time. Lancet Infect Dis 20(5), 533-534. 256
Haukioja, A., Söderling, E., Tenovuo, J., 2008. Acid production from sugars and sugar alcohols 257
by probiotic lactobacilli and bifidobacteria in vitro. Caries Res 42(6), 449-453. 258
Higgins, T.S., Wu, A.W., Illing, E.A., Sokoloski, K.J., Weaver, B.A., Anthony, B.P., Hughes, N., 259
Ting, J.Y., 2020. Intranasal Antiviral Drug Delivery and Coronavirus Disease 2019 260
(COVID-19): A State of the Art Review. Otolaryngol Head Neck Surg, 194599820933170. 261
Hoffmann, M., Kleine-Weber, H., Krüger, N., Müller, M., Drosten, C., Pöhlmann, S., 2020. The 262
novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the 263
cellular protease TMPRSS2 for entry into target cells. bioRxiv. 264
Jia, H.P., Look, D.C., Shi, L., Hickey, M., Pewe, L., Netland, J., Farzan, M., Wohlford-Lenane, 265
C., Perlman, S., McCray, P.B., Jr., 2005. ACE2 receptor expression and severe acute 266
respiratory syndrome coronavirus infection depend on differentiation of human airway 267
epithelia. J Virol 79(23), 14614-14621. 268
Komura, M., Suzuki, M., Sangsriratanakul, N., Ito, M., Takahashi, S., Alam, M.S., Ono, M., Daio, 269
C., Shoham, D., Takehara, K., 2019. Inhibitory effect of grapefruit seed extract (GSE) on 270
avian pathogens. J Vet Med Sci 81(3), 466-472. 271
Meister, T.L., Brüggemann, Y., Todt, D., Conzelmann, C., Müller, J.A., Groß, R., Münch, J., 272
Krawczyk, A., Steinmann, J., Steinmann, J., Pfaender, S., Steinmann, E., 2020. Virucidal 273
Efficacy of Different Oral Rinses Against Severe Acute Respiratory Syndrome 274
Coronavirus 2. J Infect Dis 222(8), 1289-1292. 275
Salli, K., Lehtinen, M.J., Tiihonen, K., Ouwehand, A.C., 2019. Xylitol's Health Benefits beyond 276
Dental Health: A Comprehensive Review. Nutrients 11(8). 277
Schoeman, D., Fielding, B.C., 2019. Coronavirus envelope protein: current knowledge. Virol J 278
16(1), 69. 279
Siddiqi, H.K., Mehra, M.R., 2020. COVID-19 illness in native and immunosuppressed states: A 280
clinical-therapeutic staging proposal. J Heart Lung Transplant 39(5), 405-407. 281
Storey, D., Lee, A., Bornet, F., Brouns, F., 2007. Gastrointestinal tolerance of erythritol and xylitol 282
ingested in a liquid. Eur J Clin Nutr 61(3), 349-354. 283
Su, X., D'Souza, D.H., 2011. Grape seed extract for control of human enteric viruses. Appl Environ 284
Microbiol 77(12), 3982-3987. 285
Uebanso, T., Kano, S., Yoshimoto, A., Naito, C., Shimohata, T., Mawatari, K., Takahashi, A., 286
2017. Effects of Consuming Xylitol on Gut Microbiota and Lipid Metabolism in Mice. 287
Nutrients 9(7). 288
Ur-Rehman, S., Mushtaq, Z., Zahoor, T., Jamil, A., Murtaza, M.A., 2015. Xylitol: a review on 289
bioproduction, application, health benefits, and related safety issues. Critical reviews in 290
food science and nutrition 55(11), 1514-1528. 291
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

Weissman, J.D., Fernandez, F., Hwang, P.H., 2011. Xylitol nasal irrigation in the management of 292
chronic rhinosinusitis: a pilot study. Laryngoscope 121(11), 2468-2472. 293
Xu, M.L., Wi, G.R., Kim, H.J., Kim, H.J., 2016. Ameliorating Effect of Dietary Xylitol on Human 294
Respiratory Syncytial Virus (hRSV) Infection. Biol Pharm Bull 39(4), 540-546. 295
296
297
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

Table 1. Virucidal efficacy of Xlear compounds against Rhinovirus-16 and SARS-CoV-2 298
after a 15-minute incubation with virus at 22 ± 2ºC. 299
300
301
302
303
a Log10 CCID50 of virus per 0.1 mL. The assay lower limit of detection is 0.67 Log10 304
CCID50/0.1 mL. 305
b LRV (log reduction value) is the reduction of virus compared to the virus control 306
307
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

Figure 1. a) SARS-CoV-2 dose-response inhibition. Xlear was incubated at different dilutions 308
with SARS-CoV2 (200 pfu) for 1h at 37 C. At the end of the incubation, mixtures were serially 309
diluted and added for 1h at 37°C on Vero-E6 cells. Mixtures were then removed, and cells 310
overlaid with medium containing 0.8% avicel. Cells were fixed 48hpi and plaques were counted. 311
Results are mean and SEM of 2 independent experiments performed in duplicate. b) Xlear 312
toxicity evaluation. Different dilutions of the nasal spray were incubated for 1h (followed by 313
addition of medium for 47h) or for 48h on cells in DMEM 5% FBS. At the end of the incubation 314
MTT reagent was added on cells and percentages of viability were evaluated by comparing 315
treated and untreated wells. 316
317
318
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

Figure 2. SARS CoV-2 virucidal assay. Xlear was incubated with SARS-CoV2 (5*105 pfu) for 319
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
TCID50/ml was determined. Results are mean and SD of two independent experiments. 322
323
324
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

Tested
Concentration Virus Tested Incubation
Time Virus
Titer a
LRV b
Xlear 90% Rhino-16 15-minute 5.0 0
Ethanol 90% Rhino-16 15-minute 1.5 3.17
Virus Control na Rhino-16 15-minute 4.67 na
Xlear 90% SARS-CoV-2 15-minute 3.0 0.67
GSE 0.2% in DI water 90% SARS-CoV-2 15-minute <0.67 3.0
Saline w/ 11% Xylitol 90% SARS-CoV-2 15-minute 3.5 0.17
Saline w/ 11% Erythritol 90% SARS-CoV-2 15-minute 4.3 0
Ethanol 90% SARS-CoV-2 15-minute <0.67 3.0
Virus Control na SARS-CoV-2 15-minute 3.67 na
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted November 25, 2020. ; https://doi.org/10.1101/2020.11.23.394114doi: bioRxiv preprint