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Inhibitory effects of specific combination of natural compounds against SARS-CoV-2 and its Alpha, Beta, Gamma, Delta, Kappa, and Mu variants

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  • Dr. Rath Health Foundation

Abstract and Figures

Despite vaccine availability, the global spread of COVID-19 continues, largely facilitated by emerging SARS-CoV-2 mutations. Our earlier research documented that a specific combination of plant-derived compounds can inhibit SARS-CoV-2 binding to its ACE2 receptor and controlling key cellular mechanisms of viral infectivity. In this study, we evaluated the efficacy of a defined mixture of plant extracts and micronutrients against original SARS-CoV-2 and its Alpha, Beta, Gamma, Delta, Kappa, and Mu variants. The composition containing vitamin C, N-acetylcysteine, resveratrol, theaflavin, curcumin, quercetin, naringenin, baicalin, and broccoli extract demonstrated a highest efficacy by inhibiting the receptor-binding domain (RBD) binding of SARS-CoV-2 to its cellular ACE2 receptor by 90%. In vitro exposure of test pseudo-typed variants to this formula for 1 h before or simultaneously administrated to human pulmonary cells resulted in up to 60% inhibition in their cellular entry. Additionally, this composition significantly inhibited other cellular mechanisms of viral infectivity, including the activity of viral RdRp, furin, and cathepsin L. These findings demonstrate the efficacy of natural compounds against SARS-CoV-2 including its mutated forms through pleiotropic mechanisms. Our results imply that simultaneous inhibition of multiple mechanisms of viral infection of host cells could be an effective strategy to prevent SARS-CoV-2 infection.
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Inhibitory effects of specic combination of
natural compounds against SARS-CoV-2 and its
Alpha, Beta, Gamma, Delta, Kappa, and Mu
variants
ANNA GOC, ALEKSANDRA NIEDZWIECKIp,
VADIM IVANOV, SVETLANA IVANOVA and
MATTHIAS RATH
Dr. Rath Research Institute, 5941 Optical Ct., San Jose, CA 95138, USA
Received: November 30, 2021 Accepted: December 20, 2021
Published online: January 21, 2022
ABSTRACT
Despite vaccine availability, the global spread of COVID-19 continues, largely facilitated by emerging
SARS-CoV-2 mutations. Our earlier research documented that a specific combination of plant-derived
compounds can inhibit SARS-CoV-2 binding to its ACE2 receptor and controlling key cellular
mechanisms of viral infectivity. In this study, we evaluated the efficacy of a defined mixture of plant
extracts and micronutrients against original SARS-CoV-2 and its Alpha, Beta, Gamma, Delta, Kappa,
and Mu variants. The composition containing vitamin C, N-acetylcysteine, resveratrol, theaflavin,
curcumin, quercetin, naringenin, baicalin, and broccoli extract demonstrated a highest efficacy by
inhibiting the receptor-binding domain (RBD) binding of SARS-CoV-2 to its cellular ACE2 receptor by
90%. In vitro exposure of test pseudo-typed variants to this formula for 1 h before or simultaneously
administrated to human pulmonary cells resulted in up to 60% inhibition in their cellular entry.
Additionally, this composition signicantly inhibited other cellular mechanisms of viral infectivity,
including the activity of viral RdRp, furin, and cathepsin L. These ndings demonstrate the efcacy of
natural compounds against SARS-CoV-2 including its mutated forms through pleiotropic mechanisms.
Our results imply that simultaneous inhibition of multiple mechanisms of viral infection of host cells
could be an effective strategy to prevent SARS-CoV-2 infection.
KEYWORDS
SARS-CoV-2, variants, RdRp, furin, cathepsin L
INTRODUCTION
Despite availability of vaccines and drastic public health measures, the spread of SARS-CoV-
2 infection continues [1]. According to the WHO, as of August 31, 2021, there have been
almost 217 million cases worldwide, resulting in more than 4.51 million deaths [2].
Within one year of the pandemic, several variants of SARS-CoV-2 virus have emerged
including: United Kingdom B.1.1.7 (Alpha) variant, South Africa B.1.315 (Beta), Brazil P.1
(Gamma), and mutated forms from India known as lineage B.1.617.1 (Kappa) and B.1.617.2
(Delta) [3]. All of these variants spread to other countries, with each mutation potentially
giving rise to further variants such as originated in Japan R.1 variant having a mutation
E484K located in the spike protein giving "increased resistance" to antibodies generated by
the current vaccines [4], as well as a D614G mutation giving increased infectiousness and
present in all other variants that have surpassed the Alpha strain [5]. Clinical reports also
show that the British variant can infect patients, who have received the vaccine developed
against the original SARS-CoV-2, thereby challenging any claim of its universal efcacy [6].
European Journal of
Microbiology and
Immunology
11 (2021) 4, 8794
DOI:
10.1556/1886.2021.00022
© 2021 The Author(s)
ORIGINAL RESEARCH
PAPER
pCorresponding author. Dr. Rath
Research Institute BV, 5941 Optical
Ct., San Jose, CA 95138, USA. Tel.:
þ1 408 567 5050.
E-mail: author@drrath.com
There are recent indications that the newly emerged Mu
variant and R.1 variant may escape antibodies generated by
the application of vaccines against original SARS-CoV-2 [7].
Thus, it is foreseeable that the ultimate control of the
ongoing COVID-19 pandemic caused by the rapidly
emerging mutations of SARS-CoV-2 will be compromised
by the need to develop new vaccines potentially for every
new variant, and by the related scientic, economic, and
social consequences of such a strategy.
The cellular entry mechanism of SARS-CoV-2 has been
extensively studied and found to be mediated by a spike
protein anchored on the surface of the virus. The spike
protein is a trimer, with three receptor-binding S1 heads
sitting on top of a trimeric membrane fusion S2 stalk [8].
The spike S1 protein on SARS-CoV-2 contains a receptor-
binding domain (RBD) that binds to a target cell using the
human ACE2 (hACE2) receptor present on the surface of
many types of cells and is proteolytically activated by human
proteases. Entry into host cells is an important determinant
of viral infectivity and pathogenesis [911].
Cellular infection by the SARS-CoV-2 is facilitated by
several host enzymatic proteins, including transmembrane
protease serine 2 (TMPRSS2), furin, cathepsin L, as well as
RNA-dependent RNA polymerase (RdRp) catalyzing viral
RNA multiplication [12]. The RNA-dependent RNA poly-
merase (RdRP) is a conservative in the structure viral
enzyme with no host cell homologs and as such it has been
of interest in developing selective inhibitors as a potentially
safer alternative for treating COVID-19 and not affecting
human host cells proteins directly [13].
Application of natural compounds with their wide pleio-
tropic effects provides an opportunity of controlling several
mechanisms associated with SARS-CoV-2 infection with a
high margin of safety. Since the emergence of COVID-19,
several studies have shown the efficacy of such compounds as
inhibitors [1417]. Our earlier studies also identied the ef-
cacy of plant-derived compounds on several cellular mech-
anisms of SARS-CoV-2 infectivity [1820]. In this study, we
evaluated the efcacy of a specic combination of micro-
nutrients and plant extracts against newly emerged mutated
forms of SARS-CoV-2 and the activity of key enzymes
involved in viral processing in human alveolar cells. This
approach aims at simultaneous inhibition of multiple cellular
mechanisms involved in SARS-CoV-2 infectivity, irrespective
of mutation and/or subtype, thereby opening new avenues
towards the global control of the pandemic.
MATERIALS AND METHODS
Cell lines, virial particles, and antibodies
A549 and VeroE6 cells were obtained from American Type
Culture Collection (Manassas, VA, USA). The hACE2/A549
cells (i.e., A549 cells stably overexpressing human ACE2 re-
ceptor) and eGFP-luciferase-SARS-CoV-2 spike protein
encapsulated pseudo-typed particles were purchased from
GenScript (Piscataway, NJ, USA). Pseudo-typed ΔG-luciferase
(GpΔG-luciferase) rVSV was obtained from Kerafast (Boston,
MA, USA). Alpha, Beta, Gamma, Delta, Kappa, Mu, and bald
eGFP-luciferase-SARS-CoV-2 pseudo-typed particles were
from BPS Bioscience (San Diego, CA, USA). All other com-
pounds were from Sigma (St. Louis, MO, USA) except for
broccoli extract that was from Bulk Supplements (Henderson,
NV, USA).
Plant-derived compositions
The combinations of natural compounds tested in this study
are presented in Table 1. Stock solutions of these combi-
nations were prepared in dimethylsulfoxide (DMSO) at
50 mg mL
1
and kept at 20 8C until analysis. For the ex-
periments, the stock solution was diluted with 1 3phos-
phate buffered saline (1 3PBS) or a buffer recommended
by the manufacturer of the utilized kit (enzyme activity as-
says) or corresponding cell culture medium (cell-based ex-
periments) to nal concentrations indicated in the Figures.
Binding of SARS-CoV-2 pseudo-typed virions to hACE2
receptor
The experiment followed our previously published method-
ology [18,19]. Briey, original eGFP-luciferase-SARS-CoV-2
spike protein encapsulated pseudo-virion or its variants were
incubated at 37 8Cwith0100
m
gmL
1
of MixV for either 1 h
before it was added into a monolayer of hACE2/A549 cells or
added simultaneously to hACE2/A549 cells. Cells were incu-
bated for an additional 48 h at 37 8C. After the 48-h incubation
period, the transduction efciency was quantied by recording
the luciferase activity, utilizing a luciferase assay system
(Promega, Madison, WI, USA) and a spectrouorometer
(Tecan Group Ltd., Männedorf, Switzerland). The positive
control was bald eGFP-luciferase-SARS-CoV-2 pseudo-typed
particles, and the negative control was ΔG-luciferase rVSV
pseudo-typed particles. Data are presented as a % of control
without Mix-V addition (mean ±standard deviation (SD),
n55).
In vitro RdRp activity assay
In vitro RdRp activity was evaluated using a SARS-CoV-2
RNA Polymerase Assay Kit (ProFoldin, Hudson, MA, USA)
Table 1. Combinations of plant extracts and micronutrients tested
in the study
Substance MixA MixB MixC MixV MixE
Green tea extract þþþ
Resveratrol þþþþþ
Broccoli extract þþþþþ
Curcumin þþþþþ
Quercetin þþþþþ
Naringenin þþþþþ
Baicalin þþþþþ
Theaavin þþþ
Vitamin C þþþþþ
N-acetylcysteine þþ
Fucoidan þþ
88 European Journal of Microbiology and Immunology 11 (2021) 4, 8794
according to the manufacturers protocol. Briey, 0.5
m
Lof
50 3recombinant RdRp or 20
m
L of cell lysate [to transduce
cells with eGFP-luciferase-SARS-CoV-2 RdRp lentivirus
(GenScript, Piscataway, NJ, USA), VeroE6 cells were seeded
into a 6-well plate in the presence of complete growth me-
dium and treated with 8
m
LmL
1
polybrene (Sigma, St.
Louis, MO, USA) for 30 min, followed by the addition of
eGFP-luciferase-SARS-CoV-2 RdRp lentivirus at multiplic-
ity of infection (MOI 55) [our previous preliminary results
showed an almost 100% transduction rate can be achieved
with this MOI], and spin-inoculation at 1,0003gfor 1.5 h.
After 24 h at 37 8C incubation, cells were fed with fresh
complete growth medium. After 48 h post-inoculation, cells
were detached with 1 mM EDTA, washed three times with 1
3PBS supplemented with 3% fetal bovine serum (FBS)] was
incubated with 2.5
m
Lof503buffer, 20
m
L of water (only
when recombinant RdRp enzyme was used), and 1.0
m
Lof
MixV at 0100
m
gmL
1
concentrations for 15 min at RT,
followed by the addition of the master mix containing 0.5
m
L
of 50 3NTPs and 0.5
m
Lof503template (as a single-
stranded polyribonucleotide). The reaction (25
m
L) was
incubated for 2 h at 34 8C and then stopped by the addition
of 65
m
Lof103uorescence dye, and the uorescence
signal was recorded in 10 min at extension/emission 5488/
535 nm using a uorescence spectrometer (Tecan, Group
Ltd., Männedorf, Switzerland). Positive control contained
100
m
gmL
1
remdesivir. Results are expressed as a % of
control without MixV addition (mean ±SD, n55).
Furin activity assay
Effects of MixV on furin enzymatic activity were evaluated
using a SensoLyte Rh110 Furin Activity Assay Kit (AnaSpec,
Fremont, CA, USA) according to the manufacturers pro-
tocol. Briey, MixV at 5 and 10
m
gmL
1
concentrations was
mixed with furin recombinant protein for 15 min, followed
by the addition of uorogenic Rh110 furin substrate. The
samples were incubated for 1 h at 22 8C and the uorescence
was recorded at extension/emission 5490/520 nm with a
uorescence spectrometer (Perseptive Biosystems Cytouor
4000, Framingham, MA, USA). Positive control as a peptidyl
chloromethylketone (Decanoyl-Arg-Val-Lys-Arg-CMK)
contained 0.1
m
M. Data are presented as a % of control
without MixV addition (mean ±SD, n55).
Cathepsin L activity assay
Effect of MixV on the activity of recombinant cathepsin L
was evaluated using a Cathepsin L Activity Screening Assay
Kit (BPS Bioscience, San Diego, CA, USA), according to the
manufacturers protocol. Briey, MixV at 5 and 10
m
gmL
1
concentrations was added to cathepsin L (0.2 mU/
m
L) for
15 min at 22 8C prior to uorogenic substrate (Ac-FR-AFC)
(10
m
M) addition and incubation for further 60 min at RT.
Positive control contained only cathepsin L, and negative
control contained cathepsin L and cathepsin L inhibitor E-
64 (25
m
M). The uorescence was recorded at extension/
emission 5360/480 nm with a uorescence spectrometer
(Tecan Group Ltd., Männedorf, Switzerland). Data are
presented as a percentage of control without MixV addition
(mean ±SD, n55).
Viability
Cell viability assay was performed using MTT substrate.
Briefly, 40 310
3
A549 cells per well were treated with
different concentrations of MixV alone or in combination with
vitamin D and zinc for up to 48 h. Subsequently, the wells
were washed with 1 3PBS and complete growth medium
supplemented with 5 mg mL
1
MTT was added, followed by
incubation for 4 h at 37 8C. Next, the culture medium was
aspirated and 100
m
L of methanol was added. The absorbance
was assessed at 570 nm with a uorescence spectrometer
(Molecular Devices, San Jose, CA, USA). Data are presented as
a % of control without MixV addition (mean ±SD, n58).
Statistical analysis
Data for all experiments are presented as an average value
and standard deviation from at least three independent ex-
periments. Comparison between different samples was done
by a two-tailed T-test using the Microsoft Ofce Excel
program. Differences between samples were considered
signicant at Pvalues less than 0.05.
Ethics
No human subjects were involved in this study. Therefore
IRB ethical approval and informed consent are not applicable.
RESULTS
Selection of the most effective composition of natural
compounds based on its inhibition of viral RBD binding
to hACE2 receptor
The results in Fig. 1 show the efcacy of various combina-
tions of plant extracts and micronutrients on inhibition of
SARS-CoV-2 RBD binding to epithelial pulmonary cells
over-expressing human ACE2 receptor. All ve composi-
tions displayed inhibitory effects on RBD binding at the level
of 50% and higher. The highest inhibition, i.e., by 90%
compared to control, was obtained with MixV applied at
10
m
gmL
1
concentration (Fig. 1A). The addition of vitamin
D or zinc to MixV did not further enhance the inhibition of
RBD binding to human ACE2 receptor as shown in Fig. 1B.
Effect of MixV on cellular attachment and entry of
SARS-CoV-2 and its pseudotyped variants
The effect of different pattern of applications of MixV on the
attachment and cellular entry of eGFP-luc-spike-enveloped
pseudo-typed variants was tested using A549/hACE2 cells.
The results presented in Fig. 2A show that the exposure of
pseudo-virions to MixV before their infection of human
lung cells was effective in decreasing cellular entry of all
tested SARS-CoV-2 variants. As such, the MixV applied at
10
m
gmL
1
to original SARS-CoV-2 strain and its Alpha,
European Journal of Microbiology and Immunology 11 (2021) 4, 8794 89
Beta, Gamma, Delta, Kappa, and Mu variants for 1 h before
their adding to A549/hACE2 cells, resulted in decreased viral
entry by 39%, 48%, and 42%, 57%, 51%, 39%, and 24%,
respectively. Figure 2B shows the effects of MixV on the test
variants cellular entry inhibition when it was applied
simultaneously with pseudo-typed virions and A549/hACE2
cells. In the presence of MixV at 10
m
gmL
1
concentration,
the cellular entry of Alpha virions was inhibited by 38%,
Beta by 48% and Gamma by 41%. The inhibitory effect of
the MixV on original SARS-CoV-2 virions entry was 57%
and Delta, Kappa and Mu variants 64%, 42%, and 24%,
respectively. The results presented on Fig. 3A show that
MixV applied at 5 and 10
m
gmL
1
was not affecting viability
of A549/hACE2 cells and the administration of vitamin D3
alone or with zinc did not have additional impact on their
survival (Fig. 3B).
Effect of MixV on viral RdRp polymerase
In our study we also tested whether MixV acts beyond the
binding steps of the SARS-CoV-2 infection process, by
examining its effect on other important cellular mechanisms
in viral infection, such as the activity of RdRp, which is
essential for viral replication. As presented in Fig. 4A,
enzymatic activity of recombinant SARS-CoV-2 RdRp was
signicantly inhibited by 53% in the presence of MixV at
10
m
gmL
1
. Neither Vitamin D3 nor zinc enhanced this
inhibitory effect. A similar inhibitory effect of MixV on
RdRp activity was also observed when using the lysate of
cells overexpressing RdRp protein (Fig. 4B).
Effect of MixV on host cellular proteases
It has been documented that various specific host cell pro-
teases, including furin and cathepsin L, are required to
facilitate SARS-CoV-2 cellular entry by enzymatic priming
spike proteins and viral egress. In our study, we applied cell-
free assays to study direct effects of MixV on activity of these
enzymes. As presented in Fig. 5A, we observed inhibition of
the activity of furin by 12% and 34% with MixV applied at 5
and 10
m
gmL
1
, respectively. This inhibitory effect of MixV
was further enhanced in the presence of vitamin D resulting
Fig. 1. Effect of test compositions on SARS-CoV-2 RBD-spike protein attachment to lung epithelial cells. Inhibitory effect of Mixes A, B, C, V,
and E on viral SARS-CoV-2 RBD binding to human ACE2 receptor (A). Inhibitory effect of MixV in the presence of vitamin D and zinc on
viral SARS-CoV-2 RBD binding to human ACE2 receptor (B); control 0.25% DMSO, positive and negative controls was provided by the
manufacturer of the kit; data are presented as % of control ±SD; * P0.001; VD3 25-hydroxy Vitamin D
3
at 25
m
gmL
1
,Znzinc
aspartate at 2.5
m
gmL
1
Fig. 2. Effect of MixV on SARS-CoV-2 eGFP-luciferase pseudo-typed variants entry. hACE2/A549 cells were inoculated with six different
variants of SARS-CoV-2 pseudo-virions encapsulated with spike proteins and treated with MixV at designated concentrations either as a
pre-treatment (A) or as a simultaneous exposure (B). Control 0.05% DMSO, negative control pseudo-typed ΔG-luciferase (G*ΔG-
luciferase) rVSV, positive control bald SARS-CoV-2 eGFP-luciferase pseudo-virions; # P0.05, ΔP0.01, * P0.001
90 European Journal of Microbiology and Immunology 11 (2021) 4, 8794
Fig. 4. Effect of MixV on viral RdRp. Effects of MixV alone (A) and in combination with vitamin D3 and zinc (B) on activity of SARS-CoV-2
RdRp. Puried recombinant RdRp enzyme in VeroE6 cells was incubated with MixV at designated concentrations for 15 min at RT. After
application of reaction mix composed of NTPs and RNA template incubation was carry out for 2h at 34 8C. Signal was measured in 10 min
at extension/emission 5488/535 nm with spectrouorometer. Control 0.05% DMSO, positive control 100
m
gmL
1
remdesivir;
ΔP0.01, * P0.001; VD3 25-hydroxy Vitamin D3 at 25
m
gmL
1
,Znzinc aspartate at 2.5
m
gmL
1
Fig. 5. Effect of MixV on cellular proteases. Effect of MixV on furin activity (A). Furin as a recombinant protein was incubated with MixV
alone or in combination with vitamin D and zinc at indicated concentrations for 15 min followed by addition of uorogenic Rh110 furin
substrate, and incubation for 1h at 22 8C. Effect of MixV on cathepsin L activity (B and C). Cathepsin L as a recombinant protein was
incubated with MixV alone or in combination with vitamin D and zinc at indicated concentrations for 1 h at 22 8C followed by the
application of 10
m
Mofuorogenic substrate for 30 min. Control 0.05% DMSO, positive control 25
m
gmL
1
E-64; # P0.05,
*P0.001, VD3 25-hydroxy Vitamin D
3
at 25
m
gmL
1
,Znzinc aspartate at 2.5
m
gmL
1
Fig. 3. Viability of human lung epithelial cells. Effects of MixV on survival of A549/hACE2 cells (A). Effects of MixV its combination with
vitamin D3 and zinc on survival of A549/hACE2 cells (B). Viability of A549/hACE2 cells was assessed using MTT method as described in
Experimental Procedures section; controls 0.05% DMSO; * P0.001 VD3 25-hydroxy Vitamin D
3
at 25
m
gmL
1
,Znzinc aspartate at
2.5
m
gmL
1
European Journal of Microbiology and Immunology 11 (2021) 4, 8794 91
in 54% inhibition. A combination of MixV with vitamin D
and zinc even further decreased furin activity by 78%. As
shown in Fig. 5B, the MixV, although mildly, also was
effective in decreasing activity of cathepsin L involved in
SARS-CoV-2 endosomal egress. In the presence of 10
m
g
mL
1
MixV enzymatic activity of cathepsin L was reduced
by 14%, and addition of vitamin D3 did not have a signi-
cant effect on cathepsin L inhibition.
DISCUSSION
Our study represents a new comprehensive approach to
COVID-19 with its aim at controlling not one but several
cellular mechanisms associated with SARS-CoV-2 infectivity
at once. Our results show that a defined combination of
active plant components and selected micronutrients (MixV)
can significantly affect critical steps required for cellular
infectivity of the original SARS-CoV-2 strain and its mutated
forms Alpha, Beta, Gamma, Delta, Kappa, and Mu, by
inhibiting viral attachment to cellular ACE2 receptors,
decreasing availability of these receptors on human lung cells,
and decreasing activity of viral RdRp necessary for viral
replication. In addition, these natural compounds interfered
with cellular entry of the virions by decreasing activity of
important enzymes such as furin and cathepsin L (see Fig. 6).
These findings further expand on our earlier study using
SARS-CoV-2 virion particles, which identified core in-
gredients in this combination, which were effective in
inhibiting viral binding to hACE2 receptor and decreasing
expression of ACE2 receptors on human small alveolar
epithelial cells [21]. Taking into account immediate avail-
ability and high safety prole of these natural compounds,
their application as preventive measures, e.g. as an adjunct to
vaccines and therapies of COVID, is plausible.
While all tested nutrient combinations (A, B, C, D and E)
were effective in blocking RBD binding to ACE2 by about
50%, the MixV demonstrated the highest, 90% inhibition of
viral binding. This would indicate that synergistic or at least
additive effect could play a role here, not excluding other,
e.g., allotropic, interactions between the compounds of
MixV and RBD/ACE2.
Our study investigated inhibitory effects of MixV on
cellular internalization of both SARS-CoV-2 virions and its
mutated forms using different pattern of exposure of these
virions to MixV and human lung cells. As such, we incu-
bated test virions with MixV for 1 h before their exposure to
the cells and combined MixV with virions and cells simul-
taneously. The results indicate that maximum inhibition of
cellular transduction by these virions can be obtained when
all of these elements interacted at the same time. This would
imply that this mixture affects metabolic aspects of cell-vi-
rions interactions rather than acts on the virus itself.
Although its efficacy in inhibiting viral internalization was
not virus specific, MixV was nevertheless most effective in
inhibiting the original SARS-CoV-2 and Delta variants
compared to other mutated forms.
In addition to the inhibitory effect of MixV on cellular
attachment and entry of SARS-CoV-2 virions, we also
observed that MixV affected cellular mechanisms involved
in endosomal egress and multiplication of the virus by
decreasing activity of enzymes involved in viral cellular
processing. Among the proteolytic enzymes involved in
these processes, the activity of cathepsin L and furin has
been shown to be important for pathogenicity of SARS-
CoV-2 [22,23]. It also has been shown that furin action
towards the SARS-CoV-1 spike protein is necessary for the
fusion of virions with host membranes, without directly
affecting viral infectivity [24]. It appears that effective con-
trol and treatment of SARS-CoV-2 infection might neces-
sitate simultaneous inhibition of several proteases to
effectively obstruct these pathological conversions.
Here we have shown that, in addition to impairing viral
binding to hACE2 overexpressing lung epithelial cells, the
MixV decreased activity of membrane protease furin and
endosomal cathepsin L. The reduction of the activity of these
enzymes was observed at non-toxic MixV concentrations.
This effect is significant, as the lack of the additional furin
cleavage site on the SARS-CoV-1 spike protein has a sub-
stantial influence on its infectivity [25].
Fig. 6. Schematic representation of the effects of MixV on key cellular processes associated with SARS-CoV-2 infectivity
92 European Journal of Microbiology and Immunology 11 (2021) 4, 8794
In addition to SARS-CoV-2 infection, the potential
signal link between spike protein, furin, and ACE2 has
been implied in the occurrence of adverse cardiovascular
events [26]. Since these enzymes are also involved in
various biochemical pathways, a lack of complete inhibi-
tion at the concentrations of MixV used in the study may
be benecial. We observed that vitamin D3 and zinc had
additional effect on lowering enzymatic activity of furin,
but not cathepsin L. Finally, we also recorded a substantial
inhibition of activity of viral RdRp at these concentra-
tions, with its consequences at reduced replication rate of
the virus.
Based on this study and our earlier findings [18,19,27],
this combination of plant-derived compounds and micro-
nutrients may constitute a new anti-SARS-CoV-2 strategy
by simultaneously affecting multiple aspects of viral life
cycle, including viral entry and replication. This strategy was
also implemented in our earlier studies, including those of
human inuenza H1N1, bird u H1N5, and others, which
were based on selecting natural components that simulta-
neously affect key pathology mechanisms across a wide
spectrum of infective agents [2831].
Our study implies that a simultaneous control of key
steps of the SARS-CoV-2 infectivity by a definite combina-
tion of plant-derived and biologically active compounds can
be effective in addressing complex pathologies, such as
involved in this viral infection. Taken together, with the
recently established clinical evidence that vitamin C in-
fusions can successfully combat COVID-19 even at
advanced stages [32], phyto-compounds through their
pleiotropic biological effects could represent a new approach
against SARS-CoV-2 and its mutants.
Funding: This research was supported by funding from the
non-profit Dr. Rath Health Foundation belonging to
Stichting Administratiekantoor Dr. Rath Holding, NL.
Author contributions: A.G. conceived, designed, and per-
formed the experiments, analyzed data, reviewed the paper,
and had primary responsibility for final content; V.I. and S.I.
designed and performed the experiments, analyzed data,
reviewed the paper, and had primary responsibility for final
content. A.N. and M.R. supervised, validated, wrote the
paper, and had primary responsibility for final content. All
authors had full access to all data in the study, they have
read and approved the final manuscript and take re-
sponsibility for the integrity of the data and the accuracy of
the data analysis.
Conflict of interest: Dr. Rath and Dr. Niedzwiecki are
members of the non-profit Dr. Rath Health Foundation
but do not receive any financial revenues from it. All au-
thors declare no potential conflict of interest with respect
to the research, authorship, and/or publication of this
article.
Data availability statement: All data generated or analyzed
during this study are included in this published article.
ACKNOWLEDGMENT
The authors thank Dr. Bilwa Bhanap, Vyoma Bhanap,
Waldemar Sumera, and Cathy Flowers for their valued
contribution added to this manuscript.
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creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium for non-commercial purposes, provided the
original author and source are credited, a link to the CC License is provided, and changes if any are indicated.
94 European Journal of Microbiology and Immunology 11 (2021) 4, 8794
... According to Kumar et al. [18], magnesium ascorbate has been revealed as, among 106 other nutraceticals, the most powerful inhibitor of M PRO /3CL PRO , a key protease in SARS-CoV-2, that as a nonstructural protein participates in the replication and life cycle of the virus inside the host cell. Due to its interaction with ACE2, vitamin C is also mentioned as one of the natural compounds having the inhibitory activity against different variants of coronavirus, such as Alpha-, Beta-, Gamma-, Delta-, Kappa-or Mucoronavirus [19]. As was shown by Ivanov et al. [20], AA and its specific combinations with other natural compounds lower ACE2 expression at the protein level and at the RNA level as was proved in Human Small Airways Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMEC). ...
... According to Kumar et al. [18], magnesium ascorbate has been revealed as, among 106 other nutraceticals, the most powerful inhibitor of M PRO /3CL PRO , a key protease in SARS-CoV-2, that as a nonstructural protein participates in the replication and life cycle of the virus inside the host cell. Due to its interaction with ACE2, vitamin C is also mentioned as one of the natural compounds having the inhibitory activity against different variants of coronavirus, such as Alpha-, Beta-, Gamma-, Delta-, Kappa-or Mu-coronavirus [19]. As was shown by Ivanov et al. [20], AA and its specific combinations with other natural compounds lower ACE2 expression at the protein level and at the RNA level as was proved in Human Small Airways Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMEC). ...
... As was shown by Ivanov et al. [20], AA and its specific combinations with other natural compounds lower ACE2 expression at the protein level and at the RNA level as was proved in Human Small Airways Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMEC). Goc et al. [19] showed that the defined mixture of plant extracts and micronutrients with vitamin C demonstrated a highest efficacy by inhibiting the receptor binding domain (RBD), required to bind SARS-CoV-2 to ACE2 receptor. The mixture also significantly inhibited other mechanisms of viral infectivity, including the RNA-dependent RNA polymerase (RdRP), necessary for viral replication, as well as furin and cathepsin L, important for fusion of virions with host membranes and involved in viral multiplication. ...
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Vitamin C has a number of acitvities that could contribute to its immune-modulating effects. The only question is whether we should provide ourselves with only the right level of it, or do we need much more during a pandemic? The possibility of reducing the incidence of viral diseases in a well-nourished population through the use of dietary supplements based on vitamin C is not supported in the literature. Despite this, the belief that an extra intake of vitamin C can increase the efficacy of the immune system is still popular and vitamin C is advertised as a remedy to prevent infectious disease. This article refers to the justification of the use of vitamin C in high doses as an anti-SARS-CoV-2 prophylaxis in healthy subjects. Does it make sense or not? As it turns out, any effects of vitamin C supplementation may be more prominent when the baseline vitamin C level is low, for example in physically active persons. People with hypovitaminosis C are more likely to respond to vitamin C administration. No studies regarding prevention of COVID-19 with high-dose vitamin C supplementation in healthy subjects were found.
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SARS-CoV-2 pandemic remains a challenge to human health and economy worldwide. Previously we have shown that a combination of active plant-derived compounds and plant extracts can dose-dependently inhibit binding of RBD-spike protein SARS-CoV-2 to the ACE2 receptor and its expression on human alveolar epithelial cells. Here we use eGFP-luciferase-SARS-CoV-2 spike protein pseudo-virions and SARS-CoV-2-RdRp, to show if the antiviral effectiveness of this combination of plant-derived compounds and plant extracts expands to other important key mechanisms of SARS-CoV-2 infection. Or results revealed that this combination of five plant-derived compounds inhibited the attachment of the SARS-CoV-2 pseudo-typed particles with lung hACE2/A549 cells. In addition, it down-regulated the activity of key enzymes known to be crucial for the entry of the SARS-CoV-2 virus, such as TMPRSS2, furin and cathepsin L, but not their expression at protein level. This combination did not affect ACE2 binding to and ACE2 enzymatic activity, but modestly decrease cellular expression of neuropilin-1 molecule and significantly inhibited activity of viral RdRp. This study demonstrates inhibitory effects of this combination on key cellular mechanisms of SARS-CoV-2 infection. The findings further support the use of plant-derived compounds as effective health measures against SARS-CoV-2-caused infection.