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Study Dandelion prevents spike protein from attaching

In Vitro Effect of Taraxacum officinale Leaf Aqueous Extract on
the Interaction between ACE2 Cell Surface Receptor and
SARS-CoV-2 Spike Protein D614 and Four Mutants
Hoai Thi Thu Tran 1 , Michael Gigl 2 , Nguyen Phan Khoi Le 1 , Corinna Dawid 2 and Evelyn Lamy 1,*

Citation: Tran, H.T.T.; Gigl, M.; Le,
N.P.K.; Dawid, C.; Lamy, E. In Vitro
Effect of Taraxacum officinale Leaf
Aqueous Extract on the Interaction
between ACE2 Cell Surface Receptor
and SARS-CoV-2 Spike Protein D614
and Four Mutants. Pharmaceuticals
2021, 14, 1055.
Academic Editors: Jean Jacques
Vanden Eynde and Annie Mayence
Received: 20 September 2021
Accepted: 14 October 2021
Published: 17 October 2021
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1 Molecular PreventiveMedicine, UniversityMedical Center and Faculty ofMedicine, University of Freiburg,
79108 Freiburg, Germany; (H.T.T.T.); (N.P.K.L.)
2 Food Chemistry andMolecular Sensory Science, Technical University ofMunich, 85354 Freising, Germany; (M.G.); (C.D.)
* Correspondence:; Tel.: +49-761-270-82150
Abstract: To date, there have been rapidly spreading new SARS-CoV-2 “variants of concern”. They
all contain multiple mutations in the ACE2 receptor recognition site of the spike protein, compared
to the original Wuhan sequence, which is of great concern, because of their potential for immune
escape. Here we report on the efficacy of common dandelion (Taraxacum officinale) to block protein–
protein interaction of SARS-COV-2 spike to the human ACE2 receptor. This could be shown for the
wild type and mutant forms (D614G, N501Y, and a mix of K417N, E484K, and N501Y) in human
HEK293-hACE2 kidney and A549-hACE2-TMPRSS2 lung cells. High-molecular-weight compounds
in the water-based extract account for this effect. Infection of the lung cells using SARS-CoV-2 spike
D614 and spike Delta (B.1.617.2) variant pseudotyped lentivirus particles was efficiently prevented
by the extract and so was virus-triggered pro-inflammatory interleukin 6 secretion. Modern herbal
monographs consider the usage of this medicinal plant as safe. Thus, the in vitro results reported here
should encourage further research on the clinical relevance and applicability of the extract as prevention
strategy for SARS-CoV-2 infection in terms of a non-invasive, oral post-exposure prophylaxis.
Keywords: ACE2 binding inhibitor; COVID-19; dandelion; SARS-CoV-2 prevention; S1 spike mutation
1. Introduction
In late 2019, the disease known as Corona Virus Disease 2019 or COVID-19 was
first reported [1]. It is induced by the severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2). Dry cough, fever, fatigue, headache, myalgias, and diarrhea are common
symptoms of the disease. In severe cases people may become critically ill with acute
respiratory distress syndrome [2]. The SARS-CoV-2 virus surface is covered by a large
number of glycosylated S proteins, which consist of two subunits, S1 and S2. The S1
subunit recognizes and attaches to the membrane-anchored carboxypeptidase angiotensinconverting
enzyme 2 (ACE2) receptor on the host cell surface through its receptor binding
domain (RBD). The S2 subunit plays a key role in mediating virus–cell fusion and in concert
with the host transmembrane protease serine subtype 2 (TMPRSS2), promotes cellular
entry [3]. This interaction between the virus and host cell at entry site is crucial for disease
onset and progression.
To date, there have been rapidly spreading new variants of SARS-CoV-2, Alpha
(variant B.1.1.7), Beta (variant B.1.351), and Gamma (variant P.1). Delta (variant
B.1.617.2) recently emerged and spread explosively, replacing Alpha around the world.
Most of these variants share the mutation N501Y in the spike protein [4] and SARSCoV-
2 variants with spike protein D614G mutations now predominate globally. The
Beta variant contains, besides D614G, other spike mutations, including three mutations
Pharmaceuticals 2021, 14, 1055.
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(K417N, E484K, and N501Y) in the RBD [5]. Preliminary data suggest a possible
association between the observed increased fatality rate and the mutation D614G
and it is hypothesized that a conformational change in the spike protein results in
increased infectivity [6]. Free energy perturbation calculations for interactions of the
N501Y and K417N mutations with both the ACE2 receptor and an antibody derived
from COVID-19 patients raise important questions about the possible human immune
response and the success of already-available vaccines [7]. Further, increased resistance
of the variants Beta and Alpha to antibody neutralization has been reported; for the Beta
variant this was largely due to the E484K mutation in the spike protein [8]. The Delta variant
has been associated with more severe disease, increased transmission, and breakthrough
infections in vaccinated individuals [9–12]. Liu et al. [13] found that the variants’ spike P681R
mutation augments spike processing, which leads to enhanced SARS-CoV-2 fitness over the
Alpha variant.
Interference with the interaction site between the spike S1 subunit and ACE2 has
the potential to be a major target for therapy or prevention [14]. Compounds of natural
origin may offer here some protection against viral cell entry while having no or few
side effects. Here we report on the inhibitory potential of dandelion on the binding
of the spike S1 protein RBD to the hACE2 cell surface receptor and compare the effect
of the original D614 spike protein to its D614G, N501Y, and mix (K417N, E484K, and
N501Y) mutations.
Taraxacum officinale (L.) Weber ex F.H.Wigg. (common dandelion) belongs to the
plant family Asteraceae, subfamily Cichorioideae with many varieties and microspecies.
It is a perennial herb, widely distributed in the warmer temperate zones of the Northern
Hemisphere inhabiting fields, roadsides, and rural sites. T. officinale is consumed as
food, but also used in European phytotherapy for diseases of the liver, gallbladder,
and digestive tract or for rheumatic diseases. Modern herbal monographs consider the
plant usage as safe and have evaluated the empirical use of T. officinale for gallstones
or biliary diseases with a positive outcome. Therapeutic indications for the use of
T. officinale are listed in the German Commission E, the European Scientific Cooperative
for Phytotherapy (ESCOP) monographs [15,16] as well as by the British Herbal Medicine
Association [17]. The plant contains a wide array of phytochemicals including terpenes
(sesquiterpene lactones such as taraxinic acid and triterpenes), phenolic compounds
(phenolic acids, flavonoids, and coumarins), and also polysaccharides [18]. The predominant
phenolic compound was found to be chicoric acid (dicaffeoyltartaric acid).
The other constituents are mono- and dicaffeoylquinic acids, tartaric acid derivatives,
flavone, and flavonol glycosides. The roots, in addition to these compound classes, contain
high amounts of inulin [19]. Dosage forms include aqueous decoction and infusion,
expressed juice of fresh plants, and hydroalcoholic tincture as well as coated tablets
from dried extracts applied as monopreparations [20] but also integral components of
pharmaceutical remedies. The aim of this study was to investigate whether T. officinale
aqueous leaf extracts and its high molecular weight components block the interaction of
ACE2 receptor and SARS-CoV-2 spike protein.
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2. Results
2.1. Chemical Analysis of T. officinale Leaf Extract
Metabolomics-based fingerprinting of T. officinale and Cichorium intybus L., leaf extract
was performed by untargeted UPLC-TOF-MS analysis (Figure 1) and putative metabolite
identification was done by database search of accurate mass and MSe fragmentation
patterns. FoodDB, the Plant Metabolic Network, PlantCyc, and Nature Chemistry were
used for tentative annotation. The most abundant compounds of these extracts in both
negative ion mode (ESI-) and positive ion mode (ESI+) were identified (Supplementary
Tables S1–S4). These are mainly in line with previous reports [18,19,21].
Figure 1. Metabolic analysis of T. officinale (TO) and C. intybus (CI) leaf extract using UPLC-TOF-MS.
Measurements were done in high resolution mode with negative electrospray ionization (ESI-) and
positive electrospray ionization (ESI+).
2.2. T. officinale Inhibits Spike RBD–ACE2 Binding
We first investigated the inhibition of interaction between SARS-CoV-2 spike protein
RBD and ACE2 using extracts from T. officinale leaves. In Figure 2A, the concentrationdependent
inhibition of Spike S1–ACE2 binding upon treatment with T. officinale extract
is given (EC50 = 14.9 mg/mL). Extracts from C. intybus, which is another plant from the
family Asteraceae, also showed a concentration-dependent binding inhibition, but with
less potency (EC50 = 31.4 mg/mL) (Figure 2B). We then prepared two fractions of the
extracts, separating them into a high molecular (>5kDa) and low molecular weight (<5kDa)
fraction. As can be seen from Figure 2C–D, the bioactive compounds were mostly present
in the HMW fraction. Only minor activity was seen in the LMW fraction.
Using hACE2-overexpressing HEK293 cells, the potential of the extracts to block spike
binding to cells was further investigated. As can be seen from Figure 3, pre-incubation of cells
with T. officinale for one minute efficiently blocked cell binding of spike by 76.67% 2.9, and
its HMWfraction by 62.5 13.4% as compared to the water control. The C. intybus extract
was less potent; binding inhibition was seen at 37 20% after 1 min.
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Figure 2. Effect of T. officinale and C. intybus extracts on SARS-CoV-2-Spike–ACE 2 inhibition. (A,B) Concentrationdependent
effect of T. officinale (TO) and C. intybus (CI) extract. (C,D). Effect of fractions from TO and CI leaf extract. The
extracts were freeze-dried and a molecular weight fractionation was subsequently carried out. The cut-off was set to 5 kDa
(HMW > 5 kDa, LMW < 5kDa). H+L: HMW and LMW fractions; 50 mg of dried leaves per mL water was used as reference.
HMW and LMW fraction quantities equivalent to dried leaves were used. The binding inhibition was assessed using ELISA
technique. Bars are means + SD. Solvent control: distilled water (a.d.); * p < 0.05, ** p < 0.01. Significance of difference was
calculated relative to the solvent control by one-way ANOVA.
Figure 3. Cont.
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Figure 3. Binding inhibition of S1 spike protein to human HEK293-hACE2 cells by extract pre-incubation. Cells were
pre-incubated for the indicated times with 10 mg/mL T. officinale (TO), its HMW fraction, equal to 10 mg/mL extract
(HMW), and 10 mg/mL C. intybus (CI) or solvent control (a.d.) and subsequently treated with His-tagged S1 spike protein for 1 h
without a washing step in between at 4 C. Binding inhibition was assessed using flow cytometry. N = 3, bars aremeans + SD.
Upper left: cytogram of gated HEK-hACE2 cells. Middle: overlay of representative fluorescence intensity histograms
for ACE2 surface expression. Upper right: overlay of representative fluorescence intensity histograms for spike-binding
inhibition by the extracts or a.d.; positive control: 20 g/mL soluble hACE2. Cells were stained with anti-His-tag Alexa
Fluor 647 conjugated monoclonal antibody; ** p < 0.01. Significance of difference was calculated relative to the solvent
control by one-way ANOVA.
Cell treatment with equal amounts of spike D614 and its variants D614G and N501Y
confirmed a stronger binding affinity of D614G (about 1.5-fold) and N501Y (about 3- to
4-fold) than D614 spike protein to the ACE2 surface receptor of HEK293 cells (Figure 4A).
Pre-treatment with T. officinale quickly (within 30 s) blocked spike binding to the ACE2
surface receptor (Figure 4B,C). After 30 s, this was 58.2 28.7% for D614, 88.2 4.6% for
D614G, and 88 1.3% for N501Y binding inhibition by T. officinale extract. Even though
for C. intybus extract a binding inhibition of spike could be seen, this was about 30–70%
less compared to T. officinale, dependent on the spike protein investigated. When binding
was studied at 37 C instead of 4 C, the results were comparable for T. officinale, but even
weaker for C. intybus extract in this cell line (Figure 4D). We also raised the question of
whether the extracts could replace spike binding to the ACE2 surface receptor of human
cells. For this, we first incubated the cells with D614, D614G, or N501Y spike protein and
subsequently with the extracts. As seen in Figure 4D, T. officinale could potently remove
spike from the receptor (on average 50%); C. intybus was much weaker than (on average
25%). We extended our experiments to human A549-hACE2-TMPRSS2 cells and could
confirm the results observed in HEK293-hACE2 cells for T. officinale (Figure 4D–G). This
cell line has been stably transfected with both the human ACE2 and TMPRSS2 genes
and interestingly, here the C. intybus extract was more effective as compared to HEKhACE2
cells. Upon extract pre-treatment, spike-binding inhibition to the cells was between
73.5% 5.2 (D614) to 86.3% 3.23 (N501Y) for T. officinale extract and 56.1% 5.28
(D614) to 63.07% 14.55 (N501Y) for C. intybus extract. Already at 0.6 mg/mL, T. officinale
significantly blocked binding to D614G spike protein by about 40% (IC50 = 1.73 mg/mL).
When cells were pre-incubated with the spike protein before extract treatment, results were
comparable for T. officinale extract for D614 and D614G but somewhat lower for N501Y
(Figure 4C,D). Also, in this setting, a mixture of spike mutants N501Y, K417N, and E484K
was tested, and here again, T. officinale extract blocked binding by 82.97% 6.31(extract
pre-incubation) and 79.7% 9.15 (extract post-incubation). Extracts, incubated in human
saliva for 30 min at 37 C before cell treatment had comparable effects on spike D614G
inhibition (Figure 4H) indicating a good stability of the bioactive compounds in saliva.
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Figure 4. Binding inhibition of spike D614, and its mutants D614G, N501Y or mix (N501Y, K417N and E484K) to human
HEK293-hACE2 and A549-hACE2-TMPRSS2 cells by extract pre- or post-incubation. Overlay of fluorescence intensity
histogram for (A) unstained HEK cells, staining control (anti-His-tag Alexa Fluor 647), and cells incubated with His-taglabelled
spike D614, D614G or N501Y for 1 h at 4 C. (B,C) cells pre-incubated with solvent control (a.d.), 10 mg/mL
T. officinale (TO) or 10 mg/mL C. intybus (CI) for 30–60 s, and then treated with His-tag-labelled S1 spike D614, D614G or
N501Y protein for 1 h without a washing step in between at 4 C. (D–G) Effect of extract incubation on HEK or A549 cells
either before or after incubation with His-tag-labelled spike D614, D614G, N501Y or mix (N501Y, K417N and E484K) protein
at 37 C. (H) Plant extracts were incubated in saliva from four human donors for 0.5 h at 37 C. Afterwards, cells were
pre-treated with 5 mg/mL extracts for 60 s at 37 C before incubation with His-tag-labelled spike D614 protein for 0.5 h at
37 C. Spike-binding inhibition to human cells was assessed using flow cytometric analysis of cells stained with anti-His-tag
Alexa Fluor 647 conjugated monoclonal antibody. Bars are means + SD; * p < 0.05, ** p < 0.01. Significance of difference was
calculated relative to the respective solvent control by one-way ANOVA.
2.3. T. officinale Does Not Interfere with ACE2 Enzyme Activity
To see whether T. officinale extract interferes with the catalytic activity of the ACE2
receptor or affects ACE2 protein expression, we treated A549-hACE2-TMPRSS2 cells with
the extract for 1–24 h before cell lysis and detection. No loss in cell viability was seen after
extract exposure to the cells for 84 h (Figure 5A). No enzyme activity impairment could be
detected after 1 or 24 h (Figure 5B). Spike significantly downregulated ACE2 protein after
6 h (Figure 5C, black bars), and this was also true for the extract, either alone (Figure 5C,
white bars) or in combination with spike (black bars). After 24 h, this effect was abolished
(Figure 5D).
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Figure 5. Effect of T. officinale extract on ACE2 enzyme activity and protein expression. (A) Viability
of A549-hACE2-TMPRSS2 cells was determined using trypan blue cell staining after 84 h exposure
to the extract. (B) Cells were incubated with TO extract or 500 ng/mL S1 protein and analyzed for
enzyme activity using a fluorescence kit. (C,D) Cells were exposed for 6 h or 24 h to extract without
(white bars) or with (black bars) 500 ng/mL S1 protein and analyzed for ACE2 protein expression
using a human ACE2 ELISA kit; a.d.: solvent control. Bars are means + SD, N 3 independent
experiments; * p < 0.05, ** p < 0.01. Significance of difference was calculated relative to the respective
control by one-way ANOVA.
2.4. T. officinale Blocks SARS-CoV-2 Spike D614 and Spike Delta (B.1.617.2) Variant Pseudotyped
Lentivirus Transduction
Using a SARS-CoV-2 spike D614 and spike Delta (B.1.617.2) variant pseudotyped
lentivirus, we then studied whether the extract could block virus entry via spike inhibition.
When pre-treated with the extract, spike D614 virus transduction was diminished by about
85% at 20 mg/mL, which was in the range of inhibition observed by 0.35 mg/mL of the
HMW extract (Figure 6A, left). As shown in Figure 6A (right), a substantial reduction in
Delta variant pseudovirus infection of lung cells was also observed upon treatment with
extracts from T. officinale or the HMW extract (72% at 20 mg/mL, and 69% at 0.35 mg/mL,
respectively). Anti-hACE2 antibody, which was used here as a reference, blocked virus
transduction by 74% at 100 g/mL.
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Figure 6. Viral transduction inhibition of A549-hACE2-TMPRSS2 cells by T. officinale extract. (A) Cells were pre-treated with
T. officinale (TO) or HMW extract for 0.5 h before infection with 7500 TU/mL SARS-CoV-2 spike D614 or Delta (B.1.617.2)
variant for 24 h; (B,C) Cells were transduced with 7500 TU/mL SARS-CoV-2 for (B) 3 h before addition of TO for another
21 h or (C) 24 h. After transduction, the medium was exchanged with fresh medium containing TO or HMW extract at
the indicated concentrations and post-incubated for 60 h. (D) Cells were pre-treated with 40 mg/mL TO for 3 h before
transduction with the indicated virus titer for 24 h. After that, the medium was exchanged with fresh medium and incubated
for another 48 h. Luminescence was then detected within 1 h. 0.35 mg/mL HMW extract equals to 20 mg/mL TO extract.
Transduction control: (􀀀) negative control: bald lentiviral pseudovirion; (+) positive control: firefly luciferase lentivirus;
inhibitor positive control: 100 g/mL anti-hACE2 antibody. (E) Pro-inflammatory IL-6 cytokine secretion analysis was done
either after 24 h virus transduction together with extract (left), after 24 h + 60 h post-infection with extract (middle) or after
60 h post-infection with extract (right) using multiplexing flow cytometric analysis. Solvent control: distilled water (a.d.).
N 3 independent experiments; * p < 0.05, ** p < 0.01. Significance of difference was calculated relative to the solvent
control by one-way ANOVA.
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To investigate which step of the SARS-CoV-2 pseudovirus infection was targeted by
the extract, we treated cells at different time points, such as before (pre-treatment), during
(co-treatment), or after (post-treatment) viral infection. Under the different treatment
conditions, the luminescent signal produced by spike D614 virus transduction was inhibited
at 10 mg/mL extract by 70% 16.7 (A), 58% 9.6 (B), and 53% 8.1 (C). Besides, we
addressed the effectiveness of infection prevention in cells without post-infection. It can be
seen from Figure 6D that, dependent on the initial virus load, infection was diminished
by >43% at 1500 TU/mL, and 35% at 3000 TU/mL, whereas there was a minimal change
at 7500 TU/mL. A 36% inhibition was reached by the plant extract in SARS-CoV-2 spike
Delta variant transduced cells at 1500 TU/mL (Figure 6D, right). The inhibition of virus
transduction by the extract concurred with a significant suppression of the virus triggered
inflammatory response, as determined by diminished secretion of the pro-inflammatory
cytokine IL-6 in A549-hACE2-TMPRSS2 cells (Figure 6E).
3. Discussion
Developing effective prevention and treatment strategies for SARS-CoV-2 infections
remains a challenge as new variants emerge that are likely to be more contagious and better
able to escape the immune system. Although the first vaccines have now received marketing
authorization, challenges in distribution and concerns about durable effectiveness
and risk of re-infection remain [22]. Vaccine breakthrough infections have already been
reported, especially transmission with the Delta variant [9,11,12]. This suggests a potential
waning in the protective effect of the vaccines against Sars-CoV-2 infections [23,24].
Subsequent infections may, though, possibly be milder than the first one. Booster vaccinations
to enhance the immune response may be the best strategy for risk reduction.
Besides vaccination, blocking the accessibility of the virus to membrane-bound ACE2 as
the primary receptor for SARS-CoV-2 target cell entry, represents an alternative strategy
to prevent COVID-19. Here, different approaches exist [25], but of course each of these
treatment strategies also has its fundamental as well as translational challenges which need
to be overcome for clinical utility. Technical hurdles include off-target potential, ACE2-
independent effects, stability, or toxicity [25]. Compounds from natural origin could be an
important resource here as they have already been well described and many of them have
been established as safe. While in silico docking experiments suggested different common
natural compounds as ACE2 inhibitors, spike-binding inhibition to ACE2 has not been
shown for most of them so far, which might be explained by a lack of complete coverage of
ACE2 binding residues by the compounds [26]. However, for glycyrrhizin, nobiletin, and
neohesperidin, ACE2 binding falls partially within the RBD contact region and thus, these
have been proposed to additionally block spike binding to ACE2 [20]. The same accounts
for synthetic ACE2 inhibitors, such as N-(2-aminoethyl)-1 aziridine-ethanamine [27]. In
contrast, the lipoglycopeptide antibiotic dalbavancin has now been identified as both
an ACE2 binder and SARS-CoV-2 spike-ACE2 inhibitor [28]; SARS-CoV-2 infection was
effectively inhibited in both mouse and rhesus macaque models by this compound. In
addition, with a hydroalcoholic pomegranate peel extract, blocking of spike-ACE2 interaction
was shown at 74%, for its main constituents punicalagin at 64%, and ellagic acid at
36%. Using SARS-CoV-2 spike pseudotyped lentivirus infection of human kidney-2 cells,
virus entry was then efficiently blocked by the peel extract [29]. In the present study, we
could show potent ACE2-spike S1 RBD protein inhibition by T. officinale extracts using a
cell-free assay and confirmed this finding by demonstrating efficient ACE2 cell surface
binding inhibition in two human cell lines. We observed stronger binding of the variants
D614G and N501Y to the ACE2 surface receptor of human cells, but all tested variants were
sensitive to binding inhibition by T. officinale, either used before spike protein exposure or
after. To date, several studies indicate that the D614G viral lineage is more infectious than
the D614 virus [30]. In addition, the presence of characteristic mutations such as N501Y
results in higher infectivity than the parent strain which might be due to a higher binding
affinity between the spike protein and ACE2 [31]. So our findings on T. officinale extracts
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could here be important, as with the progression of the pandemic, new virus variants
of potential concern will emerge which may also reduce the efficacy of some vaccines or
cause increased rates of reinfections. As mentioned above, an issue in the development of
products such as prophylaxis for SARS-CoV-2 infection or for slowing the systemic virus
spread, is the selectivity towards virus intrusion with low toxicity needed for the host. For
current medical indications, no case of overdose by T. officinale has been reported [15,17,20].
The recommended dosage is 4–10 g (about 20–30 mg per mL hot water) up to three times
per day (Commission E and ESCOP). Based on the information provided by the European
Medicines Agency contraindications for the use of T. officinale are hypersensitivity to the
Asteraceae plant family or their active compounds, liver and biliary diseases, including
bile duct obstruction, gallstones and cholangitis, or active peptic ulcer [20]. The plant is a
significant source of potassium [32] and thus a warning is given because of the possible
risk for hyperkalemia. The use in children under 12 years of age, or during pregnancy and
lactation has not been established due to lack of adequate or sufficient data.
While ACE2 enzyme activity was not affected by T. officinale extract in the present
study, ACE2 protein was transiently downregulated in the ACE2-overexpressing lung
cell line. ACE2 plays a key role in SARS-CoV-2 infection, so lowering ACE2 levels could
theoretically provide some initial protection [33]. However, this could also affect important
cell physiological functions. ACE2 is an important zinc-dependent mono-carboxypeptidase
in the renin–angiotensin pathway, critical in impacting the cardiovascular and immune
systems. Disruption of the angiotensin II/angiotensin-(1-7) balance by ACE2 enzyme
activity inhibition or protein decrease and more circulating angiotensin II in the system,
is recognized to promote lung injury in the context of COVID-19 disease [34]. Thus, this
observation needs more attention in ongoing studies.
The lung could be assumed to be the primary target of interest but, ACE2 mRNA
and protein expression have been found in epithelial cells of all oral tissues, especially
in the buccal mucosa, lip, and tongue [35]. These data concur with the observation of
very high salivary viral load in SARS-CoV-2-infected patients [36,37]. As an essential part
of the upper aerodigestive tract, the oral cavity is thus believed to play a key role in the
transmission and pathogenicity of SARS-CoV-2. There is high potential that prevention
of viral colonization at the oral and pharyngeal mucosa could be critical for averting
further infection to other organs and the onset of COVID-19 [38]. Commercial virucidal
mouth-rinses, povidone-iodine at the first place, have thus been suggested to potentially
reduce the SARS-CoV-2 virus load in infected persons [39,40], but significant clinical studies
do not exist to date [40]. Blocking SARS-CoV-2 virus binding to cells of the oral cavity
with T. officinale extracts might be tolerable for a consumer, if necessary only for limited
periods of time (e.g., product application after contact with infected persons or when
being infected). More physiologically relevant in vitro experiments that were carried out
by us showed that only short contact times with T. officinale extract were necessary for
efficient blocking of SARS-CoV-2 spike binding or for removing already-bound spike from
the cell surface. Further evidence of relevance was provided by demonstrating effective
protection of T. officinale against SARS-CoV-2 spike D614 and Delta variant pseudotyped
virus infection of human lung cells. The use of pseudotyped viruses does not allow us to
assess the contribution of virion properties such as membrane or envelope proteins to cell
tropism [35]. However, pseudovirus data are considered a useful tool to document the
importance of ACE2 in the spike-protein-mediated steps of cell entry.
4. Materials and Methods
4.1. Plant Material
The study was carried out using dried leaves from T. officinale (vom Achterhof,
Uplengen, Germany; batch no. 37259, B370244, and P351756). C. intybus was purchased
from Naturideen (Germany).
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4.2. Plant Extraction
Dried plantmaterialwasweighted in an amber glass vial (Carl RothGmbH,Germany) and
mixed with HPLC-grade water (a.d.), at roomtemperature (RT). Extracts were then incubated
for 1 h and centrifuged at 16,000 g (3min, RT). The supernatant was filtered (0.22 m) prior to
use for the experiments.
4.3. Ultra-Performance Liquid Chromatography (UPLC)-Time-of-Flight (TOF)-Mass Spectrometry
High resolution mass spectra were recorded on a Waters Synapt G2-S HDMS mass
spectrometer (Waters, Manchester, UK) coupled to an Acquity UPLC core system (Waters,
Milford, MA, USA) consisting of a binary solvent manager, sample manager, and column
oven. For screening of extracts, aliquots (5 L) of all samples were injected into the UPLCTOF-
MS system using a BEH C18 column (150 mm 2.1 mm, 1.7 m, Waters), operated at
a flow rate of 0.4 mL/min at 45 C. Chromatographic separation was done with a gradient
of aqueous formic acid (0.1%, A) and acetonitrile (0.1%, B). The gradient started with a
mixture of 1% B and was kept isocratic for 1 min, then, increased to 100 % B within 9 min
and held at 100 % B for 1 additional min. Measurements were performed in high resolution
mode with negative electrospray ionization (ESI􀀀) and positive electrospray ionization
(ESI+). The ion source parameters were as follows: capillary voltage +2.5 kV (ESI+) or
􀀀2.5 kV (ESI􀀀), sampling cone 50V, source offset 30V, source temperature 150 C, desolvation
temperature 450 C, cone gas 2 L/h, nebulizer gas flow 6.5 bar, and desolvation gas 850 L/h.
Data processing was handled withMassLynx 4.1 (Waters) and Progenisis QI (Waters).
4.4. Cell Lines and Culture Conditions
Human embryonic kidney 293 (HEK293) cells, stably expressing hACE2, were generously
provided by Prof. Dr. Stefan Pöhlmann (Göttingen, Germany). The cells were
maintained in Dulbecco’s modified Eagle medium (DMEM), high glucose supplemented
with 10% fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin, and 50 g/mL
zeocin (Life Technologies, Darmstadt, Germany). Human A549-hACE2-TMPRSS2 cells,
generated from the human lung A549 cell line were purchased from InvivoGen SAS
(Toulouse Cedex 4, France) and maintained in DMEM, high glucose supplemented with
10% heat-inactivated FBS, 100 U/mL penicillin/streptomycin, 100 g/mL normocin,
0.5 g/mL puromycin, and 300 g/mL hygromycin. To subculture, all cells were first
rinsed with phosphate-buffered saline (PBS) then incubated with 0.25% trypsin-EDTA until
detachment. All cells were cultured at 37 C in a humidified incubator with 5% CO2/95%
air atmosphere.
4.5. Analysis of SARS-COV2 Spike–ACE2 Interaction Inhibition Using ELISA and
Flow Cytometry
A commercially available SARS-CoV-2 Inhibitor Screening Kit (Cat#: 16605302, Fisher
Scientific GmbH, Schwerte, Germany) was used for cell free detection of SARS-CoV-2 Spike–
ACE2 interaction inhibition. This colorimetric ELISA assay measures the binding between
immobilized SARS-CoV-2 spike protein RBD and biotinylated human ACE2 protein. The
colorimetric detection is done using streptavidin-HRP followed by TMB incubation. A
SARS-CoV-2 inhibitor (hACE2) was used as method verified reference. % inhibition was
calculated relative to the solvent control (distilled water, a.d.).
Cell surface expression of ACE2 was determined by using a human ACE2 PE-conjugated
antibody (Bio-Techne GmbH,Wiesbaden-Nordenstadt, Germany) and flow cytometric analysis.
For analysis of SARS-CoV-2 S1 Spike RBD–ACE2 binding, 2 105 cells (5 x 106 cells/mL)
were pre-treated with plant extracts for different time points. Then, 500 ng/mL SARSCoV-
2 Spike S1 (Trenzyme GmbH, Konstanz, Germany), spike S1 D614G, N50Y, or a mix
of K417N, E484K, and N501Y (Sino Biological Europe GmbH, Eschborn, Germany)—His
recombinant protein were added into each sample, and samples were further incubated
for 30–60 min. In another setting, cells were pre-treated with 500 ng/mL SARS-CoV-2
Spike–His recombinant protein for 30 min prior to incubation with the plant extract for
Pharmaceuticals 2021, 14, 1055 12 of 15
30–60 s at 4 C or 37 C. The samples were incubated in PBS buffer containing 5% FBS.
Cells were then washed one time with PBS buffer containing 1% FBS at 500g, 5 min before
staining with His-tag A647 mAb (Bio-Techne GmbH,Wiesbaden-Nordenstadt, Germany)
for 30 min at RT. Subsequently, cells were washed twice as described above. The cells were
analyzed by using a FACSCalibur (BD Biosciences, Heidelberg, Germany); 10,000 events
were acquired. The median fluorescence intensity (MFI) of each sample were determined
using FlowJo software (Ashland, OR, USA). % spike-binding inhibition was calculated
relative to the solvent control (distilled water, a.d.).
4.6. Human ACE2 Enzyme Activity and ProteinQquantification
A549-hACE2-TMPRSS2 (2 105) cells were seeded in a 24-well plate in high glucose
DMEM medium, containing 10% heat-inactivated FBS, at 37 C, 5 % CO2. Cells were then
treated with T. officinale extract with/without 500 ng/mL SARS-CoV-2 S1 Spike RBD protein
for 1–24 h. Afterwards, cells were washed with PBS and lysed. 25 g protein were used
for quantification of ACE2 protein (ACE2 ELISA kit), 5 g for ACE2 enzyme activity (ACE2
activity assay kit, Abcam, Cambridge, UK) according to the manufacturer’s instructions.
4.7. Infection of A549-hACE2-TMPRSS2 Cells Using SARS-CoV-2 Spike D614 and Delta
(B.1.617.2) Variant Pseudotyped Lentivirus
SARS-CoV-2 spike D614 and Delta variant pseudotyped lentivirus particles, produced
with SARS-CoV-2 spike and SARS-CoV-2 B.1.617.2 variant spike (Genbank Accession
#QHD43416.1) as the envelope glycoproteins instead of the commonly used VSV-G, were
purchased from BPS Bioscience, (Catalog#: 79942, and Catalog #78215, respectively, Biomol,
Hamburg, Germany). These pseudovirions also contain the firefly luciferase gene driven
by a CMV promoter. Thus, the spike-mediated cell entry can be quantified via luciferase
reporter activity. The bald lentiviral pseudovirion (BPS Bioscience #79943), where no
envelope glycoprotein is expressed, was used as a negative control. The firefly luciferase
lentivirus (Puromycin) from BPS Bioscience, (catalogue#: 79692-P) was used as a positive
control for transduction. These viruses constitutively express firefly luciferase under a
CMV promoter. Anti-hACE2 antibody (Biomol, NSJ-F49433) was used as reference for
transduction inhibition assay. Lung cells were seeded at 1 105 cells/cm2 in a 96-well
plate in DMEM containing 10% heat-inactivated FBS, 100 U/mL penicillin/streptomycin,
100 g/mL normocin, 0.5 g/mL puromycin, and 300 g/mL hygromycin and left
overnight. The medium was replaced by DMEM containing 10% heat-inactivated FBS and
cells were either pre-treated with T. officinale extract at different time points before adding
the lentivirus particles or vice versa. After 24 h of virus particle incubation, the medium was
removed by washing with PBS, fresh medium was added and cells were post-transduced
with/without the addition of T. officinale extract. Luminescence was detected within 1 h
using the one-step luciferase reagent from BPS following the manufacturer´s protocol in a
multiplate reader from Tecan (Tecan Group Ltd., Crailsheim, Germany); solvent control:
10% distilled water (a.d.).
4.8. Quantification of Cytokine Release by Multiplex Bead Technique
After 24 h SARS-CoV-2 spike pseudotyped lentivirus transduction and 60 h post-infection
of A549-hACE2-TMPRSS2 cells, supernatants were collected and stored at 􀀀80 C until
analysis for cytokine secretion using the humanMACSplex cytokine 12-kit (Miltenyi Biotec
GmbH, Bergisch Gladbach, Germany) according to manufacturer’s protocol. Cytokines below
the detection limit of 3.2 pg/mL were not included in the analysis.
4.9. Molecular Weight Fractionation from Plant Extracts
Extracts from dried plant leaves were prepared by adding bidistilled water (5 mL) to
plant material (500 mg each). The samples were incubated in the dark at room temperature
(RT) for 60 min, followed by centrifugation at 16,000g for 3 min. The supernatants were
collected and membrane-filtrated (0.45 m), resulting in the extracts. Aliquots were freeze
dried for 48 h to determine their yield by weight. The extracts were then further separated
Pharmaceuticals 2021, 14, 1055 13 of 15
in a high molecular weight (HMW) and low molecular weight (LMW) fraction, using
a centrifugation tube with an insert containing a molecular weight cut-off filter (5 kDa,
Sartorius Stedim Biotech, Goettingen, Germany). Each HMW fraction was purified by
flushing with 20 mL of water, yielding the HMW fractions, as well as LMW. The fractions
were freeze dried, their yield determined by weight and stored at 􀀀20 C until use.
4.10. Determination of Cell Viability Using Trypan Blue Staining
Cell viability was assessed using the trypan blue dye exclusion test as described
before [41]. Briefly, A549-hACE2-TMPRSS2 cells were cultured for 24 h, and then exposed
to extracts or the solvent control (a.d.) for 84 h.
4.11. Statistical Analysis
Results were analyzed using the GraphPad Prism 6.0 software (La Jolla, CA, USA).
Data are presented as means +SD. Statistical significance was determined by the oneway
ANOVA test followed by Bonferroni correction. p-values < 0.05 (*) were considered
statistically significant and <0.01 (**) were considered highly statistically significant.
5. Conclusions
Developed vaccine candidates all aim to generate antibody (and T cell) responses
against the spike protein and spike sequences from the early Wuhan strain served here as
a basis [42]. However, SARS-CoV-2 is steadily mutating during continuous transmission
among humans. Virus antigenic drift is clearly shown by the recent appearance of new
variants. It is evolving in such a way that it may eventually be able to evade our existing
therapeutic and prophylactic approaches aimed at the viral spike. A growing number of
studies already report reduced efficacy of neutralizing antibodies against the SARS-CoV-2
Delta variant compared with its original form [43,44]. Interestingly, T. officinale showed
only slightly less effectiveness against the Delta variant pseudotyped virus in vitro, and
the plant extract also demonstrated effective binding inhibition of four relevant spike
mutations to the human ACE2 receptor. This could be a major advantage in prevention of
SARS-CoV-2 infection. Thus, the results encourage more in-depth analysis of T. officinales’
effectiveness and now require further confirmatory clinical evidence.
Supplementary Materials: The following are available online at
.3390/ph14101055/s1, Table S1: Chemical analysis of T. officinale leaf extract using UPLC-TOF-MS
negative ion mode (ESI􀀀), Table S2: Chemical analysis of T. officinale leaf extract using UPLC-TOF-MS
positive ion mode (ESI+). Table S3: Chemical analysis of C. intybus leaf extract using UPLC-TOF-MS
negative ion mode (ESI􀀀), Table S4: Chemical analysis of C. intybus leaf extract using UPLC-TOF-MS
positive ion mode (ESI+).
Author Contributions: Conceptualization, E.L.; methodology, H.T.T.T.; M.G., E.L. and N.P.K.L.;
validation, E.L.; H.T.T.T.; M.G. and N.P.K.L.; formal analysis, E.L.; H.T.T.T. and M.G.; resources, E.L.
and C.D.; writing—original draft preparation, E.L.; writing—review and editing, E.L., H.T.T.T. and
M.G.; supervision and funding acquisition, E.L. and C.D. All authors have read and agreed to the
published version of the manuscript.
Funding: The article processing charge was funded by the Baden-Wuerttemberg Ministry of Science,
Research and Art and the University of Freiburg in the funding program Open Access Publishing.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained in the article and Supplementary Materials.
Conflicts of Interest: The authors declare no conflict of interest.
Pharmaceuticals 2021, 14, 1055 14 of 15
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