The Hydroalcoholic Extract of Uncaria tomentosa (Cat's Claw) Inhibits the Infection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) In Vitro

The coronavirus disease 2019 (COVID-19) has become a serious problem for public health since it was identified in the province of Wuhan (China) and spread around the world producing high mortality rates and economic losses. Nowadays, the WHO recognizes traditional, complementary, and alternative medicine for treating COVID-19 symptoms. Therefore, we investigated the antiviral potential of the hydroalcoholic extract of Uncaria tomentosa stem bark from Peru against SARS-CoV-2 in vitro. The antiviral activity of U. tomentosa against SARS-CoV-2 in vitro was assessed in Vero E6 cells using cytopathic effect (CPE) and plaque reduction assay. After 48 h of treatment, U. tomentosa showed an inhibition of 92.7% of SARS-CoV-2 at 25.0 μg/mL (p < 0.0001) by plaque reduction assay on Vero E6 cells. In addition, U. tomentosa induced a reduction of 98.6% (p=0.02) and 92.7% (p=0.03) in the CPE caused by SARS-CoV-2 on Vero E6 cells at 25 μg/mL and 12.5 μg/mL, respectively. The EC50 calculated for the U. tomentosa extract by plaque reduction assay was 6.6 μg/mL (4.89–8.85 μg/mL) for a selectivity index of 4.1. The EC50 calculated for the U. tomentosa extract by TCID50 assay was 2.57 μg/mL (1.05–3.75 μg/mL) for a selectivity index of 10.54. These results showed that U. tomentosa, known as cat's claw, has an antiviral effect against SARS-CoV-2, which was observed as a reduction in the viral titer and CPE after 48 h of treatment on Vero E6 cells. Therefore, we hypothesized that U. tomentosa stem bark could be promising in the development of new therapeutic strategies against SARS-CoV-2.


Introduction
e severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused serious public health problems since it was identified in Wuhan (China) in late 2019 [1]. e World Health Organization (WHO) declared coronavirus disease with COVID-19 whereas Venezuela and Uruguay were the ultimate nations to confirm their patient zero, considering the pandemic epicenter after Europe [4]. Even though some vaccines have already been approved only with phase 3 results, currently, there is no preventive treatment or antiviral drug available against SARS-CoV-2 [5].
Nowadays, the World Health Organization (WHO) recognizes that traditional, complementary, and alternative medicine has many benefits [6]. Several candidates with possible antiviral effects have been explored from medicinal plants in the preclinical phase. Uncaria tomentosa (Willd.) DC. (U. tomentosa) belongs to the Rubiaceae family, which is also known as cat's claw and contains more than 50 phytochemicals [7]. Oxindole alkaloids (pentacyclic oxindole alkaloids (POA) and tetracyclic oxindole alkaloids (TOA)) have been recognized as a fingerprint of this species in some pharmacopeias, and several pharmacological activities are linked to this kind of alkaloids [8,9]. It has been demonstrated that U. tomentosa exerts an antiviral effect on human monocytes infected with dengue virus 2 (DENV-2) [10] and herpes simplex virus type 1 (HSV-1) [11]. In our previous studies in silico, U. tomentosa's components inhibited the SARS-CoV-2 enzyme 3CLpro and disrupted the interface of the receptor-binding domain of angiotensin-converting enzyme 2 (RBD-ACE-2) as well as the SARS-CoV-2 spike glycoprotein [12,13]. Additionally, bioactivities such as antiinflammatory [14], antiplatelet [15], and immunomodulatory [16] were reported in the literature. Furthermore, other components isolated from the stem bark such as quinovic acids, polyphenols (flavonoids, proanthocyanidins, and tannins), triterpenes, glycosides, and saponins were identified by instrumental methods [9,[17][18][19][20]. e evaluation of natural compounds to inhibit SARS-CoV-2 in preclinical studies might lead to discovering new antiviral drugs and to a better understanding of the viral life cycle [21]. Several cell lines such as human airway epithelial cells, Vero E6 cells, Caco-2 cells, Calu-3 cells, HEK293Tcells, and Huh7 cells are considered the best models in vitro to determine the antiviral activity against SARS-CoV-2 [22]. Vero E6 cells highly express the ACE-2 receptor; they produce a high titer of viral particles and do not produce interferon [22]. erefore, in vitro test in this cell line constitutes the first step at the beginning of the antiviral studies.
Although the pathophysiology of COVID-19 is not completely understood, a severe inflammatory process has been associated with the severity and progression of the disease [23]. erefore, the immune activation so far described during the course of the infection as well as the pulmonary injury could be ameliorated by U. tomentosa linked to its traditional use as an anti-inflammatory in the folk medicine from South America for years [24].
Based on its antiviral activity on other ARN viruses and our in silico findings against SARS-CoV-2, we assayed the hydroalcoholic extract of U. tomentosa stem bark from Peru as a potential antiviral agent in vitro against this severe acute respiratory syndrome coronavirus 2.

Obtaining Extract from Plant Material.
One hundred grams of the raw plant material (stem bark) of U. tomentosa was powdered and extracted with 700 ml of 70% ethanol at room temperature for 7 days. en, the extract was evaporated by using rotary evaporation to obtain a desiccated extract, which was stored at 4°C until further use.

Identification of the U. tomentosa Stem Bark Constituents by LC/MS (UHPLC-ESI + -HRMS-Orbitrap).
e identification of the main phytochemicals present within the hydroalcoholic extract of U. tomentosa was carried out on an LC Dionex UltiMate 3000 ( ermo Scientific, Germering, Germany) equipped with a degassing unit, a gradient binary pump, an autosampler with 120-vial well-plate trays, and a thermostatically controlled column compartment. e autosampler was held at 10°C, and the column compartment was maintained at 40°C. Chromatographic separation was performed on a Hypersil GOLD aQ column ( ermo Scientific, Sunnyvale, CA, USA; 100 mm × 2.1 mm id, 1.9 μm particle size) with an LC guard-column Accucore aQ Defender cartridge ( ermo Scientific, San Diego, CA, USA; 10 × 2.1 mm id, 2.6 μm particle size). e flow rate of the mobile phase containing ammonium formate (FA)/water (A) and FA/acetonitrile (B) was 300 μL/min. e initial gradient condition was 100% A, changed linearly to 100% B in 8 min, maintained for 4 min, returned to 100% A in 1 min, and maintained for 3 min. e injection volume was 1 μL. e LC was connected to an Exactive Plus Orbitrap mass spectrometer ( ermo Scientific, Bremen, Germany) with a heated electrospray ionization (HESI-II) source operated in the positive ion mode. e Vspray was evaluated at 1.5, 2.5, 3.5, and 4.5 kV. e nebulizer temperature was set at 350°C; the capillary temperature was 320°C; sheath gas and auxiliary gas (N 2 ) were adjusted to 40 and 10 arbitrary units, respectively. Nitrogen (>99%) was obtained from a generator (NM32LA, Peak Scientific, Scotland, UK). During the full scan MS, the Orbitrap-MS mass resolution was set at 70000 (full-width-at-half-maximum, at m/z 200, RFWHM) with an automatic gain control (AGC) target of 3 × 106, a C-trap maximum injection time of 200 ms, and a scan range of m/z of 100-1000. e ions injected to the HCD cell via the C-trap were fragmented with stepped normalized collision energies of 20, 30, 40, and 50 eV. e mass spectra were recorded in the AIF (all-ion fragmentation) mode for each collision energy at an RFWHM of 35000, an AGC target of 3 × 106, a

Cell Viability
Assays. e viability of Vero E6 cells in the presence of the U. tomentosa extract was evaluated using an MTT (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Briefly, Vero E6 cells were seeded at a cell density of 1.0 × 10 4 cells/well in 96-well plates and incubated for 24 h at 37°C in a humidified 5% CO 2 atmosphere. en, 100 μL of serial dilutions (1 : 2) of the U. tomentosa extract ranging from 3.1 to 50 μg/mL was added to each well and incubated for 48 h at 37°C with 5% CO 2 . After incubation, the supernatants were removed, cells were washed twice with phosphate-buffered saline (PBS) (Lonza, Rockland, ME, USA), and 30 μL of the MTT reagent (Sigma-Aldrich) (2 mg/ mL) was added. e plates were incubated for 2 hours at 37°C with 5% CO 2 , protected from light. en, formazan crystals were dissolved by adding 100 μL of pure DMSO to each well. Plates were read using a Multiskan GO spectrophotometer ( ermo) at 570 nm. e average absorbance of cells without treatment was considered as 100% of viability. Based on this control, the cell viability of each treated well was calculated. e treatment concentration with 50% cytotoxicity (the 50% cytotoxic concentration, CC50) was obtained by performing nonlinear regression followed by the construction of a concentration-response curve (GraphPad Prism). For the MTT assay, 2 independent experiments with four replicates of each experiment were performed (n � 8).

Antiviral Assay.
e antiviral activity of the U. tomentosa extract against SARS-CoV-2 was evaluated with a pre-post strategy where the treatment was added before and after the infection. Briefly, Vero E6 cells were seeded at a density of 1.0 × 10 4 cells/well in 96-well plates and incubated for 24 h at 37°C with 5% CO 2 . After incubation, 50 μL of double dilutions of cat's claw (3.1-25 μg/mL) was added to the cell monolayers for 1 h at 37°C with 5% CO 2 . en, the treatment was removed, and cells were infected with SARS-CoV-2 stock at a multiplicity of infection (MOI) of 0.01 in 50 μL of DMEM supplemented with 2% FBS. e inoculum was removed 1 hour postinfection (h.p.i), replaced by 170 μL of cat's claw dilutions, and incubated for 48 h at 37°C with 5% CO 2 . en, cell culture supernatants were harvested and stored at −80°C for virus titration by plaque assay and TCID50 assay. e supernatant of infected cells without treatment was used as infection control. Chloroquine (CQ) at 50 μM was used as a positive control for antiviral activity; 2 independent experiments with 3 replicates of each experiment were performed (n � 6).

Plaque Assay for SARS-CoV-2 Quantification.
e capacity of the U. tomentosa extract to decrease the PFU/mL of SARS-CoV-2 was evaluated by plaque assay on Vero E6 cells. Briefly, 1.0 × 10 5 Vero E6 cells per well were seeded in 24-well plates for 24 h at 37°C with 5% CO 2 . Tenfold serial dilutions of the supernatants obtained from the antiviral assay (200 μL per well) were added by duplicate on cell monolayers. After incubation for 1 h at 37°C with 5% CO 2 , the viral inoculum was removed and 1 mL of semisolid medium (1.5% carboxymethyl cellulose in DMEM 1X with 2% FBS and 1% penicillin-streptomycin) was added to each well. Cells were incubated for 5 days at 37°C with 5% CO 2 .
en, cells were washed twice with PBS. en, cells were fixed and stained with 500 μL of 4% formaldehyde/1% crystal violet solution for 30 minutes and washed with PBS. Plaques obtained from each condition were counted. e reduction in the viral titer after treatment with each concentration of the U. tomentosa extract compared to the infection control is expressed as inhibition percentage. Two independent experiments with two replicates of each experiment were performed (n � 4).

TCID50 Assay for SARS-CoV-2 Quantification.
e capacity of the U. tomentosa extract to diminish the CPE caused by SARS-CoV-2 on Vero E6 cells was evaluated by TCID50 assay. Briefly, 1.2 × 10 4 Vero E6 cells per well were seeded in 96-well plates for 24 h at 37°C with 5% CO 2 . Tenfold serial dilutions of the supernatants obtained from the antiviral assay (50 μL per well) were added by quadruplicate on cell monolayers. After 1 h incubation, at 37°C with 5% CO 2 , the viral inoculum was removed and replaced by 170 μL of DMEM supplemented with 2% FBS. Cells were incubated for 5 days at 37°C with 5% CO 2 . en, cells were washed twice with PBS and then fixed and stained with 100 μL/well of 4% formaldehyde/1% crystal violet solution for 30 minutes. Cell monolayers were washed with PBS. e number of wells positive for CPE was determined for each dilution (CPE is considered positive when more than 30% of cell monolayer is compromised). e viral titer of TCID50/mL was calculated based on the Spearman-Käerber method. e reduction of viral titer after treatment with each concentration of the U. tomentosa extract compared to infection control is expressed as Evidence-Based Complementary and Alternative Medicine inhibition percentage. A control of cells without infection and treatment was included. Two independent experiments with two replicates of each experiment were performed (n � 4).

Statistical Analysis.
e median inhibitory concentration (IC50) values represent the concentration of the U. tomentosa extract that reduces virus particle production by 50%. e CC50 values represent the cat's claw solution concentration that causes 50% cytotoxicity. e corresponding dose-response curves were fitted by nonlinear regression analysis using a sigmoidal model. e calculated selectivity index (SI) represents the ratio of CC50 to IC50. All data were analyzed with GraphPad Prism (La Jolla, CA, USA), and data are presented as mean ± SEM. Statistical differences were evaluated via Student's t-test or Mann-Whitney U test; a value of p ≤ 0.05 was considered significant, with * p ≤ 0.05, * * p ≤ 0.01, and * * * p ≤ 0.001.

Identification of Components in the Hydroalcoholic Extract of U. tomentosa by LC/MS (UHPLC-ESI + -HRMS-Orbitrap)
. Different constituents in the U. tomentosa stem bark such as spirooxindole alkaloids, indole glycoside alkaloids, quinovic acid glycosides, and proanthocyanidins were identified by LC-MS analysis (Table 1 and Supplementary materialsS1-S6).
e LC-MS data provided information on spirooxindole alkaloids as a broad peak that appeared at a retention time (t R ) of 4.82 min and showed an (M + H) + ion at m/z 369.18018 that are characteristics for speciophylline, isopteropodine, isomitraphylline, uncarine F, mitraphylline, and pteropodine. Furthermore, two peaks at 4.99 and 5.18 min, respectively, showed the [M+H] + ion at m/z 385.21127 that were identified as those isomeric spirooxindole-related alkaloids rhynchophylline and isorynchophylline. On the other side, a molecular ion peak (M + H) + of 547.22992 m/z, which eluted at 4.03 min, provided the identity of the indole glycoside alkaloid 3dihydrocadambine. As expected, LC/MS phytochemical analysis showed that the hydroalcoholic extract of U. tomentosa was comprised predominantly of five proanthocyanidins (PAs), including proanthocyanidin C1, epiafzelechin-4β-8, proanthocyanidin B2, epicatechin, and chlorogenic acid, which eluted at 3.76-4.25 min. Finally, LC-MS data along with ESI mass spectra gave characteristic protonated quasimolecular ions of isomeric quinovic acid glycosides ([M + H] + ion at m/z 957.50458). In sum, LC/MS allowed the identification of known components in the hydroalcoholic extract of U. tomentosa used, such as alkaloids, quinovic acid glycosides, and proanthocyanidins (PAs), which play important roles in the biological activities of this medicinal herb and are considered as a fingerprint for quality control that ensures fitness for therapeutic uses.

e Cell Viability Assay on Vero E6 Cells in the Presence of the U. tomentosa Extract.
e viability of Vero E6 cells in the presence of U. tomentosa was higher than 90.0% at concentrations of 25.0 μg/mL or lower, after 48 h of incubation ( Figure 1). Cell viability at 50.0 μg/mL was 17.3%; for this reason, this concentration was not included in the antiviral assay. e CC50 calculated for U. tomentosa was 27.1 μg/mL. Chloroquine at 50 μM (positive control of inhibition) did not affect the viability of Vero E6 cells (Figure 1).

Discussion
In South America, the second wave of novel coronaviruses might be more aggressive, increasing the mortality rate and new cases [26]. Medical trials are underway to determine the efficacy of several vaccines against SARS-CoV-2 [27]. Otherwise, herbal medicines could become a promising option to tackle the ongoing pandemic caused by COVID-19 [28]. Some plant extracts and phytochemicals were modeled over numerous targets of SARS-CoV-2 by using in silico studies, which is the first step in the discovery of new drugs [29]. In China, the use of herbal formulas has been included in the protocol of primary attention in COVID-19 and medical trials were carried out, and promising results to ameliorate the symptoms were reported [30].
Our previous study of U. tomentosa (cat's claw) on this novel coronavirus using in silico analysis showed that two possible mechanisms could be involved in the in vitro antiviral activity against SARS-CoV-2. ese findings revealed that 3CLpro, an essential enzyme for viral replication [31], showed key molecular interactions with speciophylline, cadambine, and proanthocyanidin B2, with high binding affinities ranging from −8.1 to −9.2 kcal/mol. [12]. On the other hand, phytochemicals of U. tomentosa such as proanthocyanidin C1, QAG-2, uncarine F, 3-  [13]. Since Vero E6 cells are commonly used to replicate SARS-CoV-2 due to the high expression level of the ACE-2 receptor and lack the ability to produce interferon [32], phytochemicals are the appropriate substrate to explore the antiviral activity of phytochemicals targeting the receptor binding as well as the SARS-CoV-2 main protease, which is a high-profile antiviral drug target, and several compounds have been discovered as main protease inhibitors [33,34]. Mechanisms of the antiviral activity of the hydroalcoholic extract of U. tomentosa, on other viruses like Dengue (DEN-2), have been elucidated; alkaloids (pentacyclic alkaloids) from U. tomentosa induced apoptosis of infected cells and reduced inflammatory mediators such as TNF-α and IFN-α with similar effects to dexamethasone [10]. e quinovic acids (33.1-60 μg/mL) inhibited the vesicular stomatitis virus (VSV) [35], and the total extract at concentrations less than 15.75 μg/mL inhibited the herpes simplex virus (HSV-1) replication when added to Vero cells at the same time compared to the virus [11].
Here, we demonstrated that U. tomentosa also has an antiviral activity in vitro against the SARS-CoV-2 by inhibiting the release of infectious particles and reducing the cytopathic effect on Vero E6 cells. e EC50 was calculated at 6.6 μg/mL (95% CI: 4.89-8.85 μg/mL) by plaque assay and at 2.57 μg/mL (95% CI: 1.05-3.75 μg/mL) by TCID50 assay, whilst the CC50 was 27.1 μg/mL. In other medicinal plants assayed against SARS-CoV-2, similar antiviral activity was shown; in particular, Echinaforce ® (an Echinacea purpurea preparation) exhibited an antiviral activity at 50 μg/mL [36]. Liu Shen capsule, a traditional Chinese medicine, inhibited the SARS-CoV-2 replication with an EC50 value of 0.6024 μg/mL and CC50 of 4.930 μg/mL [37]. Likewise, phillyrin (KD-1), a representative constituent of Forsythia  suspensa ( unb.), presented an EC50 at 63.90 μg/mL and CC50 of 1959 μg/mL [38]. Sulfated polysaccharides named RPI-27 and heparin inhibited SARS-CoV-2 in vitro with an EC50 of 8.3 ± 4.6 μg/mL and 36 ± 14 μg/mL, respectively [39]. In our study, selectivity indices of 4.1 and 10.5 were obtained by plaque assay and TCID50, respectively. According to a previous report [40], these results were classified as low selectivity (SI ≥ 2.0 and < 5) and high selectivity (SI ≥ 10), respectively. In spite of SI having a low value, theoretically having a higher value would be more effective and safer during in vivo treatment for a given viral infection. However, there is no evidence of severe toxicity of U. tomentosa, and traditionally, its popular use in the form of maceration or decoction is safe [41].
e lower concentration used of the U. tomentosa extract (3.1 μg/mL) caused a significant increase in the number of infectious viral particles compared to the infection control ( Figure 2). is result could be due to compounds present in the extract that at this concentration promote an increase in cell proliferation or regulation of metabolic pathways that regulate the expression of viral receptors or synthesis of proteins necessary for the viral replicative cycle [42,43]. ese findings demonstrate the importance of evaluating and identifying the compounds present in U. tomentosa with antiviral effect against SARS-CoV-2 and selecting the proper concentration for use.
ere is enough evidence that U. tomentosa could ameliorate a wide array of symptoms associated with COVID-19, like the severe inflammation characterized by a cytokine storm [24] causing endothelial dysfunction. According to the antiviral activity of U. tomentosa against SARS-CoV-2, several biochemical mechanisms could be involved in each phase of the viral life cycle. As previously reported in our in silico studies, U. tomentosa could interfere with viral entrance into host cells [12], affecting viral replication [13]. Furthermore, ACE-2 receptors, which are expressed in Vero E6 cells, could also be blocked by the phytochemicals of U. tomentosa during the entrance of SARS-CoV-2 into the host cells, and the aforementioned studies backed up our hypothesis [13].
Besides, it might control the hyperinflammation, via inhibition of IL-1α, IL-1β, IL-17, and TNF-α [44], reduce Evidence-Based Complementary and Alternative Medicine oxidative stress [45], and protect the endothelial barrier, via inhibition of IL-8, which is linked to the induction of permeability [46]. It also has antithrombotic potential via antiplatelet mechanism and by thrombin inhibition [15]. Furthermore, U. tomentosa modulates the immune system by extending lymphocyte survival via an antiapoptotic mechanism [47]. It is known that the 3α protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in Vero E6 cells [48]; therefore, the phytochemicals found in the hydroalcoholic extract could inhibit this process and protect against the inflammatory cascade. Interestingly, U. tomentosa bark extract reduced the lung inflammation produced by ozone in mice [49]. Based on our results, U. tomentosa is a promising medicinal herb to combat COVID-19, but it is necessary to continue with animal models followed by clinical trials to validate our results in the context of COVID-19 patients.
is study is the first approach to evaluate the potential use of U. tomentosa against SARS-CoV-2; we have to explore specific mechanisms of inhibition and propose the main molecules involved with the antiviral activity. As shown in our phytochemical analysis, the presence of chemical groups determined by LC/MS (UHPLC-ESI + -HRMS-Orbitrap), such as spirooxindole alkaloids, indole glycoside alkaloids, quinovic acid glycosides, and proanthocyanidins, suggests that they could be responsible for the described activity. Here, the mechanisms discussed about the hydroalcoholic extract of U. tomentosa are only inferred under the mechanisms evaluated in other RNA viruses reported in the literature and also our previous in silico studies on SARS-CoV-2.
In regard to the antiviral activity of U. tomentosa, the EC50 was calculated at 6.6 μg/mL, which is an indicator of a promising activity as an extract, but it cannot be taken as a reference value to reach plasma concentration because U. tomentosa extract presented several phytochemicals, which were not quantified and individually tested. Since cat's claw has been used in clinic for other diseases, there are no clinical studies carried out and reported pharmacokinetic data. However, in mice, the administration of 5 mg/Kg per oral of six Uncaria alkaloids presented a bioavailability ranging between 27.3% and 68.9% and with a maximum plasma concentration (C max ) between 305.3 ± 68.8 ng/mL and 524.5 ± 124.5 ng/mL [50]. Additionally, the recommended dose of U. tomentosa is one gram given two to three times daily [51]. A standardized extract consisting of less than 0.5% oxindole alkaloids and 8% to 10% carboxy alkyl esters has been used at doses of 250 to 300 mg in clinical studies [52]. In humans, no toxic symptoms were reported with a usual administration of 350 mg/day for 6 weeks [53,54] and 300 mg dry extract daily for 12 weeks [55]. Traditional uses such as tinctures, decoctions, capsules, extracts, and teas are prepared and, in a decoction, up to 20 g of raw bark per liter of water has been used; although this information is based on traditional practices, this equates to 4 mg oxindole alkaloids [56]. us, we hypothesized that the antiviral activity on SARS-CoV-2 is attributed to the whole extract synergized by all its phytochemicals acting by different mechanisms discussed above.

Conclusion
U. tomentosa has been widely used as an anti-inflammatory and immunomodulatory agent. Previous studies have shown that U. tomentosa has a broad spectrum of effects on several RNA viruses. In this study, we demonstrated that hydroalcoholic extract of U. tomentosa stem bark inhibited the release of SARS-CoV-2 infectious particles and reduced the cytopathic effect caused by the virus on Vero E6 cell line, underlying the importance of continuing this investigation with specific in vitro assays, followed by studies in animal models, and finally validating its use in clinical trials. Our investigation shows for the first time the antiviral effect of U. tomentosa on this novel coronavirus (SARS-CoV-2).

Data Availability
All data used to support the findings of this study can be made available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no known conflicts of interest or personal relationships that could have appeared to influence the work reported in this paper.