European Black Elderberry Fruit Extract Inhibits Replication of SARS-CoV-2 In Vitro

: Coronavirus disease-19 (COVID-19) is still affecting the lives of people round the globe and remains a major public health threat. The emergence of new variants more efﬁciently transmitted, more virulent and more capable of escaping naturally acquired and vaccine-induced immunity creates a long-term negative outlook for the management of the pandemic. The development of effective and viable prevention and treatment options to reduce viral transmission is of the utmost importance. The fruits of the European black elderberry and extracts thereof have been traditionally used to treat viral infections such as coughs, cold and ﬂu. Speciﬁcally, its efﬁcacy against the Inﬂuenza A virus has been shown in vitro as well as in human clinical trials. In the current project, we investigated the antiviral activity of a black elderberry extract, mainly containing anthocyanins and phenolic compounds, against SARS-CoV-2 and its variants of concern and explored the possible mode of action by performing time of addition experiments. The results revealed that the extract displayed a strong anti-SARS-CoV-2 activity against the Wuhan type as well as the variants of concern Alpha, Beta, Gamma, Delta and Omicron with a comparable antiviral activity. Based on cytotoxicity data, a 2-log theoretical therapeutic window was established. The data accumulated so far suggest that the viral replication cycle is inhibited at later stages, inasmuch as the replication process was affected after virus entry. Therefore, it would be legitimate to assume that black elderberry extract might have the potential to be an effective treatment option for SARS-CoV-2 infections.


Introduction
The Coronavirus disease 2019 , caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is responsible for short-and long-term complications, e.g., the need for respiratory support or persistent cardiovascular complications. To date, the pandemic has resulted in around 647 million global cases and over 6.6 million deaths [1]. An ongoing major problem is the continuous emergence and spread of SARS-CoV-2 variants determined as "Variants of Concern" (VoCs) by the WHO. These variants are able to evade vaccine-or infection-induced antiviral immune response [2,3].

Inhibitors
Liquid and dry European black elderberry (Sambucus nigra L.) extract (brand name ElderCraft ® ) was provided by IPRONA AG/SPA, Italy, and was designated as EC 3.2 and EC 14, respectively. EC 3.2 is a water extract in liquid format, standardized to a minimum of 3.2% anthocyanin while EC 14 is a spray-dried water extract standardized to minimum of 14% anthocyanin. To compare the activity of the EC 3.2 and EC 14, the EC 14 powder was diluted in water and set to the same anthocyanin content as EC 3.2.

Viruses
The "Wuhan type" virus SARS-CoV-2 PR-1 , isolated from a 61-year-old patient, was amplified in Vero B4 cells as described in [29]. The virus strains SARS-CoV-2 Alpha, Beta, Gamma and Delta were obtained from Michael Schindler (University Hospital, Tübingen, Germany). The SARS-CoV-2 Alpha variant (210416_UKv) was generated as described in [31]. SARS-CoV-2 Beta was generated as described in [42]. The Gamma variant (210504_BRv) and the Delta variant (210601_INv) were isolated from throat swabs collected in May 2021 at the Institute for Medical Virology and Epidemiology of Viral Diseases, University Hospital, Tübingen, from PCR-positive patients and generated as described in [32]. The clinical SARS-CoV-2 Omicron variant was generated as described in [43]. SARS-CoV-2 Viral titers of each variant were determined by an endpoint titration assay. For the generation of new virus stock, virus-containing cell culture supernatant was harvested 72 h post-infection (hpi) and passed through a 0.45 µm pore-size filter. All virus stocks were stored at −80 • C until further usage.
For preincubation experiments, cells were preincubated either with or without inhibitors for 2 h at 37 • C. After 1 h of infection, the inoculum was removed and cells were incubated without treatment for another 3 days. At 72 hpi, supernatants were harvested and analyzed as described above. For the co-treatment experiments, inhibitors were present during the 1 h of infection, following removement of the inoculum and incubation of the cells for another 3 days without treatment. At 72 hpi, supernatants were harvested and analyzed as described above.

Assessment of Cell Viability
Viability of infected and treated cells was assessed by the water-soluble tetrazolium salt (WST)-1 assay (Cat.: 5015944001, Roche, Penzberg, Germany) according to the manufacturer's instructions.

Determination of the Amount of Viral RNA Copies from Released Viruses by qRT-PCR
The amount of viral RNA copies in the virus-containing samples was quantified by real-time PCR Luna Universal Probe One-Step RT-PCR Kit from New England Biolabs (Cat: E3006L, Ipswich, MA, USA). This kit allows reverse transcription, cDNA synthesis and PCR amplification in a single step. Samples were analyzed by 7500 software v2.3 (Applied Biosystems, Waltham, MA, USA). As described previously in [45], PCR primers were designed and used. Thereby, the polynucleotide sequence contains parts of the SARS-CoV-2 Envelope (E) and RNA-dependent RNA-polymerase (RdRp) genes and was used as

Software and Statistics
Microsoft Word and Excel were used. GraphPad Prism 9.0 was used for statistical analyses and to generate graphs. Figures were generated with CorelDrawX7. The 7500 software v2.3 was used to evaluate the results obtained by qRT-PCR. The HPLC analysis data were captured and evaluated using Chromeleon 7 (Thermo Fisher Scientific Inc., Waltham, MA, USA).

High Performance Liquid Chromatography (HPLC) Analysis of Elderberry Extracts
The HPLC analysis was performed as published by IFU No. 71 as established by International Fruit and Vegetable Juice Association with modifications [46]. The analysis was performed on an UltiMate 3000 HPLC device coupled with a diode array detector DAD-3000 (Thermo Fisher Scientific Inc., Waltham, MA, USA). For phenolic compounds, a wavelength of 350 nm and for anthocyanins a wavelength of 520 nm was used. Total anthocyanin content was calculated using cyanidin-chloride as standard with conversion factor of 1.393 to convert the result from cyanidin-chloride into cyanidin-3-glucoside.

European Black Elderberry Extract Compositional Analysis
Initially, the main components of European black elderberry extract (EC 3.2) and European black elderberry extract dried power (EC 14; resolved in water), both used in this study, were determined. Therefore, the anthocyanin and polyphenol content of the extracts were analyzed using High Performance Liquid Chromatography (HPLC). The main anthocyanins were identified in both extracts as cyanidin-3-sambubioside-5-glucoside, cyanidin-3-sambubioside and cyanidin-3-glucoside (chromatogram of liquid extract EC 3.2 shown in Figure 1).
Initially, the main components of European black elderberry extract (EC 3.2) and European black elderberry extract dried power (EC 14; resolved in water), both used in this study, were determined. Therefore, the anthocyanin and polyphenol content of the extracts were analyzed using High Performance Liquid Chromatography (HPLC). The main anthocyanins were identified in both extracts as cyanidin-3-sambubioside-5glucoside, cyanidin-3-sambubioside and cyanidin-3-glucoside (chromatogram of liquid extract EC 3.2 shown in Figure 1).
The analysis of the content of phenolic compounds revealed that, among others, mainly rutin, chlorogenic acid and isoquercitirin were present in both black elderberry extracts (chromatogram of liquid extract EC 3.2 shown in Figure 2). Table 1 summarizes the total amounts of the identified compounds of EC 3.2 and EC 14.  The analysis of the content of phenolic compounds revealed that, among others, mainly rutin, chlorogenic acid and isoquercitirin were present in both black elderberry extracts (chromatogram of liquid extract EC 3.2 shown in Figure 2).

European Black Elderberry Extract Exhibits Efficient Antiviral Activity against SARS-CoV-2 in Different Cell Lines
In order to determine whether liquid European black elderberry extract (EC 3.2) exhibits antiviral activity against SARS-CoV-2, human Caco-2 colon carcinoma-derived

European Black Elderberry Extract Exhibits Efficient Antiviral Activity against SARS-CoV-2 in Different Cell Lines
In order to determine whether liquid European black elderberry extract (EC 3.2) exhibits antiviral activity against SARS-CoV-2, human Caco-2 colon carcinoma-derived epithelial cells [47] and Calu-3 human lung cells, the most extensively studied surrogate lung cell infection model that expresses ACE2 and TMPRSS2 endogenously [48], were infected with SARS-CoV-2 Wuhan type ( Figure 3).    Wuhan type in Calu-3 and Caco-2 cells. Calu-3 (A) and Caco-2 cells (B) were infected with the clinical isolate SARS-CoV-2PR-1 at a MOI of 2 × 10 −2 . One hour after infection and removal of input virus, cells were treated with indicated dilution steps of EC 3.2. A total of 1 μM Remdesivir (RDV) was included as a positive control. Cell culture supernatants were harvested at 3 dpi. The virions were purified and analyzed by qRT-PCR. Bars show mean values of three independent experiments ± standard deviation. Statistical analysis was performed using a multiple comparison Kruskal-Wallis test (Anova) followed by Dunn's post hoc test (*** p < 0.001; **** p < 0.0001 versus the untreated control).
To control for the potential unspecific effects of EC 3.2 treatment on cell viability, water-soluble tetrazolium salt (WST)-1 assays were performed in uninfected Caco-2 or Calu-3 cells under otherwise identical conditions as for the virus infection experiments. The results, summarized in Figure 4, demonstrate that treatment with EC 3.2 at dilutions up to 1:100, and thus in a concentration range where the replication of SARS-CoV-2 was completely blocked, had no impact on cell viability in both investigated cell types ( Figure  4). The TD50 values for EC 3.   Next, it was determined if European black elderberry dried power (EC 14) resolved in water also showed a similar antiviral activity to EC 3.2. As this dried powder is commonly used in food supplements, it is of interest whether the drying process has any negative influence on its antiviral activity. Therefore, Calu-3 cells were infected with the SARS-CoV-2 Wuhan type as described before and qRT-PCR analysis was performed ( Figure 5). Similarly to EC 3.2, EC 14 also inhibits the replication of SARS-CoV-2 in a dose-dependent manner with a comparable IC 50 value of~1:800 ( Figure 5). Nutraceuticals 2023, 3, FOR PEER REVIEW 8 Next, it was determined if European black elderberry dried power (EC 14) resolved in water also showed a similar antiviral activity to EC 3.2. As this dried powder is commonly used in food supplements, it is of interest whether the drying process has any negative influence on its antiviral activity. Therefore, Calu-3 cells were infected with the SARS-CoV-2 Wuhan type as described before and qRT-PCR analysis was performed ( Figure 5). Similarly to EC 3.2, EC 14 also inhibits the replication of SARS-CoV-2 in a dose-dependent manner with a comparable IC50 value of ~1:800 ( Figure 5).

Figure 5.
European black elderberry dried powder (EC 14) resolved in water inhibits replication of SARS-CoV-2 Wuhan type. Calu-3 cells were infected with the clinical isolate SARS-CoV-2PR-1 at a MOI of 2 × 10 −2 . One hour after infection and removal of input virus, cells were treated with indicated dilution steps of EC 14. A total of 1 μM RDV was included as a positive control. Cell culture supernatants were harvested at 3 dpi. The virions were purified and analyzed by qRT-PCR. Bars show mean values of three independent experiments ± standard deviation. Statistical analysis was performed using a multiple comparison Kruskal-Wallis test (Anova) followed by Dunn's post hoc test (* p < 0.05; ** p < 0.01; **** p < 0.0001 versus the untreated control).

EC 3.2 Exhibits Comparable Antiviral Activity against All SARS-CoV-2 Variants of Concern
In order to determine if EC 3.2 exhibits a comparable, broad antiviral activity against all described VoCs of SARS-CoV-2, Calu-3 human lung cells were infected with the VoCs Alpha, Beta, Gamma, Delta and Omicron ( Figure 6).

Figure 5.
European black elderberry dried powder (EC 14) resolved in water inhibits replication of SARS-CoV-2 Wuhan type. Calu-3 cells were infected with the clinical isolate SARS-CoV-2 PR-1 at a MOI of 2 × 10 −2 . One hour after infection and removal of input virus, cells were treated with indicated dilution steps of EC 14. A total of 1 µM RDV was included as a positive control. Cell culture supernatants were harvested at 3 dpi. The virions were purified and analyzed by qRT-PCR. Bars show mean values of three independent experiments ± standard deviation. Statistical analysis was performed using a multiple comparison Kruskal-Wallis test (Anova) followed by Dunn's post hoc test (* p < 0.05; ** p < 0.01; **** p < 0.0001 versus the untreated control).

EC 3.2 Exhibits Comparable Antiviral Activity against All SARS-CoV-2 Variants of Concern
In order to determine if EC 3.2 exhibits a comparable, broad antiviral activity against all described VoCs of SARS-CoV-2, Calu-3 human lung cells were infected with the VoCs Alpha, Beta, Gamma, Delta and Omicron ( Figure 6). Treatment with EC 3.2 led to a dose-dependent reduction of virus replication that occurred with comparable efficacy for all VoCs (Figure 6). The IC50 values varied between ~ 1:800 for the Wuhan type ( Figure 3) and ~1:200 for Alpha ( Figure 6). However, the IC90 was ~ 1:100 for all VoCs and thus in a similar range ( Figure 6). One hour post-infection, different dilutions of EC 3.2 were added to the cells. Three dpi cell culture supernatants were harvested and virus production was analyzed by qRT-PCR ( Figure 6). Treatment with EC 3.2 led to a dose-dependent reduction of virus replication that occurred with comparable efficacy for all VoCs (Figure 6). The IC 50 values varied betweeñ 1:800 for the Wuhan type ( Figure 3) and~1:200 for Alpha ( Figure 6). However, the IC 90 was~1:100 for all VoCs and thus in a similar range ( Figure 6). The IC 50 and IC 90 values for EC 3.2 following infection with the Wuhan type and respective VoCs are summarized in Table 2. Next, we calculated the IC 50 values for the main compounds of EC 3.2, based on the results of the HPLC analysis (Figures 1 and 2 and Table 1). The results are summarized in Table 3.

Treatment with EC 3.2 Does Not Affect Early Steps of the Replication of SARS-CoV-2
Next, it was analyzed if EC 3.2 blocks the replication of SARS-CoV-2 by interfering with the early steps of the viral replication cycle. Therefore, time of addition (TOA) experiments were performed. First, Calu-3 cells were preincubated with different concentrations of EC 3.2 for 2 h at 37 • C (see treatment scheme Figure 7A). Following two washing steps with PBS to remove EC 3.2, cells were infected with SARS-CoV-2 Wuhan type and cell culture supernatants were harvested after 3 days and analyzed by qRT-PCR.
The data revealed that the preincubation of the cells without further treatment during the infection and post-infection period does not interfere with SARS-CoV-2 replication ( Figure 7A). Remdesivir, an inhibitor of RNA metabolism [49], was used as a control and also exhibit no antiviral activity in this experimental setup ( Figure 7A). Nutraceuticals 2023, 3, FOR PEER REVIEW 11 was ~1:800; thus, the additional co-treatment with EC 3.2 has no further influence on the replication capacity of SARS-CoV-2 ( Figure 7C). In summary, the TOA experiments revealed that EC 3.2 does not influence early steps in the replication cycle of SARS-CoV-2 but rather exerts its antiviral activity effect during the later steps of viral replication.

Discussion
COVID-19, caused by SARS-CoV-2, is still responsible for the ongoing worldwide pandemic that led to a global health and socioeconomic crisis. Similar to the occurrence of SARS-CoV, Middle East respiratory syndrome-related coronavirus (MERS-CoV) and SARS-CoV-2, it can be expected that in the future new coronaviruses will emerge by Next, EC 3.2 was added to Calu-3 cells only during the time of infection with SARS-CoV-2 Wuhan type for 1 h and without applying further treatment to the cells afterwards (treatment scheme Figure 7B). After 1 h, the infectious supernatants were removed and the cells were incubated without further treatment for three days, followed by q RT-PCR analysis of the supernatants. As described for the pre-treatment, the co-treatment with EC 3.2 also has no influence on the replication capacity of SARS-CoV-2 ( Figure 7B).
Within a final setup, Calu-3 cells were treated during the 1 h infection period with SARS-CoV-2 Wuhan type and, additionally, during three days post-infection with EC 3.2 (treatment scheme Figure 7C). Thereby, a similar dose-dependent reduction of viral replication ( Figure 7C) was detected as with the post-treatment alone ( Figure 3A). The IC 50 was~1:800; thus, the additional co-treatment with EC 3.2 has no further influence on the replication capacity of SARS-CoV-2 ( Figure 7C). In summary, the TOA experiments revealed that EC 3.2 does not influence early steps in the replication cycle of SARS-CoV-2 but rather exerts its antiviral activity effect during the later steps of viral replication.

Discussion
COVID-19, caused by SARS-CoV-2, is still responsible for the ongoing worldwide pandemic that led to a global health and socioeconomic crisis. Similar to the occurrence of SARS-CoV, Middle East respiratory syndrome-related coronavirus (MERS-CoV) and SARS-CoV-2, it can be expected that in the future new coronaviruses will emerge by zoonotic transmission from animals to humans potentially leading to new threats. This demonstrates the need for pandemic preparedness.
Regarding viral infections, vaccines are valuable but have limitations due to the high mutation rate of viruses, especially for RNA viruses such as SARS-CoV-2. This causes the continuous appearance of escape mutants, which are able to evade vaccine-induced immunity. Thus, there is an urgent need for new therapeutics that are available within a short period of time, broadly active, safe, cost-effective and easily distributable for patients all over the world when compared to standard antivirals.
In this study, an antiviral effect of European black elderberry fruit extract against SARS-CoV-2 and its VoCs was shown for the first time. In order to analyze whether black elderberry extract exhibits antiviral activity against various VoCs, Calu-3 cells were infected with SARS-CoV-2 Alpha, Beta, Gamma, Delta and Omicron. The results showed that the replication of these variants could be blocked in a comparable concentration range ( Figure 6). This suggests an effective inhibition of viral replication independent of the current and possibly future variants of SARS-CoV-2.
Fruit extracts and juices have previously been shown to be a potential source of antiviral agents (for review, see [50]). Thereby, fruit extracts from, e.g., blackberry, blackcurrant, mulberry and pomegranate exhibit antiviral properties against various viruses such as the Dengue virus, IAV, Zika, Hepatitis C virus (HCV), Human immunodeficiency virus 1 (HIV-1) and the polio virus [50]. For black elderberry extracts, several in vitro studies show an antiviral activity against IAV and IBV, as well as FIV [34][35][36]51,52]. Moreover, an ethanolic Sambucus Formosana Nakai extract exhibits antiviral activity against the endemic human coronavirus HCoV-NL63 [37]. Most importantly, human clinical trials have shown that a Sambucus nigra extract significantly reduces the total duration and severity of upper respiratory symptoms following common cold or IAV infections [38]. In addition, black elderberry extracts exhibit an immunomodulatory effect, which seems to be attributed to the polysaccharide fraction. In this context, it was shown that Sambucus nigra fruits contain peptic polysaccharides influencing the immune system via the activation of macrophages and dendritic cells [53][54][55], which might also contribute to the therapeutic effects seen in human clinical trials.
The black elderberry extract used in this study is a standardized European black elderberry fruit extract (Sambucus nigra, variety 'Haschberg') with a total polyphenol content of 4.6% and a total anthocyanin content of 3.5%. The used extract in this study consists of a mixture of organic compounds including but not limited to polyphenols, anthocyanin, mono-and disaccharides, proteins, lipids and carbohydrates. Therefore, the observed antiviral effect of the whole elderberry extract cannot be attributed to single compounds. Further studies including the identified main compounds of the used black elderberry fruit extract are necessary to designate the anti-SARS-CoV-2 effect to specific compounds of the whole extract.
The antiviral activities of polyphenols as well as anthocyanins against various viruses were extensively described previously (for review, see [56,57]). The phenolic compounds of various plants are discussed to be effective against SARS-CoV-2 [58]. For the extracts of some plants, not including black elderberry, an antiviral activity of phenolic compounds against SARS-CoV-2 was shown in vitro [58]. In this study, we identified as the main anthocyanins cyanidin-3-sambubioside-5-glucoside, cyanidin-3-sambubioside and cyanidin-3glucoside ( Figure 1). The main polyphenols were identified as chlorogenic acid, rutin and isoquercetin ( Figure 2). This is consistent with previous reports on the phenolic content of elderberry fruits [59][60][61]. For some of these identified compounds, an antiviral activity was reported previously. For instance, isoquercitrin displayed potent antiviral activity against the Varicella Zoster virus (VZV) and human Cytomegalovirus (hCMV) [62]. In addition, a very recent study shows that isoquercitrin inhibits the replication of IAV [63]. Moreover, it was proposed that the combinational treatment of quercetin and vitamin C might be an effective therapy for the prevention and treatment of COVID-19 [64]. For chlorogenic acid, an antiviral activity was described against different viral strains of IAV as well as for Enterovirus 71 [65,66]. However, to our knowledge, the specific anti-SARS-CoV-2 activities of these compounds have not been reported yet.
Analyzing the potential antiviral mode of action of phenolic compounds, it was shown that the flavonoids present in elderberry fruits can directly bind to H1N1 Influenza virus particles, thereby inhibiting the entry of the virus into the host cells [52]. Another study indicates that an ethanolic extract of Sambucus nigra fruits can inhibit the infectious bronchitis virus (IBV), a pathogenic chicken coronavirus, at early points during replication [67]. By performing time of addition experiments, Cho et al. showed that isoquercetin inhibits viral attachment and the entry of IAV [63]. Such a mechanism inhibiting the early stages of the replication of SARS-CoV-2 is unlikely to be the case for European black elderberry extract inasmuch as the time of addition experiments performed in this study displayed antiviral activity only for the later stages of virus replication. Treatment before or during the infection with SARS-CoV-2 had no influence on the replication capacity (Figure 7), strongly indicating an inhibitory effect after virus entry.
However, there are also several reports showing an antiviral effect of anthocyanins or phenolic compounds at later stages in the viral replication cycle. For instance, the main anthocyanin present in European black elderberry fruits, i.e., cyanidin-3-sambubiocide, can bind and inhibit the active pocket of the IAV neuraminidase [51]. In another study, it was demonstrated that natural phenolic compounds are able to inhibit the papain-like protease (PLpro) of SARS-CoV-2 with an IC 50 of 4-10 µM [68]. The papain-like protease is a viral protease with multiple functions and is crucial for the virus replication of SARS-CoV-2. Such a mechanism might also be responsible for the observed antiviral effect of black elderberry fruit extract (Figures 3, 5 and 6 and Tables 1 and 2) and will be the subject of further investigations. For rutin, which is also a main phenolic compound in the black elderberry extract used in this study ( Figure 2 and Table 1), an inhibition of the second SARS-CoV-2 protease 3CLpro was shown in a low micromolar range [69][70][71], which might also, at least partially, be responsible for the antiviral effect of European black elderberry fruit extract.
Due to the history of using European black elderberry, it can be considered as safe for ingestion. The unripe berries of black elderberry can contain cyanogenic compounds such a sambunigrin, which is readily degraded during a short heat treatment. Thus, both extracts used in this study, EC 3.2 and EC 14, undergo a short heat treatment during production. This is in concert with the data in this study, which showed no toxic effect on Caco-2 and Calu-3 cells when treated with different concentrations of black elderberry extract up to a dilution of 1:50, which points towards a broad therapeutic window (Figure 4).
The results of this study suggest that European black elderberry fruit extracts could provide beneficial effects in therapeutic settings following a SARS-CoV-2 infection. Their low cytotoxicity and wide availability in nature would make them a readily distributable treatment option for current and future pandemics.

Patents
IPRONA AG/SPA has filed a PCT and EP patent entitled "Elderberry extract for use in a method of preventing or treating a SARS-CoV-2 infection" claiming the priority date of 06.12.2021.