EP4297764A1 - An extract from a plant species of the subfamily cichorioideae for use in the prevention or treatment of covid-19 - Google Patents

Abstract

The present invention is inter alia concerned with an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae for use in the prevention or treatment of coronavirus disease 2019 (COVID-19). Further, the present invention relates to a pharmaceutical composition comprising the afore-mentioned extract and optionally a pharmaceutically acceptable excipient for the above use, wherein the extract is the sole active ingredient. The present invention also relates to the use of the afore-mentioned extract for inhibiting the interaction between the spike protein or a mutant thereof of the SARS-CoV-2 virus, or a fragment thereof, and the ACE2 receptor, or a fragment thereof, wherein the extract is used outside the human or animal body or used in vitro or not for use in methods for treatment of the human or animal body, respectively.

Description

An extract from a plant species of the subfamily Cichorioideae for use in the prevention or treat ment of COVID-19

FIELD OF THE INVENTION

The present invention is in the field of COVID-19 treatment. Thus, the present invention relates to an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichori- oideae for use in the prevention or treatment of coronavirus disease 2019 (COVID-19). Further, the present invention relates to a pharmaceutical composition comprising the afore-mentioned extract and optionally a pharmaceutically acceptable excipient for use in the prevention or treat ment of coronavirus disease 2019 (COVID-19), wherein the afore-mentioned extract is the sole active ingredient. Finally, the present invention relates to the use of the afore-mentioned extract for inhibiting the interaction between the spike protein or a mutant thereof of the SARS-CoV-2 virus, or a fragment thereof, and the ACE2 receptor, or a fragment thereof, wherein the extract is used outside the human or animal body or used in vitro ox not for use in methods for treatment of the human or animal body, respectively.

BACKGROUND OF THE INVENTION

In late 2019, the disease known as Corona Virus Disease 2019 or COVID-19 was first reported (1,

2). 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 (3). 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 angiotensin-converting 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 (4). This interaction between the virus and host cell at entry site is crucial for disease onset and progression.

Up to February 2020, there were three rapidly spreading new variants of SARS-CoV-2 which were first reported in the United Kingdom (variant B.1.1.7), South Africa (variant B.1.351) and Brazil (vari ant P.1), all of which share the mutation N501Y in the spike protein (5). SARS-CoV-2 variants with spike protein D614G mutations now predominate globally. Preliminary data suggest a possible association between the observed increased fatality rate with the mutation D614G and it is hy pothesized that a conformational change in the spike protein results in the 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 ques tions about the possible human immune response and the success of already available vaccines

(7).

There is sufficient evidence now to support the concept that targeting the interaction site be tween the RBD of the spike S1 subunit and ACE2 has the potential to be a major target for ther apy or prevention (8). It is noted that ACE2 mRNA and protein expression have been found in epithelial cells of all oral tissues, especially in the buccal mucosa, lip and tongue (31). These data concur with the observation of very high salivary viral load in SARS-CoV-2 infected patients (32, 33). 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 pre vention of viral colonization at the oral and pharyngeal mucosa could be critical for averting fur ther infection to other organs and the onset of COVID-19 (34). Commercial virucidal mouth- rinses, such as e.g. povidone-iodine, have thus been suggested to potentially reduce the SARS- CoV-2 virus load in infected persons (35-37).

In view of the above, there is the need for compounds that target the afore-mentioned interac tion site between the RBD of the S1 subunit of the spike protein and the ACE2 receptor in order to provide an effective prevention or treatment of COVID-19.

CN111150792 discloses a Chinese medicine composition with activity against SARS-CoV-2, which is made of the following raw materials: Daqingye, wild chrysanthemum, coptis, annona seeds, ag- eratum, atractylodes, Bupleurum, artificial bezoar, houttuynia cordata, dandelion root, honey suckle, forsythia, Scutellaria, shegan, salvia, chuanbei, Tianshan snow lotus, astragalus, Cordyceps, Codonopsis, and Licorice. CN111150792 further discloses that certain combinations of the afore mentioned plants are extracted together and the resulting extracts are then combined in order to arrive at the Chinese medicine composition. However, CN111150792 does not provide any infor mation, let alone experimental data, which of the overall 21 different plants in fact has activity on its own against SARS-CoV-2. In other words, while CN111150792 discloses activity against SARS- CoV-2 of an extract of all 21 plants, it fails to disclose activity of a single extract of each plant of the mixture, let alone activity of a single extract from dandelion root. In yet other words, CN111150792 discloses a composition comprising dandelion but fails to disclose that dandelion as such has activity against SARS-CoV-2.

CN111298049 discloses a Chinese medicine composition with activity against SARS-CoV-2, which is made of the following raw materials: bupleurum, Scutellaria, pinellia terata, cassia twig, magno lia officinalis, almond, aster, Daqing, radix isatidis, dandelion, silver flower, forsythia, chrysanthe mum, honeysuckle, reed root, imperata cylindica, vitex vulgaris, and fried coix. However, CN111298049 does not provide any information, let alone experimental data, which of the overall 19 different plants in fact has activity on its own against SARS-CoV-2. In other words, while CN111298049 discloses activity against SARS-CoV-2 of a composition of 19 plants together, it fails to disclose activity of each single plant of the mixture, let alone single activity of dandelion. In yet other words, CN111298049 discloses a composition comprising dandelion but fails to disclose that dandelion as such has activity against SARS-CoV-2.

OBJECTS AND SUMMARY OF THE INVENTION

The inventors of the present invention have surprisingly found that an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae inhibits the interac tion between the RBD of the S1 subunit of the spike protein and the ACE2 receptor. The inventors have shown that this inhibition is effective in preventing the binding of the spike protein to the ACE2 receptor as well as in removing the spike protein from the ACE2 receptor. Importantly, the inventors have also shown that virus entry via the spike protein into cells comprising the ACE2 receptor can be blocked by the extract according to the invention.

In the first aspect, the present invention is directed to an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae for use in the prevention or treat ment of coronavirus disease 2019 (COVID-19).

Embodiments relating to the plant species

In systematic botany, there is the order of Asteralaes, which goes down to the species of interest for the present invention as follows: Order: Asteraiaes - Family: Asteraceae Subfamily: Cicho- rioideae -^Tribe: Cichorieae - Subtribes: Crepidinae, Cichoriinae and Hieraciinae - Geni: Tarax acum from the subtribe Crepidinae, Cichorium xoxx\ the subtribe Cichoriinae and Hieracium xoxx\ the subtribe Hieraciinae i^. H. Wigg). The data of the present application point into a direction that the species of the subfamily Asteroideae are less effective for COVID-19 treatment. Positive data were obtained for the species Cichorium intybus, Taraxacum officinalis and Hieracium piio- ceiia, which are all of the subfamily Cichorioideae.

In view of the above, the plant species is in a preferred embodiment of the tribe Cichorieae. In yet another preferred embodiment, the plant species is of the subtribe Cichoriinae, Hieraciinae ox Crepidinae. In a further preferred embodiment, the plant species is of the genus Cichorium or Taraxacum ox Hieracium. Thus, the plant species is preferably Cichorium spp. or Taraxacum spp. or Hieracium spp.

It is preferred that the plant species is selected from the group consisting of Cichorium intybus, Taraxacum officinalis, Hieracium piioceiia, Cichorium endivia, C re pis, Arnoseris minima, Lapsana communis, Leontodon, Hypochaeris radicata, Cicerbita macrophyiia, Lactuca saiigna, and Lactuca sativa L. It is most preferred that the plant species is Cichorium intybus ox Taraxacum officinalis ox Hier- acium piioceiia.

In view of the above, a most preferred embodiment of the first aspect relates to an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae ox use in the prevention or treatment of coronavirus disease 2019 (COVID-19), wherein the plant species is Cichorium intybusox Hieracium piioceiia or Taraxacum officinalis. It is evident from the formula tion of this most preferred embodiment of the first aspect that it is the extract as such that is ef fective, i.e. an extract from one of the three afore-mentioned plant species and not an extract from many plant species including one of the three afore-mentioned plant species. Thus, in an embodiment, the extract is the sole active ingredient.

In another embodiment, the extract for use according to the present invention is from the leaves, flowers, stalks and/or roots of a mixture of at least two plant species of the subfamily Cichorioi- deae.

In a preferred embodiment thereof, the at least two plant species are of the tribe Cichorieae. In yet another preferred embodiment, the at least two plant species are of the subtribe Cichoriinae and/or Hieraciinae. In a further preferred embodiment, the at least two plant species are of the genus Cichorium, Taraxacum and/ or Hieracium. It can be preferred that the at least two plant species are selected from the group consisting of Cichorium intybus, Taraxacum officinalis and Hieracium piioceiia. In yet another embodiment, the at least two plant species are two plant spe cies, namely Cichorium intybus and Taraxacum officinalis.

In view of the above, a most preferred embodiment of the first aspect relates to an extract from the leaves, flowers, stalks and/or roots of a mixture of at least two plant species of the subfamily Cichorioideae ox use in the prevention or treatment of coronavirus disease 2019 (COVID-19), wherein the at least two plant species are selected from the group consisting of Cichorium inty bus, Hieracium piioceiia and Taraxacum officinalis. A mixture of all three of the afore-mentioned species may of course also be used. It is evident from the formulation of this most preferred em bodiment of the first aspect that the extract from at least two plant species selected from the group consisting of Cichorium intybus, Hieracium piioceiia and Taraxacum officinalis is effective as such, i.e. an extract from two or three of the afore-mentioned three plant species and not an ex tract from many plant species including two or three of the three afore-mentioned three plant species. Thus, in an embodiment, the extract is the sole active ingredient.

Embodiments relating to the plant material

The extract for use according to the present invention is an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae.

In a preferred embodiment, the extract for use according to the present invention is from the leaves, flowers and/or stalks. In yet another preferred embodiment, the extract for use according to the present invention is from the leaves.

Embodiments relating to the extract

The extract for use according the present invention may be an aqueous extract, an alcoholic ex tract, or a mixture thereof. When it comes to an alcoholic extract, a methanolic extract, an etha- nolic extract or an isopropanolic extract can be preferred. In an embodiment, the extract is an aqueous-alcoholic extract, including an aqueous-methanolic extract, an aqueous-ethanolic ex tract and an aqueous-isopropanolic extract. Typically, the alcoholic part of the extract will be sig nificantly lower than the aqueous part. Thus, the aqueous-alcoholic extract may in particular comprise less than about 40% alcohol, preferably less than about 30% alcohol, more preferably less than about 20% alcohol and most preferably less than about 10% alcohol.

The extract for use according to the present invention may also be an extract obtained by fer menting the leaves, flowers, stalks and/or roots. A lactobacillus may e.g. be used for the fermen tation, typically with sugar also added to an e.g. fresh juice obtained from pressing the plant parts or from an initial aqueous extract.

Further, the extract for use according to the present invention may be a supercritical C0₂-extract. Here, the non-polar, lipophilic substances are extracted by supercritical C0₂ resulting in a lipo philic fraction and an hydrophilic fraction, with the latter fraction being mainly the fraction of in terest for the present invention.

Finally, the extract for use according to the present invention may be an aqueous extract com prising salts, such as in particular NaCI. Here, an isotonic NaCI solution (0.9% NaCI in water) can be preferred as solvent when producing the extract.

In the most preferred embodiment, the extract is an aqueous extract.

A concentration range of about 1 mg/ml to about 250 mg/ml (dried plant part / solvent) appears to be suitable for the use according to the present invention. A concentration of about 10 mg/ml to about 100 mg/ml may be used, where a concentration of about 20 mg/ml to about 100 mg/ml can be preferred. Of course, any other suitable concentration can easily be provided by either di luting an extract or concentrating an extract.

It is important to stress that the afore-mentioned concentration ranges are generally in line with ranges used in current medical indications for Cichorium intybus, Taraxacum officinalis and Hier- acium piioceiia (as set out below in more detail).

A typical method for obtaining an extract is the mixing of the plant material (typically in a dried state) with a solvent, preferably water, at a specific temperature (e.g. at 4°C, at room temperature or a higher temperature up to the boiling point of the solvent), wherein the temperature may be achieved by cooling or heating the solvent to the respective temperature prior to mixing. Op tionally, the extraction may comprise a step of sonication with an ultrasonic homogenizer (usually for a few minutes). Typically, the extraction is performed for a time period ranging from a few seconds to several hours, e.g. from about 30 seconds to about 20 hours. It is noted that a time period of several days may even be used. However, a time period of about 15 minutes to about 2 hours seems to be well-suited but, as shown herein, a time period of about 1 minute is already sufficient to obtain an effective extract. A step of centrifugation in order to remove the plant ma terial may then be carried out (e.g at about 16.000g for a few minutes at room temperature), op tionally followed by a filtration of the obtained supernatant (e.g. using a filter with a pore size of about 0.22 pm or about 0.45 pm).

It can further be preferred that the extract for use according to the present invention is enriched in compounds or consists essentially of compounds with a molecular weight higher than about 5 kDa. Such an enrichment or such a characteristic of the extract can e.g. be obtained by separat ing the initially obtained extract (see above) in fractions comprising compounds of lower and higher molecular weights at the cut-off molecular weight, and then using the fraction containing the compounds of the desired molecular weight, here a molecular weight higher than about 5 kDa. Typically, such a fractionation comprises the preparation of an initial extract from the plant material, optionally including a step of filtration, e.g. using a membrane with a pore size of about 0.22 pm or about 0.45 pm, followed by separation into molecular weight fractions defined by a cut-off filter. In the present case, a cut-off filter with a size of 5 kDa is preferably used. The rele vant fraction may then be purified by using a suitable solvent, here preferably water, and then optionally freeze-drying the desired fraction and storing it at -20°C, if deemed appropriate for storage reasons. Other methods to obtain an extract that is enriched in compounds or consists essentially of compounds with a molecular weight higher than about 5 kDa are known to the skilled person and may of course also be used.

Detailed protocols for preparing a suitable extract can be found in the examples of the present application. They can be used and optionally be supplemented with typical steps when preparing an extract. On a general level, the skilled person is aware of alternatives and steps that may devi ate from the protocols in the examples, which nevertheless result in an extract that can effectively be used in accordance with the present invention.

Embodiments relating to the disease

Several studies indicate that the D614G viral lineage is more infectious than the D614 virus (18). Also, the presence of characteristic mutations such as N501Y of, e. g. the so-called UK variant B.1.1.7, result in higher infectivity than the parent strain which might be due to a higher binding affinity between the spike protein and ACE2 (19). The data of the present application shows that the extract according to the present invention is not only effective against the wildtype virus but also against the afore-mentioned variants comprising the mutated spike proteins. Before the above background, it is very likely that the extract is generally effective against variants, including e.g. variant B.1.1.529.

Thus, in an embodiment, the extract for use according to the present invention is for the preven tion or treatment of COVID-19 that is caused by the severe acute respiratory syndrome corona- virus 2 (SARS-CoV-2) or a variant thereof.

The variant of SARS-CoV-2 may comprise a spike protein with a mutation selected from the group consisting of K417N, N501Y, D614G, and combinations thereof. The variant of SARS-CoV-2 may be the variant B.1.1.7 (also referred to as the "UK" variant), the variant B.1.351 (also referred to as the "South Africa" variant) or variant P.1 (also referred to as the "Brazil" variant).

The results furthermore show that the binding inhibition of spike protein and mutants thereof to human ACE2 by the extract of the present invention is achieved independent from whether the incubation with the extract was carried out prior to or after the incubation with spike protein. Fur thermore, it is shown herein that virus entry via the spike protein into cells comprising the ACE2 receptor was blocked by the extract according to the invention.

Accordingly, the disease may be prevented or treated.

Embodiments relating to the administration

In a preferred embodiment, the extract is administered to the oral cavity, the nasal cavity and/or the pharyngeal cavity. This includes e.g. flushing the oral cavity and the pharyngeal cavity with extract, ideally for a time period of at least about 5 to 10 seconds, preferably at least about 20 seconds, more preferably at least about 30 seconds to 1 minute. It also includes e.g. the spraying of the extract into the oral cavity (optionally also of the pharyngeal cavity). The same applies to the nasal cavity, where a suitable administration device may be used (such as e.g. a device for a nasal douche). The afore-mentioned administration may be carried out as a prevention, i.e. when likely being confronted with a situation where there might be a rather high SARS-CoV-2 (includ ing variants thereof) load (due to e.g. closed windows or a short distance to other persons). The afore-mentioned administration may, however, also be carried out when having SARS-CoV-2 (in cluding variants thereof) present already in the respiratory system to reduce the SARS-CoV-2 (in cluding variants thereof) load on the respective cells of the respiratory system. Since this inhibits the further systemic spread of the virus, also such an application may be referred to as preven tion.

In yet another preferred embodiment, the extract is administered to the lung or the gastrointesti nal tract or the systemic circulation. This includes e.g. the inhalation of the extract into the lung (optionally using suitable inhalation-devices) or the drinking of the extract or the swallowing of tablets comprising the dried extract in order to provide the extract in the gastrointestinal tract and/or the systemic circulation. Infusion may also be used to provide the extract in the systemic circulation. The afore-mentioned administration may be carried out as a treatment, i.e. when having SARS-CoV-2 (including variants thereof) present already in the body to reduce the SARS- CoV-2 (including variants thereof) load on the respective cells of the organs reached via the afore-mentioned administrations. As this treatment also inhibits the further spread of the virus, it may alternatively be referred to as prevention, depending on the stage of the infection and the COVID-19 symptoms, respectively.

Embodiments relating to preferred combinations of features

In a preferred embodiment, the present invention relates to an aqueous extract from the leaves, flowers and/or stalks of a plant species of the subfamily Cichorioideae for use in the prevention or treatment of coronavirus disease 2019 (COVID-19).

In another preferred embodiment, the present invention relates to an extract from the leaves, flowers, and/or stalks of a plant species of the genus Cichorium or Taraxacum or Hieracium for use in the prevention or treatment of coronavirus disease 2019 (COVID-19). It is preferred in this embodiment that the extract is an aqueous extract.

In yet another preferred embodiment, the present invention relates to an aqueous extract from the leaves, flowers and/or stalks of a plant species of the subfamily Cichorioideae for use in the prevention of coronavirus disease 2019 (COVID-19), wherein the extract is administered to the oral cavity, the nasal cavity and/or the pharyngeal cavity. It is preferred in this embodiment that the COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a variant thereof, wherein the variant is variant B.1.1.7 or variant B.1.351 or variant P.1.

In another preferred embodiment, the present invention relates to an extract from the leaves, flowers, and/or stalks of a plant species of the genus Cichorium or Taraxacum or Hieracium for use in the prevention of coronavirus disease 2019 (COVID-19), wherein the extract is administered to the oral cavity, the nasal cavity and/or the pharyngeal cavity. It is preferred in this embodiment that the extract is an aqueous extract. It can further be preferred in this embodiment that the COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a variant thereof, wherein the variant is variant B.1.1.7 or variant B.1.351 or variant P.1.

In a preferred embodiment, the present invention relates to an aqueous extract from the leaves, flowers and/or stalks of a plant species of the subfamily Cichorioideae for use in the prevention or treatment of coronavirus disease 2019 (COVID-19), wherein the plant species is Cichorium inty- bus or Hieracium piioceiia or Taraxacum officinalis.

In yet another preferred embodiment, the present invention relates to an aqueous extract from the leaves, flowers and/or stalks of a plant species of the subfamily Cichorioideae for use in the prevention of coronavirus disease 2019 (COVID-19), wherein the plant species is Cichorium inty- bus or Hieracium piioceiia ox Taraxacum officinalis, and wherein the extract is administered to the oral cavity, the nasal cavity and/or the pharyngeal cavity. It is preferred in this embodiment that the COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a variant thereof, wherein the variant is variant B.1.1.7 or variant B.1.351 or variant P.1.

In yet another preferred embodiment, the present invention relates to an aqueous extract from the leaves, flowers and/or stalks of a plant species of the subfamily Cichorioideae for use in the treatment of coronavirus disease 2019 (COVID-19), wherein the plant species is Cichorium intybus or Hieracium pilocella or Taraxacum officinalis, and wherein the extract is administered to the lung or the gastrointestinal tract or the systemic circulation. It is preferred in this embodiment that the COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a variant thereof, wherein the variant is variant B.1.1.7 or variant B.1.351 or variant P.1.

In the second aspect, the present invention is directed to a pharmaceutical composition compris ing an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae and optionally a pharmaceutically acceptable excipient for use in the prevention or treatment of coronavirus disease 2019 (COVID-19), wherein the plant species is Cichorium intybus or Hieracium pilocella ox Taraxacum officinalis, and wherein the extract is the sole active ingredi ent.

An embodiment related to the second aspect is concerned with a pharmaceutical composition comprising an extract from the leaves, flowers, stalks and/or roots of a plant species of the sub family Cichorioideae and optionally a pharmaceutically acceptable excipient for use in the pre vention or treatment of coronavirus disease 2019 (COVID-19), wherein the plant species is Cicho rium intybus.

Yet another embodiment related to the second aspect is concerned with a pharmaceutical com position comprising an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae and optionally a pharmaceutically acceptable excipient for use in the prevention or treatment of coronavirus disease 2019 (COVID-19), wherein the plant species is Hi eracium pilocella.

Yet another embodiment related to the second aspect is concerned with a pharmaceutical com position consisting of an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae and at least one pharmaceutically acceptable excipient for use in the prevention or treatment of coronavirus disease 2019 (COVID-19), wherein the plant species is Ta raxacum officinalis and wherein the extract is preferably an aqueous extract.

All of the embodiments disclosed above for the extract for use in the first aspect apply for the second aspect as well. Reference is thus made to the embodiments relating to i) the plant spe cies, ii) the plant material, iii) the extract, iv) the disease, v) the administration, and vi) the combi nation of features. When it comes to the pharmaceutical composition for use of the second aspect, which comprises the extract as defined in the first aspect as the sole active ingredient, the pharmaceutical compo sition may be a solution or an optionally coated tablet comprising dried extract. When a solution is concerned, the solution may be selected from the group consisting of an ointment, a lotion, decoction, an infusion, a juice made of a fresh plant and a hydroalcoholic tincture. Preferably, the solution is administered orally, topically or via infusion, whereas the coated tablet is administered orally.

The pharmaceutically acceptable excipient may be a substance/substances like sucrose, mannitol, sorbitol, starch and starch derivatives, lactose, cellulose and lubricating agents such as magne sium stearate, disintegrants and buffering agents, in particular if the pharmaceutical composition is a tablet.

When it comes to Taraxacum officinale, it should be noted that the EMA in the Community herbal monograph on Taraxacum officina/eWJeber ex Wigg., radix cum herba dated 20 Novem ber 2019 refers to herbal preparations in solid or liquid dosage forms for oral use and commi nuted herbal substance as herbal tea for oral use. Accordingly, corresponding pharmaceutical compositions are already known and used for other, unrelated indications. For these unrelated indications, the posology and method of administration is given in the afore-mentioned mono graph as a) comminuted dried root with herb, 3-4 g as a decoction or 4-10 g as an infusion up to 3 times daily, b) 1 coated tablet, 300 mg dry extract, 2 times daily or 1-2 coated tablets, 150 mg dry extract each, 3 times daily, c) liquid extract 90 drops (90 drops = 3.15 ml = 3.31 g), 3 times daily, d) liquid extract 35 drops (35 drops = ca. 1 ml = 1 g), 3 times daily, and e) expressed juice from fresh flowering Taracaci radix cum herba.

When it comes to Cichorium intybus and Hieracium pitosetta, it should be noted that both are also listed by the EMA (EMA/HMPC/113041/2010 and EMA/HMPC/680374/2013). Hieracium pilo- sella leaves and/or roots are traditionally used as herbal tea or solid dosage, with a dosage is up to 4 g in 250 ml of boiling water, 3 times daily or 1.3 g daily dosage of powdered herbal sub stance. Cichorium intybus leaves and/or roots are traditionally used as herbal tea (infusion or de coction). The daily dosage is up to 6 g used as infusion or decoction in two glasses of water (38). In the German Commission E, an average daily dose of 3 g herb is recommended.

In principle, the already known pharmaceutical compositions may be used for the treatment of coronavirus disease 2019 (COVID-19) and may include the posology and method of administra tion, which, however, might need to be adapted depending on the specific composition and route of administration. The embodiments given above in the first aspect, in particular the em bodiments relating to the extract and the administration, are a suitable guidance for the skilled person when adapting the posology and method of administration.

In the third aspect, the present invention is directed to the use of an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae for inhibiting the in teraction between the spike protein or mutants thereof of the SARS-CoV-2 virus, or a fragment thereof, and the ACE2 receptor, or a fragment thereof, wherein the plant species is Cichorium intybusox Hieracium pilocella or Taraxacum officinalis and wherein the extract is used outside the human or animal body. This may alternatively be formulated as the use of an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae for inhibiting the interaction between the spike protein or mutants thereof of the SARS-CoV-2 virus, or a frag ment thereof, and the ACE2 receptor, or a fragment thereof, wherein the plant species is Cicho rium intybusox Hieracium pilocella or Taraxacum officinalis and wherein the extract is used in vitro. Still alternatively, this may be formulated as the use of an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae for inhibiting the interaction between the spike protein or mutants thereof of the SARS-CoV-2 virus, or a fragment thereof, and the ACE2 receptor, or a fragment thereof, wherein the plant species is Cichorium intybusox Hieracium pilocella ox Taraxacum officinalis and wherein the extract is not for use in methods for treatment of the human or animal body.

The following embodiments disclosed above for the extract for use in the first aspect apply for the extract of the third aspect as well: the embodiments relating to i) the plant species, ii) the plant material, and iii) the extract.

It can be preferred that the extract is used in the third aspect in a reconstituted biochemical sys tem or a cell culture system. In such a system, it is e.g. possible to identify or screen for further suitable inhibitors of the interaction between the spike protein and the ACE2 receptor, wherein the extract according to the present invention may be used in binding competition experiments.

The biochemical system preferably comprises recombinant proteins including at least the S1 sub unit with the RBD of the spike protein, if not the complete spike protein, and the ACE2 receptor, or a fragment thereof. A cell culture system preferably at least comprises cells expressing the ACE2 receptor on the cell surface and a suitable measure for infection, such as e.g. a lentiviral system expressing at least the S1 subunit with the RBD of the spike protein, if not the complete spike protein. An exemplary suitable lentiviral system is shown in the example section of the pre sent application.

DESCRIPTION OF THE FIGURES

Figure 1 - Collected fresh dandelion ( taraxacum officinal e) plant leaves.

The plant leaf samples (1A-C) were collected at three different places in the region of Freiburg i. Br., Germany on 12.7.2020. The leaves were washed and subsequently dried at 50°C in an oven for 5h. The common dandelion has deeply serrated large leaves (5-40 cm long) that are either light or dark green and are clustered in a rosette at the base of the plant. The flowering stalks (5- 40 cm long) are upstanding, and carrying a solitary, terminal inflorescence (16).

Figure 2 - Dried dandelion ( taraxacum officinale ?) and common chicory ( cichorium intybu^ plant parts. 2A) dandelion plant leaves (vom Achterhof, Uplengen, Germany; batch no. 37259), 2B) pulverized dandelion roots, collected in Germany (HNK Steviamarkt Semrau, TangerhQtte, Germany), 2C) dandelion flowers, collected in Poland (Naturix24, Germany) and 2D) chicory leaves, collected in Germany (Naturideen, Germany).

Figure 3 - Effect of dandelion and chicory on Sars-CoV-2-Spike - ACE 2 inhibition.

A) Dandelion plant leaves (sample A-C) were collected at different places in Freiburg, Germany. Each bar represents the result of one experiment. B) Concentration dependent effect by dande lion leaf extract. Bars are means +SD. C) Effect of dandelion flowers and root powder. Bars are means + SD. D) Concentration dependent effect of chicory (chicoryum intybus) plant extract. Bars are means + SD. Extraction was done using UAE/1h or 16h extraction time. E) Chamomille (Matri caria chamomilla) and F) calendula (Calendula officinalis) flowers showed at best a small (<20%) effect. Binding inhibition was assessed using ELISA technique. A. d.: distilled water

Figure 4 - Effect of extraction conditions on the capacity of dandelion to inhibit Sars-CoV-2- Spike - ACE 2 interaction.

Dandelion leaves were extracted for 1 or 16h, with or without UAE and/or HWE. Binding inhibition was assessed using ELISA technique. Each bar represents the result of one experiment. A. d.: dis tilled water

Figure 5 - Effect of fractions from dandelion leaves on Sars-CoV-2 Spike - ACE 2 inhibition.

The extracts made from dandelion (A) or common chicory (B) were freeze dried and a molecular weight fractionation 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 refer ence. HMW and LMW fraction quantities equivalent to dried leaves were used. The binding inhi bition was assessed using ELISA technique. N=3, bars are means + SD.

Figure 6 - Binding inhibition of S1 spike protein to human HEK293-hACE2 cells by extract pre incubation.

Cells were pre-incubated for the indicated times with the extract from 10 mg/ml dandelion (TO), its HMW fraction, equal to 10 mg/ml extract (HMW) and 10 mg/ml chicory (Cl) or solvent control: a.d. and subsequently treated with HIS-tagged S1 spike protein for 1h without a washing step in between at 4°C. Binding inhibition was assessed using flow cytometry. N=3, bars are means +

SD. Upper left: cytogram of gated HEK-hACE2 cells. Middle: overlay of representative fluores cence intensity histograms for ACE2 surface expression. Upper right: overlay of representative fluorescence intensity histograms for spike binding inhibition by the extracts or solvent control: a.d.; positive control: 20 pg/ml soluble hACE2. Cells were stained with anti-His-tag Alexa Fluor 647 conjugated monoclonal antibody.

Figure 7 - Binding inhibition of spike D614, and its mutants D614G and N501Y to human HEK293- hACE2 cells by extract pre- or post-incubation.

Overlay of fluorescence intensity histogram for A) unstained cells, staining control (anti-His-tag A647), and cells incubated with His-tag labelled spike D614, D614G or N501Y for 1h at 4°C. B) cells pre-incubated with solvent control (a.d.), 10 mg/ml dandelion (TO) or 10 mg/ml chicory (Cl) for 30-60 sec., and then treated with His-tag labelled S1 spike D614, D614G or N501Y protein for 1h without a washing step in between at 4°C. In figure C), means of 3 independent experiments are given + SD. D) Effect of 10 mg/ml extract incubation for 60 sec. at 37°C either before (left) or af ter (right) incubation with His-tag labelled spike D614, D614G or N501Y protein for 0.5h at 37°C. Bars are mean + SD. 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.

Figure 8 - Binding inhibition of spike D614, and its mutants D614G and N501Y to human A549- hACE2-TMPRSS2 cells by extract pre- or post-incubation and effect of human saliva.

A) Cells were pre-incubated with solvent control (a.d.), 10 mg/ml dandelion (TO) or 10 mg/ml chicory (Cl) for 60 sec., and then treated with His-tag labelled S1 spike D614, D614G or N501Y protein for 0.5h without a washing step in between at 37°C. B) Concentration dependent inhibi tory effect of TO pre-treatment on spike D614G mutant binding. Cells were pre-treated with TO extract for 60 sec., and then treated with His-tag labelled S1 spike D614G for 0.5h without a wash ing step in between at 37°C. C) Cells were pre-incubated with His-tag labelled S1 spike D614, D614G or N501Y protein for 0.5h and then treated with 10 mg/ml TO or Cl for 60 sec. without washing step in between at 37°C. D) Concentration dependent inhibitory effect of TO post-treat ment on spike D614G mutant binding. Cells were pre-incubated with His-tag labelled S1 spike D614, D614G or N501Y protein for 0.5h and then treated with 10 mg/ml TO or Cl for 60 sec. with out washing step in between at 37°C. E) Plant extracts were incubated in saliva from 4 human donors for 30 min. at 37°C. Afterwards, cells were pre-treated with 5 mg/ml extracts for 60 sec. at 37°C before incubation with His-tag labelled spike D614 protein for 0.5h at 37°C. Bars are mean + SD.

Figure 9 - Inhibition of viral transduction of A549-hACE2-TMPRSS2 cells by dandelion extract.

Cells were pre-treated with a.d. or 10 mg/ml dandelion (TO) extract for 0.5 h before transduction with 5 pi SARS-CoV-2-Spike pseudotyped lentivirus (Luc reporter). After 24 h of transduction, the medium was changed to fresh medium and cells incubated for another 60 h. Luminescence was detected after 1h. (-) negative control: bald lentiviral pseudovirion; (+) positive control: firefly lu- ciferase lentivirus. N=1

Figure 10 - Influence of the incubation time and temperature on the inhibitory activity of the ex tract.

Dried plant material was weighted in an amber glass vial (Carl Roth GmbH, Germany) and mixed with HPLC-grade water (a.d.), either at RT or at 100°C (HWE) or at 4°C and incubated for 1 min or for 60 minutes as indicated. Extracts were then centrifuged at 16.000g (3 min, RT). The superna tant was filtered (0.22 pm) prior to use in the experiments. DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in more detail in the example section, the following definitions are introduced.

1. Definitions

As used in the specification and the claims, the singular forms of "a" and "an" also include the corresponding plurals unless the context clearly dictates otherwise.

The term "about" in the context of the present invention denotes an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in ques tion. The term typically indicates a deviation from the indicated numerical value of ±10% and preferably ±5%.

It needs to be understood that the term "comprising" is not limiting. For the purposes of the pre sent invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined to comprise at least a certain number of embodi ments, this is also meant to encompass a group which preferably consists of these embodiments only.

The terms " Taraxacum officinalis , " Taraxacum officinaid' and “common dandelion" are used in terchangeable herein. Dandelion ( Taraxacum spp .) belongs to the plant family Asteraceae, sub family Cichorioideae. It is a perennial herb, native distributed in the warmer temperate zones of the Northern Hemisphere inhabiting fields, roadsides and ruderal sites. The common dandelion is consumed as vegetable food but also employed in European phytotherapy to treat disorders from the liver, gallbladder, digestive tract or rheumatic diseases. Modern herbal monographs consider the plant usage as safe and have evaluated the empiric use of Taraxacum with a positive outcome. Therapeutic indications for the use of Taraxacum officinale are listed in the German Commission E, the European Scientific Cooperative for Phytotherapy (ESCOP) monographs (9, 10) as well as in the British Herbal Medicine Association (11). The plant contains a wide array of phy tochemicals including terpenes (sesquiterpene lactones such as taraxinic acid and triterpenes), phenolic compounds (phenolic acids, flavonoids, and coumarins) and also polysaccharides (12). The predominant phenolic compound was found to be chicoric acid (dicaffeoyltartaric acid). The other were mono- and dicaffeoylquinic acids, tartaric acid derivatives, flavone and flavonol glyco sides. The roots, in addition to these compound classes, contain high amounts of inulin (13). Dos age forms comprising Taraxacum officinale include aqueous decoction and infusion, expressed juice of fresh plant, hydroalcoholic tincture as well as coated tablets from dried extracts applied as monopreparations (14) but also integral components of pharmaceutical remedies.

Both, Cichorium iniybus and Hieracium piioseiia ex listed by the EMA (EMA/HM PC/113041/2010 and EMA/HMPC/680374/2013). Hieracium pitosetta leaves and/or roots are traditionally used as herbal tea or solid dosage for treatment of urinary tract complaints and to achieve flushing of the urinary tract. The dosage is up to 4 g in 250 ml of boiling water, 3 times daily or 1.3 g daily dos age of powdered herbal substance. No case of overdose has been reported. Cichorium intybus leaves and/or roots are traditionally used as herbal tea (infusion or decoction) for the treatment of digestive disorders and to promote renal and digestive elimination functions. The daily dosage is up to 6 g used as infusion or decoction in two glasses of water (38). In the German Commis sion E, an average daily dose of 3 g herb is recommended. No treatment related toxicity was ob served in a subchronic toxicity study in rats using up to 1 g/kg/day (39).

The term "aqueous extract" is to be understood such that the solvent is essentially only water. Accordingly, traces of further solvents or other ingredients (such as e.g. salts) may be present but in a manner that does not substantially influence the properties of water as a solvent to be used for the extraction.

When referring to "an amount of a plant part", this typically refers to the amount of the plant part in the dried state.

The term "inhibiting the interaction" as used herein in the third aspect of the invention means that preferably no interaction at all (at least not to a detectable level) between the spike protein and the ACE2 receptor takes place any more. However, when a given interaction between these two proteins (set to 100%) is greatly reduced, e.g. to a level of about 70%, about 60%, about 50%, about 40%, about 30%, preferably about 20%, more preferably about 10% or most prefera bly about 5% or less, such a reduced interaction is still encompassed by the term "inhibiting the interaction". When reference is made herein in the third aspect to a fragment of the spike protein or a mutant thereof of the SARS-CoV-2 virus, this is to be understood such that at least the RBD of the S1 subunit of the spike protein or a mutant thereof is present. Accordingly, when reference is made in the third aspect to a fragment of the ACE2 receptor, this is to be understood such that at least the spike protein binding domain is present.

The term "pharmaceutically acceptable excipient" as used herein refers to compounds commonly comprised in pharmaceutical compositions, which are known to the skilled person.

The term "the sole active ingredient" as used herein means that the extract referred to is the sole pharmaceutically active ingredient. In the present context, this means that the extract is the sole pharmaceutically active ingredient in the prevention or treatment of COVID-19. Thus, the extract as referred to herein comprises the at least one compound or at least one active agents or at least one API that has in fact activity in the prevention or treatment of COVID-19. Within the con text of a pharmaceutical composition comprising the extract as the sole active ingredient, this means that no further compounds or active agents or APIs for use in the prevention or treatment of COVID-19 are present, in particular no extracts from other plant species with such activity.

The term "prevention" as used herein means that a subject will not suffer from COVID-19 or will at least not suffer from severe symptoms of COVID-19, e.g. as the spread of the SARS-CoV-2 vi rus in the body could be inhibited and/or the SARS-CoV-2 virus load could be reduced. Thus, "prevention of COVID-19" may alternatively be referred to as "prevention of COVID-19 or a symptom, in particular a severe symptom, thereof".

The term "treatment" as used herein includes any type of a beneficial effect, e.g. amelioration of at least one symptom of COVID-19. Such a symptom may e.g. be the multisystem inflammatory syndrome, or any other symptom which accompanies or follows COVID-19 induced by SARS- CoV-2. Thus, "treatment of COVID-19" may alternatively be referred to as "treatment of COVID- 19 or a symptom thereof".

2. Preferred embodiments of the present invention Preferred embodiments of the present invention relate to:

1. An extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae for use in the prevention or treatment of coronavirus disease 2019 (COVID-19).

2. The extract for use according to embodiment 1, wherein the plant species is of the tribe Cichorieae, preferably of the subtribe Cichoriinaeox Hieraciinaeox Crepidinae, more prefera bly of the genus Cichorium or Hieracium or Taraxacum.

3. The extract for use according to embodiment 1 or 2, wherein the plant species is Cichorium intybusox Hieracium piioceiia or Taraxacum officinalis.

4. The extract for use according to any one of embodiments 1 to 3, wherein the extract is from the leaves, flowers and/or stalks.

5. The extract for use according to any one of embodiments 1 to 4, wherein the extract is ad ministered to the oral cavity, the nasal cavity and/or the pharyngeal cavity.

6. The extract for use according to embodiment 5, wherein the extract is for use in the preven tion of COVID-19.

7. The extract for use according to any one of embodiments 1 to 4, wherein the extract is ad ministered to the lung or the gastrointestinal tract or the systemic circulation.

8. The extract for use according to embodiment 7, wherein the extract is for use in the treat ment of COVID-19.

9. The extract for use according to any one of the preceding embodiments, wherein the COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a variant thereof. 10. The extract for use according to embodiment 9, wherein the variant is variant B.1.1.7 or vari ant B.1.351 or variant P.1.

11. A pharmaceutical composition comprising an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae and optionally a pharmaceutically ac ceptable excipient for use in the prevention or treatment of coronavirus disease 2019 (COVID-19).

12. The pharmaceutical composition for use according to embodiment 11, wherein the pharma ceutical composition is a solution or an optionally coated tablet comprising dried extract.

13. The pharmaceutical composition for use according to embodiment 12, wherein the solution is selected from the group consisting of a decoction, an infusion, a juice of a fresh plant and a hydroalcoholic tincture.

14. Use of an extract from the leaves, flowers, stalks and/or roots of a plant species of the sub family Cichorioideae for inhibiting the interaction between the spike protein or a mutant thereof of the SARS-CoV-2 virus, or a fragment thereof, and the ACE2 receptor, or a frag ment thereof, wherein the extract is used outside the human or animal body.

15. The use according to embodiment 14, wherein the extract is used in a reconstituted bio chemical system or a cell culture system.

3. Examples

The following Examples are merely illustrative and shall describe the present invention in a fur ther way. These Examples shall not be construed to limit the present invention thereto.

Example 1: Materials and methods

Plant Materials: The study was carried out using different parts (leaves, flowers or root) of plants from the genus taraxacum [Order: Asteraiaes- Family: Asteraceae Subfamily: Cichorioideae -^Tribe: Cichorieae (also called Lactuceae) - Subtribe: Crepidinae - Genus: Taraxacum F. H. Wigg.]. According to (15), the genus Taraxacum includes approximately 30-57 varieties with many microspecies, divided into nine sections. In figure 1 and figure 2A-C pictures of sample ma terial are given. To investigate whether other plants from the same tribe ( Cichorieae ) could exert similar effects as observed for dandelion, the common chicory ( Cichorium intybus) from the sub tribe Cichoriinae, which contains 6 geni in total, was also tested (see figure 2D).

Cell lines and culture conditions: Human embryonic kidney 293 (HEK293) cells, stably expressing hACE2, were used. The cells were maintained in Dulbecco^'s modified Eagle medium (DMEM), high glucose supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin/streptomycin and 50 pg/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-inacti vated FCS, 100 U/ml penicillin/streptomycin, 100 pg/ml normocin, 0.5 pg/ml puromycin and 300 pg/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% C02/95% air atmosphere.

Plant extraction: For plant extraction different methods, i. e. hot water extraction (HWE) and ultra- sonic-assisted extraction (UAE), were investigated as indicated in the examples. Dried plant mate rial was weighted in an amber glass vial (Carl Roth GmbH, Germany) and mixed with HPLC-grade water (a.d.), either at 4°C, RT, 37°C or at 100°C (HWE). For UAE, samples were sonicated for 3 min., at 20 kHz uniform sonic waves with constant sound radiation using a Bandelin SONOPULS ultrasonic homogenizer (Berlin, Germany). Extracts were then incubated for different time points and centrifuged at 16.000g (3 min, RT). The supernatant was filtered (0.22 pm) prior to use for the experiments.

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 inter action 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 was used as method verified reference. 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, 2x105 cells (5x106 cells/ml) were either a) pre-treated with plant extracts for different time points. Then, 500 ng/ml SARS-CoV-2 Spike Sl-His recombinant protein was added into each sample and further incu bated for 30-60 min or b) pre-treated with 500 ng/ml SARS-CoV-2 Spike Sl-His recombinant protein for 30-45 min prior to incubation with the plant extract for 30-60 sec at 4°C or 37°C. The samples were incubated in PBS buffer containing 5% FCS. Cells were then washed one time with PBS buffer containing 1% FCS at 500 x g, 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 analysed by using a FACSCalibur (BD Biosci ences, Heidelberg, Germany), 10 000 events were acquired. The median fluorescence intensity (MFI) of each sample were determined using FlowJo software (Ashland, Oregon, USA).

Infection of A549-hACE2-TMPRSS2 cells using SARS-CoV-2 pseudotyped lentivirus: SARS-CoV-2 spike pseudotyped lentivirus particles, produced with SARS-CoV-2 spike (Genbank Accession #QHD43416.1) as the envelope glycoproteins instead of the commonly used VSV-G, were pur chased from BPS Bioscience, (Catalog#: 79942, Biomol, Hamburg). These pseudovirions also con tain 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 Fire fly Luciferase Lentivirus (Puromycin) from BPS Bioscience (catalogue#: 79692-P) was used as pos itive control for transduction. These viruses constitutively express firefly luciferase under a CMV promoter. The above-mentioned lung cells were seeded at 0.1x106 cells/cm2 in 96-well plate in DMEM containing 10% heat-inactivated FCS, 100 U/ml penicillin/streptomycin, 100 pg/ml normocin, 0.5 pg/ml puromycin and 300 pg/ml hygromycin overnight. The medium was replaced by DMEM + 10% heat-inactivated FCS and cells pre-treated with a.d. or 10 mg/ml TO extract for 30 min. Afterwards, 5 pi of the lentivirus particles were added for 24 h. The medium was re moved by washing with PBS, fresh medium was added and cells incubated for another 60 h. Lu minescence was detected within 1h using the one-step luciferase analysis reagent from BPS fol lowing the manufacturer's protocol in a multiplate reader from Tecan (Tecan Group Ltd, Crail- sheim, Germany).

Molecular weight fractionation from plant extracts: Extracts from dried plant leaves were pre pared by adding destilled water (5 mL) to plant material (500 mg each). The samples were incu bated in the dark at RT for 60 min, followed by centrifugation at 16.000 g for 3 min. The superna tants were collected and membrane filtrated (0.45 pm), resulting in the extracts. Aliquots were freeze dried for 48h to determine their yield by weight. The extracts were then further separated in a high molecular weight (HMW) and low molecular weight (LMW), 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.

Statistical analysis: Results were analysed using the GraphPad Prism 6.0 software (La Jolla, Califor nia, USA). Data were presented as means + SD. Statistical significance was determined by the one-way ANOVA test followed by Bonferroni correction. P values <0.05 (*) were considered sta tistically significant and <0.01 (**) were considered highly statistically significant.

Example 2: Dandelion inhibits Spike S1 - ACE2 binding

The inhibition of interaction between SARS-CoV-2 spike protein RBD and ACE2 was investigated using extracts (25 mg of dried leave material per ml water) from dandelion leaves, collected at three different places in Freiburg, Germany. As seen in figure 3A, there was some variation be tween the three samples starting from 54.6% (sample 1A), 37.8% (sample 1B) and 69.4% (sample 1C) after 1h extraction. Extension of extraction time to 16h increased the inhibitory effect of the plants by about 10% to 68.6% (A), 47.3 (B) and 85.3 % (C). In figure 3B, the concentration de pendent inhibition of Spike S1 - ACE2 binding upon treatment with dandelion leave extract is given. The EC50 of dandelion was calculated in this assay at 12 mg/ml. The efficacy of other plant parts was also investigated in the binding assay. Dandelion flowers showed comparable effects as compared to the leaves (figure 3C), while the effect of root powder was weaker under the same extraction conditions. Interestingly, extracts from the subfamily Cichorioideae, the common chicory ( Cichorium intybus), and Hawkweed (Hieracium pilocella), also showed a concentration dependent binding inhibition, but with less potency than dandelion (data not shown for Hawkweed). The EC50 of chicory was calculated at 30 mg/ml (figure 3D). In contrast, chamomille ( Matricaria chamomiHa) and calen dula ( Calendula officinalis flowers, which belong to the subfamily Asteroideae, had either no or only marginal (<20%) effect in this assay (figures 3E and 3F).

In figure 4, results of different extraction methods are given. A 10 mg dried leaves per ml water extract, derived by using UAE at RT showed a 41.8% (1h) and 54.5% (16h) Spike-ACE2 binding in hibition. No further increase in the inhibitory potential could be seen for 50 mg/ml extract under these conditions. Compared to that, the extract generated after 1h without sonication could in hibit the Spike-ACE2 binding by 58.3% and 64.5% (extracted at RT and 100°C, respectively).

Example 3: The high molecular weight fraction is more effective than the low molecular weight fraction

Two fractions of the dried dandelion leaves and also of the chicory plant were prepared, separat ing the extract into a high molecular (>5kDa) and low molecular weight (<5kDa) fraction. As can be seen from figure 5, the bioactive compounds were mostly present in the HMW fraction.

Using hACE2 overexpressing HEK293 cells, the potential of dandelion and chicory extracts to block spike binding to cells was further investigated. As can be seen from figure 6, pre-incubation of cells with dandelion for one minute efficiently blocked cell binding of spike by 76.67% ± 2.9, and its HMW fraction by 62.5 ± 13.4% as compared to water control. After 3h, inhibition was still at 50 ± 13.6% for the extract, and 35.0 ± 20% for the HMW fraction of dandelion. For the chicory extract, binding inhibition was observed at 37 ± 20% after 1min. and 5.6 ± 9.9%.

Example 4: Effect on spike protein variants and block of virus entry

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 5-fold) than D614 spike protein to the ACE2 surface receptor of HEK293 cells (figure 7A). Pre-treatment with dandelion quickly (within 30 sec.) blocked spike binding to the ACE2 surface receptor (figure 7B). After 30 sec., this was 58.2 ± 28.7% for D614, 88.2 ± 4.6% for D614G, and 88 ± 1.3% for N501Y binding in hibition by dandelion extract. Even though for chicory extract a binding inhibition of spike could also be seen, this was about 30-70% less compared to dandelion, dependent on the spike pro tein investigated. When binding was studied at 37°C instead of 4°C, the results were comparable for dandelion, but seemed to be weaker for chicory extract in this cell line (figure 7D). For dande lion and chicory extracts the inhibition of spike binding was 47.90 ± 14.72 and 13.12 ± 12.37 (D614), 68.42 ± 14.53 and 8.86 ± 15.29 (D614G), 71.66 ± 7.66 and 37.56 ± 16.14 (N501Y), respec tively. The question was also raised whether the extracts could replace spike binding to the ACE2 surface receptor of human cells. For this, the cells were first incubated with D614, D614G or N501Y spike protein and subsequently with the extracts. As given in figure 7C, dandelion could potently remove spike from the receptor, with chicory being slightly weaker.

The experiments were extended to human A549-hACE2-TMPRSS2 cells and confirmed the results observed in HEK293-hACE2 cells for dandelion (figure 8A). This cell line has been stably trans fected with both, the human ACE2 and TMPRSS2 genes, and, interestingly, here the chicory ex tract was much more effective as compared to HEK-hACE2 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 dan delion extract and 56.1 ± 5.28 (D614) to 63.07 ± 14.55 (N501Y) for chicory extract. Already at 0.6 mg/ml, dandelion blocked binding to D614G spike protein by about 40%. When cells were pre incubated with the spike protein before extract treatment, results were comparable for dandelion extract for D614 and D614G but somewhat lower for N501Y (figure 8E). Extracts, incubated in hu man saliva for 30 min at 37°C before cell treatment had comparable effects on spike D614G inhi bition (figure 8B) indicating a good stability of the bioactive compounds in saliva.

Different extraction times and temperatures were tested in the setup of the human A549-hACE2- TMPRSS2 cells as discussed above and shown in figure 8A. The results of these conditions are shown in figure 10 and it can be concluded from the results that an extraction time of 1 minute is already sufficient to extract active compounds. Further, the extraction can successfully be carried out at temperatures ranging from 4°C to 100°C.

Using a SARS-CoV-2 spike pseudotyped lentivirus, it was then investigated whether the extract is capable of blocking virus entry via spike inhibition. As can be seen in figure 9, virus transduction was diminished by the extract at about 80%. Even though the use of pseudotyped viruses does not allow to assess the contribution of virion characteristics, such as membrane or envelope pro teins, on the cell tropism (17) they are a useful tool to document the relevance of ACE2 for the cell entry steps mediated by the spike protein. It could thus be shown that dandelion also blocks virus uptake.

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Claims

Claims

1. An extract from the leaves, flowers, stalks and/or roots of a plant species of the sub family Cichorioideae for use in the prevention or treatment of coronavirus disease 2019 (COVID-19), wherein the plant species is Cichorium intybusox Hieracium pilocella or Taraxacum officinalis.

2. The extract for use according to claim 1, wherein the extract is from the leaves, flowers and/or stalks.

3. The extract for use according to claim 1 or 2, wherein the extract is administered to the oral cavity, the nasal cavity and/or the pharyngeal cavity.

4. The extract for use according to claim 3, wherein the extract is for use in the preven tion of COVID-19.

5. The extract for use according to claim 1 or 2, wherein the extract is administered to the lung or the gastrointestinal tract or the systemic circulation.

6. The extract for use according to claim 5, wherein the extract is for use in the treat ment of COVID-19.

7. The extract for use according to any one of the preceding claims, wherein the COVID- 19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a variant thereof.

8. The extract for use according to claim 7, wherein the variant is variant B.1.1.7 or variant B.1.351 or variant P.1.

9. A pharmaceutical composition comprising an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae and optionally a phar maceutically acceptable excipient for use in the prevention or treatment of corona virus disease 2019 (COVID-19), wherein the plant species is Cichorium intybusox Hier acium pilocella ox Taraxacum officinalis and wherein the extract is the sole active ingre dient.

10. The pharmaceutical composition for use according to claim 9, wherein the pharma ceutical composition is a solution or an optionally coated tablet comprising dried ex tract.

11. The pharmaceutical composition for use according to claim 10, wherein the solution is selected from the group consisting of a decoction, an infusion, a juice of a fresh plant and a hydroalcoholic tincture.

12. Use of an extract from the leaves, flowers, stalks and/or roots of a plant species of the subfamily Cichorioideae for inhibiting the interaction between the spike protein or a mutant thereof of the SARS-CoV-2 virus, or a fragment thereof, and the ACE2 recep tor, or a fragment thereof, wherein the plant species is Cichorium intybusox Hier- acium pilocella or Taraxacum officinalis and wherein the extract is not for use in meth ods for treatment of the human or animal body.

13. The use according to claim 12, wherein the extract is used in a reconstituted biochem ical system or a cell culture system.