CN116867503A - Therapeutic compositions based on DHODH inhibiting leaves of aster in the treatment of RNA viral infections - Google Patents

Abstract

A therapeutic composition comprises mother liquor of tincture of leaf extract of plants of Nelumbo genus Nelumbo nucifera (Nelumbo tena) and Nelumbo nucifera (lobata) for inhibiting human dihydroorotate dehydrogenase (DHODH).

Description

Therapeutic compositions based on DHODH inhibiting leaves of aster in the treatment of RNA viral infections

RNA genome viruses cause many human pathologies such as influenza, dengue fever, hepatitis C, measles, infant bronchiolitis, or even the coronavirus recently called Covid-19.

Conventional therapies against RNA genome viruses include targeting the activity of viral proteins necessary for the viral cycle. These essential proteins may include, inter alia, RNA polymerase, integrase, helicase or protease. However, although these viral proteins can be conserved in several RNA genome viruses, the development of broad-spectrum therapeutic molecules active against a variety of viruses remains relatively limited.

In addition, the plasticity of the viral genome, as well as the adaptability and evolution of RNA genome viruses, facilitate the rapid emergence of escape mutants. These mutants render treatment with broad-spectrum therapeutic molecules targeted to viral proteins necessary for the viral cycle ineffective.

In order to circumvent these therapeutic target mutation problems, a new therapeutic approach has been developed in recent years that targets host cells rather than the virus itself. The principle of this new method is to block the cellular mechanisms necessary for viral replication, thereby preventing viral proliferation by stimulating an innate immune response in cells infected with RNA genome viruses.

One of the cellular mechanisms necessary for viral replication is known as the pyrimidine biosynthesis cycle. In fact, pyrimidines are critical to the survival of human cells, especially when the human cells are host cells for pathogens (especially RNA genome viruses).

More specifically, in mammalian and human cells, pyrimidine synthesis proceeds through two biosynthetic pathways: "pyrimidine de novo synthesis pathway", and another "salvage pathway" that proceeds under specific physiological conditions. Most human parasites do not possess a "salvage pathway" for pyrimidine synthesis. However, pyrimidine is necessary for the manufacture of pyrimidine nucleotides. These pyrimidine nucleotides are essential for cell survival and proliferation. Thus, blocking the "pyrimidine de novo synthetic pathway" is considered to be an effective therapeutic approach to selectively target human parasites without affecting the function of the human host and its normal cells.

In other words, all human pathogens, including RNA genome viruses, lack a "salvage pathway" for pyrimidine biosynthesis. Thus, the "pyrimidine de novo synthesis pathway" of a host cell is a targeted pathway for the elimination of these pathogens in therapy, particularly to block replication of RNA viruses in the host cell.

The pyrimidine de novo biosynthesis pathway is a synthetic pathway that proceeds in several sequential steps. This path and its steps are shown in figure 1 of the accompanying drawings.

More specifically, the fourth step of the pyrimidine de novo biosynthetic pathway involves the dehydrogenation of dihydroorotate, known as "DHO", which leads to the formation of orotate "ORO".

The oxidoreductase type enzyme that catalyzes the dehydrogenation reaction of dihydroorotate is the dihydroorotate dehydrogenase called "DHODH".

During the dehydrogenation of DHO to ORO, there is electron transfer between two cofactors, one of which is an electron donor and the other is an electron acceptor. For example, during this dehydrogenation of dihydroorotic acid, electron transfer is by means of the mononucleotide flavin FMN/FMNH ₂ Redox coupling and ubiquinone QH ₂ The coupling of/Q or nicotinamide adenine NAD+/NADH is completed. DHODH binds to its cofactor FMN and to ubiquinone to catalyze the oxidation of dihydroorotic acid to orotic acid.

The pyrimidine de novo biosynthetic pathway ensures the synthesis of uridine 5-monophosphate, known as UMP. UMP can be used as a precursor for other pyrimidine nucleotides. The latter is essential and indispensable for cell division and metabolic activity of host cells infected with RNA genome viruses. Thus, inhibition of DHODH in host cells has been shown to result in a decrease in the amount of pyrimidine in the infected cells, thereby amplifying the innate anti-viral immune response.

DHODH enzymes are known to fall into two groups, DHODH class 1 and DHODH class 2. These two classes of DHODH are established based on their sequence similarity, their binding sites, their cellular locations and their preferred substrates.

DHODH class 1 is a cytoplasmic enzyme present in protozoan pathogens.

DHODH class 2 is a monomeric protease that binds to the inner membrane of eukaryotic mitochondria.

In other words, DHODH belongs to class 2 in humans, which is a mitochondrial protein located on the outer surface of the mitochondrial inner membrane. Human DHODH has two domains, consisting of an alpha/beta barrel domain containing the active site and an alpha-helical domain, the latter forming a slit opening to the active site.

There are several known human DHODH inhibitors such as buquinate, teriflunomide or leflunomide.

For example, leflunomide is used to treat rheumatoid arthritis or multiple sclerosis. The immunosuppressive effects of leflunomide are due to consumption of pyrimidine uptake by T cells or more complex pathways mediated by interferons or interleukins. Inhibition of human DHODH by leflunomide is due to the immobilization of leflunomide on the alpha-helical domain, which forms a cleft opening to the active site of DHODH. Although leflunomide is marketed as a pharmaceutically active ingredient, it has side effects, especially diarrhea, nausea, vomiting, canker sores, abdominal pain, colon inflammation, headache, tendon inflammation, exacerbation of natural hair loss, eczema, dry skin, elevated transaminases or reduced leukocytes, in 1% -10% of patients.

Inhibition of human DHODH by buquinate is achieved by the same mechanism as leflunomide, i.e., by occupying the cleft opening to the active site. Buconazole was used as an anticancer drug at the end of the 1980 s; however, it has not been accepted as a drug because it causes various adverse side effects. In addition, buconazole is known to be highly toxic to human cells. Therefore, the idea of using it in therapy to combat viral infections is rapidly abandoned by the medical community.

Thus, known DHODH inhibitors appear not to be suitable for treating viral symptoms in patients during pathological processes associated with RNA genomic viral infection, in particular due to their side effects and cytotoxic effects on cells, regardless of cell type.

That is why in the case of viral infections, in particular RNA viral infections, it is necessary to find alternative solutions to known DHODH inhibitors such as buquinate, teriflunomide or leflunomide. In addition to blocking the pyrimidine de novo synthesis pathway by inhibiting DHODH enzymatic activity, this alternative solution is also expected to be non-cytotoxic to all human cells with minimal side effects when administered in pharmaceutical form.

Furthermore, in the present context, most patients depend on the source and design of the drug. In general, it is desirable for the patient to reduce the proportion of synthetic components constituting the drug as much as possible. These synthetic components may have side effects like the active components. Thus, in addition to inhibiting human DHODH, the present invention aims to find a DHODH inhibitor that is as natural in origin as possible, of simple design, with a low carbon footprint, so as to limit side effects while treating RNA viral infections and related symptoms.

The object of the present invention is to overcome the drawbacks of the prior art by proposing a therapeutic composition comprising a mother liquor of tincture of leaf extract of the genus chamomile (Neurolaena), the species of schizo She Maiju (lobata), for use as a medicament for inhibiting human dihydroorotate dehydrogenase (DHODH).

The advantage of this therapeutic composition is that the tincture mother liquor containing said split She Maiju (Neurolaena lobata) as active ingredient inhibits the activity of DHODH. The tincture mother liquor has the advantages of convenient and quick preparation and low cost in industrial scale. Moreover, the mother liquor of the tincture of the therapeutic composition of the invention is advantageously of natural origin, i.e. non-synthetic, while being recognized in the literature as non-cytotoxic in vivo.

Thus, the naturally derived active ingredients of the therapeutic composition cause little to no side effects, and are not cytotoxic. The therapeutic compositions of the present invention inhibit DHODH activity without cytotoxic effects after administration to a patient. Administration of the therapeutic composition containing the tincture stock results in an increase in the innate immune response in patients whose immune system is impaired by pathogens or any other disease.

Furthermore, according to other features of the invention, the therapeutic composition is used as a medicament for treating symptoms associated with RNA genomic viral infection.

The therapeutic composition, containing a mother liquor of a tincture of echinacea (Neurolaena lobata), has the characteristics of inhibiting DHODH activity and blocking pyrimidine from the head synthesis pathway, and is suitable for the case of viral infection caused by RNA viruses, especially for the case of RNA positivity.

In fact, RNA viruses lack a "pyrimidine salvage pathway". The cellular infection mechanism of RNA viruses should employ the host cell's "de novo synthesis pathway" to synthesize pyrimidine, which is necessary for the cellular replication mechanism, i.e., the proliferation of the virus. Thus, inhibition of the de novo synthesis pathway of host cells blocks cellular replication, i.e., viral replication of RNA viruses. It is therefore appropriate to use the therapeutic compositions of the invention as a medicament for the treatment of symptoms associated with RNA genomic viral infection.

According to a preferred embodiment, the therapeutic composition of the invention is used as a medicament for the treatment of symptoms associated with infection by an RNA genome virus selected from the viral families in the list: coronaviridae, flaviviridae, orthomyxoviridae, or togaviridae.

Particularly preferably, the invention also relates to a process for preparing a dry extract of a mother liquor of a tincture of dry leaves of split She Maiju (Neurolaena lobata), characterized in that it comprises the following steps:

i) Preparing a mixture based on dry leaves of echinacea (Neurolaena lobata) in a mass concentration of between 15 and 20g/L in 50 ° of policosanol;

ii) the mixture is immersed for about 21 days while stirring;

iii) After impregnation, the mixture was filtered on a "filter" with a porosity of 50-75 μm and a filtrate and solid retentate of the mother liquor of the tincture corresponding to the split She Maiju (Neurolaena lobata) dry leaf was obtained;

iv) diluting the liquid tincture mother liquor by 4 times by adding an aqueous solution and then obtaining an aqueous-alcoholic solution;

v) evaporating the alcohol contained in the solution by means of a rotary evaporator until an aqueous solution is obtained;

vi) freezing the aqueous solution thus obtained, followed by lyophilization, to obtain a dry extract of diluted tincture stock of split She Maiju (Neurolaena lobata) dry leaves.

Advantageously, the mass concentration of the mixture prepared in step i) is from 16 to 17g of split She Maiju (Neurolaena lobata) dry leaf/liter (L) 50 DEG policosanol, and preferably equal to 16g/L.

Particularly preferably, in step iv), the tincture mother liquor is diluted by mixing a volume of filtrate equal to 0.75L with a volume of water equal to 2.25L.

The invention also relates to a method for preparing a liquid solution of a dry extract of diluted lyophilized tincture of dry leaves of split She Maiju (Neurolaena lobata), obtained according to the above method, obtained by diluting the dry extract in water or in a pharmaceutically acceptable aqueous solvent, at a concentration of 6500 to 20000ng dry extract/mL aqueous solvent, preferably between 6667 and 20000 ng/mL.

In the present application, a pharmaceutically acceptable solvent means a solvent which can be used for preparing a pharmaceutical composition and has characteristics of being non-toxic and biologically acceptable for veterinary drugs as well as for human drugs.

The application also relates to a diluted mother liquor extract of a tincture of dry leaves of the schizophyllum (Neurolaena lobata), in particular a 4-fold dilution of the mother liquor of the tincture, which is in solution and has a final concentration of 6500 to 20000ng/ml (mass of the lyophilized dry extract of the diluted mother liquor/volume of the aqueous solution), for the treatment of a viral infection caused by SARS-CoV-2 virus causing Covid 19.

The diluted mother liquor extract of tincture of the split She Maiju (Neurolaena lobata) dry leaf as described above can be obtained by the above method.

The tincture mother liquor extract of split She Maiju (Neurolaena lobata) leaves is particularly suitable for use as a medicament for reducing cytokine production, particularly IL-6 and IP-10 production, in the treatment of severe viral infections caused by the SARS-CoV-2 virus causing Covid 19.

Other features and advantages of the application will be elucidated by the following detailed description of non-limiting embodiments of the application, with reference to the accompanying drawings, in which:

[ FIG. 1]: FIG. 1 schematically shows steps of the de novo pyrimidine production biosynthetic pathway in human cells, wherein the fourth step comprises a dehydrogenation reaction, i.e. the effect of the dihydroorotate dehydrogenase "DHODH" to convert dihydroorotate "DHO" to orotate ORO;

[ FIG. 2]: FIG. 2 schematically illustrates the inhibition of human mitochondrial DHODH by a composition of the present invention in a human host cell infected with an RNA genomic virus;

[ FIG. 3]: FIG. 3 shows a graph of optical density as a function of contact time between DHODH and its substrate for a sample consisting of a tincture mother liquor of a leaf extract of Prinsepia utilis or Prinsepia utilis (Neurolaena lobata), called "H1", or another tincture mother liquor of a leaf extract of Prinsepia utilis (Neurolaena lobata), called "H2", measured at 600nm at a concentration of 0.01 μg/mL;

[ FIG. 4]: FIG. 4 shows a graph of optical density as a function of contact time between DHODH and its substrate for a sample consisting of buconazole, or "H1", or "H2", measured at 600nm at a concentration of 0.1 μg/mL;

[ FIG. 5]: FIG. 5 shows a graph of optical density as a function of time of contact between DHODH and its substrate for a sample consisting of buconazole, or "H1", or "H2", measured at 600nm at a concentration of 1 μg/mL;

[ FIG. 6]: FIG. 6 shows the percentage inhibition of the activity of DHODH on its substrate observed between 0 and 275 seconds in the presence of a sample, as a function of the concentration of sample H1 or H2;

[ FIG. 7]: FIG. 7 shows the percent inhibition of DHODH on its substrate activity observed between 0 and 275 seconds in the presence of a sample, as a function of the concentration of sample H1, H2 or buconazole;

the invention relates to a therapeutic composition comprising a mother liquor of a tincture of leaf extract of plants of the genus Neurolaena, neurolata (lobata) as a medicament for inhibiting human dihydroorotate dehydrogenase (DHODH).

Figure 1 schematically shows a pyrimidine de novo synthesis pathway comprising several steps, wherein step 4 involves the action of human dihydroorotate dehydrogenase "DHODH".

In the pyrimidine de novo synthesis pathway, precursors to the pyrimidine nucleus are glutamine, aspartic acid and CO ₂ . For example, as shown in FIG. 1, in a first step, a carbamoyl phosphate is formed by a carbamoyl phosphate synthetase. In the second step, the aspartate transcarbamylase catalyzes the formation of carbamoylaspartate. The latter is converted in a third step to dihydroorotic acid "DHO" by means of a dihydroorotase. Then in a fourth step, the dihydroorotate dehydrogenase "DHODH" will catalyze the conversion of "DHOTo orotic acid "ORO", which will act as a reaction precursor to obtain uridine monophosphate "UMP". UMP is useful for the RNA polymerization mechanism required for cell proliferation.

According to the invention, the mother liquor of the tincture of leaf extract of plants of the genus Nelumbo (Nelumbo ena), schizophragma She Maiju (lobata) is advantageously of natural origin. In fact, split She Maiju (Neurolaena lobata) is a Compositae plant found in the West Indian islands and Central America, especially the Cyamopsis citrusca. In these areas, such plants are easy to plant and harvest due to the favourable agroecological and soil conditions.

Thus, the use of the extract of echinacea (Neurolaena lobata), due to its natural origin, advantageously allows to reduce the risk of side effects when it is used in pharmaceutical compositions for the treatment of diseases.

Furthermore, to support this view, several research and scientific publications have demonstrated that split She Maiju (Neurolaena lobata) extracts do not have any in vivo toxicity (Gracioso J.S. et al, J.Pharm.Pharmacol.1998, 50:1425-1429;Gracioso J.S. Et al, phytomedecine,2000, 7 (4), pages 283-289). In particular, in mice, no physiological toxicity was observed after days following oral ingestion of a 5000mg/kg dose of an aqueous alcoholic extract of the aerial parts of split She Maiju (Neurolaena lobata).

Thus, in addition to its natural origin, the extract of split She Maiju (Neurolaena lobata) is chosen at a selected concentration as the active ingredient of a therapeutic composition intended to inhibit DHODH activity, advantageously in accordance with the purpose of the present invention of being non-cytotoxic to all human cells, in particular those cells involved in an immune response.

According to the invention, the mother liquor of the tincture of the leaf extract of the split She Maiju (Neurolaena lobata) consists of an aqueous alcoholic solution.

According to a preferred embodiment, the tincture mother liquor is made up of only the plant leaf extract of the split She Maiju (Neurolaena lobata).

According to the invention, the therapeutic composition is in the form of a galenic form for oral ingestion. For example, the therapeutic composition is in a liquid form, such as a syrup form, or in a solid form, such as a tablet form.

According to the invention, the therapeutic composition contains as active ingredient the mother liquor of the split She Maiju (Neurolaena lobata) tincture having DHODH activity inhibitory effect, together with other excipients allowing the galenic formulation thereof. The possible interactions between these excipients and the mother liquor of the tincture do not interfere nor affect the inhibition of DHODH.

Preferably, the therapeutic composition of the present invention comprises only excipients of natural origin which do not cause side effects or cause few side effects when formulated with the split She Maiju (Neurolaena lobata) tincture mother liquor.

According to a preferred embodiment, the therapeutic composition is used as a medicament for the treatment of symptoms associated with viral infections.

More specifically, the therapeutic composition of the invention is used as a medicament for the treatment of symptoms associated with infection by an RNA genome virus selected from the viral families in the list: coronaviridae, flaviviridae, or togaviridae:

In the coronaviridae family, mention may be made, as an example, of SARS-CoV-2 causing Covid 19;

in the flaviviridae family viruses, the genus hepacivirus will be cited, the only representatives of which are the viruses responsible for hepatitis C, and the viruses of the genus flaviviridae or Zika viruses responsible for yellow fever, and,

among the torgaviridae viruses, the viruses causing chikungunya fever will be cited.

In fact, RNA viruses require the pyrimidine head synthesis pathway of the host cell to function in order to replicate the virus within the host cell. These viruses lack pyrimidine salvage pathways. However, if the cellular pathway is blocked by the use of the therapeutic compositions of the present invention that inhibit DHODH activity, RNA viruses will no longer be able to replicate in the host cell, and these viruses lack both the pyrimidine de novo and pyrimidine salvage synthetic pathways.

FIG. 2 schematically shows the mechanism of action of the composition of the present invention in inhibiting DHODH in human cells infected with RNA genomic viruses. Due to the tincture mother liquor of the split She Maiju (Neurolaena lobata) leaf extract of the composition of the present invention, DHODH induced dehydrogenation does not occur in mitochondria. Pyrimidine is blocked from the de novo synthesis pathway and the salvage synthesis pathway that it lacks is not available. UMP and the nucleotide pyrimidinyl cannot be used by RNA viruses to initiate viral replication.

Thus, even after introduction of the RNA viral genome into the host cell, the host cell is unable to produce viral particles. Inhibition of DHODH prevents viral proliferation of RNA genome viruses and prevents the production and escape of viral particles out of the host cell membrane. Thus, the use of the composition of the present invention having DHODH activity inhibitory effect can prevent the development of symptoms caused by the proliferation of viruses and the presence of pathogens in the body. The compositions of the invention are therefore useful therapeutic means for the treatment of viral infections, in particular for the treatment of RNA genomic viral infections.

Thus, the use of the therapeutic compositions of the invention as a medicament for treating symptoms associated with RNA genome viral infection, including solutions to combat infection and limit viral proliferation.

The therapeutic compositions of the present invention are a good alternative to existing treatment regimens aimed at inhibiting viral diseases of DHODH. This particular use allows preventing viral replication in host cells by inhibiting pyrimidine de novo synthesis pathways while enhancing immune responses against cells and without cytotoxic effects.

The therapeutic compositions of the present invention thus include an alternative to allow inhibition of DHODH, i.e., blocking the pyrimidine de novo synthesis pathway necessary for RNA virus replication, which does not attack and destroy cells involved in the immune response against the pathogen.

The therapeutic composition of the present invention also has the advantage of being easy to manufacture while being as natural as possible in the eyes of the consumer and the patient in whom it will be used.

The following test results are intended to illustrate the inhibition of DHODH by the therapeutic compositions of the present invention.

The other in vitro test results obtained, detailed below, in particular illustrate the effect of the therapeutic compositions of the invention, in particular on the viral infection severity caused by the SARS-CoV-2 virus causing Covid-19.

In fact, these results indicate that the therapeutic extract of the present invention, containing the mother liquor of the tincture of the leaf extract of the schizophyllum (Neurolaena lobata), has a particularly interesting effect on reducing the ratio of certain cytokines released by cells after infection with the SARS-CoV-2 virus.

Cytokines are proteins that are naturally synthesized by immune cells to mediate the immune response following infection by pathogens. They promote a natural inflammatory response to protect the infected body against pathogens.

However, in some cases of SARS-CoV-2 infection, cytokines are released in large amounts, especially in lung cells, so as to trigger a "cytokine storm". Such immune system overreactions can lead to hyper-inflammatory reactions, possibly damaging tissues, causing acute respiratory distress syndrome, leading to physiological deterioration and even being fatal to the person triggering such reactions.

As shown below, the results of in vitro experiments with therapeutic compositions according to the invention employing tincture mother liquor containing extract of leaves of the plants of the Dioscorea schizophyllum (Neurolaena lobata) demonstrate that cytokine production can be significantly reduced by the action of said compositions.

To demonstrate the test results related to DHODH inhibition, therapeutic compositions of the present invention containing tincture mother liquor of a leaf extract of a stevia (Neurolaena lobata) plant were tested.

The tincture mother liquor forms a sample to be tested. The measurement of inhibition of the substrate effect by DHODH was performed in a transparent-wall conventional multiwell plate. The hole contains the sample to be tested, DHODH and the substrate thereof.

To evaluate the inhibition of DHODH by the samples, an optical density parameter called "DO" was used. In practice, DO at 600nm was measured in each well at several time intervals within 5 minutes.

More specifically, each well contains a diluted or undiluted sample, DHODH enzyme and its colorimetric substrate diluted in a test buffer. The colorimetric substrate comprises DHO, which can be converted to ORO by the action of DHODH.

Over time, the consumption of DHODH enzyme versus color substrate DHO appears as a decrease in DO. This decrease means that colored DHO is converted to colorless ORO due to DHODH activity. In other words, DHO is consumed, which results in a change in the measured DO as the activity of DHODH is reduced to ORO.

In the case of DHODH activity inhibition by the sample, DO remained stable over time. Indeed, under inhibition, the colorimetric DHO substrate is not converted to ORO by DHODH, so DO remains as DO of the original DHO.

For the test, the following were used:

class 1 enzyme solutions called "rhDHODH" for "recombinant human DHODH",

-consists of 50mM Tris, 150mM KCl and 0.1%X-100, pH 8,

-a substrate mixture of DHODH comprising: 2mM L-dihydrowhey, called "DHO", 0.2mM dechlorobenzoquinone, called "Q", and 0.12mM 2.6-dichloroindophenol sodium salt hydrate, called "DPIP", were dissolved in test buffer,

diluted or undiluted samples of the mother liquor of tinctures of plant leaf extracts She Maiju (Neurolaena lobata) were dissolved in a dilution buffer of dimethyl sulfoxide called "DMSO".

In the substrate mixture of DHODH, L-dihydroorotate called "DHO" is a colorimetric substrate used for DHODH during the dehydrogenation reaction.

Among the substrate mixtures, dechlorobenzoquinone known as "Q" and dichloroindoxyl sodium salt hydrate known as "DPIP" are electron acceptors and electron donors. The transfer of these electrons allows the dehydrogenation reaction to be carried out by DHODH oxidoreductase.

Preparation of tincture mother liquor of the extract of the leaves of the plants of the split She Maiju (Neurolaena lobata) type constituting sample H1：

Tincture mother liquor H1 of the split She Maiju (Neurolaena lobata) plant leaf extract was obtained by the following steps of the protocol:

harvesting the split She Maiju (Neurolaena lobata) leaves,

drying the said blades and/or vanes of the plant,

crushing the dry leaves until a powder is obtained,

immersing the powder in the policosanol solution for 21 days until an immersion liquid is obtained, the dosage ratio being 1

g powder with 62.5mL of policosanol solution,

the impregnation is filtered until a filtrate, called H1, is obtained.

In the above method, according to a preferred embodiment, the blade may be dried under a stream of hot air, preferably at a temperature below 40 ℃, for about 120 hours until its residual moisture content is about 6% to 9%, preferably 6.5% to 9%.

The water content is determined by any suitable method known to those skilled in the art. For example, the moisture content may be measured using a dryer installed in a room having a temperature below 40 ℃ and a relative humidity below 85% without direct exposure to sunlight, air flow, or vibration.

For example, the XM60 dryer sold by pressa MOLEN, france, has a standard accuracy of 1mg at high resolution and a temperature range of 30 ℃ to 230 ℃ in 1 ℃ increments, and can be used to measure the residual moisture content of the blade.

Preferably, in the above protocol, the powder is immersed in a solution of policosanol at a temperature of 25 ℃ to 30 ℃, preferably 30 ℃, for about 21 days, with slow stirring for 12 hours per day.

Preparation of tincture mother liquor of the extract of the leaves of the plants of split She Maiju (Neurolaena lobata) constituting sample H2

Tincture mother liquor H2 of the split She Maiju (Neurolaena lobata) plant leaf extract is obtained by the following steps of the method:

recovering split She Maiju (Neurolaena lobata) leaves,

-drying the said blades and/or vanes,

100g of dry leaves are immersed in 1L of pure ethanol solution for 7 days until an immersion liquid is obtained,

filtering the impregnation solution on celite until a filtrate is obtained,

-concentrating the filtrate by means of a rotary evaporator.

The concentrated filtrate is dried under pressure, in particular using a vacuum tank, until sample H2 is obtained.

Analysis protocol for the inhibition of DHODH by compositions of the present invention。

To demonstrate the inhibition of DHODH by the therapeutic compositions of the present invention, two mother liquors, designated H1 and H2, respectively, were prepared.

Mother liquor H1 was obtained by carrying out the above method after harvesting of split She Maiju (Neurolaena lobata) plant leaves.

Mother liquor H2 is obtained by carrying out the above-described process.

To test the effect of samples on DHODH inhibition and to understand the "dose response effect", each sample H1 and H2 was diluted with DMSO buffer.

Diluting samples H1 and H2 allows the following concentrations to be obtained: unit is μg sample/mL total solution in well: 0.01 μg/mL;0.1 μg/mL;1 μg/mL;10 μg/mL;100 μg/mL; 1000. Mu.g/mL, as shown in tables 1 and 2, respectively, below.

To initiate this protocol, dilutions of sample H1 or H2 were placed in wells in the presence of rh DHODH enzyme and substrate mixture. To obtain an average of DO measurements for a concentration of sample H1 or H2, three measurements were made for each diluted concentration of H1 and H2.

More specifically, in order to obtain the following results, the following steps of the embodiments are performed:

adding the rh DHODH enzyme prepared on the same day in the test buffer to each well containing a dilution of sample H1 or H2,

placing at 37℃for 6 minutes in the presence of enzyme and sample,

then 50. Mu.L of the above-mentioned coloring substrate mixture is added, in this case for a time of 0,

the DO at 600nm was measured in each well every 55 seconds at regular time intervals by immediate interpretation within 275 seconds.

The amount of enzyme added to each well is the same. In this protocol, enzyme was added to the wells so as to obtain a concentration of 0.06 μg rh DHODH enzyme per mL total solution in the wells.

Table 1 below shows the results obtained for sample H1:

Table 1: inhibition of DHODH activity by sample H1

In Table 1, the first column gives the OD measurement time interval at 600 nm. In other words, this corresponds to the contact time of sample H1 with rh DHODH in the presence of the substrate mixture.

In table 1, the second row gives the concentration of sample H1 in the well. The unit of this concentration is μg of total solution in the wells of sample H1/mL.

Each column of table 1 gives the DO average of three measurements at 600nm at the same concentration of sample H1 and over the specified contact time with rh DHODH and its substrate.

For example, as shown in Table 1, the DO average value measured for three wells of the same volume after a sample H1 at a concentration of 0.1 μg/mL was contacted with rh DHODH and a colored substrate mixture for 110 seconds was 0.315.

The row with symbol Δ in table 1 shows the difference between the DO average value measured when the samples H1, rh DHODH, and their substrates coexist for 0 seconds and the DO average value measured when they coexist for 275 seconds.

The symbol delta represents a decline in DO between 0 and 275 seconds of coexistence, that is, the ability to convert DHO to ORO by the effective activity of DHODH.

In table 1, the last two rows show the percentage of activity of converting colored DHO to ORO by the activity of rh DHODH at each concentration of sample H1, and the percentage of inhibition of rh DHODH activity by sample H1.

At a given concentration of sample H1 (then H2), the percentage activity is calculated as follows:

percent activity= (Δh1x100)/Δ of negative control.

For example, for sample H1 of 0.01 μg/mL: percent activity= (0.092x100)/0.208= 44.231.

Percent inhibition is calculated by the formula: 100-value of percent activity.

To verify the effective activity of rh DHODH to convert its DHO substrate during the test, two negative controls were prepared. The negative control is mainly used for verifying the activity of DHODH on a substrate thereof and measuring the activity percentage and the inhibition percentage of a sample.

The negative control columns give the DO average measured at 600nm at various measurement intervals (units: seconds).

The negative control comprises: rh DHODH, indicated in Table 1 as "E", was present in the total solution in the well at a concentration of 0.06. Mu.g/mL, containing 50. Mu.L of the colored substrate mixture, indicated as "S", and sample H1 was replaced with DMSO-only buffer, indicated as "T".

The results showed a decrease in the DO average measured at 600nm between 0 and 275 seconds.

Thus, the colored substrate DHO is fully converted to ORO due to the reducing activity of the rh DHODH enzyme. The rh DHODH enzyme works well for substrate mixtures. In addition, neither the DMSO buffer nor the test buffer to dilute the DHO substrate affected the dehydrogenation activity of the rh DHODH enzyme.

As shown in Table 1, sample H1 of the present invention inhibited the activity of DHODH on its substrate. The percent inhibition was between 51% and 56% at all tested concentrations of H1. It was also found that for the tested concentrations, the increase in the concentration of sample H1 did not equate to an increase in the percentage of inhibition of DHODH activity.

The same protocol and the same DO measurement allow quantification of the percent inhibition of sample H2.

Table 2 below gives the results obtained for sample H2 at different concentrations tested.

Table 2: inhibition of DHODH activity by sample H2

As shown in Table 2, sample H2 of the present invention inhibited DHODH activity.

The percent inhibition was between 48% and 67% at all tested concentrations of H2.

Unlike sample H1, it appears to have a dose-responsive effect. Indeed, at a concentration of 1000. Mu.g/mL, the percent inhibition appears to be significantly higher than at a concentration of 0.01. Mu.g/mL.

In other words, in the presence of the enzyme, the percent inhibition increases as the concentration of sample H2 increases. Sample H2 differs from sample H1 in the preparation protocol of the leaf extract of split She Maiju (Neurolaena lobata). Thus, it appears that the cause of the rh DHODH inhibitory activity is concentrated on the leaf, and the inhibitory activity varies depending on the preparation method of the leaf tincture mother liquor.

Figures 3 to 5 show the change in optical density measured at 600nm for H1, H2 or buconazole samples at a given concentration over time.

The graph in FIG. 3 shows the measurement of DO values over time at 600nm for samples of total solution in wells at a concentration of 0.01. Mu.g/ml.

Also, the curve in FIG. 4 is a sample having a concentration of 0.1. Mu.g/mL, and the curve in FIG. 5 is a sample having a concentration of 1. Mu.g/mL.

In any case, the DP at 600nm remains substantially stable over time for the known DHODH inhibitors, regardless of the concentration of the sample. Thus, buquinate is a DHODH inhibitor.

For samples H1 and H2, a decrease in DO measured at 600nm was observed. This decrease means a decrease in rh DHODH activity. However, it was found that for each sample, DO drops sharply starting from 110 seconds of contact.

Between 0 and 110 seconds, the DO measured at 600nm is fairly stable. This seems to mean that the DHODH enzyme is not immediately active. In the first 110 seconds, DHODH did not or substantially did not undergo dehydrogenation in the presence of sample H1 or H2. Thus, samples H1 and H2 appear to slow down the triggering of DHODH on its substrate activity.

To effectively verify the reduction activity of the rh DHODH enzyme on its substrate, two positive controls were prepared. The positive control well contains, instead of the diluted samples H1 or H2, buquinate, which is a known inhibitor of DHODH activity.

At the same time as measuring sample DO, DO of the bupquinate solution diluted with DMSO buffer was measured in the presence of rh DHODH and its substrate.

In the same manner as the samples, the inhibition of rh DHODH by the buconazole solutions of different concentrations was measured.

To verify the activity of rh DHODH on its substrate, three positive controls were prepared at the same time as the measurement protocol for buconazole inhibition of DHODH. The positive control wells contained rh DHODH, termed "E", and its substrate mixture, termed "T", and DMSO buffer instead of buconazole, termed "T".

Table 3 below shows the results obtained for the buconazole samples at the different concentrations tested.

Table 3: inhibition of DHODH activity by buquinate

In Table 3, the activity of rh DHODH for converting its substrate was verified as a positive control. In fact, a decrease in DO measured at 600nm was effectively observed over time. This decrease is due to the conversion of DHO to ORO due to the dehydrogenating activity of rh DHODH. Neither DMSO buffer nor test buffer affected the activity of the rh DHODH enzyme. Thus in this measurement regime of buconazole inhibiting DHODH, the rh DHODH enzyme acts.

Table 3 shows that the various tested concentrations of buconazole have inhibitory activity against rh DHODH, ranging from 92% to 100%.

To demonstrate the inhibitory activity of samples H1 and H2, FIG. 6 shows a plot of the relative percent inhibition as a function of concentration for samples H1 and H2 after 275 seconds of contact with DHODH and its substrate.

In FIG. 6, H1 was found to be relatively stable in percent inhibition of rh DHODH at concentrations between 0.01 and 1000 μg/mL.

In contrast, when the concentration exceeds 100. Mu.g/mL, the percentage of inhibition by H2 increases significantly. In particular, the percent inhibition of rh DHODH advantageously reaches 66.3% at a concentration of 1000. Mu. g H2/mL of total solution in the wells.

Likewise, fig. 7 shows the percent inhibition as a function of concentration for samples H1, H2 and buconazole after 275 seconds of contact with DHODH and its substrate. Samples H1 and H2, like buconazole, were found to be inhibitors of the activity of DHODH on its substrate. In fact, although the inhibition of DHODH by samples H1 and H2 did not reach 100% and was as effective as buconazole, it did exist.

Thus, based on the results obtained, the natural source of split She Maiju (Neurolaena lobata) tincture mother liquor samples H1 and H2 had potent and significant inhibitory activity on DHODH enzyme.

Thus, the therapeutic composition of the present invention comprising a mother liquor of a tincture of leaf extract of a plant of the genus Neurolaena (Neurolaena) genus, neurolaena She Maiju (lobata) species has an effect of inhibiting DHODH activity.

Thus, a therapeutic composition comprising any of these samples, namely a mother liquor of a tincture based on leaves of the schizophyllum (Neurolaena lobata), is a natural, non-toxic product, and is expected to treat diseases aimed at inactivating DHODH.

In particular, the therapeutic compositions of the invention are viable methods for treating diseases caused by viral pathogen infection, particularly RNA genome viral infection.

In vitro tests were also performed to demonstrate the antiviral and virucidal efficacy of therapeutic compositions based on tincture mother liquor of echinacea (Neurolaena lobata), as well as to determine inhibition of certain cytokine release.

Preparation of the sample to be tested：

Several samples were prepared using split She Maiju (Neurolaena lobata) plant leaves to conduct these tests.

The sample is denoted by "TOTUM".

"TOTUM 3" corresponds to a tincture mother liquor obtained from leaves of Echinacea paradoxa (Neurolaena lobata); the preparation method comprises the following steps:

i) Mixing 3.6kg of dry leaf of split She Maiju (Neurolaena lobata) with a volume of 225L of 50 ° policosanol, which corresponds to a mass concentration of 16g of split She Maiju (Neurolaena lobata) dry leaf/liter policosanol; more broadly, a mixture of split She Maiju (Neurolaena lobata) dry leaf/liter 50 ° policosanol is prepared at a mass concentration of 15 to 20g/L, preferably 16 to 17g/L, more preferably equal to 16 g;

Preferably, as in the preparation of sample H1 described above, the leaves may be dried under a stream of hot air, preferably at a temperature below 40℃, for about 120 hours until their residual moisture content is about 6% to 7%. The water content can be measured by the same method as H1.

ii) the mixture was immersed with stirring at a temperature of about 30℃for about 21 days, with slow stirring every day for 12 hours.

iii) After impregnation, the mixture is filtered on a "filter" or filter bag with a porosity of 50-75 μm and a filtrate and retentate are obtained;

iv) sampling 3L of filtrate;

v) the alcohol contained in the filtrate was evaporated three times by means of a rotary evaporator until an aqueous solution of about 1L in volume was obtained.

vi) the aqueous solution thus obtained is frozen and then lyophilized.

Finally, a lyophilisate with a mass equal to 12.3g is obtained.

"TOTUM 4" is a diluted sample of the mother liquor of the tincture.

More specifically, in order to obtain such a sample, in step iv) of the protocol to obtain TOTUM 3 below, 0.75L of filtrate is sampled instead of 3L, said filtrate being diluted with a volume of water equal to 2.25L. Thus obtaining a hydroalcoholic solution with a volume equal to 3L. In general, the filtrate or the mother liquor of the tincture obtained in step iii) above is diluted 4-fold.

Then, the following step of obtaining the TOTUM 4 is similar to the step performed for obtaining the TOTUM 3, and as described above, namely:

v) evaporating the alcohol contained in the hydroalcoholic solution, preferably in a volume equal to 3L, by means of a rotary evaporator, for example in three times, until an aqueous solution is obtained, the volume of which is approximately equal to 1L.

vi) the aqueous solution thus obtained is frozen and then lyophilized.

Finally, a dry extract of a diluted (quadruple) tincture mother liquor was obtained with a mass equal to 5.6g of lyophilizate and corresponding to split She Maiju (Neurolaena lo-bata) dry leaves.

As a negative control, a sample called "TOTUM 2" was prepared using Mu Sa dried banana (Musa sapentium) pulp.

More specifically, to obtain this sample, 150g of dry banana pulp was diluted in 5L of policosanol at 50 °.

The mixture was immersed for about 5 days, stirred for 2 to 3 hours per day, and then filtered on a filter paper having a porosity of 10 to 20 μm to obtain 4L of hydroalcoholic filtrate.

The filtrate was then evaporated by means of a rotary evaporator until a dry extract with a mass equal to 6g was obtained.

Evaluation of TOTUM2, 3, 4 during treatment of human lung epithelial cells (Calu-3) and renal cells (VeroE 6-TMPRSS 2) Inhibition of SARS-CoV2 virus amplification causing Covid19

The SARS-CoV2 virus strain used to conduct these tests is the European strain (mutation of the D614G strain) corresponding to the SARS-CoV-2 reference strain Slovakia/SK-BMC5/2020.

The strain is provided by the European virus archive Global (Evag) (https:// www.european-virus-archive. Com /) platform.

SARS-Cov2 strain was amplified by Oncodestin on VeroE6 TMPRSS2 cell line and titrated.

Two cell lines were used in these evaluation tests: namely the following cell lines:

calu-3, human lung adenocarcinoma (from ATCC-American type culture Collection);

vero E6-TMPRSS2, non-human primate kidney epithelial cells (from NIBSC-british national biological institute).

Calu-3 cell model has been fully described in the literature of Sars-CoV (see C. -T.K.Tseng, J.Tseng, L.Perrone, M.Worthy, V.Popov and C.J.Peters, "apical entry and release of Severe acute respiratory syndrome-associated coronavirus in polarized Calu-3 lung epithelial cells", J Virol, vol. 79, pp. 15, 9470-9479, month 8 2005, doi: 10.1128/JVi.79.15.9470-9479.2005).

Calu-3 cells were grown in monolayers in a humidified atmosphere (5% CO2, 95% air) at 37℃in corresponding cell culture media (MEM+1% pyruvic acid+1% glutamine+10% bovine fetal serum).

In a humidified atmosphere (5% CO) ₂ 95% air), vero E6-TMPRSS2 cells were grown in monolayers in the corresponding cell culture medium (dmem+1% pyruvic acid+1% antibiotic mixture (penicillin, streptomycin and geneticin) +2% foetal calf serum) at 37 ℃.

Cells of both cell lines adhere to plastic vials. For the cell passaging procedure, cells were isolated from the flask by trypsin-vinylic treatment for 20 minutes (Vero-line cells) or 5 minutes (callu-line cells) and were neutralized by adding complete medium. In this study, cells were deposited on 96-well plates.

Cells were counted and their viability was assessed using a Vi-cell counter.

In a first set of tests, termed "CAS1", cells of the two cell lines are contacted with the test compound (especially TOTUM 2, 3, 4) for 24 hours prior to exposure to the SARS-CoV-2 virus strain. The first group, "CAS1", allows the study of antiviral effects, i.e., cells were treated with compounds prior to infection.

In a second set of tests, termed "CAS2", the SARS-CoV-2 strain is contacted with various test compounds, including TOTUM 2, 3, 4, for 30 minutes at room temperature prior to contacting the cells with the virus. The second set of "CAS2" allows the study of virucidal effects, i.e., the virus is contacted with the compound prior to contact with the cells.

Test protocol for CAS1 testing Using Calu-3 and VeroE6TMPRSS2 cell linesCounting cellsAnd its viability was assessed using a Vi-CELL analyzer.

Cells were seeded to achieve fusion:

-Vero E6 TMPRSS2-30000 cells/well;

calu-3-90000 cells/well.

A10 mg/mL DMSO stock solution was prepared using the lyophilizates and dry extracts of TOTUM 2, 3 and 4. Using these stock solutions, seven concentrations of test compounds were prepared in complete growth medium and added to the cells: 10000. 3333, 1111, 370, 123, 41, 14ng/mL.

These concentrations were used for the first biological replication (n=1).

A second biological replication was performed with different concentrations (n=2). In fact, the concentration of the test was adjusted after analysis of the results of the first biological replication.

Thus, for n=2 replicates, the following concentrations were employed: TOTUM 3 is 100000, 33333, 11111, 3704, 1235, 412 and 137ng/mL, TOTUM 2 and TOTUM 4 is 20000, 6667, 2222, 741, 247, 82 and 27ng/mL.

As a reference control compound or positive control, active metabolites of adefovir are used. Seven concentrations of adefovir (20000, 6667, 2222, 741, 247, 82, 27 nM) were prepared and added to cells.

The active metabolite of adefovir is provided by oncostign as a 20mM DMSO stock solution.

Plates were incubated at 37℃for 24 hours.

Then, a volume of 10 μl of virus preparation, equivalent to MOI (multiplicity of infection) =0.01, was added and incubated at 37 ℃ for 48 hours (VeroE 6TMPRSS2 cells) or 72 hours (Calu-3 cells).

A portion of the supernatant (50. Mu.L) was collected and stored at a temperature equal to-20℃to determine viral load.

A portion of the supernatant (200. Mu.L of triplicate: 2X 50. Mu.L + remaining volume) was collected and stored at a temperature equal to-20℃to determine cytokine dose.

Using Calu-3 and VeroE6TMPRSS2 cell linesTest scheme for CAS2 testCELLs were counted and their viability was assessed using a Vi-CELL analyzer.

Cells were seeded to achieve fusion:

-Vero E6TMPRSS 2-35000 cells/well;

calu-3-80000 cells/well.

Seven concentrations of test compounds were prepared in fresh growth medium using lyophilisates and dry extracts of total 2, 3 and 4: 10000. 3333, 1111, 370, 123, 41, 14ng/mL.

These concentrations were used for the first biological replication (n=1).

As with CAS1, a second biological replication (n=2) was performed at different concentrations.

Thus, for n=2 replicates, the following concentrations were employed: TOTUM 3 is 100000, 33333, 11111, 3704, 1235, 412 and 137ng/mL, TOTUM 2 and TOTUM 4 is 20000, 6667, 2222, 741, 247, 82 and 27ng/mL.

Similarly, seven concentrations of reference or positive control, active metabolite of adefovir (20000, 6667, 2222, 741, 247, 82, 27 nM) were prepared.

A volume of 10 μl of the virus preparation, corresponding to moi=0.01, was mixed with the test compound and incubated for 30 minutes at room temperature.

The compound/virus mixture is then added to the cells.

Cells were incubated at 37℃for 48 hours (VeroE 6 TMPRSS2 cells) or 72 hours (Calu-3 cells).

A portion of the supernatant (50. Mu.L) was collected and stored at-20℃to determine viral load.

A portion of the supernatant (200. Mu.L of triplicate aliquots: 2X 50. Mu.L + remaining volume) was collected and stored at-20℃to determine cytokine doses.

Note that: viral-free plates were made to assess the cytotoxicity of the test compounds against both cell types. Cell viability was assessed under all conditions by CellTiter Glo test according to manufacturer's recommendations (Promega, G7570).

For CAS1 and CAS2, viral load quantification was performed by RTqPCR targeting the ORF1ab viral gene at the end of the experiment.

The extraction of viral RNA was accomplished by Macherey Nagel Viral RNA kit and RNA was frozen at-80℃until RT-qPCR was performed.

Using SuperScript ^TM The One-Step qRT-PCR system kit performs complete RT-qPCR by primer and ORF1ab gene targeting qRT-PCR condition. Amplification was performed using Bio-Rad CFX384 ^TM The device and corresponding software.

Also for CAS1 and CAS2, cytotoxicity tests or CellTiter-Luminescent cell viability test to assess cytotoxicity of the test sample.

CellTiter-The luminescent cell viability test is a general method for determining the number of living cells in a culture, which is based on the quantification of the presence of ATP (an indicator of metabolically active cells).

The method was used on VeroE6-TMPRSS2 and Calu-3 cells in the absence of virus to determine cytotoxicity of each test compound.

The method is also used to infect VeroE6-TMPRSS2 cells 48 hours later in the presence of virus in the presence of cytopathic effects (active virus); viruses are present in the Vero cell model and appear cytopathic after use of the cell apparatus, while viruses are continuously produced in the Callu model.

Testing was performed according to the supplier's protocol.

After removal of all supernatants for PCR reactions and cytokine assays, 100 μl of fresh cell culture medium was added to 100 μl of reagent and incubated for at least 15 minutes after mixing until luminescence was recorded.

For CAS1 and 2, cell culture supernatants collected 48 hours and 72 hours post-infection of Vero and Calu-3 cell lines were subjected to cytokine dosimetry, particularly IL-6, MCP1 and IP10, by ELISA using a commercially available kit.

Results: cytotoxicity assessment of TOTUM (no Virus)

The toxicity of "TOTUM" test samples and of the active metabolite of Readefovir on Calu-3 cells not exposed to the virus was assessed by cell viability measurements performed 96 hours after exposure to the compound.

The results are shown in the following table, with cell viability expressed as a percentage of untreated cells after exposure to Calu-3 cell line compounds, ET corresponding to standard deviation:

the results of biological replication after treatment with the adefovir dipivoxil active metabolite were similar, with an average cell viability of 91% (27 nM, n=1) to 116% (247 nM and 20000nM, n=1).

Cell viability after treatment with total 2, 3, 4 at seven concentrations of 14ng/mL to 10000ng/mL at biological replication n=1 was similar to that obtained after treatment with Jing Ruide civir active metabolite.

Similar results were obtained for TOTUM 2 and 4 at concentrations of 137ng/mL to 100000ng/mL for biological replication N=2 (see Table below).

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However, at a concentration of 100000ng/ml, a decrease in cell viability was observed when TOTUM 3 was used.

Toxicity of the TOTUM 2, 3, 4 and Ruidexivir active metabolites on VeroE6-TMPRSS2 cells not exposed to virus was assessed by cell viability measurements performed 72 hours after exposure to the compounds.

The results are shown in the following table, with cell viability expressed as percent of untreated cells after exposure to VeroE6 cell line compounds, ET corresponding to standard deviation:

the results of biological replication after treatment with the adefovir dipivoxil active metabolite were similar, with an average cell viability of 92% (27 nm, n=1, left table below) to 109% (20000 nm, n=2, right table below).

At n=1, cell viability after total 2, 3, 4 treatment was similar to that obtained after Jing Ruide ciwei active metabolite treatment at seven concentrations of 14ng/mL to 10000 ng/mL.

Similar results were obtained for TOTUM 2 and 4 at concentrations of 137ng/mL to 100000ng/mL for the test at N=2.

In addition, for TOTUM 3, the concentration was 33333ng/mL to 100000ng/mL, which was cytotoxic.

Results: antiviral action of CAS 1-Compounds

The purpose of these tests is to evaluate the antiviral effect of the compounds to be analyzed, i.e. cells are treated with the compounds prior to infection.

First, viral RNA was quantified by targeting the ORF1ab gene, and viral load was assessed on the Calu-3 cell line. The infected control cells were 100% reference.

The results of biological replication after treatment with the active metabolite of adefovir are similar. Viral load decreases with increasing compound concentration, wherein:

at n=1, the percentage relative to infected and untreated cells is 84% (27 nM radciclovir) to 0% (2222 nM, 6667nM and 20000 nM) (viral load is undetectable),

at n=2, the percentage relative to infected and untreated cells is 92% (55 nM radciclovir) to 0% (4444 nM, 13 333nM and 40 000 nM) (viral load is undetectable),

for the test compounds, the viral load was 67% (370 ng/mL of total 3) to 280% (10000 ng/mL of total 2) at n=1.

At n=2, the result of total 2 is similar to n=1 at seven concentrations. Viral loads of 59% and 49% were observed for TOTUM 4 at 6667ng/mL and 20000 ng/mL.

In addition, no detectable viral load (0%) was observed for TOTUM 3 at 100000 ng/mL.

Three cytokine (IL 6, IP10 and MCP 1) dosimetry was performed on cell culture supernatants (units: ng/mL) using Calu 3 lung cell line inoculated with SARS-CoV-2. Cells were treated with the test product for 24 hours and then inoculated with the strain for 72 hours. The compound concentration of the active metabolite of rituximab is in nM and the compound concentration of the test TOTUM is in ng/mL.

Il6 is a pro-inflammatory cytokine with basal levels of 300pg/mL. In the case of infection, the level was greatly increased by about 2000pg/mL. Chemokine IP10 was involved in the inflammatory process and basal levels were undetectable. In the case of infection, the level was greatly increased by about 400pg/mL.

No cytokine MCP1 was detected in the study performed. Cytokines have transient expression; thus, in performing dose measurements, cytokines may have been expressed or will be expressed, as single point interpretation, rather than kinetic interpretation, is performed 48h and 72h post-infection for Vero and Callu models, respectively.

For IL6 (the left table below is first replication n=1, the right table is second replication n=2), a dose-response effect of the adefovir active metabolite was observed as expected, with a decrease in cytokine concentration with increasing compound concentration.

Dose response effects were also observed for chemokine IP10 (n=1 replicates for the left table below and n=2 replicates for the right table).

Of particular interest, during IL6 dosimetry, similar results, i.e., dose response effects, were observed for total 3 and 4 at n=1.

N=2 is the same.

The dose response effect was also very pronounced during the test with TOTUM 3 and 4 in the IP10 dosimetry at N=1 replicates:

N=2 is the same:

no dose-response effect of total 2 was observed in either IL6 dosimetry or IP10 dosimetry (results not shown).

Viral RNA was quantified by targeting the ORF1ab gene and viral load was assessed on VeroE6-TMPRSS2 cell line.

The results of biological replication after treatment with the active metabolite of adefovir are similar. Viral load decreases with increasing compound concentration, wherein:

at n=1, the percentage relative to infected and untreated cells is 101% (27 nM radciclovir) to 0% (6667 nM and 20000 nM) (viral load is undetectable),

-n=2, the percentage relative to infected and untreated cells is 70% (27 nM radciclovir) to 0% (6667 nM and 20000 nM) (viral load is undetectable):

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n=1, at seven concentrations of 14ng/mL to 10000ng/mL, the results obtained after treatment with TOTUM 2, 3, 4 are similar to those obtained after treatment with adefovir active metabolite, relative to the viral load of infected and untreated cells; antiviral activity does not appear to decrease.

These results are not described in detail herein.

At n=2, the results for total 2 were similar to n=1 at seven concentrations of 137ng/mL to 100000ng/mL (no decrease in viral load was caused and thus total 2 had no antiviral activity).

Furthermore:

after treatment with TOTUM 3 at a concentration of 33333ng/mL to 100000ng/mL, viral loads of 9% and 0% respectively were observed, i.e. undetectable:

after treatment with 6667ng/mL of TOTUM 4, a viral load of 78% was observed relative to the infected and untreated cells, whereas after treatment with a concentration of 20000ng/mL, a viral load reduction to 40% was observed:

TOTUM 3 caused a significant reduction in viral load at concentrations of 33333ng/mL and 100000ng/mL, even undetectable.

TOTUM 4 caused a significant reduction in viral load at a concentration of 20000 ng/mL.

Results: virucidal effects of CAS 2-Compounds

The purpose of these tests is to evaluate the virucidal effect of the compound to be analyzed, i.e., the virus is incubated with the compound for 30 minutes, and then the cells are incubated with the pretreated inoculum.

First, viral RNA was quantified by targeting the ORF1ab gene, and viral load was assessed on the Calu-3 cell line.

The results of biological replication after treatment with the active metabolite of adefovir are similar. Viral load decreases with increasing compound concentration, wherein:

-at n=1, the percentage relative to infected and untreated cells is 113% (27 nM radciclovir) to 0% (6667 nM and 20000 nM):

-n=2, the percentage relative to infected and untreated cells is 109% (27 nM radciclovir) to 0% (2222 nM, 6667nM and 20000 nM):

N=1, at seven concentrations ranging from 14ng/mL to 10000ng/mL, the results obtained after treatment with TOTUM 2, 3, 4 were similar to those obtained after treatment with adefovir active metabolite (results not shown) with respect to viral load of infected and untreated cells.

At n=2, the results of TOTUM 2 were similar to n=1 at seven concentrations of 27ng/mL to 20000ng/mL and 14ng/mL to 10000 ng/mL.

Furthermore:

after treatment with TOTUM 3 at a concentration of 100000ng/mL, a viral load of 0% was observed (viral load was undetectable);

after TOTUM 4 treatment at concentrations 2222ng/mL and 6667ng/mL, viral loads of 46% and 43%, respectively, were observed;

for IL6 and IP10, three cytokine dosimetry was performed on supernatant samples of cell cultures.

At n=1 replicates (left below table) and n=2 replicates (right below table), a dose-response effect of the adefovir active metabolite was observed for IL6, and cytokine concentrations decreased with increasing compound concentrations.

In IL6 dosimetry, similar results were observed for tum 3 and 4 at n=1:

n=2 replicates were identical:

dose response effects were also found for IP10 when virus was inoculated with adefovir at n=1 replicates (left table below) and n=2 replicates (right table below).

Similar results, i.e. dose response effects, were observed for tum 3 and 4 at n=1 during IP10 dosimetry.

N=2 replicates were identical:

viral RNA was quantified by targeting the ORF1ab gene and viral load was assessed on VeroE6-TMPRSS2 cell line.

The results of biological replication after treatment with the active metabolite of adefovir are similar. Viral load decreases with increasing compound concentration, wherein:

at-n=1, the percentages relative to infected and untreated cells were 97% (27 nM radciclovir) to 0% (6667 nM) and 1% (20000 nM), respectively (viral load is undetectable):

-n=2, the percentage relative to uninfected and untreated cells is 100% (27 nM radciclovir) to 0% (2222 nM, 6667nM and 20000 nM) (viral load is undetectable):

n=1, at seven concentrations ranging from 14ng/mL to 10000ng/mL, the viral load of the infected and untreated cells after treatment with TOTUM 2, 3, 4 was similar or higher than that obtained after treatment with the adefovir active metabolite (results not shown, no virucidal effect before).

At n=2, at seven concentrations ranging from 27ng/mL to 20000ng/mL, a result similar to n=1 was observed for TOTUM 2 (results not shown, TOTUM 2 had no virucidal effect).

Furthermore:

after treatment with TOTUM 3 at a concentration of 33333ng/mL, a viral load of 16% was observed; after treatment with a concentration of 100000, no detectable viral load was observed (0%):

after treatment with TOTUM 4 at a concentration of 20000ng/mL, a viral load of 47% was observed:

conclusion(s)

The results obtained allow to demonstrate the following:

in one aspect, the compound, ruidexivir, is tested in vitro as a reference active metabolite against SARS-CoV-2 virus, which fully demonstrates antiviral and virucidal activity against the virus without significant toxicity to cells. The viral load in CAS1 and CAS2 decreases with increasing concentration of adefovir. Furthermore, for cytokines IL6 and IP10, a dose-response effect of the active metabolite of adefovir was observed, wherein the concentration of the cytokine decreased with increasing concentration of the metabolite, as expected. These results allow verification of the test scheme employed.

However, while having significant in vitro activity against SARS-CoV-2 virus, redeSivir has well known nephrotoxic effects and may be detrimental to Covid patients.

The reference sample, TOTUM 2, was obtained from dried banana pulp. The results obtained in the tests carried out indicate that this extract has no antiviral or virucidal effect. Furthermore, no dose-response effect on cytokine release was observed for this sample.

Moreover, these test results, which are expected to be negative, support the implemented embodiment.

With respect to the sample called TOTUM 3, it was obtained from the tincture mother liquor of concentrated split She Maiju (Neurolaena lobata), while the reference sample named TOTUM 4, corresponding to the dilution of the tincture mother liquor, also allows to obtain said TOTUM 3, as detailed in the obtaining schemes of these TOTUM 3 and 4 described above.

The results obtained with TOTUM 3 as detailed above indicate that, on the one hand, the compound concentrations were 14 to 10000ng/mL, with no toxicity to Calu-3 and VeroE6 cells.

On the other hand, when the compound concentration is 100000ng/mL, attention is paid to cytotoxicity against Calu-3 line cells. This is also true for VeroE6 line cells, where cytotoxicity is present, at compound concentrations of 33333ng/mL and 100000 ng/mL.

Meanwhile, for TOTUM 3 at a concentration of 100000ng/mL, no detectable viral load was observed for the Calu-3 cell line. However, this concentration has been shown to have cytotoxic effects. For the VeroE6 cell line, no detectable viral load was observed at compound concentrations of 33333ng/mL and 100000 ng/mL. However, cytotoxicity still exists.

It appears that the concentration of active compound in such samples is too high and results in cytotoxicity.

The results obtained with TOTUM 4, which is a four-bit dilution of the mother liquor containing the tincture, are particularly interesting as such, compared to the above-mentioned TOTUM 3.

It is noted that in the sample preparation method, in step iv) of obtaining TOTUM 3 according to the tincture mother liquor, 0.75L of filtrate was sampled, said filtrate being diluted with a volume of water equal to 2.25L. A total volume of hydroalcoholic solution equal to 3L was then obtained, corresponding to a quadruple dilution of the mother liquor of the tincture of the dry leaves of split She Maiju (Neurolaena lobata).

The results obtained for TOTUM 4 indicate that, on the one hand, it does not exhibit any cytotoxicity on model cell lines, regardless of the concentration tested, even at the highest concentration.

While not cytotoxic, the antiviral activity of TOTUM 4 was demonstrated at concentrations of 6667ng/mL and 20000ng/mL, with viral loads of 59% and 49% observed for the Calu-3 cell line, respectively. Antiviral activity was also detected by VeroE6 line after treatment with total 4 at concentrations 6667, 10000ng/mL and 20000ng/mL, with viral loads of 78%, 66% and 40% respectively being observed.

It is also noted that of particular interest, in the dosimetry of cytokines IL6 and IP10 in CAS1 and CAS2, a dose-response effect of TOTUM 4 was observed, in which the cytokine concentration decreased with increasing compound concentration.

Thus, based on the above results, particularly regarding the dosimetry of cytokines IL6 and IP10 in the lung cells, it was confirmed that diluted mother liquor extract of tincture, especially 4-fold dilution of mother liquor of tincture, was split up She Maiju (Neurolaena lobata) and lyophilized, in the form of a liquid solution having a concentration of 6500 to 20000ng/mL (mass of lyophilized extract of diluted mother liquor/volume of aqueous solution), preferably 6667 to 20000ng/mL, having antiviral and virucidal activity against SARS-CoV-2 virus causing Covid 19, and being effective against the severe symptoms of the disease.

In fact, the results indicate that TOTUM 4 has a dose-dependent effect on the release of cytokines IL-6 and IP10, and that this extract of the mother liquor of the tincture of Echinacea (Neurolaena lobata) is particularly suitable for avoiding possible cytokine storms, especially in the lungs of Covid critically ill patients, in order to cope with viral infections.

As used herein, a "Covid-19 critically ill patient" refers to a patient hospitalized for anti-Covid-19 and undergoing oxygen therapy.

Claims (8)

1. A therapeutic composition comprises a mother liquor of tincture of leaf extract of plants of the genus Nelumbo (Nelumbo) and Nelumbo nucifera (lobata), use as a medicament for inhibiting human dihydroorotate dehydrogenase (DHODH) for the treatment of infection by an RNA genome virus, characterized in that said RNA genome virus is selected from the list of:

the virus of the family coronaviridae,

hepatitis C-causing virus in the flaviviridae family, yellow fever-causing virus or Zika virus,

-a virus causing chikungunya fever by togaviridae.

2. The therapeutic composition of claim 1, wherein the RNA genome virus is a SARS-CoV-2 virus that causes Covid 19.

3. A method of preparing a dry extract of diluted tincture of dry leaves of split She Maiju (Neurolaena lobata) mother liquor, the method comprising the steps of:

i) Preparing a mixture based on dry leaves of echinacea (Neurolaena lobata) in a mass concentration of between 15 and 20g/L in 50 ° of policosanol;

ii) the mixture is immersed for about 21 days while stirring;

iii) After impregnation, the mixture was filtered on a "filter" with a porosity of 50-75 μm and a filtrate and solid retentate of the mother liquor of the tincture corresponding to the split She Maiju (Neurolaena lobata) dry leaf was obtained;

iv) diluting the liquid tincture mother liquor by 4 times by adding an aqueous solution and then obtaining an aqueous-alcoholic solution;

v) evaporating the alcohol contained in the solution by means of a rotary evaporator until an aqueous solution is obtained;

vi) freezing the aqueous solution thus obtained, followed by lyophilization, to obtain a dry extract of diluted tincture stock of split She Maiju (Neurolaena lobata) dry leaves.

4. Process for the preparation of a dry extract of diluted tincture mother liquor of dry leaves of split She Maiju (Neurolaena lobata) according to the preceding claim, characterized in that the mass concentration of the mixture prepared in step i) is 16 to 17g split She Maiju (Neurolaena lobata) dry leaves/liter (L) 50 ° policosanol, and preferably equal to 16g/L.

5. The method of preparing a dry extract of diluted tincture mother liquor of dry leaves of split She Maiju (Neurolaena lobata) according to claim 3 or 4, characterized in that in step iv) the tincture mother liquor is diluted by mixing a volume of filtrate equal to 0.75L and a volume of water equal to 2.25L.

6. A method for preparing a liquid solution of a dry extract of diluted lyophilized tincture mother liquor of split She Maiju (Neurolaena lobata) dry leaves, obtained according to the method of any one of claims 3 to 5, characterized in that the liquid solution is obtained by diluting the dry extract in water or in a pharmaceutically acceptable aqueous solvent, the concentration of the liquid solution being between 6500 and 20000ng dry extract/mL aqueous solvent, preferably between 6667 and 20000 ng/mL.

7. A diluted mother liquor extract of a tincture of dry leaves of echinacea (Neurolaena lobata), in particular a 4-fold dilution of the mother liquor of the tincture, which is a solution and has a final concentration of 6500 to 20000ng/ml (mass of lyophilized dry extract of diluted mother liquor/volume of aqueous solution), for use in the treatment of a viral infection caused by the SARS-CoV-2 virus causing Covid 19.

8. Tincture mother liquor extract of split She Maiju (Neurolaena lobata) leaves according to the preceding claim for use in reducing cytokine production, in particular IL-6 and IP-10 production, in the treatment of severe viral infections caused by the SARS-CoV-2 virus causing Covid 19.