System-biology based pharmaceutical composition comprising a mixture of herb extracts as active ingredients, use of said composition as a medicament, particularly in the treatment of human coronavirus infections such as covid-19.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition. More specifically, the invention relates to a pharmaceutical composition comprising a mixture of herb extracts as active ingredients. The pharmaceutical composition is useful in prophylaxis, treatment and posttreatment of human coronavirus infections, in particular Covid-19.

BACKGROUND OF THE INVENTION

This invention is based on “system biology” (SB) approach to cope with Covid-19 pandemic.

SB is based on both holistic approach and scientific method. SB has been one of the synergic achievements of the science and technology, especially the integration and interface of biology, medical and systems sciences, and information technology, in the last five decades. The nature of SB is a combination of disciplinary, multi-disciplinary, interdisciplinary, trans-disciplinary, and meta-disciplinary. SB opens novel and promising horizons in medical and pharmaceutical sciences. In this invention by applying SB, a meta-study is performed on published sources to develop novel anti-coronavirus compositions.

Thus the invention relates to a novel antiviral composition to inhibit a group of coronavirus proteins which bind a set of human proteins. The composition has high inhibitory, antiinflammatory and immune-enhancing capacity. The composition is provided in proper dosage forms to be used as a “staging medicine” to treat corona viral infections as a “staging disease”. The “staging medicine” comprises a set of products and a set of methods to treat Covid-19 patients.

Covid-19 is a “staging disease” with 5 stages. That is, in Stage 1, a person is in high-risk to be afflicted with a viral load (VL) of less than 650 copies per ml. Stage 2 includes patients having positive PCR test with a VL about 10 ⁴ copies per ml, but without symptoms. Patients in Stage 3 also have positive PCR tests with a VL about 10 ⁷ copies per ml and mild symptoms. Short breath will be added in Stage 4 with a VL of 1.34xlO ⁿ copies per ml. Finally, acute respiratory syndrome appears in Stage 5 with a VL more than 1.34xlO ⁿ (Park, Jay J H et al. “Clinical trials of disease stages in COVID 19: complicated and often misinterpreted”. The Lancet Glob Health. 8.10, (2020): el249-el250.

Adverse effects of Covid-19 are costly, severe, long-lasting and cause a “post-Covid stage”. Survivors of Covid-19 suffer from various disabilities and subsidiary diseases such as dementia and memory related lapses, muscle disease and weakness due to the impairments regarding their cognitive and mental health or physical function, shortness of breath, mental disorders, which include sleeping, mood, anxiety and concentrate related issues. In acute cases palpitations and mental injury outcomes are reported. Recovered patients more exposed to show syndromes of autoimmune diseases like Guillain-Barre syndrome and pediatric inflammatory multisystem syndrome besides stork occurrences and cardiovascular disease were reported. Gastrointestinal outcomes are another long-term side effect. Also new onset of diabetes and hypertension is observed. However, the Covid-19 symptoms like loss of taste and smell, sore throat and difficulties to swallow, and skin rash are also observed in long time after infection. Recovery from adverse effects of “post-Covid stage” and recapture normal health are the main concern of the survivors (Yelin, Dana, et al. "Long-term consequences of COVID-19: research needs." The Lancet Infectious Diseases 20.10 (2020): 1115-1117).

A proper treatment or medicine for Covid-19 is to be “staging”. The treatment for Stages 1 and 2 is to be prophylaxis. In Stage 3 out-patient treatment is needed. Patients in Stage 4 require to be hospitalized in wards. Finally, patients in Stage 5 are treated in intensive care units (ICUs).

This invention provides a “staging medicine” corresponding to Stages 1 to 4 of Covid-19. For prophylaxis cases, two antiviral sprays (nasal-sinuses and oral-pharynx) are developed to remove and prevent the proliferation of coronavirus colonies in nasal-sinuses and throat-pharynx areas.

In Stage 3, an antiviral tablet dosage form is added to remove coronaviruses in the patient’s body. For treatment of patients in Stage 4, an antiviral aromatherapy drop is developed for removal of coronavirus from patient’s respiratory system.

For “post-Covid stage”, the tablet and drop doses help to mitigate and improve the long-lasting adverse effects.

Human coronavirus first appeared in 1961(Monto, Arnold S., Benjamin J. Cowling, and JS Malik Peiris. "Coronaviruses." Viral infections of humans. Springer, Boston, MA, 2014. 199- 223). There exist extensive researches and studies on human coronavirus and the relevant diseases such as SARS, MERS, and Covid-2 in the last six decades. The core point that incurs infection is the binding among corona viral proteins, on one side, and human proteins, on the other. 19656 proteins are known in human body (Piovesan, Allison, et al. "Human protein-coding genes and gene feature statistics in 2019." BMC research notes 12.1 (2019): 1-5). 332 of human proteins are bound by 27 coronaviral proteins. Of these 27 coronaviral proteins, 19 coronaviral proteins are structural and non-structural proteins (NSPs). The first group or structural proteins includes 4 proteins that are specified as spike (S), envelop (E), membrane (M), nucleocapsid (N) of coronavirus. The second group includes 15 NSPs that are specified as NSP1 to NSP15 with two added sub-indexes (Gordon, David E., et al. "A SARS-CoV-2 protein interaction map reveals targets for drug repurposing." Nature 583.7816 (2020): 459-468).

Structural proteins and NSPs of coronavirus bind to 256 (out of 332) human proteins. To cope with coronavirus disease directly requires mainly to inhibit all viral bindings and also to treat the effects of previous bindings (Gordon, David E., et al. "A SARS-CoV-2 protein interaction map reveals targets for drug repurposing." Nature 583.7816 (2020): 459-468).

A thorough meta-study of the last six-decade researches and studies, indicates 786 compounds have been declared with ability to prevent ailment, cope with coronavirus, and/or remedy its effects. In fact, these compounds act as inhibitor, antiviral, and anti-inflammatory and/or immune-enhancing agents.

The 786 compounds inhibit 15 (out of 19) proteins of coronavirus. Therefore, there exists a remarkable research deficiency about the inhibitory compounds to the 4 remaining NSPs. This opens a broad horizon for future research subjects to cope with coronavirus disease.

In this invention the inhibitory compounds to the coronaviral proteins are provided from herbs.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a pharmaceutical composition comprising a mixture of herb extracts. It is a further object of the invention to provide a pharmaceutical composition comprising a mixture of herb extracts to be used as a medicament, in particular a medicament to be used in the treatment of coronavirus infections. Thus, a first aspect of the invention provides a pharmaceutical composition, comprising a mixture of at least six extract of herbs selected from the group consisting of Urtica Dioica, (common nettle), Echinacea purpurea (eastern purple coneflower), Artemisia annua (sweet wormwood), Glycyrrhiza glabra (licorice), Scutellaria baicalensis (Chinese skullcap), Rheum palmatum (Chinese rhubarb), Hyssopus officinalis (hyssop), Rosmarinus officinalis (rosemary), Nigella sativa (black cumin), Eucalyptus alba (white gum), Pterocarpus santalinus (red sandalwood), Curcuma longa (turmeric), and Panax Ginseng (Asian ginseng) as active ingredients.

In one embodiment of the first aspect of the invention, the at least six extract of herbs are Echinacea purpurea (eastern purple coneflower), Glycyrrhiza glabra (licorice), Rheum palmatum (Chinese rhubarb), Hyssopus officinalis (hyssop), Rosmarinus officinalis (rosemary), and Panax Ginseng (Asian ginseng). Said mixture of at the least six extract of herbs may further comprise one or more extract of herbs selected from the group consisting of Urtica Dioica, (common nettle), Artemisia annua (sweet wormwood), Scutellaria baicalensis (Chinese skullcap), Nigella sativa (black cumin), Eucalyptus alba (white gum), Pterocarpus santalinus (red sandalwood), and Curcuma longa (turmeric) as active ingredients.

In yet another embodiment of the first aspect of the invention, the mixture of extract of herbs comprises the following extract of herbs: Urtica Dioica, (common nettle), Echinacea purpurea (eastern purple coneflower), Artemisia annua (sweet wormwood), Glycyrrhiza glabra (licorice), Scutellaria baicalensis (Chinese skullcap), Rheum palmatum (Chinese rhubarb), Hyssopus officinalis (hyssop), Rosmarinus officinalis (rosemary), Nigella sativa (black cumin), Eucalyptus alba (white gum), Pterocarpus santalinus (red sandalwood), Curcuma longa (turmeric), and Panax Ginseng (Asian ginseng) as active ingredients.

According to the invention, the proporsion of the individual herb extract may vary. Preferably, the proporsion of the individual herb extract in said mixture of extract of herbs is in the range of about Urtica dioica (common nettle) 16 - 20%, Echinacea purpurea (eastern purple coneflower) 12-19%, Artemisia annua (sweet wormwood) 22-28%, Glycyrrhiza glabra (licorice) 6-14%, Scutellaria baicalensis (Chinese skullcap) 6-9%, Rheum palmatum (Chinese rhubarb) 5-10%, Hyssopus officinalis (hyssop) 2.5-4.5%, Rosmarinus officinalis (rosemary) 2.5-4.5%, Nigella sativa (black cumin) 1-5%, Eucalyptus alba (white gum) 2.5 - 5%, Pterocarpus Santalinus (red sandalwood) 1.5-3%, Curcuma longa (turmeric) 0.6- 1.5%, and Panax ginseng (Asian ginseng) 0.3-1.5% by weight based on the total weight of the mixture of herbal extracts. In one preferred embodiment of the invention, the proporsion of the individual herb extract in said mixture of extract of herbs are Urtica dioica (common nettle) about 18.5%, Echinacea purpurea (eastern purple coneflower) about 15%, Artemisia annua (sweet wormwood) about 25%, Glycyrrhiza glabra (licorice) about 10%, Scutellaria baicalensis (Chinese skullcap) about 7.5%, Rheum palmatum (Chinese rhubarb) about 7.5%, Hyssopus officinalis (hyssop) about 3.5%, Rosmarinus officinalis (rosemary) about 3.5%, Nigella sativa (black cumin) about 2.5%, Eucalyptus alba (white gum) about 3.3%, Pterocarpus Santalinus (red sandalwood) about 2%, Curcuma longa (turmeric) about 1%, and Panax ginseng (Asian ginseng) about 0.5% by weight based on the total weight of the mixture of herbal extracts.

The mixture of herbs present in the composition of the invention, may be used as it is, i.e. as ethanol extracts or further formulated into any suitable pharmaceutically acceptable dosage forms such as solid, liquid, semi-solid, gel, creme etc.. Thus, the pharmaceutical composition of the invention may further comprise pharmaceutically acceptable excipients. Preferred dosage forms according to the invention are tablets, nasal-sinuses spray, oral-pharynx spray, or inhaling droplets.

According to a second aspect of the invention, the pharmaceutical composition may be used as a medicament. In particular, the pharmaceutical composition according to the invention may be used in treatment of a coronavirus infection. The coronavirus infection may be selected from the group of MERS and SARS viruses, preferably the SARS-CoV-2 virus causing COVID-19 disease.

In yet another aspect, the invention provides a method for treatment of coronavirus infections, comprising the step of administering to a mammal that has been exposed to or are in risk of being exposed to coronavirus, a safe and effective amount of the pharmaceutical composition of the invention.

The present invention will become apparent from the detailed description given below. The detailed description and specific examples disclose preferred embodiments of the invention by way of illustration only. Those skilled in the art understand from guidance in the detailed description that changes and modifications may be made within the scope of the invention as defined in the appended claims. Hence, it is to be understood that the terminology used herein is for purpose of describing particular embodiments only, and is not intended to be limiting. It should be noted that, as used in the specification and the appended claim, the articles "a", "an", "the", and "said" are intended to mean that there are one or more of the elements unless the context explicitly dictates otherwise. Furthermore, the words "comprising", "including", "containing" and similar wordings does not exclude other elements or steps.

In the context of the present invention, the terms pharmaceutical composition, herbal composition, therapeutic herbal composition, novel medicine and novel antiviral composition etc. are used interchangeably.

The term “herb extract” and “extract of herb” are used interchangeably. Consequently, the term “mixture of extract of herbs” and “mixture of herb extracts” are used interchangeably.

In the context of the present invention, the dosage regimen disclosing effective amount in “mg” refers to the amount of total active ingredients of herbs in the composition.

In this context, treatment of coronavirus is to be understood to encompass prophylactic treatment, treatment of coronavirus infections itself, and post-treatment of adverse effects caused by the coronavirus infection. Also included is use of the composition as daily supplement. Adverse effects following coronavirus diseases e.g. Covid-19 disease, are typically respiratory disorders, which may be long lasting. Thus, the pharmaceutical composition of the invention may be used to inhibit coronavirus infection, i.e. as prophylactic treatment, as a pharmaceutical composition to treat the coronavirus infection itself and as a pharmaceutical composition to treat adverse effects following coronavirus disease, e.g. SARS or Covid-19.

BRIEF DESCRIPTIONS OF THE DRAWINGS

The above objects, as well as additional objects, features and advantages of the present disclosure, will be more fully appreciated by reference to the following illustrative and nonlimiting detailed description of example embodiments of the present disclosure, when taken in conjunction with the accompanying drawings. Figure 1 shows the mechanism of action of the novel medicine as a “compound-protein interaction network”. The graph contains 3 types of nodes “red”, “purple”, and “cyan” nodes for “human proteins”, “virus proteins”, and “natural inhibitory compounds”, respectively. The 15 proteins are categorized into 4 groups comprising: 1- structural proteins, 2- non-structural proteins, 3- accessory proteins and 4-replication related proteins.

Figure 2 shows the mean reduction rates of viral load for case and control groups 4 and 8 days after treatment.

Figure 3 indicates the “cumulative probability distribution function (CDF) of treatment duration" of patients in “case” group.

Figure 4 indicates the “cumulative probability distribution function (CDF) of treatment duration" of patients in “control” group.

Figure 5 indicates the cumulative probability distribution of the of treatment duration of treatment and control groups.

DETAILED DESCRIPTION OF THE INVENTION

First of all, to nominate the most effective herbs, we made a dataset comprising all the available inhibitory compounds against different kinds of coronavirus. Some of them had IC50 value and some do not. As a first filtration criterion, we omitted all the compounds without IC50 values. By this filtration 340 out of 786 compounds remained. Then we determined the natural resources of these 340 compounds. As a result, 28 herbs were defined. We cross-checked these 28 herbs to discern more effective ingredients for treatment of coronavirus infection due to references. Finally, 13 herbs were nominated also based on their frequencies of being reported in literatures.

We extracted the major compounds of these 13 herbs to construct the mechanism of action of a novel medicine. Comparing major compounds of each herb with 786 total inhibitory compounds, we determined the inhibitory compounds existing in each herb. The inhibitory compounds of each herb are shown in Table 1 the total number of inhibitory compounds of these 13 herbs are 54 compounds. Table 1- Inhibitory Compounds of Herbs

In the next step, the coronavims proteins that are inhibited by those 54 compounds were determined. The matching graph presented in Figure 1 indicates that the compounds inhibit 15 (out of 19) different proteins of coronavims. The mechanism of action of the novel medicine as a

“compound-protein interaction network” is shown in Figure 1. The graph contains 3 types of nodes “red”, “purple”, and “cyan” nodes for “human proteins”, “vims proteins”, and “natural inhibitory compounds”, respectively. The 15 proteins are categorized into 4 groups comprising: 1- structural proteins, 2- non-stmctural proteins, 3- accessory proteins and 4-replication related proteins.

Echinacea is a genus of herbaceous plants in the family Asteraceae, informally referred to as purple coneflowers. Echinacea species are specifically very beneficial for immune system acting as immunomodulator. Furthermore, studies confirm that Echinacea species can strengthen and stimulate the human immune system, helps anti-inflammation and sterilization, and opposing virus. Meanwhile, the effectiveness of Echinacea species for prevention of diseases such as fever, influenza, SARS, anaphylaxis, arthritis and throat inflammation has been proved. The therapeutic activity of the E. purpurea has been attributed to its content of caffeic acid, chlorogenic acid, echinacoside and lipophilic derivative compounds alkylamides. Studies show that these compounds act as inhibitors of corona virus proteins (Signer, Johanna, et al. "In vitro virucidal activity of Echinaforce ^® , an Echinacea purpurea preparation, against coronaviruses, including common cold coronavirus 229E and SARS-CoV-2." Virology journal 17.1 (2020): 1- 11).

Scutellaria baicalensis georgi belongs to the family of Labiatae, informally referred to as Chinese skullup. An aqueous extract of Scutellaria spp has been shown to exhibit antiviral activity against viral respiratory infections such as SARS-Cov2. Besides, this plant is very useful for treatment of diarrhea, dysentery, hypertension, hemorrhaging, insomnia, and inflammation. Plant flavonoids isolated from Scutellaria spp, have also been shown to exhibit antiviral activity. Most of these flavones such as baicalin, wogonoside and their aglycones baicalein wogonin are the major bioactive compounds extracted from the root of S. baicalensis. Baicalin and its derivatives represent a novel class of compounds with potential for the development of safe drugs for therapy of diseases associated with viruses such as SARS infection or other related infections. As a result, baicalin is a recommended for antiviral prophylaxis or treatment (Liu, Hongbo, et al. "Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro." Journal of enzyme inhibition and medicinal chemistry 36.1 (2021): 497-503).

Curcuma Longa L. is mainly produced in Sichuan, Zhejiang, Guangdong, and Guangxi provinces in China, and harvested in winter or spring. It belongs to the family of Zingiberaceae. The major constituent in the rhizome of Curcuma longa (Zingiberaceae) is “curcumin”. Curcumin and its analogues or flavonoid compounds are very beneficial because of their high antioxidant activity, low toxicity and abundance in nature, and their anti-inflammatory, anti-NFKB, and cyclooxygenase suppression. Aqueous extract and hydroalcoholic extract of Curcuma inhibited the secretion of cytokine IL-6 and/or IL-8 in human keratinocytes cultures with an activity similar to betametasone- 17-valerate. The aqueous and hydroalcoholic extracts of Curcuma have been seen to inhibit cell proliferation. The extract therefore shows cytostatic activity. Furthermore, curcumin is one of inhibitory compounds that could inhibit specific proteins in SARS-Cov2 (Manoharan, Yamuna, et al. "Curcumin: A wonder drug as a preventive measure for COVID19 management." Indian Journal of Clinical Biochemistry 35.3 (2020): 373-375). Panax ginseng species belongs to the Araliaceae family and is distributed or cultivated in the Far Eastern region of Asia. Polysaccharides of Panax ginseng have immune-stimulating activities. In addition, numerous studies have reported the beneficial effects of ginseng on diverse diseases such as cancer, immune system disorder, neuronal disease, and cardiovascular disease.

Furthermore, ginseng and its purified components have also been shown to possess protective activities against microbial infections. Although ginseng itself can exert direct antiviral effects by inhibiting viral attachment, membrane penetration, and replication, the foremost antiviral activities of ginseng are attributed to the enhancement of host immunity. It was found that the main component of plants of the genus Panax is saponin. The main panax ginseng saponins are ginsenosides Rbl , Rb2 , Rc, Rd, Rgl and Re. All of them have different biological activity, depending on the chemical structure. Studies show that ginsenoside mostly have anticancer and immunoregulation effects. Ginsenoside has also certain antivirus action, especially SARS-CoV2 virus and can inhibit certain viral proteins (Chikhale, Rupesh V., et al. "Sars-cov-2 host entry and replication inhibitors from Indian ginseng: an in-silico approach." Journal of Biomolecular Structure and Dynamics (2020): 1-12).

The root extract from Glycyrrhiza glabra has antiulcer, antiviral, antihepatotoxic, alkalizing antifungal and anti-SARS properties and belongs to Fabaceae family. Licorice originates in Turkey, Iran, Spain, Greece and North China. The most outstanding chemical compound found in licorice ( Glycyhrrhizza glabra) is glycyrrhizin that is a triterpene glucoside. The structure of glycyrrhizin comprises triterpene part (glycyrrhetinic acid) and two iduronic acid residues. Glycyrrhizin can stimulate interferon to generate and cause phlegm and coughing suppression. Glycyrrhizin is also known to be strengthening the immune system, stimulates the adrenal gland, and is diuretic and laxative. Considering the wide use of the molecule in traditional system of medicine and the level of toxicity well tested, this can be used along with the antiviral drugs to enhance their efficacy for inhibiting different protein targets in viruses (Maurya, Dharmendra Kumar. "Evaluation of Yashtimadhu (Glycyrrhiza glabra) active Phytochemicals Against Novel Coronavirus (SARS-CoV-2)." (2020)).

Red sandalwood ( Pterocarpus macrocarpus) is a pterocarpous plant of Papilionaceae. The origin of the red sandalwood is in the countries of India, Indonesia, Philippines, Burma, and also have distribution in Yunnan, Guangdong and Guangxi in China. Research shows that different polarity solvent extraction parts of leaves, roots and stems of the Pterocarpous indicus show wide antiviral activity because of its major inhibitory compounds specially against SARS-Cov2 viral proteins. Pterostilbene (trans-3,5-dimethoxy-4'-hydroxystilbene), originally derived as an extract from red sandalwood, also holds an anti-inflammatory (and ultimately anti-neoplastic) properties, particularly when in combination with a synergistic component such as curcumin (Kumar, Dinesh. "Anti-inflammatory, analgesic, and antioxidant activities of methanolic wood extract of Pterocarpus santalinus L." Journal of pharmacology & pharmacotherapeutics 2.3 (2011): 200).

The stinging nettle ( Urtica dioica ) is best known as a member of the Urtica genus, which has been widely used by herbalists around the world for centuries. In Brazil and Peru herbal medicine the entire plant is used for various disorders, including viral infections, diabetes and inflammatory conditions. Lectins, a carbohydrate-binding protein with agglutinating properties in Urtica dioica are example of a natural substance that has therapeutic properties and potentials. Lectins are produced by Urtica dioica, mainly in their roots or rhizomes, has been shown to inhibit HIV-1-, HIV-2-, CMV-, RSV-, influenza A and SARS. It is thought that the carbohydrate-binding plant proteins exert their antiviral action. These agents have been shown to inhibit the entry process of the virus. Other major compounds of Urtica dioica such as caffeic acid, kaempferol and quercetin also have inhibitory properties against SARS and SARS-Cov2 viral proteins (.Kumaki, Yohichi, et al. "Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin." Antiviral research 90.1 (2011): 22-32).

Rhubarb is the dried root and root of Rheum palmatum L., a perennial herb belonging to Polygonaceae family. In oriental medicine, it is used for the treatment of thyroid sarcoma, hepatic degenerative degeneration, hepatic vein congestion, constipation, jaundice, acute enteritis, acute conjunctivitis, respiratory viral infection, cholangiocarcinoma, etc. Recently, it has been found that rhubarb has a similar action to interferon. Rhubarb major compounds are sennoside A, B, C, D, E, F (sennoside A, B, C, D, E, F), anthraquinone derivative, chrysophanol, emodine (emodin), and aloe-emodin. Various studies have been performed based on the pharmacological action of rhubarb major compounds. These studies indicated that these compounds can inhibit plenty of proteins of SARS-Cov2(Schwarz, Silvia, et al. "Emodin inhibits current through SARS-associated coronavirus 3a protein." Antiviral research 90.1 (2011): 64- 69).

Hyssopus officinalis or hyssop is a shrub in the Lamiaceae family native to Southern Europe, the Middle East, and the region surrounding the Caspian Sea. Due to its properties as an antiseptic, cough reliever, expectorant, phlegm eliminator, dampness remover, diaphoresis detoxification and anti-inflammatory detumescent. It has been used in traditional herbal medicine. Thus, Hyssopus officinalis can be very useful for treatment of viral respiratory infections such as MERS and SARS infections. Studies show plenty of hyssop bioactive compounds can target SARS and SARS-COV2 structural proteins (shown in Table 1) and inactive coronavirus as a result. In addition, Hyssopus officinalis can suppress IL-2, IL-6, ET-1, TNF-a, Ly, Eos, Neu secretion and alleviate the inflammation caused by different respiratory disorders (da Silva,

Joyce Kelly R., et al. "Essential oils as antiviral agents, potential of essential oils to treat sars- cov-2 infection: An in-silico investigation." International journal of molecular sciences 21.10 (2020): 3426). Rosmarinus officinalis (Rosemary) belongs to Lamiaceae family. Rosemary originates from Spain and the Mediterranean region, and is a plant with a unique aroma similar to camphor. Its leaves include monoterpene hydrocarbons, power fountains, and Bonoleo. Radical scavenging capacities and antioxidant activities of methanolic extracts from the leaves of Rosmarinus officinalis were studied by various assays. It contains ingredients such as camosol, camosic acid, rosmarinic acid, mononole, cinellonole, linaronore, benolebenenore, phenenolic acid, strong norenoic acid (rosmarinesin), triterpenic acid, saponoside and tannin. In herbal medicine, Rosemary is used as a choleretic, a diuretic, an antispasmodic, a hair-growth stimulator, to treat gastrointestinal symptoms and antioxidant. Besides, Rosemary is well-known as a natural antiviral (like anti-coronavirus) due to the antiviral properties of its major compounds (Silva, Estela Fernandes, Paula Fernandes e Silva, and Timoteo Matthies Rico. "Anti-Sars-CoV effect of Rosemary (Rosmarinus officinalis): A blind docking strategy." (2020).

Nigella sativa also known as black cumin is an annual flowering plant in the Ranunculaceae family, native to eastern Europe (Bulgaria, Cyprus and Romania) and western Asia (Turkey, Iran and Iraq). Main phytochemicals reported from different varieties of N. sativa include sterols and saponins, phenolic compounds, alkaloids, novel lipid constituents and fatty acids, and volatile oils of varying composition. Among these phytochemicals the abundantly constituents identified are trans-anethole, p-cymene limonene, carvone, a-thujene, thymoquinone (TQ), thymohydroquinone (THQ), dithymoquinone, carvacrol, and b-Pinene with various concentration. The volatile oil of black cumin has dose-reliant anti-inflammatory activity. The anti-inflammatory action of TQ might be related to inhibition of the oxidative product of arachidonic acid formation, such as thromboxane B2 and leukotriene by blocking both cyclooxygenase and lipoxygenase enzymes. The various extracts, oil, and active constituent (a- hederin) of N. sativa also showed an improvement of tracheal responsiveness and significant anti-inflammatory activity via decreasing the release of histamine and leukotrienes while increasing the PGE2 from the mast cells. These preclinical and clinical studies evidenced the potential anti-asthmatic effects of N. sativa but further investigations are required to assure its efficacy. In addition, N. sativa seed oil was found to have antiviral power against different type of coronavirus. The possible mechanism of action of this antiviral property is related to inhibitory nature of N. sativa compounds that are capable of inhibiting main proteins of coronavirus (Ahmad, Sajjad, et al. "Molecular docking, simulation and MM-PBSA studies of nigella sativa compounds: a computational quest to identify potential natural antiviral for COVID-19 treatment." Journal of Biomolecular Structure and Dynamics (2020): 1-9).

Artemisia annua, also known as sweet wormwood is a common type of wormwood native to temperate Asia and belongs to the plant family of Asteraceae. Artemisia annua extracts have been used in ancient Chinese medicine for a number of treatments. It was specifically recommended for fevers in Chinese ancient herbal medicine references. The antimalarial activity of Artemisia annua was rediscovered in China in 1972, and the antimalarial active principal of Artemisia annua was named “artemisinin”. Recently, however, this extract has been used for the treatment of malaria. Moreover, it has been disclosed that artemisinin is the active agent that delivers the antimalarial benefits of Artemisia annua extracts. Artemisinin is a naturally occurring substance, obtained by purification from Artemisia annua. Artemisinin and its analogs are sesquiterpene lactones with a peroxide bridge, and are characterized by very low toxicity and poor water solubility. Artemisinin is known as a humoral immunosuppressive agent. Artemisinin stimulates cell-mediated immunity, and yet decreases abnormally elevated levels of polyamine regulatory proteins. It also markedly inhibits nucleic acid and protein syntheses. Further, it affects cellular membrane functions and decreases hepatic cytochrome oxidase enzyme system activity. Still further, it is virustatic against influenza and coronavirus through inhibiting their proteins. Plant extracts of Artemisia species also show anti-inflammatory, antirheumatic and antimicrobial properties (Cao, Ruiyuan, et al. "Anti-SARS-CoV-2 potential of artemisinins in vitro." ACS infectious diseases 6.9 (2020): 2524-2531).

Eucalyptus is one of the most commonly used essential oils. It is currently used in many allopathic medical preparations. It is one of the three best oils for use with any respiratory tract problems because the component eucalyptol is mucolytic (it relaxes the flow of mucous) and it excretes the eucalyptol out though the lung surface. As it is inhaled it gives an immediate effect. It prevents the initial infection of the human respiratory system by pathogens causing diseases such as colds, flus and pneumonia. The inhaled oils soak into the cells within the respiratory system provides additional protection against infection by pathogens. The oils reduce any preexisting infection caused by pathogens, and therefore, in conjunction with the immune system, again provides additional protection against infection by pathogens. The oils help mucous membranes repair themselves, and so this effect also provides additional protection against infection by pathogens. The oils help repair many types of existing damage and therefore, this effect also provides additional protection against infection by pathogens. Prophylactic use of inhaled eucalyptus oil and/or tea tree oil can prevent opportunistic infections of the human respiratory system. Moreover, studies show that eucalyptol can have antiviral properties against viruses like MERS, SARS and SARS-Cov2 by inhibiting certain viral proteins (Muhammad, Ibrahim Ahmad, et al. "A Computational Study to Identify Potential Inhibitors of SARS-CoV-2 Main Protease (Mpro) from Eucalyptus Active Compounds." Computation 8.3 (2020): 79).

The geographical origin, soil specification, genetic identity, harvest time, maturity of herbal plants, certain plant part utilized (such as leaf, stem, seed and root), type and rate of fertilizers, and other pre-harvest factors, also post-harvest processing method, have a significant impact on the chemical composition of any particular herb. In this invention, the optimum pre- and postharvest conditions for selecting herbs are considered.

All the ingredients of the present composition are reported as safe ingredients at specific dosages in literature and by experiments. The products of the invention in the dosage forms tablet, nasal- sinuses spray, oral-pharynx spray, aromatherapy drop have been proven to be safe by different toxicity studies and physicochemical experiments.

Pharmaceutical compositions made from natural herbs have been known and used for thousands of years to cure or ameliorate various viral infections. Some of these herbal compositions have been disclosed for having medicinal properties for curing or ameliorating symptoms associated with HIV infection. The 13 individual herbs include Rosmarinus officinalis, Hyssopus officinalis, Scutellaria baicalensis, Panax ginseng, Eucalyptus globulus, Glycyrrhiza glabra, Echinacea purpurea, Nigella sativa, Artemisia annua, Rheum officinale, Urtica dioica, Curcuma longa and Ptercarpus santalinus. These herbs are further described to be able to exhibit anti-SARS-CoV activities (Bouyahya, Abdelhakim, et al. "Therapeutic Strategies of COVID-19: from Natural Compounds to Vaccine Trials." Biointerface Research in Applied Chemistry 11.1 (2020)).

The present invention discloses a therapeutic herbal composition comprising extract of herbs selected from Rosmarinus officinalis, Hyssopus officinalis, Scutellaria baicalensis, Panax ginseng, Eucalyptus globulus, Glycyrrhiza glabra, Echinacea purpurea, Nigella sativa,

Artemisia annua, Rheum officinale, Urtica dioica, Curcuma longa and Ptercarpus santalinus, along with pharmaceutical acceptable excipients, useful in the treatment of symptoms associated with SARS-Cov-2, SARS, MERS, and some other viral infections (Farshi, Parastou, et al. "A comprehensive review on the effect of plant metabolites on coronavimses: focusing on their molecular docking score and IC50 values." (2020)).

The pharmaceutical names, botanical or zoological names, family names, common descriptions, and major ingredients of the herbs used in the present invention are shown in Table 2.

Table 2- Herbs of the Compositions of the invention

Thus, the present invention provides a pharmaceutical composition, comprising a mixture of at least six extract of herbs selected from the group consisting of Urtica Dioica, (common nettle), Echinacea purpurea (eastern purple coneflower), Artemisia annua (sweet wormwood), Glycyrrhiza glabra (licorice), Scutellaria baicalensis (Chinese skullcap), Rheum palmatum (Chinese rhubarb), Hyssopus officinalis (hyssop), Rosmarinus officinalis (rosemary), Nigella sativa (black cumin), Eucalyptus alba (white gum), Pterocarpus santalinus (red sandalwood), Curcuma longa (turmeric), and Panax Ginseng (Asian ginseng) as active ingredients.

The 13 individual herb extracts of the composition of the invention may be grouped into two subgroups of “core” ingredients and “additional” ingredients.

The “core” ingredients contribute to the primary efficacy and healing effects such as antiviral, anti-inflammatory and immunomodulatory properties of the composition are extracts of: Echinacea purpurea, Glycyrrhiza glabra, Rheum palmatum, Hyssopus officinalis. Rosmarinus officinalis, and Panax Ginseng. Thus, in one embodiment of the invention, the at least six extract of herbs are Echinacea purpurea (eastern purple coneflower), Glycyrrhiza glabra (licorice), Rheum palmatum (Chinese rhubarb), Hyssopus officinalis (hyssop), Rosmarinus officinalis (rosemary), and Panax Ginseng (Asian ginseng).

The “additional” ingredients used to enhance the antiviral properties and pharmaceutical effects resulted from the “core” ingredients are extracts of: Urtica Dioica, Artemisia annua, Scutellaria baicalensis, Nigella sativa, Eucalyptus alba, Pterocarpus santalinus, and Curcuma longa,

Thus, in one embodiment of the invention, the mixture of at the least six extract of herbs may further comprise one or more extract of herbs selected from the group consisting of Urtica Dioica, (common nettle), Artemisia annua (sweet wormwood), Scutellaria baicalensis (Chinese skullcap), Nigella sativa (black cumin), Eucalyptus alba (white gum), Pterocarpus santalinus (red sandalwood), and Curcuma longa (turmeric) as active ingredients.

The mixture of herbs may also comprise extract of all 13 herbs identified by the present invention. Thus, in one embodiment , the mixture of extract of herbs comprises the following extract of herbs; Urtica Dioica, (common nettle), Echinacea purpurea (eastern purple coneflower), Artemisia annua (sweet wormwood), Glycyrrhiza glabra (licorice), Scutellaria baicalensis (Chinese skullcap), Rheum palmatum (Chinese rhubarb), Hyssopus officinalis (hyssop), Rosmarinus officinalis (rosemary), Nigella sativa (black cumin), Eucalyptus alba (white gum), Pterocarpus santalinus (red sandalwood), Curcuma longa (turmeric), and Panax Ginseng (Asian ginseng) as active ingredients.

The determination of the proportion of each herb as ingredient in the composition is based on the following criteria: 1 - the IC50 value of main compound of each herb, 2- the number of inhibitory compounds of each herb.

The proportions of the herbal extracts used in the process of making the pharmaceutical composition of the present invention may vary. Preferred proportions are detailed in Table 3.

Table 3- Composition of the invention shown as preferred ranges and preferred proportion

Thus, in one embodiment of the invention, the proportion of the individual herb extract in said mixture of extract of herbs is in the range of about Urtica dioica (common nettle) 16 - 20%, Echinacea purpurea (eastern purple coneflower) 12-19%, Artemisia annua (sweet wormwood) 22-28%, Glycyrrhiza glabra (licorice) 6-14%, Scutellaria baicalensis (Chinese skullcap) 6-9%,

Rheum palmatum (Chinese rhubarb) 5-10%, Hyssopus officinalis (hyssop) 2.5-4.5%, Rosmarinus officinalis (rosemary) 2.5-4.5%, Nigella sativa (black cumin) 1-5%, Eucalyptus alba (white gum) 2.5 - 5%, Pterocarpus Santalinus (red sandalwood) 1.5-3%, Curcuma longa (turmeric) 0.6- 1.5%, and Panax ginseng (Asian ginseng) 0.3-1.5% by weight based on the total weight of the mixture of herbal extracts.

More preferably, the proportion of the individual herb extract in said mixture of extract of herbs are Urtica dioica (common nettle) about 18.5%, Echinacea purpurea (eastern purple coneflower) about 15%, Artemisia annua (sweet wormwood) about 25%, Glycyrrhiza glabra (licorice) about 10%, Scutellaria baicalensis (Chinese skullcap) about 7.5%, Rheum palmatum (Chinese rhubarb) about 7.5%, Hyssopus officinalis (hyssop) about 3.5%, Rosmarinus officinalis (rosemary) about 3.5%, Nigella sativa (black cumin) about 2.5%, Eucalyptus alba (white gum) about 3.3%, Pterocarpus Santalinus (red sandalwood) about 2%, Curcuma longa (turmeric) about 1%, and Panax ginseng (Asian ginseng) about 0.5% by weight based on the total weight of the mixture of herbal extracts.

As a starting point for the preparation of the herb extracts, any part of the herb may be used. In one embodiment, the entire plant is used including leaf, stem, seed and root. In other embodiments, the leaf, stem, root or seed, or any combination thereof are used. Preferably the different herb extract originate from: (1) leaf Rosmarinus officinalis (Rosemary), (2) leaf of Hyssopus officinalis (Hyssop), (3) root of Scutellaria baicalensis (Chinese skullcap), (4) root of Panax ginseng (Asian ginseng), (5) leaf of Eucalyptus alba (White Gum), (6) root of Glycyrrhiza glabra (Licorice), (7) leaf of Echinacea purpurea (eastern purple coneflower), (8) seed of Nigella sativa (Black cumin), (9) leaf of Artemisia annua (sweet wormwood), (10) root of Rheum palmatum (Chinese rhubarb), (11) leaf of Urtica dioica (Common Nettle), (12) root of Curcuma longa (turmeric), and (13) leaf of Pterocarpus Santalinus (red sandalwood).

The mixture of herbs present in the composition of the invention, may be used as it is, i.e. as ethanol extracts or further formulated into any suitable pharmaceutically acceptable dosage forms such as solid, liquid, semi-solid, gel, creme etc.. Thus, the pharmaceutical composition of the invention may further comprise pharmaceutically acceptable excipients. The excipients will be selected dependent on the dosage form to be provided. Exemplary suitable dosage forms include, effervescent tablet, caplet, pill, capsules coated capsules, powder, troche, lozenge, sachet, pencil sachet, four stitching sachet, suppository, roll, cream (wax or no-wax-based), dry powder sachet, chewing products, soft-gel, syrup, concentrated syrup, oral or nasal spray (vacuum or pressured), topical spray, liquid-filled capsule, gel-cap, liquid sachet, ampoule, injectable liquid as intravenous or intramuscular or intraperitoneal or oral liquid vial, suspension, rinsing and gargle products and other forms known to the artisan of ordinary skill in the pharmaceutical sciences. Preferred dosage forms according to the invention are tablets, inhalatory spray or droplets. In particular, tablets for oral administration, inhalatory spray for nasal-sinuses or oral-pharynx administration and/or aromatherapy droplets for topical administration.

Different preferred dosage forms may be provided in as a set of dosage forms, called “Package”. The set may contain tablets, nasal-sinuses spray, oral-pharynx spray, and aromatherapy inhaling drop in suitable dosages and units.

According to a second aspect of the invention, the pharmaceutical composition may be used as a medicament. In particular, the pharmaceutical composition according to the invention may be used in the treatment of a coronavirus infection. The coronavirus infection may be selected from the group of MERS and SARS viruses, preferably the SARS-CoV-2 virus causing COVID-19 disease.

In this context, treatment of coronavirus diseases, as “staging diseases” encompasses prophylactic treatment, treatment of coronavirus infections, and post-treatment of adverse effects caused by the coronavirus infection. The disease are typically with respiratory disorders and certain symptoms, e.g., symptoms defined in Case Report Form of World Health Organization for Covid-19. Some of these symptoms maybe long lasting.

The composition of the invention is “staging medicaments” with a synergy of antiviral, antiinflammatory, and immune-enhancing capacity to inhibit coronavirus infection, as prophylactic treatment agent, a composition to treat the coronavirus infection, and a pharmaceutical composition to treat adverse effects following coronavirus disease, and similarly to cope with SARS, MERS, and similar viral infections such as various types of influenza.

In yet another aspect, the invention provides a method for treatment of coronavirus infections, comprising the step of administering to a mammal that has been exposed to or are in risk of being exposed to coronavirus, a safe and effective amount of the pharmaceutical composition of the invention.

In an embodiment, the composition of the present invention is in the dosage form of nasal- sinuses spray (e.g. SaliraVira Nasal ^® ) with a prophylaxis effective amount of 100 to 160 mg per event (i.e., risk of being infected such as being in crowds or having interaction with covid patients), and oral-pharynx (e.g. SaliraVira Oral ^® ) with a prophylaxis effective amount of 100 to 160 mg per event for “pre-exposure stage or Stage 1” to treat the subjects. In an embodiment, the composition of the present invention is in the dosage form of nasal- sinuses spray (e.g. SaliraVira Nasal ^® ) with a therapeutic effective amount of 200 to 320 mg per day and oral-pharynx (e.g. SaliraVira Oral ^® ) with a therapeutic effective amount of 200 to 320 mg per day for “post-exposure stage or Stage 2” to treat the subjects.

In an embodiment, the composition of the present invention is in the dosage form of nasal- sinuses spray (e.g. SaliraVira Nasal ^® ) with a therapeutic effective amount of 200 to 320 mg per day, oral-pharynx spray (e.g. SaliraVira Oral ^® ) with a therapeutic effective amount of 200 to 320 mg per day, and antiviral tablet (e.g. SaliraVira Tablet ^® ) with a therapeutic effective amount of 2600 to 3100 mg per day for “mild symptoms stage or Stage 3” to treat the subjects.

In an embodiment, the composition of the present invention is in the dosage form of nasal- sinuses spray (e.g. SaliraVira Nasal ^® ) with a therapeutic effective amount of 200 to 320 mg per day, oral-pharynx spray (e.g. SaliraVira Oral ^®) with a therapeutic effective amount of 200 to 320 mg per day, and antiviral tablets (e.g. SaliraVira Tablet ^®) with a therapeutic effective amount of 2600 to 3100 per day, and with antiviral drops (SaliraVira Drop ^®) with a therapeutic effective amount of 1900 to 2300 mg per day for “severe symptoms (with short-breath) stage or Stage 4” to treat the subjects.

In an embodiment, the composition of the present invention in any suitable pharmaceutically acceptable dosage forms, cited before, to be used in intensive care units such as suppository, ampule, nasal-sinuses spray, oral-pharynx spray, tablets, or drops for “acute respiratory symptoms stage or Stage 5” to treat the subjects so that the total therapeutic effective amounts 4900 to 6040 mg in addition to the required routine treatments in intensive care units.

In an embodiment, the composition of the present invention is in the dosage form of tablets (e.g. SaliraVira Tablet ^® ) with antiviral, anti-inflammatory and immune-enhancing properties, in a therapeutic effective amount of 2600 to 3100 mg per day, and with antiviral, anti-inflammatory and immune-enhancing drops (e.g. SaliraVira Drop ^® ) with a therapeutic effective amount of 1900 to 2300 mg per day, or with alternative dosage forms of the said composition, for “post- Covid-19 recovery stage” to treat the subjects.

As apparent from above, the composition of the present invention may be formulated by alternative proportions and/or different combinations of the herbal extracts, or by adding and/or omitting some compounds and/or some herbs, cited as major ingredients in Table 2 and in the various dosage forms described in the foregoing, with changed efficacies.

The invention will now be further illustrated with reference to the following non-limiting examples.

EXAMPLE 1

Pharmaceutical Preparation

The kinds and proportions of the herbal ingredients used in the process of making the pharmaceutical composition of the present invention are described in Table 2. The pharmaceutical composition is in the brand name "SaliraVira ^® ”. It is formulated and provided as products in the current forms of tablet, nasal-sinuses spray, oral-pharynx spray, aromatherapy drop, and also extendable to other requisite dosage forms as cited in above.

Quality control tests carried out for each individual raw material were according to conventional methods used in the herbal pharmaceutical field, which include, but are not limited to, physical appearance, loss on drying, total ash, acid insoluble ash, alcohol extracts, water extracts, TLC, HPLC, heavy metals, microbial counts and residual pesticides.

For manufacturing process, each individual herb was pretreated according to common procedures. The herbs were weighed and certain amount of each herb was separated.

In order to manufacture the tablets (SaliraVira ^® tablets) and aromatherapy drops, each of the individual herbs with certain amounts were chopped in very small pieces and thoroughly mixed together. The mixed herbs were placed in an extractor and ethanol 96% was added and then percolated for 24 hours. The extracted solution of 13 herbs were passed through a set of filters. This extracted solution was called Extract I (“SaliraVira ^® Extract I”).

Extract I was directly used for aromatherapy drops. The degree of purity used for manufacturing the aromatherapy drops are same as Extract I.

To produce the tablets, Extract I is fed in granularization process with following steps:

1- The auxiliary materials (mainly calcium hydrophilic dihydrogen, microcrystalline cellulose and starch) were passed through the manual sieve of mesh 20 in large quantities and mixed. 2- The mixture is slowly added to the powders in rapid mixer granulator (RMG) and are mixed in a low speed. Then, proportional to the size of RMG and the amount of the mixture, water is added gradually to the powder inside the pot at a slow speed so that to allow it to rotate inside the pot. This process takes 10 minutes. At this stage, mixing continued for 2.5 minutes without chopper and 2 minutes with chopper.

3- The resulting dough was passed through a granulator and transferred to the Fluid Bed Processor (FBP). Then, PVP K30 binder powder was added to the solvent tank in the required amount and standardized “SaliraVira ^® Extract I” is added to the upper tank.

4- The granules were dried in FBP with an inlet temperature of 80°C, an outlet temperature of 60°C and a tablet temperature of 40°C to bring the humidity below one percent. The dried granules were passed through a Freut mill with 12 mesh. Finally, the homogeneous powder with the same granules was taken out of the product tank of the device for tableting and packaged into tablets, identified SaliraVira ^® Tablet.

For manufacturing of nasal or oral sprays; the individual herb was cut into small pieces and thoroughly mixed together. The mixed herbs were placed in an extractor and ethanol 70% was added and then percolated about 24 hours. The extracted solution of 13 herbs were passed through a set of consequent filters for several times. This extracted solution was called Extract II (“SaliraVira ^® Extract II”)

For nasal-sinuses spray, lecithin, glycerol and MCT oil or other modifiers are added to the Extract II. In the process the ethanol is reduced to about 40% v/v. The solution will be mixed and then will be ready for filling and capping stages and finally can be packaged as nasal-sinuses spray, identified SaliraVira ^® Nasal spray.

For oral-pharynx spray, glycerol is added to Extract II. In the next step, the degree of ethanol was reduced to about 60% v/v and the materials are thoroughly mixed together. In this stage, the solution is ready to be filled and capped and eventually be packaged as oral spray, identified SaliraVira ^® Oral spray

EXAMPLE 2

Physicochemical analyses of the pharmaceutical composition of the invention

The liquid Extract I and the solid composition of the tablets were submitted to analysis. Extracts with various alcohol contents is used for nasal-sinuses spray, oral-pharynx spray, and aromatherapy drop. Based on FDA Guidance for Nasal Spray and Inhalation Solution, Suspension, and Spray Drug Products Section IV.L, the ethanol alcohol as solvent or and preservative is variable between 10-95%. The content of alcohol in aromatherapy drop is 94 percent v/v. In the aromatherapy process it will be inhaled in 500 ml hot water. The alcohol content of nasal-sinuses and oral-pharynx spray is less than 40 percent. All the alcohol content in SaliraVira package is under permitted daily exposure (PDE) based APPENDIX 6. TOXICOLOGICAL DATA FOR CLASS 3 SOLVENTS of the FDA Guidance. pH test of the liquid extract was performed three times in 15 and 25 Celsius degrees with calibrated digital pH meter. The pH was 6 in the permissible range of 5 to 7 for respiratory extracts.

Total flavonoids have been determined for the compositions in dosage forms nasal-sinuses spray, oral-pharynx spray and aromatherapy drop. This assay done according to Aluminum chloride colorimetric method. Extracts I and II (0.5 ml of 1 : 10 g ml-1) in methanol were separately mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. It remained at room temperature for 30 min; the absorbance of the reaction mixture was measured at 415 nm with a double beam Perkin Elmer UV/Visible spectrophotometer. The calibration curve was prepared by preparing Rutin (Standard flavonoid) solutions at concentrations 1 to 100 mg ml-1 in methanol. The result was 3.65 mg in ml of each dosage form.

Total flavonoids determined, based on Hyperoside is 0.048 mg/ml.

Based on BP 2017 and using standard oven, the dry residue of 100 gr of extract is 2.81 w/w.

Using pycnometer, the density of the solution is 0.82 kg/liter.

Based on USP-40, the microbial tests of the extract indicate, the numbers of all bacteria are zero and the amount of yeast in extract is less than 10. EXAMPLE 3

Acute and Chronic Toxicity Study of the pharmaceutical composition of the invention in mice

Purpose: The following experiment was conducted at Salari Institute of Cognition and Behavorial disorders in Alborz Province in Iran to examine in vivo acute and chronic toxicity of the pharmaceutical composition of the invention.

Methods: To evaluate the effects of the pharmaceutical composition of the invention, 20 C57BL/6 inbred mice were studied for acute and chronic prescription of the composition. Mice were housed in the Animal House for 1 week, provided with proper light, temperature and moisture and fed with standard mouse food pellets and water ad libitum. Seven-week-old mice weighting 23 ± 5 g were used for experiments.

The mice were randomly divided into four groups, twenty mice were included in control groups and twenty mice were included in treatment group for evaluating acute and chronic toxicity, respectively. The pharmaceutical composition of the invention in the form of ethanol extracts has been taken by mice via gavage for 48 hours at dose of lOOOmg/kg BW/day. To follow-up chronic prescription of SaliraVira ^® , a dose of lOOmg/kg BW/day of the drug have been taken by mice via gavage, once a day for 7 days.

During treatment, weight, mortality, any abnormal behavior, anorexia, drowsiness, inactivity, decreased response to physical stimuli were studied.

At the end of the treatment, hematological factors, liver enzymes and histopathology of brain, liver and kidney tissues were examined. Blood samples were taken from the heart and the tissues of the brain, liver and kidney were removed from the body of the mice in accordance with ethical and scientific principles to evaluate the rate of inflammation, cell death and tissue damage.

Results: The study of hematological factors in the test of control groups in both acute and chronic treatment did not show a significant difference. Also, among the liver factors, none of the studied enzymes showed a significant difference in activity between the treatment and control groups Pathological studies did not show a significant difference in inflammation, necrosis and degeneration between the control and treatment groups in both acute and chronic prescriptions. It is noteworthy that the results of studies indicate that the use of SaliraVira ^® cause no pathological injuries of the brain, liver and kidney. In addition, it indicates that the use of SaliraVira ^® reduces the inflammation of the brain and liver in treatment groups comparing to relative to the control groups.

Conclusion: Behavioral, hematological and pathology studies of the pharmaceutical composition of the invention did not show any significant differences between control and treatment groups in acute and chronic categories. The results of studies show that the pharmaceutical composition of the invention is safe. In addition, taking the pharmaceutical composition of the invention in different periods has reduced inflammation of the brain and liver.

EXAMPLE 4

In vivo bone marrow assay on the pharmaceutical composition of the invention

Purpose: The following experiment was conducted at the CytogGnome Medical Genetics Laboratory in Tehran Province in Iran to examine the clastogenic effects of the pharmaceutical composition of the invention.

Methods: To evaluate the clastogenic effects of the pharmaceutical composition of the invention, femurs dissected from seven-week-old male NMRI mice were purchased from Pasteur Institute of Iran, Alborz province, Iran. They were housed in the Animal House for 1 week, provided with proper light, temperature and moisture and fed with standard mouse food pellets and water ad libitum. Seven-week-old mice weighted 23 ± 5 g.

Five mice were allocated to control group and 10 mice were allocated for treatment. All animal experiments in this study were carried out with the prior approval of the Institutional Ethics Committee, strictly adhering to the Helsinki guidelines provided for the care and treatment of animals. Mice were treated with acute dose of 1000 mg/kg BW/day by administering the pharmaceutical tablet of the invention dissolved in water by gavages.

Treated and untreated mice were sacrificed 72 hours after drug treatment. The mice were killed by cervical dislocation; their femoral bone marrow was flushed out by means of fetal calf serum, and a cell suspension from both femurs was prepared. The suspension was centrifuged for 5 min at 1,000 rpm. After centrifuging, the supernatant was removed and cells were re-suspended in the remaining serum, and a smear was prepared on glass slides, fixed with methanol and stained in May Gnmwald-Giemsa (Merck, Darmstadt, Germany). In this method of staining, polychromatic erythrocytes (PCEs) are stained blue-violet, while normochromatic erythrocytes (NCEs) are stained in yellow- orange.

In order to study the cytotoxic effects of the pharmaceutical composition of the invention on the proliferation of the bone marrow cells, the ratio of PCE/ NCE) was calculated. This ratio is an indicator of the proliferation rate and turnover of PCE to NCE which should be similar at normal situation, but decline in the case of cytotoxicity.

Results: Results are summarized and provided as Table 4. As seen in the table the frequency of micronuclei induced by treatment of acute dose of the pharmaceutical composition of the invention is nearly similar to those animals not given treatment (p-value, P=0.462).

Table 4- study the cytotoxic effects of the pharmaceutical composition of the invention

Conclusion: These results imply no genotoxic or clastogenic effects have been occurred in bone marrow of treated mice with the pharmaceutical composition of the invention. Moreover, no change of the ratio of PCE/NCE is observed, indicating no cytotoxic effect of the pharmaceutical composition of the invention in bone marrow (p-value, P= 0.672).

EXAMPLE 5

Clinical Study of the pharmaceutical composition of the invention on Covid-19 patients

General aspects

Purpose: This clinical study is to evaluate the efficacy of the pharmaceutical composition of the invention based on improving symptoms and reducing viral load in treatment of Covid-19 patients.

Permissions: This clinical study is initiated, performed, and fulfilled according to ethic code: IR.SBMU.PHARMACY.REC.1399.276 and clinical trials code: IRCT20201220049771N1.

Material: Covid-19 outpatients were treated with a set of dosage forms of the pharmaceutical composition of the invention, called “package”. The package contains SaliraVira Tablet ^® as tablet, SaliraVira Nasal ^® as nasal-sinuses spray, SaliraVira Oral ^® as oral-pharynx spray, and SaliraVira Drop ^® as aromatherapy inhaling drop.

Dosage regimen: Each patient randomly prescribed to take nasal-sinuses spray (2 puffs in each nostil, 5 times/day) and oral-pharynx spray (2 puffs in pharynx vial oral, 5 times/day)), one tablet in each 6 hours, 4 times and inhaling the drop (10 to 15 drops in 500 ml, in 8 hour timespan) in 56 Celsius degree 2 times daily.

Method: Covid-19 patients are separated in “case” (or “treatment”) and “control” groups. The patients in both “case” and “control” groups are treated by routine treatment in hospital. In parallel, the patients in “case” group are treated with the package. The infliction of the patients was confirmed by PCR test with “cycle threshold” value or approval of physician based on their symptoms. Then the “dosage regimen” was performed

Standard forms: The Covid-19 Case Report Form, World Health Organization (WHO CRF) is for the symptoms of hospitalized patients (in Stages 4 and 5 of disease). This clinical study is for Covid-19 outpatients (Stages 2 and 3). It required to modify the WHO CRF to be adjusted for Covid-19 outpatients. Finally, we used a “modified WHO CRF” with 14 symptoms for this clinical trial (CT).

Supervisory: The Covid-19 patients were diagnosed and introduced by the physicians of Covid- 19 Section, Department of Infectious Diseases and Tropical Medicine (DIDTM), Imam Khomeini Hospital Complex (IKHC), Affiliated to Tehran University of Medical Sciences (TUMS).

Sample size: The clinical study comprised 170 patients, including 87 patients (treated by the pharmaceutical composition of the invention), 56 patients as control group, and 27 patients did not complete CT.

Viral load evaluation

Viral load (VL) efficacy of “case” and “control” groups are tested. Patients' “cycle threshold” values indicate VL values. Drug efficacy with respect to VL is determined by “mean reduction rate” of the patients in “case” and “control” groups. “Cycle threshold” values of the patients are tested in the 1 ^st , 4 ^th , and 8 ^th days of treatment period.

“Efficacy” of the pharmaceutical composition of the invention used by each case or control group is determined by the respect probability. “Significancy” of the drug used by each case or control group is determined by the respect p-values.

The comparison of VL mean reduction rates for case and control groups are shown in Figure 2. In the 4 ^th day, the probability of mean reduction rates of VL for patients is 27% and 10% for patients in case and control groups, respectively. Also, in the 8 ^th day, the mean reduction rates of VL for the patients in case and control group are 60% and 40%, respectively.

Intra- and inter-group comparisons of VL in “case” and “control” groups are used to state the efficacy and significancy of VL reduction following treatment with the pharmaceutical composition of the invention.

For intra-group comparisons of VL, the p-values, which indicates the significancy of the difference for each case and control groups in dayl-to-day4 period, are 0.09 and 0.26, respectively. That is, the latter p-values indicate the significancy of drug efficacy. Same p-values for case and control groups in dayl-to-day8 period are 0.0009 and 0.0016, respectively. Also, the inter-group comparison of VL stated by p-value of case group versus control group in day8case- to-day8control is 0.005. This indicates that the pharmaceutical composition of the invention has superior efficacy with a probability of 99.5% to routine treatment.

Symptom testing

Based on “modified WHO CRF” for Covid-19 out-patients, 14 symptoms of the patients in both “case” and “control” groups were tested daily in 8 -day timespans of treatment. The “significancy” and “efficacy” of treatment are measured by the relevant “p-values” and “probability” of changes in symptoms in both groups, respectively. The p-value in each group is one-sided and it compare the probability distribution of the Covid-19 symptoms in the n ^th (i.e., 4 ^th , 6th) day to the 1 ^st day of treatment. The results are shown in Table 5 below.

Table 5- Integrated results of symptoms of Covid-19 patients in “case” and “control” groups

According to Table 5, in the 4 ^th day of treatment, with a probability of at least 95% (or p-value < 0.05) the improvement of 3 out of 14 symptoms of Covid-19 (i.e., joint-pain, muscle-aches, wheeze) in “case” group were significantly and efficaciously preferred to “control” group.

According to Table 5, in the 6 ^th day of treatment, with a probability of at least 90% (or p-value < 0.1) the improvement of 7 out of 14 symptoms of Covid-19 (i.e., sore-throat, headache, shortness-of-breath, decreased-smell, abdominal-pain, inability-to-walk, and rhinorrhea) in “case” group were significantly preferred to “control” group. Also, 4 out the 7 symptoms (i.e., headache, abdominal-pain, inability-to-walk, rhinorrhea) in “case” group efficaciously preferred to “control” group.

According to Table 5, in the 6 ^th day of treatment, with a probability of at least 95% (or p-value < 0.05) the improvement of 5 out of 14 symptoms of Covid-19 (i.e., sore-throat, headache, shortness-of-breath, abdominal-pain, inability-to-walk) in “case” group were significantly preferred to “control” group. Also, 4 out the 5 symptoms (i.e., sore-throat, headache, abdominal- pain, inability-to-walk) in “case” group efficaciously preferred to “control” group.

According to Table 5, in the 7&8-day treatment timespan, with a probability of at least 90% (or p-value < 0.1) the improvement of 4 out of 14 symptoms of Covid-19 (i.e., decreased-taste, fatigue, altered-consciousness-confusion, chest-pain) in “case” group were significantly preferred to “control” group. Also, 2 out the 4 symptoms (i.e., fatigue, altered-consciousness- confusion) in “case” group efficaciously preferred to “control” group.

According to Table 5, in the 7 ^th &8 ^th -day treatment timespan, with a probability of at least 95% (or p-value < 0.05) the improvement of 2 out of 14 symptoms of Covid-19 (i.e., fatigue, altered- consciousness-confusion) in “case” group were significantly and efficaciously preferred to “control” group.

Results from testing the symptoms of Covid-19 patients of “case” an “control” groups in 8-day treatments with a probability of 90% (or p-value < 0.1) indicate that SaliraVira ^® is more significant for all 14 symptoms and more significant and efficaciously for 10 out of 14 symptoms.

Results from testing the symptoms of Covid-19 patients of case a control groups in 8 -day treatments with a probability of 95% (or p-value < 0.05) indicate that the pharmaceutical composition of the invention is more significant for 10 symptoms and more significant and efficaciously for 8 out of 10 symptoms.

Figure 3 indicates the “cumulative probability distribution function (CDF) of treatment duration" of patients in “case” group with a mean of 8.8 and standard deviation of 5.5 days. With a probability of 87% the patients treated by the pharmaceutical composition of the invention were recovered by the 10 ^th day of treatment with the composition of the invention. The difference of the treatment duration of “case” and “control” with a p-value < 0.05 is tested by both one-sided hypothesis test and mutually inclusiveness of their domains.

Figure 4 indicates the “CDF of treatment duration" of patients in “control” group with a mean of 13.7 and standard deviation of 3.3 days. With a probability of 87% the patients were recovered by the 17th day of drug use.

“CDFs of treatment duration" of patients in “case” and “control” groups are compared in Figure 5. It indicates that the treatment duration of “case” group has “statistical dominance” (Type I) to “control” group. The treatment of patients using the package of the invention is in average 4.9 days less than other routine treatments. Further, the results in the 10 ^th day of the treatment, the patients using the package of the invention will be recovered with a probability of 87% comparing to the probability of 12% for other patients using routine treatment.

Overall conclusion

Clinical trials indicate that the pharmaceutical composition of the invention has significant efficacy as pharmaceutical composition to accelerate reducing coronavirus, shortening treatment period by 4.9 days in average, and speed up improving all 14 symptoms of Covid-19 outpatients in 8-day period. None of the patients in “case” needed to be hospitalized or died, where 28% of the “control” group needed hospital.