DESC:FIELD OF THE INVENTION
The present invention relates to the field of phytopharmaceutical medicine. More particularly, it relates to ayurvedic composition comprising herbal extracts and zinc oxide (Jasad Bhasma). Herbal extracts contain phytochemicals that inhibit SARS-CoV-2 spike (S) protein docking with ACE2 receptor. The phytochemicals also inhibit virus replication by inhibiting RNA-dependent RNA polymerase. Additionally the invention relates to a method of preparation of the oral herbal composition with zinc oxide (Jasad Bhasma).

BACKGROUND OF THE INVENTION
COVID-19 is associated with both pulmonary pathology and extra pulmonary manifestations in multiple vital systems, including hematologic-immune, cardiovascular, renal, gastrointestinal and hepatobiliary, endocrine-neurologic, and ophthalmic dysfunctions. SARS-CoV-2 binds to the angiotensin-converting enzyme-2 (ACE-2) receptors present on the cell surface which undergo endocytosis, and the virus enters inside the cell. Upon entering the cell, it synthesizes RNA using the host metabolic processes in which enzyme RNA dependent RNA polymerase (RdRp) is essential. Due to cellular damage caused by the virus, the alveolar macrophages generate various proinflammatory cytokines like interleukin-6 (IL-6), resulting in alterations in the vascular permeability. There is leakage of cytokines, promoting more inflammation with dysfunctional immune responses and infiltration of inflammatory cells like monocytes, macrophages, and T-cells, ultimately producing cytokine storm. Uncontrolled cytokine storm causes lung inflammation and multi organ inflammation leading to respiratory distress and organ failure, resulting in death.

US8846114B1 discloses a composition for treating herpes and cold sores. A herpes treatment capsule consisting essentially of therapeutically effective amounts of olea europaea leaf extract, olea europaea fruit oil, hypericum performatum extract, propolis extract and maleluca alternifolia extract.

US5837257 This invention relates to compositions derived from Chinese herbal medicines, medicinal plants and extracts thereof, and to their use for the treatment of animals infected with viruses, especially with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). A therapeutically effective amount of a composition which comprises at least one herbal medicine selected from the group consisting of: Solani herba, prepared from the whole plant of Solanum nigrum; Lespedezae herba, prepared from the whole plant of Lespedeza cuneata; Senecinis herba, prepared from the whole plant of Senecio scandens; and Ligustri fructus, prepared from the mature fruit of at least one plant selected from the group consisting of Ligustrum lucidum and Ligustrum japonicum.

IN750/CHE/2008 The process for preparing herbal remedy invention comprising Zingiber Officinalis, Piper Nigrum and Piper Longum, Ficus Racemosa, Solanum Tribolatum, Acalypha Indica, Terminalia Chebula, Cynodon Dactylon, Erythrina Variegata, Ficus Bengalensis, Phyllanthus Amalus, Murraya Koenigii, Trigonella Foenum, Centella Asiatica, Emblica Officinalis, Azadirachta Indica, Moringa Oleifera seeds, Cuminum Cyminum, Rosa Chinensis and Ficus religiosa which has been used to treat all Common cold, Continuous cough.

Ramesh K. Goyal, Jaseela Majeed, Rajiv Tonk, Mahaveer Dhobi, Bhoomika Patel, Kalicharan Sharma, and Subbu Apparsundaram “Current targets and drug candidates for prevention and treatment of SARS-CoV-2 (COVID-19) infection” Rev. Cardiovasc. Med. 2020 vol. 21(3), 365–384, discusses on Current targets and drug candidates for prevention and treatment of SARS-CoV-2 (COVID-19) infection.
Bachir Benarba and Atanasio Pandiella, “Medicinal Plants as Sources of Active Molecules Against COVID-19 Frontiers in Pharmacology” 11:1189. doi: 10.3389/fphar.2020.01189, discusses on the different herbal products (extracts) and purified molecules may exert their anti-SARS-CoV-2 actions by direct inhibition of the virus replication or entry.

Shimon Ben-Shabat & Ludmila Yarmolinsky & Daniel Porat & Arik Dahan, “Antiviral effect of phytochemicals from medicinal plants: Applications and drug delivery strategies”, Drug Delivery and Translational Research, Given the challenges and possibilities of antiviral treatment, this review provides the verified data on the medicinal plants and related herbal substances with antiviral activity, as well as applied strategies for the delivery of these plant extracts and biologically active phytochemicals.

Beverly C David, Sudarsanam G “Ethnomedicinal plant knowledge and practice of people of Javadhu hills in Tamilnadu” Asian Pacific Journal of Tropical Biomedicine. September 2011; Objective: To conduct an ethnobotanical survey and collect information from local people about the medicinal plants used in the treatment of diseases by the tribes of Javadhu hills.

Hence, there is a need for an efficacious prophylactic and therapeutic treatment for COVID-19.

OBJECT OF THE INVENTION
It is primary object of the present invention to provide an oral herbal composition comprising phytochemicals.

It is another object of the present invention to provide a phytopharmaceutical medicine for the treatment of COVID-19.
It is another object of the present invention to provide an oral herbal composition or a phytomedicine that inhibits SARS-CoV-2 spike interaction or docking with the ACE2 receptor.

SUMMARY OF THE INVENTION
One or more problems of the conventional prior arts may be overcome by various embodiments of the present invention.

It is the primary aspect of the present invention to provide an herbal composition for the treatment of COVID-19 by targeting ACE2 receptor and immunity enhancement in humans, consisting extract of:
Solanum nigrum – 15 to 35% w/w;
Acalypha indica – 15 to 35% w/w;
Ocimum sanctum – 4 to 15% w/w;
Adathoda vasica – 4 to 15% w/w;
Withania somnifera – 4 to 15% w/w;
Glycyrrhiza glabra – 4 to 15% w/w;
Piper nigrum – 4 to 15% w/w;
Zinc oxide (Jasad bhasma) – 3 to 8% w/w; and
pharmaceutical excipients – 8 to 16% w/w.

It is another aspect of the present invention to provide the herbal composition for the treatment of COVID-19 by targeting ACE2 receptor and immunity enhancement in humans, wherein the pharmaceutical excipient includes starch.

It is another aspect of the present invention to provide the herbal composition for the treatment of COVID-19 by targeting ACE2 receptor and immunity enhancement in humans, wherein composition preferably comprises extract of Solanum nigrum – 33.33% w/w; Acalypha indica – 33.33% w/w; Ocimum sanctum – 4% w/w; Adathoda vasica – 4% w/w; Withania somnifera – 4% w/w; Glycyrrhiza glabra – 4% w/w; Piper nigrum – 4% w/w; zinc oxide (Jasad bhasma) – 3.33% w/w; and Pharmaceutical excipients – 10% w/w.

It is another aspect of the present invention to provide the herbal composition for the treatment of COVID-19 by targeting ACE2 receptor and immunity enhancement in humans, wherein the extract comprises one selected from a whole plant extract, leaf extract, root extract, fruit extract and a combination thereof.

It is another aspect of the present invention to provide the herbal composition for the treatment of COVID-19 by targeting ACE2 receptor and immunity enhancement in humans, wherein the extract is prepared in solid powder or capsule form.

It is another aspect of the present invention to provide the herbal composition for treatment of COVID-19 by targeting ACE2 receptor and immunity enhancement in humans, wherein the extract is administered one form selected from the group consisting of a solid powder, capsule and a combination thereof.

It is another aspect of the present invention to provide the herbal composition for treatment of COVID-19 by targeting ACE2 receptor and immunity enhancement in humans, wherein the composition comprising the extract of Solanum nigrum, Acalypha indica, Ocimum sanctum, Adathoda vasica, Withania somnifera, Glycyrrhiza glabra, and Piper nigrum; and zinc oxide (Jasad bhasma) is administered at a dosage of 4500 mg per day administered as 1500 mg thrice daily.

It is another aspect of the present invention to provide the herbal composition for treatment of COVID-19 by targeting ACE2 receptor and immunity enhancement in humans, wherein the unit dosage strength of the composition comprising the extract of Solanum nigrum, Acalypha indica, Ocimum sanctum, Adathoda vasica, Withania somnifera, Glycyrrhira glabra, and Piper nigrum; and zinc oxide (Jasad bhasma) is 750mg per capsule.

It is another aspect of the present invention to provide the herbal composition for treatment of COVID-19 by targeting ACE2 receptor and immunity enhancement in humans, wherein the extract is hydro-alcohol extract comprising ratio of in a range between alcohol 50 and 90%: water in a range between 10 and 50 %.

It is another aspect of the present invention to provide a process for formulation of herbal composition for treatment of COVID-19 by targeting ACE2 receptor and immunity enhancement in humans, comprising:
grinding of the dried plants Solanum nigrum, Acalypha indica, Ocimum
sanctum, Adathoda vasica, Withania somnifera, Glycyrrhiza glabra, and Piper
nigrum to a size of 3 to 4 mm;
preparing a solvent extract of the plants by heating at a temperature range
between 70 and 80 degree Celsius with continuous extraction under vaccum
400-600 mmHg maintaining the temperature between 70 and 80 degree
Celsius for 4-5 hours;
filtering the material through 100 mesh filter and concentrate the extract using
multiple-effect evaporators at a temperature between 75 and 80 degree Celsius
for 4-5 hours to obtain required Total solids 25-35% under vaccum 400-600
mmHg;
subjecting the extract to heat treatment at a temperature 121±2°C for 20
minutes and cooling the content to below 40°C;
subjecting the concentrated extract to vacuum drying/ spray drying/ freeze-
drying to get dry powder;
adding of zinc oxide (Jasad bhasma) and starch, and mixing for a period of 15
minutes;
sieving of mixed material using a 40 mesh sieve; and
loading of above composition into a gelatin capsule to obtain capsules,
wherein the herbal composition comprises extract of Solanum nigrum
– 15 to 35% w/w; Acalypha indica – 15 to 35% w/w; Ocimum
sanctum – 4 to 15% w/w; Adathoda vasica – 4 to 15% w/w; Withania
somnifera – 4 to 15% w/w; Glycyrrhiza glabra – 4 to 15% w/w; Piper
nigrum – 4 to 15% w/w; Zinc oxide (Jasad bhasma) – 3 to 8% w/w;
and pharmaceutical excipients – 8 to 16% w/w.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1A shows a schematic of the experimental protocol. Viral entry: ACE2 receptor assay.
Figure 1B shows a representative data of a dose response using different concentrations of CoroQuil-Zn assed to HEK293T cells and SARS-CoV-2 virus Spike (S) protein containing virions; CoroQuil-Zn reduces viral entry.
Figure 2A illustrates graphical representation of relationship between SARS-CoV-2 genome equivalent and CoroQuil-Zn (mg/ml).
Figure 2B illustrates graphical representation of relationship between viability of lung cells and CoroQuil-Zn (mg/ml).
Figure 3A and 3B illustrates results obtained using the concentration matrix of Remdesivir and CoroQuil-Zn presented as 2D and 3D synergy visual maps.
Figure 4A and 4B illustrates results obtained using the concentration matrix of Favipiravir and CoroQuil-Zn presented as 2D and 3D synergy visual maps.
Figure 5 illustrates graphical representation of relationship between percentage of control and CoroQuil-Zn (mg/ml).
Figure 6 illustrates graphical representation of reduced viral load in the CoroQuil-Zn group suggesting that CoroQuil-Zn may offer protection from SARS-CoV-2 infection.

DETAILED DESCRIPTION OF THE INVENTION
The source and geographical origin of the biological materials:
Acalypha indica dried - whole plant, Solanum nigrum dried - whole plant, Yashthimathu dried - roots, Vasaka dried – leaves, Piper nigrum – dried fruits, Aswagandha dried – roots, and Tulasi dried – leaves were obtained from MRP Nutraceuticals, No. 74/9-H2, Shagunthalaamma complex, Bagalur road, Hosur – 635109, Tamil Nadu.

The present invention is an ayurvedic composition (CoroQuil-Zn) containing zinc oxide (Jasad Bhasma) and herbal extracts. Herbal extracts contain phytochemicals that inhibit SARS-CoV-2 Spike (S) protein docking with ACE2 receptor. The phytochemicals also inhibit virus replication by inhibiting RNA-dependent RNA polymerase.

The present invention attempted to develop an efficacious prophylactic and therapeutic treatment for COVID-19. The phytoconstituents present in different plants were identified and selected using in silico molecular docking and cell-based antiviral assays for their ability to interact with the host ACE2 receptor and viral RNA-dependent RNA polymerase (RdRp). The in-silico molecular docking studies revealed that flavonoids that interact with both ACE2 and RdRp proteins. CoroQuil-Zn is formulated based on these molecular docking and cell-based studies of phytochemicals.

CoroQuil-Zn contains plant extracts derived from Acalypha indica and Solanum nigrum mainly. In addition, CoroQuil-Zn also contains Zinc oxide (Jasad bhasma), Adathoda vasica, Glycyrrhiza glabra, Ocimum sanctum, Withania somnifera, and Piper nigrum. Phytochemicals in CoroQuil-Zn reduces the entry of virus into the cell by docking at the interface of SARS-CoV-2 and ACE2 interface.

Based on the molecular docking studies and cell-based studies Acalypha indica and Solanum nigrum plant extracts have been selected mainly due to the composition of their phytoconstituents. CoroQuil-Zn capsules formulation contains plant extracts Acalypha indica and Solanum nigrum. None of the existing AYUSH products contain these plant extracts in combination.

CoroQuil-Zn formulation comprises of the following ingredients in percentage by weight basis:
Solanum nigrum: 15 to 35% w/w; Acalypha indica: 15 to 35% w/w; Ocimum sanctum: 4 to 15% w/w; Adathoda vasica: 4 to 15% w/w; Withania somnifera: 4 to 15% w/w; Glycyrrhiza glabra: 4 to 15% w/w; Piper nigrum: 4 to 15% w/w; Zinc oxide (Jasad bhasma): 3 to 8% w/w; and pharmaceutical excipients: 8 to 16% w/w.

Manufacturing process:
Basic process of separating active ingredients from herbs/ botanicals, involves extraction with water, organic solvents like alcohol and combinations thereof. Value addition comes from concentration of the extract and removal of inactive materials i.e., Marc.

Process description:
In majority of the cases, processing of herbs/medicinal plants for extracting the active ingredients goes through the following stages.

Size reduction and pelletization:
The dried plant material is disintegrated/ powdered by feeding it into a hammer mill or a disc pulverizer which has built-in services. Usually, the plant material is reduced to a size between 3 and 4 mm size reduction maximizes the surface area, which in turn enhances the mass transfer of active principle from plant material to the solvent. Sometimes, the powdered material is pelletized to avoid caking.
Hydro-alcohol extraction:
Methanol or Ethanol 50% to 90% and water 10% to 50%
After ensuring the extractor are cleaned, charge the required quantity of material into the extractor and pump the hydro alcohol as required based on raw material volume. Close the manhole of the extractor and apply steam into the extractor jacket. Heat the content until reaches 70-80 degree with continuous extraction under vaccum 400-600 mmHg.

Maintain the temperature between 70-80 degrees for 4-5 hours with the continuous circulation and record the temperature every one hour and record it during process.

After completion of process filter the material through 100 mesh filter cloth and collect it into Reactor/Rising film evaporator (RFE) for concentration.
Note: Check the Total solids of material and ensure that the expected yield is achieved. If the yield is found lesser than expectation continue one more wash, after discussing with the In-charge.

Concentration:
The enriched extract is concentrated in multiple-effect evaporators (in case of extraction with water or the solvent is distilled out to get the concentrate).

Ensure that the reactor used for the concentration is completely cleaned and free from previous products.

Check the TS% of the initial concentrate feed and concentrate material with the temperature 75-80 degree for 4-5 hours with continuous circulation to meet required TS 25-30% under vacuum 400-600 mmHg.
Note: Once desired TS value is attained, autoclave the material.

Autoclave:
Switch off the agitator, apply the steam into reactor jacket and heat the content until the temperature reaches 121°C. Maintain the temperature at 121±2°C for 20 minutes and the content to below 40°C, transfer for drying.

Drying:
The concentrated extract is subjected to vacuum drying/spray drying/ freeze drying to get the dry powder.

Spray drying:
Slowly charge extract into spray dryer feed tank.

Operate the spray drier and, start the heating and wait until the inlet temperature reaches 185°C.

Once the temperature reaches 185°C, start water feeding through the funnel and maintain the inlet temperature between 185-190°C and outlet powder material temperature between 95-100°C.

After temperature gets stabilized within the above mentioned range, close the water feeding. Open the feed inlet valve and start drying within the specified temperature.
Note: If found any lumps in spray dried material go for milling and sifting.

Milling:
Mill the spray dried material by using multi mill.

Sifting:
Sieve the multi milled material through 40#.

Packing:
Unload the extract in double lined LDPE poly bags tie with cord and place within HDPE drums.

EXAMPLE 1
Standard operating procedure for formulation of Coroquil-Zn capsules:
The following is the SOP for the formulation protocol of 50,000 capsules of 750 mg Coroquil-Zn capsules. Given below is the percentage contribution of each of the ingredients for the formulation.

S.No Sanskrit name Scientific name Common name Part used Quantity
1 Kakamachi (Makoi) Solanum nigrum Black nightshade Whole plant 33.33%
2 Haritamanjari Acalypha indica Indian nettle Whole plant 33.33%
3 Tulsi Ocimum sanctum Holy basil Leaf 4%
4 Adathoda Adathoda vasica Malabar nut Leaf 4%
5 Ashwagandha Withania somnifera Indian ginseng Root 4%
6 Yashtimadhu Glycyrrhiza glabra Liquorice Root 4%
7 Maricha Piper nigrum Black pepper Fruit 4%
8 Jasad bhasma Zinc oxide NA Powder 3.33%
9 Excipient (Starch) NA NA Powder 10%
Step 1:
The hydroalcoholic extracts of ingredients S.no: 1-7 were weighed in a calibrated electronic weighing balance. They were weighed as follows:
Makoi – 12.5 kg; Haritamanjari – 12.5 kg; Tulasi – 1.5 kg; Adathoda vasica – 1.5 kg; Ashwagandha – 1.5 kg; Yashtimadhu – 1.5 kg; Maricha- 1.5 kg; Jasad bhasma- 1.25 kg; and Starch (excipient) – 3.75 kg.

Step 2:
The above mixture was mixed in a sigmoidal mixing machine for a period of 15 minutes.

Step 3:
The mixed material was carefully collected and sieved by hand using a 40 mesh sieve. The entire process was completed in 35 minutes.

Step 4:
The entire lot was weighed again to determine if there was any loss during weighing and sieving.

Step 5:
Empty hard gelatin capsules were loaded onto capsule filling trays using an automatic capsule loading machine. After each tray was loaded, the mixture was filled using a semi-automatic hand filling machine.

Step 6:
Filled capsules were chosen at random for quality control checks to determine uniformity of weight and pH. Capsules that did not meet the specifications were discarded.

Step 7:
After the entire lot was filled and in-process quality checks were completed satisfactory, capsules were filled in bottles with each bottle containing 60 capsules.

Step 8:
Ten bottles of filled CoroQuil-Zn were sent to an NABL accredited lab for QC testing for the following parameters:
a) Acid-insoluble ash
b) Heavy metals (Lead, Arsenic, Cadmium, Mercury)
c) Total aflotoxins
d) Microbial load which measured total microbial count, total yeast and mould, E.coli, S.aureus, P.aeruginosa, C.albans.
e) Polynuclear aromatic hydrocarbons
f) Polychlorinated biphenyls
g) Organochlorine pesticides
h) Organophosphorus pesticides

Step 9:
Upon passing of all QC parameters, CoroQuil-Zn is considered as final finished product (FFP) and is ready for shipping.

CoroQuil-Zn Pre-clinical studies:
Procedure:
Remedium therapeutics conducted a detailed literature and in silico molecular docking studies and arrived at a set of phytochemicals that could potentially serve as an entry inhibitor for SARS-CoV-2 with the Spike (S) protein. Based on list of phytochemicals, Remedium did cell culture study to find out which phytochemical had an activity as the entry inhibitor for SARS-CoV-2 Spike protein. After the discovery of a set of phytochemicals through this process, Remedium investigated the plants/herbs that have these phytochemicals in reasonable quantities. After isolating the potential plants/herbs (7 plants), Remedium performed the extraction process as prescribed in the Ayurvedic pharmacopeia of India so that the final form can be powder that can be used in the formulation of the drug CoroQuil-Zn. Remedium formulated the drug using the plant extracts as prescribed in the API so that the safety profile is accounted for. Remedium again performed a cell culture assay and animal studies with CoroQuil-Zn formulation to see if the same effect of inhibiting SARS-CoV-2 Spike protein is exhibited in the CoroQuil-Zn formulation. After confirmation from both the cell culture studies and animal studies Remedium concluded with the formulation and froze the specifications to take it to the Clinical trials.

Introduction
It is well established that the binding of Spike (S) protein of SARS-CoV-2 virus to the host cell ACE2 receptors triggers a series of molecular events resulting in the entry of the virus into the host cells. This viral entry activates an immune response in the host for resolving the infection. Depending on the magnitude and duration of the viral load and the activation of immune response, the SARS-CoV-2 infection may result in asymptomatic, mild, moderate, and severe COVID-19 disease and in some critically ill patients may cause death.

CoroQuil-Zn was designed mainly to counter the binding of SARS-CoV-2 to ACE2 receptors, thereby blocking the entry of the virus into the host cells. We have developed a series of cell-based experiments to study the activity of the plant extracts to formulate CoroQuil-Zn. The results derived from these cell-based studies are presented below. We also have conducted a pilot experiment in a hamster model. All the preclinical laboratory experiments were carried out in collaboration with the Molecular Interactions Dept of University of Missouri, USA.

1. Potency of CoroQuil-Zn in Pseudo-typed viral entry assay
For determining the potency of CoroQuil-Zn on the ACE2-mediated entry of SARS-CoV-2 into host cells, a pseudo-typed viral entry assay was performed. This study used human HEK293T cells that express recombinant ACE2 receptors and SARS-CoV-2 virus Spike (S) protein. HEK293T cells (96 well plates; 1×104 cells/well) were incubated with different concentrations of plant extracts for 15 minutes. Then, virions that are pseudo-typed with SARS-CoV-2 “S” protein were added to the cells for 48 hours. A Bright-Glo luciferase reagent (luminescence) was added to the cells, and the luciferase activity was determined using a Veritas microplate luminometer. Luminescence measured in the absence of plant extracts was used as 100% control for quantitation.

Figure 1A shows a schematic of the experimental protocol. In this experiment paradigm, the virus enters the cells by binding to the ACE2 receptors and the virus is unable to dock to the ACE2 receptor, enter into the cell and replicate. Therefore, the intensity of luminescence “Lights ON” in the Figure 1A schematic corresponds directly to the virus entering the host cells. When the experiment was repeated by adding CoroQuil-Zn to the HEK293T cells, there is reduction in the luminescence, which is being depicted in Figure 1A as “Lights OFF”. This confirms a 100% inhibition of viral entry into the HEK293T cells through the ACE2 receptor.

Figure 1B shows a representative data of a dose response using different concentrations of CoroQuil-Zn added to HEK293T cells and SARS-CoV-2 virus Spike (S) protein containing virions. CoroQuil-Zn produced a concentration-dependent reduction in viral entry with an IC50 of 840 µg/ml.

2. Potency of CoroQuil-Zn in SARS-CoV-2 viral infection assay
After confirming that CoroQuil-Zn inhibits viral entry via ACE2 receptor-mediated mechanism, we tested the effect of CoroQuil-Zn in Calu-3 cells, a human lung cell line expressing native ACE2 receptors. Calu-3 cells were plated in 96-well plates (2×104 cells/well) and incubated with test compounds for 2 hours. Cells were then infected (MOI=0.01) with the SARS-CoV-2 virus for 1 hour, and then the culture medium was replaced with a fresh medium with test compounds for 48 hours. MTT was used to measure cell survival and metabolic activity. Supernatants were collected and SARS-CoV-2 genome levels were quantified.

Treatment of Calu-3 cells with CoroQuil-Zn produced a concentration-dependent reduction in SARS-CoV-2 viral genome equivalent with an IC50 of 420 µg/ml (Figure 2A). This reduction in viral genome was accompanied by a concentration-dependent increase in the cell viability with an IC50 of 680 µg/ml (Figure 2B), showing the survival of cells due to reduced viral infection. Both results together suggest that CoroQuil-Zn has antiviral activity against SARS-CoV-2. Based on the above assays, we can reasonably conclude that CoroQuil-Zn reduces the SARS-CoV-2 viral load and increased the viability of lung cells, thereby confirming that CoroQuil-Zn can possibly be used as a therapy for SAR2-CoV-2 infection.

Assay CoroQuil-Zn IC50 µg/ml
Pseudo-typed viral entry assay 840
Calu-3 viral load assay 420
Calu-3 Cell viability assay 680

3. Efficacy of CoroQuil-Zn in Combination Treatments
Both Remdesivir and Favipiravir are known to inhibit viral mRNA replication by inducing sequence errors during replication executed at RNA-dependent RNA polymerase (RdRp). Considering that CoroQuil- Zn can inhibit viral entry via ACE2 receptor, a test was conducted to study the combined effect of CoroQuil-Zn with Remdesivir and Favipiravir independently in cell-based viability assays.
Human liver Huh-7 cells were plated in 96-well plates (1×104 cells/well) and incubated with test compounds for 2 hours. Cells were then infected (MOI=0.01) with the SARS-CoV-2 virus for 1 hour, and the culture medium was replaced with a fresh medium with test compounds for 48 hours. ToxGlo was used to measure cell survival. Both Remdesivir (RDV) and Favipiravir (FPV) were tested at 0-5 nM, with CoroQuil-Zn (CQZ) at 0-100 mg/ml in combination with Remdesivir and Favipiravir in two independent experiments. Cell survival was measured and data analyzed using the SynergyFinder software to obtain the Loewe synergy score. A Loewe synergy score of > 10 is considered a significant synergistic effect.

Remdesivir and CoroQuil-Zn
Results obtained using the concentration matrix of Remdesivir and CoroQuil-Zn are presented as 2D and 3D synergy visual maps in Figure 3A and Figure 3B, respectively. The red region depicts the enhanced activity of Remdesivir and CoroQuil-Zn at various concentrations of Remdesivir and CoroQuil-Zn as depicted in the matrix. The composite Loewe synergy score for the combination treatment was 14.93.

Favipiravir and CoroQuil-Zn
Results obtained using the concentration matrix of Favipiravir and CoroQuil-Zn are presented as 2D and 3D synergy visual maps in Figure 4A and Figure 4B, respectively. The red region depicts the enhanced activity of Favipiravir and CoroQuil-Zn at various concentrations of Favipiravir and CoroQuil-Zn as depicted in the matrix. The composite Loewe synergy score for the combination treatment was 20.48.

The larger Loewe scores for both Remdesivir and Favipiravir demonstrate a pronounced combined effect of CoroQuil-Zn when administered along with Remdesivir and Favipiravir independently. These findings suggest that inhibiting RdRp and ACE2 receptor may lead to synergistic action, leading to a rapid reduction in viral load and speed up the recovery of SARS-CoV-2 infected patients.

4. CoroQuil-Zn inhibits SARS-CoV-2 variant N501Y
Genetic variants of SARS-CoV-2 have been emerging throughout the COVID-19 pandemic. The “UK,” “Brazil,” and “South Africa” variants of the SARS-CoV-2 virus are more contagious than the previous variants and all of these variants have a common mutation N501Y in the Spike protein. We designed a pseudo-typed virus to study if this mutation affects the activity of CoroQuil-Zn. In the pseudo-typed virus entry assay model, CoroQuil-Zn inhibited entry of N501Y variant with a magnitude comparable to the wildtype virus providing evidence that this mutation did not affect the inhibitory activity of CoroQuil-Zn (Figure 5). We have done assays in Delta variant which is B.1.617 and found to be effective.

5. Efficacy of CoroQuil-Zn in SARS-CoV-2 infection in Syrian hamster model
Having confirmed the inhibitory effect of CoroQuil-Zn in 3 cell types, we have initiated animal studies to further understand the effect of CoroQuil-Zn in the SARS-CoV-2 hamster model. In a pilot experiment, Syrian Hamsters were intranasally infected with SARS-CoV-2 aerosol in a BSL-3 facility. Two groups were divided, with one as a Control group consisting of two Syrian Hamsters (n=2) and another CoroQuil-Zn group consisting of eight Syrian Hamsters (n=8). Eight Hamsters in the CoroQuil-Zn group were intranasally infected with SARS-CoV-2. The first dose of CoroQuil-Zn (300 mg/kg of CoroQuil-Zn) was administered via oral gavage 2.5 hours prior to infecting the hamster with SARS-CoV-2. The Control group was given a placebo. Treatment continued in both groups every 12 hours for 4 consecutive days. Animals were euthanized on day 4, and lungs were harvested. The viral load was determined by RT-qPCR. The initial viral load on Day 1 was determined using oral swab samples.

We observed that SARS-CoV-2 infection did not produce a significant weight loss in any group. The control group hamsters appeared lethargic, but the CoroQuil-Zn treated group hamsters were active. The viral load in both groups was comparable on day 1. However, on day 4, we observed reduced viral load in the CoroQuil-Zn group suggesting that CoroQuil-Zn may offer protection from SARS-CoV-2 infection (Figure 6).

Conclusion:
It is known that most of the current drugs for curing SARS-CoV-2 virus are repurposed. It is also known that these drugs produce only limited benefits in combating the SARS-CoV-2 virus. We have been focused on identifying ACE2 inhibitors and have formulated a drug that could eventually reduce the viral load in mild and moderate COVID-19 patients thereby putting the patients on a recovery path within a short span of time. We are continuing our research to find out more about this SARS-CoV-2 virus and are confident that we would be able to help the humanity and save precious lives in this unforeseen pandemic situation.

Phase 3 clinical trials:
Brief summary:
This was an open-label, two-arm, parallel design, prospective, single-centre, phase-3 study to assess the safety and efficacy of CoroQuil-Zn when compared to the standard of care [Mild cases: Azithromycin 500 mg tab (once daily) + Corticosteroids (Twice daily) + Paracetamol (Twice daily) + Aspirin 500 mg (Twice daily); Moderate cases: Piperceillin tazobactum 4.5 mg inj. or Ceftrixone 1 gm inj. (Once daily) +Corticosteroi (Twice daily) + Enoxiparin 40 (Twice daily) + Paracetamol 650 mg (Twice daily); IV infusion if required (only in case of weakness); All antibiotics for 6 days and other medications based on the reports and PI discretion] for the treatment of mild to moderate COVID-19 infection.

• 120 patients screened and 114 randomized in this study.
• Both inpatients and outpatients were enrolled in this study.
• 57% males and 42% females were enrolled in this study.
• The median age group was 31 years in this study.
• The study drug was well-tolerated by the subjects and no adverse drug reactions were observed.
• There were no deaths reported in both the treatment groups.
• Clinical response and clinical cure at the day 7 visit: 98% of the patients in the CoroQuil-Zn group showed a clinical response when compared to the standard of care group (82%) which is statistically significant (p-value: 0.0035).
• The median time to clinical response and clinical cure was 3 days in the CoroQuil-Zn group and 6 days in the standard of care group which is statistically significant (p-value: <0.0001).
• Virological cure at day 7 visit: Majority of the patients in the CoroQuil-Zn arm (95%) and showed virological cure as compared to the standard of care group (59%) which is statistically significant (p-value: <0.0001).
• Virological cure at day 14 visit: Majority (97%) of the patients in the CoroQuil-Zn group showed virological cure when compared to the standard of care group (86%) which is statistically significant (p-value: 0.0372).
• The median time for virological cure was 6 days for the CoroQuil-Zn group and 7 days for the standard of care group which is statistically significant (p-value: <0.0001).
• The severity of Dyspnea at the day 7 visit: There is a significant reduction in dyspnea with CoroQuil-Zn group as compared to the standard of care group which is statistically significant (p-value: <0.0001).
• The severity of Dyspnea at the Day 14 visit: There is no significant reduction is Dyspnea with the CoroQuil-Zn group as compared to the standard of care group on day 14.
• Baseline IL-6 levels compared to Day 7 and Day 14 visit: There is a statistically significant reduction in IL-6 levels at day 7 (p-value: <0.0001) and Day 14 (p-value: <0.0001) in the CoroQuil-Zn group which is not seen in the standard of cure group when compared to the baseline visit. There is statistically significant change seen between the two treatment groups at day 7 (p-value: 0.002) and day 14 visit (p-value: 0.0046) when compared to the baseline visit.
• Baseline CRP, D-Dimer and serum ferritin levels compared to day 7 and day 14 visit: There is no statistically significant reduction observed between the two treatment groups at day 7 and day 14 visits when compared to the baseline visit.
• No clinically significant findings were observed in the physical examination, vitals, clinical laboratory tests, 12-lead ECG, chest X-ray and routine urine analysis.
• A total of 05 adverse events (AEs) were reported in this study. All AEs were mild in severity, which resolved without sequelae.

Overall, at Day 7 visit in the CoroQuil-Zn treatment group, a statistically significant Clinical response, Clinical cure, Virological cure and reduction in the severity of Dyspnea was observed.

Conclusion:
During the primary analysis, a numerically higher positive trend was observed in the clinical response. Throughout the trial, a statistically significant improvement was seen in the CoroQuil-Zn treatment group for clinical response, clinical cure, and virological cure, reduction in the severity of dyspnea and recovery rate in the secondary analysis. The median time to clinical response, clinical cure, and virological cure was also statistically significant in the CoroQuil-Zn treatment group.
From the analysis of this trial data, it is evident that capsule CoroQuil-Zn 750 mg benefits mild to moderate COVID-19 patients. Administering CoroQuil-Zn 750 mg will reduce the patient load on the hospital, recovery at home and may fulfill the current unmet need during this ongoing pandemic. ,CLAIMS:WE CLAIM:
1. An herbal composition for the treatment of COVID-19 by targeting ACE2 receptor in humans, consisting extract of:
Solanum nigrum –15to 35% w/w;
Acalypha indica –15to 35% w/w;
Ocimum sanctum – 4 to 15% w/w;
Adathoda vasica – 4 to 15% w/w;
Withania somnifera – 4 to 15% w/w;
Glycyrrhiza glabra – 4 to 15% w/w;
Piper nigrum – 4 to 15% w/w;
Zinc oxide (Jasad bhasma) – 3 to 8% w/w; and
pharmaceutical excipients – 8 to 16% w/w.

2. The herbal composition for the treatment of COVID-19 by targeting ACE2 receptor in humans as claimed in claim 1, wherein the pharmaceutical excipient includes starch.

3. The herbal composition for the treatment of COVID-19 by targeting ACE2 receptor in humans as claimed in claim 1 and 2, wherein composition preferably comprises extract of Solanum nigrum – 33.33% w/w; Acalypha indica – 33.33% w/w; Ocimum sanctum – 4% w/w; Adathoda vasica – 4% w/w; Withania somnifera – 4% w/w; Glycyrrhiza glabra – 4% w/w; Piper nigrum – 4% w/w; zinc oxide (Jasad bhasma) – 3.33% w/w; and Pharmaceutical excipients – 10% w/w.

4. The herbal composition for the treatment of COVID-19 by targeting ACE2 receptor in humans as claimed in claim 1, wherein the extract comprises one selected from a whole plant extract, leaf extract, root extract, fruit extract and a combination thereof.

5. The herbal composition for the treatment of COVID-19 by targeting ACE2 receptor in humans as claimed in claim 1, wherein the extract is prepared in solid powder or capsule form.

6. The herbal composition for treatment of COVID-19 by targeting ACE2 receptor in humans as claimed in claim 1, wherein the extract is administered one form selected from the group consisting of a solid powder, capsule and a combination thereof.

7. The herbal composition for treatment of COVID-19 by targeting ACE2 receptor
in humans as claimed in claim 1, wherein the composition comprising the extract of Solanum nigrum, Acalypha indica, Ocimum sanctum, Adathoda vasica, Withania somnifera, Glycyrrhiza glabra, and Piper nigrum; and zinc oxide (Jasad bhasma) is administered at a dosage of 4500 mg per day administered as 1500 mg thrice daily.

8. The herbal composition for treatment of COVID-19 by targeting ACE2 receptor in humans as claimed in claim 1, wherein the unit dosage strength of the composition comprising the extract of Solanum nigrum, Acalypha indica, Ocimum sanctum, Adathoda vasica, Withania somnifera, Glycyrrhira glabra, and Piper nigrum; and zinc oxide (Jasad bhasma) is 750mg per capsule.

9. The herbal composition for treatment of COVID-19 by targeting ACE2 receptor in humans as claimed in claim 1, wherein the extract is hydro-alcohol extract comprising ratio of alcohol in a range between 50 and 90%: water in a range between 10 and 50%.

10. A process for formulation of herbal composition for treatment of COVID-19 by targeting ACE2 receptor in humans, comprising of steps:
grinding of the dried plants Solanum nigrum, Acalypha indica, Ocimum
sanctum, Adathoda vasica, Withania somnifera, Glycyrrhiza glabra, and Piper
nigrum to a size of 3 to 4 mm;
preparing a solvent extract of the plants by heating at a temperature between
70 and 80 degree Celsius with continuous extraction under vaccum 400-600
mmHg maintaining the temperature between 70 and 80 degree Celsius for 4-5
hours;
filtering the material through 100 mesh filter and concentrate the extract using
multiple-effect evaporators at a temperature between 75 and 80 degree Celsius
for 4-5 hours to obtain required Total solids 25-35% under vacuum 400-600
mmHg;
subjecting the extract to heat treatment at a temperature 121±2°C for 20
minutes and cooling the content to below 40°C;
subjecting the concentrated extract to vacuum drying/ spray drying/ freeze-
drying to get dry powder;
adding of zinc oxide (Jasad bhasma) and starch, and mixing for a period of 15
minutes;
sieving of mixed material using a 40 mesh sieve; and
loading of above composition into a gelatin capsule to obtain capsules,
wherein the herbal composition comprises extract of Solanum nigrum
– 15 to 35% w/w; Acalypha indica – 15 to 35% w/w; Ocimum
sanctum – 4 to 15% w/w; Adathoda vasica – 4 to 15% w/w; Withania
somnifera – 4 to 15% w/w; Glycyrrhiza glabra – 4 to 15% w/w; Piper
nigrum – 4 to 15% w/w; Zinc oxide (Jasad bhasma) – 3 to 8% w/w;
and pharmaceutical excipients – 8 to 16% w/w.