DESC:RELATED APPLICATION
The present application claims the benefit of priority to Indian Provisional Patent Application no. 202021026362 filed on June 22, 2020 and the entire provisional specification.

FIELD OF THE INVENTION

The present invention relates to a composition of inhalable active compounds isolated and purified from root of a plant by a selective method. In particular, the invention relates to a dry powder inhalation composition of compounds which have been isolated and purified from root of a plant such as licrorice. More particularly, the invention relates to a dry powder inhalation composition comprising Glabridin and Glycyrrihizin containing licorice extracts for the treatment of COVID-19 infection.

BACKGROUND OF THE INVENTION

In January 2020, the World Health Organization (WHO) declared the outbreak of a new coronavirus disease, COVID-19, to be a Public Health Emergency of International Concern. Severe acute respiratory syndrome (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and a novel coronavirus (2019-nCoV) that emerged in Wuhan, China belong to the same class of virus that affect humans. Globally, authorities predict that the coronavirus pandemic will last for extended period of time. It continues to remain a large threat until approximately two-thirds of the world’s population is immune. This presents an immediate need for alternative therapeutic strategies that offer population an immunity to this virus.

It is postulated that the spike glycoprotein (S glycoprotein), which attaches the COVID-2 virions to the host cell membrane, plays a dominant role in host range restriction utilizing angiotensin converting enzyme 2 (ACE2) as a receptor. ACE2 receptor is abundantly present in humans in the epithelia of the lung and small intestine, endothelial cells of the liver. It has been reported that about 2–11% of patients with COVID-19 had liver comorbidities. Further, 14–53% cases reported abnormal levels of alanine aminotransferase and aspartate aminotransferase (AST). On a more wary note, during disease progression, liver injury is more prevalent in severe cases than in mild cases of COVID-19.

Currently there are limited treatment options for COVID-19, and therapeutic strategies to deal with the infection are largely focused on sustaining a patient's physiological well-being. Experience clinicians are relying on their knowledge in treating SARS, MERS, and other previous influenza viruses for current COVID-19 patients. Some of the drugs being used include Fabiravir and ribavirin, Lopinavir/ritonavir, Remdesivir, Arbidol, Ivermectin, Chloroquine and hydroxychloroquine, Cyclosporin A, interferons, Tocilizumab, and plasma therapy; each of them having its own limitations and efficacy success. This goes to reiterate a lack of effective therapy strategy and options for the COVID-19 infection. Additionally, issues such as high doses of these drugs and their shortages only add to the patient burden. This has created a great need for a viable alternative to current therapeutic options.

Several herbal drugs exhibit an effect on viral replication, viral adsorption, cell-to cell spread, viral polymerase activity. A large section of the global population uses herbal medicines for their primary health care. Herbal drugs are less toxic and have fewer side effects, and therefore present more attractive prospects in treatment of infections. Since many of the plant materials lack solubility in aqueous media and are prone to oxidation and degradation by low gastric pH, it is postulated that the availability of the drug may be enhanced when they are supplied via alternate routes of delivery, such as pressurized metered-dose inhalers (pMDIs) and dry powder inhalers (DPIs). Additionally, DPI devices are popularly used for the direct drug delivery to the lungs to treat respiratory disorders such as chronic obstructive pulmonary disease. DPIs for suitable plant extracts to treat coronavirus in the lungs can significantly alter the current gap in treatment options.

Glycyrrhiza glabra Linn (licorice) belongs to the Leguminosae family and is cultivated in Italy, Russia, France, UK, USA, Germany, Spain, China, and Northern India. Known for its anti-viral, anti-inflammatory, anti-tumor, anti-microbial, and anti-oxidant properties; Glycyrrhiza glabra is also used to treat cough due to its demulcent and expectorant properties. It has two major constituents – Glabridin and Glycyrrhizin. While, Glycyrrihizin (polar entity) exhibits wide and versatile properties such as anti-viral and anti-microbial activity against viruses such as HIV, HCV, influenza HSV, rotavirus, coxsackievirus, HRSV, and HBV, the other constituent Glabridin (non-polar flavonoid) has been reported to have anti-viral activity against HBV. Studies have reported that Glycyrrhiza glabra extracts can down-regulate the expression of pro-inflammatory cytokines, tumor necrotic factor alpha (TNFa), interleukin 1 (IL-1), and interleukin 6 (IL-6), the levels of which are increased in COVID patients. Further, Glycyrrhiza glabra root exhibits a protective antioxidant effect on bronchial cells in the lungs. Thus, a DPI composition comprising Glycyrrihizin, and Glabridin extracts will enable the effect to reach the lungs. This will significantly help reduce the progression of COVID when used alone or in combination with other standard drugs.

High amount of nitrogen oxide (NO) released during viral and bacterial infections, accelerating mutation of viral RNA. Typically, oral administration of Glabridin reduces the amount of NO. Glabridin shall serve as a potential anti-COVID activity and reduce the progression of viral infection in COVID patients. Glabridin and Glycyrrihizin shall be supplied as a pure compound or as extracts of Glycyrrhiza glabra with a known amount of Glabridin and Glycyrrihizin estimated in the finished dosage form. Glycyrrhiza glabra root extract reduces liver inflammation and alleviates hepatic injury, which normalizes the level of hepatic enzymes; therefore, the use of Glycyrrhiza glabra extract benefits COVID patients. Because NO production is essential in the pathogenesis of COVID, it shall be useful to administer Glabridin and Glycyrrihizin in therapeutic doses.

It is therefore desirable to have a composition derived from a plant source, which provides a safe, well- tolerated and effective treatment of COVID-19 and which moreover, overcomes the shortcomings and limitations of the current treatments has been disclosed. The present invention is based on the unexpected observation that a DPI composition of Glabridin and Glycyrrihizin has substantial effect in enhancing the treatment of conditions caused by SARS-CoV, MERS-CoV, novel coronavirus (2019-nCoV).

According to the present invention there is therefore provided a composition comprising of Glabridin and Glycyrrihizin extracts in optimal ratios wherein the composition is a dry powder inhaler. Further, the composition is designed to treat patients affected with severe acute respiratory syndrome caused by SARS-CoV, MERS-CoV, novel coronavirus (2019nCoV).

SUMMARY OF THE INVENTION
In view of the foregoing, for overcoming the disadvantages of the prior art, the object of the invention is to provide a novel dry powder inhalation composition comprising a therapeutically effective amount of Glabridin and Glycyrrihizin extracts for the treatment of COVID-19 related infections.

Further object of the present invention is to provide a composition for treatment of severe acute respiratory syndrome, which is safe, well-tolerated, non-toxic, with minimal and reversible adverse reactions or side effects, and most importantly, which minimizes relapse or recurrence of the disease following completion of a treatment regimen, suitable for an inhalable application. Another object of the present invention is to provide a composition, which is selective, simple, efficient and cost-effective.

Accordingly, the present inventors report the Glabridin and Glycyrrihizin extracts obtained from the roots of Glycyrrhiza glabra plant can be formulated into a novel composition, suitable for dry powder inhalation and thereby, providing a method for treatment of severe acute respiratory syndrome caused by SARS-CoV, MERS-CoV, novel coronavirus (2019-nCoV).

One embodiment of the invention is an anti-viral herbal composition for the treatment of COVID-19 comprising of purified Glycyrrihizin extract and purified Glabridin extract and pharmaceutically acceptable carriers.

In another embodiment of the invention, the amounts of Glycyrrihizin and Glabridin extracts are 88% w/w of the composition and pharmaceutically acceptable excipients.

In one embodiment of the invention, the said composition is an inhaler composition. In a preferred embodiment of the invention, the said inhaler composition is in form of a dry powder inhaler. According to an alternate embodiment, the composition is administered as pressurized metered-dose inhaling composition.

In one embodiment of the invention, the composition further comprises a lubricant, a glidant, an anticoagulant. In a preferred embodiment of the invention, the lubricant is magnesium stearate, the glidant is Aerosil 200, the anticoagulant is citric acid.

In another embodiment of the invention, the composition is formulated in a capsule. In another embodiment of the invention, the capsule is stored in a blister pack or a high-density polyethylene container.

In a preferred embodiment of the invention, the anti-viral herbal composition for the treatment of COVID-19 comprises 88% w/w purified Glycyrrihizin extract and purified Glabridin extract; 5% w/w magnesium stearate; 4% w/w Aerosil 200; 3% w/w citric acid.

In another embodiment of the invention is a process for preparing an anti-viral herbal composition comprising: a) washing all the herbal extracts with water and drying the herbal extracts; b) powdering the herbal extracts with pulveriser or grinder to obtain the powdered form; c) sieving the powdered form of the herbal extracts; d) mixing the herbal extracts with excipients and drying sufficiently to obtain the final product; e) formulating the final product into capsules; f) sealing and storing the capsules in blisters or containers.

According to one embodiment, the composition is administered using a suitable dry powder inhaler device.

According to one embodiment, the drug shall be administered as acute therapy. According to one embodiment, the drug shall be administered as chronic therapy. According to another embodiment, the composition shall be used for treating patients suffering from severe acute respiratory syndrome caused by SARS-CoV coronavirus, MERS-CoV coronavirus, the novel coronavirus (2019-nCoV).

According to an alternate embodiment, the composition may be used in combinations of various appropriate ratios with allopathic drugs being used for COVID treatment for patients comprising of Dexamethasone, Ivermectin, Favipiravir and ribavirin, Lopinavir/ritonavir, Remdesivir, Arbidol, Chloroquine and hydroxychloroquine, Cyclosporin A, interferons, Tocilizumab.

DETAILED DESCRIPTION OF THE INVENTION
To clarify the above and other purposes, features, and advantages of this invention, specific embodiment of this invention is especially listed and described in detail with the examples as follows. The principal and mode of operation of this invention have been described and illustrated in its embodiment. At the outset, a person skilled in the art will appreciate that this invention may be practiced otherwise than is specifically described and illustrated. The invention should not be limited by the above-described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention. In the following description of the invention, certain terminology may be used for the purpose of reference only, and is not intended to be limiting.

Central to this invention is a composition to be used for treating patients suffering from severe acute respiratory syndrome caused by SARS-CoV coronavirus, MERS-CoV coronavirus, the novel coronavirus (2019-nCoV). An important aspect of the composition is providing extracts of Glabridin and Glycyrrihizin in optimal ratios which are therapeutically effect for treating the infections.

The term " Glycyrrhiza glabra" disclosed herein comprises the cultivated or naturally grown plant and commercially available plant, but not intended to limit thereto herein. Glycyrrhiza glabra, commonly known as licorice. Glycyrrhiza glabra is known for its anti-viral, anti-inflammatory, anti-tumor, anti-microbial, and anti-oxidant properties. However, specific activities of the plant against the novel coronavirus (2019-nCoV) have not been reported. Similarly, the therapeutic doses of Glabridin and Glycyrrihizin for treating said infections is also not disclosed in the prior art. The instant composition, thus, has original attributes in that it discloses the anti-COVID-19 activity of the composition in addition to the optimal amounts of both Glabridin and Glycyrrihizin required to achieve this effect.

While therapeutic benefits of both Glabridin and Glycyrrihizin are known, it is also evident in the literature that in many instances Glabridin is more potent than Glycyrrihizin. This was further substantiated by the present inventors in a separate investigative study. In said study, the present inventors explored Glycyrrhiza glabra’s activity for mammalian hexokinase inhibition by in-vitro studies. This study was conducted in the light of the recent understanding of benefit of hexokinase inhibitor in the treatment of COVID-19 infections. It was observed that the instant licorice DPI composition at 0.11% concentration demonstrated more hexokinase inhibition (60.65% inhibition) as compared to 2-deoxy-2-glucose at 0.8% concentration (37.25% inhibition), currently approved for treating COVID-19 infections in humans. This was particularly noteworthy because a comparative analysis of the activity by pure Glabridin and pure Glycyrrihizin, in the same study, demonstrated that the hexokinase inhibition activity was completely attributed to Glabridin. While this does not throw any effect on their anti-viral activity as demonstrated further in this application, it definitely enhances the therapeutic benefit of the instant composition.

Glycyrrhiza glabra comprises of Glycyrrhizin and Glarbridin compounds, both of which exhibit broad anti-viral properties. Glycyrrhizin is polar while Glabridin is non-polar. As a result, the compounds exhibit different properties while being used in a composition. Accordingly, the instant composition shall comprise of two extracts: a) an aqueous extract comprising of Glycyrrihizin and b) an acetone extract comprising of Glabridin.

The primary organ that is affected by COVID-19 infection is the lungs. The present inventors report for the first time that Glabridin and Glycyrrihizin potentially alleviate COVID related biochemical changes in affected patients and prove beneficial for clinicians to treat COVID-19 patients. Particularly for the purpose of this patent, the inventors present a composition with combination of Glabridin and Glycyrrhizin to enhance the indicated activity of the composition.

Central to this invention, is the use of Glabridin and Glycyrrihizin. Glabridin and Glycyrrihizin for use in the compositions and methods of the present invention can also be prepared from crude Glycyrrhiza glabra root. The extracts obtained from the roots of Glycyrrhiza glabra plant, comprising of Glabridin and Glycyrrihizin shall be vital for administering to patients with severe acute respiratory syndrome. Thus, stressing on the optimal extraction of the compounds from the plant. Even though, several solvents including water can be used for fractionation of the extracts obtained from the roots of Glycyrrhiza glabra plant the present inventors have found that solvents like water, ethyl acetate, acetone, dicholormethane, ethanol, provide distinct fractions.

The present inventors report a process for preparation of the aqueous, acetone extract obtained from the roots of Glycyrrhiza glabra plant comprising the steps of extraction of Glabridin and Glycyrrhizin from the roots of Glycyrrhiza glabra plant with water, ethyl acetate, dichloromethane, acetone or mixtures thereof respectively, and drying of the extracts to give a powder. The present invention reports significantly higher yields of Glabridin using non-polar solvents as compared to those reported in the literature.

In fact, the extract of the invention can be prepared by a number of extraction procedures. Suitable procedures include multiple steps, for example maceration, percolation, and extraction using supercritical fluids, liquefied gases or suitable solvents. Typically, the extraction procedure provides an extract containing substantially all the water-soluble compounds present in the plant. Also, typically, suitable solvents for extraction of the roots of the plant are water and alcohols, such as ethanol or mixtures of water and the alcohol. Even though, several solvents including water can be used for fractionation of the extracts obtained from the roots of Glycyrrhiza glabra plant the present inventors report that solvents like ethyl acetate, acetone, dicholormethane, ethanol, provide distinct fractions. The bioactive extract of Glycyrrhiza glabra contains Glycyrrhizin, Glabridin, and other compounds, such as alkaloids, flavonoids, organic acids, amino acids, sugars/glycosides and salts, which are reported to have low bioavailability due to their properties such as low water solubility, poor absorption and rapid metabolism. Glabridin can be extracted in the non-aqueous phase. Extract of Glycyrrhizin can be obtained in a similar manner using water separately or using the spent medium after acetone evaporation and precipitation with suitable acids and dried after water wash.

The choice of solvent and mixtures thereof, coupled with optimal temperature and time of extraction are critical for acquiring high yields of compounds. Non-polar solvents have been used in the literature to extract Glabridin, in comparison the present invention reports significantly higher yields i.e., at 5% yield using ethyl acetate and at least 7% yield using dichloromethane. Glabridin is extracted using non-polar solvents comprising of dicholoromethane, ethyl acetate, acetone, mixtures thereof.

Also, central to the invention is a composition of Glabridin and Glycyrrihizin for treatment of severe acute respiratory syndrome, which is safe, well-tolerated, non-toxic, with minimal and reversible adverse reactions or side effects, and most importantly, which offers a treatment regimen, suitable as an inhalation administration. Further the composition shall be selective, simple, efficient and cost-effective.

It is imperative to use therapeutically effective amount of Glabridin as the active compound. Glabridin may be present in amorphous or crystalline form. Further, the purity level of Glabridin suitable for the composition shall be determined and incorporated accordingly. Similarly, Glycyrrhizin of suitable purity levels shall be present in amorphous or crystalline form. Accordingly, an important embodiment of the invention is providing Glabridin and Glycyrrihizin extracts in optimal ratios. The present inventors report that based on the aforementioned yields, the ratio of Glabridin and Glycyrrihizin in a total of 22 mg amount in the composition, shall be 0.2mg: 0.1mg to 0.5 mg: 0.3mg. In a preferred embodiment, based on the aforementioned yields, the ratio of Glabridin and Glycyrrihizin in a total of 22 mg amount in the composition, shall be 0.385 mg: 0.22 mg.

Also critical to the composition is the characterization of fine particles, including size, surface area, density, porosity, and rugosity. These physical properties impact all aspects of dry powder delivery, from bulk powder properties including flow, fluidization, and dispersibility, to aerosol performance, to absorption, and, indirectly, to distribution, metabolism, and excretion of the drug product. DPIs work efficiently when the drug particles are micronized. Therefore, particle size of Glycyrrhiza glabra extracts suitable for the composition shall be determined and incorporated accordingly and have been described further in the examples. Similarly, suitable particle size for Glycyrrhizin shall be obtained wherever applicable. Appropriate particle formation technologies may be employed to achieve this.

At the core of the invention is the use of Glabridin and Glycyrrihizin for alleviating the symptoms of the disease. With this in mind, the present invention comprises of combination of Glabridin and Glycyrrhizin in order to enhance the efficiency of the said composition.

Central to the invention is a DPI composition with Glabridin and Glycyrrihizin as the active compounds. DPIs have been providing effective treatments for patients worldwide. Dry powder for inhalation composition was selected as the approach to deliver the purified active compounds extract to lungs owing to the numerous advantages of DPI over other pulmonary compositions such as nebulizers. DPIs exhibit better environmental sustainability due to absence of propellants in the composition and also show higher stability as they are at a lower energy state in dry form. DPIs are able to deliver a wide range of doses to the lungs and can be manufactured by different techniques comprising freeze drying, spray drying so as to achieve desired composition characteristics.

Excipients are critical components in the composition of inhaled drug products. In one embodiment of the invention, the composition further comprises a lubricant, a glidant, an anticoagulant. In a preferred embodiment of the invention, the lubricant is magnesium stearate, the glidant is Aerosil 200, the anticoagulant is citric acid.

In another embodiment of the invention, the instant DPI composition shall be formulated into a capsule form. Capsules offer a single dose container for the inhalation powders. For the purpose of this patent, the composition has been formulated into hard gelatin capsules and Hydroxypropyl Methylcellulose (HPMC) capsules. However, the DPI composition can also be formulated into an alternate suitable form. Further, in order to wholly analyze the physical aspects of the composition, it needs to be investigated for its stability properties. This has a direct correlation with its packaging environment. In one embodiment of the invention, ana appropriate container shall be provided for storage of capsules so as to preserve their physical and chemical stability. Accordingly, for the purpose of this patent, the instant capsules were packaged in blister packs such as Alumpack blister, and high-density polyethylene containers. In a preferred embodiment, the capsules were packaged in Alumpack blisters.

It should be noted that the present invention i.e., the composition of Glabridin and Glycyrrihizin can alternatively be administered as a pressurized metered-dose inhaler with propellants.

It is proposed that the composition may be administered as a part of acute therapy and/or chronic therapy against severe acute respiratory syndrome caused by SARS-CoV coronavirus, MERS-CoV coronavirus, the novel coronavirus (2019-nCoV). Further, it is proposed that the composition itself is sufficient as a part of treatment. However, wherever required the composition shall be administered in combination with another drug.

While a broad anti-viral activity of Glabridin and Glycyrrihizin is known, their specific activity against the novel coronavirus (2019-nCoV) has not been reported in the literature. To this effect, it is not possible to linearly apply the doses of said compounds from the literature to the instant composition of the treatment of COVID-19 infections. The instant composition is a result of a carefully calibrated combination of dose and ratio of Glabridin and Glycyrrihizin in therapeutic amounts required for the indicated purpose. The present inventors report the efficacy of the instant composition for treatment of severe acute respiratory syndrome. Additionally, the composition considered to be safe, well-tolerated, non-toxic, and most importantly, allows for a treatment regimen, suitable for an inhalable application. A person skilled in the art shall appreciate that the complexity and sophistication of this composition is in the fact that it is selective, simple, efficient and cost-effective.

The yields, concentration, amount of the ingredients of the composition, the process for making the composition is illustrated through non-limiting working examples. Optimal DPI dose is finalized based on results of in-vitro assays. Stability of the DPI composition has been demonstrated in comparative packaging environments. The efficiency of the composition for the purpose of treating COVID-19 infections is illustrated through non-limiting working examples of in-vitro assays. Further, this activity of the instant composition has been compared with Remdesivir, currently well-established therapy against the infection. The present inventors report the activity of the instant composition is comparable to that of Remdesivir.

EXAMPLES

The following examples are provided for illustrative purposes only, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of the claims provided herein.

Example 1: Preparation of Glycyrrhiza glabra root extracts
The plant raw material was commercially procured. The roots of Glycyrrhiza glabra were procured from Nature & Nature health care limited (Batch No: GG012001). 100 g Glycyrrhiza glabra roots raw material was extracted with 4 volumes of fresh acetone followed by three volumes of the solvent two times (each extraction for 3 h) at 45-50oC. After extractions, all extractions were pooled and concentrated on a rotary evaporator under vacuum at 45-50 oC to obtain a dry powdered acetone extract of Glycyrrhiza glabra. Similar protocol was followed to obtain extract with water or 30% methanol water extract. For extraction with dichloromethane, the extraction temperature was maintained between 30-35oC, below the boiling point of the dichloromethane solvent, keeping all other extraction parameters constant. Amounts (% w/w) of Glabridin and Glycyrrihizin were measured using HPLC. The response for the Glabridin peak at 254nm and Glycyrrhizinic acid peak at 254nm were recorded.
Table 1: Glabridin and Glycyrrihizin extracts quantified using HPLC
Extraction solvent Extract yield (g%) % Glabridin % Glycyrrihizin
Ethyl acetate 5.1 5.59 Not detectable
Acetone 5.6 5.09 0.16
Dichloromethane 3.5 6.65 0.12
Ethanol (100%) 6.7 3.73 0.68

Example 2: Preparation of DPI formulation of Glycyrrhiza glabra root extracts
Herbal extracts of Glycyrrhiza glabra were prepared as per Example 1
Excipients such as magnesium stearate, Aerosil 200 and citric acid were accurately weighed and mixed using a mortar and pestle. Glycyrrhiza glabra extracts comprising of Glabridin and Glycyrrihizin were obtained using acetone and water; both the extracts were sifted through with excipients through 60# sieve. After sifting all the mixtures were blended into the Alphie three-dimensional mixture made at 25 rpm for 30 minutes. The above blend was micronized using air jet mill using the following parameters: milling pressure – 9.0 bar; feeding pressure – 5.0 bar; feeder pressure – 2.0 bar; feed rate – 2.0 gm/min. After milling, micronized powder was passed through a 60# sieve and mixed in to Alphie three-dimensional mixture at 25 rpm for 30 minutes.

The particle size distribution of mixture of micronized Glabridin and Glycyrrihizin in the composition were analyzed using Malvern Mastersizer. D10, D50, and D90 values were observed to be 1.38, 3.69, and 9.18 respectively.
Table 2: Ingredients in DPI formulation of Glycyrrhiza glabra DPI
Ingredient %w/w
Glycyrrhiza Glabra acetone extract 44
Glycyrrhiza Glabra water extract 44
Magnesium stearate 5
Aerosil 200 4
Citric acid 3

Example 3: Stability of DPI formulation
The Glycyrrhiza glabra DPI formulation was prepared as per Example 2. Studies were conducted to confirm stability of the formulation and the effect of lubricant content on the capsule shell. For this, hard gelatin capsules and Hydroxypropyl Methylcellulose (HPMC) capsules were prepared comprising the DPI formulation. The HPMC capsules were not coated and packed in Alumpack blisters and polyethylene (HDPE) containers to compare their stability. A comparative study was performed at 2-8°C, 30°C and 75% RH, and 40°C and 75% RH for 90 days. It was observed that the capsules exhibited better stability in the Alumpack blisters as compared to the HDPE containers (Table 2).

Hard gelatin capsules were coated with magnesium stearate. As the HPMC capsules demonstrated better stability in Alumpack blisters, both hard gelatin capsules were packed in Alumpack blisters only. Stability of magnesium stearate coated and uncoated hard gelatin capsules in Alumpack blisters was compared. The study was performed at 30°C and 75% RH for 30 days. It was observed that hard gelatin capsules exhibited better stability when coated with magnesium stearate (Table 3). Overall, the stability of capsules in Alumpack blisters was considered superior.

Table 3: Stability of Glycyrrhiza glabra DPI HPMC capsules in Alumpack blisters and HDPE containers over 30 days
Capsule Type Time Glabridin Fraction Glycyrrihizin Fraction
Uncoated capsules in HDPE container Day 0 50 47
Day 15 32 32
Day 30 28 31
Uncoated capsules in Alumpack blisters Day 0 50 47
Day 15 40 40
Day 30 23 25

Table 4: Stability of hard gelatin capsules in Alumpack blisters over 30 days
Capsule Type Time Glabridin Fraction Glycyrrihizin Fraction
Capsules coated with magnesium stearate Day 0 48 45
Day 15 39 39
Day 30 40 40
Uncoated capsules Day 0 42 37
Day 15 20 21
Day 30 18 20

Table 5: Stability of capsules in Alumpack blisters and HDPE over 90 days at different temperatures
Pack Type Time Glabridin Fraction Glycyrrihizin Fraction
Alumpack blister pack 2-8 °C 38 36.5
30°C and 75% RH 36.5 36
40°C and 75% RH 29 33
HDPE container 2-8 °C 43.2 41.7
30°C and 75% RH 42.6 41.7
40°C and 75% RH 9.9 14.9

Example 4: Anti-viral activity of DPI formulation
In vitro analysis was conducted using the antiviral screening method. The assay was done in a 96-well plate format in 3 wells for each sample. 1x10e4 VeroE6 cells were plated per well and incubated at 37 °C overnight for the monolayer formation. Subsequently, cells were incubated with the Glabridin and Glycyrrihizin at concentration of 1 µg/ml each. The control cells were incubated with 1% ethanol for ethanol-soluble compounds. The cells without Glabridin and Glycyrrihizin were the control for water-soluble compounds. The cells were infected with SARS-CoV2 at a MOI of 0.01. After 24 and 48 hours, viral RNA was extracted from 100 µl culture supernatant and subjected to qRT-PCR (in duplicates) where Ct values for N and E gene sequence were determined. Inhibition of virus replication was determined based on the fold change in the Ct value in Glabridin and Glycyrrihizin -treated cells compared to the control. Remdesivir was used as a positive control for viral inhibition.

It was observed that the inhibition of virus replication by Glabridin and Glycyrrihizin was comparable with that by Remdesivir. In fact, Glabridin and Glycyrrihizin demonstrated comparable activity of inhibition of virus replication at lower concentrations as compared to Remdesivir.

Table 6: Antiviral screening for Glabridin and Glycyrrihizin
Compound Concentration % Inhibition of virus replication
24 hr post infection 48 hr post infection
E N E N
Glabridin and Glycyrrhizin 1 µg/ml each 88 88 91 91
Remdesivir 10 µg/ml 72 77 99 99
,CLAIMS:CLAIMS

1. An anti-viral herbal composition for the treatment of COVID-19 comprising of Glycyrrihizin extract, Glabridin extract and pharmaceutically acceptable carriers.

2. The composition of Claim 1, wherein Glycyrrihizin and Glabridin extracts are present in 88% w/w of the composition and pharmaceutically acceptable excipients.

3. The composition of claim 1, wherein the said composition is an inhaler composition.

4. The composition of claim 5, wherein the said inhaler composition is in form of a dry powder inhaler.

5. The composition of claim 1 further comprising a lubricant, a glidant, an anticoagulant.

6. The composition of claim 7, wherein the lubricant is magnesium stearate, the glidant is Aerosil 200, the anticoagulant is citric acid.

7. The composition of claim 1, wherein the composition is formulated in a capsule.

8. The composition of claim 7, wherein the capsule is stored in a blister pack or a high-density polyethylene container.

9. An anti-viral herbal composition for the treatment of COVID-19 comprising of 88% w/w purified Glycyrrihizin extract and purified Glabridin extract; 5% w/w magnesium stearate; 4% w/w Aerosil 200; and 3% w/w citric acid.

10. A process for preparing the anti-viral herbal composition of claim 1 comprising: a) washing all the herbal extracts with water and drying the herbal extracts; b) powdering the herbal extracts with pulveriser or grinder to obtain the powdered form; c) sieving the powdered form of the herbal extracts; d) mixing the herbal extracts with excipients and drying sufficiently to obtain the final product; e) formulating the final product into capsules; f) sealing and storing the capsules in blisters or containers.