DESC:FUNCTIONALIZED ENCAPSULATED COMPOSITIONS FOR ENVELOPED VIRUSES INCLUDING SARS-COV-2

FIELD OF THE INVENTION

The present invention relates to encapsulated compositions of fractionated plant extracts targeted towards enveloped viruses including SARS-CoV-2, more particularly, to a fractionated extracts from the peel of Punica granatum fruit and its encapsulation in biodegradable polymers with suitable surface functionalisation to target the desired tissues.

BACKGROUND OF THE INVENTION

Punica granatum, commonly known as pomegranate, is a deciduous shrub of the family Lythraceae, native to central and western Asia, and grows to 6-20 feet tall. It has long been cultivated for its orange-sized edible fruit and its attractive ornamental plant features, and has been used popularly as a medicinal plant since ancient times for several purposes.

Punica granatum extract and a fraction containing punicalagin as major compound are promising source of antiviral compounds against many viruses. Immunofluorescence analysis demonstrated virucidal effect of pomegranate extracts and transmission electron microscopy reveals damage in viral particles treated with this extract. Punicalagin, the main ellagitannin from pomegranate fruits, targets and inactivates herpes simplex virus 1 viral particles and can prevent binding, penetration, and cell-to-cell spread, as well as secondary infections. A pomegranate polyphenol extract with punicalagin as a major compound was found to inhibit the influenza virus.

However, plant extracts and their metabolites exhibit poor solubility, bioavailability and stability and hence low efficacy. Punica granatum extracts are degraded in the gastro-intestinal tract to other forms of metabolites which are further susceptible to action by the gut flora. Hence to preserve the integrity of the bioactives and deliver desired concentration to specific tissues, the present invention aims at developing encapsulated and functionalised versions of the fractions.

Application of drug loaded nanosized carriers such as polymeric nanocapsules (such as PLGA, PEG, Chitosan, Silk, PLA, etc.), liposomes, dendrimers are used to overcome the above mentioned problems, and hence enhance bioavailability and thereby efficacy. Immobilisation of proteins or ligands which target specific enzymes/proteins in the disease pathways is designed to achieve targeted drug delivery.

The present invention discloses a unique polymer and/ or a blend of polymers used to encapsulate the extracts and designed to deliver it to a particular site in the human body or else concentrated into a particular class of tissues leading to enhanced bioavailability and extended duration of action. For example, a particular fraction of the bioactives from Punica granatum rind through suitable encapsulation and functionalisation is designed to get concentrated or targeted towards the epithelial cells of the airways for increased bioactivity against SARS-CoV-2 which infects the respiratory pathway.

The present invention discloses a unique method to use a novel encapsulation coating which is biocompatible with the fractional Punica granatum extract demonstrating antiviral activity against SARS-CoV-2.

OBJECTIVES OF THE INVENTION

The primary objective of the present invention is to develop anti-viral compositions to ameliorate, mitigate, cure and possibly prevent infections/ diseases caused by enveloped RNA viruses, including, SARS-CoV-2.

Another objective of the present invention is to develop compositions comprising extracts of Punica granatum demonstrating antiviral activity with enhanced efficacy and bioavailability.

Yet another objective of the present invention is to develop anti-viral compositions comprising fractionated extracts from the peel of Punica granatum encapsulated in a blend of biodegradable polymers with suitable surface functionalisation to target the desired tissues/ organs.

SUMMARY OF THE INVENTION

One of the embodiments of the present invention discloses anti-viral compositions primarily comprising extracts of Punica granatum peel encapsulated in a biodegradable polymer or combination of polymers with suitable surface functionalisation.

Another embodiment of the present invention discloses anti-viral compositions primarily comprising extracts of Punica granatum peel encapsulated in a biodegradable polymer of combination of polymers with suitable surface functionalisation such as ligands.

According to yet another embodiment of the present invention, the composition comprises of a particular fraction of the hydroalcoholic extracts (Ethanol & Water comprising different proportion of 50:50 / 70:30 / 80:20) of the Punica granatum peel.

According to yet another embodiment of the present invention, the selected extracts are suitably encapsulated in polymeric coating, the said polymer- selected from a group comprising of Zymosan, Silk, Laminarin, Oligomannose glycans and/or a combination thereof and Lactoferrin as the common ligand with other biodegradable materials.

According to yet another embodiment, the encapsulated compositions are suitably functionalised with ligands selected from a group comprising of Lactoferrin proteins that can be immobilized / fixed to the polymer surface, polymers that can be coated on to the encapsulating polymer blend, to target sites of interest which may be the sites of entry or viral replication of the enveloped RNA viruses including SARS-CoV-2.

According to still another embodiment, the encapsulated compositions developed are in the nano delivery format which may be in oral form (viz. tablet, capsule, soft gel capsules, mouth dissolving tablets, buccal delivery formulations etc.).

According to yet another embodiment, the compositions of the present invention are administered orally or in the form of an aerosol or inhalant or a fine liquid spray through the mouth or nose.

Yet another embodiment of the present invention discloses methods of preparation of compositions from fractionated extracts of the peel of plant Punica granatum encapsulated in biodegradable polymers with suitable surface functionalisation to facilitate concentration of the encapsulated particles to prevent or retard the disease progression or cure the disease arising out of the enveloped viruses including but not limited to Influenza-A, Hepatitis-B, Hepatitis-C, Human Immunodeficiency Virus (HIV), Herpes Simplex Virus Type-1 &2,Zika, Dengue Virus (Serotypes 1 through 4), SARS-CoV-2 etc.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described in the detailed description that follows, by reference to the noted drawings by way of illustrative embodiments of the invention, in which like reference numerals represent similar parts throughout the drawings. The invention is not limited to the precise arrangements and illustrative examples shown in the drawings:

Figure 1A illustrates graphical representation of LCMS-MS Hydroalcoholic extract of Punica granatum in Positive scan; and Figure 1B illustrates graphical representation of LCMS-MS Hydroalcoholic extract of Punica granatum in Negative scan;

Figure 2A illustrates graphical representation of Particle size Distribution of Zymosan-Laminarin or Zymosan-Oligomannose complex; and Figure 2B illustrates graphical representation of Particle size Distribution of Silk-Laminarin or Silk-Oligomannose complex;

Figure 3 shows SEM images of Zymosan-Laminarin Complex (3A); Zymosan-Oligomannose complex (3B); and Silk-Laminarin complex (3C);

Figure 4 illustrates graphical representation of drug release profile of antiviral encapsulation;

Figure 5 illustrates FTIR spectroscopy of Zymosan-Laminarin polymeric complex (5A); and Silk fibroin-Laminarin polymeric complex; and

Figure 6 illustrates X-ray diffraction curves for Zymosan-Laminarin complex (6A); Silk-Laminarin complex(6B).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the exemplary embodiment(s) of the present invention. Before describing in detail embodiments that are in accordance with the present disclosure, it should be observed that the embodiments reside primarily in combinations of different components of the composition.

In this document, the terms "comprises," "comprising," or “including” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a composition, system, method, article, device or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such compositions, system, method, article, device, or apparatus. An element proceeded by "comprises ...a" does not, without more constraints, preclude the existence of additional identical elements in the process, product, method, article, device or apparatus that comprises the element.

Any embodiment described herein is not necessarily to be construed as preferred or advantageous over other embodiments. All of the embodiments described in this detailed description are illustrative, and provided to enable persons skilled in the art to make or use the disclosure and not to limit the scope of the disclosure, which is defined by the claims.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit and scope of the invention. Thus, the present invention covers the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

In the following description, for the purpose of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention can be practiced without these specific details.

One of the embodiments of the present invention discloses anti-viral compositions primarily comprising extracts of Punica granatum peel encapsulated in a biodegradable polymer or combination of polymers with suitable surface functionalisation.

Another embodiment of the present invention discloses anti-viral compositions primarily comprising extracts of Punica granatum peel encapsulated in a biodegradable polymer of combination of polymers with suitable surface functionalisation such as ligands.

According to yet another embodiment of the present invention, the composition comprises of a particular fraction of the hydroalcoholic extracts (Ethanol & Water comprising different proportion of 50:50 / 70:30 / 80:20) of the Punica granatum peel.

According to yet another embodiment of the present invention, the selected extracts are suitably encapsulated in polymeric coating, the said polymer- selected from a group comprising of Zymosan, Silk, Laminarin, Oligomannose glycans and/or a combination thereof and Lactoferrin as the common ligand with other biodegradable materials.

According to yet another embodiment, the encapsulated compositions are suitably functionalised with ligands selected from a group comprising of Lactoferrin proteins that can be immobilized / fixed to the polymer surface, polymers that can be coated on to the encapsulating polymer blend, to target sites of interest which may be the sites of entry or viral replication of the enveloped RNA viruses including SARS-CoV-2.

According to still another embodiment, the encapsulated compositions developed are in the nano delivery format which may be in oral form (viz. tablet, capsule, soft gel capsules, mouth dissolving tablets, buccal delivery formulations etc.).

According to yet another embodiment, the compositions of the present invention are administered orally or in the form of an aerosol or inhalant or a fine liquid spray through the mouth or nose.

Yet another embodiment of the present invention discloses methods of preparation of compositions from fractionated extracts of the peel of plant Punica granatum encapsulated in biodegradable polymers with suitable surface functionalisation to facilitate concentration of the encapsulated particles to prevent or retard the disease progression or cure the disease arising out of the enveloped viruses including but not limited to Influenza-A, Hepatitis-B, Hepatitis-C, Human Immunodeficiency Virus (HIV), Herpes Simplex Virus Type-1 &2,Zika, Dengue Virus (Serotypes 1 through 4), SARS-CoV-2 etc.

Preparation of the Punica granatum extracts:

The extracts as used in the present invention are hydro-alcoholic extracts (viz. Water : Ethanol) of Punica granatum peel in the ratio as tabulated below in Table-I.

Table-I
Water 10% 20% 30% 40% 50%
Ethanol 90% 80% 70% 60% 50%
W:E ratio 1:9 1:4 1:2.33 1:1.5 1:1

In another embodiment of the present invention, the hydro-alcoholic extracts used can be water-Acetone mixture (range of 1:1to 1:9) and water-n-Butyl mixture (range of 1:1to 1:9).

Extraction and Fractionation Protocol for Rind of Punica granatum:

Material required: Punica granatum shade dried rind powder was obtained by passing the dried rind powder under 80 mesh. All solvents used is of Analytical grade / HPLC grade and the regents and standards sourced from reputed suppliers with CoA accompanying the reagents. The permission from National Biodiversity Authority is also obtained for the study and the copy of permission is submitted herewith.

Extraction methodology: The hydroalcoholic extraction from crude peel powder is processed in different proportions (as mentioned in Table-I) of water and ethanol mixture.

The dried rind powder is weighed and 200 grams of powder mixed with the chosen hydroalcoholic solvents in a ratio of 1:10 and subjected to shaking for 20 – 28 hours under 34-40 degree centigrade in an orbital shaker with 200+20 RPM. This is followed by sonication for 30 minutes at 34-40 degree centigrade at 50+10 kHz. The mixture is then filtered through Whatman No.1 Filter paper. The filtrate is vacuum distilled by rotavapor under 60+10 degree centigrade for removal of solvents and extracts weighed to calculate the yield. The extracts are then freeze dried and stored under -18 degrees in amber coloured glass bottles for further analysis.

4 Fractionation protocol: The hydroalcoholic extracts are subject to column separation through a mixture of XAD-7 and Silica resin beds in a proportion of 5:2. A combination of solvents are used in the order of polarity: Hexane > Chloroform > Ethyl acetate > n-Butanol to get different fractions. These are labelled 1 to 10 and used for the antiviral assays. Each of the fraction thus obtained are subjected to the characterization protocol.

Characterisation tests: The hydroalcoholic extracts are subject to the following characterization tests:
1. Determination of total phenolic content in gallic acid equivalents;
2. Determination of total flavonoid content in quercetin equivalents;
3. Determination of hydrolysable tannins content in tannic acid equivalent;
4. Determination of condensed tannins content in catechin equivalents; and
5. Antioxidant properties through DPPH and ABTS protocol.

The above tests would be carried out as per well studied protocols in published literature and suitably modified. Each of the fractions would be subject to HPLC and spectrophotometer to characterize and identify the bioactives as per method of analysis developed in house. The fractions showing promising antiviral activity are then characterized using HRLCMS-QTOF to identify the constituents and potential bioactives for formulation development. Please refer Figure 1-A and 1B
6. Substantiation of different
Invitro testing of the extracts for its antiviral properties: The different fractions of the hydroalcoholic extracts are subject to in-vitro tests done under BSL-3 facility at approved Government Labs to determine the efficacy against SARS-CoV-2 virus known to have caused Covid-19. For EC50 estimation, the Vero cells are infected with SARS-CoV2-19 as per the protocol developed inhouse. After infection, the cells are treated with different concentrations of the extract viz. (3.125µg/ml, 6.25µg/ml, 12.5µg/ml, 25µg/ml and 50µg/ml respectively) followed by collection of respective supernatants at 22 hpi.

The results for a particular fraction of the hydroalcoholic extract are as tabulated below in Table-II. It is noted that the IC50 value of the given extract against SARS-CoV-2 is 15.93 µg/ml.

Table – II: Efficacy of hydroalcoholic extract against SARS-CoV-2 (in vitro)
Sl. No. Compound Concentration Mean Ct Val Copy No./ ML % reduction
1 Infected only - 26.442 21959.18167 -
2 CLS- Test extract 3.125 µg/ml 26.444 21937.53738 0.1
3 CLS- Test extract 6.25 µg/ml 26.853 16765.27983 23.6
4 CLS- Test extract 12.5 µg/ml 27.216 13205.88742 39.8
5 CLS- Test extract 25.0 µg/ml 28.685 5025.718422 77.1
6 CLS- Test extract 50.0 µg/ml 33.376 230.0545683 98.9

Rationale for encapsulation of extracts: The bioactive extracts are subject to simulated human digestive process comprising a gastric phase (pH 3.2) followed by intestinal phase (pH 7.4) as described by Gullon B, et al. 2015. J. Functional Foods. 19:617-620. The GI digested extracts (GID) are again evaluated on various parameters to evaluate the effect of degradation arising out of the passage through Human GI tract.

Table-III: Functional characteristics degradation on GI tract passage (in vitro)

Sl.
No Description TPC
(Total Phenolic Content) TFC
(Total Flavonoid Content)
DPPH
(Antioxidant activity) %
Baseline Digested Degradation Baseline Digested Degradation
1 CLS-fraction-1 645.0 143.7 77.7 % 1243.0 225.1 81.9 % Antioxidant activity of baseline reduced from 76.9% to 11.6%
2 CLS-fraction-2 616.7 150.6 75.6 % 651.8 152.8 76.5 % Antioxidant activity of baseline reduced from 76.9% to 3.7%
3 CLS-fraction-3 614.1 220.2 64.1 % 600.3 318.8 46.9 % Antioxidant activity of baseline reduced from 79.7% to 6.9%

Table-IV: Bioaccessible characteristics on GI tract passage (in vitro)
Sl.
No Extract Code Description TPC
(Total Phenolic Content) TFC
(Total Flavonoid Content) DPPH3
(Antioxidant activity) %
1 CLS-fraction-1 Bio accessible fraction (Plasma) 46.32 88.3 11.6
2 Insoluble fraction (Undigested/Feces) 97.36 136.8 7.7
Total Bioactive content 143.68 225.1 -
3 CLS-fraction-2 Bio accessible fraction (Plasma) 29.61 36.8 3.7
4 Insoluble fraction (Undigested/Feces) 121.00 116.0 3.0
Total Bioactive content 150.61 152.8 -
5 CLS-fraction-3 Bio accessible fraction (Plasma) 47.50 166.5 6.9
6 Insoluble fraction (Undigested/Feces) 172.70 152.3 17.1
Total Bioactive content 220.20 318.8 -

From Table-III and Table-IV as provided above, the stability studies clearly establish that the bioactive extracts demonstrating antiviral activity in the invitro setup has the possibility of extensive degradation in the GI tract and the bio-accessible fraction indicating systemic bioavailability is reduced to a small proportion of the total bioactive component.

Further, encapsulation of the extracts in a suitable biocompatible polymer system would preserve the integrity of the bioactives and significantly increase the bioavailability and consequently, the efficacy against the viruses.

Encapsulation and surface functionalisation of bioactive extracts:

The selected extracts are suitably encapsulated in polymeric coating, the said polymer being selected from a group comprising of Zymosan, Silk, Laminarin, Oligomannose glycans and combinations thereof with other excipients. The resulting polymer complex is then surface functionalised with Lactoferrin.

Example 1:

In one of the Polymeric combinations, Zymosan and Laminarin polymeric blend in a ratio of (1: 0.3) to (1: 0.51), is used for providing biodegradable polymeric encapsulation of the extracts of Punica granatum. In other words, (50 ± 5 mg) of Zymosan are blended with (20 ± 3 mg) of Laminarin in the ratio disclosed above. The polymeric blend is then surface functionalised and immobilised with Lactoferrin which acts to target viral proteins and deliver the extracts preferentially at the local sites of infection.

Similarly, Zymosan with Oligomannose glycans act as different polymeric blends with Lactoferrin as the ligand to serve different vehicles of encapsulation for the hydroalcoholic extracts for use as an antiviral for enveloped RNA viruses. The different Zymosan polymeric blends are depicted in the table below:

Table-V: Polymeric blend of Zymosan with 2 different polymers

Polymer combination Specification Ratio of mixture
Zymosan + Laminarin 50 ± 5 mg of Zymosan with 20 ± 3 mg of Laminarin (1: 0.3) ranging to (1: 0.51)
Zymosan + Oligomannose glycans 50 ± 5 mg of Zymosan with 20 ± 3 mg of Oligomannose glycans (1: 0.3) ranging to (1: 0.51)

Example 2:

In one of the polymeric combinations, a polymeric blend of Silk and Laminarin or Silk with Oligomannose glycans respectively duly surface functionalised with Lactoferrin. The polymeric blends were considered in the ratio of (1:0.3) to (1:0.51) for providing the encapsulation of the extracts of Punica granatum.

Table-VI: Polymeric blend of Silk with 2 different polymers

Polymer combination Specification Ratio of mixture
Silk + Laminarin 50 ± 5 mg of Silk with 20 ± 3 mg of Laminarin (1: 0.3) ranging to (1: 0.51)
Silk + Oligomannose glycans 50 ± 5 mg of Silk with 20 ± 3 mg of Oligomannose glycans (1: 0.3) ranging to (1: 0.51)

Preparation of Zymosan and Laminarin Polymeric blend with Lactoferrin surface functionalisation :

Zymosan is a large complex and aqueous insoluble polymer which possess hollow and porous surface that is exploited for drug loading, thereby acting as microcarriers. (50 ± 5 mg) of Zymosan is mixed with (50 ± 6 mg) of extracts of bioactives overnight (viz. 10 ± 2 hours), centrifuged to obtain particles that are lyophilized. The lyophilized particles are incubated with (20 ± 3) mg Laminarin dissolved in water for (30 ± 3) min to coat the particles. The Laminarin coated Zymosan particles are again centrifuged at 14,000 rpm at (4 ± 0.5 °C) for 10 ± 2 min, followed by the Polymeric-extract mix being allowed to stir for (8 ± 2) hours at 25 degrees centigrade at 80 rpm. Lactoferrin is dissolved in water (1:1w/w) and the solution is added to the Polymeric-extract mix slowly. This creates a nanocomplex embedding the extract with immobilised Lactoferrin acting as the ligand. This complex is lyophilized and stored in (5 ± 2) degree centigrade for use.

Preparation of Zymosan and Oligomannose glycans Polymeric blend with Lactoferrin surface functionalisation:

(50 ± 5 mg) of Zymosan is mixed with (50 ± 6 mg) of the Punica granatum extract overnight (10 ± 2 hours), centrifuged to obtained particles that are then lyophilized. The lyophilized particles are incubated with (20 ± 3) mg Oligomannose glycan dissolved in water for (30 ± 3) min to coat the particles. The Oligomannose glycan coated Zymosan particles are again centrifuged at 14,000 rpm at (4 ± 0.5 °C) for 10 ± 2 min, followed by the Polymeric-extract mix being allowed to stir for (8 ± 2) hours at 25 degrees centigrade at 80 rpm. Lactoferrin is dissolved in water (1:1w/w) and the solution added to the Polymeric-extract mix slowly. This creates a nanocomplex embedding the extract with immobilised lactoferrin acting as the ligand. This complex is lyophilized and stored in (5 ± 2) degree centigrade for use.

Preparation of Silk and Laminarin polymeric blend with Lactoferrin surface functionalisation:

Silk fibroin (2%) was dissolved in a mixture of CaCl2 and ethanol in a concentration of 0.76gram/ml at 50 degree centigrade and to the aqueous solution obtained, the plant extract is added slowly over continuous stirring. The solution is allowed to be stirred overnight at 60-70°C. The solution was centrifuged, followed by dialysis (10 kDa cutoff) against water for one week (water changed 3 times each day). Final silk liquid was lyophilized and the weight of material is measured to calculate the concentration of silk in final solution. The said solution, Silk:Hydroalcoholic extract is allowed for continuous stirring (10 ± 2hrs) at 60 rpm. The solution is added dropwise to acetone in a ratio of (1:10), followed by centrifugation at 14000 rpm for 30 min. The resultant Polymeric-extract nanoparticle is then incubated with (20 ± 3) mg Laminarin dissolved in water for (30 ± 3) min to coat the particles. The Laminarin coated silk particles are again centrifuged at 14,000 rpm at (4 ± 0.5 °C) for 10 ± 2 min. As the final step, Lactoferrin is dissolved in water (1:1w/w) and the solution added to the Polymeric-extract mix slowly. This creates a nanocomplex embedding the extract with silk encapsulation with the immobilised lactoferrin acting as the ligand. This complex is lyophilized and stored in (5 ± 2) degree centigrade for use.

Preparation of Silk and Oligomannose Polymeric blend with Lactoferrin surface functionalisation:

Silk fibroin (2%) iss dissolved in a mixture of CaCl2 and ethanol in a concentration of 0.76gram/ml at 50 degree centigrade and to the aqueous solution obtained, the plant extract is added slowly over continuous stirring. The solution is allowed to be stirred overnight at 60-70°C. The solution is centrifuged, followed by dialysis (10 kDa cutoff) against water for one week (water changed 3 times each day). Final silk liquid is lyophilized and the weight of material is measured to calculate the concentration of silk in final solution. The said solution, Silk: hydroalcoholic extract is allowed for continuous stirring (10 ± 2hrs) at 60 rpm. The solution is added dropwise to acetone in a ratio of (1:10), followed by centrifugation at 14000 rpm for 30 min. The resultant Polymeric-extract nanoparticle is then incubated with (20 ± 3) mg Oligomannose glycan dissolved in water for (30 ± 3) min to coat the particles. The Oligomannose glycan coated silk particles are again centrifuged at 14,000 rpm at (4 ± 0.5 °C) for 10 ± 2 min. As the final step, Lactoferrin is dissolved in water (1:1w/w) and the solution added to the Polymeric-extract mix slowly. This creates a nanocomplex embedding the extract with silk encapsulation with the immobilised lactoferrin acting as the ligand. This complex is lyophilized and stored in (5 ± 2) degree centigrade for use.

Different characterization parameters of the encapsulated polymer complexes:

1. Encapsulation efficiency and Drug loading capacity –
Lyophilized nanoparticles are dissolved in 5% HCl solution to completely release the encapsulated extract from the polymer matrix, and then measured using UV–vis spectrophotometry (Jasco) to determine the loaded amount of extract. Percentage of efficiency of encapsulation is evaluated by determining the content of free extract (unencapsulated) or bound extract (embedded in the nanoparticles). The encapsulation efficiency and drug loading capacity of the nanoparticles are tabulated below in Table-VII.

Table-VII: Encapsulation characteristics of different encapsulated formulations

Nanoparticles Encapsulation efficiency % Drug Loading (µg/mg) Zeta potential
Zymosan-laminarin polymeric complex with Lactoferrin based formulation 33.65±2.67 404.64±5.66 28.98
Zymosan-oligomannose glycan polymeric complex with Lactoferrin based formulation 30.45±1.47 369.60±5.43 26.98
Silk-laminarin polymeric complex with Lactoferrin based formulation 25.69±4.84 309.1±5.82 - 28.26
Silk- oligomannose glycan polymeric complex with Lactoferrin based formulation 27.79±4.24 310.53±3.22 - 24.14

2. Particle Size Analysis and Zeta potential -
One milligram of particles is homogenously suspended in 1.0 mL of distilled water and the corresponding size distribution of the particles in each sample is analyzed using a Zetatrac-Zeta potential particle size analyzer (Microtrac Inc.). The superficial electrical charge of particles used as an index of nanoparticle stability. A stable suspension has a higher zeta potential whereas a lower value indicates colloidal instability and nanoparticle aggregation. Nanoparticles with a zeta potential between ± 30 mV are stable particles. Figure 2A shows that the Zymosan-Laminarin nanoparticles fall in the stable range of zeta potential. It is to be noted that the Zymosan-Laminarin polymeric encapsulated particle is in the range of 2835-3770 nm with a mean size of 3284 nm. Similarly, Figure 2B shows that the particle size distribution of Silk based nano formulations with Laminarin or Oligomannose glycan depicts 300-400 nm range.

3. Scanning Electron Microscopy (SEM) -
The prepared polymer encapsulated particles are detected using a scanning electron microscope (FEI Quanta FEG 200-High Resolution). The SEM images of Zymosan-Laminarin polymeric particles and Zymosan-Oligomannose glucan polymeric particles alongwith the SEM images of Silk-Laminarin polymeric complex and Silk- Oligomannose glucan polymeric particles are shown in Figure 3.

4. Drug release studies -
The release of extract from nanoparticles is carried out under two conditions, viz. with phosphate buffered saline (PBS) solution at pH = 7.4 and pH = 3.2. About 5 mg of extract loaded different polymeric nanoparticles is dispensed in 5 ml of PBS at 25°C/200 rpm. The supernatant (1 ml) is collected and measured by UV–vis spectroscopy (Jasco) to determine the release amount of extract. The cumulative release of extracts from nanoparticles is defined as the amount of extract released into solution in terms of time. Figure 4 shows release of extracts at pH 3.2 and pH 7.4 by nanoparticles. It is to be noted that higher release of extracts is observed at pH 7 for Zymosan-Laminarin polymeric particles followed by Silk-Laminarin polymeric complex.

5. Fourier-transform infrared spectroscopy (FTIR) –
The chemical interactions between in the complex are analyzed by an IR/ATR Spectrophotometer (PERKIN ELMER SPECTRUM). FTIR spectra of particles ground along with KBr powder in the frequency range of 4000–400?cm-1 at a resolution of 1?cm-1 are measured.Two intense broad bands at 3394 indicate the O–H stretch of the hydroxyl groups in Zymosan in the Zymosan-Laminarin polymeric complex (refer Figure 5 A). Bands at 2922 cm–1 indicates the C–H stretch and CH2OH stretch, respectively. The band at 1372 cm–1 could be attributed to the C–H band, and the band at 1637 cm–1 could be attributed to the C=O group in Zymosan-Laminarin polymeric complex. The band at 1042 cm–1 could be attributed to the C–O–C stretch. Laminarin overlapping which can be seen is attributed to its overlapping with the hydroxyl group peak.

The Characteristic amide-1 peak of Silk fibroin is seen at 1638 cm-1 (refer Figure 5B)where the nano particles contain 53% of betasheet content, 20% helix and random coils and turns. This helps to make it amorphous and better degradability.

6. X-ray diffraction -
XRD analysis is carried out to investigate the polymorphism of the nanoparticle prepared. The physical nature and interactions within the nanoparticles are examined by an Enraf Nonius CAD4-MV31 single crystal diffractometer. The X-ray diffractograms of nanoparticles are obtained in the range of 10–80° with a scan step size of 2° at ambient temperature. All the polymer complexes of Zymosan with Laminarin or Oligomannose glucan (refer Figure 6-A) shows a broad peak at 2?=22° which indicates the amorphous nature of the encapsulated complexes. The Silk Fibroin based polymeric nano formulations containing Laminarin or Oligomannose glucan (refer Figure 6-B) shows a broad peak at 2?=20.17° which indicates the good drug entrapment capacity compared to the crystalline nature of silk fibre.

7. Invivo (Mice) study demonstrating improved outcomes after encapsulation –
The bioactive extracts are subjected to an in-vivo study to determine the bioavailability of the bioactive extracts after encapsulation. A mice (C57BL/6, male) pharmacokinetic study is conducted to understand the systemic bioavailability of Punicaligin and Ellagic acid, the major biomarkers of the bioactive extracts from the encapsulated formulations. These biomarkers help in estimating the difference in bioavailability of the encapsulated extracts compared to that of unencapsulated extracts. Both the type of formulations are administered via oral route with twice daily (bid) dosing for 4 days. After 4 days of dosing, plasma, liver and feces are collected from animals and analyzed by HPLC. The concentrations of Punicalagin and Ellagic acid in the plasma, liver and feces samples are tabulated in Table-VII and Table-VIII respectively.

Table-VII: Distribution of drug- Punicalagin
Formulations No. of Animals Punicalagin Concentration (µg/mL) - Mean values
Plasma Liver Feces
(Zymosan-Laminarin) + Lactoferrin 6 1.4 BLQ 3.8
(Zymosan-Oligomannose) + Lactoferrin 6 1.1 BLQ 3.0
(Silk-Laminarin) + Lactoferrin 6 1.0 BLQ 4.2
(Silk-Oligomannose) + Lactoferrin 6 0.6 BLQ 6.2
Unencapsulated extracts 6 0.5 BLQ 4.4
Note: BLQ refers to Below 0.3 µg/mL

Table-VII: Distribution of drug- Ellagic Acid
Formulations No. of Animals Ellagic acid Concentration (µg/mL) - Mean values
Plasma Liver Feces
(Zymosan-Laminarin) + Lactoferrin 6 17.5 BLQ BLQ
(Zymosan-Oligomannose) + Lactoferrin 6 7.5 4.2 30.0
(Silk-Laminarin) + Lactoferrin 6 9.7 2.2 34.2
(Silk-Oligomannose) + Lactoferrin 6 9.6 4.4 28.2
Unencapsulated extracts 6 6.9 2.4 36.6

The two drawbacks associated with administration of extracts in oral form- (1) degradation in efficacy due to low bioavailability and (2) excretion of bioactives in feces are, accordingly, overcome with encapsulated formulations.

Punicalagin is one of the major bioactives and a dependable biomarker in the Punica granatum extracts which has been demonstrated to exhibit antiviral characteristics [1-3]. The invivo test results show that after encapsulation, the bioavailability of Punicalagin in the plasma has increased by 20% to 180% depending on the formulation deployed. Increase of bioavailability in the plasma which would help to confer enhanced antiviral activity. Moreover, the loss of bioactives in feces have been brought down by 4.5% to 30% depending on the formulation deployed which can be still decreased by improved formulation parameters.

Ellagic Acid is also one of the major bioactives in the Punica granatum extracts, showing antiviral properties against enveloped viruses in insilico modelling studies [4]. Our invivo experiments show that the bioavailability of Ellagic Acid has increased by 8.7% to 153% depending on the formulation deployed, while the loss of bioactives in feces have been brought down by 6.5% at the least which can be still decreased by improved formulation parameters.

Hence, encapsulated formulations are distinct improvement in the functional parameters compared to crude peel extracts administration.

References:

1. Salles, T.S., Meneses, M.D.F., Caldas, L.A. et al. Virucidal and antiviral activities of pomegranate (Punica granatum) extract against the mosquito-borne Mayaro virus. Parasites Vectors 14, 443 (2021). https://doi.org/10.1186/s13071-021-04955-4
2. Houston DMJ, Bugert JJ, Denyer SP, Heard CM (2017) Potentiated virucidal activity of pomegranate rind extract (PRE) and punicalagin against Herpes simplex virus (HSV) when co-administered with zinc (II) ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV. PLoS ONE 12(6): e0179291. https://doi.org/10.1371/journal.pone.0179291
3. Tito, A. et al. (2021). Pomegranate Peel Extract as an Inhibitor of SARS-CoV-2 Spike Binding to Human ACE2 Receptor (in vitro): A Promising Source of Novel Antiviral Drugs. Frontiers in Chemistry, 9. doi:10.3389/fchem.2021.638187
4. Bupesh G. et al.(2014). Antiviral activity of Ellagic Acid against envelope proteins from Dengue Virus through Insilico Docking. International Journal of Drug Development and Research, 6.
5. Seo DJ,Choi C.Viral Disease and Use of Polyphenolic Compounds; Chapter 25,Polyphenols:Prevention and Treatment of Human Disease,Elsevier,2018, pp 301-311
6. Liu C et al. Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases. ACS Cent.Sci. 2020, https://dx.doi.org/10.1021/acscentsci.0c00272
7. Conzelmann C et al. Antiviral activity of plant juices and green tea against SARS-CoV-2 and influenza virus in vitro. bioRxiv preprint doi: https://doi.org/10.1101/2020.10.30.360545.
,CLAIMS:I/WE CLAIM:

1. An antiviral composition comprising fractionated extracts of the peel of Punica granatum encapsulated with atleast one biodegradable polymer suitably functionalized with atleast one ligand immobilized to the polymer surface.

2. The antiviral composition as claimed in Claim 1 wherein it comprises of a particular fraction of hydroalcoholic extracts of Punica granatum peel.

3. The antiviral composition as claimed in Claim 2 wherein the hydroalcoholic extracts include ethanol and water in the proportion of 50:50, 70:30 and 80:20.

4. The antiviral composition as claimed in Claim 1 wherein the encapsulated polymer is selected from a group comprising of Zymosan, Silk, Laminarin, Oligomannose glycan and/or a combination thereof.

5. The antiviral composition as claimed in Claim 1 wherein the encapsulated polymer is suitably functionalized with Lactoferin proteins.

6. The antiviral composition as claimed in Claim 1 wherein the composition is in nanodelivery format to be administered in oral form.

7. An antiviral composition comprising fractionated extracts of the peel of Punica granatum encapsulated with atleast one biodegradable polymer suitably functionalized with a ligand immobilized to the polymer surface, wherein polymer is selected from a group comprising of Zymosan, Silk, Laminarin, Oligomannose glycan and/or a combination thereof.

8. The antiviral composition as claimed in Claim 7 wherein the encapsulated polymer is suitably functionalized with Lactoferin proteins.

9. An antiviral composition comprising fractionated extracts of the peel of Punica granatum encapsulated with a blend of Zymosan and Laminarin immobilized/ coated with Lactoferin.

10. An antiviral composition comprising fractionated extracts of the peel of Punica granatum encapsulated with a combination of Zymosan and Oligomannose immobilized/ coated with Lactoferin.

11. An antiviral composition comprising fractionated extracts of the peel of Punica granatum encapsulated with a combination of Silk and Laminarin immobilized/ coated with Lactoferin.

12. An antiviral composition comprising fractionated extracts of the peel of Punica granatum encapsulated with a combination of Silk and Oligomannose immobilized/ coated with Lactoferin.