DESC:FIELD OF THE INVENTION

The invention relates to fractionated extracts from the peel of plant Punica granatum and its encapsulation in biodegradable polymers with suitable ligands in order to target the concentration of the encapsulated particles principally to the liver, more particularly, to fractionated extracts and its method of preparation to prevent and cure malaria arising out of different parasites such as P.falciparum, P.vivax, P.malariae and P.ovale.

BACKGROUND OF THE INVENTION

Malaria is one of the major public health problems globally and also impacts India heavily. India is ranked fourth in the global burden index. As per World Malaria Report 2022, 247 million confirmed cases resulted in 0.62 million deaths. Anopheles stephensi is the chief mosquito vector that harbours P. falciparum and P. vivax plasmodium, responsible for the particular types of malaria, accounting for around 99% of all malaria cases. Similar picture holds good for India too. The P. falciparum infections otherwise called cerebral malaria or brain malaria are known to lead to severe malaria, with high mortality, if timely treatment with effective drugs is not administered. At present, malaria affects all population groups in the country regardless of gender or age although children and pregnant women are at high risk.

Since 2000, there has been a significant increase in the number of countries that have moved towards malaria elimination. In the same period, India also has made considerable progress in reducing its malaria burden and consequently formulated a National Framework for Malaria Elimination in India (2016-2030). This envisions a malaria free country by 2027 and elimination by 2030. Presently, the most effective options include only vector control through LLINs (Long Lasting Insecticide Nets) and IRS (Indoor Residual Spray) which does not address the critical factor of host plasmodium reservoirs in the human liver. In most endemic areas, sub-microscopic and asymptomatic reservoirs of parasites in humans have shown to be efficient source of continued transmission. The presumptive/empirical antimalarial treatment of an entire population to clear the subclinical parasite reservoir is a potential strategy that can help accelerate malaria elimination.

Considering the short-comings of the current anti-malarial formulations, compounds isolated from natural source like Punica granatum have been demonstrated to exhibit anti-malarial activity and the inhibition of pro-inflammatory mechanism seen in the onset of cerebral malaria. Although a majority of the different serovars or cultivars of Punica granatum exhibit different degrees of anti-malarial activity, only a few cultivars belonging to the wild phenotypes of Punica granatum have the desired bioactivity for commercial use.

The liver stages of Plasmodium, constituting a severe bottleneck in the host life cycle, presents a unique opportunity for prophylactic intervention. When a mosquito takes a blood meal, only tens to a few hundred sporozoites are transmitted to the host — each sporozoite is potentially able to initiate hepatocyte infection and form a liver schizont. At this juncture the load is substantially smaller than that in the blood stage infection, which can exceed billions. Eliminating parasites in the liver prevents disease progression in the individual and eliminates transmission, as parasites never reach the blood stage. Furthermore, it also reduces the propensity for resistance development due to the fewer parasites present in the liver. Our objective for this invention is to develop hepatic targeted formulations using nano-technology options and deliver the extracts to the liver to neutralize the sporozoites before they can reach the blood stage.

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 the liver, the present invention aims at developing encapsulated and functionalised versions of the formulations loaded with the bioactive fractions.

Application of drug loaded nanosized carriers such as polymeric nanocapsules (including zymosan, pectin, aminated curdlan, etc.), liposomes, dendrimers are used to overcome the above-mentioned problems, thereby enhancing bioavailability and efficacy of the bioactive plant extract. Immobilisation of proteins or ligands which target sites in the disease pathways is designed to achieve targeted drug delivery.

The present invention discloses an encapsulated formulation comprising USFDA approved polymer blend to encapsulate the bioactive fraction and deliver it to the human liver leading to enhanced bioavailability and antimalarial potency at the liver site. For example, a particular fraction of the bioactives from Punica granatum rind through suitable encapsulation and functionalisation is targeted towards the liver cells and designed to get concentrated for increased bioactivity against P.falciparum and possibly P.vivax which cause malaria after completion of its growth phase in the host liver.

The present invention also discloses a unique method to use a novel encapsulation coating which is biocompatible with the fractional Punica granatum extract demonstrating antimalarial activity. The production methods for such compositions are described with a view to cure and possibly prevent malaria arising out of different parasites including P.falciparum, P. vivax, P. malariae and P. ovale.

OBJECTIVES OF THE INVENTION

The primary objective of the present invention is to develop anti-malarial composition to ameliorate, mitigate, cure and possibly prevent infections/ diseases caused by different parasites including P.falciparum, P. vivax, P. malariae and P. ovale.

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

Yet another objective of the present invention is to develop hepatic targeted formulations using nano-technology options and deliver the extracts to the liver site

Still another objective of the present invention is to develop anti-malarial compositions comprising fractionated extracts from the peel of Punica granatum encapsulated in a blend of biodegradable polymers with suitable surface functionalisation to target the liver site to neutralize the sporozoites before they can reach the blood stage.

SUMMARY OF THE INVENTION

One of the embodiments of the present invention discloses anti-malarial 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-malarial compositions primarily comprising extracts of Punica granatum peel encapsulated in a biodegradable polymer or a 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 ethanolic extracts 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 Chitosan, Pectin, Aminated curdlan and/or a combination thereof and Pectin or Lactobionic Acid as the ligand with other biodegradable materials and/or excipients.

According to yet another embodiment, the encapsulated compositions are suitably functionalised with ligands selected from a group comprising of Pectin and Lactobionic Acid that can be immobilized / fixed to the polymer surface, polymers that can be coated on to the encapsulating polymer blend, to target the liver site to neutralize the sporozoites before they can reach the blood stage.

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

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 caused by different parasites including P.falciparum, P. vivax, P. malariae and P. ovale.

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 1 illustrates a flow chart of process of fractionation of Punica granatum rind powder;

Figure 2 shows graphical representation of anti-malarial activity of each extract of Punica granatum (IC50 values) against P. falciparum;

Figure 3 shows chromatographic fingerprinting of the anti-malarial fraction;

Figure 4 shows SEM images of encapsulated polymer complexes of (a) Chitosan , Pectin and Lactobionic acid based formulation; (b) Chitosan and Pectin based formulation; and (c) Aminated Curdlan and Pectin based formulation;

Figure 5 shows Fourier Transform Infra Red Spectroscopy (FTIR) images of (a) Lactobionic acid linked Chitosan coacerved with Pectin; (b) FTIR of Chitosan coacerved with Pectin with extract; and (c) Aminated Curdlan encapsulated extract coacerved with Pectin;

Figure 6 shows X-ray Diffraction (XRD) images /curves of (a) Chitosan+Pectin+Lactobionic Acid based formulation and Chitosan+Pectin formulation which exhibit similar amorphous nature; and (b) XRD curve of Aminated Curdlan+Pectin based formulation which also exhibit amorphous nature;

Figure 7 shows Differential Scanning Calorimetry (DSC) images of (a) Chitosan+Pectin+Lactobionic Acid based formulation; (b) Chitosan+Pectin based formulation; and (c) Aminated Curdlan+Pectin based formulation; and

Figure 8 shows Thermal Gravimetric Analysis (TGA) images of (a) Chitosan+Pectin+Lactobionic Acid based formulation; (b) Chitosan+Pectin based formulation; and (c) Aminated Curdlan+Pectin based formulation.
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-malarial 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-malarial compositions primarily comprising extracts of Punica granatum peel encapsulated in a biodegradable polymer or a 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 ethanolic extracts 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 Chitosan, Pectin, Aminated curdlan and/or a combination thereof and Pectin or Lactobionic Acid as the ligand with other biodegradable materials and/or excipients.

According to yet another embodiment, the encapsulated compositions are suitably functionalised with ligands selected from a group comprising of Pectin and Lactobionic Acid that can be immobilized / fixed to the polymer surface, polymers that can be coated on to the encapsulating polymer blend, to target the liver site to neutralize the sporozoites before they can reach the blood stage.

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

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 caused by different parasites including P.falciparum, P. vivax, P. malariae and P. ovale.

Preparation of the Punica granatum extracts:

The extraction and fractionation protocol from the rind of Punica granatum is described in the following lines:

1. Raw Material required: Punica granatum shade dried peel powder was obtained by selecting the chosen cultivar and passing the dried peel powder under 80 mesh. All solvents used was of analytical grade / HPLC grade and the regents and standards used was from reputed suppliers with CoA accompanying the reagents.

2. Extraction methodology: The extraction from the crude peel powder was done using Ethanol (70% to 99% purity) as a solvent. The dried peel powder from selected serovars of Punica granatum was weighed and mixed with Ethanol (1:10 ratio w/w) and subjected to Soxhlet extraction for 24 hours under a range of temperature ranging from 37 degrees to 60 degrees centigrade. The extracted crude was subject to microwave treatment or sonication or loaded onto an orbital shaker with 220+20 RPM. The mixture was then filtered through Whatman No.1 Filter paper. The filtrate was vacuum distilled by Rotavapor under 50 to 70 degree centigrade for removal of solvents and the extracts weighed to calculate the yield which was noted. The extracts were further freeze dried and stored under minus 20+3 degrees in amber coloured glass bottles to avoid photo-degradation for further tests.

3. Fractionation Protocol: Referring to Figure 1, the freeze-dried extracts were then subjected to consecutive liquid/liquid partitioning with different solvents of increasing polarity, starting with least polar Hexane > Chloroform > Ethyl acetate > n-Butanol in that increasing order. This partitioning process was undertaken in column involving different resin beds comprising but not limited to XAD-7, XAD-14 and XAD-16 systems. The crude extracts were allowed sufficient time for adsorption following by elution for a specified time period ranging from 10+3 minutes to 20+3 minutes through a mixture of Chloroform, and Ethanol in a fixed proportion (1:3 to 1:7) under room temperature. Each of the fractionated extract was then purified by passing through filter paper and subjected to vacuum filtration and freeze dried for storage under minus 20 degree centigrade. For each of the fractional extracts, the following phyto-functional tests were carried out and the results are tabulated in Table - I.
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.

Table - I
Sl.
No Description TPC1 (SD) TFC2 (SD) DPPH3 %
1 Methanolic Extract 645.05 (1.39) 1243.00 (2.18) 76.9
2 Ethyl Acetate Fraction of Ethanolic extract 616.70 (2.96) 651.80 (1.94) 76.9
3 n-Butanol Fraction of Ethanolic extract 614.14 (3.63) 600.30 (0.59) 79.7
4 Ethanolic extract 554.57 (3.33) 717.00 (1.10) 77.9
5 Ethyl Acetate extract 418.25 (2.71) 562.80 (2.55) 78.2
6 Residual Fraction of Ethanolic extract 346.14 (2.49) 409.80 (1.41) 67.3
7 Hydroalcoholic (EtoH:H2O) extract 484.07 (0.66) 618.00 (2.49) 74.6
8 Hydroalcoholic (MeoH:H2O) extract 552.86 (2.08) 816.30 (3.58) 79.0
9 Aqueous extract 393.18 (4.98) 731.00 (2.15) 73.6
10 Chloroform Fraction of Ethanolic extract 226.21 (1.45) 1125.00 (1.53) 62.6
11 n-hexane Fraction of Ethanolic extract 115.69 (0.74) 1587.00 (2.24) 74.6

1TPC (Total Phenolic Content) has been calculated based on Gallic Acid Equivalent and expressed in mg/ g of extract; SD= Standard Deviation. The TPC of each extract was performed using the Folin–Ciocalteu’s reagent (Singleton RS & Rossi JA. 1965. American Journal of Enology and Viticulture. 16:144–158).

2TFC (Total Flavonoid Content) has been calculated based on Quercetin Equivalent and expressed in mg/ g of extract; SD= Standard Deviation. The TFC, was based on Blasa et al. (2005) was used. (Blasa M, Candiracci M, Accorsi A, et al. 2005. Food Chemistry. 97:217–222).

3DPPH (Antioxidant activity) for the reference standard (Ascorbic Acid) found to be 80.8 %. The antioxidant activity of different lyophilised samples was measured in terms of radical scavenging ability, using the stable radical DPPH (Brand-Williams W, Cuvelier ME, & Berset C. 1995. LWT-Food Science and Technology. 28:25–30).

The above tests were carried out as per well studied protocols in published literature and suitably modified. Each of the fractions was subjected to HPLC and HRLCMS/MS to characterize and identify the bioactives as per method of analysis developed in house. These bioactive fractions were screened for invitro antimalarial efficacy against P.falciparum strain (3D7) at CSIR-CCMB. The anti-malarial activity of P. grantum viz. IC50 values for each extract against P. falciparum (3D7) asexual blood stage parasites conducted at CSIR-CCMB are provided in Table - II. Figure 2 shows the graphical representation of anti-malarial activity of each extract of Punica granatum (IC50 values) against P. falciparum.

Table - II
Sl.No. Description (Mean IC50 (S.D.)
1 Methanolic Extract 1.31 (0.28)
2 Ethyl Acetate Fraction of Ethanolic extract 1.52 (0.06)
3 n-Butanol Fraction of Ethanolic extract 1.64 (0.06)
4 Ethanolic extract 1.74 (0.25)
5 Ethyl Acetate extract 2.15 (0.02)
6 Residual Fraction of Ethanolic extract 3.15 (0.05)
7 Hydroalcoholic (EtoH:H2O) extract 3.89 (0.21)
8 Hydroalcoholic (MeoH:H2O) extract 3.89 (0.98)
9 Aqueous extract 4.83 (0.26)
10 Chloroform Fraction of Ethanolic extract 7.92 (0.52)
11 n-hexane Fraction of Ethanolic extract 15.89 (0.74)

The fractions showing promising antimalarial activity against P.falciparum was then taken up for further development for encapsulation and hepatic targeting via ligands for formulation development. Care was taken to only consider those extracts which comply with Class-3 solvents of ICH guidelines based on safety and toxicology studies. Based on the consistency of antimalarial activity, the best performing extract was chosen to be taken up for encapsulation and hepatic delivery functionalization. The chemical fingerprinting was obtained through HPLC data. The chromatographic fingerprinting of the anti-malarial fraction is shown in Figure 3.

Encapsulation and surface functionalisation of bioactive extracts:

Punica granatum extracts are known to get degraded in the GI tract to other forms of metabolites which are further metabolised by the gut flora. Hence to preserve the integrity of the bioactives and to deliver the desired concentration of the bioactive fractions to the liver, encapsulated and functionalised versions of the P. granatum fractions have been formulated.

USFDA approved biopolymers that had passed the biocompatibility tests of carcinogenicity, immunotoxicity, pro-inflammatory responses, genotoxicity and cytotoxicity were considered and a novel blended polymer was created which would be compatible with the chosen bioactive fraction derived from P. granatum peel. The efficiency parameters considered for encapsulation was based on certain criteria including encapsulation efficiency, drug loading capacity, size and zeta potential/ charge.

Accordingly, a shortlist of three novel polymer blends were created for encapsulation development. The three nano-encapsulation system with ligand for that were designed for delivering the antimalarial fraction derived from P. granatum peel to the liver are:

• Chitosan + Pectin + Lactobionic Acid based formulation
• Chitosan + Pectin based formulation
• Aminated Curdlan + Pectin based formulation

The Analysis of the encapsulating polymer complexes were carried out on the parameters as shown in Table – III.
Table – III
Nanoparticles Encapsulation efficiency (EE) % Drug Loading
(µg/mg) Mean Particle size
(nM) Zeta potential
(mV)
Chitosan + Pectin + Lactobionic Acid based formulation 57.45±2.63 174.47±6.33 520 14.80
Chitosan + Pectin based formulation 64.28±4.96 354.86±7.25 401.6 -30.50
Aminated Curdlan + Pectin based formulation 49.38±5.32 282.42±6.19 921 -26.10

Encapsulation efficiency and Drug loading capacity:

Lyophilized nanoparticles were dissolved in 5% HCl solution to completely release the encapsulated extract from the polymer matrix which was measured using UV–vis spectrophotometry (Jasco) to determine the loaded amount of extract. %EE (Encapsulation Efficiency) was evaluated by determining the content of free extract (unencapsulated) or bound extract (embedded in the nanoparticles).

Particle Size Analysis:

The superficial electrical charge of particles is 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. Table - III above shows that all the polymer nanoparticles fall in the stable range of zeta potential. One milligram of particles was homogenously suspended in 1.0 mL of distilled water and the corresponding size distribution of the particles in each sample was analyzed using a Zetatrac-Zeta potential particle size analyzer (Microtrac Inc.).

Preparation of Chitosan + Pectin + Lactobionic Acid based formulation:

Preparation of this formulation system is a two-step process wherein the first step being linking Chitosan with Lactobionic acid followed by the preparation of lactobionic acid linked Chitosan and Pectin nano-encapsulate. Lactobionic acid is used as a multifunctional galactosylation agent that possesses biodegradability, cell biocompatibility, specificity, selectivity and self-assembly properties. Nanoencapsulations with lactobionic acid as specific ligand recognizes asialoglycoprotein receptor (ASGPR) present in hepatocytes specifically promoting hepatocyte-matrix interactions with increased liver specific activities during hepatocyte proliferation. Lactobionic acid is conjugated to nanocarriers by covalent amide coupling between carboxyl functional group of Lactobionic acid and the amine groups of polymers. Lactobionic acid is linked to Chitosan through amine coupling using ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) mediated polymerization. Briefly 358 ± 30 mg of Lactobionic acid is mixed with 115 ± 21 mg of NHS and 310 ± 35 mg of EDC, to which 80 + 8 mg of Chitosan (in 1% HCl) was added. The complete mixture was stirred for 72+3 hrs at room temperature. The resultant solution was dialyzed against distilled water using a 12 kDa MWCO dialysis tubing for 2+1 days. The distilled water is changed for every 8 + 0.5 hours. The dialyzed product is lyophilized to obtain Lactobionic acid linked chitosan polymer. Lactobionic acid linked chitosan polymer complex (50 ± 5 mg) was dissolved in 1% acetic acid was mixed with (100 ± 11 mg) of the selected P.granatum fractionated extract with continuous stirring for 12+2 hours. Pectin (50 ± 6 mg) dissolved in water was mixed with the Lactobionic acid linked Chitosan polymer together with the bioactive extract to form coacervate nanoencapsulated formulation. The nanoparticles were centrifuged at 14,000+3000 rpm at 4 ± 0.5°C for 10+1 min. The pellet obtained was lyophilized and stored for further analysis. The encapsulated product therefore is a composite formulation consisting of Lactobionic acid, Chitosan, Pectin and the fractionated extract in the specified ratio (w/w) to form the hepatic targeted formulation whose bioactivity has been proven to be a potent antimalarial in invitro as well as invivo systems. The novelty lies in formulation of a composite product comprising Chitosan: Pectin: Lactobionic Acid in the ratio as tabulated below in Table – III.
Table – III
Polymer Combination Specification Ratio of Mixture
Chitosan + Pectin + Lactobionic Acid 80 + 8 mg of Chitosan with 50 ± 6 mg of Pectin with 358 ± 30 mg of Lactobionic Acid (1.63 : 1 : 7.45) ranging to (1.57 : 1 : 6.92)

Preparation of Chitosan + Pectin based formulation:

Preparation of this formulation system is a two-process wherein the first step being the Chitosan linking with selected P.granatum fractionated extract in a ratio of 1:2 with continuous stirring for 12+2 hours. This is followed by the second step of linking the Chitosan-extract complex with Pectin, the water-soluble and biocompatible anionic polysaccharide. Pectin is rich in galacturonic acid which acts as a ligand for the ASGPR receptors that targets hepatocytes. Chitosan (50± 6 mg) was dissolved in 1% acetic acid followed by addition of the selected P.granatum fractionated extract (100 ± 12 mg) in a ratio of 1:2 (w/w) and being continuously stirred overnight. This was followed by pectin (50 ± 6mg) mixed with the above complex which forms a coacervate nanocomplex. The nanoparticles were centrifuged at 14,000+3000 rpm at at 4 ± 0.5°C for 10+1 min. The pellet obtained was lyophilized and stored for further analysis. The encapsulated product therefore is a composite formulation consisting of Chitosan and Pectin with the fractionated extract in the specified ratio (w/w) to form the hepatic targeted formulation whose antimalarial bioactivity has been proven under invitro and invivo conditions. The novelty lies in formulation of a composite product comprising Chitosan:Pectin in the ratio as tabulated below in Table-IV.

Table-IV
Polymer Combination Specification Ratio of Mixture
Chitosan + Pectin 50 + 6 mg of Chitosan with 50 ± 6 mg of Pectin (1: 1) ranging to (1.27 : 0.78)

Preparation of Aminated Curdlan + Pectin based formulation:

Preparation of this formulation system is a three-step process wherein the first step being production of aminated curdlan followed by the second step of creation of a complex of aminated curdlan and P.granatum fractionated extract. The third step is conjugation of the aminated curdlan and P.granatum fractionated extract with pectin to form a nanoencapsulate system which exhibits antimalarial efficacy with hepatic targeting. Curdlan is an anionic polymer which is rendered cationic with the addition of amine groups. Curdlan (1± 0.1 g) was dissolved in 20 ± 2 ml of 5M NaOH and continuously stirred at 4± 0.5 °C for 30 + 5 min to activate hydroxyl groups. To the curdlan solution, 2-dimethylaminoethyl chloride (5 ± 0.25 g) was added in a ratio of 1:5 (w/w) and incubated at 50+2°C for 1 ± 0.1 hr. The resultant mix was washed with diethyl ether thrice and the pH was adjusted to 7.0± 0.1. The solution was dialyzed (12 kDa MWCO tubing) against water for 3 ± 0.1 days followed by lyophilization of the product. Thus the Aminated curdlan-extract complex is now ready for conjugation with pectin. Aminated curdlan which possess positively charged amines is completely coacerved with negatively charged pectin polymer. No external crosslinker is added to the solution. In this nanoencapsulate system pectin acts as the ligand for hepatocyte targeting. As the third step, Aminated curdlan (50 ±5 mg) is dissolved in water in a ratio of 1:2 (w/w) and the resultant solution is mixed with (100 ± 11 mg) P.granatum fractionated extract in a ratio of 1:1 (v/v) and stirred for 24+4 hours. The addition of Pectin (50 ± 6 mg) to aminated curdlan-extract solution was done by way of ionic gelation process. The nanoparticles were centrifuged at 14,000+3000 rpm at 4 ± 0.5°C for 10+1 min. The pellet obtained was lyophilized and stored for further analysis. The encapsulated product therefore is a composite formulation consisting of Aminated Curdlan, Pectin and the fractionated extract in the specified ratio (w/w) to form the hepatic targeted formulation whose antimalarial bioactivity has been proven under invitro and conditions. The novelty lies in formulation of a composite product comprising Aminated Curdlan:Pectin in the ratio as tabulated below in Table – V.

Table - V
Polymer Combination Specification Ratio of Mixture
Aminated curdlan + Pectin 50 + 5 mg of Aminated curdlan with 50 ± 6 mg of Pectin (1: 1) ranging to (1.25 : 0.80)

Structural characterization of the encapsulated polymers:
• Scanning Electron Microscope(SEM) Images: The prepared polymer encapulated particles were analysed using a Scanning Electron Microscope (FEI Quanta FEG 200-High Resolution). All the three encapsulation systems have been prepared with coacervation process, leading to hydrogel structures with porous nature. The SEM images are provided in Figure 4.

• Fourier Transform Infra Red Spectroscopy (FTIR) Images: The chemical interactions in the Polymer complex were 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 were measured. The SEM images are provided in Figure 5. These images establish the actual manufacture of the polymer complexes and therefore help to differentiate the outcome when the components vary.

• X-Ray Diffraction (XRD) Images: The physical nature and interactions within the nanoparticles were examined by an Enraf Nonius CAD4-MV31 single crystal diffractometer. The X-ray diffractograms of nanoparticles were obtained in the range of 10–80° with a scan step size of 2° at ambient temperature. XRD analysis was carried out to investigate the polymorphism of the nanoparticle prepared. All the polymer complexes show a broad peak at 2?= 22° which indicates the amorphous nature of the encapsulated complexes.The XRD images are provided in Figure 6.

• Differential Scanning Calorimetry (DSC) Images: DSC can be used to determine the thermodynamic variations of nanoparticles related to their morphological changes, where the melting and crystallization behaviour of particles were measured. DSC analysis was carried out using a DSC-Q200 (TA Instruments, USA). A heating rate of 10?°C/min was used with a constant nitrogen flow (20?ml/min). Samples (3–5?mg) were placed in a hermetic aluminum pan and sealed, while a sealed empty aluminum pan was used as a reference. The DSC images are provided in Figure 7. A wide endothermic peak from 70 °C to 110 °C, corresponding to the dehydration of the polymer establish the nature of the components.

• Thermal Gravimetric Analysis (TGA) Images: The heat stability of the nanoparticles was determined using a Q500 HI-RES TGA (TA INSTRUMENTS). The experiments were performed from 25?°C to 600?°C at a heating rate of 10?°C/min under a constant nitrogen flow (20?ml/min). The TGA images are provided in Figure 8. From the TGA images it is established that attaching Lactobionic acid to Chitosan increases its thermal stability with the main degradation event between 150-360°C (47.82% weight loss). The presence of dual ligand, Lactobionic acid and Pectin attached to Chitosan, increases the stability of the complex. In case of Chitosan+Pectin polymer complex, Pectin has the main degradation step at 234°C and Chitosan at 292°C. The coacervation of Chitosan and Pectin lead to two degradation steps at 236°C and 493°C, resulting in better thermal stability of Chitosan-Pectin blend in comparison to the individual biopolymers. In case of Aminated Curdlan+Pectin polymer system, two degradation peaks at 240°C and at 600°C is observed while pure Curdlan decomposes only 53%. Amination, encapsulation with extract and coacervation with pectin increases the thermal stability of the nanomaterial.

Pilot scale pharmacokinetic study to determine tissue distribution of the encapsulated formulations benchmarking Ellagic Acid as the functional bioactive compound:

The three different encapsulated formulations of the extracts viz. Chitosan + Pectin + Lactobionic Acid based formulation; Chitosan + Pectin based formulation; and aminated Curdlan + Pectin based formulation were studied in a limited pharmacokinetic model invivo in C57/BL6 male mice. The objective was to determine the tissue distribution and bioavailability after multiple administration (4 days of dosing, twice a day) of the different nano-encapsulated formulations at the liver (site of interest). The unencapsulated extracts were also administered to understand the differences in tissue concentration and validate a key hypothesis of targeted encapsulation to be a superior mode of drug delivery compared to unencapsulated extracts. The below Table -VI shows invivo model Pharmacokinetic parameters of Encapsulated formulations compared to Unencapsulated extracts.
Table – VI
Formulations No. Of Animals Ellagic Acid (biomarker) concentration (µg/mL)
Plasma Liver Feces
Unencapsulated extracts 5 6.9 2.4 36.6
Chitosan+Pectin+Lactobionic Acid based formulation 5 BLQ 4.6 BLQ
Chitosan+Pectin based formulation 5 17.3 5.2 30
Aminated Curdlan+Pectin based formulation 5 BLQ 4.2 28.2
BLQ (Below Limits of Quantification) refers to concentration less than 0.3 µg/mL

All the three formulations have exhibited significantly higher concentration of the functional biomarker (Ellagic Acid which acts as the primary antimalarial agent) at the Liver, which is the objective of the invention.
,CLAIMS:I/WE CLAIM:

1. A synergistic antimalarial composition comprising ethanolic fractionated extracts of the peel of Punica granatum encapsulated with atleast one biodegradable polymer, selected from a group consisting of Chitosan, Pectin, and Aminated curdlan and/or a combination thereof with other excipients, functionalized with Pectin or Lactobionic Acid immobilized to its surface.

2. The synergistic antimalarial composition as claimed in Claim 1, wherein the composition is in nano-delivery format to be administered in oral form.

3. A synergistic antimalarial composition comprising ethanolic fractionated extracts of the peel of Punica granatum encapsulated with a mixture of Chitosan and Pectin and immobilized with Lactobionic Acid.

4. The synergistic antimalarial composition as claimed in Claim 3, wherein it comprises of ethanolic fractionated extracts of the peel of Punica granatum encapsulated with a mixture of Chitosan and Pectin , and immobilized with Lactobionic Acid, in the ratio ranging between (1.63:1:7.45) to (1.57:1:6.92) respectively.

5. A synergistic antimalarial composition comprising ethanolic fractionated extracts of the peel of Punica granatum encapsulated with Chitosan and immobilized with Pectin.

6. The synergistic antimalarial composition as claimed in Claim 5, wherein it comprises ethanolic fractionated extracts of the peel of Punica granatum, encapsulated with Chitosan and immobilized with Pectin, in the ratio ranging between (1 : 1) to (1.27 : 0.78) respectively.

7. A synergistic antimalarial composition comprising ethanolic fractionated extracts of the peel of Punica granatum encapsulated with Aminated curdlan and immobilized with Pectin.

8. The synergistic antimalarial composition as claimed in Claim 7, wherein it comprises of ethanolic fractionated extracts of the peel of Punica granatum encapsulated with Aminated curdlan and immobilized with Pectin, in the ratio ranging between (1 : 1) to (1.25 : 0.80) respectively.