1. Rutin loaded mucoadhesive nanoparticles comprising rutin, one or more stabilizer and pectin.

2. The rutin loaded mucoadhesive nanoparticles as claimed in claim 1, wherein rutin present in an amount ranging from 7.41% to 11.77%, one or more stabilizers present in an amount ranging from 88.24% to 92.59% and pectin present in an amount ranging from 1% to 3%.

3. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein the stabilizers are a mixture of non-ionic surfactants.

4. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein the non-ionic surfactant is selected from Kolliphor.

5. The rutin loaded mucoadhesive nanoparticles as clamed in claim 4, wherein the Kolliphor is selected from Kolliphor® RH 40, Kolliphor® 188 (Pluronic F-68) and Kolliphor® P407 (Pluronic F-127).

6. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein the particle size of nanoparticles ranges from 100 nm to 500 nm.

7. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein the particles have a Polydispersibility index from 0.1 to 1.

8. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein the particles have a zeta potential from -30 mV to -20 mV.

9. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein the particles have rutin content of 96% to 99.82%.

10. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein mucin binding efficiency of the nanoparticle is in the range of 40% to 80%.

11. A process of preparing rutin loaded mucoadhesive nanoparticles as claimed in claim 1, said process comprising (a) preparing an aqueous solution of one or more stabilizer, (b) adding methanolic solution of rutin to the aqueous solution of stabilizer to obtain a rutin- stabilizer mixture, (c) sonicating the rutin- stabilizer mixture at amplitude between 25 W - 45 W for a period of 2 min to 10 min at a temperature range between 2°C to10 °C to obtain rutin nanosuspension, (d) adding pectin to the rutin nanosuspension and stirring at 500 rpm to 1000 rpm for a period of 2 h to 8 h followed by probe sonication and lyphilization.

12. A rutin loaded mucoadhesive nanoparticles composition for management of asthma, said composition comprising rutin loaded mucoadhesive nanoparticles of claim 1 with or without pharmaceutically acceptable surfactant and/or excipient.

Description:
FIELD OF THE INVENTION
The present invention relates to pectin stabilized rutin loaded mucoadhesive nanoparticles for use in asthma management. More particularly, the present invention relates to pharmaceutical compositions comprising the pectin stabilized rutin loaded mucoadhesive nanoparticles. The present invention also relates to a process for the preparation of pectin stabilized rutin loaded mucoadhesive nanoparticles.

BACKGROUND OF THE INVENTION
Asthma is a persistent inflammatory heterogeneous condition manifesting reversible bronchoconstriction and hyper-responsiveness of airways. Globally, it has impacted more than 300 million individuals and by 2025 number is anticipated to increase by 400 million. The pathophysiological changes like wheezing, shortness of breath and cough are usually associated with pulmonary oedema, airway muscle hypertrophy and mucus hypersecretion resulting in airway remodelling during asthma. It can impact any age group, race or nation. Furthermore, asthma greatly generates burden on social, financial and healthcare systems.
Conventional asthma management options are corticosteroids, beta-2-agonists, and leukotriene receptor antagonists. The recurrence and/ or other pathologic situations due to side effects associated with prolonged use of conventional therapies and the progression of asthma to chronic obstructive pulmonary disease (COPD) is still an issue in asthma patients. Therefore, inclination of both patients and clinicians is extending towards the use of phytotherapies due to their therapeutic effectiveness with higher safety index and cost effectiveness. These observations have propelled the researchers to develop newer phytotherapies to tackle precisely underlying asthma pathophysiology and limitations of existing therapies. Among phytoconstituents, rutin has promising therapeutic potential in a variety of chronic diseases, including bronchial asthma due to its antioxidant and anti-inflammatory activities. In particular, rutin ability to inhibit NF-?ß pathways and suppress chemokines production attenuates airway inflammation. However, immensely low aqueous solubility limits its absorption which peculiarly hampers its therapeutic benefits. Furthermore, susceptibility of rutin to undergo photodegradation and enzymatic degradation in gastrointestinal tract (GIT) also imposes major challenge in contemporary use of rutin and its pharmaceutical product development.
Commonly employed techniques to amplify dissolution rate of poorly soluble drugs are micronization, nanosizing or transformation of crystalline form to an amorphous state. However, amorphous solids are generally less stable both physically and chemically due to their higher free energy and entropy. Nanonization remarkably enhances effective surface area of particles to interact with solvent which correspondingly increases their dissolution rate and saturation solubility. Literature has reported a variety of formulations like liposomes, self-emulsifying systems, solid lipid nanoparticles, conversion into amorphous nanoparticles using supercritical anti-solvent process, and micronization etc. to overcome pharmaceutical impedance of rutin and enhance its bioavailability. However, most of rutin-loaded carrier systems suffer stability and safety issues and there is lack of information on pharmacokinetic and pharmacodynamic profile. In addition, poor drug loading and hasty drug release are also the notable hurdles during scale-up, restricting their clinical evolution. Antisolvent precipitation technique is a widely utilized simple and easy technique to prepare nanoparticless. The ultrasonication aids in formation of smaller particles by minimizing size of newly formed particles during antisolvent nanoprecipitation and suppress the aggregation of fine particles by cavitation. However, notable amplification of Gibb’s free energy of particles in nanometer range often leads to aggregation and particle growth. Therefore, use of stabilizer becomes essential to control particle size and morphology by providing steric hindrance between particles to assure enduring stability.
Federica De Gaetano et al., Molecules 2021, 26(4), 1039; https://doi.org/10.3390/molecules26041039 aimed to prepare and characterize solid lipid nanoparticles loading rutin (RT-SLNs) for the treatment of oxidative stress-induced diseases. Phospholipon 80H® as a solid lipid and Polysorbate 80 as surfactant were used for the SLNs preparation, using the solvent emulsification/diffusion method. The spherical RT-SLNs were obtained with low sizes, ranging from 40 to 60 nm (hydrodynamic radius) for the SLNs prepared starting from 2% and 5% (w/w) theoretical amount. All prepared formulations showed negative zeta-potential values. RT was efficiently encapsulated within SLNs, obtaining high encapsulation efficiency and drug content percentages, particularly for SLNs prepared with a 5% theoretical amount of RT. In vitro release profiles and analysis of the obtained data applying different kinetic models revealed Fickian diffusion as the main mechanism of RT release from the SLNs. The morphology of RT-SLNs was characterized by scanning electron microscopy (SEM), whereas the interactions between RT and the lipid matrix were investigated by Raman spectroscopy, evidencing spectral modifications of characteristic bands of RT due to the establishment of new interactions. Finally, antioxidant activity assay on human glioblastoma astrocytoma (U373) culture cells showed a dose-dependent activity for RT-SLNs, particularly at the highest assayed dose (50 µM), whereas the free drug showed the lesser activity.
Nartaya Thirawong et al., European Journal of Pharmaceutics and Biopharmaceutics 2007, examined mucoadhesive performance of various pectins with different degrees of esterification and molecular weights with porcine gastrointestinal (GI) mucosa, i.e. buccal, stomach, small intestine and large intestine, using a texture analyzer equipped with mucoadhesive platform. The instrumental parameters and test conditions such as pre-hydration time of pectin disc, contact time, contact force, test speed of probe withdrawal, GI tissue and test medium were also studied. Two parameters derived from texture analysis, namely maximum detachment force (Fmax) and work of adhesion (Wad), were used as parameters for comparison of mucoadhesive performance. The results indicated that degree of hydration of pectin disc affected the mucoadhesive properties. The mucoadhesion of pectin increased with the increased contact time and contact force, but not by the increased probe withdrawal speed. Tissue from different parts of GI tract and test medium also influenced the mucoadhesion. Pectins showed a stronger mucoadhesion on large intestinal mucosa than on small intestinal mucosa. The mucoadhesive properties of pectins on gastric mucosa depended on pH of the medium; a higher Fmax and Wad in a pH 4.8 medium than a pH 1.2 medium was revealed. Additionally, pectin showed a significantly higher mucoadhesion than carbomer934P in most of the GI mucosa tested. The results also demonstrated that the mucoadhesive performance of pectins largely depended on their characteristics, i.e. higher degree of esterification and molecular weight gave a stronger mucoadhesion. These findings suggest that pectin can be used as a mucoadhesive carrier for GI-mucoadhesive drug delivery systems.
Rutin exhibit substantial therapeutic benefits. However, its poor oral bioavailability and extensive metabolism in gut limits its application in clinics. Cellulosic polymers, polyvinyl pyrrolidone, pectin and solubilizers are widely used as stabilizer alone or in combination. The selection of appropriate stabilizers to a particular drug depends upon its capability of aiding in particle size reduction apart from stabilizing efficiency, biocompatibility and safety. Henceforth, the primary aim of present disclosure is to nanonize rutin to enhance its dissolution rate, bioavailability and therapeutic effectiveness in management of asthma.

OBJECTS OF THE INVENTION
In order to obviate the drawbacks in the existing state of the art, the main object of the present disclosure is to provide pectin stabilized rutin loaded mucoadhesive nanoparticles.
Another object of the present disclosure is to provide pectin stabilized rutin loaded mucoadhesive nanoparticles capable of being used in asthma management.
Yet another object of the present disclosure is to provide pharmaceutical compositions comprising the pectin stabilized rutin loaded mucoadhesive nanoparticles.
Yet another object of the present disclosure is to provide pharmaceutical compositions comprising the pectin stabilized rutin loaded mucoadhesive nanoparticles showing enhanced therapeutic efficacy.

Yet another object of the present disclosure is to provide a process for the preparation of pectin stabilized rutin loaded mucoadhesive nanoparticles.

Yet another object of the present disclosure is to provide a process for the pectin stabilized rutin loaded mucoadhesive nanoparticles capable of being used in the asthma management.

SUMMARY OF THE INVENTION
It will nevertheless be understood that no limitation of the scope of the invention is thereby intended by way of embodiments and examples. Such alterations and further modifications in the present invention, and such further applications of the principles of the invention as would normally occur to those skilled in the art are to be construed as being within the scope of the present invention.

It will be understood by those skilled in the art that the summary of the invention provided herein is exemplary and explanatory of the invention and are not intended to be restrictive thereof. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The composition, methods, and examples provided herein are only illustrative and not intended to be limiting.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Similarly, one or more steps of method or components proceeded by "comprises... a" does not, without more constraints, preclude the existence of other, steps or components. Appearances of the phrase "in a preferred embodiment”, “in an embodiment", “in another embodiment” and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.

To obviate the drawbacks in the existing state of the art, there is provided mucoadhesive pectin stabilized rutin mucoadhesive nanoparticless (RNPs). The RNPs are prepared by antisolvent precipitation method assisted with ultrasonication to improve rutin bioavailability and therapeutic effectiveness in asthma management. RNPs are optimized in regard of type and concentration of stabilizer, amplitude and time of sonication and concentration of pectin. Particle size, size distribution and mucoadhesivity were opted as quality parameters. Optimized RNPs exhibited 98.82 ± 4.63 % drug content with particle size in nanometric range (130.41±11.21 nm) with homogeneous dispersibility (0.27± 0.03) and 63.38±3.25% mucin binding efficiency. DSC and pXRD confirmed reduced crystallinity of rutin in RNPs while SEM established nanometric size of optimized formulation. Dissolution study indicated almost complete drug release in 24h from optimized formulation. Optimized RNPs exhibited significant colloidal stability at different environmental conditions. Pharmacokinetic study revealed 2.13-fold increase in oral rutin bioavailability after its incorporation in pectin stabilized RNPs. The remarkable suppression of pulmonary oedema, airway hyperresponsiveness i.e., elevation of bronchospasm onset time and reduction in recovery time are observed in RNPs treated group compared to pure rutin. In conclusion, pectin based mucoadhesive RNPs of the present disclosure provides promising potential in asthma management with ease of scalability. The present invention also provides a process to prepare RNPs, and a composition thereof.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1 depicts scanning electron monographs of optimised RNPs.
Figure 2 depicts ATR-FTIR spectrum of rutin, Kolliphor-RH 40, pectin, physical mixture and optimized formulation respectively.
Figure 3 depicts DSC thermogram of rutin, Kolliphor-RH 40, pectin, physical mixture and optimized formulation respectively.
Figure 4 depicts pXRD analysis spectra of rutin (A), Kolliphor-RH 40 (B), pectin (C), physical mixture (D) and optimized formulation (E) respectively.
Figure 5 depicts dissolution profile of rutin and optimized formulation respectively.
Figure 6 depicts effect of storage conditions on colloidal parameters (A) particle size, (B) PDI, (C) zeta potential and (D) percentage drug content of optimized formulation respectively.
Figure 7 depicts plasma concentration profile of rutin and optimized formulation respectively.
Figure 8 depicts effect of different treatment on (A) bronchospasm onset time, (B) percentage recovery time, (C) percentage protection and (D) wet/dry weight ratio respectively.

DETAILED DESCRIPTION OF THE INVENTION WITH NON-LIMITING EMBODIMENTS AND ILLUSTRATIONS
The present disclosure provides novel pectin stabilized rutin loaded mucoadhesive nanoparticles used in the management of asthma. More specifically, the disclosure provides novel pectin stabilized rutin loaded mucoadhesive nanoparticles (RNPs) having enhanced bioavailability, therapeutic efficacy, stability and improved drug loading capacity.

The present invention also provides a process to prepare novel pectin stabilized rutin loaded mucoadhesive nanoparticles. A methanolic solution (W1) of rutin is added to an aqueous solution of stabilizer at the rate of 1 ml/ min. The resultant solution is then sonicated under specific conditions to obtain optimized rutin nanosuspension. The type and amount of stabilizer, sonication time and amplitude are optimized to prepare stable rutin nanosuspension. Pectin at specific concentration is added to optimized rutin nanosuspension and stirred at 700 rpm for 6 h followed by probe sonication at 4°C for 5 min. The developed hydrogel is lyophilized at -80 ºC and 0.05 mbar for 36 h utilizing a cryoprotective agent (Table 1). Lyophilized hydrogel formulations are kept in desiccator at 4°C to perform further studies.

In an embodiment of the present disclosure, the specific conditions for sonication to obtain optimized rutin nanosuspension are an amplitude of 45W for 5 min at 4 °C.

In an embodiment of the present disclosure, the specific concentration of pectin is in the range of 1% to 3% w/v.

The nanonization of drugs usually demands high energy inputs during sizing. Therefore, modified anti-solvent nanoprecipitation ultrasonication method was utilized to fabricate rutin nanoparticles. The reduced solubility of rutin in aqueous medium facilitated its abrupt crystallization and formation of drug crystals of average particle size 5140.23 ± 93.67 nm with high PDI (0.95 ± 0.26). This indicated that rutin dispersion formed was thermodynamically unstable due to crystal growth facilitated by nucleation and agglomeration even under sonication. Therefore, stabilizer (pluronic F 68) was added to aqueous medium for steric stabilization of nanoparticles and to impede the interaction of nanosized dispersed drug molecules. Stabilizer addition facilitated the formation of nanoparticles with average size below 200 nm with better dispersibility in contrast to their absence (Table 1). Micelles produced due to adsorption of stabilizer at interface of nanosized particle surfaces might have reduced the surface free energy and retarded their aggregation or coalescence.

In order to select a suitable stabilizer, different stabilizers (Kolliphor RH-40, Pluronic-F68 and Pluronic-127) of varying HLB were screened. Among the stabilizers, kolliphor RH-40 facilitated the formation of smaller-sized particles with uniform dispersibility. This indicated that Kolliphor RH-40 bestowed the generation of stable mechanical and thermodynamic barriers at the interface of drug particles and aqueous medium.

On varying amount of Kolliphor RH-40, variation in average particle size, PDI and zeta potential was noticed from 130.41± 11.21 nm to 198.63 ± 13.22 nm, 0.27±0.03 to 0.61±0.05 and -27.01± 2.48mV to -20.23 ± 3.54 mV respectively. Larger particle size and PDI at lower Kolliphor RH-40 concentration indicated the paucity of stabilizers to turn down the surface free energy of newer surfaces produced and their agglomeration. The results displayed decrease in size and PDI with an increase in zeta potential on varying Kolliphor RH-40 amount (Table 1). However, further increase in stabilizer amount (500 mg) increased the particle size and PDI confirming heterogeneous particle size distribution. The outcome of studies confirmed that a suitable amount of stabilizer was needed to retard or prevent the crystal growth by imparting steric stabilization.

Process variables like sonication amplitude and sonication time had imparted remarkable consequences on particle size, PDI and ZP (Table 1). Probe sonication induced cavitation and shear to cleave larger particles and their agglomerates. The increase in sonication time from 7.5 to 10 min notably diminished the particle size and PDI of RNPs (Table 1). However, further enhancement in sonication time (12.5 min) had greatly made the system thermodynamically unstable by distorting the stabilizer barrier layer and hastened the aggregation contributing to increase in particle size and PDI (Table 1).

Similarly, an increase in sonication amplitude from 25 to 45 W had remarkable consequences on particle size diminution and their PDI due to the increased probability of particles remaining in the area of higher power density. However, drastic increase in nanoparticles size and reduced zeta potential was observed with increase in sonication amplitude to 65 W (Table 1). The accelerated particle collision rate due to increased energy input in contrast to stabilizer adsorption rate over newer surfaces generated might have escalated the recoalescence rate.

PDI of batches prepared varied from 0.27 ±0.03 to 0.61±0.05 (Table 1). Batches with PDI value <0.3 indicated the homogeneity of size distribution. Zeta potential of batches varied from -20.23 ± 3.54 mV to -27.01 ± 2.48 mV. Batches with an absolute value of zeta potential close to -30 mV are regarded as being more stable (Table 1). Higher zeta potential of optimized formulation also confirmed optimization of both formulation and process variables to fabricate stable rutin-loaded nanoparticles. Meanwhile, high percentage yield (90.30 ± 6.43% to 98.78 ± 4.72%) and rutin content (96.08 ± 5.45% to 99.82 ± 4.67%) were achieved for all batches. The processing losses during RNPs fabrication might have contributed to the loss in both yield and drug content. So, depending upon the particle size, PDI and zeta potential, RNC batch prepared by sonicating drug dispersion in Kolliphor RH-40 (400 mg) solution for 10 min at 45W amplitude was selected for further studies.
Different batches of rutin nanosuspension were analysed for particle size, PDI and zeta potential utilizing photon correlation spectroscopy.
Lyophilized formulations were dispersed in methanol and centrifuged to collect supernatant which was evaluated for percentage drug content. Yield of lyophilized rutin formulations was determined by utilizing following formula.
Percent Drug Content = Actual Drug Content ?100
Total Drug Amount Taken

Percent yield = Weight of Lyophillized Formulation ?100
Total weight of all solids

Table 1: Effect of different formulation and process variables on quality parameters of various batches prepared respectively.

The ability of uncoated and pectin coated rutin nanoparticless to bind with mucin was determined by turbidimetric method. Briefly, equal amount of porcine mucin suspension was blended with equal amount of nanoparticles and incubated for 60 min at 37 °C. Successively, mixture was centrifuged to collect supernatant. Supernatant was assessed for free mucin content spectrophotometrically at 253 nm. The mucin binding proficiency of formulations was estimated by subsequent formula
Mucin binding proficiency (%) = Total Mucin amount – Free Mucin amount X 100
Total Mucin amount

Optimized lyophilized RNCs exhibited collapsed appearance and poor re-dispersion in aqueous medium manifesting crystal growth due to aggregation of micelles during lyophilisation (Table 2). In order to construct a stable and readily re-dispersible form of RNPs, pectin coating was done over nanoparticles and lyophilized. The use of pectin facilitated the formation of slightly collapsed formulations with higher particle size and PDI with improved re-dispersibility (Table 2). Pectin being a hydrophilic polymer on adsorption on surface of rutin nanoparticles via van der Waals forces and/ or hydrogen bonding might have furnished good dispersion of hydrophobic nanoparticles and prevented agglomeration by steric stabilization. However, higher pectin concentration (3 % w/v) reduced re-dispersibility due to enhanced thickness coat and wetting time. In addition, pectin imparted mucoadhesivity to nanoparticles (Table 2). Increase in pectin concentration to 2 % w/v enhanced the mucoadhesion of nanoparticles. However, further increase in pectin concentration (3 % w/v) had no significant impact on re-dispersibility and mucoadhesion (Table 2). The increase in particle size of RNPs might have reduced the effective surface area of mucin adsorption and solvent interaction.

Table 2: Effect of pectin coating on particle size, zeta potential, PDI, re-dispersibility and mucin binding efficiency respectively.
Pectin amount (% w/v) Appearance

Particle size (nm±SD) PDI±SD

ZP
(mV±SD) Yield
(% ± SD) Rutin content
(% ± SD) Re-
dispersibility time (s ± SD) Mucin binding efficiency (% ± SD)

0 Collapsed cake 130.41±11.21 0.27±0.03 -25.83± 3.35 98.78± 4.72 99.82± 4.67 157.42±
14.15 7.85±3.13
1 Partially collapsed cake 151.42±
16.98 0.26±
0.04 -22.90± 2.64 97.12 ± 9.23 97.35± 5.36 90.13±
10.17 45.01±5.89
2 Uniform 183.61±10.21 0.26±0.06 -27.51± 3.42 96.18±6.02 99.56± 6.74 39.45±
6.11 76.38±4.36
3 Slightly collapsed cake 407.47±12.40 0.42±0.13 -22.24± 2.79 93.47±5.41 97.56± 4.33 62.37 ±
8.73 56.14±5.89
Solubility determination
Shake flask method was used to estimate solubility of rutin alone and in presence of stabilizers. In brief, an excess amount of rutin and optimized R-NPs were added to distilled water, 0.1 N HCl (pH 1.2) and phosphate buffer pH 6.8 and shaken at 100 rpm on an orbital isothermal shaker for 24 h at 37 ± 2 °C. Subsequently, each sample was centrifuged for 10 min at 12,000 rpm. Supernatants collected were suitably diluted and analyzed utilizing UV-Visible spectrophotometer at 363 nm.

The immense increase in solubility of rutin by RNPs around 15, 18 and 12-fold at pH 1.2, phosphate buffer pH 6.8 and pH 7.4 respectively was observed. The ability of rutin to form molecular dispersion with kolliphor RH-40 and hydrophilic coat of pectin has synergistically boosted the drug solubility by furnishing good dispersion of hydrophobic nanoparticles with enhanced wetting.

Solid state characterization
Morphology of optimised lyophilised formulation was evaluated utilizing SEM (MIRA3, TESCAN). Sample was glued, mounted on an aluminium plate, dried and laminated with gold-palladium alloy under an inert environment of argon prior to imaging. RNPs were analyzed at 20 kv under suitable magnification and resultant microphotographs were captured.

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was utilized to estimate the intermolecular interactions among rutin and formulation components respectively using Bruker EQUINOX 55 FTIR spectrophotometer. Rutin, kolliphor RH40, pectin, physical mixture and optimized formulation were placed over ATR lens and scanned within a spectral range of 4000-400 cm-1 at a scanning frequency of 45 times respectively.

Thermograms of rutin, kolliphor RH40, pectin, physical mixture and optimized formulation were recorded utilizing differential scanning calorimeter. Samples were packed in standard aluminium pans, hermetically sealed and heated over a temperature range of 30-300 °C at 10°C min-1 under an atmosphere of nitrogen pursed at a flow rate of 60ml min-1.

X-ray diffractograms of rutin, kolliphor RH40, pectin, physical mixture and optimized formulation were recorded by powder X-ray diffractometer. Samples were scanned at a current of 40 mA and a voltage 40kV in the diffraction scanning range of 0-100°, 2?.

SEM images of optimised pectin coated RNPs showed homogenously distributed quasi-spheroidal particles having size in the range of 100-150 nm (Figure 1). The results of SEM imaging are in good agreement with observation of photon correlation spectroscopy.

ATR-FTIR spectrum of pure rutin revealed typical peaks at 3468.35 cm-1 and 3384.54 cm-1 due to O-H stretching vibration while 2691.03 cm-1 represents C-H banding. The peaks at 1651.03 cm-1 represent stretching vibration of asymmetric C=O and 1360.44 cm-1 due to C-OH vibrations. Additionally, peaks at 1061.36 cm-1 and 1010.39 cm-1 verify the presence of an aromatic group. Kolliphor-RH 40 showed an intense band at
2859.17 cm-1, 2920.83 cm-1, 1732.24 cm-1, 1460.16 cm-1, 1348.88 cm-1 due to asymmetric alkyl C-H stretching, C=O stretching (aldehyde), and asymmetric C-H stretching and bending of alkane group respectively. The characteristic bands of pectin were observed at 3389 cm-1, 2939.98 cm-1, 2863.49 cm-1, 1733.80 cm-1, 1424.68 cm-1, and 1064.09-1005.65 cm-1 due to hydroxyl group of pectin skeleton, C-H bond of -CH2 alkyl group, -C=O bond of the ester carbonyl group, C-H bond associated with primary amide groups and stretching band of C-O-C bond respectively. The spectrum of optimised formulation exhibited characteristics peak of rutin at 3356.56 cm-1, 2640.27 cm-1, 1741.25 cm-1, 1460.79 cm-1, 1343.63 cm-1 and 1016.86 cm-1 indicating asymmetric O-H stretching, C-H stretching, carbonyl stretching, C-OH vibrations and verified the presence of aromatic group respectively. No remarkable difference in peak positioning in spectrum of formulation was observed (Figure 2) indicating the uniform molecular dispersion of drug along with physicochemical stability of rutin with excipients in the formulation.

Thermograms of coarse rutin, Kolliphor RH-40, pectin, physical mixture and optimised formulation are displayed in Figure 3. Rutin showed a relatively broad endothermic peak at 172 °C whereas Kolliphor RH-40 and pectin showed endothermic peak at 115 °C and 210 °C respectively. Thermogram of physical mixture exhibited characteristic peaks of drug and Kolliphor RH-40 nearly at the same position demonstrating the absence of any specific interaction between rutin and stabilizers. A shift in endothermic peak of rutin to 165 °C was observed in formulation thermogram (Figure 3). The adsorption of Kolliphor-RH 40 and pectin on rutin surface might have attributed to slight shift in endothermic peak to lower temperature.

Diffractograms of rutin, Kolliphor RH-40, pectin, physical mixture and optimised formulation are displayed in Figure 4. Diffraction pattern of Kolliphor RH 40 and pectin showed diffused peaks in the spectrum of pectin indicating their amorphous nature. The sharp distinctive peaks at 10.65°, 12.17°, 25.96°, 31.31°, 32.64°, 36.65°, 38.84° and 40.40° were observed in rutin diffractogram ascertaining its crystalline nature. A similar diffraction pattern was detected in physical mixture. The presence of characteristic peaks of rutin in optimized formulations’ diffractogram with reduced peak intensity was observed. The surface adsorption of Kolliphor-RH 40 and pectin on nanosized drug might be attributing to reduced crystallinity of rutin in optimized formulation.

Dissolution study
In-vitro release of rutin from optimized formulation was estimated using dialysis bag diffusion method. Optimized formulation equivalent to 50 mg rutin was kept in dialysis bag and immersed in pH progressive dissolution fluid (200 ml, 0.1N HCl pH 1.2 to mimic the stomach pH conditions for 2 h followed by phosphate buffer pH 6.8) maintained at 37 ± 0.5 ºC and 100 rpm respectively. Tween-80 (0.5% w/v) was added to dissolution media to attain sink conditions. Aliquots (3 ml) were withdrawn at predestined time intervals, filtered and analysed spectrophotometrically respectively. Volume of dissolution media was maintained constant by immediately replacing with fresh buffer after each removal. The cumulative percentage drug release versus time curve was plotted. Data acquired from dissolution study was analyzed by curve fitting to distinct kinetic models (zero order, first order, Higuchi diffusion model and Korsmeyer Peppas equation) to determine rutin release behaviour. The highest numerical value of regression coefficient (R2) was employed to describe the best-fit model.

Around 16% drug was released in simulated gastric fluid in 2h followed by sustained drug release upto 24 h from formulation. The initial drug release in 2 h might be attributed to release of drug adsorbed on the surface of particles followed by diffusion across the swelled polymeric matrix. On the contrary, pure drug showed 3% drug release in 2 h and ~48% in 12 h respectively (Figure 5). The water-adsorbing behaviour of pectin providing a saturated aqueous environment might be contributing to good wetting and dispersibility of nanosized formulation with a faster drug release rate compared to pure drug. The results indicated that rutin dissolution was restricted by its poor wetting and aqueous solubility. The presence of Kolliphor RH-60 at the interface of rutin and aqueous phase might have lessened surface tension and enhanced the dissolution rate of rutin. In addition, kolliphore RH 40 facilitated formation of multimolecular micelles which have attributed the sustained drug release for 24 h. The interaction of rutin with hydrophobic domain of Kolliphor RH-40 via Vander Waals forces might have de-accelerated the rutin partitioning and diffusion from multimolecular micelles. The release data was fitted to different mathematical kinetic models to obtain insight into drug release mechanism from optimized formulation. The value of correlation coefficient (R2) for zero-order, first-order, Higuchi, and Korsemeyer model was 0.87, 0.96, 0.92 and 0.76 respectively. The highest R2 value was obtained for the Higuchi model indicating that drug release follows diffusion and dissolution-controlled model.

Storage stability
Optimized RNPs were exposed to real time (25 ± 2 °C / 60±5% RH) and accelerated (40 ± 2 °C / 75 ± 5 % RH) stability conditions for 12 months and 6 months respectively according to ICH guidelines. Optimised RNPs were stored in amber colored glass vials sealed via screw caps. Colloidal stability of formulation was measured at a predetermined time interval of 0 day, 1, 3, 6, 9 and 12 months for long term stability while upto 6 months at accelerated conditions respectively.
Optimized RNPs stored at room temperature and accelerated storage conditions manifested no significant variation in their colloidal quality parameters like particle size, zeta potential, PDI and % drug content (Figure 6). The observations revealed that all formulation variables were adequately optimized to maintain colloidal characteristics for effective long-term use. No remarkable variation in color of optimized RNPs was observed on visual evaluation at different storage conditions.

In-vivo studies
Healthy wistar rats (180 - 250g) and guinea pigs (200-230g) acquired from animal house of Banasthali Vidyapith were kept under disease free standard laboratory conditions with maintenance of temperature (22±2°C), humidity (55± 2%) and light-dark cycle of 12 h each. Animals were fed with standard chow diet and provided water ad libitum. In vivo experiments were sanctioned by Institutional Animal Ethical Committee (IAEC, 574/GO/ReBi/S/02/CPCSEA) of Banasthali Vidyapith and executed in accordance with CPCSEA guidelines of the Ministry of Animal Welfare, Government of India.

Pharmacokinetics study
Twelve healthy Wistar rats (180-250g) kept on overnight fasting with free access to water prior to start of experiments were arbitrarily distributed into 2 groups (n=6). Animals of groups I and II were orally administered with free rutin (15 mg/Kg) and RNPs (equivalent to 15 mg/Kg rutin) respectively. Blood samples (200 µl) were withdrawn from rat tail vein at preset time intervals (0.5, 1, 2, 4, 6, 8, 12 and 24 h after drug administration) in heparinised tubes and immediately centrifuged at 5000 rpm for 10 min at 4 °C. Plasma was extracted with methanol: acetonitrile (2:1 v/v), centrifuged at 12,000 rpm for 10 min at 4 °C. Supernatant collected was estimated for rutin content via HPLC analysis using Phenomenex C18 column (250 mm X 4.6mm, 5µm) at room temperature with a mobile phase of methanol: acetonitrile: water (45:15:40 v/v/v). Sample (50 µl) was injected and system was run isocratically at 0.6 ml/min to detect rutin at 359 nm.

Plasma concentration-time profile and pharmacokinetic parameters of rutin and pectin coated RNPs after single oral dose equivalent to 15 mg/Kg rutin have been elucidated in Figure 7 respectively. The proportionately higher Cmax (4.06-fold) and AUC0-24h (2.17-fold) was observed on oral administration of optimized RNPs in comparison to rutin. Higher intrinsic solubility offered by optimized RNPs and prevention of rutin precipitation might be accounting for faster and higher extent of absorption in comparison to rutin dispersion. Significantly lower Tmax of formulation (~3-fold) than pure rutin indicated faster rutin absorption. In addition, optimized RNPs attained higher plasma concentration throughout its 24 h profile (Figure 7). Substantial paracellular, transcellular and phagocytic uptake of colloidal-sized particles along with their lymphatic drainage from the small intestine might have extended half-life (28.71±0.18 h) and MRT (26.82±0.22 h) of optimized RNPs. Prolonged drug release behaviour of optimized RNPs and diffusion down the concentration gradient from cells and lymphatic system into blood might be maintaining higher plasma concentration for longer duration as demonstrated by their higher bioavailability (2.17-fold) compared to rutin dispersion.

Pharmacodynamics study
Fifteen healthy guinea pigs were sensitized by intraperitoneal injections of OVA and alum solution on 1st and 7th day respectively. Subsequently on 14th day, OVA booster dose was administered to accomplish the sensitization challenge. Naïve animals (n=3) were sham sensitized with normal saline similar to OVA-sensitized animals. OVA-sensitized guinea pigs were randomly distributed into four groups (n=3) respectively. Guinea pigs of group I were treated with normal saline (positive control), while group II, III and IV received rutin dispersion (15mg/Kg), optimised formulation (equivalent to 15mg/Kg rutin) and chlorphenamine maleate (15mg/Kg) as standard drug orally for 14 days. Animals of each group were subjected to 1% histamine dihydrochloride solution exposure after 2 weeks treatment. Animals were sharply observed to record onset of bronchospasm and recovery time. The onset of bronchospasm refers to the duration between being exposed to histamine aerosol and experiencing breathing difficulties that may lead to convulsions. The recovery time is the time taken by OVA-sensitized animals with preconvulsive dyspnoea to recover after being placed in fresh air. The results were noted and compared with saline-treated group. The percentage protection offered against bronchospasm onset by treatment was calculated by using formula:


Where, Ta= bronchospasm onset time before drug treatment,
Tb = onset bronchospasm time after treatment

Where Ra = Recovery time before drug treatment,
Rb = Recovery time after drug treatment

On 29th day, animals were sacrificed to collect lung and trachea. The left lung was excised, blotted dry and instantly weighed. Subsequently lung was re-weighed after being dried for 48 h at 60°C and wet-to-dry weight ratio was estimated.

Data herein has been disclosed as mean ± SD for each group (n=3). Means were statistically compared utilizing one-way ANOVA followed by Bonferroni’s multiple comparison tests employing GraphPad Prism version 5.0 (GraphPad Software, California, USA). p<0.05 was set as level of statistical significance in all studies.

OVA-sensitized guinea pigs showed a significant increase in hyper-responsiveness when exposed to histamine. Clinical parameters such as onset of bronchospasm, frequency of jerky episodes and difficulty in breathing after exposure to histamine were measured (Figure 8). OVA-sensitized animals showed significantly higher bronchial hyper-responsiveness in response to histamine compared to saline-sensitized animals (p<0.05). Observations showed remarkable postpone in bronchospasm induction and frequency of jerks in animals treated with rutin, chlorpheniramine and optimised formulation in comparison to control group (p<0.05). Both pure drug and optimized formulation inhibited the jerk induction and reduced the severity of spasms. However, protection and recovery after treatment with formulation was remarkably enhanced compared to pure drug (p<0.05). This might be attributed to higher drug bioavailability as observed during pharmacokinetic study.

In order to assess the magnitude of OVA-induced pulmonary oedema, wet/dry ratio of lungs was estimated. The massive leukocytes and neutrophils infiltration into lungs might have resulted in a significantly higher wet/dry ratio in the sensitized group compared to pure rutin and optimized formulation (p<0.05) (Figure 8). However, a significantly (p<0.05) higher reduction in wet/dry ratio was observed with optimized formulation indicating a remarkably higher protective effect in suppressing pulmonary oedema via reducing inflammatory cell accumulation.

In one embodiment, the rutin loaded mucoadhesive nanoparticles of the present invention shows improved pharmacokinetics and bioavailability, improved therapeutic effects and improved rutin dissolution along prolonged drug release for 24 h.

In one embodiment, the rutin loaded mucoadhesive nanoparticles of the present invention shows enhanced rutin solubility at simulated gastric pH 1.2, intestinal pH 6.8 and 7.4.

In one embodiment, the pectin boosted good dispersibility of drug along with Kolliphore by enhancing wetting.

In one embodiment, there is provided a rutin loaded mucoadhesive nanoparticles composition for management of asthma, said composition comprising rutin loaded mucoadhesive nanoparticles with or without pharmaceutically acceptable surfactant and/or excipient.

The present invention successfully confers the development of pectin stabilized rutin nanoparticles using anti-solvent nanoprecipitation-ultrasonication method. The outcomes of physicochemical characterization depicted enhanced dissolution and environmental stability of optimized R-NPs. Pharmacokinetic study demonstrated that R-NPs could significantly improve bioavailability and enhance rutin uptake efficiency of lungs. Besides, oral administration of R-NPs showed better efficacy in suppressing airway hyerresponsiveness with remarkably higher protection and shorter recovery time by suppressing oxidative stress in lungs and BAL fluid respectively.
, C , Claims:We Claim:
1. Rutin loaded mucoadhesive nanoparticles comprising rutin, one or more stabilizer and pectin.
2. The rutin loaded mucoadhesive nanoparticles as claimed in claim 1, wherein rutin present in an amount ranging from 7.41% to 11.77%, one or more stabilizers present in an amount ranging from 88.24% to 92.59% and pectin present in an amount ranging from 1% to 3%.
3. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein the stabilizers are a mixture of non-ionic surfactants.
4. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein the non-ionic surfactant is selected from Kolliphor.
5. The rutin loaded mucoadhesive nanoparticles as clamed in claim 4, wherein the Kolliphor is selected from Kolliphor® RH 40, Kolliphor® 188 (Pluronic F-68) and Kolliphor® P407 (Pluronic F-127).
6. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein the particle size of nanoparticles ranges from 100 nm to 500 nm.
7. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein the particles have a Polydispersibility index from 0.1 to 1.
8. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein the particles have a zeta potential from -30 mV to -20 mV.
9. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein the particles have rutin content of 96% to 99.82%.
10. The rutin loaded mucoadhesive nanoparticles as clamed in claim 1, wherein mucin binding efficiency of the nanoparticle is in the range of 40% to 80%.
11. A process of preparing rutin loaded mucoadhesive nanoparticles as claimed in claim 1, said process comprising
(a) preparing an aqueous solution of one or more stabilizer,
(b) adding methanolic solution of rutin to the aqueous solution of stabilizer to obtain a rutin- stabilizer mixture,
(c) sonicating the rutin- stabilizer mixture at amplitude between 25 W - 45 W for a period of 2 min to 10 min at a temperature range between 2°C to10 °C to obtain rutin nanosuspension,
(d) adding pectin to the rutin nanosuspension and stirring at 500 rpm to 1000 rpm for a period of 2 h to 8 h followed by probe sonication and lyphilization.
12. A rutin loaded mucoadhesive nanoparticles composition for management of asthma, said composition comprising rutin loaded mucoadhesive nanoparticles of claim 1 with or without pharmaceutically acceptable surfactant and/or excipient.