EVALUATION OF ANTI-INFLAMMATORY POTENTIALS OF PONGAMIA PINNATA LOADED PHYTOSOMES

Abstract

Abstract The present study emphasis on the phytosomes nanoformulation for the management of inflammatory conditions. A phytosomal nanoformulation comprising of Flavonoid rich Pongamia pinnata (1.5% w/v), wherein the concentration of Flavonoid rich Pongamia pinnata is 1.5 mglml in the phytosomal nanoformulation; Soya lecithin (0.5 % w/v) and Cholesterol (0.25 % w/v). The phytosomal formulation of Pongamia pinnata demonstrates significantly enhanced anti-infliV:nf!Iatory potential compared to the plain extract and the pure form of the marketed drug diclofenac. This stable nanoformulation of phytosomes effectively reveals potent anti-inflammatory act'ivity for managing inflammation.

Specification

EVALUATION OF ANTI-INFLAMMATORY POTENTIALS OF PONGAMIA
PINNATA LOADED PHYTOSOMES
FIELD OF THE INVENTION
The present invention pertains to the development of innovative drug delivery system
particularly focusing on the use of phytosomes for enhancing the anti-inflammatory potential
of natural compounds derived from Pongamia pinna/a by effectively targeting inflammation
at the cellular level. The formulation is evaluated for its efficacy using in vitro HRBC- (Human
Red Blood Cell) method. The invention could have significant implications for treating various
inflammatory disorders, offering a natural and efficient alternative to synthetic antiinflammatory
agents, thereby promoting a holistic approach to health and wellness.
BACKGROUND OF THE INVENTION
Inflammation, a fundamental immune response, plays a pivotal role in the. body's defense
against harmful stimllli, such as pathogens, injuries, or irritants. It's a complex biological
process arranged by various cells and molecules, aiming to eliminate the inciting agent and
initiate tissue repair. While acute inflammation is typically beneficial, chronic inflammation
can lead to detrim~ntal effects, contributing to various diseases like rheumatoid arthritis,
cardiovascular disorders and even certain cancers.
Addressing this issue, research into anti-inflammatory activity has garnered significant
attention in the medical field. Anti-inflammatory agents, whether natural or synthetic, work by
modulating the body's immune response to control inflammation. Various natural compounds
have demonstrated potent anti-inflammatory properties. Moreover, exploring novel
compounds and therapeutic strategies continues to drive advancements in the field, holding
promise for improved management of inflammatory disorders in the future.
Phytosomes offer significant advantages over conventional herbal extracts, primarily due to
their enhanced bioavailability, which improves absorption in the gastrointestinal tract. This
increased bioavailability allows a higher proportion of the active phytoconstituent to reach
systemic circulation, maximizing therapeutic effects. Additionally, phytosomes have diverse
applications across pharmaceutical, nutraceutical and cosmetic industries. They can be
formulated into various dosage forms, including tablets, capsules, creams, and gels, facilitating
convenient administration and targeted delivery of bioactive compounds to specific tissues or
organs. Phytosome technology also supports the development of combination products, where
multiple phytoconstituents with complementary mechanisms of action are co-encapsulated to
produce synergistic effects, thereby improving therapeutic efficacy.
The present study aims to formulate, optimize, and evaluate a novel phytosome loaded with
flavonoid-rich Pongamia pin nata extract for the management of inflammatory conditions. This
innovation capitalizes on the advantages of phytosome technology to enhance drug delivery
and therapeutic efficacy, ultimately improving outcomes for patients with inflammation-related
disorders.
Objectives of study
)> To prepare flavonoid rich Pongamia pi1mata extract.
}> To formulate and evaluate phytosomes loaded with flavonoid fraction of Pongamia
pinna/a.
}> To evaluate anti-inflammatory potential of flavonoid -fraction of Pongamia pinna/a
loaded ph}'tosomes.
Statement of the Invention
The _present study focuses on developing r-ihytosomes loaded with flavonoid_-rich Pongamia
pinnata extract to manage inflammation. After .subjecting the plant for extraction and
fractionation the yield of the extract obtained was 8.55% w/w. The phytosomes loaded with
Flavonoidal rich Pongamia pinna/a extract were successfully formulated using the thin-film
hydration method. Optimization of the nanoformulation was achieved using a three-factor, two'
level (32) design, resulting in nine batches, as outlined in Table I. These batches were prepared
with phospholipids-specifically, cholesterol and soya lecithin-and were assessed for particle
size, zeta potential, and PDI, with results presented in Table 2.
Response on Particle size
The particle size of Pongamia pinnata loaded phytosomes ranged from 141.8 to 360.lnm,
indicating the uniform size as shown in Table 2. The final particle size (PS) analysis equation,
expressed in terms of coded factors, emphasizes the concentrations of soya lecithin (XI) and
cholesterol (X2), with data analysed to fit a full linear model incorporating interaction terms to
correlate the studied responses with these variables. As depicted in 3D plot , it is indicated that
higher the levels of both independent variables had the positive impact on the particle size,
whereas reducing the level exhibits the negative effect.
PS = +237.04 +71.15 X1+24.50X2 Equation 1
Response on Zeta Potential
Zeta potential was analyzed to assess the surface characteristics essential for predicting the
colloidal stability of the dispersion. The analysis showed that the phytosonies had a negative
surface charge, with average values ranging from -25.1 to -76.1 mY, as presented in the table
above. The 30 plots and the generated polynomial equation confirm that the independent
variables negatively impact zeta potential; As the concentrations of soya lecithin and
cholesterol increase, a corresponding reduction in zeta··ptJtential is observed, and conversely,
lower concentrations lead to an elevation in zeta potential. The equation below represents the
correlation between these variables.
The model's relevance was vahdated through F and P values apphed to the results, as detailed
in Table 3: The effects of soya lecithin and cholesterol on particle size and zeta potential ar~
illustrated by the response surface and contour plots in Figures 1 and 2.
Formulation Optimization
The Design· expert® software provided a range of suggestions that optimally satisfied the set
constraints of the selected formula (Xt: SL: 50% w/v and Xz: CH: 25 w/v %). Table 4 displays
predicted value for the response variables of the optimized Phytosomes. Figure 3 displays the
Overlay plot for the optimized batch.
Morphological analysis by Scanning electron microscopy (SEM) and transmission
electron microscopy (TEM)
The morphology of the Phytosomes formulations is shown in the SEM and TEM micrographs
in Figure 5 and 6. The phytosomes were observed to be spherical, with an average particle size
of 141.8 nm.
Stability analysis
The PS and ZP of the optimized phytosomes was studied at two different temperatures, (25 ±
2°C) and ( 4 ± 2°C), for the stability study and stored for 3 months at room temperature (25 ±
2°C) as well as refrigerated conditions ( 4 ± 2°C).
Anti-inflammatory Studies
The flavonoid-rich Pongamia pinna/a-loaded phytosomes demonstrated promising in vitro
anti-inflammatory activity, achieving 80.96±0.36% inhibition of hemolysis at I mg/mL,
compared to the extract's 113.997±0.82%. Diclofenac, a standard anti-inflammatory drug,
exhibited a maximum activity of 58.1988±0.51% at the same concentration. Initially,
phytosomes showed a higher percentage of inhibition at 250 mg/mL compared to the extract,
with a more significant increase in activity observed in the phytosomes. The HRBC membrane
stabilization method indicated that the stability of the red blood cell membrane, similar to
lysosomal membranes, is crucial for preventing inflammation and tissue damage by inhibiting
the release of inflammatory enzymes. This study revealed that hemolysis of RBCs was
influenced by the hypotonicity ofhyposaline, which causes cell membrane lysis and the release
of cellular fluids and electrolytes. Both the flavonoid-rich Pongamia pinnata -loaded
phytosomes and the extract effectively stabilized the RBC membrane, preventing the release
of lytic enzymes and inflammatory mediators. The phytosomes exhibited anti-inflammatory
activity comparable to that of diclofenac, while previous studies have only reported the antiinflammatory
effects of the Pongamia pinnata extract using the protein denaturation assay.
Table 6: Results of anti-inflammatory activity for Pongamia pinnata extract, Pongamia
pinnata loaded phytosomes and Pure Diclofenac drug.
Brief description of figures
Figure 1: Response surface plot showing the effect of concentration of Soya lecithin and
Cholesterol on particle size.
Figure 2: Response surface plot showing the effect of concentration of Soya lecithin and
·cholesterol on zeta potential.
Figure 3: Overlay plot showing optimize box-hehnken experimental design conditions· as a
flag within the design space.
Figure 4: Peak report from zetasizer showing particle size of 141.8 nm (A) and zeta potential
of -25.1 mV (B) for the optimized phytosome.
Figure 5: Scanning electron microscopy of optimized Flavonoid rich Pongamia pinnata
phytosomes.
Figure 6: Transmission electron microscopy of optimized Flavonoid rich Pongamia pin nata
phytosomes.
Figure 7: In-vitro anti-inflammatory activity of Flavonoid rich Pongantia p_innata phytosomes,
Pongamia pinnata extract and pure Diclofenac drug.
Materials required:
I. Flavonoid rich Pongamia pinna/a extract
2. Soua lecithin
3. Cholesterol
4 . Methanol
5. Diclofenac
6. Chloroform
7 . Pet ether
8. Ethanol
Equipment and apparatus:
I. Electronic balance
2. UV spectroscopy
3. Fourier transform infrared spectroscopy (FTIR)
4. Differential scanning calorimetry (DSC)
5. Ultra probe sonicator
6. Homogenizer
7. Rota Evaporator
8. Milli Q water system
9. Magnetic stirrer
I 0. Particle Size analyser
II. Stability Chamber
Design methodology:
Extraction and fractionation of plant material
Extract was prepared by using maceration and Soxhlet technique utilizing ethanol as a solvent,
further the extract was subjected for roiary evaporation to obtain the crude extract. Further the
crude extract was subjected for fractionation method as described by Cos et a/. with a few
minor adjustmentsto get the flavonoid-rich fraction.
Phytosomes formulation
Phytosomes were prepared by using different ratios of soya lecithin and cholesterol by thin
film hydration method. The drug was dissolved in methanol, while the soya_ lecithin and
cholesterol were diss·olved in dich!oromethane. The above mixture was placed in a round
bottom flask and evaporated througli'a rotary evaporator at 40oc and 180 rp~ until all of the
solvent had evaporated and a thin layer had formed on the RBF. For up to 24 hours, the flask
was stored in refrigerator. Mixture of ethanol and water (1:1) was used to hydrate the film for
I hour at 40oc in a rotary evaporator. The particle size was decreased by sonication for 30
minutes after the producing the phytosomes suspension. Table I summarizes the different ratios
of formulation components.
Design of Experiment
A three-level factorial design was implemented using Design-expert® softwarewhich required
an experiment to be carried out at all possible combinations of the three levels of each of the
factors considered. The independent variables used were the amount of Cholesterol (XI) and
the amount of soya lecithin (X2). The independent variables were screened using a multilevel
factorial design (32
), and nine formulations of Pongamia pinna/a phytosomes were obtained.
All the formulations were prepared using the thin film hydration method and then evaluated
particle size (Yl) and zeta potential (Y2), to determine the optimized formulation.
Particle Size Distribution, Polydispersity Index and Zeta potential.
Particle size distribution and zeta potential of phytosomes formulas were measured using
Dynamic light scattering (DLS) particle size analyser with a computerized system (Malvern,
Zetasizer). Zeta potential and polydispersity index measurements were also performed.
Scanning eiectron microscopy (SEM) analysis
"Scanning electron microscopy has been used to determine particle size distribution and surface
morphology of the complexes. Samples were studied using (FEI, Quanta 200-Netherland)
Scruining microscope (Japan). Approximately 5 11L of the sample was transformed to a cover
slip, which in turn was mounted on a specimen tab: The samples were allowed to dry at room
temperature. Then the particle size of the formulation was viewed and photographed using
Scanning Electron Microscope. The particles were coated with platinum by using vacuum
evaporator and thus, the coated samples were viewed and photographed. Digital image~ of
phytosomes were taken by random scanning of the stub at different magnifications.
Transmission elect~n microscopy (TEM) analysis
Vesicles morphology ofphyto.somes was observed visually with a (FEI, Tecnail2-Netherland)
Transmission Electron Microscopy (TEMj\ A total volume of5 ml sample was dispersed before
the sample was analysed. The mixture was then stirred and a drop of the sample was p'laced on
the specimen. The 400 mesh grid was placed over the specimens and allowed to stand for I
minute.· Residual droplets on the grid were cleaned using a filter paper. A drop of 5 uranyl
acetate was dropped over the grid and the rest of the excess solution was removed using a filter
paper. The grid was left for 30 minutes and the films were then viewed on a transmission
electron microscope and photographed.
Stability Studies:
To determine the stability of the optimized phytosomes, short term stability studies were
conducted in compliance with ICH GCP guidelines. The prepared formulation was stored in
glass vials within a humidity controlled oven maintained at a temperature of 25±2°C and
relative humidity of 65±5%. Additionally, it was refrigerated at 4±20°C with relative humidity
of 65±5%. At regular intervals 0, 30, and 90 days, a sample was extracted for analysis.
/11 vitro Anti-inflammatory activity
H..R. BC method was used for the estimation of in vitro anti-inflammatory activity. Blood was
collected from healthy volunteers and was mixed with equal volume of sterilized Alsevers
solution. This blood solution was centrifuged at 3000 rpm and the packed cells were separated.·
The packed cells were washed with isosaline solution and a I 0% v/v suspension was made with
isosaline. This HRBC suspension was used for the estimation of anti-inflammatory property.
Different concentrations of extract, reference sample and control were separately mixed with
lmL of phosphate buffer, 2 mL ofhyposaline and 0.5 mL ofHRBC suspension. All the assay
mixtures were incubated at 37°C for 30 minutes and centrifuged at 3000 rpm. The supernatant
liquid was decanted and the hemoglobin content was estimated by a spectrophotometer at 560
.nin. The percentage hemolysis was estimated by assuming the hemolysis produced in the
control as I 00%.
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ethnopharmacology. 2001 ;78(2-3): 151-7 Summary
This study highlights the significant potential of Flavonoid rich Pongamia pinna/a-loaded
phytosomes in enhancing the anti-inflammatory effects of the plant extract The optimized
formulation, designed using a 32 factorial approach, includes Pongamia pin nata, soya lecithin,
and cholesterol, resulting in improved particle size and zeta potentiaL SEM and TEM analyses
confirmed the morphology of the phytosomes dispersion. The formulation demonstrated
sustained drug release over several hours. Short-term stability and anti-inflammatory studies
indicated that Pongamia pinna/a phytosomes exhibit stronger anti-inflammatory activity,
suggesting their efficacy at lower doses. In conclusion, the incorporation of Pongamia pinna/a
into a phytosomes formulation represents a promising advancement in herbal medicine,
offering a novel strategy to enhance the therapeutic potential of natural products as a viable
alternative or complement to conventional anti-inflammatory drugs. Further clinical research
is essential to validate its safety and effectiveness in human healthcare .

WE CLAIM,
I. A phytosomal nanoformulation comprising of Flavonoid rich Pongamia pinnata (1.5
% w/v), wherein the concentration of Flavonoid rich Pongamia pinnata is 1.5 mg/ml in
the phytosomal nanoformulation; Soya lecithin (0.5 % w/v) and Cholesterol (0.25 %
w/v).
2. A phytosomal nanoformulation is claimed in Claim I, wherein the phytosomal
formulation is highly potential for the anti-inflammatory activity compared to plain
extract of Pongamia pinnata and marketed diclofenac pure drug.

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