Description:Technical Field of the Invention
The present invention relates to a phytosomal formulation. This invention is more specifically related to Phytosomes of p-coumaric acid with enhanced drug release activity.
Background of the Invention
Oxidative damage in human body is caused by an excess of reactive oxygen species. The treatment procedures include carriers like phytosomes are promising systems to optimize absorption of medicinal extracts [Direito, R., Reis, C., Roque, L., Gonçalves, M., Sanches-Silva, A., Gaspar, M.M., Pinto, R., Rocha, J., Sepodes, B., Rosário Bronze, M. and Eduardo Figueira, M., 2019. Phytosomes with persimmon (Diospyros kaki l.) extract: Preparation and preliminary demonstration of in vivo tolerability. Pharmaceutics, 11(6), p.296; Biswas, S., Mukherjee, P.K., Kar, A., Bannerjee, S., Jana, S.N., Haldar, P.K. and Sharma, N., 2021. Enhanced permeability and photoprotective potential of optimized p-Coumaric acid-phospholipid complex loaded gel against UVA mediated oxidative stress. Journal of Photochemistry and Photobiology B: Biology, 221, p.112246; Direito, R., Reis, C., Roque, L., Gonçalves, M., Sanches-Silva, A., Gaspar, M.M., Pinto, R., Rocha, J., Sepodes, B., Bronze, M.R. and Figueira, M.E., 2019. Phytosomes with persimmon (Diospyros kaki l.) extract. Pharmaceutics, 11(6); Indian Patent
Application Number 202111002294]. Although there are several works available of phytosomes containing bioactive plant extracts still there is a need to develop enhanced drug delivery phytosomes.
Objects of the Invention
The main object of the present invention is to provide a phytosomal formulation.
Yet another object of the present invention is to provide phytosomes of p-Coumaric acid.
Still another object of the present invention is to provide a process for the preparation of phytosomes of p-Coumaric acid.
Another object of the present invention is to provide phytosomes of p-Coumaric
acid with enhanced drug release activity.
Summary of the Invention

The present invention provides a phytosomal formulation. In an embodiment of the present invention, the phytosomal formulation with enhanced drug release activity, comprising of p-Coumaric Acid; pharmaceutically acceptable excipients; Wherein pharmaceutically acceptable excipients are a combination of Phosphatidylcholine dissolved in dichloromethane; and extract dissolved in methanol; wherein the ratio of Phosphatidylcholine and extract ranges from 0.5:1 to 3:1 by weight. The present invention provides a process for the preparation of phytosomal formulation. In an embodiment of the present invention, the process for the preparation of phytosomal formulation with enhanced drug release activity, comprising of dissolving extract in methanol; separately dissolving Phosphatidylcholine in dichloromethane; mixing extract solution with the Phosphatidylcholine solution and pouring in a 200 ml round bottomed flask; obtaining concentrate by refluxing the mixture for different durations of 1-4 hours and at various temperature 45-65°C; evaporating the resulting clear solution and drying under vacuum at 40°C; obtaining the residues and storing in desiccator. The phytosomal formulation with enhanced drug release activity as claimed in claim 1, wherein the aqueous solubility of phytosomal formulation 79.48 ± 0.63µg/mL n-Octanol solubility of phytosomal formulation 543.34 ±0.63µg/mL. The phytosomal formulation with enhanced drug release activity, wherein the mean particle size of phytosomal formulation 465.3± 20.0 nm; 20 % Intensity 92.2; Width 367.6 r.nm and polydispersity index of phytosomal formulation 0.437±0. The phytosomal formulation with enhanced drug release activity, wherein the rate of dissolution 89.87 at the end of 12 hours. The phytosomal formulation with enhanced drug release activity, wherein the formulation is useful for faster release of drug.
Brief Description of drawings
In the drawings accompanying the specification, Figure 1 shows Particle size analysis of Coumaric Acid
In the drawings accompanying the specification, Figure 2 shows Particle size analysis of Phytosomes of p-Coumaric acid.
In the drawings accompanying the specification, Figure 3 shows Zeta Potential of Phytosomes of p-Coumaric Acid and p-Coumaric acid.

In the drawings accompanying the specification, Figure 4 shows XRD spectra of XRD spectra of PC.
In the drawings accompanying the specification, Figure 5 shows XRD spectra of XRD spectra of PM (p-Coumaric Acid + PC).
In the drawings accompanying the specification, Figure 6 shows XRD spectra of Phytosomes of p-Coumaric acid.
In the drawings accompanying the specification, Figure 7 shows Differential scanning calorimetry of p-Coumaric Acid.
In the drawings accompanying the specification, Figure 8 shows Differential scanning calorimetry of PC.
In the drawings accompanying the specification, Figure 9 shows Differential scanning calorimetry of PM.
In the drawings accompanying the specification, Figure 10 shows Differential scanning calorimetry of Phytosomes of p-Coumaric acid.
In the drawings accompanying the specification, Figure 11 shows FTIR Spectra of p- Coumaric Acid and FTIR Spectra of PC.
In the drawings accompanying the specification, Figure 12 shows FTIR Spectra of Physical Mixture (PM).
In the drawings accompanying the specification, Figure 13 shows FTIR spectra of Phytosomes of p-Coumaric acid.
In the drawings accompanying the specification, Figure 14 shows SEM of Phytosomes of p-Coumaric acid.
In the drawings accompanying the specification, Figure 15 shows In vitro dissolution study of p-Coumaric acid & Phytosomes of p-Coumaric acid.
Detailed description of the Invention
The present invention provides a Phytosomal formulation. The Phytosomal formulation with enhanced drug release activity, comprising of p-Coumaric Acid; pharmaceutically acceptable excipients; Wherein pharmaceutically acceptable excipients are a combination of Phosphatidylcholine dissolved in dichloromethane; and extract dissolved in methanol; wherein the ratio of Phosphatidylcholine and extract ranges from 0.5:1 to 3:1 by weight. The present invention provides a process for the preparation of phytosomal formulation. The process for the preparation of phytosomal formulation with enhanced drug release activity, comprising of dissolving extract in methanol; separately dissolving Phosphatidylcholine in dichloromethane; mixing extract solution with the Phosphatidylcholine solution and pouring in a 200 ml round bottomed flask; obtaining concentrate by refluxing the mixture for different durations of 1-4 hours and at various temperature 45-65°C; evaporating the resulting clear solution and drying under vacuum at 40°C; obtaining the residues and storing in desiccator. The phytosomal formulation with enhanced drug release activity as claimed in claim 1, wherein the aqueous solubility of phytosomal formulation 79.48 ± 0.63µg/mL n-Octanol solubility of phytosomal formulation 543.34 ±0.63µg/mL. The phytosomal formulation with enhanced drug release activity, wherein the mean particle size of phytosomal formulation 465.3± 20.0 nm; % Intensity 92.2; Width 367.6 r.nm and polydispersity index of phytosomal formulation 0.437±0. The phytosomal formulation with enhanced drug release activity, wherein the rate of dissolution 89.87 at the end of 12 hours. The phytosomal formulation with enhanced drug release activity, wherein the formulation is useful for faster release of drug at the end of dissolution period. MATERIALS AND METHODS
Preparation of Phytosomes
The phytosomes were prepared by refluxing followed by solvent evaporation technique. Phytosomes were prepared in different ratios, i.e., 0.5:1, 0.75:1, 1:1, 2.5:1 and 3:1 of Phosphatidylcholine to Extracts. Phosphatidylcholine and Extracts were dissolved in dichloromethane and Methanol respectively. Both the solutions were mixed and pour in a 200 ml round bottomed flask. The mixture was refluxed for different duration i.e., 1-4 hr and at various temperature 45-65°C. The resulting clear solution was evaporated, dried under vacuum (40°C); the residues were gathered and stored in desiccators for further use.
Physico-chemical characterization of prepared phytosomes Apparent Solubility
The apparent solubility of extracts and phytosomes was determined by adding excess of extract and phytosome to 5ml of water or n-octanol in sealed glass containers at room temperature (25-30ºC). The liquids were agitated for 24 hours then centrifuged for 20 min at 1,000 rpm to remove excess of extract. The supernatant was filtered through a membrane filter (0.45 m) then 1 ml filtrate was diluted with 9 ml of distilled water or n- octanol and these samples were measured spectrometrically at respective maximum absorbance of the extracts using UV spectrophotometer.
Apparent Solubility
The apparent solubility of the pure Coumaric acid, the physical mixture of coumaric acid and PC, and the prepared Coumaric acid-PC complex (PCP) are shown in Table 1. It was observed that the pure Coumaric acid had poor aqueous solubility (4.18 µg/mL), and a relatively higher solubility in n- Octanol (289.65 µg/mL). The prepared Coumaric acid showed a significant increase in the aqueous solubility.
This increase in the solubility of the prepared complex may be explained by reduced molecular crystallinity of the drug and amphiphilic nature of the phytosome.
Table 1: Apparent solubility study of p-Coumaric acid, PM and Phytosomes of p-coumaric acid in water and n-octanol

SN
Sample Aqueous Solubility
(µg/mL)a n-Octanol solubility
(µg/mL)a
1 p-Coumaric acid 4.18 ± 0.32 289.65 ± 0.54
2 PM 7.12 ± 0.45 364.21± 0.35
3 PCP 79.48 ± 0.63 543.34 ±0.63
Entrapment efficiency
Entrapment efficiency (EE) was measured using UV visible spectrophotometer (UV- 3000+, Lab India). Weighed quantities of Phytosomes equivalent to 10 mg of extracts were added to 50 ml methanol in a 100 ml beaker. The contents were stirred on a magnetic stirrer for 4 hours and then allowed to stand for one hour. Clear liquid was decanted and centrifuged at 5000 rpm for 15 minutes. After centrifugation the supernatant was filtered through 0.45 µm Whatman filter paper and after suitable dilution absorbance was measured in UV; the concentration of drug was measured. All measurements were performed in triplicate.
Drug Content
Drug content evaluation Phytosomes (10mg) was taken and dissolved in small quantity of methanol, shaken vigorously for 2 hrs after adjusting volume to 100ml using methanol there after the resultant solution was filtered and 1ml of sample was withdrawn and diluted to 10 ml in a volumetric flask and the absorbance was recorded by UV spectrophotometer.
Particle size distribution
The particle size analysis of the prepared samples was carried out using photon correlation spectroscopy, with dynamic light scattering on Zetasizer nano (Model: Nano series, S90 Zeta sizer, Malvern). The phytosomes were dispersed in isopropyl alcohol by stirring on a magnetic stirrer for 10 minutes. The dispersion was analysed in size analyser.
The mean particle size of p-Coumaric acid and Phytosomes of p-Coumaric acid were showed in figure. The mean particle size of Coumaric acid and Phytosomes of p-Coumaric acid was found to be 780.8± 20.0 nm and 465.3± 20.0 nm respectively. The polydispersity index of Coumaric acid and Phytosomes of p- coumaric acid was found to be 0.505±0.03 and 0.437±0.21respectively.
Figure 1 shows Particle size analysis of Coumaric Acid.
Figure 2 shows Particle size analysis of Phytosomes of p-Coumaric acid.
Figure 3 shows Zeta Potential of Phytosomes of p-Coumaric Acid and p-Coumaric acid.
Table 2 shows Particle size analysis of Coumaric acid and Phytosomes of p-
Coumaric acid.
SN Material Avg Size (r.nm) % Intensity Width PD
1 p-Coumaric acid 780.8 85.2 710.1 0.505
2 Phytosomes of
p-Coumaric acid 465.3 92.2 367.6 0.437
X-Ray diffraction (XRD) study
The polymorphic states of samples were evaluated using X ray diffractometer (D- 8 Advance Bruker, USA). The operating conditions were: voltage 45 kV; current
0.8 mA and scanning speed 1/min. The samples were scanned with the diffraction angle over a range of 5–60° (2? angle), using the Cu-Anode X-ray tube and scintillation detector.
The powder x-ray diffraction (PXRD) patterns of (A) p-Coumaric Acid, (B) PC,
(C) PCP and (D) PM are shown in figures. XRD of p-Coumaric Acid shows intense crystalline peaks in figure indicating higher crystallinity of drug than phyto-
phospholipid complex PCP. Phytosomes of p-Coumaric acid exhibited least number of crystalline peaks, and it was interpreted that Phytosomes of p-Coumaric acid would be more amorphous than p-Coumaric Acid. The broad peak of PC masked the sharp peaks of phytosomes of p-Coumaric acid. The XRD pattern of phytosomes of p-Coumaric acid exhibited the broad peak signifying amorphous
character. The disappearance of the peaks was associated with the formation of drug phospholipid complex.
Figure 4 shows XRD spectra of XRD spectra of PC.
Figure 5 shows XRD spectra of XRD spectra of PM (p-Coumaric Acid + PC). Figure 6 shows XRD spectra of Phytosomes of p-Coumaric acid.
Differential scanning calorimetry (DSC)
The thermal analyses of samples were carried out using differential scanning calorimeter Perkin Elmer (USA) (Model JADE DSC). The analysis was performed under a purge of dry nitrogen gas with high-purity medium was used to calibrate the heat flow and the heat capacity of the instrument. The samples (~5 mg) were held in closed metal pans. Each sample was subjected to a single heating cycle from 0°C to 300°C at a heating rate of 10°C/min.
Differential scanning calorimetry of p-Coumaric Acid, PC, the physical mixture of p-Coumaric Acid with PC (PM), and the prepared Phytosomes of p-Coumaric acid was shown in figures. The p-Coumaric Acid revealed broad endothermic peaks at 134°C. PC shows endothermic peak at 131.2ºC. In physical mixture peak observe at 128.8ºC. The DSC thermograms of Phytosomes of p-Coumaric acid showed endothermic peaks at 125.1°C. The original peaks of P-Coumaric Acid and PC disappeared from thermogram of complex, thus indicating the successful formation of complex.
Figure 7 shows Differential scanning calorimetry of p-Coumaric Acid .
Figure 8 shows Differential scanning calorimetry of PC
Figure 9 shows Differential scanning calorimetry of PM
Figure 10 shows Differential scanning calorimetry of Phytosomes of p-Coumaric acid.
Fourier Transform Infrared spectroscopy (FTIR) Study
Fourier transform infrared spectrophotometer (Model: IR Prestige-21, Shimadzu, Japan) was employed to study the interaction between extracts and PC and to establish the structure and chemical stability of prepared phytosomes. The IR spectra of samples were obtained by the potassium bromide (KBr) method. KBr pellets were prepared by gently mixing small quantity of sample (1 mg) with 100 mg KBr. A small quantity of sample was placed just below the probe on to which the probe was tightly fixed and scanned in the wave number region 4000-500 cm-. The obtained IR spectra were interpreted for functional groups at their respective wave number (cm-1). Fourier transform infrared spectroscopy (FTIR) analyses of the P-Coumaric Acid, PC, the physical mixture of p-Coumaric Acid with PC (PM), and the prepared Phytosomes of p-Coumaric acid were studied interaction between p-Coumaric Acid and PC. The FTIR spectrum of p-Coumaric Acid showed broad peak at 3500 cm-1 representing the aliphatic alcoholic (-OH) group, 2900 cm-1 (CH stretching), 1650 cm-1 (C=O stretching). FTIR spectrum of PC (Figure) revealed the characteristic absorption at 2921 and 2850 cm-1 (CH stretching), 1775 cm-1 (C=O stretching), 1235 cm-1 (P=O stretching), 1081cm-1 (P–O–C stretching) and 975 cm-1 (C-C-N stretching). The FTIR spectrum of the prepared Phytosomes of p-Coumaric acid (Figure 11) is quite different from that of p-Coumaric Acid and PC.
Figure 11 shows FTIR Spectra of p-Coumaric Acid and FTIR Spectra of PC
Table 3: Interpretation of IR spectra for p-Coumaric Acid

SN Stretching Peak (cm-1)
1 O-H stretching 3500
2 Aro C-H stretching 2900
3 Ali C-H stretching 2850
4 C=O stretching 1650
5 C-O-C stretching 1332
6 P=O stretching 1243
Table 4: Interpretation of IR spectra for Phosphatidylcholine

SN Stretching Peak (cm-1)
1 O-H stretching 3280
2 Ali C-H stretching 2927
3 C=O stretching 1590
4 P-O-C stretching 1102
5 P=O stretching 1235
6 C-C-N stretching 975
Figure 12 shows FTIR Spectra of Physical Mixture (PM)
Table 5: Interpretation of IR spectra of physical mixture (PM)

SN Stretching Peak (cm-1)
1 O-H stretching 3244
2 Aro C-H stretching 2922
3 Ali C-H stretching 2851
4 C=O stretching 1630
5 C-O-C stretching 1330
6 P=O stretching 1250
Figure 13 shows FTIR spectra of Phytosomes of p-Coumaric acid
Table 6: Interpretation of IR spectra for phytosomes of p-Coumaric Aacid
SN Stretching Peak (cm-1)
1 O-H stretching 3345
2 Aro C-H stretching 2976
3 Ali C-H stretching 2882
4 C=O stretching 1643
5 O-H bonding 1100
6 C-O-C stretching 1400
7 P=O stretching 1150
8 C=O carboxyl 1690
6 C-C-N stretching 975
Scanning electron microscopy (SEM)
Samples were coated with gold in a Fine Coat Ion Sputter (S-4800 TYPE II, Hitachi, Japan). Analysis was done on the coated sample by placing a pinch of sample in the Scanning electron microscope and surface morphology was viewed and photographed to observe their particle shape and surface morphology.
SEM photographs of p-Coumaric acid and Phytosomes of p-Coumaric acid are shown crystalline state of P-Coumaric acid. It was visualized in the SEM photograph as numerous crystals. In figure 14 drugs was completely converted in to phyto-phospholipid complex where p-Coumaric acid was physically enwrapped by PC imparting amorphous nature to the complex due to which crystals disappeared.
SEM photographs of p-Coumaric Acid and Phytosomes of p-Coumaric acid are shown crystalline state of p-Coumaric Acid. It was visualized in the SEM photograph as numerous crystals. In figure 14 & 15 drugs was completely converted in to phyto-phospholipid complex where p-Coumaric Acid was physically enwrapped by PC imparting amorphous nature to the complex due to which crystals disappeared.
Figure 14 shows SEM of p-Coumaric Acid
Figure 15 shows SEM of Phytosomes of p-Coumaric acid
Dissolution Study (In-vitro drug release)
The in vitro dissolution profiles of prepared phytosomes were obtained. The dissolution studies were carried out in a Labindia DS 8000, eight station dissolution test apparatus, type II at 100 rpm and 37°C. An accurately weighed amount of phytosome 50 mg was put in to 900 ml of pH 6.8 phosphate buffer. Samples (3 ml each) of dissolution fluid were withdrawn at different time intervals and replaced with an equal volume of fresh medium to maintain sink conditions. Samples were withdrawn and filtered through a 0.45 µm membrane filter, diluted suitably and then analyzed spectrophotometrically at 268 nm to determine drug release from the complex and the drug.
The results of in vitro drug release studies are shown in Table 7. The 12-hours dissolution in the phosphate buffer (pH 6.8) revealed that, the pure p-Coumaric acid showed the slowest rate of dissolution, i.e., at the end of the dissolution period only about 40.32% w/w of p-Coumaric acid was dissolved. The prepared Phytosomes of p-coumaric acid revealed a significantly faster release of Phytosomes of p- coumaric acid at the end of dissolution period. At the end of 12 h, over 89.87% w/w p-Coumaric acid was observed to be released from the Phytosomes of p-Coumaric acid. Figure 15 shows In vitro dissolution study of p-Coumaric acid & Phytosomes of p-Coumaric acid.
Table 7: In vitro dissolution study of p-Coumaric Acid and Phytosomes of p- Coumaric acid
SN Time (Hr) p-Coumaric acid Phytosomes of p-Coumaric acid
01 1 22.08 34.76
02 2 24.44 39.75
03 3 25.32 42.02
04 4 26.41 47.13
05 5 28.13 53.54
06 6 30.17 62.16
07 7 32.18 69.57
08 8 33.12 72.43
09 9 34.23 76.32
10 1 36.43 79.21
11 1 38.56 84.37
12 1 40.32 89.87
, C , Claims:We claim:
1. A phytosomal formulation with enhanced drug release activity, comprising:
a) p-Coumaric Acid;
b) pharmaceutically acceptable excipients;
Wherein pharmaceutically acceptable excipients are a combination of:
a) Phosphatidylcholine dissolved in dichloromethane; and
b) extract dissolved in methanol;
wherein the ratio of Phosphatidylcholine and extract ranges from 0.5:1 to 3:1 by weight.
2. A process for the preparation of phytosomal formulation with enhanced drug release activity, comprising:
a) dissolving extract in methanol;
b) separately dissolving Phosphatidylcholine in dichloromethane;
c) mixing extract solution of step a) with the Phosphatidylcholine solution of step b) and pouring in a 200 ml round bottomed flask;
d) obtaining concentrate by refluxing the mixture for different durations of 1-4 hours and at various temperature 45-65°C;
e) evaporating the resulting clear solution and drying under vacuum at 40°C;
f) obtaining the residues and storing in desiccator.
3. The phytosomal formulation with enhanced drug release activity as claimed in claim 1, wherein the aqueous solubility of phytosomal formulation 79.48 ± 0.63µg/mL n-Octanol solubility of phytosomal formulation 543.34 ±0.63µg/mL.
4. The phytosomal formulation with enhanced drug release activity as claimed in claim 1, wherein the mean particle size of phytosomal formulation 465.3± 20.0nm; % Intensity 92.2; Width 367.6 r.nm and polydispersity index of phytosomal formulation 0.437±0.
5. The phytosomal formulation with enhanced drug release activity as claimed in claim 1, wherein the rate of dissolution 89.87 at the end of 12 hours.
6. The phytosomal formulation with enhanced drug release activity as claimed in
claim 1, wherein the formulation is useful for faster release of drug at the end of dissolution period.
Dated this 7th day of October, 2023

To be signed digitally by (Sanchita Tewari) Agent for the Applicant
Patent Agent (IN/PA 2711)