Formulation and Evaluation of a Phytosomal Drug Delivery System of Cyperus Rotundus in Ointment Form

Herbal remedies have attracted a lot of interest because of their enormous therapeutic potential and minimal adverse effects.  Nagarmotha (Cyperus rotundus) root is one such medicinal herb long renowned for its analgesic, antioxidant, and anti-inflammatory properties. [1] Arthritis, characterized by joint inflammation, stiffness, edema, and restricted movement, principally includes osteoarthritis and rheumatoid arthritis.  Although NSAIDs and corticosteroids relief pain, their long-term use leads to detrimental effects, driving the quest for safer, natural alternatives. Although terpenoids, flavonoids, and essential oils are present in Nagarmotha, its hydrophilic nature prevents it from penetrating deeper skin layers when applied topically [2,3].

Phytosome technology provides a solution by creating lipid-compatible complexes of phospholipids with water-soluble phytochemicals, enhancing solubility, permeability, and overall bioavailability.  Due to their high molecular size or poor lipid solubility, several herbal ingredients show poor absorption [4,5].  Phytosomes promote membrane permeability through phosphatidylcholine-based amphiphilic complexes, allowing effective absorption across lipid-rich biological membranes [6].  The scientific justification for creating Nagarmotha Phytosomes is amply supported by numerous research studies that show better anti-inflammatory and anti-arthritic results using Phytosomal formulations as opposed to free extracts [7,8].

An ointment dosage form was chosen for targeted and localized relief of arthritic inflammation.  Longer contact times, improved skin penetration, fewer systemic side effects, deeper diffusion into inflammatory joints, and a soothing protection base that lessens pain and stiffness are some of the medicinal benefits of Nagarmotha root extract.[9]

Additionally, during application, massage increases local blood flow, which facilitates better drug absorption.  Every excipient was selected to ensure sustained skin retention and maintain compatibility with the Phytosomal complex.  Nagarmotha Phytosomes were prepared with different extract-to-phospholipid ratios and evaluated for particle size, zeta potential, entrapment efficiency, and drug content in order to identify the optimal formulation. [10]

MATERIALS AND METHOD

Materials- Nagarmotha roots extract, Soy lecithin.  Dichloromethane, N-hexane, Phosphate buffer, Tween 80, Span 80, White soft paraffin, Liquid paraffin, Cetyl alcohol, Polyethylene glycol (PEG 400), Methyl and Propyl paraben.

Methods

A rotary evaporation technique was used to create the Nagarmotha Phytosome complex.

When phospholipids (soy lecithin) interact with the water-soluble bioactive components of Nagarmotha root extract, such as flavonoids and terpenoids, a lipid-compatible molecular complex known as the "Nagarmotha Phytosome complex" is formed.  The goal of this combination is to improve the hydrophilic Nagarmotha extract’s topical bioavailability by making it more soluble and permeable through lipid-rich biological membranes.

Extract and phospholipid ratios ranging from 1:1 to 1:6 were prepared using the rotary evaporation method. Both were dissolved in 30 mL of dichloromethane and refluxed for two hours at 50°C. After solvent evaporation a thin film was formed. 20ml n-hexane was added to precipitate the Phytosome. The precipitate was collected dispersed with Tween 80 and phosphate buffer pH 7.4 and stored at room temperature. [11]

Nagarmotha phytosomal ointment preparation (Oil in water)-

Cetyl alcohol, span 80, liquid paraffin, and white soft paraffin were melted at 700C to create the oily phase. Methyl paraben, propyl paraben, PEG 400, and the Nagarmotha Phytosomes were separately dissolved in filtered water at 700C to create the aqueous phase. To create a consistent, stable ointment, the aqueous phase was then gradually added to the oily phase while being constantly stirred.[12]

Formulation tables-

Table no. 01 Formulation table of Nagarmotha phytosomes

Table no. 02 Formulation table of phytosomal ointment

Evaluation Parameters of Nagarmotha Phytosomes-

1. Particle size-

The mean particle size and polydispersity index (PDI) of the Nagarmotha Phytosomes were determined using a Microtrac particle size analyzer and the Dynamic Light Scattering (DLS) technique. A little aliquot of the formulation was diluted with distilled water before being tested at a scattering angle of 90° at 25°C in order to ensure the correct particle concentration and avoid multiple scattering. ^[13]

2. zeta potential -

The greater stability of any dispersion is due to strong particle electrostatic repulsion. Zeta potentials are considered to have high physical stability if they are greater than +20 mV or less than -20 mV. A small sample was dissolved in clean water, sonicated for one minute, and then placed into omega corvettes to measure the zeta potential. ^[14]

3. Entraptment efficiency-

Centrifuging the Phytosomes for 45 minutes at 12,000 rpm and -4°C released the unentrapped medication. By measuring absorption at 267 nm, a UV-Visible spectroscopy was used to determine the free medication concentration in the supernatant. The ratio of drug encapsulate was calculated using the specific formula.^[15]

Formula:

Entraptment efficiency(%)=Total amount of drug - Amount of free drugTotal amount of drug 100

D. Drug content-

To determine the amount of drug incorporated in the Phytosome formulation, 1ml of the solution was diluted with nine ml of phosphate buffer (p^H 7.4). The drug was extracted and filtered using Whatman filter paper following a 15-minute sonication. The clear filtrate was then analysed at the extract's λ_max (267nm) using UV-visible spectroscopy. ^[16]

Formula:

Drug content (%)=Practical Drug Content Theoretical Drug Content 100

E. Compatibility study

An FTIR compatibility investigation was conducted. The material's selective absorption in the infrared spectrum is what this technique depends on. The infrared spectrum is made up of absorption bands created by molecular vibrations brought on by absorbed infrared light. These bands aid in determining the composition of unknown materials by revealing the type of bonds and functional groups present.^[16]

F. In vitro drug release study

A dialysis tube containing Nagarmotha Phytosomes were placed in 250 mL of phosphate buffer (pH 7.4) at 37 ± 0.5 °C with agitation at 100 rpm in order to study drug release using a modified cellophane membrane. At intervals of up to 8 hours, samples (5 mL) were taken out and replaced with new buffer. An ultraviolet spectrophotometer was used for the analysis.^[17]

G. SEM

Scanning electron microscopy is utilized to identify the Phytosome Particle size distribution and surface morphology. For an electron microscope, the samples were mounted on a brass stub after being coated with gold using an ion sputter. Using a random scan of the stub, the loaded Phytosome was photographed digitally at magnifications of 1,000, 5,000, 10,000, and 30,000 X. ^[18]

Evaluation of Nagarmotha Phytosomal ointment-

A. Organoleptic characteristics

The physical properties of the drug-loaded and blank formulations, including phase separation, texture, color, and homogeneity, were examined visually. To check for texture and homogeneity, a small amount was rubbed between fingers; any stiffness, roughness, or greasiness was noted.^[19]

B. Viscosity-

The Brookfield viscometer, which used spindle number seven, was used to determine the viscosity of the mixtures at 100 RPM.

C. p^H-

1gm of sample was diluted in 50 ml of Purified water and constantly stirred to create a homogenous dispersion in order to determine the p^H of the ointment. The p^H of this dispersion was identified by using a calibrated digital p^H meter.^[20]

D. Spreadability-

The time (in seconds) required for two glass slides to separate under a constant weight is known as spreadability. The ointment was compacted into a uniform layer, and the separation time was recorded, in order to assess spreadability.^[22]

S = (M × L) / T

Where, L=

M=

T=

E. Washability-

Following application to the skin, the formulation's simplicity of rinsing with water was tested.

F. Solubility-

In order to determine solubility, the necessary amounts of the produced ointments were dissolved in 10 ml of solvent, such as water, boiling water, alcohol, ether etc.

G. Loss on drying -

The moisture content was determined by heating 1 g of ointment to 105°C and monitoring weight loss over time. This indicates volatile material and validates the stability assessment.^[23]

H. Drug diffusion study-

Drug diffusion was investigated by using a Franz diffusion cell, which had a dialysis membrane separating the phosphate buffer in the receptor chamber from the ointment in the donor chamber. The temperature was maintained at 37?±?0.5?°C with constant stirring. The samples were collected at specified intervals and evaluated using UV spectrophotometry. Cumulative drug release was plotted over time to assess diffusion.

I. Stability study -

Following ICH guidelines, a four-week stability study was conducted on the herbal ointment at 2°C, 25°C, and 37°C. The optimized formulation did not exhibit colour shift, texture variation, or phase separation at any temperature. Table 4 confirms consistent appearance, pH, viscosity, spreadability, and drug release, demonstrating good physical stability under both normal and accelerated conditions.^[24]

RESULTS AND DISCUSSION-

Table no. 03 Results of Nagarmotha phytosomes

Table no. 03 Results of phytosomal ointment

Fig. no. 01 SEM image under microscope