Formulation and Evaluation of Phytosomes Loaded Polyherbal Gel for Pharyngitis

Pharyngitis, also referred to as sore throat, is the inflammation of the pharynx, which leads to a painful throat. Therefore, pharyngitis should be understood as a manifestation of an underlying ailment, rather than a standalone condition itself.¹ This syndrome commonly occurs due to viral and/or bacterial infections, such as the common cold and flu (both viral disorders) or infection with the Streptococcus bacterium (strep throat). Pharyngitis may sometimes manifest in conjunction with mononucleosis, which is an alternative term for the viral infection often known as “mono”.² Fungal pharyngitis typically happens when a person’s immune system is weakened or when they have been using steroids or antibiotics for a long time. Occasionally, allergies such as hay fever or allergic rhinitis can cause throat discomfort. Inadequate humidity levels in indoor spaces and chronic mouth breathing, especially in the winter months, can lead to recurring throat irritation, particularly upon waking up in the morning.³

Novel vesicular drug delivery systems aim to administer the medication at a pace determined by the body’s requirement during the treatment time, while also routing the active ingredient to the site of action. To accomplish targeted and strictly controlled medication delivery, several innovative vesicular drug delivery systems with varying routes of administration have emerged. Phytosome is a novel developing technology used in phytopharmaceuticals that incorporates phytocomponents of herbal extracts that are enveloped and bound by lipids.⁴ Traditional medicines and phytomedicines have been used therapeutically for health maintenance purposes since ancient times. Developments in the discipline of medicinal product delivery commenced recently intending to effectively handle human ailments. Phytosomes are a distinctive botanical formulation and drug-delivery technology that produces lipophilic molecular complexes to increase the absorption and bioavailability of phytoconstituents.^5,6

The herbaceous annual Acalypha indica features cup-shaped involucres around tiny flowers and catkin-like inflorescences. It is best known for its root’s attraction to domestic cats and for its multiple medicinal properties like anti-inflammatory, anti-microbial and analgesics. It is found across the Tropics. The chief constituents present in this plant are acalypin, acalyphamide, aurantiamide, succinimide and flindersin.^7,8 Pergularia daemia is common in tropical and subtropical regions. It is a perennial twining plant that produces milky sap. The plant has some active principles like calactin, lupeol, oleanolic acid, corotoxigenin, calotropin, β-sitosterol, daucosterol, etc. It has anti-inflammatory, antioxidant and anti-diabetic properties.^9,10 Plectranthus amboinicus/Indian Borage is another name for Coleus amboinicus. It is from the Lamiaceae family. Indian Borage is commonly grown in Asian nations such as India, Sri Lanka, Malaysia, Indonesia and others. They are used to treat whooping cough, phlegm, microbial infections and inflammation. The pharmacological action of aerial plant parts is investigated. This plant is well-known as oregano, which improves the flavour of food. Chemical constituents like Thymol, Cis-caryophyllene and P-cymene was present in the plant.^11–14

The main objective of this study is to develop a combination of Phytosomes derived from Acalypha indica, Pergularia daemia and Coleus amboinicus in order to enhance the bioavailability in comparison to traditional drugs. Additionally, the study aims to examine the properties of the phytosomes and to develop and evaluate a polyherbal phytosomal gel for the treatment of Pharyngitis.

Standard plant drugs were bought from Yucca Enterprises, Mumbai, India. We purchased the soy lecithin, PEG 400 and Carbopol 934 from Anandsupra Chemicals in Chennai, India. All other chemicals and reagents were of analytical grade.

Acalypha indica, Pergularia daemia and Coleus amboinicus fresh leaves were collected, dried and roughly pulverized. In a Soxhlet apparatus, dried coarsely powdered leaves were extracted with 99.9% ethanol (60-70°C). Antisolvent Precipitation was used to precipitate the extracts.^15,16

Using standard methods, ethanolic extracts from each plant species were examined for alkaloids, glycosides, carbohydrates, proteins, phenolic compounds, saponins, tannins, flavonoids, steroids, terpenoids, gums and mucilage.^17–19

The combined phytosomes were prepared by the antisolvent precipitation method which involves the controlled addition of a non-solvent (antisolvent) to a solution containing a solute. Initially, the combined extracts were prepared by blending equal proportions of 1:1:1 ratio from all three plant extracts. The appropriate quantity of combined extracts and soy lecithin (1:1) were added to a 100 mL round bottom flask and refluxed for 2 hr at a temperature under 60°C with 20 mL of dichloromethane. The mixture is reduced to 5-10 mL. The precipitate was produced by carefully adding 20 mL of hexane while stirring continuously. The precipitate was then filtered, combined and overnighted in vacuum desiccators. An amber-colored glass bottle is used to keep the crushed dried precipitate at room temperature Table 1.^20–22

The phytosomes were evaluated based on several parameters, including percentage yield, entrapment efficiency, particle size determination, zeta potential and SEM analysis. These parameters were measured to assess the quality and stability of the phytosomes.^23–25

The gels containing phytosome complexes and plant drugs were formulated utilizing the various excipients and solvents outlined in Table 2. 10 mL of distilled water was used to dissolve the required amount of propyl and methylparaben in the beaker. The mixture was then supplemented with carbopol 934P while being vigorously stirred. The Carbopol dispersion was mixed with the phytosome complex solution, which had been dissolved in 0.1 mL of ethanol in a separate beaker. Gels were developed by adding triethanolamine to the dispersion. Utilizing varying concentrations (1%, 3% and 5%) of prepared combined phytosomes, multiple formulations, such as F1, F2 and F3, were developed. For further investigation, the developed gels were stored at room temperature in appropriate containers.^26–28

The gel was then evaluated for various quality parameters such as homogeneity, pH, drug content, Viscosity, spreading and extruding properties. The homogeneity test ensured uniform distribution of the phytosomes within the gel. The pH was measured to ensure compatibility with the skin. Drug content analysis determined the amount of active phytosomal compounds in the gel. Spreading and extruding properties assessed the gel’s ease of application.²⁹

Pharyngitis, a prevalent condition distinguished by inflammation of the larynx, presents considerable obstacles on account of its complex aetiology. Inflammation and microbial invasion are fundamental to its pathogenesis, necessitating the implementation of efficacious therapeutic interventions. This research aims to accomplish endeavour is to assess the potential therapeutic effects of novel compounds that possess anti-inflammatory and antimicrobial properties on pharyngitis. Orchestrated by a variety of mediators including cytokines and chemokines, the inflammatory response in pharyngitis significantly contributes to the severity of symptoms and tissue injury. Our objective in evaluating the anti-inflammatory properties of our compounds is to ameliorate patient discomfort and mitigate this response. Furthermore, the presence of pharyngitis, which is frequently caused by bacterial or viral pathogens, highlights the critical need for antimicrobial interventions. By employing meticulous microbial assays, our objective is to identify compounds that possess the ability to selectively bind to and eradicate these pathogens, thereby impeding their growth and alleviating the spread of infection. By identifying novel therapeutics, our research has the potential to significantly impact the management of pharyngitis, providing millions of individuals afflicted with this incapacitating condition with respite.

The antimicrobial activity of the polyherbal phytosomal gel was assessed using the agar well diffusion technique. Plates of Mueller Hinton agar was prepared and sterilized by placing them in an autoclave set at 121°C for 15 to 20 min. The next step was to pour the sterile media onto the previously sterilized Petri plates and then to wait for them to cool to room temperature. The bacterial suspension containing Streptococcus mutans and Staphylococcus aureus was uniformly distributed on the agar plates using sterile cotton swabs. Using a sterile polystyrene point, we formed wells with a diameter of 9 mm in the agar plates. The wells were subsequently filled with polyherbal phytosomal gel at varying concentrations (25 μg, 50 μg, 100 μg). The plates were placed in an incubator set at a temperature of 37°C for 24 hr. The antibacterial activity was assessed by measuring the diameter of the zone of inhibition surrounding the wells. The diameter of the zone of inhibition was measured using a ruler and recorded in mm and the value was used to compute the zone of inhibition.^30,31

Figure 1:
SEM images of the phytosome.

Figure 2:
Zeta Potential of Phytosomal complex.

Three tests, including the Bovine Serum Albumin Denaturation Assay, the Egg Albumin Denaturation Assay and the Membrane Stabilisation Method, were used to evaluate the polyherbal phytosomal gel’s anti-inflammatory effectiveness.

About 0.05 mL of polyherbal phytosomal gel at various doses (10-50 g/mL) was combined with 0.45 mL of bovine serum albumin. The pH was adjusted to 6.3. Following a 10 min interval at room temperature, it was incubated in a water bath that was heated to 55°C for 30 min. The standard group utilised was diclofenac sodium, while the control group used was dimethyl sulphoxide. The samples were then spectrophotometrically analysed at 660 nm. The protein denaturation percentage was calculated.³²

Figure 3:
Particle Size Determination of Phytosomal Complex.

Figure 4 A:
Anti-Microbial Test performed against Staphylococcus aureus.

The egg albumin denaturation experiment was conducted using 2.8 mL of phosphate buffer and 0.2 mL of fresh egg albumin. Various amounts of polyherbal phytosomal gel (10-50 g/mL) were added to the reaction mixture. We adjusted the pH to 6.3. After 10 min at room temperature, it was incubated in a water bath at 55°C for 30 min. The standard group utilized was diclofenac sodium, while the control group used was dimethyl sulphoxide. The samples were then spectrophotometrically analyzed at 660 nm. The protein denaturation percentage was calculated.³³

Take fresh human blood samples in sterile tubes with an anticoagulant. Separate the RBCs from other blood components by centrifuging the blood at a 1000 g rate for 10 min at room temperature. Take away the supernatant, then PBS-wash the RBCs three times. To obtain a 10% (v/v) RBC suspension, resuspend the RBCs in the Tris-HCl buffer. 1mL of the RBC suspension should be pipetted into each centrifuge tube. Each tube was then filled with varying quantities of polyherbal phytosomal gel. Gently mix the tubes and incubate them at 37°C for 30 min. To pellet the RBCs, centrifuge the tubes at 1000 g for 10 min at room temperature. Using a UV-Vis spectrophotometer, determine the absorbance of the supernatant at 540 nm.³⁴

All the measurements were performed in triplicates and the results were expressed as the mean±standard error of mean using GraphPad Prism 5.0. Two-way Analysis of Variance (ANOVA) was performed and a p value of less than 0.05 was deemed significantly different.

The extractions were performed for all the plant drugs by using the Soxhlet apparatus the physical nature of the drug was semisolid which yields 6.2%, 6.88% and 7.82% respectively. Preliminary phytochemical screening of extract for the presence of multiple chemical components were performed and results were tabulated in Table 3. Combined phytosomes were prepared by the antisolvent precipitation method. The formulated phytosomes contain phospholipids and drug extracts. The evaluation parameters were performed and the results were discussed. Vesicle development could be observed in the phytosomes’ microscopical image, which was noted. It was found that the produced vesicles were all similar in size and shape. No clumsy particles were found. The prepared phytosomes showed better percentage yield, entrapment efficiency, particle size and Zeta potential. Data for these parameters are 72.5%, 86.94%±0.3, 158 nm and -8 mv respectively and results were tabulated in Table 4. Figures 1 and 2 shows the reports of particle size and zeta potential. Figure 3 displays the surface morphology, shape and structure of the combined phytosomes under various magnifications. It was found that the phytosomes are irregular in shape with a rough surface.

All the developed gel formulations passed a visual evaluation and it was found that they all had an acceptable look and homogeneity. The pH of the gel formulations was between 5.4 and 5.1, which is between the skin’s normal pH range and won’t cause irritation to the skin. Using spectrophotometry, the drug content of the gel formulations was calculated between 200 and 600 nm. All the formulations’ drug contents were found to range from 82.37% to 90.09%, with the best formulation F3 having the highest drug concentration at 90.09%. The formulations’ viscosities were measured and the formulation with the best viscosity F3 measured at 35123 centipoises. The developed gel formulations’ spreadability was tested and it was determined that all of the formulations displayed a respectable spreadability. The best formulation, F3, had a spreadability coefficient of 4.1 cm. It was discovered that the concentration of the gelling ingredients affected the extrudability of the manufactured gel formulation. With an increase in gelling agent concentration, extrudability decreased. The best formulation F3’s extrudability was determined to be 9.2 gm/cm². By comparing three formulations the F3’s extrudability was determined to be 9.2 gm/cm² which is better than F1 and F2. As a result, the prepared gel has the best extrudability and the data were tabulated in Table 5.

Figure 4 B:
Graphical representation of Polyherbal gel against Staphylococcus aureus.

Figure 5 A:
Anti-microbial test performed on Streptococcus Mutants.

Figure 5 B:
Graphical representation of Polyherbal gel Against Streptococcus mutants.

Figure 6 A:
Bovine Serum Albumin Denaturation Assay. Each value represents the mean±SEM of three replicates.

Figure 6 B:
Egg Albumin Denaturation Assay. Each value represents the mean±SEM of three replicates.

Antimicrobial in vitro experiments demonstrated that formulations F1, F2 and F3 were effective against S. aureus and S. mutans as shown in the Figures 4 and 5. Specifically, the antibacterial activity of formulation F3 was found to be 30% higher than that of F1 and 20% higher than that of F2. This observation highlights the strong antimicrobial effects of F3, which inhibits the growth of S. aureus and S. mutans.

Formulation F3 outperforms the other formulations in the antimicrobial assay. Thus, the F3 formulation is evaluated in the in vitro anti-inflammatory assay and it is deemed the optimized batch. Various doses ranging from 10 to 50 μg/mL were used to analyze the F3 formulation. To reduce the body’s inflammatory response, anti-inflammatory medicines act by blocking specific biochemical pathways or mediators. In order to determine the anti-inflammatory effect, this study employed the BSA assay, which measures the ability of a substance to prevent protein denaturation. This involves maintaining the functional form of proteins by stabilizing their structure, preventing misfolding by minimizing protein aggregation and even providing insights into how chemicals protect proteins like BSA from harm during inflammation by interfering with inflammatory processes. Figure 6 A shows the comparison of anti-inflammatory activities and it can be shown that the newly produced Phytosomal polyherbal gel, which is mediated by Acalypha indica, Pergularia daemia and Coleus amboinicus, had anti-inflammatory activity that was comparable to the standard. A specific attribute was evaluated by analyzing the samples at 660 nm. The standard reference compound, diclofenac, was examined at concentrations varying from 10 to 50 μg/mL, while the produced phytosomal gel was evaluated at concentrations that were equivalent to it. In comparison to the reference, which demonstrated an anti-inflammatory action of 75.10±0.30% at a concentration of 40 μg/mL, the Phytosomal gel demonstrated 73.50±0.40%. In comparison to the standard’s 89.10±0.40% activity, the Phytosomal gel showed an anti-inflammatory activity of 86.03±0.80% at 50 μg/mL. The anti-inflammatory activity of the phytosomal gel was found to be significantly equivalent to that of standard Diclofenac at concentrations from 10 to 50 μg/mL.

Figure 6 C:
Membrane Stabilization Assay. Each value represents the mean±SEM of three replicates.

In Egg Albumin denaturation assay, egg albumin is heated or acidified at various degrees and its functional and structural modifications are observed. We can gain insight into the stability and response of egg albumin to various environmental circumstances by assessing its denaturation. Protein activity in biological systems can be better understood using this data. The reference substance, diclofenac and Phytosomal gel were tested at doses ranging from 10 to 50 μg/mL as shown in Figure 6B. The Phytosomal gel had a significant anti-inflammatory effect of 69.90±0.30% compared to the reference, which showed an anti-inflammatory action of 71.80±0.40% at a concentration of 40 μg/mL.

In Membrane stabilization assay, the Phytosomal gel exhibited an anti-inflammatory activity of 84.40±0.80% at a concentration of 50 μg/mL, which is slightly less than the standard’s activity of 88.50±0.30%. The phytosomal gel demonstrated an equivalent degree of anti-inflammatory effectiveness to the standard Diclofenac at dosages ranging from 10 to 50 μg/mL. The test evaluates a drug’s ability to prevent cell contents from leaking through broken membranes. Drugs protect cells’ structural integrity, vital to their function and health, by stabilizing these membranes. The phytosomal gels display 75.90±0.20% at 40 μg/mL, while the standard yields 79.80±0.60%. At 50 μg/ mL, the gel will likely show 84.92±0.30%, while the standard yields 89.80±0.54% as shown in Figure 6C. In contrast to the highest-quality medication, these findings show that the phytosomal gel is much more efficacious.

The current investigation focused on formulating and assessing a polyherbal phytosomal gel using extracts derived from Acalypha indica, Pergularia daemia and Coleus amboinicus. The Soxhlet extraction method yielded substantial quantities of semi-solid extracts from these plants, verifying the extraction process’s efficacy. The preliminary phytochemical screening revealed the existence of various bioactive components, which are essential for the healing properties of the extracts.

The phytosomes were prepared using the antisolvent precipitation method, which effectively combined phospholipids with drug extracts. The assessment of phytosomes demonstrated encouraging outcomes, with a substantial percentage yield of 72.5% and an entrapment efficiency of 86.94%±0.3. The phytosomal formulation demonstrated good stability and uniformity, as evidenced by a particle size of 158 nm and a zeta potential of -8 mV.³⁵ SEM examinations provided additional verification of the morphological features, demonstrating the presence of vesicles with irregular shapes and a coarse surface, which can augment the available surface area for interaction with biological membranes.^36,37

Formulation F3’s promising topical uses are highlighted by its outstanding performance in the gel matrix. Essential for successful cutaneous distribution, Formulation F3 displayed ideal viscosity and Spreadability in addition to high drug content (90.09%), appropriate pH and excellent homogeneity. Because of its extrudability, it is easy to administer and increases patient compliance, two important factors in patient care settings.

Results from the antimicrobial test showed that F3 formulation was highly effective against S. aureus and S. mutans.³⁰ It is suggested that the plant extracts and phospholipids interact synergistically, increasing the antibacterial efficacy by 30% compared to other formulations. Investigating the causes and possible synergies with conventional antibiotics in the fight against multidrug-resistant infections should be the focus of future research. The strong anti-inflammatory effects of formulation F3 were confirmed by in vitro anti-inflammatory assessments, which showed a notable reduction of protein denaturation and membrane stabilization. As a natural alternative to diclofenac, it shows promise in inflammatory conditions, with comparable efficacy throughout studied concentrations. To better understand its precise action mechanisms, possible interactions with inflammatory mediators and safety profile over the long term, additional research is necessary.³³

To sum up, the produced polyherbal phytosomal gel has a lot of potential in the pharmaceutical and dermatological fields, especially in formulation F3. Further preclinical and clinical investigations are warranted due to its improved bioavailability, powerful antibacterial efficacy and potent anti-inflammatory properties. Thorough clinical trials to confirm its therapeutic effectiveness and safety in varied patient groups should be the focus of future research, along with optimising formulation parameters, investigating synergistic effects with conventional medicines and scaling up manufacturing.

This study focused on the formulation and evaluation of combined phytosomes derived from Acalypha indica, Pergularia daemia and Coleus amboinicus for enhanced bioavailability and targeted drug delivery in the treatment of Pharyngitis. The developed phytosomal system demonstrated the successful formation of uniform vesicles with desirable characteristics such as high yield, excellent entrapment efficiency, small particle size and appropriate zeta potential. These properties indicate the potential of the phytosomes for the efficient delivery of herbal extracts. Furthermore, the formulated polyherbal phytosomal gel exhibited suitable attributes for topical application, including pH compatibility with the skin and optimal viscosity. The in vitro evaluation of the polyherbal phytosomal gel demonstrated significant anti-inflammatory and anti-microbial activity, comparable to the standard drug, in various assays. This suggests the potential therapeutic effectiveness of the developed formulation in managing Pharyngitis conditions. The utilization of phytosomes as a drug delivery system holds promise for improved bioavailability and targeted action, which may enhance the overall therapeutic outcomes. Overall, the findings of this study support the potential of combined phytosomes derived from Acalypha indica, Pergularia daemia and Coleus amboinicus as an innovative approach for effective drug delivery in the treatment of Pharyngitis. Further studies, including in vivo experiments and clinical trials, are warranted to validate the efficacy and safety of the developed formulation for broader clinical applications.