Piper Cubeba Seeds Based Herbal Nasal Spray: A Novel Antimicrobial and Anti-Inflammatory Formulation for Respiratory Health

Respiratory health refers to the proper structural and functional integrity of the respiratory system, enabling efficient ventilation, gas exchange, and protection against environmental pollutants and infectious agents [1]. The upper respiratory tract plays a vital role in maintaining respiratory homeostasis by filtering, warming, and humidifying inhaled air while serving as the first barrier against airborne pathogens and particulate matter [2]. Several defense mechanisms operate in the nasal cavity, including the mucociliary clearance system, epithelial barrier, and innate immune responses that prevent microbial invasion into the lower respiratory tract. In addition, the resident microbiota of the upper airway contributes to immune regulation and pathogen resistance, thereby maintaining respiratory balance [3].

Disruption of these protective mechanisms due to inflammation, microbial colonization, or environmental pollutants can compromise mucosal defense and increase susceptibility to respiratory infections. Sinusitis, also known as rhinosinusitis, is one of the most common inflammatory disorders affecting the paranasal sinuses. It is characterized by symptoms such as nasal congestion, facial pain, mucosal edema, and impaired mucociliary clearance. The condition contributes significantly to global morbidity and represents a major subset of upper respiratory tract disorders [4]. Epidemiological studies indicate that sinusitis affects approximately 10–15% of adults worldwide, while respiratory infections remain among the leading causes of illness and mortality globally [5].

These conditions impose a considerable healthcare burden, particularly in developing countries. The pathophysiology of sinusitis involves both microbial colonization and dysregulated inflammatory responses. Bacterial pathogens such as Staphylococcus aureus and Escherichia coli are commonly associated with acute and chronic sinus infections. These microorganisms can form biofilms, evade host immune defenses, and induce persistent mucosal inflammation, thereby contributing to chronic sinus disease [6]. Following microbial invasion, respiratory epithelial cells recognize pathogen-associated molecular patterns through Toll-like receptors, which activate intracellular signaling pathways including the nuclear factor kappa B (NF-κB) pathway. Activation of this pathway regulates the expression of pro-inflammatory mediators such as tumor necrosis factor-α, interleukin-1β, and interleukin-6, leading to vasodilation, leukocyte recruitment, mucosal swelling, and obstruction of sinus drainage pathways [7].

Evaluation of anti-inflammatory activity in vitro commonly involves assays that measure the inhibition of protein denaturation and suppression of inflammatory mediators. The protein denaturation assay is widely used as an initial screening method for anti-inflammatory compounds. During inflammatory conditions, proteins may undergo structural denaturation due to heat, chemical stress, or inflammatory mediators, which can lead to the production of auto-antigens and further inflammatory responses. Compounds capable of stabilizing proteins and preventing denaturation are therefore considered to possess anti-inflammatory potential. The bovine serum albumin (BSA) protein denaturation assay is frequently employed to evaluate the ability of natural extracts or formulations to inhibit heat-induced protein denaturation, thereby indicating potential anti-inflammatory activity [8].

Nitric oxide (NO) is another important mediator involved in inflammatory responses associated with respiratory infections. During immune activation, inducible nitric oxide synthase (iNOS) is expressed in macrophages and other immune cells, resulting in the production of nitric oxide from L-arginine [9]. Although moderate levels of nitric oxide contribute to antimicrobial defense, excessive production can lead to oxidative and nitrosative stress. Nitric oxide may react with superoxide radicals to generate peroxynitrite, a highly reactive nitrogen species capable of causing lipid peroxidation, protein modification, and DNA damage. These processes amplify mucosal inflammation and contribute to tissue injury [10]. Therefore, inhibition of nitric oxide production is commonly used as an indicator of anti-inflammatory activity in pharmacological studies.

Plant-derived bioactive compounds have attracted significant attention as potential alternatives to conventional antibiotics due to their broad pharmacological properties and lower risk of adverse effects. Phytochemicals such as terpenoids, flavonoids, alkaloids, and essential oils have demonstrated antimicrobial, antioxidant, and anti-inflammatory activities through various mechanisms, including disruption of microbial membranes, inhibition of biofilm formation, and modulation of inflammatory signaling pathways [11].

Among medicinal plants, Piper cubeba has been widely recognized for its therapeutic value in traditional medicine. Extracts from its seeds have demonstrated antimicrobial, anti-inflammatory, and antioxidant activities. Previous studies have shown that Piper cubeba extracts can reduce inflammatory mediators such as nitric oxide, tumor necrosis factor-α, and interleukin-6 by modulating inflammatory pathways. Additionally, the plant exhibits strong antioxidant activity through free radical scavenging mechanisms that help protect tissue from oxidative stress [12]. Since respiratory disorders often involve microbial infection, inflammation, and oxidative damage simultaneously, plant-based formulations capable of targeting multiple pathways may provide effective therapeutic benefits.

Advances in computational pharmacology have further strengthened the process of identifying potential therapeutic compounds from medicinal plants. Molecular docking studies are widely used to predict the interaction between bioactive phytochemicals and specific biological targets involved in disease pathways. Cyclooxygenase-2 (COX-2) is one of the key enzymes responsible for the synthesis of prostaglandins during inflammatory responses, making it an important target in anti-inflammatory drug development. Inhibition of COX-2 activity can reduce the production of inflammatory mediators and thereby alleviate inflammation and pain. Similarly, bacterial DNA gyrase is an essential enzyme involved in DNA replication and transcription in bacteria and is considered a major target for antimicrobial agents. Computational docking techniques allow the evaluation of binding affinity, interaction patterns, and stability of phytochemical compounds with these protein targets, thereby providing insight into their potential therapeutic mechanisms before experimental validation [13].

MATERIALS AND METHODS

1. MATERIALS

Dried seeds of Piper cubeba L. were procured from a local herbal supplier and authenticated based on standard pharmacognostic characteristics. The seeds were cleaned, shade-dried, and powdered using a mechanical grinder for extraction. Distilled water was used as the extraction solvent and for formulation of the herbal nasal spray.

All chemicals and reagents used in the study were of analytical grade. Bovine Serum Albumin (BSA) was used for the protein denaturation assay to evaluate anti-inflammatory activity. Sodium nitroprusside and Griess reagent were used for nitric oxide scavenging assay.  Reagents for phytochemical screening such as Mayer’s reagent, Wagner’s Reagent, Ferric chloride, Molisch reagent, and foam test reagents were used according to standard procedures.Microbiological media including nutrient agar and nutrient broth were prepared for anti-microbial testing. Bacterial strains used for anti-microbial evaluation included Staphylococcus aureus and Escherichia coli, which are commonly associated with respiratory infections.

For molecular docking studies, the three-dimensional structures of cyclooxygenase-2 (COX-2) proteins (PDB IDs: 5IKR, 1VQQ, 2XCT) and DNA gyrase were retrieved from the Protein Data Bank. Ligands identified from GC-MS analysis of Piper cubeba extract such as 4-epi-cubedol, β-phellandrene, furanone derivatives, benzene derivatives, and ledol were used for docking.

2. METHODS

2.1 PREPARATION OF Piper cubeba SEED EXTRACT

The dried seeds of Piper cubeba refer were washed with distilled water to remove impurities and shade-dried as shown in Figure 1. The dried seeds were then powdered using a mechanical grinder. The powdered material was subjected to aqueous extraction by soaking the powder in ethanol and allowing it to stand for an appropriate period with intermittent stirring. The extract was filtered using Whatman filter paper to remove solid residues. The filtrate obtained was concentrated and stored at 4°C until further use for phytochemical analysis and formulation as depicted in Figure 2.    

Figure 1. Dried Seeds of Piper cubeba

Figure 2. Aqueous Extract of Piper cubeba

2.2 QUALITATIVE PHYTOCHEMICAL SCREENING

Preliminary phytochemical screening of the aqueous extract of Piper cubeba seeds was carried out to identify the presence of major secondary metabolites using standard qualitative methods .

The extract was tested for alkaloids, flavonoids, terpenoids, steroids, carbohydrates, proteins, amino acids, saponins, and phenolic compounds using appropriate chemical reagents. The presence of each phytochemical constituent was determined based on characteristic color changes or precipitate formation during the reactions. The results were recorded as absent, low (+), moderate (++), or high (+++) presence pf phytochemicals.

Detection of Alkaloids

The extract was treated with dilute hydrochloric acid and filtered [14]. The filtrate was tested with Wagner’s reagent. Formation of reddish-brown precipitate indicated the presence of alkaloids [15].

Detection of Carbohydrates

Fehling’s test was performed by heating the extract with Fehling’s solutions I and II. Formation of red precipitate indicated presence of sugars.

Detection of Glycosides

The extract was hydrolysed with HCl and subjected to Borntrager’s test. Pink coloration indicated presence of glycosides [16].

Detection of Saponin

Persistent foam formation indicated presence of saponins[17].

Detection of Proteins

Formation of pink/violet color indicated presence of proteins[18].

Detection of Amino Acids

Purple coloration confirmed presence of amino acids[19].

Detection of Phenolic Compounds

Dark green coloration indicated phenolic compounds[20].

Detection of Flavonoids

Yellow coloration after addition of ammonia and sulphuric acid indicated flavonoids.

Detection of Terpenoids

Reddish-brown interface formation indicated terpenoids.

Detection of Steroids

Blue-green coloration confirmed presence of steroids.

2.3 GC-MS ANALYSIS

Gas chromatography - mass spectrometry (GC-MS) analysis of the Piper cubeba seed extract was performed to identify the bioactive compounds present in the extract. The sample was injected into the GC-MS instrument equipped with a suitable capillary column-under controlled temperature condition.

The compounds were identified by comparing the obtained mass spectra with the spectral data available in standard libraries such as NIST database. Major compounds identified from the analysis included 4-epi-cubedol, β- phellandrene, furanone derivatives, benzene derivatives, and ledol, which were further used for molecular docking.

2.4 MOLECULAR DOCKING STUDIES

Molecular docking studies were performed to evaluate the interaction of the identified phytochemicals with target proteins associated with inflammation. The crystal structures of Cyclooxygenase-2(COX-2) with PDB ID’s 5IKR, 1VQQ, and 2XCT, along with DNA gyrase, were obtained from the Protein Data Bank.

The ligand structures corresponding to the compounds identified in the GC-MS analysis were prepared and optimized prior to docking. Docking simulations were performed using appropriate molecular docking software to determine the binding affinity and interaction patterns between the ligands and the target proteins. Binding energies and interaction residues were analyzed to evaluate the potential anti-inflammatory and anti-microbial mechanisms of the compounds.

2.5 FORMULATION OF HERBAL NASAL SPRAY

The herbal nasal spray formulation was prepared using the aqueous extract of Piper cubeba seeds. The extract was incorporated into physiologically compatible aqueous base containing suitable excipients to maintain isotonicity and stability. The formulation was mixed thoroughly and filled into sterile nasal spray containers under hygienic conditions. The composition of the herbal nasal spray formulation is shown in Table 1. 

TABLE 1. Composition of Piper cubeba Herbal Nasal Spray

INGREDIENT	AMOUNT
Purified water (USP)	90 mL
Sodium Chloride	0.90 g
Glycerin	1.50 g
Propylene glycol	0.50 g
Citric acid (Anhydrous)	0.02 g
Sodium Hydroxide (1N solution)	~ Q.S. to pH 6.2
Purified water	Q.S. to 100 mL

2.6 PHYSICOCHEMICAL EVALUATION

The prepared nasal spray formulation was evaluated for various physicochemical parameters including pH, viscosity, isotonicity, clarity, and spray pattern to determine its suitability for nasal administration.

The pH of the formulation was measured using a digital pH meter. Viscosity was determined using a viscometer, while isotonicity was evaluated to ensure compatibility with nasal mucosa. Spray pattern and uniformity were examined to assess the performance of the nasal delivery system.

2.7 ANTIMICROBIAL ACTIVITY

The antimicrobial activity of the formulated nasal spray was evaluated against Staphylococcus aureus and Escherichia coli using the agar well diffusion method. Sterile nutrient agar plates were inoculated with bacterial cultures, and wells were created in the agar medium.

Different concentrations of the extract or formulation were introduced into the wells, and the plates were incubated under suitable conditions. After incubation, the zones of inhibition were measured in millimeters to determine the antibacterial-effectiveness of the formulation.

2.8 DETERMINATION OF MIC AND MBC

Minimum Inhibitory Concentration (MIC) was determined using broth dilution method. The lowest concentration inhibiting visible growth was recorded as MIC.

Minimum Bactericidal Concentration (MBC) was determined by subculturing onto agar plates. The lowest concentration showing no bacterial growth was recorded as MBC.

2.9 PROTEIN DENATURATION ASSAY

The anti-inflammatory of the formulation was evaluated using Bovine Serum Albumin (BSA) protein denaturation assay [21]. The reaction mixture consisted of BSA solution and different concentrations of the test sample. The mixture was incubated and then heated to induce protein denaturation was calculated to determine the anti-inflammatory potential of the formulation.

2.10 NITRIC OXIDE SCAVENGING ASSAY

The nitric oxide scavenging activity of the extract was determined using the sodium nitroprusside-method. Sodium nitroprusside in aqueous solution generates nitric oxide, which reacts with oxygen to form nitrite ions. These ions react with Griess reagent to produce a colored complex that can be measured spectrophotometrically. Different concentrations of the sample were incubated with sodium nitroprusside, followed by addition of Griess reagent. The absorbance was measured, and the percentage inhibition of nitric oxide production was calculated to evaluate the anti-inflammatory and anti-oxidant activity of the formulation.

2.11 MUCOSAL IRRITATION STUDY

Mucosal irritation study was evaluated using an artificial membrane model, which eliminates need for animal or human tissues. Circular pieces of the membrane were prepared, rinsed with phosphate-buffered saline (PBS, pH7.4), and hydrated at room temperature. The test formulation was applied onto the membrane surface, with PBS as a negative control and 0.1% sodium lauryl sulfate as a positive control. The membranes were incubated. Observations were made at 0 to 48 hours intervals.

RESULTS

1. PHYTOCHEMICAL ANALYSIS

Preliminary qualitative phytochemical screening of the aqueous extract of Piper cubeba seeds was carried out to determine the presence of major classes of secondary metabolites. The analysis revealed the presence of several bioactive phytochemicals including Terpenoids, Alkaloids, Saponins, Steroids, Flavonoids, and amino acids.

Semi-quantitative evaluation indicated that carbohydrates, terpenoids, and steroids were present in high amounts (+++), whereas alkaloids, proteins, amino acids, and flavonoids were detected in moderate amounts (++). The saponins were present in low concentration (+). However, phenolic compounds and glycosides were not detected (-) in the aqueous extract. The results of phytochemical screening are evidenced in Table 2 and Figure 3.

TABLE 2 . Qualitative phytochemical screening of Piper cubeba seed extract

Figure 3.  Phyto-chemical Screening Test Tubes

2. GC-MS ANALYSIS

Gas chromatography - mass spectrometry (GC-MS) analysis of the Pier cubeba seed extract revealed the presence of multiple volatile phytoconstituents. The chromatogram showed several distinct peaks corresponding to different compounds as observed in Figure 4, identified by comparison with the NIST spectral library.Major compound identified in the extract included 4-epi-cubedol, β-phellandrene, ledol, 1H-cyclopenta[1,3] cyclopropa[1,2]benzene derivatives, and 2(3H)- furanone derivatives. The retention times of these compounds ranged from 4.6977 to 20.1958 minutes, indicating the presence of both monoterpenes and sesquiterpenes as major constituents. These compounds were selected for further molecular docking analysis to evaluate their potential biological activities.

Figure 4.  GC-MS Chromatogram

3. MOLECULAR DOCKING STUDIES

Molecular Docking analysis was performed to investigate the interaction of the identifies phytochemicals with target proteins involved in inflammation and microbial survival, including cyclooxygenase (COX-2), Penicillin- binding protein 2a (PBP2a), and DNA gyrase. Docking results demonstrated that the selected compounds exhibited favorable binding interactions with the active sites of the target proteins. In the COX-2 enzyme, several ligands interacted with key amino acid residues including Tyr385, Leu352, Phe518, Met522, Ala527, Ser530, Arg120, Val523, Trp387 and Leu384, suggesting potential inhibition of inflammatory pathways. The 3D binding conformation of COX-2 with the targeted proteins are illustrated in Figure 5.  

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Figure 5.  (a) Molecular Docking Interaction of 4-epi-cubedol with COX-2, (b) Molecular Docking Interaction of β- phellandrene with COX-2, (c) Molecular Docking Interaction of benzene derivatives with COX-2, (d) Molecular Docking Interaction of furanone derivatives with COX-2, (e) Molecular Docking Interaction of Ledol with COX-2.

Similarly, docking with 1VQQ revealed interactions with residues such as Arg129, Arg219, Glu217, His271, Val255, Glu128, Tyr201, Leu202, and Thr194, indicating possible antibacterial activity through interference with bacterial cell wall synthesis. The 3D binding conformation of 1VQQ with the targeted proteins are illustrated in Figure 6.          

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Figure 6. (a) Molecular docking interaction of 4-epi-cubedol with 1VQQ, (b) Molecular docking interaction of β- phellandrene with 1VQQ, (c) Molecular docking interaction of benzene derivative with 1VQQ, (d) Molecular docking interaction of furanone derivative with 1VQQ, (e) Molecular docking interaction of ledol with 1VQQ.

For DNA gyrase, the identified compounds showed interactions with residues including Asp668, Thr315, Tyr287, Ile317, Lys316, Asp210, Ser207, Gln133, Tyr336, and His267, suggesting potential inhibition of bacterial DNA replication mechanisms. The 3D binding conformation of DNA gyrase with the targeted proteins are illustrated in Figure 7.

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Figure 7. (a) Molecular docking interaction of 4-epi-cubedol with 2XCT, (b) Molecular docking interaction of β- phellandrene with 2XCT, (c) Molecular docking interaction of benzene derivative with 2XCT, (d) Molecular docking interaction of furanone derivative with 2XCT, (e) Molecular docking interaction of ledol with 2XCT.

These docking interactions indicate that phytochemicals present in Piper cubeba may contribute to both anti-inflammatory and anti-microbial activities.

4. PHYSICOCHEMICAL CHARACTERIZATION OF NASAL SPRAY

The developed Piper cubeba nasal spray formulation was evaluated for several physicochemical parameters to determine its suitability for nasal administration. The formulation appeared clear and colorless, indicating the absence of visible particulate matter. The pH of the formulation was 6.2 which falls within the acceptable physiological range for nasal preparations. The formulation also exhibited isotonic properties, ensuring compatibility with nasal mucosa. Viscosity measurements indicated an appropriate consistency for nasal delivery. Spray pattern evaluation demonstrated uniform atomization and formation of a fine mist, suggesting efficient dispersion of the formulation during administration.

5. BIOLOGICAL EVALUATION
   1. ANTIMICROBIAL ACTIVITY

The antimicrobial activity of the formulated nasal spray was evaluated against Staphylococcus aureus and Escherichia coli using the agar well diffusion method. The formulation exhibited concentration-dependent antibacterial activity against both testes micro-organisms. The zones of inhibition observed at concentration of 20 μg and 40μg are presented in Table 3 and illustrated in Figure 8.

The formulation showed stronger inhibition against Staphylococcus aureus compared to Escherichia coli, suggesting better activity against Gram - positive bacteria.

TABLE 3 . Antimicrobial activity of the Piper cubeba nasal spray formulation

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Figure 8. (a) Antimicrobial Activity of Piper cubeba Nasal Spray Formulation Against Staphylococcus aureus Showing Zone of Inhibition in Agar Well Diffusion Assay, (b) Antimicrobial Activity of Piper cubeba Nasal Spray Formulation Against Escherichia coli Showing Zone of Inhibition in Agar Well Diffusion Assay.

1. 2. MIC AND MBC

The Minimum Inhibitory Concentration (MIC) represents the lowest concentration of the extract required to inhibit visible growth of microorganisms. The Minimum Bactericidal Concentration (MBC) represents the lowest concentration required to completely kill the bacteria.

In this study, Staphylococcus aureus exhibited lower MIC (50 µg/mL) and MBC (100 µg/mL) values compared to Escherichia coli, which showed MIC (75 µg/mL) and MBC (150 µg/mL) as given in Table 4 and  Figure 9. This indicates that the extract is more effective against Gram-positive bacteria than Gram-negative bacteria. The higher resistance observed in E. coli may be due to the presence of an outer membrane that limits the penetration of bioactive compounds.

TABLE 4. MIC and MBC of the formulated herbal extract against bacterial strains

Figure 9.   Graphical representation of MIC and MBC values of the formulation against selected bactcerial strains

1. 3. ANTI-INFLAMMATORY ACTIVITY   

PROTEIN DENATURATION ASSAY

The anti-inflammatory activity of the formulation was evaluated using the Bovine Serum Albumin (BSA) protein denaturation assay. The formulation demonstrated concentration-dependent inhibition of protein denaturation, indicating its ability to stabilize proteins and reduce inflammatory responses. Maximum inhibition was observed at 500 μg per mL, with an IC₅₀ value indicating strong anti-inflammatory potential. The quantitative values are summarized in Table  5 and the trend is graphically illustrated in Figure  10.

TABLE  5. Percentage Inhibition of Bovine Serum Albumin (BSA) Protein Denaturation by Piper cubeba Nasal Spray Formulation at Different Concentrations.

Figure 10. Concentration-Dependent Inhibition of Protein Denaturation by Piper cubeba Nasal Spray Formulation.

NITRIC OXIDE SCAVENGING ASSAY

Nitric oxide Scavenging Activity was evaluated using the sodium nitroprusside method. The formulation showed significant inhibition of nitric oxide production in a dose-dependent manner. Maximum inhibition was observed at higher concentrations, indicating the potential of the formulation to reduce nitric oxide- mediated inflammatory responses. The quantitative values are summarized in Table  6 and the trend is graphically illustrated in Figure 11.

TABLE  6. Percentage Inhibition of Nitric Oxide Radicles by Piper cubeba Nasal Spray Formulation Determined using the Nitric Oxide Scavenging Assay.

Figure 11.  Dose Dependent Nitric Oxide Scavenging Activity of Piper cubeba Nasal Spray Formulation.

1. 4. MUCOSAL IRRITATION STUDY

The mucosal irritation study indicates that the formulation is safe for nasal use. No irritation was observed at 0 hr. Mild irritation was seen at 1 hr and 24 hr, which completely subsided by 48 hr as given in Table 7 and Figure 12. This suggests the formulation causes only transient and reversible irritation.

TABLE 7. Mucosal irritation scores of the formulated nasal spray at different time intervals

Figure 12. Graphical representation of mucosal irritation scores over time

DISCUSSION

Respiratory tract infections are frequently associated with microbial invasion and inflammatory responses that contribute to tissue irritation, mucosal swelling, and oxidative stress within the respiratory tract. Natural products have long been explored as alternative therapeutic agents due to their diverse phytochemical composition and pharmacological activities. In the present study, herbal nasal spray formulation based on Piper cubeba seed extract was developed and evaluated for its phytochemical composition, molecular interactions, physicochemical characteristics, anti-microbial activity and anti-inflammatory potential. The findings demonstrate that the formulation possesses multiple biological activities that may support its potential application in the management of respiratory infections and associated inflammatory conditions.

Preliminary phytochemical screening revealed the presence of several classes of bioactive secondary metabolites, including alkaloids, carbohydrates, flavonoids, terpenoids, steroids, saponins, proteins, and amino acids. Among these, terpenoids and steroids were detected in higher concentrations, while alkaloids and flavonoids were present in moderate amounts. The predominance of terpenoids compounds in Piper cubeba seeds has been reported in previous phytochemical studies, which describe the plant as a rich source of essential oils and sesquiterpene derivatives. Terpenoids are widely recognized for their antimicrobial, anti-inflammatory, and anti-oxidant properties. These compounds can exert antimicrobial activity by disrupting microbial cell membranes, altering membrane permeability, and interfering with intracellular metabolic processes. In addition, terpenoids are capable of modulating inflammatory signaling pathways by inhibiting the production of inflammatory mediators such as prostaglandins and nitric oxide.

Steroids and phytosterols identified in the extract may also contribute to the anti-inflammatory activity observed in this study. Plant-derived steroids are known to stabilize cellular membranes and reduce release of inflammatory mediators, thereby limiting inflammatory responses. Similarly, flavonoids are well documented for their anti-oxidant activity and their ability to scavenge reactive oxygen species.

Oxidative stress is closely associated with inflammatory processes and respiratory tissue damage; therefore, the presence of flavonoids may provide protective effects against oxidative injury. Alkaloids, which were detected in moderate amounts, have also been reported to possess antimicrobial properties by interfering with bacterial DNA replications and protein synthesis. Collectively, the presence of these phytochemical constituents suggests that Piper cubeba extract contains multiple bioactive compounds capable of exerting synergistic pharmacological effects.

GC-MS analysis further confirmed the presence of several volatile phytoconstituents in the extract, including 4-epi-cubedol, β- phellandrene, ledol, furanone derivatives, and benzene derivatives. Many of these compounds belong to the sesquiterpene class, which is commonly found in essential oils of medicinal plants. Sesquiterpenes such as 4-epi-cubedol and ledol have been reported to exhibit anti-microbial and anti-inflammatory activities. Their lipophilic nature allows them to interact with biological membranes and penetrate microbial cells, there-by enhancing their pharmacological activity. The presence of both hydrocarbon and oxygenated sesquiterpenes in the extract suggests a chemically diverse composition that may contribute to the overall biological activity of the formulation.

To further explore the potential mechanisms underlying these biological activities, molecular docking studies were preformed against important protein targets associated with inflammation and bacterial survival. The selected targets included cyclooxygenase-2 (COX-2), penicillin- binding protein 2a, and DNA gyrase. COX-2 is a key enzyme involved in the synthesis of prostaglandins, which are major mediators of inflammation and pain. Docking analysis revealed that several identified compounds, particularly 4-epi-cubedol and ledol, demonstrated favorable interaction with important amino acid residues within the active site of the COX-2 enzyme. Interactions with residues such as Tyr-385, Ser-530 and Arg-120 are particularly significant because these binding and catalytic-activity during prostaglandin synthesis. The ability of the compounds to interact with these catalytic residues suggests that they may inhibit COX-2 activity by blocking substrate access or interfering with the catalytic mechanism, thereby reducing inflammatory responses.

Docking studies against penicillin- binding protein 2a (PBP2a) revealed interactions involving residues such as Arg-129, Arg-219, Glu-217, and Val-255. PBP2a is an important enzyme involved in bacterial cell wall synthesis and is associated with antibiotic resistance in certain bacterial strains, particularly Staphylococcus aureus. The observed interactions suggest that the identified phytochemicals may interfere with the structural integrity of the bacterial cell wall or alter the conformation of the enzyme, thereby inhibiting bacterial growth. Similarly, molecular docking against DNA gyrase demonstrated interactions with residues including Asp-668, Thr-315, Tyr-287, and Ile-317. DNA gyrase is an essential enzyme responsible for DNA replication and supercoiling in bacteria. Inhibition of this enzyme disrupts DNA replication processes and ultimately leads to bacterial cell death. The presence of stable ligand-protein interactions within the binding pocket supports the potential antimicrobial activity of the compounds identified in the extract.

The formulated nasal spray also demonstrated suitable physicochemical characteristics for intranasal administration. The formulation appeared clear and colorless, indicating a homogeneous preparation without particulate matter. The pH of the formulation was found to be 6.2, which falls within the physiological range of the nasal cavity and is therefore unlikely to cause mucosal irritation. Maintaining an appropriate pH is an important requirement for nasal formulations to ensure patient comfort and mucosal compatibility. Isotonicity testing confirmed that the formulation was compatible with nasal fluids, thereby reducing the risk of osmotic irritation. In addition, the formulation exhibited appropriate viscosity, allowing effective spray formation while maintaining adequate residence time on the nasal mucosa. Spray pattern evaluation revealed a fine and uniform mist, suggesting that the formulation can achieve effective distribution across the nasal cavity, which is essential for optimal therapeutic performance.

The antimicrobial activity of the formulation was evaluated against two bacterial strains commonly associated with respiratory infections, Staphylococcus aureus and Escherichia coli. The results demonstrated measurable zones of inhibition against both micro-organisms, indicating that the formulation possesses antibacterial properties. A concentration -dependent increase in antimicrobial activity was observed against Staphylococcus aureus, suggesting that the formulation may be particularly effective against Gram-positive bacteria. Gram-positive bacteria are frequently implicated in respiratory tract infections, including sinusitis and upper respiratory infections. Although the activity against Escherichia coli was comparatively lower, the presence of inhibitory zones still indicates moderate antibacterial efficacy against Gram-negative bacteria may be attributed to the presence of an additional outer membrane in these organisms, which often limits the penetration of antimicrobial compounds. Lower MBC and MIC values were observed for Staphylococcus aureus, indicating higher susceptibility of Gram-positive bacteria. The higher resistance in Escherichia coli may be due to its outer membrane barrier.

The anti-inflammatory activity of the formulation was further confirmed through the protein denaturation inhibition assay. Protein denaturation is an important mechanism involved in inflammatory processes, as denatured proteins can trigger immune response and promote the release of inflammatory mediators. In the present study, the formulation demonstrated strong inhibition of protein denaturation in a concentration-dependent manner, indicating significant anti-inflammatory potential. This activity may be attributed to the presence of terpenoids, flavonoids, and phytosterols within the extract, which are known to stabilize proteins and cell membranes while inhibiting inflammatory mediator release.

Nitric oxide scavenging activity was also evaluated to assess the anti-oxidant potential of the formulation. Nitric oxide is an important signaling molecule involved in immune response; however, excessive production of nitric oxide can contribute to oxidative stress and tissue damage during inflammatory conditions. The results of the nitric oxide inhibition assay demonstrated that the formulation exhibited strong scavenging activity, suggesting that it may help reduce oxidative stress in inflamed tissues. The antioxidant activity observed in this study is likely associated with the presence of flavonoids and terpenoids, which are well known for their free radical scavenging properties.

 The mucosal irritation study revealed no irritation at the initial stage and only mmild, transient irritation at later time points, which subsided within 48 hours. No severe inflammation or damage was observed , indicating that the formulation is safe and well-tolerated for nasal administration.

Overall, the findings of the present study indicate that the Piper cubeba seed-based nasal spray formulation possesses multiple pharmacological activities, including anti-microbial, anti-inflammatory and anti-oxidant effects. The combination of these particularly beneficial for respiratory therapy, as respiratory infections typically involve both microbial invasion and inflammatory responses.  By targeting multiple biological pathways simultaneously, the formulation may provide a comprehensive therapeutic approach for the management of respiratory infections and inflammation.

Furthermore, the favorable physicochemical characteristics of the formulation support its suitability for intranasal administration, enabling effective delivery of active compounds directly to the site of action.

CONCLUSION

The present study successfully developed and evaluated a Piper cubeba herbal nasal spray formulation intended for the management of respiratory infections and associated inflammatory conditions.

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