Development and Characterization of Proniosomal Formulation of Nepeta cataria L. Extract for Enhanced Antiallergic Activity

Table 2.1: Composition of different type of surfactant & carrier in proniosomal formulations containing Nepeta cataria

Table 2.2: Composition of different proniosomes formulations containing Nepeta cataria

2.8 Evaluation of Proniosomes

The prepared proniosomes were evaluated for physical appearance, particle morphology by optical microscopy, flow properties (angle of repose, bulk density, tapped density, Carr's index, and Hausner ratio), vesicle size, polydispersity index (PDI), zeta potential, entrapment efficiency, and transmission electron microscopy (TEM). Particle size and zeta potential were determined using dynamic light scattering, while vesicle morphology was examined using TEM.

2.9 In Vitro Drug Release

In vitro release studies were performed using USP Dissolution Apparatus II containing 900 mL of phosphate buffer (pH 6.8) maintained at 37 ± 0.5°C and stirred at 50 rpm. Samples were withdrawn at predetermined intervals and replaced with fresh medium. Drug concentration was determined spectrophotometrically.

2.10 Drug Release Kinetics

The release data were fitted to Zero-order, First-order, Higuchi, and Korsmeyer–Peppas kinetic models to determine the release mechanism and kinetics of the optimized proniosomal formulation.

2.11 Statistical Analysis

All experiments were performed in triplicate (n = 3), and results were expressed as mean ± standard deviation (SD). Statistical analysis was carried out using one-way ANOVA followed by Tukey's post hoc test. A value of p < 0.05 was considered statistically significant.

2.12 Percentage Yield of Various Extracts of Nepeta cataria Leaves

The extraction of phytoconstituents from medicinal plants is a crucial step in herbal drug research, as the efficiency of extraction directly influences the recovery of bioactive compounds responsible for pharmacological activity. In the present investigation, dried leaves of Nepeta cataria were subjected to successive Soxhlet extraction using solvents of increasing polarity, namely petroleum ether (60–80°C), ethyl acetate, and distilled water. The successive extraction method was adopted to ensure the selective extraction of compounds based on their polarity and to maximize the recovery of phytochemicals.

The percentage yield obtained from different solvent extracts is presented in Table 2.3. Among the three extracts, the aqueous extract exhibited the highest percentage yield (15.8% w/w), followed by the ethyl acetate extract (10.6% w/w), whereas the petroleum ether extract showed the lowest yield (4.2% w/w).

Table 2.3 Percentage yield of various extracts of Nepeta cataria leaves

The relatively low yield observed with petroleum ether can be attributed to its non-polar nature, which mainly extracts lipophilic constituents such as fixed oils, waxes, chlorophyll pigments, and certain terpenoids. Since Nepeta cataria contains comparatively lower amounts of non-polar constituents, the recovery obtained with petroleum ether was limited. Similar observations have been reported in previous phytochemical investigations of members of the Lamiaceae family, where petroleum ether extracts generally exhibit lower extraction efficiency than semi-polar and polar solvents.

The ethyl acetate extract produced a moderate extraction yield (10.6% w/w), indicating the presence of a considerable amount of semi-polar phytoconstituents. Ethyl acetate is particularly effective for extracting flavonoids, phenolic acids, tannins, coumarins, and triterpenoids. These classes of compounds are recognized for their antioxidant, anti-inflammatory, antihistaminic, and immunomodulatory activities. Since the present research aimed to develop a proniosomal formulation for antiallergic activity, the recovery of these biologically active constituents was considered more important than obtaining the highest extraction yield.

The aqueous extract demonstrated the highest extraction yield (15.8% w/w), reflecting the abundance of hydrophilic constituents such as carbohydrates, proteins, amino acids, glycosides, polysaccharides, and certain phenolic compounds. Although the aqueous extract yielded the greatest quantity of extractable material, phytochemical screening revealed that many of these constituents are not directly responsible for the desired antiallergic activity.

The difference in extraction yield clearly demonstrates the influence of solvent polarity on the extraction process. Polar solvents generally possess higher penetration capacity into plant tissues and effectively dissolve polar metabolites through hydrogen bonding interactions. In contrast, non-polar solvents are selective for lipophilic constituents and therefore produce lower extraction yields.

The present findings are consistent with previous studies, which reported that methanol, ethanol, and water extracts of Nepeta cataria exhibit significantly higher extraction yields than petroleum ether extracts because of the predominance of phenolic compounds and flavonoids in the plant. Similar extraction behaviour has also been reported for other medicinal plants belonging to the family Lamiaceae.

Overall, the extraction study indicates that although the aqueous extract produced the highest percentage yield, the ethyl acetate extract contained a greater concentration of pharmacologically active constituents. Therefore, the ethyl acetate extract was selected for further phytochemical investigation, isolation of bioactive compounds, and proniosomal formulation development.

2.13 Preliminary Phytochemical Screening of Various Extracts

Preliminary phytochemical screening is one of the most important steps in herbal drug research because it provides qualitative information regarding the chemical constituents present in plant extracts. Identification of phytochemical classes helps establish a relationship between the observed pharmacological activity and the bioactive constituents responsible for the therapeutic effect.

In the present study, qualitative phytochemical analysis was carried out using standard chemical tests to detect the presence of alkaloids, carbohydrates, glycosides, steroids, triterpenoids, flavonoids, tannins, proteins, saponins, coumarins, and fixed oils in different solvent extracts of Nepeta cataria. The results are summarized in Table 2.4.

The petroleum ether extract tested positive only for fixed oils, while all other phytochemical tests were negative. This observation indicates that petroleum ether primarily extracted non-polar constituents, including lipids and fatty substances. These compounds contribute little to the intended antiallergic activity and therefore the petroleum ether extract was not considered suitable for further investigation.

The ethyl acetate extract showed positive reactions for flavonoids, triterpenoids, tannins, and saponins. The presence of these phytoconstituents is of considerable pharmacological significance because each class is associated with important biological activities.

Flavonoids are among the most extensively studied natural antioxidants. They inhibit mast-cell degranulation, suppress histamine release, reduce eosinophil infiltration, and inhibit the synthesis of inflammatory mediators such as prostaglandins and leukotrienes. Their ability to stabilize mast cells makes them valuable candidates for the treatment of allergic disorders.

Triterpenoids possess potent anti-inflammatory properties through inhibition of cyclooxygenase (COX) and lipoxygenase pathways. They also suppress the production of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6. These mechanisms contribute significantly to the reduction of allergic inflammation.

Tannins exhibit excellent antioxidant activity by scavenging reactive oxygen species generated during inflammatory responses. Oxidative stress plays a major role in allergic diseases, and therefore tannins indirectly contribute to the reduction of tissue damage associated with chronic inflammation.

Saponins possess immunomodulatory activity and are reported to regulate both humoral and cell-mediated immune responses. Several studies have demonstrated that saponins reduce inflammatory edema and inhibit the release of allergic mediators from activated mast cells.

The aqueous extract showed positive tests for carbohydrates, proteins, and saponins. These constituents contribute to the nutritional and medicinal value of the plant but are considered less significant for the targeted antiallergic activity when compared with phenolic compounds and flavonoids.

Among all three extracts, the ethyl acetate fraction demonstrated the richest composition of pharmacologically important phytoconstituents. Therefore, it was selected for further chromatographic isolation of active compounds and subsequent formulation studies.

The phytochemical profile obtained in the present investigation agrees with previously published reports on Nepeta cataria. Several researchers have identified flavonoids, rosmarinic acid, caffeic acid derivatives, nepetalactones, iridoids, and phenolic compounds as the major bioactive constituents responsible for the plant's antioxidant, anti-inflammatory, antimicrobial, and antiallergic activities.

These findings validate the selection of the ethyl acetate extract as the most suitable candidate for the development of a proniosomal drug delivery system. The abundance of flavonoids and phenolic compounds in this extract is expected to contribute significantly to improved therapeutic efficacy against allergic disorders.

Table 2.4: Preliminary phytochemical screening of various extracts of Nepeta cataria leaves

+: present, -: absent

2.14 Characterization of the Isolated Compound (NC-1)

Following phytochemical screening, the ethyl acetate extract was subjected to column chromatography for the isolation of bioactive constituents. Fractions showing similar thin-layer chromatographic (TLC) profiles were pooled, concentrated, and purified to obtain the isolated compound designated as NC-1.

The isolated compound was characterized using ultraviolet (UV) spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. These analytical techniques provide valuable information regarding chromophoric groups and functional groups present in the isolated molecule.

The UV spectrum of NC-1 exhibited a characteristic absorption maximum corresponding to conjugated aromatic systems, indicating the presence of a phenolic compound. The observed UV absorption pattern was consistent with hydroxycinnamic acid derivatives reported in the literature.

FTIR spectral analysis further confirmed the chemical identity of the isolated compound. A broad absorption band observed around 3398 cm⁻¹ indicated hydroxyl (–OH) stretching vibration, confirming the presence of phenolic hydroxyl groups. The absorption peak at 1670 cm⁻¹ corresponded to carbonyl (C=O) stretching of the carboxylic acid group. Characteristic aromatic C=C stretching bands were observed at 1601 cm⁻¹ and 1510 cm⁻¹, while peaks between 1314–1212 cm⁻¹ represented C–O stretching vibrations. Additional absorption bands in the fingerprint region further supported the presence of substituted aromatic structures.

Comparison of the obtained spectral data with published literature revealed excellent agreement with the reported spectra of p-coumaric acid. The melting point of the isolated compound also corresponded to the standard melting range of p-coumaric acid, confirming its identity.

The isolation of p-coumaric acid is of particular significance because this naturally occurring phenolic acid exhibits strong antioxidant, anti-inflammatory, and antihistaminic properties. It has been reported to inhibit mast-cell activation, suppress histamine release, reduce oxidative stress, and regulate inflammatory signaling pathways such as NF-κB. These pharmacological actions strongly support its role as one of the active constituents responsible for the traditional medicinal use of Nepeta cataria in allergic and inflammatory disorders.

The successful isolation and characterization of p-coumaric acid also validated the effectiveness of the extraction and chromatographic procedures employed in the present study. Moreover, the identification of this bioactive marker compound provides scientific evidence for the therapeutic potential of Nepeta cataria and justifies its incorporation into a proniosomal drug delivery system aimed at enhancing bioavailability and sustained drug release

3. Preformulation Studies

3.1 UV–Visible Spectral Analysis

The UV–visible spectrum of the isolated compound (NC-1) exhibited a characteristic absorption maximum corresponding to the conjugated phenolic structure of the molecule (Figure 3.1). The observed λmax was consistent with the reported spectral characteristics of p-coumaric acid, indicating the presence of an aromatic cinnamic acid derivative. The UV spectral data, together with the melting point and FTIR analysis, supported the preliminary identification of NC-1 as p-coumaric acid.

Figure 3.1: UV spectra of Nepeta Cataria Extract

3.2 FTIR Analysis

The FTIR spectrum of NC-1 (Figure 3.2) confirmed the presence of the characteristic functional groups of p-coumaric acid. A broad absorption band at 3398.16 cm⁻¹ corresponded to hydroxyl (–OH) stretching, while peaks at 2819.69 and 2576.05 cm⁻¹ were assigned to the O–H stretching vibration of the carboxylic acid group. The strong peak at 1670.88 cm⁻¹ represented carbonyl (C=O) stretching of the carboxylic acid. Aromatic C=C stretching vibrations appeared at 1601.83 and 1510.15 cm⁻¹, whereas peaks between 1314.46–1212.98 cm⁻¹ corresponded to C–O stretching vibrations. The fingerprint region showed characteristic aromatic C–H bending peaks at 977.73, 940.08, 831.70, and 517.70 cm⁻¹ (Table 3.1). These spectral features were in agreement with published data for p-coumaric acid, confirming the identity of NC-1.

3.3 FTIR analysis

Figure 3.2: IR Spectra of NC-1

Table 3.1: IR spectral data results of NC-1

The chemical structure of NC-1 was characterized as p-coumaric acid based on melting point, UV, IR spectral data.

4. Evaluation of Nepeta cataria Proniosomes

4.1 Physical Appearance and Morphology

Figure 4.1: Nepeta cataria proniosome powder

Discussion: The above figure 4.1 shows, that Nepeta cataria proniosome is off white, free flowing powder.

1. 2. Optical microscopy

Figure 4.2: Optical Microscopy of Nepeta cataria proniosome powder

The prepared proniosomal formulation was obtained as an off-white, dry, and free-flowing powder without evidence of aggregation (Figure 4.2). Optical microscopic examination revealed predominantly spherical particles with a relatively uniform size distribution and slightly rough surface morphology. The absence of particle agglomeration suggested successful preparation of stable proniosomal powder.

4.3 Entrapment Efficiency

Table 4.1: Drug Entrapment of formulations (F1-F8)

Figure 4.3: Drug Entrapment of formulations (F1-F8)

The percentage entrapment efficiency of formulations F1–F8 ranged from 65.28 ± 0.15% to 88.97 ± 0.17%. Formulation F3 exhibited the highest entrapment efficiency (88.97 ± 0.17%), whereas F1 showed the lowest (65.28 ± 0.15%). The enhanced entrapment observed in F3 may be attributed to the optimized surfactant-to-cholesterol ratio, which improves bilayer organization and minimizes drug leakage. The results indicate that formulation composition significantly influenced drug encapsulation efficiency, with F3 demonstrating superior vesicle-forming ability.

4.4 Particle Size and Zeta Potentia

Figure 4.4: Particle size of proniosomes

Figure 4.5: Zeta Potential of proniosomes

Dynamic light scattering analysis showed that the optimized proniosomal formulation possessed an average particle size of 268.5 nm, indicating the formation of nanosized vesicles suitable for enhanced topical delivery. The zeta potential was determined to be −32.5 mV, suggesting good colloidal stability due to sufficient electrostatic repulsion between vesicles. The nanoscale particle size combined with high zeta potential indicates that the optimized formulation is expected to exhibit improved physical stability and efficient drug delivery.

4.5 Micromeritic Properties

Characterization of different formulations of proniosome powder containing drug was given in table 5.5.

Table 4.2: Characterization of different formulations of proniosome powder

Figure 4.6: Characterization of proniosome powder

The micromeritic properties of proniosomal powder formulations are presented in Table 4.2. The angle of repose ranged from 26.80° to 39.85°, indicating acceptable to excellent flow properties. Bulk density varied between 0.41 and 0.62 g/cm³, while tapped density ranged from 0.50 to 0.80 g/cm³. Hausner's ratio and Carr's compressibility index ranged from 1.07–1.32 and 7.50–22.10%, respectively.

Among all formulations, F6 exhibited the lowest Hausner's ratio (1.07) and Carr's index (7.50%), indicating excellent flowability and compressibility. Although F7 showed the lowest angle of repose (26.80°), the overall micromeritic evaluation suggested that F6 possessed the most favourable powder flow characteristics. The acceptable micromeritic properties observed for all formulations indicate their suitability for pharmaceutical processing and handling.

1. 6. Transmission electron microscope (TEM)

Figure 4.7: TEM Image of Formulation F3

1. 7. In vitro drug release studies

Table 4.3: In vitro drug release of Nepeta cataria proniosome formulation F3 & pure drug

Figure 4.8: Percentage drug release of Nepeta cataria proniosome formulation F3 &Gel

Discussion: The in-vitro drug release data of the pure drug (control gel) and the proniosomal formulation (F3) of Nepeta cataria demonstrate a clear distinction in their release behaviour across different dissolution media.

In 0.1 N HCl (acidic medium), the control gel exhibited a rapid and immediate drug release pattern. Approximately 15.842 ± 0.712% drug was released within 0.25 h, which increased sharply to 95.873 ± 1.084% within 2 hours, indicating that the pure drug lacks any release-retarding mechanism and is readily available for dissolution in gastric conditions.

In contrast, the proniosomal formulation F3 showed a significantly slower release in the same medium. Only 8.964 ± 0.845% drug was released at 0.25 h, gradually increasing to 24.986 ± 1.102% at 2 h. This reduced release in acidic pH can be attributed to the encapsulation of the drug within proniosomal vesicles composed of surfactant and cholesterol. These vesicles act as a barrier, restricting drug diffusion and protecting the drug from immediate release in the gastric environment.

4.8 Drug release kinetic studies

4.8.1 Zero order kinetics

Figure 4.9: Zero order graph of formulation F3

4.8.2 First order kinetics

Figure 4.10: First order graph of formulation F3

4.8.3. Higuchi’s Model

Figure 4.11: Higuchi order graph of formulation F3

4.8.4 Korsmeyer-Peppas Model

Figure 4.12: Korsmeyer-Peppas order graph of formulation F3

Table 4.4: Kinetic equation parameter of formulation F3