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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access Full Text Article   Research Article

Formulation and evaluation of Niosomal cream from moringa leaf extract for enhanced Antifungal Activity

Pratiksha K. Hajare *, Mahesh R. Reddy, Yogesh D. Somawanshi , Vedashree A. Dhumekekar 

Department of Pharmacology PES’s Modern College of Pharmacy Nigdi, India

 

 

INTRODUCTION:

The increasing prevalence of fungal infections represents a critical challenge to global health systems, affecting populations through both superficial skin conditions and severe systemic diseases¹. Cutaneous infections such as ringworm and yeast-related candidiasis impact millions annually, while immunocompromised groups—including HIV/AIDS patients, organ transplant recipients, and individuals undergoing chemotherapy—face elevated risks of invasive infections like aspergillosis and cryptococcal meningitis ². Epidemiological studies reveal that superficial fungal infections affect nearly 1.7 billion people worldwide, with dermatophyte infections alone impacting 20-25% of the global population³.  

Diagnostic complexities and escalating antifungal resistance, fueled by genetic adaptations and biofilm production, hinder effective management of these infections⁴. Conventional therapies face limitations due to adverse effects; azole antifungals, for example, carry risks of liver damage and drug interactions through cytochrome P450 enzyme inhibition⁶. Nephrotoxic effects plague polyene drugs like amphotericin B, while allylamines such as terbinafine often trigger gastrointestinal or cutaneous side effects⁷. Rising resistance patterns further diminish treatment efficacy, with fluconazole-resistant Candida strains now reported in 20-30% of cases in some regions⁸. Topical drug delivery remains suboptimal due to the skin’s lipid-rich stratum corneum barrier, which restricts penetration of antifungal agents into deeper tissue layers⁹.  

Fungal pathogens pose distinct therapeutic challenges compared to bacterial or viral infections. Their eukaryotic biology shares cellular processes with human hosts, narrowing the scope for selective drug targets and increasing treatment-related toxicity risks¹⁰. Chronic infection courses demand extended therapies, amplifying concerns about medication adherence and cumulative side effects¹¹.  

Moringa oleifera, a plant with extensive traditional medicinal applications, shows promise in addressing these challenges. Its leaves contain bioactive compounds—including isothiocyanates (e.g., sulforaphane), flavonoids (quercetin, kaempferol), saponins, and alkaloids—that demonstrate antifungal properties through multiple mechanisms, such as cell membrane disruption and inhibition of critical fungal enzymes^13,14. Laboratory studies confirm its activity against Candida, Aspergillus, and dermatophytes, with animal models supporting its therapeutic potential¹⁵.  

This plant’s selection for antifungal research is supported by its global availability, eco-friendly cultivation, favorable safety profile, and broad-spectrum activity¹⁶. Its suitability for topical formulations addresses the need for localized treatment of superficial infections while minimizing systemic exposure, offering a potential solution to current therapeutic limitations¹⁷.

Niosomal Drug Delivery Systems

Niosomes, vesicular systems formed from non-ionic surfactants, have emerged as an innovative approach for topical drug delivery due to their biocompatibility and versatility ¹⁸. These biodegradable structures can encapsulate both water-soluble and lipid-soluble compounds, making them effective carriers for plant-based therapeutics like Moringa extracts ¹⁹. Compared to liposomes, niosomes demonstrate superior stability and scalability, with surfactants such as Span and Tween enabling customization of vesicle characteristics ^20,21. By enhancing drug solubility and promoting fusion with skin lipids, niosomes overcome the stratum corneum barrier, improving localized delivery while protecting encapsulated agents from degradation ^22,23.  

MATERIALS:

Table 1: Ingredients and its roles

Ingredients	Role
Moringa leaf extract	API
Span 60	Surfactant
Cholestrol	Membrane stabilizer
Ethanol	Solvent
Beeswax	Emulsifier
Borax	Stabilizer
Triethanolamine	pH adjuster
Methyl paraben	Preservative
Rose oil	Fragnance
Distilled water	Vehicle

 

Table 2: ingredients with optimized batch

Ingredient	F1	F2	F3	Optimized
Moringa leaf extract	0.5	0.6	0.7	0.6
Span 60	1	1.2	1.4	1.2
Cholestrol	0.4	0.5	0.6	0.5
Beeswax	1	0.9	1	0.9
Borax	0.2	0.2	0.2	0.2
Triethanolamine	0.3	0.2	0.1	0.2
Methyl paraben	0.2	0.2	0.2	0.2
Liquid paraffin	0.8	0.9	1	0.9
Rose oil	1	1	1	1
Distilled water	q.s.	q.s.	q.s.	q.s.

 

 

Methodology:

1. Preparation of Moringa Leaf Extract  

Fresh Moringa oleifera leaves were authenticated, air-dried in shaded conditions (25 ± 2°C) for one week to preserve thermolabile constituents ²⁴, and pulverized into a fine powder using an electric grinder. The powder was sieved through a 60-mesh screen to ensure uniformity ²⁵. For extraction, 100 g of powder underwent cold maceration in 500 mL of 95% ethanol for 72 hours with periodic agitation ²⁶. The mixture was filtered through Whatman No. 1 filter paper, and the residue was re-extracted twice with fresh solvent to maximize phytochemical recovery ²⁷. The combined filtrates were concentrated using a vacuum rotary evaporator (Büchi Rotavapor R-210) at 40°C under reduced pressure, followed by desiccation in a vacuum chamber to yield a solvent-free extract ²⁸. The dried extract (yield calculated gravimetrically) was stored in sealed containers at 4°C ²⁹. 

2. Niosome Synthesis (Thin-Film Hydration)  

1. Span 60, cholesterol, and Moringa extract were dissolved in 10 mL ethanol within a round-bottom flask ³⁰.  

2. Ethanol was evaporated using a rotary evaporator (60 ± 2°C, 25 mmHg, 120 rpm) to form a thin lipid film ³¹.  

3. The film was hydrated with 10 mL phosphate-buffered saline (pH 7.4) under gentle agitation (room temperature, 1 hour) to form niosomes ³². 

4. The dispersion was sonicated (Sonics Vibra Cell probe sonicator) for 5 minutes (40% amplitude, 2s on/1s off pulses) to homogenize vesicle size ³³.   

5. The final niosomal suspension was refrigerated (4°C) for stability ³⁴.   

 

Figure 1: Moringa leaf extract

 

Figure 2: Niosome preparation by rotary evaporator

3. Cream Development 

Oil Phase: Beeswax and liquid paraffin were melted (70 ± 2°C water bath) ³⁵.  

Aqueous Phase: Borax and methyl paraben were dissolved in heated distilled water (70 ± 2°C) ³⁶.  

Emulsification: The aqueous phase was gradually incorporated into the oil phase under high-shear mixing (Remi Motors stirrer, 1500 rpm) ^37.  

Cooling: Triethanolamine was added during cooling (room temperature) with continuous stirring to form a stable emulsion ³⁸.  

Niosome Integration: Niosomal dispersion was blended into the cream base using gentle spatulation to preserve vesicle integrity ³⁹.  

Finalization: Rose oil (fragrance) was incorporated, followed by homogenization (IKA T25 homogenizer, 3000 rpm, 5 minutes) ^40. The cream was stored in airtight containers at ambient conditions ⁴¹.  

Phytochemical Analysis of Extract

Test for Alkaloids  

-Dragendroff’s Test: A volume of 2 mL of the extract solution was mixed with several drops of Dragendroff’s reagent (potassium bismuth iodide solution). The appearance of an orange-red precipitate was interpreted as evidence of alkaloid presence ⁴².  

- Hager’s Test: To 2 mL of the extract solution, a few drops of Hager’s reagent (saturated picric acid solution) were added. The formation of a yellow precipitate was indicative of alkaloids ⁴³.  

Test for Flavonoids  

A 3 mL aliquot of the extract solution was combined with 10 mL of distilled water and shaken. Subsequently, 1 mL of 10% sodium hydroxide was added. The emergence of a yellow hue confirmed the presence of flavonoids ⁴⁴.  

Test for Saponins  

Frothing Test: The extract solution (3 mL) was diluted with 2 mL of distilled water in a test tube and vigorously shaken for 5 minutes. A stable foam layer at the surface signaled the presence of saponins ⁴⁵.  

Test for Steroids  

Chloroform (2 mL) was added to 2–3 mL of the extract solution, followed by careful layering of 1–2 mL of concentrated sulfuric acid. A reddish-brown interface between the layers indicated steroids. Further addition of 1–2 mL of acetic anhydride produced a reddish-pink hue, confirming steroid presence ⁴⁶.   

Test for Tannins  

The extract solution (2 mL) was gently heated for 2 minutes, cooled, and treated with three drops of 5% ferric chloride. A greenish-black or bluish-black hue confirmed tannin content ⁴⁷. 

 

 

Figure 3: Phytochemical test of extract

 

Characterization of Niosomal Cream  

Particle Size  

The niosomal dispersion was diluted 1:100 with filtered deionized water to prevent multiple scattering effects. Measurements were conducted at 25°C with a 90° detection angle, with triplicate analyses performed for each sample ⁴⁸.  

Entrapment Efficiency (EE)  

EE was assessed via ultracentrifugation to separate unentrapped extract from niosomes. The supernatant was analyzed spectrophotometrically to calculate the percentage of entrapped extract ^49,50.  

Zeta Potential (ZP)  

ZP was measured using a Zetasizer Nano ZS (Malvern Instruments, UK). Samples were diluted 1:100 with filtered deionized water and analyzed at 25°C under 20 V/cm field strength. ZP values exceeding ±30 mV was considered indicative of stable formulations ⁵¹.  

Figure 4: Graph of zeta potential

pH   

The pH of a 5% aqueous cream solution was measured using a digital pH meter (Eutech pH 700, Singapore) to ensure alignment with the skin’s natural pH range (5.0–6.5) ⁵².  

Viscosity  

Viscosity was determined using a Brookfield viscometer (LVDV-II+Pro, USA) with spindle #64 at 25 ± 1°C. Rheological behavior was evaluated across varying shear rates ⁵³.  

Spreadability  

A fixed quantity of cream was placed between two glass plates, and the spread diameter was measured under standardized weight to assess spreadability ⁵⁴.  

Washability  

A small cream sample applied to the skin was rinsed with water to evaluate ease of removal⁵⁵.  

In Vitro Drug Diffusion Study  

Figure 5: Invitro cell diffusion study

An egg membrane model was prepared by acid treatment and equilibration in phosphate buffer. Drug release kinetics were analyzed using a diffusion cell setup, with samples collected at intervals and quantified spectrophotometrically ^56,57  

Stability Studies  

Optimized formulations were stored at 25 ± 2°C (room temperature) and 4 ± 2°C (refrigeration) for three months to assess physical and chemical stability ⁵⁸.  

Antifungal Activity  

Antifungal effects against Candida albicans were tested via agar diffusion on Sabouraud dextrose agar, with inhibition zones measured to gauge efficacy ⁵⁹.

Figure 6: Antifungal study of formulation

RESULT AND DISCUSSION:

Calibration Curve: 

Table 3: Calibration curve

Concentration	Absorbance
2	0.4234
4	0.5281
6	0.6202
8	0.7497
10	0.8397

 

Figure 7: Graph of calibration curve

Comparative Study:

Table 4: Comparative study of formulations

Parameter	Normal cream	Standard	Niosomal Cream
Appearance	Smooth	smooth	Smooth
pH	5.5	5.4	5.5
Viscosity(cpa)	1820	980	570
Particle Size	180nm	200nm	155nm
Entrapment efficacy	NA	70	74
Zeta potential	+18	+20	+25
Spreadability	Good	Good	Good
Irritancy	None	None	None
Washability	Easily washable	Easily washable	Easily washable
Stability	Stable	Stable	Stable
Invitro drug release	65.94	75	79
Antifungal activity (Zone of inhibition)	1.53	1.9	2.5

 

 

Acknowledgement: NA

Conflict of Interest: The authors declares there is no conflict of interest. 

Author Contributions: All authors have equally contributed.

Source of Support: Nill

Funding: NA

Informed Consent Statement: Not applicable.

Ethics Approval: Not Applicable 

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