DEVELOPMENT AND IN-VITRO CHARACTERIZATION OF LIPOSOMAL GEL FORMULATION CONTAINING DRIED LEAF EXTRACT OF PLANTS ACACIA LUCOPHLOLEA AND EUPHORBIA CYTHOPHORA AND ITS COMPARATIVE ANTIBACTERIAL STUDY

DEVELOPMENT AND IN-VITRO CHARACTERIZATION OF LIPOSOMAL GEL FORMULATION CONTAINING DRIED LEAF EXTRACT OF PLANTS ACACIA LUCOPHLOLEA AND EUPHORBIA CYTHOPHORA AND ITS COMPARATIVE ANTIBACTERIAL STUDY

Shifa Mansuri *, Prithu Pathak, Trapti Shrivastava and Kuldeep Ganju

Sagar Institute of Pharmacy & Technology, Bhopal, Madhya Pradesh, India.

ABSTRACT: Background: Plant-derived bioactive offer promising alternatives for antimicrobial therapy, but their poor solubility and stability often limit topical application. Liposomal encapsulation can enhance their therapeutic potential. Objective: This study aimed to develop and characterize liposomal gel formulations containing ethanolic extracts of Acacia leucophloea and Euphorbia cyathophora leaves, and to evaluate their antibacterial efficacy. Methods: Leaves of both plants were extracted using ethanol and petroleum ether. Phytochemical screening identified alkaloids, glycosides, flavonoids, tannins, carbohydrates, and saponins predominantly in ethanolic extracts. Liposomes were prepared and characterized for particle size, zeta potential, and morphology. The optimized liposomal formulation (F5) was incorporated into a topical gel and evaluated for physicochemical properties, spreadability, and antibacterial activity using the agar well diffusion method. Results: The ethanolic extract yields were 2.31% for A. leucophloea and 2.16% for E. cyathophora. Formulation F5 showed the smallest particle size (42.53 nm), suitable zeta potential (-13.8 mV), and spherical morphology. The liposomal gel exhibited a pH of 6.8, viscosity of 1549 ± 0.26 cps, and good spreadability (34.26 g·cm/sec) without skin irritation. Antibacterial activity was significantly enhanced, with inhibition zones of 12 mm (against E. coli) and 8 mm (against S. aureus), compared to 5 mm and 3 mm for crude extracts. Conclusion: The developed liposomal gel demonstrated superior antibacterial efficacy and favorable physicochemical properties, supporting its potential as a plant-based topical therapeutic formulation.

Keywords: Acacia leucophloea, Euphorbia cyathophora, Liposomal gel, Phytochemical screening, Antibacterial activity, Liposome characterization, Topical formulation

INTRODUCTION: Liposomes have been extensively studied as drug carrier systems for parenteral delivery and, more recently, for topical applications such as dermal and ophthalmic treatments. When incorporated into topical dosage forms, liposomes can enhance localized drug delivery, improve solubility of poorly soluble drugs, and provide sustained release with minimal systemic absorption ¹.

Their ability to act as penetration enhancers and micro-reservoirs makes them promising vehicles for various therapeutic agents, including antibiotics, antifungals, and anticancer drugs ². Euphorbia cyathophora is a medicinal plant known for its diverse pharmacological activities, attributed to its rich content of bioactive compounds such as flavonoids, terpenoids, alkaloids, and phenolic acids.

Extracts from Euphorbia species have demonstrated significant anti-inflammatory, antioxidant, antimicrobial, and antitumor properties, highlighting their therapeutic relevance and potential for drug development ^{3, 4}. Similarly, Acacia leucophloea (Roxb.) Willd., belonging to the family Mimosaceae, has been widely used in traditional and Ayurvedic medicine. Various parts of this plant possess antimicrobial, anti-inflammatory, and wound-healing properties. The bark and leaves are particularly valued for treating skin infections, ulcers, and respiratory ailments due to their astringent and antibacterial effects ^{5, 6}. Considering the therapeutic potential of these plants, liposomal encapsulation may enhance the stability, solubility, and skin penetration of their bioactive constituents. Therefore, the objective of this study was to develop and characterize liposomal gel formulations containing ethanolic extracts of Acacia leucophloea and Euphorbia cyathophora leaves and to evaluate their physicochemical properties and antibacterial activity for potential topical application.

MATERIALS AND METHODS:

Chemicals: Nitroprusside, ammonia, and sodium hydroxide were procured from Merck (India). Ethanol (analytical grade) was obtained from Molychem. Concentrated sulfuric acid (H₂SO₄) was purchased from Fizmerck. All other solvents, reagents, and chemicals used were of analytical reagent (AR) grade and procured from Rankem, Clorofiltind, and HiMedia Laboratories.

Plant Collection and Authentication: Fresh leaves of Acacia leucophloea and Euphorbia cyathophora were collected, cleaned, and shade-dried at room temperature for three days. The dried leaves were stored in airtight glass containers under cool, dry conditions to prevent contamination and degradation. The plant materials were authenticated by a qualified taxonomist, and voucher specimens were deposited for future reference ⁷.

Extraction Process: Acacia leucophloea and Euphorbia cythophora plant leaves were extracted using a systematic approach that involved a continuous hot percolation process with ethanol and petroleum ether in a Soxhlet apparatus. The plant leaves were initially dried in shade before being powdered into a fine powder. A weighed amount of powdered material (300g) was placed in a porous thimble and loaded into the Soxhlet extractor. Petroleum ether was accustomed to extract non-polar compounds at 60°C, followed by ethanol for polar compounds. A round-bottom flask was filled with 300-500 mL of relevant solvent and heated in a water bath. As the solvent evaporated, it condensed in the reflux condenser and permeated the plant material, dissolving bioactive substances. The procedure of extraction was continued for 6-8 hours until the solvent in the siphon tube became colourless. After extraction, using a rotary evaporator, the solvent was removed, and the concentrated extract was dried at 40 °C in a rotating vacuum evaporator (Buchi type). The dried extract was weighed, and the percentage yield was calculated using the following formula:

% Yield = Weight of extract / Weight of plant material used × 100

The dried extract was then held at low temperatures for further phytochemical examination and organoleptic properties (percentage yield, color, and smell) ⁸.

The Organoleptic Studies: Organoleptic studies of Acacia leucophloea and Euphorbia cythophora both plant extract such as general appearance, color, odour, and condition, were conducted and observed.

Solubility Study: The qualitative solubility of Acacia leucophloea and Euphorbia cythophora both plant extract in various solvents was examined utilizing Indian pharmacopoeia. Acacia leucophloea and Euphorbia cythophora both plant extract were weighed and placed into separate 10 ml test tube, where it was dissolved in the appropriate solvents (1 ml each of methanol, DMSO, distilled water, chloroform, and acetone).

Preliminary Phytochemical Screening: Qualitative phytochemical tests were performed to detect alkaloids, flavonoids, tannins, saponins, glycosides, phenols, terpenoids, and steroids using standard procedures. Changes in color or precipitate formation confirmed the presence of specific phytoconstituents ⁹.

Preparation of Liposomes Formulation: Liposomes were prepared by the thin-film hydration technique using a rotary evaporator. Five formulations (LSF1–LSF5) were prepared by varying the concentrations of soya lecithin (200–600 mg) and cholesterol (50–250 mg), while maintaining a constant extract ratio (1:1, 500 mg each of A. leucophloea and E. cyathophora).

The lipid mixture (lecithin, cholesterol, and extracts) was dissolved in 8 mL chloroform and 2 mL ethanol and evaporated under reduced pressure at 40°C (900 mm Hg, 80 rpm) for 30 minutes to form a thin lipid film. The film was hydrated with 10 mL phosphate buffer (pH 6.8) for 2 hours to form a milky liposomal suspension, which was centrifuged at 3000 rpm for 30 minutes and stored in airtight containers ¹⁰.

Compositions:

TABLE 1: COMPOSITION OF LIPOSOME FORMULATION

Characterization Parameters of Liposomes Formulation:

Physical Properties of Liposomes by Visual Observation: After preparation, the liposomal suspension was examined for color, homogeneity, and phase separation ¹¹.

Size Distribution or Particle size: The resulting liposome formulations particle size was examined utilizing a Malvern Zetasizer based on Dynamic Light Scattering (DLS) ¹².

Zeta Potential: A Malvern Zetasizer was used to analyze the manufactured liposome formulations zeta potential in order to evaluate surface charge and stability.

Scanning Electron Microscopic (SEM): The morphological properties of optimized liposome were determined using an electron beam from a scanning electron microscope. A vacuum sputter coater was then utilized to apply a thin (2-20 nm) layer of metal, such as platinum, palladium, or gold, to the liposome. The pretreatment specimen was then bombarded by an electron beam, which generated secondary electrons known as augers. Rutherford and Kramer's Law was accustomed to select and process just the electrons scattered at a 90° angle from this interaction between the electron beam and the specimens atoms to obtain surface topography images ¹³.

Formulation of Liposomal Gel: The optimized liposomal formulation (LSF5) was incorporated into a gel base using Carbopol-940 and Carboxymethyl cellulose. Carbopol-940 (1 g) was soaked in 50 mL warm water and stirred for 2 hours. A separate dispersion of CMC (1 g), methyl paraben (0.2 mL), and propylene glycol (0.5 mL) was prepared and mixed with the Carbopol dispersion. The pH was adjusted with triethanolamine (TEA) to form a smooth gel, and 10 mL of liposomal suspension was incorporated with continuous stirring until uniform.

TABLE 2: COMPOSITION OF LIPOSOMAL GEL FORMULATION

Characterization Parameters of Extract Loaded Liposomal Gel Formulation:

Physical Appearance: The formulation was inspected for color, clarity, uniformity, and the existence of any phase separation, grittiness, or particulate matter ¹⁴.

Measurement of pH: A calibrated digital pH meter was used to measure the extract-loaded liposomal gels pH.

Determination of Viscosity: A Brookfield viscometer was used to measure the liposomal gels viscosity ¹⁵.

Spreadability of Extract Loaded Liposomal Gel: A topical gel that is placed or rubbed over the skin surface should have a high spreading coefficient. About 1 g of formulation was put on a glass slide to evaluate this. The gel was sandwiched between the two glass slides and distributed at a predetermined distance by placing a 50 mg bulk on top of another glass slide that was the same length as the first one. It was noted how long it took the gel to move a specific distance from its starting point.

The spread ability was determined by the following formula.

S= M×L/T

Where, S-Spread ability, g.cm/s M-Weight put on the glass slides upper L-length T-Time for spreading gel in sec ¹⁶.

Anti-microbial Activity of Liposomal Gel Formulation by well Diffusion Assay:

Preparation of Nutrient Agar Media: 2.8 g of Nutrient Media was dissolved in 100 mL of distilled water. The pH of the media was measured before sterilization.

To ensure disinfect the media, it was autoclaved for 15 min at 121 °C under 15 pounds of pressure. After pouring nutritional medium into plates, the plates were placed in laminar air flow until the agar became solid.

Well Diffusion Assay: E. coli and S. aureus bacterial suspensions were added to the shaker at concentration of 108 CFU/ml. After that, 100µl of the broth's inoculums (108 CFU/ml) were taken out using micropipette and inoculated onto a new, sterile, solidified Agar Media Plate. To inoculate the agar plate, the inoculums were applied to entire sterile agar surface using a sterilized spreader. A sterile cork-borer was used to bore four 6-mm wells into the solidified Agar Media Plate. Plant extract loaded gel (1 mg/ml) the solution was made and put into the wells.

Incubation Period for Observe Zone of Inhibition on Agar Plates: The bacteria on the Agar Media Plate were allowed to disperse for about half an hour at room temperature prior to being cultured for 18 to 24 hours at 37°C. Plates were inspected after incubation to determine whether a clear zone had developed around the well, signifying the antibacterial properties of the studied composition.

Zone of inhibition (ZOI) millimeters were measured and examined. Using a ruler resting on the back of an inverted Petri plate, zones were measured to the closest millimeter. A dark, non-reflective background was positioned a few inches above the Petri plate. The well's diameters and the zone of total inhibition (as perceived by the naked eye) were measured ¹⁷.

RESULT AND DISCUSSION:

Plant Collection: Leaves of Acacia leucophloea and Euphorbia cyathophora were collected, shade-dried, and used for extraction. The details of plant materials are summarized in Table 3.

TABLE 3: PLANT COLLECTION

Percentage Yield: The extraction yields varied depending on the solvent used Table 4 and Table 5. Ethanolic extracts showed higher yields than petroleum ether extracts, indicating a greater presence of polar phytoconstituents. Specifically, A. leucophloea yielded 2.31% (ethanol) and E. cyathophora 4.50% (ethanol).

TABLE 4: PERCENTAGE YIELD OF CRUDE LEAVES EXTRACTS OF ACACIA LEUCOPHLOEA EXTRACT

TABLE 5: PERCENTAGE YIELD OF CRUDE LEAVES EXTRACTS OF EUPHORBIA CYTHOPHORA

Preliminary Phytochemical Study: The qualitative phytochemical analysis Table 6 and 7 confirmed the presence of major secondary metabolites including alkaloids, glycosides, flavonoids, tannins, carbohydrates, and saponins in both plant extracts, particularly in the ethanolic fractions.

TABLE 6: PHYTOCHEMICAL TESTING OF ACACIA LEUCOPHLOEA LEAVES EXTRACT

TABLE 7: PHYTOCHEMICAL TESTING OF EUPHORBIA CYTHOPHORA LEAVES EXTRACT

Organoleptic Properties: Organoleptic observations Table 8 revealed characteristic color and odor for both extracts, confirming their identity.

TABLE 8: THE ORGANOLEPTIC STUDIES OF ACACIA LEUCOPHLOEA AND EUPHORBIA CYTHOPHORA   LEAVES EXTRACT

Solubility Study: Solubility studies Table 9 demonstrated good solubility in ethanol and petroleum ether, supporting their suitability for liposomal encapsulation.

TABLE 9: SOLUBILITY STUDY OF ACACIA LEUCOPHLOEA AND EUPHORBIA CYTHOPHORA

Evaluation Parameter of Liposomes Formulation: All five liposomal formulations (F1–F5) were milky and homogeneous Table 10.

The particle size and zeta potential results are presented in Table 11 and 12.

TABLE 10: VISIBLE OBSERVATION OF PREPARED LIPOSOMES FORMULATION

Particle size: The particle size ranged between 42.53 and 118.36 nm Table 11. The smallest vesicle size was observed in formulation F5 (42.53 nm), which is desirable for enhanced skin penetration and stability.

FIG. 1: PARTICLE SIZE (F1)          

FIG 2: PARTICLE SIZE (F2)

FIG 3: PARTICLE SIZE (F3)                

FIG 4: PARTICLE SIZE (F4)          

FIG 5: PARTICLE SIZE (F5)

TABLE 11: SIZE DISTRIBUTION OF LIPOSOME

FIG. 6: GRAPHICAL REPRESENTATION OF SIZE DISTRIBUTION OF LIPOSOME

Zeta Potential: Zeta potential values ranged from –4.1 to –17.5 mV Table 12. F5 (–13.8 mV) exhibited moderate stability due to electrostatic repulsion between vesicles.

FIG. 7: ZETA POTENTIAL (F1)   

FIG. 8: ZETA POTENTIAL (F2)

FIG. 9: ZETA POTENTIAL (F3) 

FIG. 10: ZETA POTENTIAL (F4) 

FIG. 11: ZETA POTENTIAL (F5

TABLE 12: ZETA POTENTIAL OF LIPOSOME

FIG. 12: GRAPHICAL REPRESENTATION OF ZETA POTENTIAL OF LIPOSOME

Scanning Electron Microscopic (SEM): SEM images of F5 Fig. 3 revealed spherical vesicles with smooth surfaces, confirming uniform morphology typical of stable liposomal systems.

FIG. 13: SEM (F5)

Evaluation Parameter of Liposomal Gel Formulation:

Physical Properties: The optimized liposomal formulation (F5) was incorporated into a Carbopol-based gel. The gel was milky white, semisolid, and smooth Table 13.

TABLE 13: PHYSICAL PROPERTIES OF LIPOSOMAL GEL

Measurement of Liposomal Gel Formulation: Physicochemical analysis Table 14 indicated pH 6.8, viscosity 1549 ± 0.26 cps, and excellent spreadability (13.26 g·cm/s). No skin irritation was observed, confirming topical safety.

TABLE 14: VISCOSITY, pH, SKIN IRRITATION STUDY AND SPREADABILITY TEST OF GEL FORMULATION

Results of Antimicrobial Activity of Liposomal Gel F5 formulation:

Antimicrobial Activity of Liposomal Gel: Antibacterial testing against Escherichia coli and Staphylococcus aureus showed significantly enhanced activity of liposomal gel compared to crude extracts Table 15, Fig. 4.

TABLE 15: ANTIMICROBIAL ACTIVITY OF LIPOSOMAL GEL AGAINST E.COLI AND S. AUREUS

FIG. 14: PHOTOGRAPH SHOWING ZONE OF INHIBITION OF EXTRACT LOADED LIPOSOMAL GEL AGAINST A: ESCHERICHIA COLI, B: STAPHYLOCOCCUS AUREUS

FIG. 15: ZONE OF INHIBITION OF LIPOSOMAL GEL USING PLANT EXTRACT AGAINST E. COLI AND S. AUREUS MICROORGANISMS

The improved antibacterial effect of the liposomal gel is attributed to enhanced solubility, sustained release, and improved penetration of phytoconstituents through bacterial membranes. These findings align with previous studies reporting superior antimicrobial action of liposomal formulations ^{1, 2}.

Overall Discussion: The study confirmed that ethanol is an effective solvent for extracting polar phytochemicals from A. leucophloea and E. cyathophora. Phytochemical analysis verified the presence of bioactive components with known antimicrobial properties. The optimized liposomal formulation (F5) demonstrated ideal particle size, stability, and morphology. Incorporation into a topical gel resulted in a skin-compatible, stable, and effective antibacterial formulation. The synergistic action of both plant extracts in the liposomal gel offers a promising approach for herbal-based topical therapy.

CONCLUSION: The study successfully developed and characterized a liposomal gel formulation containing ethanolic extracts of Acacia leucophloea and Euphorbia cyathophora. The optimized formulation (F5) demonstrated desirable physicochemical properties, including small particle size, suitable zeta potential, and good stability, making it suitable for topical application.

Phytochemical screening confirmed the presence of bioactive compounds responsible for antibacterial activity. The liposomal gel showed significantly enhanced inhibition against E. coli and S. aureus compared to crude extracts, indicating improved drug delivery and efficacy. These findings suggest that liposomal gel systems are an effective approach for enhancing the therapeutic potential of plant-derived antimicrobials and can serve as promising candidates for future topical antibacterial formulations.

ACKNOWLEDGEMENTS: Nil

CONFLICTS OF INTEREST: Nil

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    How to cite this article:

    Mansuri S, Pathak P, Shrivastava T and Ganju K: “Development and in-vitro characterization of liposomal gel formulation containing dried leaf extract of plants Acacia lucophlolea and Euphorbia cythophora and its comparative antibacterial study”. Int J Pharm Sci & Res 2026; 17(3): 1015-24. doi: 10.13040/IJPSR.0975-8232.17(3).1015-24.

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