Abutilon Indicum Leaf Extract–Loaded Novel Liposomal Formulation for Anti-Leprosy Activity

Medicinal plants have been an integral component of traditional healthcare systems for centuries and continue to serve as an important source of novel therapeutic agents1. Various plant parts, including roots, leaves, bark, seeds, and flowers, are known to exhibit a wide range of pharmacological activities such as antioxidant, anti-inflammatory, analgesic, antimicrobial, and antiulcer effects2. These therapeutic properties are primarily attributed to the presence of secondary metabolites such as alkaloids, flavonoids, phenolic compounds, glycosides, and terpenoids, which possess diverse chemical structures and biological functions3. Such bioactive constituents not only contribute to disease prevention but also play a crucial role in the development of alternative and complementary medicines4.

Among these medicinal plants, Abutilon indicum (L.) Sweet, commonly known as Atibala, has gained considerable attention due to its broad spectrum of pharmacological activities5. Belonging to the family Malvaceae, it is a perennial shrub widely distributed in tropical and subtropical regions, particularly in India6. Traditionally, A. indicum has been extensively used in Ayurvedic medicine for its laxative, demulcent, anti-inflammatory, analgesic, and antidiabetic properties7. The leaves, in particular, are employed in the treatment of inflammatory conditions, wound healing, and certain infectious diseases8. Ethnopharmacological reports also indicate its use in managing scorpion stings and psychosomatic disorders, highlighting its multifaceted therapeutic potential. The pharmacological efficacy of A. indicum is largely associated with its rich phytochemical composition, which includes flavonoids, tannins, saponins, and phenolic compounds known for their antimicrobial and antioxidant activities9-10.

Leprosy, also referred to as Hansen’s disease, is a chronic infectious disorder caused by Mycobacterium leprae, an obligate intracellular pathogen that primarily affects the skin, peripheral nerves, and mucosal tissues11. The disease is characterized by progressive nerve damage, skin lesions, and, in severe cases, permanent disability and social stigma12. Despite significant advancements in treatment, leprosy remains a global health concern, particularly in developing countries13. India alone contributes a substantial proportion of the global disease burden, followed by countries such as Brazil and Indonesia14. The currently recommended multidrug therapy (MDT), although effective, is associated with several limitations including prolonged treatment duration, adverse drug reactions, and issues related to patient compliance. Additionally, the emergence of drug resistance and relapse cases further necessitate the exploration of alternative therapeutic strategies15.

In this context, plant-derived bioactive compounds offer a promising avenue for the development of safer and more effective anti-leprosy agents16. However, a major limitation associated with herbal extracts is their poor solubility, low bioavailability, and instability under physiological conditions. To overcome these challenges, advanced drug delivery systems have been explored to enhance the therapeutic potential of phytoconstituents17.

Liposomes, one of the most widely investigated nanocarrier systems, have demonstrated significant potential in improving drug delivery efficiency18. These vesicular structures, composed of phospholipid bilayers enclosing an aqueous core, are capable of encapsulating both hydrophilic and lipophilic compounds19. Liposomal systems offer several advantages, including improved drug solubility, protection of active compounds from degradation, reduced toxicity, and enhanced targeted delivery20. Furthermore, their ability to provide controlled and sustained drug release makes them particularly suitable for the treatment of chronic infectious diseases such as leprosy21.

The integration of herbal medicine with nanotechnology-based delivery systems represents a novel and effective approach to overcome the limitations of conventional therapies22. Encapsulation of Abutilon indicum leaf extract into liposomes is expected to enhance its antimicrobial efficacy by improving penetration, stability, and bioavailability of the active constituents23.

Therefore, the present study aims to develop and characterize a novel liposomal formulation of Abutilon indicum leaf extract and to evaluate its anti-leprosy activity. This work seeks to establish a scientific basis for the use of plant-based nanocarrier systems as potential alternatives in the management of leprosy.

MATERIAL METHODOLOGY

Collection and Preparation of Plant Material

Fresh and healthy leaves of Abutilon indicum (L.) Sweet were collected from a suitable natural habitat during the appropriate growing season to ensure maximum phytochemical content. Care was taken to select disease-free and mature leaves to maintain consistency in the quality of the plant material. The collected samples were immediately transported to the laboratory in clean, sterile polyethylene bags to prevent contamination and degradation.

Upon arrival, the leaves were thoroughly washed with running tap water followed by rinsing with distilled water to remove adhering dust particles, soil, and other extraneous matter. The cleaned plant material was then subjected to shade drying at ambient room temperature (25–30°C) for several days. Shade drying was specifically employed to prevent the degradation of heat-sensitive and photosensitive bioactive compounds such as flavonoids and phenolics.

After complete drying, as confirmed by constant weight, the leaves were mechanically reduced in size and coarsely powdered using a grinder. The powdered material was passed through a suitable sieve to obtain uniform particle size, which facilitates efficient extraction of phytoconstituents. The dried powder was then stored in an airtight, moisture-free container and kept in a cool, dry place until further use to prevent microbial growth and oxidative degradation.

Table 1: Botanical Profile of Abutilon indicum

Authentication of Plant Material

Fresh, healthy leaves of Abutilon indicum (L.) Sweet were collected from a suitable natural source and transported to the laboratory under clean conditions. The collected material was thoroughly washed with tap water followed by distilled water to remove dust and impurities. The leaves were then shade-dried at room temperature (25–30°C) to preserve thermolabile phytoconstituents. After complete drying, the material was coarsely powdered, sieved for uniformity, and stored in an airtight container for further experimental use.

The plant material was authenticated by a qualified botanist based on its morphological characteristics. A voucher specimen was prepared and deposited in a recognized herbarium for future reference. The authenticated sample was properly labeled and used for subsequent extraction and formulation studies.

Preparation of Plant Extract

The dried and powdered leaves of Abutilon indicum were subjected to extraction using both Soxhlet extraction and maceration techniques to ensure efficient recovery of phytoconstituents with varying solubility profiles.

1.Soxhlet Extraction Technique

Approximately 25 g of the powdered plant material was placed in a thimble and extracted using a Soxhlet apparatus with a methanol–water mixture (50:50 v/v) as the solvent system. The extraction process was carried out continuously for about 72 hours, allowing repeated cycles of solvent evaporation, condensation, and percolation through the plant material to achieve exhaustive extraction.

After completion of the extraction process, the liquid extract was filtered to remove solid residues, resulting in a clear filtrate. The filtrate was then transferred to a china dish and subjected to solvent evaporation on a water bath to remove excess solvent. The concentrated extract obtained was further dried to a semi-solid mass and stored in a refrigerator, protected with aluminum foil to prevent light-induced degradation, until further use.

Figure 1: Soxhlet Extraction Setup for Abutilon indicum Leaf Extract

2. Maceration Technique

In the maceration method, 25 g of powdered Abutilon indicum leaves was soaked in a methanol–water mixture (100:90 v/v) in a clean container. The container was sealed with aluminum foil and kept at room temperature for a period of 7 days to allow adequate extraction of bioactive compounds. During this period, the mixture was intermittently stirred to enhance solvent penetration and facilitate the diffusion of phytoconstituents.

Following the extraction period, the mixture was filtered to separate the plant residue from the liquid extract. The filtrate was then transferred to a china dish and concentrated by evaporating the solvent on a water bath until a thick extract was obtained. The final extract was collected, stored in a tightly closed container, and preserved under refrigeration for subsequent experimental analysis.

Figure 2: Maceration Process of Abutilon indicum Leaf Powder in Solvent

Table 2: Extraction Conditions for Abutilon indicum Leaves

Preparation of Liposomal Formulation

The liposomal formulation of Abutilon indicum leaf extract was prepared using the thin-film hydration method, a widely employed technique for the development of vesicular drug delivery systems. In this method, the lipid components, namely soya lecithin (phospholipid) and cholesterol, were accurately measured and dissolved in a mixture of chloroform and methanol in a ratio of 2:1 (v/v) to obtain a clear and homogeneous solution.

The lipid solution was transferred into a round-bottom flask and subjected to solvent evaporation under reduced pressure using a rotary evaporator. The process was carried out with the water bath maintained at approximately 40°C, which facilitated the gradual removal of organic solvents and resulted in the deposition of a thin, uniform lipid film along the inner surface of the flask. To ensure complete elimination of residual solvents, the flask was kept undisturbed for about one hour.

Subsequently, the dried lipid film was hydrated with an aqueous solution of Abutilon indicum leaf extract prepared in distilled water. Hydration was performed at a temperature range of 40–50°C with gentle rotation or shaking of the flask to promote uniform dispersion. This step enabled the detachment of the lipid film and spontaneous formation of vesicular structures.

The process ultimately resulted in the formation of multilamellar vesicles (MLVs), indicating successful encapsulation of the plant extract within the liposomal system. The prepared formulation was further utilized for characterization and evaluation studies.

Table 3: Composition of Liposomal Formulation

Characterization of Liposomal Formulation

The prepared liposomal formulation was characterized to evaluate its physicochemical properties. Particle size analysis was performed to determine the average vesicle diameter, which plays a crucial role in drug delivery efficiency and stability. Zeta potential measurement was carried out to assess the surface charge and stability of the formulation. Additionally, the polydispersity index (PDI) was determined to evaluate the uniformity and distribution of vesicle size within the formulation.

Evaluation of Anti-Leprosy Activity

The anti-leprosy activity of the formulated liposomes was evaluated using the zone of inhibition method. The test sample was assessed against Mycobacterium leprae to determine its antibacterial efficacy. The diameter of the inhibition zone was measured and compared with that of a standard drug to evaluate the effectiveness of the formulation.

Experimental Procedure:

• The inoculum of the microorganism was prepared from the bacterial cultures. 15ml of nutrient agar (Hi media) medium was poured in clean sterilized Petri plates and allowed to cool and solidify.
• 100 µl of broth of bacterial strain was pipette out and spread over the medium evenly with a spreading rod till it dried properly.
• Wells of 6mm in diameter were bored using a sterile cork borer. Solutions of the compounds (100µl/ml) were prepared in Ethanol and 100µl of prepared test solutions (1mg/ml) and standard was added to the wells. The petri plates incubated at 37⁰C for 24 h.
• Streptomycin (1mg/ml) was prepared as a positive control and DMSO was taken as negative control.
• Antibacterial activity was evaluated by measuring the diameters of the zone of inhibitions (ZI).

RESULT AND DISCUSSION

The developed Abutilon indicum leaf extract-loaded liposomal formulation was evaluated for its physicochemical characteristics and anti-leprosy activity. The obtained results indicate successful formulation with desirable properties.

Zeta Potential

Table 4: Zeta Potential and Electrophoretic Mobility of Abutilon indicum Liposomal Formulation

Table 4 shows that the liposomal formulation exhibited a mean zeta potential of −1.4 mV with an electrophoretic mobility of −0.000011 cm²/Vs. The negative surface charge indicates the presence of electrostatic repulsion between vesicles, which contributes to preventing aggregation. However, the relatively low magnitude suggests moderate stability of the formulation.

Figure 3: Zeta Potential Distribution of Liposomal Formulation

Above graph shows that the liposomal formulation exhibits a zeta potential value centered around −1.4 mV, indicating a slight negative surface charge. The narrow distribution suggests uniform particle behavior, while the negative charge contributes to electrostatic repulsion between vesicles, helping to reduce aggregation. However, the low magnitude indicates moderate stability of the formulation. Generally, zeta potential values greater than ±30 mV indicate high stability; however, the observed value suggests moderate stability, possibly stabilized by steric hindrance due to phospholipids.

Particle Size

T

able 5: Particle Size Distribution of Abutilon indicum Liposomal Formulation

The liposomal formulation exhibited a mean particle size of 228.7 nm with a standard deviation of 53.9 nm and a mode value of 207.5 nm. The presence of a single prominent peak (S.P. area ratio = 1.00) indicates a uniform and monodisperse distribution of vesicles. The nanoscale particle size confirms successful formation of liposomes and suggests their suitability for enhanced drug delivery and improved bioavailability.

Figure 4: Particle Size Distribution Curve of Abutilon indicum Liposomal Formulation

Above graph shows that the liposomal formulation exhibits a narrow particle size distribution with a prominent peak around 200–250 nm, indicating uniform vesicle size. The sharp curve suggests a monodisperse system, confirming successful formation of liposomes with consistent size, which is favorable for improved drug delivery and stability.

Anti-Leprosy Activity

Table 6: Antibacterial Activity of Abutilon indicum Liposomal Formulation Against Mycobacterium leprae

Figure 5: Anti-Leprosy Activity of Abutilon indicum Liposomal Formulation