Development and Experimental Evaluation of Silver Nanoparticles with Anti-Microbial Activity for Extract of Abutilon Indicum

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Abutilon Indicum Plant

Abutilon indicum (L.) Sweet ssp. Indicum (Family: Malvaceae), commonly known as Atibala, is a significant traditional medicine used for various ailments. It has been traditionally used as a laxative, emollient, analgesic, anti-diabetic, anti-inflammatory, and blood tonic, and for treating conditions like leprosy, urinary diseases, jaundice, piles, and wounds. Studies have confirmed its antioxidant, antibacterial, analgesic, anti-inflammatory, anti-cancer, hepatoprotective, immunomodulatory, and larvicidal activities.

MATERIALS & METHODS: -

MATERIALS: -

Pet ether Chloroform (Solvent) Ethyl acetate (Solvent) Water (Vehicle) Silver Nitrate (Reducing agent)

Equipments: -

The equipment utilized in the study are:

Weighing balance (SHIMADZU AUX220)

Magnetic stirrer (REMI 2MLH)

Soxhlet extraction apparatus

Rotary evaporator apparatus (EV11 AGK.050)

UV-spectrophotometer (Jasco V-630)

FTIR (Jasco M-41000)

Experimental Work: -

Dried Leaves of Abutilon Indicum

Dried Leaves of Abutilon Indicum

Collection of Plant Material Abutilon indicum (Indian mallow) was collected from Kavathe Mahankal in Sangli, Maharashtra. The collection focused on preserving the plant's integrity and efficacy, ensuring correct species identification (perennial, heart-shaped leaves, yellow flowers, round fruits). Harvesting occurred during the active growth period, typically in the morning, using clean, sharp tools to minimize damage and avoid over-harvesting. Gentle handling prevented bruising. Dried material was stored in airtight containers in a cool, dark place to maintain potency.

Drying and Grinding of Plant Material

Drying: Fresh, healthy leaves were selected, washed gently under cool running water, and patted dry. They were arranged in a single layer on a clean, dry surface with good airflow. Drying took 4-5 days in a well-ventilated area away from direct sunlight to preserve color and essential oils. Leaves were flipped regularly for uniform drying and were ready when brittle. Dried leaves were stored in airtight containers away from light and moisture.

Grinding: Dried leaves were inspected for contaminants, then carefully placed into a clean grinder. Grinding was done gently to avoid overheating and preserve medicinal properties. The fine powder was transferred to a clean, airtight container and labeled appropriately.

Phytochemical Evaluation of Plant Material Qualitative chemical tests were performed to screen for various phytochemicals including carbohydrates, proteins, amino acids, alkaloids, flavonoids, steroids, tannins, glycosides, and terpenoids.

Test for Steroids: Reddish-brown color indicates presence.

Test for Alkaloids (Dragendoff’s test): Orange to reddish precipitate confirms presence.

Test for Glycosides (Killer Killani test): Reddish-brown color at the junction and a bluish upper layer indicate presence.

Test for Flavonoids: Intense yellow color with dilute NaOH, becoming colorless upon acid addition, indicates presence.

Test for Tannin: Formation of blue-black precipitate with FeCl3 indicates presence.

Test for Carbohydrate (Molish test): - Purple color indicates presence.

Test for Protein (Biuret test): - Pink color indicates presence.

Extraction of Plant Material with Different Solvents :-Coarse powdered leaves were extracted using a Soxhlet extractor for 72 hours with Pet ether, Chloroform, and Ethyl acetate. Water extraction was done by cold maceration for 7 days. All extracts were filtered, concentrated under reduced pressure at 40°C using an EQUITRON Rotary evaporator, and stored at 4-8°C.

Extraction Process of Abutilon Indicum Leaves Using Different Solvents

Percentage Yield of Different Solvents: -The percentage yield of desired compounds was calculated using the formula: Percentage Yield = (Actual Yield / Theoretical Yield) × 100%.

Conformation of Preliminary Phytochemicals by TLC:-Phytochemical screening of Abutilon indicum root, leaf, stem, and fruits with seeds showed positive results for alkaloids, saponins, terpenoids, and phenols via thin-layer chromatography. Root extracts had the highest alkaloid content, while saponins were in leaves, stems, and seeds. Phenols were abundant in leaves and stems, indicating potential antioxidant and antimicrobial properties.

Antimicrobial Activity of Plant Extract with Staphylococcus aureus:- A swab of pure bacterial culture was evenly spread over Mueller-Hinton broth and agar plates. The treated product sample was placed on the media plate and incubated for 18-24 hours at 36°C with optimal bacterial growth conditions. The antibacterial activity was tested against Staphylococcus aureus.

Selection of Potent Extract for Activity Based on antimicrobial activity: - The aqueous extract of Abutilon indicum leaves showed the maximum zone of inhibition, particularly at 800mg concentration. Considering solubility and physicochemical tests, the water extract had high solubility of active ingredients, making it the potent extract.

Preparation of Silver Nanoparticles: - Silver nanoparticles were prepared using a bioreduction method (green synthesis).

Preparation of silver nitrate solution: - A fresh solution of silver nitrate was prepared by dissolving a specific quantity in double-distilled water.

Preparation of green silver nanoparticles: - 10 ml of plant water extract was added to 90 ml of silver nitrate solution with constant stirring for bioreduction. The color changed to reddish-brown, indicating nanoparticle formation. Complete reduction occurred within 25-30 minutes, followed by 24-hour incubation at room temperature.

Isolation of polyherbal silver nanoparticles: - The solution was centrifuged at 20000 rpm for 20 minutes. The supernatant was discarded, and this step was repeated thrice to collect the silver nanoparticles

Evaluation of Silver Nanoparticles: -

Particle Size: - Vesicle size was determined using a Horiba Analyser. 1 ml of the formulation was diluted with 10 ml distilled water and placed in a polystyrene cuvette in a thermostatic chamber at 25°C. Detection was at a scattering angle of 90°. The size of the particle in diameter 106 nm were noticed.

UV Spectroscopy: -

UV-visible spectra of silver nanoparticles were taken in water medium. The strong absorption peaks at 435 nm. are associated with the silver nanoparticles. It can be assumed that leaf protein acts as a template for the synthesis of silver nanoparticles and as well as stabilizing of nanoparticle

FTIR Spectroscopy Analysis: -

The silver nanoparticles were analyzed by FT-IR spectroscopy. The FT-IR spectrum was taken in the mid IR region of 400-4,000 cm. The spectrum was recorded using Attenuated Total Reflectance (ATR) technique. The sample was directly placed in the zinc selenide crystal and the spectrum was recorded in the transmittance mode.

The antimicrobial activity of silver nanoparticles is reported to a large extent. The silver nanoparticles are synthesized and also show antimicrobial activity against S aureus bacteria. Silver nanoparticles was dissolved in aqueous extract having concentration of 600mg and antimicrobial activity was performed.

RESULT & DISCUSSION

Preliminary Test: -

Selection of the Herb: -

The plant selected was Abutilon indicum, commonly known as Atibala, belonging to the Malvaceae family. The leaves were used for their reported anti-microbial and anti-inflammatory properties.

Phytochemical Investigation: -

The phytochemical investigation revealed the presence of alkaloids, amino acids, carbohydrates, flavonoids, phenolics compounds, triterpenoids, and saponins. Tannins and steroids were absent.

Antimicrobial Activity of Silve Nanoparticles: -

Synthesized silver nanoparticles also exhibited antimicrobial activity against Staphylococcus aureus bacteria. Silver nanoparticles dissolved in aqueous extract at 600mg concentration showed an average zone of inhibition of 3.04 cm² and 3.17 cm².