Design And Evaluation of Anti-Folliculitis Cream Loaded with Silver Nanoparticle of Boerhavia Diffusa (L.)

Wound healing is a dynamic process involving four overlapping phases: coagulation and hemostasis, inflammation, proliferation, and maturation. These stages ensure tissue regeneration and restoration of skin integrity. Boerhavia diffusa(L.), a medicinal plant from the Nyctaginaceae family, is traditionally used for its antimicrobial, anti-inflammatory, and hepatoprotective properties. Rich in bioactive compounds like flavonoids, alkaloids, and glycosides, it has shown potential for therapeutic applications. This study explores the plant's extract for antimicrobial properties, which may contribute to improved wound healing.

A novel drug delivery system enhances the efficacy of herbal medicines by ensuring controlled and targeted drug release. These systems include liposomes, phytosomes, nanoparticles, and transdermal methods, which improve bioavailability, stability, and therapeutic effects while minimizing toxicity. Among these, nanoparticles—ranging from 1 to 100 nm—are promising carriers due to their high surface area and reactivity. They can be organic, inorganic, or carbon-based, and their morphology influences their properties and applications.

Silver nanoparticles (AgNPs) are particularly notable for their antimicrobial effects, making them useful in wound healing and drug delivery. Their mechanism of action involves disrupting bacterial cell membranes, generating reactive oxygen species, and interfering with cellular processes, ultimately leading to bacterial death.

Creams, as a common topical formulation, provide an effective way to deliver bioactive compounds for skin treatment. They are categorized into oil-in-water (O/W) and water-in-oil (W/O) emulsions, each with distinct properties suited for various applications. Due to their ease of application, reduced systemic side effects, and direct action on affected areas, creams are widely used in treating wounds, burns, and other skin conditions.

This study aims to develop a silver nanoparticle-based cream using Boerhavia diffusa(L.) extract, integrating modern nanotechnology with traditional medicine to enhance wound healing efficacy.

MATERIAL AND METHODOLOGY

MEDICINAL PLANT COLLECTION AND IDENTIFICATION

Boerhavia diffusa (L). is a medicinal plant which is commonly known as punarnava or spreading hog weed, belong to the family Nyctaginaceae. The whole plant or some parts of plant (stem or route) has been used for formulation. The plant was collected from the natural habitat, Aynkalam, Malappuram, Kerala and the plant was dried about two weeks and confirmed by the botanist Dr .P .Sunoj Kumar, who is a professor in the Department of Botany at Calicut University, Kerala and stored as her barium at the Calicut University, Kerala.

EXTRACTION OF THE CRUDE DRUG

The plant Boerhavia diffusa (L). were collected and shade dried about two weeks and grinded into particle. The powdered plants (20g) were extracted by using maceration with water and ethanol in the ratio (1:1) for 6hr. The obtained extract was concentrated to dryness by using heating mantle. As a result, a greasy extract was extracted.

PHYTOCHEMICAL SCREENING

The phytochemical screening of the extract was done to identify the presence of alkaloids, flavonoids, tannins, and saponins.

SYNTHESIS OF NANOPARTICLES

• Synthesis of 0.1mM Silver Nitrate

To 0.017g of silver nitrate, 100ml of water was added in dark condition to avoid oxidation

• Synthesis of Silver Nanoparticle

To 10ml of crude plant extract was added with 30ml silver nitrate solution in a drop wise manner, which is placed in magnetic stirrer for efficient mixing by using magnet at 500 rpm in room temperature until the colour changed from light brown to brick red. The study was conducted in an experimental setting to prevent light reduction and centrifuged for 1 hour for 4000rpm. The formed pellet was collected and stored for further studies.

CHARACTERISATION OF EXTRACT AND Boerhavia diffusa(L).  SYNTHESIZED SILVER NANOPARTICLE

• UV-Visible Spectroscopy Analysis

The synthesis of Boerhavia diffusa(L). measured by using absorption spectra in the wavelenght range of 210-800nm using UV spectrometer and maximum absorption length was determined.

• Transmission electron microscopy (TEM)

The size, shape, and morphology of nanoparticles can be determined through its use. Images with high resolution are produced by TEM which sends a stream of electrons through a thin sample.

• Scanning electron microscopy (SEM)

The job of SEM is to characterize the morphology and topography of particles down to nanometer level. In this technique, a narrow beam of electrons is used instead of light to produce high-resolution pictures of specimen surfaces.

• X-ray diffraction (XRD)

The job of SEM is to characterize the morphology and topography of particles down to nanometer level. In this technique, a narrow beam of electrons is used instead of light to produce high-resolution pictures of specimen surfaces.

• Fourier transform infrared spectroscopy (FTIR)

It finds the chemical bonds and functional groups that are present on the surface of the nanoparticle. FTIR analyzes the sample's absorption of infrared light at various wavelengths.

• Energy dispersive x-ray analysis (EDAX)

Energy dispersive X-ray analysis is to confirm the elemental analysis of Boerhavia diffusa(L.)silver nanoparticles.

• Zeta potential analysis

It evaluates the stability and surface charge of suspended nanoparticles. In a colloidal system, the electric potential close to a nanoparticle's surface is measured by the zeta potential.

Each of these techniques provides complementary information, and various methods are frequently used to gain a thorough understanding of the properties of silver nanoparticles.

PREPARATION OF FORMULATION

• Cream formulation

All the excipients and API were weighed accurately by using analytical balance. The oil phase was prepared by using emulsifying wax heated in the water bath, added with white soft paraffin and liquid paraffin with continuous stirring at 600C.The aqueous phase was prepared by using a preservative that is dissolved in glycerin with few drops of silver nanoparticle extract at 600C. Then the aqueous phase was added to the oil phase at a constant temperature with continuous stirring. Finally, rose oil was mixed with it.

EVALUATION OF FORMULATION

• Physical Evaluation of Cream

 The physical properties of the cream like colour and consistency were evaluated by visual examination.

 • pH Measurement

A small quantity of cream was taken in beaker and dissolved in water. pH was determined by using pH paper.

 • Spreadability

Spreadability was determined by taking small quantity of formulation on a glass slide and covered with another glass slide. A weight of 50gm was placed above it and determine it’s diameter.

S= m(l/t)

Where,

S = spreadability

m= mass

l= diameter

 t= time.

ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLE AND FORMULATION BY USING S.aureus and E.coli

• Preparation of MH agar solution and Culturing of Bacteria

The antimicrobial activity of AgNPs (silver nanoparticles) and Boerhavia diffusa (L.) against Staphylococcus aureus was evaluated using the well diffusion method. Nutrient agar plates were prepared, sterilized, and inoculated with 0.1% of the test pathogen culture using a sterile cotton swab. Wells were created in the agar, and approximately 100 µL of each sample (AgNPs and Boerhavia diffusa (L.)extract) was added to the wells. For an entire day, the plates were incubated at 37°C. After incubation, the zones of inhibition around the wells were measured in millimeters to determine antibacterial activity.

SCRATCH ASSAY

The in-vitro wound healing activity of Boerhavia diffusa (L.)-assisted AgNPs was evaluated using a scratch assay. Fibroblast cells were cultured to form a confluent monolayer in six-well plates. A scratch was made in the monolayer using a 200 µL pipette tip, and the cells were washed with PBS to remove debris. A control group was treated with 0.2% PBS, while the experimental group was treated with 50 µg of Boerhavia diffusa(L.)-assisted AgNPs. Initial images of the scratch were taken using a phase-contrast microscope. Three plates per group were incubated at 37°C for 48 hours, and cell migration into the scratch area was assessed at 0 and 48 hours using microscopy.

RESULT AND DISCUSSION

PHYTOCHEMICAL SCREENING

1. TEST FOR ALKALOIDS

• Dragendroff’s test: 2 ml Dragendroff’s reagent were added to few ml extract as result it form brown precipitate.
• Wagner’s test:  2 ml Wagner’s reagent were added to few ml extract as a result it form reddish precipitate.

2. TEST FOR PHENOLS

• Lead acetate test: Few drops of 10% lead acetate solution were added to few ml extract as a result it produce yellow precipitate.
• Shinoda test: 5 ml of alcohol and Mg ribbon were added to few ml of extract as a result if produce pink colour.

3. TEST FOR TANNINS

• Ferric chloride test: Few drops of 5%FeCl3 solution were added to few ml of extract as a result it forms black color.
• Gelatin test: 1 % gelatin solution (with a little NaCl) was added to few ml of extract as a result white precipitate is formed.

4. TEST FOR SAPONINS

• 5 ml sample were shaken with small quantity of water for 15 minutes and kept aside for 30 minutes as a result a stable form was produced.

Fig: 1, 2 Test for Alkaloids                                    

Fig: 3, 4 Test for flavonoids

Fig: 5, 6 Test for Tannins

Fig: 7 Test for Saponins

On addition of silver nitrate to the Boerhavia dffusa(L.) extract, a visual colour change is observed that brown to reddish brown appearance is which confirms the formation of silver nanoparticles. Further nanoparticles were collected by centrifugation at 6000rpm for 10 minutes. The nanoparticles were dried at room temperature and subjected to charecterization.

Fig: 8 AgNP synthesis      

Fig: 9 Brown colour          

Fig: 10 AgNP           

Fig: 11 Centrifugation

TRANSMISSSTION ELECTRON MICROSCOPY

TEM was performed. The size range of nanoparticles has spherical shape. As per the TEM analysis it was determined that it is a polycrystalline material.

SCANNING ELECTRON MICROSCOPY

The scanning electron microscopy was performed. FE-SEM morphology of Boerhavia dffusa(L.) silver nanoparticles showed the particles are spherical in shape and the size ranges from 45 to 75nm.

The image shows the crystalline nature of the sample. Several sharp and intense peaks are observed, confirming a highly crystalline structure. The most intense peak appears at approximately 20 ≈ 30°, indicating a dominant crystallographic plane. The maximum peak is 27.6660°.

FOURIER TRANSFORM INFRARED SPECTROSCOPY

FTIR of BD extract

FTIR of BD Nps

SIZE DISTRIBUTION

ENERGY DISPERSIVE X-RAY ANALYSIS (EDAX)

We used EDAX spectroscopy to establish the elemental composition of the        surfaces of nanomaterials that are biosynthesised. It confirms the presence of Ag, O and C with other trace elements in nanoparticles. The presence of oxygen supports metal-oxide bond formation nanoparticles, which can enhance anti-microbial properties and efficiency.

EVALUATION OF SILVER NANOPARTICLES

ANTI-MICROBIAL ACTIVITY

Plant extract showed 6mm, formulation showed 8mm and AgNPs showed 10mm against Staphylococcus aureus. Whereas, standard drug ciprofloxacin showed 25mm against Staphylococcus aureus.

Antimicrobial activity of NPs

Antimicrobial activity of formulation

SCRATCH ASSAY

The scratch closure at 12 and 24 hrs, as compared to the untreated cells, underscored the nanoparticles^’ capacity to enhance the migratory capabilities of fibroblast. Overall, this study paves the way for considering Boerhavia diffusa(L.) assisted silver nanoparticles as a promising candidate for therapeutic interventions in regenerative medicine and dermatology.

FORMULATION OF CREAM

Among 3 different batches of cream (f1, f2, f3); f3 formulation is selected.

EVALUATION OF CREAM

The cream is pale brown in colour. Its was smooth and light to spread. And the spreadability was found to be 3.7 Cm/s. and the pH of the cream was found to be 5.2.

DRUG RELEASE KINETICS PARAMETERS