Development and Evaluation of Gel Incorporated with Synthesized Silver Nanoparticle from Ocimum gratissimum for the Treatment of Acne Vulgaris

Acne vulgaris is one of the most prevalent skin diseases which affect almost 80% of adolescents in the world during their lifetime. Development of specific association between antibiotic and bacteria during repeated treatment develops antibiotic resistance. Medicinal and aromatic plants are widely used as medicine in ancient time. Treatment of acne has been considered as a major research area in pharmaceutical and personal cosmetic care industries. The aim of the present work was to evaluate the phytochemical composition of Ocimum gratissimum, green synthesis of silver nanoparticle and to develop herbal topical gel formulation to treat acne. Ocimum gratissimum is selected based on its antibacterial activity. Phytochemical analysis revealed phytoconstituents such as alkaloids, flavonoids, tannins and saponins are present in the extract. Silver nanaoparticle was synthesized using 1 mM aqueous silver nitrate solution from the extracts of Ocimum gratissimum and formation of silver nanoparticle was confirmed by UV spectroscopy. The synthesized silver nanoparticles were stable, spherical shape with average particle size of 207.6 nm and the polydispersity index was found to be 0.256. Synthesized silver nanoparticles was incorporated into gel base and evaluated for its physical properties such as pH, viscosity, spreadability and antibacterial activity against Propionibacterium acne, Staphylococcus aureus and Escherichia coli. The results obtained in the developed formulation showed no lumps, had uniform color dispersion and were free from any fiber and particle. It was also observed to have easy washablity, good spreadability, pH was found to be 6.72 and 6.80 similar to pH of the skin. The antibacterial study of the developed formulation showed inhibitory activity against Propionibacterium acne, Staphylococcus aureus and Escherichia coli. Synthesized silver nanoparticle of Ocimum gratissimum showed higher activity than the extract. Hence, silver nanoparticle of Ocimum gratissiumum in aqueous gel-base can be used as an appropriate formulation for treatment of acne vulgaris.


INTRODUCTION
Skin in the human being is the most susceptible part for entering various microorganisms into the body 1 .
Acne vulgaris is one of the most prevalent skin diseases which affect the young adults in the age group between 11 and 30 years. Among these age  11,13,14 . The plant Ocimum gratissimum possesses several phytoconstituents and having many potential therapeutic activities in traditional system. Ocimum gratissimum, it has been used for various skin infections due to availability of abundant amount of volatile oil [14][15][16][17] . Taking into consideration of the cost and easy availability of this medicinal plant, our present study was designed to explore systematically and analyze the phytoconstituents, synthesis nanoparticle by using plant extract then formulate topical gel containing silver nanoparticle and evaluate for its physicochemical properties and antibacterial activity.

Plant Materials
The specimen for the proposed study was collected and confirmed by Dr 19,20 .

Synthesis of Nanoparticle
In the single step green synthesis, 5 ml of leaf extract was added to 95 ml of 1 mM aqueous silver nitrate solution and kept in the dark place at room temperature for 24 h. Silver nanoparticles are formed by reduction of pure silver ions and it was monitored by measuring absorption of the reaction medium in the wavelength range of 300-700 nm using UV spectrophotometry. The synthesized silver nanoparticle (AgNP) was purified by centrifugation at 10000 rpm for 15 min. The supernatant was transferred to a clean dry beaker for further settlement of particles and repeated centrifugation was carried using cooling microfuge to get dried, purified and characterized the AgNPs 14 .

UV-Visible Spectral Analysis
Silver nanoparticles were formed by reduction of silver ion; it was monitored by measuring the absorption spectra in the wavelength range of 300-700 nm using Shimadzu UV-1800 Spectrophotometer. The spectrum was recorded and the maximum absorption wavelength was determined 14 .

Particle Size Distribution and Polydispersity Index
The mean particle size of the silver nanoparticle was determined by Photon Correlation Spectroscopy (PCS) using a Zetasizer 3000 (Malvern Instruments, UK). This analytical result reveals the mean diameter of the particle at 25°C, and at an angle of 90 degree (n=10). The PCS analysis yields a mean diameter (z-average) as a light intensity-weighed size of bulk population and the polydispersity index as a measurement for the width of a particle size distribution 14 .

Zeta Potential
The zeta potential of the synthesized silver

Preparation of Topical Formulation
Topical gel formulations were prepared by cold

Evaluation of Extracts
The color of the extracts was found to be dark green. The extracts are tested for its phytochemical analysis like alkaloids, carbohydrate, glycoside, saponins, phytosterols, phenol, tannin and flavonoids. The phytochemical analysis of the extract result is shown in (Table 1).

Green Synthesis of AgNPs
Green synthesis of AgNPs was prepared from plant extract. On mixing plant extract with the silver nitrate solution, a change in the solution color from pale yellow to dark brown was observed which indicates the reduction of silver ions and formation of silver nanoparticle.
Formation of silver nanoparticle is shown in (Figure 1).

Characterization of Silver Nanoparticles
The UV absorption spectrum of Ocimum gratissimum has shown a peak specific in the range between 400 and 450 nm. Peak specific in this region might be due to Mie scattering effect 24 . The UV spectrum of silver nanoparticle is shown in (Figure 2).

Particle Size, Polydispersity Index and Zeta Potential
The particle size analysis was done by measuring the average particle size in the aqueous reaction mixture after the completion of reaction using the zeta sizer in dynamic light scattering mode. It was observed that the average particle size was 207.6 nm which confirmed that the silver ions were reduced into nanoparticles. The polydispersity index (PDI) of the silver nanoparticle was found to be 0.256; which indicates the broad distribution of globules and its homogencity. The zeta potential was determined by using Malvern zetasizer, it was found to be -23.9 mV. The particle size and polydispersity index is shown in (Figure 3).

Physicochemical Evaluation of Gel Formulation
Among the various topical formulations, gel is preferred both in cosmetic and in pharmaceutical preparations due to its faster release rate of drug substances. Gel has various advantages because of its thixotropic property, greaseless, easily spreadable, easily removable, emollient, nonstaining and compatible with several excipients. indicates the topical formulation has better spreadability. The spreadability value was found to be 6.9 ± 0.1 (g.cm/sec) and 7.1 ± 0.1 (g. cm/ sec) for gel prepared by HPMC and Carbopol.
The observed results were comparable with the earlier literature 22 .

Antibacterial Activity of the Formulation
The antibacterial activity study results of the formulated herbal gel showed antibacterial activity against acne causing bacteria such as  (Figures 4a-4c).  Silver nanoparticle 0.02 g Silver nanoparticle 0.02 g 4 Water upto 100 g Water upto 100 g