INTRODUCTION:
Nanoparticles are fundamental building block of nanotechnology. The most important and distinct property of nanoparticles is their exhibit large surface area to volume ratio. The metallic nanoparticles are useful as they have showed good antimicrobial efficacy against bacteria, viruses, and other eukaryotic microorganisms. A number of approaches are available for the synthesis of silver nanoparticles for example facile method, thermal decomposition of silver compounds, electrochemical, sono chemical, and microwave assisted process and recently via green chemistry path[1].
An eco friendly plant mediated synthesis of in organic nanoparticles is a fast growing research in the field of nanotechnology. Biological organisms like bacteria, fungi and actinomycetes were also utilized for the synthesis of AgNPs. Biosynthetic methods employing plant extracts have drawn attention as a simple and viable alternative approach to chemical and physical method. Silver nanoparticles (AgNPs) can be synthesized from various parts of the herbal plants like bark of Cinnamomum zeylanicum, Azadirachta indica (neem) leaves, Citrus limon, tannic acid, Parthenium hysterophorus leaf, Murraya koenigii (curry leaf), Lantana camara leaf and various plant leaves[2,3]. Considering the vast potentiality of plants as sources this work aims to apply a biological green technique for the synthesis of silver nanoparticles as an alternative to conventional methods. In this regard, aqueous extract of Kinnow mandarian (Citrus reticulate) peels family Rutaceae was used for bioconversion of silver ions to nanoparticles. The mechanism behind the synthesis of silver nanoparticles was not known exactly but it is hypothesized that the enzymes NADH-dependent nitrate reductase were involved in the reduction of silver ions [4].
MATERIAL AND METHODS:
All chemicals used in the experimentation were of highest purity grade and were obtained from sigma, Bangalore, India and Merck, Mumbai, India. Kinnow fruits for the preparation of silver nanoparticles were purchased from the local market of Rajpura, Patiala, India and were authenticated from Punjabi university Patiala.
Green synthesis of silver nanoparticles:
The fresh peels of kinnow fruits were taken and thoroughly washed with distilled water and cut into small pieces. 4gm of these peels were weighed and taken in 40 ml of distilled water and boiled for 2minutes. The extract so obtained was filtered using Whatman filter paper. 1ml of the prepared extract was added to 5ml of 1Mm solution of silver nitrate and kept in microwave oven for 1minute at room temperature for the required colour change. The sample was subjected to several short burst of microwave irradiation at frequency of 2.45 GHz, at power output of about 100 W [5].
Characterization of silver nanoparticles:
UV Spectral analysis:
The reduction of pure Ag+ ions was monitored by measuring the UV Visible spectra of the resulting reaction mixture at different interval of time within the wave length range between 300nm- 600nm using Systronic UV double beam spectrophotometer- 2202.
Fourier Transform Infrared Spectroscopy:
Infrared spectroscopy gives information on the vibrational and rotational modes of motion of a molecule and hence an important technique for identification and characterization of a substance. The silver nanoparticles were analyzed under FTIR for the size conformation. All samples of the synthesized nanoparticles were scanned in the range of 3500– 500 nm by using Bruker Alpha-T instrument.
Scanning Electron Microscopy (SEM):
This technique was employed to visualize the size and shape of silver nanoparticles synthesized using kinnow fruit peel extract. The colloidal dispersion of silver nanoparticles was mounted on a copper coated grid, extra solution was removed using filter paper and then allowed to dry prior measurement. Finally images of prepared silver nanoparticles were taken using Joel, Japan, Model- LV 6501 at excitation voltage of 2 KV, magnification at 5000X.
Formulation of antidandruff shampoo:
Herbal anti-dandruff shampoo was formulated by adding the weighed amounts of herbal ingredients as per the composition given in the [Table 1]. All ingredients were taken in motar and pestle mixed to form a homogenous blend. Methi seeds extract was used to prevent hair fall, amla, shikakai and reetha extract was added as cleanser, lemon oil to give aroma, gaur gum was added to maintain consistency and Ag nanoparticles were added to impart antidandruff property to the shampoo formulation [6].
Table 1: Composition of silver nanoparticles loaded antidandruff shampoo.
|
Ingredients |
F1 |
F2 |
F3 |
F4 |
|
Methi seed extract |
2g |
2g |
2g |
2g |
|
Amla extract |
1g |
1.5g |
2 |
2.5g |
|
Shikakai extract |
1g |
1.5g |
2 |
2.5g |
|
Reetha extract |
1g |
1.5g |
2 |
2.5g |
|
Lemon oil |
1ml |
1ml |
1ml |
1ml |
|
Gaur gum |
1% |
1% |
1% |
1% |
|
Methyl paraben |
1ml |
1ml |
1ml |
1ml |
|
AgNP |
- |
100 uL |
80 uL |
40 uL |
Evaluation of Shampoo containing silver nanoparticles:
Physical appearance:
The formulated shampoo formulations were evaluated for the colour, transparency, odor and foam producing ability.
Determination of pH:
The higher pH level may damage the hairs and make them rough, the ideal pH level for shampoos is between 5-7. The pH of the shampoo formulations was measured by preparing 10% v/v shampoo solution in distilled water pH was determined using deluxe pH meter-101 at room temperature.
Determination of percentage of solid contents:
For the estimation of percentage solid content in a previously clean, dry and weighed evaporating dish, 4 grams of formulated shampoo were taken. It was then weighed again to confirm the exact weight of the shampoo. The liquid portion of the shampoo was evaporated by placing the dish on hot plate. The weight and percentage of the solid contents of shampoo left after complete drying was taken to estimate percentage of solid content [7].
Foaming ability:
Cylinder shake method was used to test for the foaming ability. 50 ml of the 1% formulated shampoo solution was placed into a 250 ml graduated cylinder, covered with one hand and shaken for 10 times. After 1 min of shaking, the total volume of the foam content was recorded. Foam stability was valued by recording the foam volume after 1 min and 4 min of shake test.
Surface tension measurement:
Surface active agents are added into shampoo formulations to lower down the surface tension and to adsorb oil and dirt from the formulation so as to increase the efficiency of formulation. The surface tension of 10% w/v solution of shampoo in distilled water was measured using stalagmometer at room temperature using drop count method.
Dirt dispersion test:
To measure the dirt dispersion ability of shampoo two drops of formulated shampoo were added to 10 ml of distilled water taken in a test tube. To this solution, one drop of India ink was added and the test tube was stoppered and shaken ten times. The amount of ink in the foam was indicated as None, Light, Moderate or Heavy [8].
Wetting time test:
A canvas paper was cut into 1-inch diameter discs having an average weight of 0.44 g. The smooth surface of disc was placed on the surface of 1% v/v shampoo solution and the stopwatch was started. The time required for the disc to sink into shampoo solution was noted down as the wetting time. [8, 9]
Antimicrobial assay of silver nanoparticles loaded shampoo:
The antimicrobial activity of the prepared silver nanoparticles and shampoo loaded with silver nanoparticles was studied against various pathogenic microorganisms such as E. coli and C. albicans using well diffusion method [10]. Penicillin and amphotericine was used as the positive control for the study. Sterile nutrient agar plates were prepared using 24 hr. grown cultures of test micro organisms. The wells of 10mm diameter were cut into plates using sterile cork borer. The prepared silver nanoparticles, plain shampoo and shampoo formulation loaded with silver nanoparticles were loaded into the wells and incubated at 37ºC for 24 hrs; the antimicrobial activity was estimated by measuring the diameter of zone of inhibition produced by the penicillin G pellet used as control, shampoo formulation and shampoo loaded with silver nanoparticles[11].
RESULTS AND DISCUSSIONS:
The formation of brownish colored solution [Figure 1] indicated the formation of silver nanoparticles.
 |
Figure 1: Silver nano particles synthesized using fresh peels of kinnow fruit
The bioreduction of Ag+ ion in aqueous solution was monitored with the help of UV– visible spectroscopic analysis represented in [Figure 2]. The UV-visible spectrum of silver nanoparticles synthesized with the help of kinnow fruit peel extracts as a reducing agent gave characteristics surface Plasmon absorption band at 412-449 nm.
FTIR analysis was carried to check the possibility of any interaction between the bio molecules and silver ions during the formation of silver nanoparticles by bio reduction reactions. The FTIR images of kinnow mandarian peel extract and silver nanoparticles containing kinnow peel extract are shown in the [Figure 3and 4] respectively. The FTIR of silver nanoparticles showed intense peaks at 3245.38, 2937.77, 1637.13, 1301.12, 1082.27 and 1019.98 cm-1 the FTIR of kinnow peel extract showed intense peaks at 3243.28, 2933.70, 2895.89, 1635.65 and 1456.63 cm-1 the shift in the positions of bands clearly indicates that flavonoids, proteins and sugar present in the extract play important role in the bioreduction of silver ions and have strong ability to bind with silver nanoparticles
SEM images of green synthesized silver nanoparticles are shown in [Figure 5] which indicated the formation of polydispersed silver nanoparticles and confirmed the formation of silver nanostructure.
Figure 2: UV visible
spectra of green synthesized silver nanoparticles
Figure 3: FTIR spectrum of green synthesized silver nanoparticles
Figure 4: FTIR spectrum of kinnow mandarins peel extract
Figure 5: SEM image of silver nanoparticles
Characterisation of shampoo containing silver
nanoparticles:
The results of physical appearance of all indicated that all formulation have good appearance with uniform dispersion. All formulations had the good foam forming ability. The pH of shampoos has been shown to be important for improving the qualities of hair and to minimizing irritation to the eyes and scalp. As seen from [Table 2], all the shampoos were acid balanced with pH ranged 5.0-5.5, which is near to the skin pH. If the shampoo has too many solids it will be hard to work into the hair or too hard to wash out. The result of percent of solids contents was found between 19-24% which means good consistency and spreadibility which is easy to wash out [12]. It has been mentioned that a proper shampoo should be able to decrease the surface tension of pure water. The reduction in surface tension of water from 72.8 dynes/cm to 32.4 dynes/ cm by the herbal shampoos is an indication of their good detergent action. Dirt dispersion test was carried out to evaluate the cleansing action of the shampoo formulations; all shampoos ensure satisfactory cleansing action. Results of evaluation of silver nanoparticles loaded shampoo are given in [Table 2].
Table 2. Characterization of silver nanoparticles loaded shampoo formulation.
|
Parameters |
F1 |
F2 |
F3 |
F4 |
|
pH |
5.0 |
5.1 |
5.5 |
5.2 |
|
Foam ability(ml) |
158 |
165 |
160 |
148 |
|
Solid content% |
19 |
24 |
20 |
22 |
|
Surface tension(dynes/cm) |
33.3 |
32.4 |
32.2 |
34.1 |
|
Dirt dispersion |
satisfactory |
satisfactory |
satisfactory |
satisfactory |
|
Wetting time |
142 |
180 |
172 |
151 |
Results of antibacterial and antifungal activity measured in term of zone of inhibition measured[Figure 7] at different interval of time indicated that shampoo formulations containing different concentrations of silver nanoparticles exhibit enhanced antimicrobial activity as compared with plain shampoo formulation and control penicillin G pellet. The enhanced anti microbial activity may be due to the release of silver cations from silver nanoparticles which act as reservoir for bactericidal agent [13]. Results of zone of inhibition are shown in the [Figure 5 and 6] which indicated that formulation F2 showed maximum antibacterial and antifungal activity against the studied pathogens.
After 24 hrs
After 48 hrs
After 120 hrs
After 10 days
Figure 5: antimicrobial activity of silver nanoparticles loaded shampoo against Escherichia coli
After 24 hrs
After 48 hrs
After 120 hrs
After 10 days
Figure 6: antifungal activity of silver nanoparticles loaded shampoo against Candida. albicans