INTRODUCTION:

Nanoparticles are being viewed as fundamental building blocks of nanotechnology. The most important and distinct property of nanoparticles is that they exhibit larger surface area to volume ratio¹. The nanoparticles are finding their applications in various fields such as biomedical, tissue engineering, health care, environmental, drug delivery, gene delivery, optics, mechanics, non-linear optical devices, food industry, space industry and many more to count on, in fact in every field many more to count on¹.

Exploring the waste materials in different area of human applications has given much attention in recent years. Use of plant waste materials (e.g. fruit peel) for value added discoveries for identification of major biological potentials is one such area where scientists are focusing with zeal. Synthesis of metal nanoparticles also one such area where plant materials such as peels, leafs, seeds and roots were extensively utilized in these days. Utilization of these waste materials in the synthesis of nanoparticles is studies under the light of green chemistry². Nanoparticles are regarded fundamental of nanotechnology. In recent past, noble metal nanoparticles have been main subject of research owing to their excellent electronic, optical, mechanical, magnetic and chemical properties. Metal nanoparticles have immense applications in the area of diagnosis, biological and catalysis Ag, Pt, Au and Zn are among major metal used in the formation of nanoparticles³.

Citrus sinensis is genus of flowering plants in the family Rutaceae. Orange peels are a boon for skin, as they possess anti-microbial, anti-inflammatory and anti-fungal properties. The dried peels can be powdered and used to scrub and exfoliate skin. It is a great cleanser, helps to cure acne and pus filled pimples and remove blackheads, dark spots and pigmentation⁴.

Orange peel can effectively lower cholesterol levels. Orange peel contains a flavonoid known as hesperidine, which is said to prevent colon cancer and osteoporosis. Consuming the peel of this fruit also ensures healthy liver functions⁵.

Silver nanoparticles are used in various applications such as biomedical devices, biosensors, catalysis, electronics and pharmaceuticals. Many of these materials are not suitable for critical applications such as in medicine. Furthermore, they represent an environmental hazard. Eco-friendly methods for the synthesis of metal nanoparticles are needed to avoid or minimize such problems. Nowadays, metal nanoparticles are synthesized using eco-friendly natural sources such as plant extracts, fruits, fungi, honey and microorganisms⁶.

The recent reports include the biosynthesis of silver nanoparticles by using plant parts like Punica granatum peel, Citrus sinensis peel, Annona squamosa peel, lemon peel, banana peel and mango peel. Silver nanoparticles prepared using biological materials have the properties of a high surface area, smaller in size and high dispersion⁷. Silver particles and fruit peel of orange it’s self have antimicrobial property and when we use combination of silver particles and above mention fruit peel extract may show synergetic effect. So, in this research work our intension is to come out with good economical and highly targeted drug delivery formulation system. In this area as per literature survey and our knowledge nothing more precise and advanced studies are not done. So, here we are representing the advanced research work in relating to the objectives provided.

MATERIAL AND METHODOLOGY:

Material used:

Orange peels were collected from local market, Bellur. Chemicals like Silver nitrate and Sodium borohydride were obtained from S. D Fine chemicals, Mumbai, India. Milli Q water was used throughout the experiment.

Method used:

Size reduction:

Fruit of orange were washed well using tap water. The peels are separated and were washed thoroughly with tap water. The washed peels were cut in to small pieces [1-5 cm] and air dried in sunlight for 20 days. The dried fruit peels were grinded properly using a mortar and pestle and later using a grinder, to obtain the powdered form and then passed through sieve no. 40 to get uniform powder and stored at room temperature. The peel powder was stored separately in air tight bottles in refrigerator.

Preparation of Extract:

100g powdered sample was extracted with 800ml ethanol at room temperature by Soxhlet extraction method for 6 hours. The mixture was filtered through a Whatman filter paper (No.2) for removal of peel particles. The residue was re-extracted twice under the same condition to ensure complete extraction. The extracts were filtered and evaporated to dryness under reduced pressure at 60°C by a rotary flask evaporator (Buchi, Japan). The extracts were placed in dark bottles and stored in refrigerator at 4°C until further use.

UV-visible spectrum analysis:

Fruit peel extract were diluted in distilled water and after dilution the samples were observed by UV-visible spectrophotometer (Shimadzu, UV-1800). The sample solution was colored, hence scanning was done in the range of 400-800 nm.

Qualitative and Quantitative analysis of phyto-chemical constituents:

Qualitative analysis:

The obtained peel extract were subjected to various qualitative analysis.

Test for alkoloids:

A pinch of crude power was mixed with 1% HCl and about 6 drops of Mayor’s reagents. A creamish or pale yellow precipitate indicated the presence of respective alkaloids⁶.

Test for amino acids:

A pinch of crude extract was treated with few drops of Ninhydrin reagent. Appearance of purple color shows the presence of amino acids⁶.

Test for tannins:

A pinch of crude extract was treated with few drops of 0.1% ferric chloride and observed for brownish green or a blue-black coloration⁶.

Test for anthraquinones (Borntrager’s test):

A pinch of crude extract was hydrolyzed with diluted Conc. H₂SO₄ extracted with benzene. 1 ml of dilute ammonia was added to it. Rose pink coloration suggested the positive response for anthraquinones⁶.

Test for saponins:

Froth test for saponins was used. 1g of the sample was weighed into a conical flask in which 10ml of sterile distilled water was added and boiled for 5 min. The mixture was filtered and 2.5ml of the filtrate was added to 10ml of sterile distilled water in a test tube. The test tube was stopped for about 30 second. It was then allowed to stand for half an hour. Honeycomb froth indicated the presence of saponins⁶.

Test for protein:

A pinch of crude powder was treated with 4% Sodium Hydroxide and few drops of 1% Copper Sulphate was added. Violet or pink colour apper the presence of protein⁶.

Test for terpenoids (Salkowski test):

A pinch of crude powder was mixed in 2 ml of chloroform, and concentrated H₂SO₄ (3 ml) was carefully added to form a layer. A reddish brown coloration of the inter face was formed to show positive results for the presence of terpenoids⁶.

Test for cardiac glycosides (Keller-Killani test):

A pinch of crude powder was treated with 2ml of glacial acetic acid containing one drop of ferric chloride solution. This was underlayed with 1ml of concentrated sulphuric acid. A brown ring of the interface indicates a deoxysugar characteristic of cardenolides⁶.

Quantitative analysis:

Equivalent weight determination:

Crude extract (0.5 g) was weighed into a 250 ml conical flask and moistened with 5 ml ethanol, 1.0 g sodium Chloride was added to the mixture followed by 100 ml distilled water and few drops of phenol red indicator. Care was taken at this point to ensure that all the extract had dissolved and that no clumping occurred at the sides of the flask before the solution was then slowly titrated (to avoid possible de-esterification) with 0.1 M NaOH to a pink colour at the endpoint. Equivalent weight was calculated using the equation below:

Equivalent Weight = (Weight of extract/ Volume of Alkali (cm³) × Molarity of Alkali) × 100%

Ash content determination:

Five grams of each of the sample was accurately weighed into a weighed empty crucible separately. The crucible was transferred to a furnace set at 60°C to burn off all the organic matter. The carbon charred and then burnt off as carbon dioxide, leaving a dark ash; this process lasted for 24 hour. The crucible was taken out of the furnace and placed in a desiccator to cool. The crucible after cooling was reweighed again. This was calculated using:

Ash Content (%) =

(Weight of Ash / Weight of Sample) × 100

Moisture content determination:

A dried empty petridish was dried in an oven, cooled in a desiccators and weighed. Five grams of the extract was transferred into the crucibles in the oven which was set at 130°C for 1h thereafter the petridish was removed, cooled in a desiccators and weighed. This process was repeated once (Aina VO et al., 2012). The moisture content was calculated using:

Moisture Content (%) = (Weight of the Residue/ Weight of the Sample) × 100%

pH: pH of obtained crude extract was determined by using calibrated pH meter (Techno scientific products, Bangalore).

Nanoparticles Preparation:

To a 50ml of freshly prepared 0.001M Silver nitrate solution, 5ml of 0.002M Sodium borohydride solution was added with continuous stirring and kept it aside for 15 minutes in a clean 250 ml beaker till a clear and slightly dark solution is obtained. Further, this clear solution is heated and maintained in water bath at 45^OC for 30 min (solution A). Orange peel extract in different concentration (200mg, 300mg and 500mg) was dissolved in 4ml of Milli Q water separately in test tube and heated slightly to get orange yellow colored solution. This solution was added drop wise into solution A with continuous stirring using glass rod for 30-45 min till orange colour solution is obtained. The obtained solution is cooled to room temperature and 0.5ml of 0.5 mcg/ml PVP solution was added as a stabilizer and filtered to get clear orange colored solution of silver nanoparticles. This solution was stored in a dark place in a well closed container until further use. Then prepared nanoparticles were evaluated for FTIR, particle size, zeta potential, surface morphology by SEM, X-ray diffraction and energy dispersive spectrometry^7,
8.

RESULTS AND DISCUSSION:

Orange peel extracts were subjected to various tests to confirm the presence of photochemical constituents. The UV-spectroscopic analysis showed that orange peel extract, a colored solution showed maximum absorbance at 280 nm (figure 1), wave length. Hence same wave length will be used for further studies. Results of qualitative analysis showed that orange peel extract showed positive results for alkaloids, tannin and saponins. The results were tabulated in table 1. Table 2 showed the equivalent weight, percentage moisture content and pH was found to be 500.10 mg/ml, 96.12 % and 3.8, respectively. The obtained peel extract was acidic in nature i.e., pH 3.8.

Fig. 1: UV-Spectrum of orange peel extract

Table 1: Phyto-chemical Analysis of orange peel extract by Soxhlet Apparatus

Sl. No	Phyto-chemicals	Status*
1	Alkaloids	+
2	Amino acid	_
3	Tannin	+
4	Anthraquinones	_
5	Saponins	+
6	Protein	–
7	Terpenoids	_
8	Cardiac glycosides	_

+ = Present, - = Absent

Table 2: Quantitative analysis of orange peel extract

Sl. No	Parameters	Values
1	Equivalent weight	500.10
2	Ash content	40%
3	Moisture content	96.12%
4	pH	3.8

The FT-IR spectrum of orange peel extract showed the distinct peak in the range of 1649, 3390, 2355 and 771cm⁻¹. The absorption peaks located mainly at 3036 cm⁻¹ are generally attributed to aromatic or aliphatic C–H stretching, 2928 cm⁻¹ are generally assigned to the alkyl C–H stretching, whereas peaks at 1734, 1375 and 1332 cm⁻¹ are due to C–O–O stretching bands, 1102 and 1050 cm⁻¹are due to C–C stretching vibrations, 713 and 624 cm⁻¹are due to acetylenic C–H bending vibrations in the region of 40–4000 cm⁻¹. All the spectrum of orange peel extracts is present in the orange peel extract nanoparticles. Hence there was no any shift of functional groups are seen in orange peel extract nanoparticles (figure 2 and 3).

The orange peel extracts silver nanoparticles were subjected to Zeta Potential analysis to determine the surface charge of the nanoparticles and to find out the aggregation behavior. The values of zeta potential for orange peel extract silver nanoparticles was found to be– 32.0 mV to -21.4 mV for orange O50 and orange O20, respectively (figure 4a, and 4b). Hence all batches of nanoparticles having higher surface charge which indicates there is least chance of aggregation.