International Journal of Drug Development and Research

Keywords

Nano-particles, Gold, Silver, anti-microbial activity

Introduction

Nano-particles, previously known as "ultrafine particles" [1] can be defined as per IUPAC guidelines as any particles whose dimension lies between the range of 1X10-9 and 1X10-7 m. [2,3]. The study concerned with synthesis of various nano-particles and its total characterization is known as Nanotechnology which is amongst the newest branch of Science and Technology [4]. Amongst various nano-particles of different chemical entities, metal nano-particles are amongst the most studied in this field. Metal nanoparticles such as silver nano-particles, gold nanoparticles, along with some other metal nanoparticles like Nickel, copper etc. have been synthesized and characterized. Other types of nano-particle are magnetic nano-particles, nanoshells, ceramic nano-particle etc.[5]:

Green Approach of Nano-particle synthesis

Amongst various approaches utilized in the synthesis of nano-particles, green approach i.e. using plant extracts for the synthesis of nanoparticles, the active compounds which helps in reduction of metallic ions to corresponding nanoparticles get enveloped around the synthesized nano-particles via very weak bonds like Wander waal's forces of attraction. When the IR spectrum of such nano-particles is studied, the peaks corresponding to the functional groups present in those organic molecules can be observed. Let A be the active organic molecule which conducts the reduction of metallic ions (say silver ions) to corresponding nano-particles (in this example, silver nano-particles), A be the active organic molecule responsible for the bio-reduction of metallic ions to corresponding nano-particles, then the reaction can be depicted as (scheme 1):

Generation of nano-particles using plant extracts are amongst the most researched topics in nanotechnology. There are various advantages of using plant extracts for nano-particles synthesis such as:

a) It eliminates the tedious and time consuming process of maintaining cell cultures [6, 7].

b) The process is simple and eco-friendly [8]

Different Methods for Nano-particle Synthesis

Different methods have been utilized for the synthesis of various nano-particles. Some methods include utilization of various reducing agents like tannic acid, citric acid etc [9,10]. Microorganisms have also been tested and positive results have been obtained. Microorganisms like Klebsiella pneumoniae etc have been fruitfully utilized for the synthesis of gold nano-particles. [11]. Amongst others, different plant extracts prepared in different solvents have also been utilized for the production of metal nanoparticles. Plants and its parts (given in brackets) like Saraca asoca(leaf), Zingiber officinalis (root), Citrus limonium (fruit), Cinnamomum verum (bark), Syzygium aromaticum (Flower bud) have been used successfully for generation of gold nano-particles in aqueous medium [12] while gold particles using alcoholic extracts of Amaranthus spinosus Linn have been successfully synthesized. [13].

Another chemical method for the synthesis of gold nano-particles involves L-tryptophan ( an essential amino acid) as a reducing agent[14]. The reaction by which the reduction of nanoparticles occurs via electrons generated by the reduction of L-Tryptophan. The process can be represented as (Scheme 2):

In another method given by Kemp et al., gold and silver nano-particles were prepared by using heparin and hyaluronan as both reducing and stabilizing agents[15]. Heparin being an excellent anticoagulant while hyaluronan being a glycosaminoglycan. The particles show stability under physiological conditions and narrow size distributions for heparin particles and wider distribution for hyaluronan particles. Studies showed that the heparin nano-particles exhibited anticoagulant properties. Additionally, either gold- or silver-heparin nano-particles exhibited local anti-inflammatory properties as well.

In this paper, we have tried to synthesize nanoparticles at much faster rates using green approach. In our study, the UV-Vis spectra's for prepared nano-particles was taken 3hrs. after stirring was discontinued while color change in the solution was observed within minutes after adding the corresponding salt solution in the aqueous plant extracts. Thus, we can say that the rate of biosynthesis of nano-particles is faster using the method utilized.

Materials and Instrumentation required:

Auric Chloride was purchased from Thomas Baker, India, Silver nitrate was purchased from Merck, India, Absolute Alcohol (Emsure®) was purchased from Merck, Germany, Double Distilled water was prepared within the lab. using Double water distillation unit, model:WDU-2000 (Patent NO.-175513) . All UV–Visible spectra ware measured using UV-VIS Spectrophotometer (Shimadzu, model UV-1601), Ultra Centrifugation machine, FTIR.

Collection of plant materials:

Three types of plants viz. datura, vinca and neem were collected from local area and were evaluated for their authentication using various organoleptic evaluation methods. Before making extracts from the collected samples, the samples was properly washed under running water so as to remove any foreign particles and were dried using technique used for herbarium preparation.

Collection of Microbial Strains:

All microbial strains were purchased from Microbial Type Culture Collection (MTCC 448), Chandigarh.

Preparation of Plant extracts:

500 mg. of dried leaves of the collected samples were accurately weighted using analytical weighing balance and were then subjected to crushing with 25 ml of Double distilled water in a mortar pestle, the paste was then collected in a suitable container and was stirred for 1hr. at 70?C using magnetic stirrer with in-built hot plate. After keeping the extract for a while, the extract was collected in centrifugation tubes and was allowed to centrifuge using Ultracentrifugation machine at 10,000 rpm for 10 min. the supernatant was collected and was diluted suitably for further analysis.

Dilution of aqueous plant extract:

3ml of Supernatant as collected above wasdiluted with 4 ml of DDW (double distilled water) and this diluted solution (see fig. 1) was subjected for both spectral analysis as well as for preparation of nano-particles.

Preparation of 2% Auric chloride (AuCl4) solution

40mg of AuCl4 was accurately weighted and was dissolved in 2000 μl of water. the solution was shaken properly and was subjected to sonication for 15 sec. so as to get a homogenous solution.

Preparation of 2% Silver nitrate (AgNO3 ) solution

40mg of AgNO3 was accurately weighted and was dissolved in 2000 μl of water. The solution was shaken properly and was subjected to sonication for 15 sec. so as to get a homogenous solution. This solution was stored in containers, well covered with aluminum foil so as to prevent its photo-oxidation.

Synthesis and Characterization of Gold Nanoparticles:

Dilution as specified above was used for the preparation of gold nano-particles. To the diluted aqueous solution of plant extracts, 200 μl of 2% Auric chloride solution was added and it was subjected for stirring for 1hr using magnetic stirrer. There was a sharp change in color of the extract solution (see fig. 2). After Suitable interval of time (3 hrs), the solution was subjected for spectral analysis using UV-Vis Spectrophotometer and peak was observed for possible generation of nano-particles. Peak at around 560 nm was recorded in the spectra which corresponded to the possible generation of gold nano-particles. This λmax value comply with the values as reported by Skirtach et al. (2005) in which he synthesized gold nano-particles using P. aeruginosa. The prepared nano-particles were collected using centrifugation machine kept at 10,000 rpm for 9 min. The supernatant was discarded and the nano-particles collected at the bottom of the centrifugation tube were collected, washed (3 times using DDW), dried and subjected for further analysis.

Synthesis and Characterization of Silver Nanoparticles:

Dilution as specified above was used for the preparation of silver nano-particles. To the diluted aqueous solution of plant extracts, 200 μl of 2% silver nitrate solution was added and it was subjected to stirring for 1hr using magnetic stirrer. There was a sharp change in color of the extract solution (see fig. 3). After suitable interval of time, the solution was subjected for spectral analysis using UV-Vis Spectrophotometer and peaks were observed for possible generation of nanoparticles. Peak at around 420 nm was recorded in the spectra which corresponded to the possible generation of silver nano-particles. the prepared nano-particles were collected using centrifugation machine kept at 10,000 rpm for 9 min. the supernatant was discarded and the nano-particles collected at the bottom of the centrifugation tube were collected, washed (3 times using DDW), dried and subjected for further analysis.

FTIR analysis of collected nano particles:

FTIR analysis was done to identify possible biomolecules which are responsible for the reduction of metal salts to corresponding nano-particles. Nano-particles synthesized by different plant extracts were collected using ultra centrifuge technique, washed three times with double distilled water and dried at room temperature and then were subjected for FTIR analysis. KBr disk method was used for obtaining the spectra. A small amount of dried nano-particles was triturated with 2.5 mg of dry potassium bromide (KBr) in a mortar pestle. The powder so obtained was punched to form a sample loaded KBr pallet. The pallet was then loaded onto the FTIR instrument set at 26°C ± 1°C. The samples were scanned in the range of 4,000 to 400 cm−1 using Fourier transform infrared spectrometer. The spectra thus obtained was compared with the reference chart to identify the possible functional groups present in the sample which caused the reduction of the gold and silver ions into corresponding nano-particles.

Antibacterial activity of biosynthesized silver using Disc Diffusion method:

Antimicrobial activity of bio synthesized silver nano-particles was conducted in-vitro by disc diffusion assay method against E. coli (MTCC 448) and the result of which were compared with 2% Silver Nitrate solution as standard. Following composition of nutrient broth (see Table No. 1) was used for preparation of culture plates. Microbial strain for the antibacterial activity was purchased from MTCC. The Microbes were first revived as per the indications given by MTCC. Sub-culturing was done to the microbes using nutrient broth. The subculture thus obtained was used to test the antimicrobial activity of the nano-particles.

Results and Discussion

Nano-particles which were prepared using neem, vinca and datura plant extracts as reducing agents were collected, washed and dried. The collected and dried nano-particles were then subjected for analysis using different analytical and non-analytical methods. Analytical methods like UV-VIS spectroscopy, FT-IR spectroscopy were among the basic analytical technique for the characterization of the nano-particles. In-vitro analysis of silver nano-particles by disc diffusion method was also employed for deducing its antibacterial activity.

UV-VIS SPECTROSCOPY:

The synthesis of the reduced metal nano-particles (gold and silver) in the colloidal solution was monitored by UV-Vis spectrophotometer analysis. UV-Vis spectroscopic technique is one of the simplest techniques to identify the formation and stability of the gold and silver nano-particles in aqueous solution. Gold and silver nano-particles were known to exhibit maximum absorption in range of 500-600 nm and 400-500 nm respectively. The synthesis of gold nano-particles was monitored by observing the UV-VIS spectra after 3 hrs. of addition of metal salt solution to aqueous plant extract and stirring the mixture for half an hour. By marking the difference in the UVVIS spectra of the plant extract (Fig 1) and the colloidal solution obtained after adding the salt solution demonstrated the synthesis of nanoparticles (fig. 2 and Fig. 3). Maximum absorption at around 550 nm to which Auric chloride solution was added marked the synthesis of gold nanoparticles. on the similar basis maximum absorption at around 430 nm to which silver nitrate solution was added marked the synthesis of silver nano-particles.

FTIR analysis was done to identify possible biomolecules which get trapped on the surface of the nano-particles and are responsible for the reduction of metal salts to corresponding nanoparticles. FT-IR spectra of the nano-particles thus obtained by bio-molecular reduction process i.e. reduction using plant extract are as follows. (Fig. 4,5,6). Various peaks associated with various stretching and bending can be assigned to different functional groups like In Fig 4. -OH stretch for peak at 3422.63 cm-1, peak broadening of -OH group depicts Hydrogen bonding, C-H stretch in alkanes at 2926.20 cm-1, C=O stretch at 1637.40 cm-1. Similarly, in Fig.5, - OH stretch at 3468 cm-1 , C-H stretch of alkanes at 2926 cm-1, C=O (carboxylic acid stretch) at 1736.86 cm-1 and in Fig. 6. -OH stretch at 3433.95 cm-1 , C-H stretch of alkanes at 2925.95 cm-1, C=O (carboxylic acid stretch) at 1635.97 cm-1 respectively.

Anti microbial activity

The prepared silver nano-particles were subjected for their antimicrobial activity against E.coli (MTCC 448). Different concentrations of silver nano- particles were used for studying the effect of increasing the concentration of silver nano-particles and variation in the zone of inhibition by nano-particles. On a similar basis, 2% silver nitrate solution (standard in this case) was also checked for its anti-microbial activity (see Table No. 2) . It was observed that nano-particles produced using plant extracts as reducing agents exert higher zone of inhibition than the silver nitrate solution. A steep increment in the ZOI was observed when the concentration of silver nano-particles was increased from 100 μg to 200 μg. Afterwards a gradual change in ZOI was noticed.

Conclusion:

From this study we conclude that Neem, vinca and datura have good reducing properties and their aqueous extracts can be used to prepare gold nano-particles. In case of silver nanoparticles, Neem has shown the best results as reducing agent. On comparison of silver nanoparticles with silver nitrate solution, an increase inthe antimicrobial activity was obtained. this depicts that silver nano-particles have better antimicrobial activity than Silver nitrate solution.

Acknowledgement

The author would like to thank Dr. Rakesh kumar Sharma, Assistant Professor, Department of Chemistry, University of Delhi for providing the necessary facilities for carrying out the work in his research laboratory.

Tables at a glance

Table 1 Table 2

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10       Scheme 1 Scheme 2 Scheme 3

References

1. 1) Granqvist C, Buhrman R, Wyns J, Sievers A: Far- Infrared Absorption in Ultrafine Al Particles. Physical Review Letters 1976; 37(10): 625.
2. 2) MacNaught, Alan D, Wilkinson, Andrew R, ed.: Compendium of Chemical Terminology: IUPAC Recommendations. 2nd ed. Blackwell Science; 1997.
3. 3) Alemán, J. Chadwick, A. V. He, J. Hess, M. Horie, K. Jones et al.:Definitions of terms relating to the structure and processing of sols, gels, networks, and inorganic-organic hybrid materials (IUPACRecommendations 2007)". Pure and Applied Chemistry 2007;79(10).
4. 4) Elumalai EK, Prasad TNVKV, Hemachandran J, Viviyan S, Therasa, Thirumalai T and David E: Extracellular synthesis of silver nanoparticles using leaves of Euphorbia hirta and their antibacterial activities. J. Pharm. Sci. & Res. 2010; 2 (9): 549-554.
5. 5) SankarKalidasSivaraman, IniyanElango, SanjeevKumar ,VenugopalSanthanam: A green protocol for room temperature synthesis of silver nanoparticles in seconds: CURRENT SCIENCE2009; 97(7): 1055-1059.
6. 6) ChelladuraiMalarkodi, ShanmugamRajeshkumar, MahendranVanaja, KanniahPaulkumar, GnanadhasGnanajobitha, GurusamyAnnadurai: Eco-friendly synthesis and characterization of gold nanoparticles using Klebsiellapneumoniae: Journal Of Nanostructure in Chemistry2013; 3(30).
7. 7) Sumit.S.Lal, P.L.Nayak: International Journal of Science Innovations and Discoveries2012; 2 (3): 325-350.
8. 8) Ratul Kumar Das, NayanmoniGogoi, PunuriJayasekharBabu, Pragya Sharma, ChandanMahanta et al. : The Synthesis of Gold Nanoparticles Using Amaranthusspinosus Leaf Extract and Study of Their Optical Properties: Advances in Materials Physics and Chemistry2012; 2(1): 275-28.
9. 9) Storhoff JJ, Elghanian R, Mucic RC, Mirkin CA, Letsinger RL: One pot colorimetric differentiation of polynucleotides with single base imperfections using gold nanoparticle probes. J Am Chem Soc1998; 120:1959- 64.
10. 10) Klaine SJ, Alvarez PJ, Batley GE, Fernandes TF, Handy RD et. al: Nanomaterials in the environment: behavior, fate, bioavailability and effects. Environ Toxicol Chem2008; 21: 1825–51.
11. 11) Agaru: The ayurvedic pharmacopoeia of India .The Controller of Publications Government of India AYUSH, Delhi, Vol. II: 25–27;2004
12. 12) Shankar SS, Rai A, Ahmad A, Sastry M: Rapid synthesis of Au, Ag, and bimetallic Au core-Ag shell nanoparticles using Neem (Azadirachta indica) leaf broth, J. Colloid Interface Sci2004; 275: 496–502.
13. 13) https://www.discoverymedicine.com/Monica- Tang/files/2009/08/tang_38_tab-1.jpg
14. 14) AzimAkbarzadeh, DavoodZare, Ali Farhangi, Mohammad Reza Mehrabi, DariushNorouzian et al,: Synthesis and Characterization of Gold Nanoparticles by Tryptophane:. American Journal of Applied Sciences 2009; 6 (4): 691-695.
15. 15) Melissa M. Kemp, Ashavani Kumar, ShaymaaMousa, Tae-Joon Park, PulickelAjayan et al.: Synthesis of Gold and Silver Nanoparticles Stabilized with Glycosaminoglycans Having Distinctive Biological Activities Biomacromolecules2009; 10: 589–595.