INTRODUCTION:

Biosynthesis of nanoparticles has received considerable attention due to increasing need to develop ecofriendly benign technologies in material synthesis. Use of parts or whole plants in nanoparticles synthesis is an exciting possibility that is relatively unexplored and under exploited. Synthesis of metallic nanoparticles using plants can potentially eliminate the problem of toxicity by making them biocompatible. Use of plant extracts for the synthesis of metallic nanoparticles could be advantageous over other environmentally benign biological processes by eliminating elaborate process of maintaining cell cultures. Thus synthetic method by plant extracts exemplifies the promising application of green synthesis of metallic nanoparticles.

Very recently green silver nanoparticles have been synthesized using various plant extracts like Tulsi (Ocimum tenuifloram)¹, Jatropha curcas², Murraya koenigii³, Capsicum annuum L⁴., Argemone mexicana⁵, Ocimum⁶, Terminalia chebula⁷, Hibiscus rosa sinesis⁸, Artocarpus heterophyllus Lam.⁹, Carob¹⁰, Mimusops elengi¹¹, Macrotylum unilorum¹², Cycas¹³, coffee and tea¹⁴, etc. Present work was designed to synthesize, characterize and investigate the antibacterial activity of silver nanoparticles using aqueous leaf extract of Arisaema tortuosum . Arisaema tortuosum (Araceae) is also known as Bag mungri and commonly found in moist-shady localities of montane and sub-montane Himalaya. It is a annual-perennial, large glabrous herbs and upto 1m high. Leaves are two, pedately compound but leaflets (9-16) are unequal and ovate lanceolate. Berries are orange-red and crowded in fruiting spadix. Paste of tuber is applied on burns and plant is poisonous to cattle¹⁵. The seed oil of A. tortuosum was studied to know the contents of the compounds of fatty acids (lauric acid, myristic acid, palmitic acid, hexadecenoic acid, stearic acid, oleic acid, linoleic acid, etc.) present in the seed oil¹⁶. A. tortuosum tuber might be used as a promising and potent antioxidant, anti-inflammatory and antiproliferative agent¹⁷. This plant is used in Rajasthan for treating bone fractures¹⁸.

Fig. 1. Arisaema tortuosum

MATERIALS AND METHODS:

All the chemicals were provided by Department of Chemistry, HNB Garhwal University, Campus Pauri-Uttarakhand (India).

Synthesis of Silver Nanoparticles

Healthy leaves of Arisaema tortuosum(GUH20755) were collected and washed several times with deionized distilled water to remove any of the adhering pathogens/ dust particles and subsequently incised into very small pieces, placed in a hot air oven at 40⁰C and then mashed to the powder form using mortar-pestle. 05g of dried finely cut Arisaema tortuosumleaves were taken in a 250 ml Erlenmeyer conical flask containing 100 ml deionized distilled water and heated for 20-25 min at 70⁰C. Then the leaf extract was filtered in a separate conical flask and stored for further research work.

At room temperature, 1:9 mixture of Arisaema tortuosumleaf extract and 1mM Silver nitrate solution was prepared in conical flask. The mixed solution was kept for 24h in the dark place so that configuration of silver nanoparticles could take place. In the beginning, light red colour of solution appeared but after 12 and 24h, it was observed that the colour of the solution changed to dark red and greyish black respectively, which indicated that complete configuration of silver nanoparticles has been taken place and silver nanoparticles were formed there. Resulted solution was centrifuged for 10 minutes at 5000 rpm, then with water and ethanol, to remove unreacted/uncoordinated material. Finally, centrifuged material was dried in the oven at 65⁰C and greyish black pellets of silver nanoparticles was collected, but, to get a finer and uniform nanoparticles for characterization, pellets were mashed using mortar-pestle.

Fig. 2. (a) A. tortuosum leaf extract, (b) 1:9 Mixture, (c) after 12h and (d) finally after 24h

Antibacterial Activity

For antibacterial test, Soyabean casein digest agar of Hi Media Pvt. Bombay, India was used. The bacteria were inoculated into Soyabean casein digest agar and incubated at 37⁰C for 18h and suspension was checked to provide approximately, 10⁸ CFU/ml..

Pure cultures of test bacterial organisms viz. Bacillus subtilis, Staphylococcus aureus,Pseudomonas aeruginosaand Lactobacillus plantarumwere used for the research work.

Agar well diffusion method¹⁹ was modified. Soyabean casein digest agar medium (SCDM) was used for bacterial cultures and culture medium was inoculated with the bacteria separately suspended in nutrient broth. A total of 8mm diameter wells were punched into the agar and filled with nanoparticles solution prepared in DMSO and solvent blanks. DMSO was used as negative control. Standard antibiotic (Erythromycin, 1mg/ml) was simultaneously used as the positive control. The plates were then incubated at 37⁰C for 18h. The antibacterial activity was evaluated by measuring the diameter of zone of inhibition. The procedure for assaying antibacterial activity was performed in triplicates to confirm the readings.

RESULTS AND DISCUSSION:

Fig. 3 represents the UV-Vis spectra of aqueous solution of leaf extract and silver nanoparticles as a function of time variation. Silver nanoparticles have free electrons, which gives surface plasmon resonance absorption band due to joint vibrations of electrons of silver nanoparticles in resonance with light waves.

Leaf extract solution was kept as blank and sharp absorption bands of silver nanoparticles at 430 nm were observed at different intervals of time. Intensity of absorption band was increased with passage of time and after 24h, no such increase was observed that means complete configuration of silver nanoparticles has been taken place.

Crystalline nature of silver nanoparticles was further confirmed by XRD analysis (Fig. 4). Three distinct peaks at 37.02⁰, 43.18⁰ and 63.40⁰ were observed which can be indexed to (111), (200) and (220) of cubic face centred silver nanoparticle.

TEMimages {Fig. 5 (a) and (b)} confirmed that the silver nanoparticles were spherical in nature and size of obtained silver nanoparticles were in the range of 8-10 nm with average size less than 10 nm. And FTIR (Fig. 6) measurements were carried out to identify the bio-functional groups for encircling and stabilization of silver nanoparticles. Peaks at 3295.45, 1588.82 and 1399.90 cm^-1 are representing the -OH, C=C and Ar-OH functional groups, respectively. These bio-functional groups encircled and stabilized the synthesized silver nanoparticles using A. tortuosum leaf extract.

Fig 3 UV-Vis spectrum of Ag nanoparticles at different intervals of time.

Fig.4 XRD pattern of Ag nanoparticles.

(a)

(b)

Fig. 5 (a) and (b) TEM images of Ag nanoparticles.

Fig. 6 FTIR spectrum of Ag nanoparticles.

Antibacterial Activity

Biosynthesized silver nanoparticles were found to be very toxic against multi drug resistant pathogenic bacteria at a concentration of 100 µl (Table 1). Silver nanoparticles synthesized using aqueous leaf extract of A. tortuosum, exhibited antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosaandLactobacillus plantarum. Nanoparticles showed much better results than the positive control (Erythromycin- 1mg/ml).

CONCLUSION:

It can be concluded that configuration of silver nanoparticles can be exhibited by using aqueous leaf extract of A. tortuosum. Well defined, crystalline, spherical and size less than 10 nm, silver nanoparticles were biosynthesized. Apart from this, study proved that biosynthesized silver nanoparticles using leaf extract can be assumed to have great potential in biomedical applications.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 15.12.2017 Modified on 04.01.2018

Asian J. Research Chem. 2018; 11(2):419-422.

DOI:10.5958/0974-4150.2018.00076.7

S.No.	Sample/Controls (100 µl)	Diameter of zone of inhibition (mm)
S.No.	Sample/Controls (100 µl)	*Bacillus subtilis* (NCFT.583.08)	*Staphylococcus aureus* (NCFT.576.08)	*Pseudomonas aeruginosa* (NCFT.645.11)	*Lactobacillus plantarum* (NCFT. 623.34)
1.	Nanoparticles	25.0	38.0	43.0	28.0
Positive control	Erythromycin (1mg/ml)	45.0	34.0	38.0	35.0