Accepted Manuscript Green synthesis of silver nanoparticles from the extract of the inflorescence of Cocos nucifera (Family: Arecaceae) for enhanced antibacterial activity R. Mariselvam, A.J.A. Ranjitsingh, A. Usha Raja Nanthini, K. Kalirajan, C. Padmalatha, P. Mosae Selvakumar PII: DOI: Reference:
S1386-1425(14)00477-6 http://dx.doi.org/10.1016/j.saa.2014.03.066 SAA 11899
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
24 January 2014 10 March 2014 21 March 2014
Please cite this article as: R. Mariselvam, A.J.A. Ranjitsingh, A. Usha Raja Nanthini, K. Kalirajan, C. Padmalatha, P. Mosae Selvakumar, Green synthesis of silver nanoparticles from the extract of the inflorescence of Cocos nucifera (Family: Arecaceae) for enhanced antibacterial activity, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.03.066
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Green synthesis of silver nanoparticles from the extract of the inflorescence of Cocos nucifera (Family: Arecaceae) for enhanced antibacterial activity R.Mariselvam1, A.J.A.Ranjitsingh1*, A.Usha Raja Nanthini1, K.Kalirajan2, C.Padmalatha3 and P.Mosae Selvakumar4 1 2
Department of Zoology, Sri Paramakalyani College, Alwarkurichi, Tamilnadu, India
Department of Chemistry, Sri Paramakalyani College, Alwarkurichi, Tamilnadu, India 3
The Principal, M.V.M. Govt. College for women, Dindigul, Tamilnadu, India.
4
Department of Chemistry, Karunya University, Coimbatore, Tamilnadu, India * Corresponding Author
Email: [email protected]
Abstract: Green synthesis of nanoparticles using plant source has been given much importance. In the present study, silver nanoparticles (AgNPs) were synthesized using the ethyl acetate and methanol (EA: M 40:60) extracts of the inflorescence of the tree Cocous nucifera. The synthesized nanoparticles were characterized by UV-visible Spectroscope, FTIR and TEM analysis. The particle size of the synthesized AgNPs was 22nm as confirmed by TEM. The qualitative assessment of reducing potential of the extracts of inflorescence indicated the presence of reducing agents. Synthesized AgNPs exhibited significant antimicrobial activity against human bacterial pathogens viz., Klebsiella pneumoniae, Bacillus substillus, Pseudomonas aeruginosa and Salmonella paratyphi. Key words: Cocos nucifera; Silver nanoparticles; Antimicrobial activity; Phytochemicals; Green synthesis.
1. Introduction: The metal nanoparticles are finding rich applications in optoelectronics, nanodevices, nano electronics, nano sensors, information storage and catalysis [1]. Among various metal nanoparticles, silver nanoparticles have attracted more attention because of their antimicrobial activity [2-5]. Jai dev and Narasimha [6] had reported that binding of silver nanoparticles of size equal/less than 5nm to gp120 protein of HIV virus prevented virus from attaching itself to the host tissue cells. Nanoparticles of noble metals have unique chemical and physical attributes that differ from the bulk substance [7]. The extremely small size and large surface area relative to their volume makes them useful for applications in nonlinear 3
optics, spectrally selective coating for solar energy absorption, optical receptors, catalysis in a chemical reaction, bio labelling, water purification and as antibacterials [8-10]. The production of metal based nanoparticles by chemical and physical methods are not eco friendly. Hence biological and biomimetic method of synthesis of nanoparticles are given much attention. In this approach green synthesis of metal nanoparticles has received an importance, as this approach does not involve any hazardous material. Synthesis of inorganic nanoparticles by biological synthesis makes them more biocompatible and environmentally benign [11]. Plant extract mediated synthesis of metal nanoparticles has an edge over microbial mediated biosynthesis of nanoparticles because the green synthesis of nanoparticles takes place extracellularly. Further, this process is quick and suitable for large scale synthesis [12, 13]. The present study focuses on the synthesis of silver nanoparticles using the extracts of the inflorescences of coconut tree (Cocos nucifera). Also attempts were made to characterize the synthesized nanoparticles using UV-visible Spectroscopy, FTIR, and TEM study. The inherent antibacterial potential of synthesized silver nanoparticles and crude extract of the inflorescence were explored. In the present work we present a rapid method for nanoparticles production using the inflorescence extracts of coconut tree, Cocos nucifera.
2. Materials and Methods: 2.1. Cocos nucifera inflorescence and crude extract preparation: The inflorescence of 25 years old coconut trees growing in the fertile lands of Kanyakumari district, Tamilnadu, India were collected. The collected inflorescences were shade dried and powdered. Methanol was used as a solvent to extract the bioactive compounds present in the inflorescence. The extracts were filtered using Whatman No 1 filter paper. The excess of methanol was removed using rotary evaporator and water bath.
2.2. Purification of crude extract: Five grams of crude extract was dissolved in equal amount of methanol (M) and to which equal amount of silica gel (60-120 mesh) was added to make column chromatograpical separation. Different combination of ethyl acetate (EA) and methanol (EA 100%, EA: M 90:10%............... to M 100%) were used to separate the bioactive compounds in the crude extract. The EA: M 40:60 fraction was found to have a good antibacterial activity and this fraction selected for further study.
4
2.3. Phytochemical screening of selected fraction of the extract (EA: M 40:60): Phytochemicals screening was made to find out the presence of carbohydrates, protein, amino acid, alkaloid, flavanoid, tannins, saponins, terpenoids, aromatic acids, phenolic compounds, Xanthoprotein, reducing sugar and triterpenoids using standard procedure.
2.4. Synthesis of silver nanoparticles (AgNPs): To ten millilitres of selected fraction (10%) (100ml of EA:M 40:60%) of inflorescence extracts and ninety millilitre of silver nitrate solution (1mM; 0.169grams of silver nitrate dissolved in 1000ml Double Distilled Water) was added and mixed well. The mixture was kept at room temperature (36±1oC) undisturbed for 24h. After 24h the bio reduced silver nitrate solution became brown in colour indicating the synthesis of silver nanoparticles.
2.5. Characterization of synthesized silver nanoparticles: The synthesized NPs were characterized using a UV-visible spectrophotometer, FTIR and Transmission Electron Microscope (TEM). The UV-visible analysis was performed in the absorption wavelength of 200 to 1000nm. The synthesized silver nanoparticles were studied
using FTIR (Perkin-Elmer GX FT-IR spectrometer). Transmission Electron Microscopy was used to observe the size, shape and morphology of the synthesized nanoparticles. Samples for TEM observation were prepared by casting a drop of the silver nanoparticles on a carbon coated copper grid and the excess solution was removed by tissue paper and allowed to air dry at room temperature for overnight.
2.6. Assessment of antibacterial activity: The antibacterial activity of green synthesized AgNPs was tested against ten bacterial isolates using Agar well diffusion method (Ahmad and Beg, 2001). Nutrient Agar plates were inoculated with 100µl of standardized culture (1.5x108 CFU/ml) of each bacterium (in triplicates) and spread with sterile swabs. Wells of 8 mm size are made in the Agar plates containing the bacterial lawn. From the synthesized AgNPs, 25µl, 50µl, 75µl and 100µl volume were poured into the wells made in the bacterial culture plates. Standard chemical (AgNO3) solution was used as a negative control. The plates thus prepared were left at room temperature for ten minutes for allowing the diffusion of the extract into the agar bacterial 5
lawn. After incubation for 24 h at 37oC, the plates were observed. The zone of inhibition was measured and expressed in millimetres. The antibacterial activity was expressed in term of the diameter of the zone of inhibition and 18mm as very active [14, 15].
2.7. Minimum Inhibitory Concentration (MIC) of the AgNPs: The minimum inhibitory concentration at which a silver nanoparticles exhibits the antimicrobial activity, was determined by using a 96 wells titer plate. Each well in the plate was filled with 250µl of Nutrient broth and inoculated with different microorganisms selected for the assay. Different concentrations (5µl, 10µl, 15µl, 20µl, 25µl, 30µl, 35µl and 40µl) of AgNPs were added in to the wells and incubated at 37o C for 24 h. The growth rate of each bacterium was determined by the turbidity method using ELISA reader at 600nm wavelength.
2.8. Mechanism of action of AgNPs on microorganisms using the simple staining method: One ml of 24 h bacterial culture was mixed with 1ml of synthesized silver nanoparticles incubated at 37oC for 24 h. After incubation the bacterial cultures were Gram stained and the colony characteristics were observed using a light microscope.
3. Results and Discussion: The phytochemical contents in the selected fraction of the extracts of coconut inflorescence are summarized in the Table 1. The presence of Tannin, alkaloids, carbohydrates, terpenoids, saponins, phenolic compounds and reducing sugar were observed in the extracts. Table 1: Phytochemical components in the extracts of Coconut tree inflorescences (crude fraction EA: M 40:60): S.No
Test for
Presence/Absence
1
Carbohydrate
+
2
Protein
-
3
Amino acid
-
4
Alkaloids
+
5
Flavanoid
-
6
6
Terpenoids
+
7
Tannins
+
8
Saponins
+
9
Aromatic acids
-
10
Phenolic compounds
+
11
Xanthoprotein
-
12
Reducing sugar
+
13
Triterpenoids
-
14
Phlobatinins
-
Formation of AgNPs by the reduction of AgNO3 during treatment with the extracts of coconut tree inflorescence is evident from the change in color of the reaction mixture. The change in color of the reaction mixture after 2 hours is presented in figure 1, which indicated the formation of AgNPs. This reaction indicates that silver ions in reaction mixture had been converted to elemental silver having the size of nanometric range. Phytochemical screening of the inflorescence extract revealed the presence of carbohydrates, alkaloids, terpenoids, tannins, saponins, phenolic compounds and reducing sugar. Shankar et al., [16] had reported that the reducing sugars are responsible for the reduction of silver nitrate to silver nanoparticles. Natural antioxidants and phytochemical like caffeine had also been reported to induce the reduction of AgNO3 to AgNPs [13]. The presence of different phytochemicals in the EA: Methanol faction of Cocous nucifera inflorescence (Alkaloid, Tannin, Saponins, Terpenoids, reducing sugar and carbohydrates) had influenced the reduction of AgNO3 to AgNPs.
Figure 1: Synthesis of AgNPs.
7
The UV- vis absorption spectra (Fig 2) of silver nanoparticles indicated the absorption peak in the visible range 427-428nm. A single peak at the wavelength 427-428 suggested the spherical size of the nanoparticles as per Mie theory. The spherical size of the AgNPs was further confirmed by TEM study. The periodic observation of prepared AgNPs during the storage period (2 months) did not show any variation in the absorption spectrum. This indicated the constancy of particle size even during storage.
Fig 2: UV - visible Spectrum of the synthesized AgNPs: FTIR analysis showed strong bands at1021cm-1, 1256 cm-1 and 3340 cm-1. The band at 1021 cm-1 corresponds to C-O stretching frequency. The band at 1256 cm-1 corresponds to C-X stretching frequency. 3340 cm-1 corresponds to N-H stretching frequency. The strong band at 1021 cm-1 represents C-O stretching of ether. A strong band at 1638 cm-1 (-C=O-) established the formation of silver nanoparticles. FTIR reports indicated that the peaks were more characteristic of terpenoids and the stabilization is achieved by phenolic as well as aromatic compounds present in the extract.
8
Fig 3: FTIR image of Selected fraction of coconut inflorescence extracts (EA: M (40:60)) and synthesized silver nanoparticles: Figure 4 shows TEM images of AgNPs. The images confirmed that the silver nanoparticles are spherical in shape and the particle size was 22nm.
Fig 4: TEM micrograph of AgNPs:
9
3.1. Antibacterial activity: The antibacterial potential of silver is well known. The synthesized AgNPs were tested for their antibacterial action using ten types of human bacterial pathogens (Table 2). The bacterial growth inhibitory potential of AgNPs was compared with standard antibiotics. At a 100µl dose the inhibitory action of AgNPs was high. The maximum inhibition was observed for K. pneumoniae (24mm), P. shigelloids (21mm), Vibrio alginolyticus (19mm) and Salmonella paratyphi (16mm). The crude extract of the inflorescence and silver nitrate were also tested separately for antibacterial activity. Bioreduced silver nanoparticles showed a higher antibacterial activity than the crude extract. Table 2: Table showing the bacterial growth inhibition potential of green synthesized AgNPs, crude extracts of inflorescence and un reduced AgNO3: S.No Microorganisms
Zone of Inhibition (mm) AgNPs AgNPs AgNPs AgNPs Unreduced Inflorescence 25µl
50µl
75µl 100 µl
AgNO3
Ampicilin
Extract (crude) (10mg/ml) (100 µl)
1
Vibrio alginolyticus
11
16
18
19
13
12
10
2
P. shigelloides
16
18
20
21
20
9
9
3
K .pneumoniae
15
17
21
24
16
12
11
4
Salmonella paratyphi
0
12
14
16
11
15
9
5
P. aeruginosa
10
11
12
14
11
10
10
6
Vibrio harveyi
10
12
12
14
15
13
12
7
Bacillus substillus
0
12
12
14
11
12
11
8
E.coli
0
10
11
12
10
10
15
9
Vibrio mimicus
0
0
0
0
0
9
13
10
Staphylococcus aureus
0
0
0
0
0
10
8
10
3.2. The Minimum Inhibitory Concentration (MIC): The MIC of silver nanoparticles dispersed in the micro titer plate are summarized in the bar diagram as a 24 h OD value of the microbial growth. The growth rate was calculated using the standard or control. Fifteen µl of silver nanoparticles was the MIC for following organisms viz., Vibrio alginolyticus, Klebsiella pnemoniae, Pseudomonas aeruginosa, Bacillus substillis and Plesiomonas shigelloides. For other organisms MIC was 25 µl. (Vibrio harveyi, Salmonella paratyphi Vibrio mimicus and Vibrio alginolyticus). The result of the MIC value indicates the high inhibitory potential of AgNPs on gram +ve bacterial pathogens. For E.coli, V.mimicus and S. aureus there was poor inhibitory action. But, the crude extract of the inflorescence and standard antibiotics showed inhibition. This indicates that the bioactive compounds mediated AgNPs have a good antibacterial capacity. The OD values of the mixture containing AgNPs and bacterial inoculums showing the bar diagram. Bar Diagram 1: MIC for different bacterial pathogen after 24 h. 1.4
MIC for different bacterial pathogen after 24 h
1.2
5µl
Optical density
1
10 µl
0.8
15 µl 0.6
20 µl 25 µl
0.4
30 µl 0.2
35 µl
0
40 µl 1
2
3
4
5
6
7
8
9
10
Control
Growth of different microorganisms as shown by OD value of culture
1- Vibrio alginolyticus, 2- P. shigelloides, 3- K .pneumoniae, 4- Salmonella paratyphi, 5- P. aeruginosa, 6- Vibrio harveyi, 7- Bacillus substillus, 8- E.coli, 9- Vibrio mimicus, 10Staphylococcus aureus
11
Fig 5: Mechanism of action of AgNPs on Microorganisms: 5A photomicrograph showing freely distributed K .pneumoniae population. 5B photomicrograph showing E.coli cells after treating with nanoparticles. 5C photomicrograph showing a mass agglutinated but alive Vibrio alginolyticus bacteria. 5D photomicrograph showing scattered P.shigellioids bacterial aggregation. 5E photomicrograph showing P. aeruginosa aggregation process with silver nanoparticles addition. The Figure 5 represent to the Gram of bacterial strains to show the mechanical action of silver nanoparticles that bind on the bacterial cell wall. Gram staining of AgNPs treated bacteria showed the disintegration of cellular organisation in bacteria. The bacterial cells were ruptured after binding with AgNPs. The study indicated that the green synthesized AgNPs interfere with the molecular built up of bacterial cell wall. The antibacterial activity of the synthesized AgNPs showed varying degree of inhibition in relation to bacterial species and concentration. The optical density value revealed that 15µl dose of AgNPs is the MIC for V. alginolyticus, K.pneumoniae, P.aeroginosa, B.sustillus and P.shigelloids.
For the bacteria V.harveyi, S.paratyphi,
V.mimicus and V.alginolyticus the MIC was 25µl. Silver nanoparticles were found to be less active in killing S.aureus and E.coli. Okafor et al [17] had also reported that the antibacterial sensitivity of AgNPs on the Gram
+ve
S.aureus was greatly lower than that of the Gram-ve
E.coli due to the presence of thick peptidoglycan layer that protects against toxins and chemicals. But in the present investigation both the Gram positive S.aureus and Gram 12
negative E.coli exhibited low sensitivity. So the mechanism of the silver nanoparticles on bacteria is still unclear. The antibacterial activity was examined by comparing colonyforming capability of different bacteria treated by various concentration of AgNPs. Antibacterial activity is studied qualitatively by agar medium by well diffusion and quantitatively interms of the MIC. A lower MIC (OD value) corresponds to higher antibacterial effectiveness. As given in Bar diagram 1 AgNPs could effectively kill bacteria in a concentration-dependent manner. Luo et al., [18] had reported that the nanoparticles induce oxidative stress to bacteria and induce ROS production. Further, under normal circumstances cells are able to defend themselves against ROS damage with antioxidant enzymes. But when the nanoparticles are inside the cell, the nanoparticles could restrain antioxidative enzymes to inhibit the capability of removing ROS. Priester et al., [19] also reported that the nanoparticles break the balance of oxidant/antioxidant and generate the accumulation of ROS in bacteria. It has also been reported that when silver nanoparticles are attached to the surface of cell membrane, the respiratory function and permeability of the bacterial cell become unstable. Other studies suggest that when bacteria are treated with silver ions, DNA tends to lose its ability to replicate. However exact mechanism of AgNPs on different bacterial cells needs a further study.
4. Conclusion: In the present study silver nanoparticles were synthesized using the extracts of inflorescence of the Cocos nucifera. The green synthesized silver nanoparticles were characterized using UV-visible Spectroscopy, FT-IR Spectroscopy and TEM analysis. The phytochemical study of selected fraction of the inflorescence extract showed in the presence of reducing sugar. So the reducing sugar was found to be major agent for silver nanoparticles synthesis. The synthesized silver nanoparticles were tested against ten bacterial strains among these the V. alginolyticus, K.pneumoniae, P.aeroginosa, B.sustillus and P.shigelloids were highly sensing to synthesized silver nanoparticles. Microscopical images of Gram strains of bacterias show that the mechanical action of silver nanoparticles that bind on the bacterial cell wall.
5. References: [1]
J. Ramyadevi, K. Jeyasubramanian, A. Marikani, G. Rajakumar, A.A. Rahuman. Mater. Lett. 71 (2012) 114.
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[2]
A. Dror-Ehre, H. Mamane, T. Belenkova, G. Markovich, A. Adin. J. Colloid Interface Sci. 339 (2009) 521.
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D. Jain, H. Kumar Daima, S. Kachhwaha, S.L. Kothari. Digest Journal of Nanomaterials and Biostructures. 4 (2009) 557.
[4]
V.K. Sharma, R.A. Yngard, Y. Lin. Adv. Colloid Interface Sci. 145 (2009) 83.
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D.K. Bozanic, B. Dimetrijevic, N. Bibic, A.S. Luyt, V. Djokovic. Carbohydr. Polym. 83 (2011) 883.
[6]
L.R. Jaidev, G. Narasimha. Colloids Surf., B. 81 (2010) 430.
[7]
D.L. Feldheim, C.A. Foss jr. Marcel Dekker. New York. (2002).
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M. Rai, A. Yadav, A. Grade. Biotechnology Advances. 27 (2009) 76.
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J. Musarrat, S. Dwivedi, B.R. Singh, A.A. Al-Khedhairy, A.A.A. Naqvi. Bio resour Technol. 101 (2010) 8772.
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A.K. Sherly, D. Arun, Paulraj Mosae Selvakumar. Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry. 43 (2013) 1068.
[11]
K. Govindaraju, S. Tamilselvan, V. Kiruthiga, G. Shankaravelu. Journal of Biopesticides. 3 (2010) 394.
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S.S. Shankar, A. Rai, A. Ahamad, M. Sastry. J. Colloid Interface Sci. 275 (2004) 496.
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Charusheela
Ramteke, Tapan
Chakrabarti, Bijaya
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Sarangi, Ram-Avatar
Pandey. Journal of Chemistry. (2013) 01. [14]
Junior, C. Zanil. Brazilian Journal of Sciences. 95 (2000) 367.
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R. Mariselvam, K. Kalirajan, A.J.A. Ranjit Singh. Journal of Applied Pharmaceutical Science. 02 (2012) 210.
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S.S. Shankar, A. Rai, A. Ahamad, M. Sastry. Chem. Mater. 17 (2005) 566.
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F. Okafor, A. Janen, T. Kukhtareva, V. Edwards, M. Curley. International Journal of Environmental Research and Public Health. 10 (2013) 5221.
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Z. Luo, Q. Wu, M. Zhang, P. Li, Y. Ding. J. Colloid Interface Sci. 362 (2011) 100.
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J.H. Priester, P.K. Stoimenov, R.E. Mielke, S.M. Webb, C. Ehrhardt, J.P. Zhang, G.D. Stucky, P.A. Holden. Environmental Science and Technology. 43 (2009) 2589.
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Green synthesis of silver nanoparticles from the ethyl acetate: methanol extract of the inflorescence of Cocos nucifera (Family: Arecaceae) for enhanced antibacterial activity R.Mariselvam1, A.J.A.Ranjitsingh1*, A.Usha Raja Nanthini1, K.Kalirajan2, C.Padmalatha3 and P.Mosae Selvakumar4 1 2
Department of Zoology, Sri Paramakalyani College, Alwarkurichi, Tamilnadu, India
Department of Chemistry, Sri Paramakalyani College, Alwarkurichi, Tamilnadu, India 3
The Principal, M.V.M. Govt. College for women, Dindigul, Tamilnadu, India.
4
Department of Chemistry, Karunya University, Coimbatore, Tamilnadu, India * Corresponding Author
Email: [email protected]
Preparation of AgNPs using the selected fraction of the ethyl acetate – methanol
extracts of the inflorescence of Cocos nucifera. Synthesis of silver nanoparticles by using plant extract as reducing agent. The green synthesized AgNPs were characterized by using UV/Visible spectroscopy, FTIR and TEM study. The Green synthesized nanoparticles exhibited a good antibacterial activity against selected bacterial human pathogens.
The mechanism of action of AgNPs on bacterial strain was analysed by using Gram straining procedure.
15
Green synthesis of silver nanoparticles from the ethyl acetate: methanol extract of the inflorescence of Cocos nucifera (Family: Arecaceae) for enhanced antibacterial activity R.Mariselvam1, A.J.A.Ranjitsingh1*, A.Usha Raja Nanthini1, K.Kalirajan2, C.Padmalatha3 and P.Mosae Selvakumar4 1 2
Department of Zoology, Sri Paramakalyani College, Alwarkurichi, Tamilnadu, India
Department of Chemistry, Sri Paramakalyani College, Alwarkurichi, Tamilnadu, India 3
The Principal, M.V.M. Govt. College for women, Dindigul, Tamilnadu, India.
4
Department of Chemistry, Karunya University, Coimbatore, Tamilnadu, India * Corresponding Author
Email: [email protected]
16
S1386-1425(14)00477-6 http://dx.doi.org/10.1016/j.saa.2014.03.066 SAA 11899
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
24 January 2014 10 March 2014 21 March 2014
Please cite this article as: R. Mariselvam, A.J.A. Ranjitsingh, A. Usha Raja Nanthini, K. Kalirajan, C. Padmalatha, P. Mosae Selvakumar, Green synthesis of silver nanoparticles from the extract of the inflorescence of Cocos nucifera (Family: Arecaceae) for enhanced antibacterial activity, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.03.066
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Green synthesis of silver nanoparticles from the extract of the inflorescence of Cocos nucifera (Family: Arecaceae) for enhanced antibacterial activity R.Mariselvam1, A.J.A.Ranjitsingh1*, A.Usha Raja Nanthini1, K.Kalirajan2, C.Padmalatha3 and P.Mosae Selvakumar4 1 2
Department of Zoology, Sri Paramakalyani College, Alwarkurichi, Tamilnadu, India
Department of Chemistry, Sri Paramakalyani College, Alwarkurichi, Tamilnadu, India 3
The Principal, M.V.M. Govt. College for women, Dindigul, Tamilnadu, India.
4
Department of Chemistry, Karunya University, Coimbatore, Tamilnadu, India * Corresponding Author
Email: [email protected]
Abstract: Green synthesis of nanoparticles using plant source has been given much importance. In the present study, silver nanoparticles (AgNPs) were synthesized using the ethyl acetate and methanol (EA: M 40:60) extracts of the inflorescence of the tree Cocous nucifera. The synthesized nanoparticles were characterized by UV-visible Spectroscope, FTIR and TEM analysis. The particle size of the synthesized AgNPs was 22nm as confirmed by TEM. The qualitative assessment of reducing potential of the extracts of inflorescence indicated the presence of reducing agents. Synthesized AgNPs exhibited significant antimicrobial activity against human bacterial pathogens viz., Klebsiella pneumoniae, Bacillus substillus, Pseudomonas aeruginosa and Salmonella paratyphi. Key words: Cocos nucifera; Silver nanoparticles; Antimicrobial activity; Phytochemicals; Green synthesis.
1. Introduction: The metal nanoparticles are finding rich applications in optoelectronics, nanodevices, nano electronics, nano sensors, information storage and catalysis [1]. Among various metal nanoparticles, silver nanoparticles have attracted more attention because of their antimicrobial activity [2-5]. Jai dev and Narasimha [6] had reported that binding of silver nanoparticles of size equal/less than 5nm to gp120 protein of HIV virus prevented virus from attaching itself to the host tissue cells. Nanoparticles of noble metals have unique chemical and physical attributes that differ from the bulk substance [7]. The extremely small size and large surface area relative to their volume makes them useful for applications in nonlinear 3
optics, spectrally selective coating for solar energy absorption, optical receptors, catalysis in a chemical reaction, bio labelling, water purification and as antibacterials [8-10]. The production of metal based nanoparticles by chemical and physical methods are not eco friendly. Hence biological and biomimetic method of synthesis of nanoparticles are given much attention. In this approach green synthesis of metal nanoparticles has received an importance, as this approach does not involve any hazardous material. Synthesis of inorganic nanoparticles by biological synthesis makes them more biocompatible and environmentally benign [11]. Plant extract mediated synthesis of metal nanoparticles has an edge over microbial mediated biosynthesis of nanoparticles because the green synthesis of nanoparticles takes place extracellularly. Further, this process is quick and suitable for large scale synthesis [12, 13]. The present study focuses on the synthesis of silver nanoparticles using the extracts of the inflorescences of coconut tree (Cocos nucifera). Also attempts were made to characterize the synthesized nanoparticles using UV-visible Spectroscopy, FTIR, and TEM study. The inherent antibacterial potential of synthesized silver nanoparticles and crude extract of the inflorescence were explored. In the present work we present a rapid method for nanoparticles production using the inflorescence extracts of coconut tree, Cocos nucifera.
2. Materials and Methods: 2.1. Cocos nucifera inflorescence and crude extract preparation: The inflorescence of 25 years old coconut trees growing in the fertile lands of Kanyakumari district, Tamilnadu, India were collected. The collected inflorescences were shade dried and powdered. Methanol was used as a solvent to extract the bioactive compounds present in the inflorescence. The extracts were filtered using Whatman No 1 filter paper. The excess of methanol was removed using rotary evaporator and water bath.
2.2. Purification of crude extract: Five grams of crude extract was dissolved in equal amount of methanol (M) and to which equal amount of silica gel (60-120 mesh) was added to make column chromatograpical separation. Different combination of ethyl acetate (EA) and methanol (EA 100%, EA: M 90:10%............... to M 100%) were used to separate the bioactive compounds in the crude extract. The EA: M 40:60 fraction was found to have a good antibacterial activity and this fraction selected for further study.
4
2.3. Phytochemical screening of selected fraction of the extract (EA: M 40:60): Phytochemicals screening was made to find out the presence of carbohydrates, protein, amino acid, alkaloid, flavanoid, tannins, saponins, terpenoids, aromatic acids, phenolic compounds, Xanthoprotein, reducing sugar and triterpenoids using standard procedure.
2.4. Synthesis of silver nanoparticles (AgNPs): To ten millilitres of selected fraction (10%) (100ml of EA:M 40:60%) of inflorescence extracts and ninety millilitre of silver nitrate solution (1mM; 0.169grams of silver nitrate dissolved in 1000ml Double Distilled Water) was added and mixed well. The mixture was kept at room temperature (36±1oC) undisturbed for 24h. After 24h the bio reduced silver nitrate solution became brown in colour indicating the synthesis of silver nanoparticles.
2.5. Characterization of synthesized silver nanoparticles: The synthesized NPs were characterized using a UV-visible spectrophotometer, FTIR and Transmission Electron Microscope (TEM). The UV-visible analysis was performed in the absorption wavelength of 200 to 1000nm. The synthesized silver nanoparticles were studied
using FTIR (Perkin-Elmer GX FT-IR spectrometer). Transmission Electron Microscopy was used to observe the size, shape and morphology of the synthesized nanoparticles. Samples for TEM observation were prepared by casting a drop of the silver nanoparticles on a carbon coated copper grid and the excess solution was removed by tissue paper and allowed to air dry at room temperature for overnight.
2.6. Assessment of antibacterial activity: The antibacterial activity of green synthesized AgNPs was tested against ten bacterial isolates using Agar well diffusion method (Ahmad and Beg, 2001). Nutrient Agar plates were inoculated with 100µl of standardized culture (1.5x108 CFU/ml) of each bacterium (in triplicates) and spread with sterile swabs. Wells of 8 mm size are made in the Agar plates containing the bacterial lawn. From the synthesized AgNPs, 25µl, 50µl, 75µl and 100µl volume were poured into the wells made in the bacterial culture plates. Standard chemical (AgNO3) solution was used as a negative control. The plates thus prepared were left at room temperature for ten minutes for allowing the diffusion of the extract into the agar bacterial 5
lawn. After incubation for 24 h at 37oC, the plates were observed. The zone of inhibition was measured and expressed in millimetres. The antibacterial activity was expressed in term of the diameter of the zone of inhibition and 18mm as very active [14, 15].
2.7. Minimum Inhibitory Concentration (MIC) of the AgNPs: The minimum inhibitory concentration at which a silver nanoparticles exhibits the antimicrobial activity, was determined by using a 96 wells titer plate. Each well in the plate was filled with 250µl of Nutrient broth and inoculated with different microorganisms selected for the assay. Different concentrations (5µl, 10µl, 15µl, 20µl, 25µl, 30µl, 35µl and 40µl) of AgNPs were added in to the wells and incubated at 37o C for 24 h. The growth rate of each bacterium was determined by the turbidity method using ELISA reader at 600nm wavelength.
2.8. Mechanism of action of AgNPs on microorganisms using the simple staining method: One ml of 24 h bacterial culture was mixed with 1ml of synthesized silver nanoparticles incubated at 37oC for 24 h. After incubation the bacterial cultures were Gram stained and the colony characteristics were observed using a light microscope.
3. Results and Discussion: The phytochemical contents in the selected fraction of the extracts of coconut inflorescence are summarized in the Table 1. The presence of Tannin, alkaloids, carbohydrates, terpenoids, saponins, phenolic compounds and reducing sugar were observed in the extracts. Table 1: Phytochemical components in the extracts of Coconut tree inflorescences (crude fraction EA: M 40:60): S.No
Test for
Presence/Absence
1
Carbohydrate
+
2
Protein
-
3
Amino acid
-
4
Alkaloids
+
5
Flavanoid
-
6
6
Terpenoids
+
7
Tannins
+
8
Saponins
+
9
Aromatic acids
-
10
Phenolic compounds
+
11
Xanthoprotein
-
12
Reducing sugar
+
13
Triterpenoids
-
14
Phlobatinins
-
Formation of AgNPs by the reduction of AgNO3 during treatment with the extracts of coconut tree inflorescence is evident from the change in color of the reaction mixture. The change in color of the reaction mixture after 2 hours is presented in figure 1, which indicated the formation of AgNPs. This reaction indicates that silver ions in reaction mixture had been converted to elemental silver having the size of nanometric range. Phytochemical screening of the inflorescence extract revealed the presence of carbohydrates, alkaloids, terpenoids, tannins, saponins, phenolic compounds and reducing sugar. Shankar et al., [16] had reported that the reducing sugars are responsible for the reduction of silver nitrate to silver nanoparticles. Natural antioxidants and phytochemical like caffeine had also been reported to induce the reduction of AgNO3 to AgNPs [13]. The presence of different phytochemicals in the EA: Methanol faction of Cocous nucifera inflorescence (Alkaloid, Tannin, Saponins, Terpenoids, reducing sugar and carbohydrates) had influenced the reduction of AgNO3 to AgNPs.
Figure 1: Synthesis of AgNPs.
7
The UV- vis absorption spectra (Fig 2) of silver nanoparticles indicated the absorption peak in the visible range 427-428nm. A single peak at the wavelength 427-428 suggested the spherical size of the nanoparticles as per Mie theory. The spherical size of the AgNPs was further confirmed by TEM study. The periodic observation of prepared AgNPs during the storage period (2 months) did not show any variation in the absorption spectrum. This indicated the constancy of particle size even during storage.
Fig 2: UV - visible Spectrum of the synthesized AgNPs: FTIR analysis showed strong bands at1021cm-1, 1256 cm-1 and 3340 cm-1. The band at 1021 cm-1 corresponds to C-O stretching frequency. The band at 1256 cm-1 corresponds to C-X stretching frequency. 3340 cm-1 corresponds to N-H stretching frequency. The strong band at 1021 cm-1 represents C-O stretching of ether. A strong band at 1638 cm-1 (-C=O-) established the formation of silver nanoparticles. FTIR reports indicated that the peaks were more characteristic of terpenoids and the stabilization is achieved by phenolic as well as aromatic compounds present in the extract.
8
Fig 3: FTIR image of Selected fraction of coconut inflorescence extracts (EA: M (40:60)) and synthesized silver nanoparticles: Figure 4 shows TEM images of AgNPs. The images confirmed that the silver nanoparticles are spherical in shape and the particle size was 22nm.
Fig 4: TEM micrograph of AgNPs:
9
3.1. Antibacterial activity: The antibacterial potential of silver is well known. The synthesized AgNPs were tested for their antibacterial action using ten types of human bacterial pathogens (Table 2). The bacterial growth inhibitory potential of AgNPs was compared with standard antibiotics. At a 100µl dose the inhibitory action of AgNPs was high. The maximum inhibition was observed for K. pneumoniae (24mm), P. shigelloids (21mm), Vibrio alginolyticus (19mm) and Salmonella paratyphi (16mm). The crude extract of the inflorescence and silver nitrate were also tested separately for antibacterial activity. Bioreduced silver nanoparticles showed a higher antibacterial activity than the crude extract. Table 2: Table showing the bacterial growth inhibition potential of green synthesized AgNPs, crude extracts of inflorescence and un reduced AgNO3: S.No Microorganisms
Zone of Inhibition (mm) AgNPs AgNPs AgNPs AgNPs Unreduced Inflorescence 25µl
50µl
75µl 100 µl
AgNO3
Ampicilin
Extract (crude) (10mg/ml) (100 µl)
1
Vibrio alginolyticus
11
16
18
19
13
12
10
2
P. shigelloides
16
18
20
21
20
9
9
3
K .pneumoniae
15
17
21
24
16
12
11
4
Salmonella paratyphi
0
12
14
16
11
15
9
5
P. aeruginosa
10
11
12
14
11
10
10
6
Vibrio harveyi
10
12
12
14
15
13
12
7
Bacillus substillus
0
12
12
14
11
12
11
8
E.coli
0
10
11
12
10
10
15
9
Vibrio mimicus
0
0
0
0
0
9
13
10
Staphylococcus aureus
0
0
0
0
0
10
8
10
3.2. The Minimum Inhibitory Concentration (MIC): The MIC of silver nanoparticles dispersed in the micro titer plate are summarized in the bar diagram as a 24 h OD value of the microbial growth. The growth rate was calculated using the standard or control. Fifteen µl of silver nanoparticles was the MIC for following organisms viz., Vibrio alginolyticus, Klebsiella pnemoniae, Pseudomonas aeruginosa, Bacillus substillis and Plesiomonas shigelloides. For other organisms MIC was 25 µl. (Vibrio harveyi, Salmonella paratyphi Vibrio mimicus and Vibrio alginolyticus). The result of the MIC value indicates the high inhibitory potential of AgNPs on gram +ve bacterial pathogens. For E.coli, V.mimicus and S. aureus there was poor inhibitory action. But, the crude extract of the inflorescence and standard antibiotics showed inhibition. This indicates that the bioactive compounds mediated AgNPs have a good antibacterial capacity. The OD values of the mixture containing AgNPs and bacterial inoculums showing the bar diagram. Bar Diagram 1: MIC for different bacterial pathogen after 24 h. 1.4
MIC for different bacterial pathogen after 24 h
1.2
5µl
Optical density
1
10 µl
0.8
15 µl 0.6
20 µl 25 µl
0.4
30 µl 0.2
35 µl
0
40 µl 1
2
3
4
5
6
7
8
9
10
Control
Growth of different microorganisms as shown by OD value of culture
1- Vibrio alginolyticus, 2- P. shigelloides, 3- K .pneumoniae, 4- Salmonella paratyphi, 5- P. aeruginosa, 6- Vibrio harveyi, 7- Bacillus substillus, 8- E.coli, 9- Vibrio mimicus, 10Staphylococcus aureus
11
Fig 5: Mechanism of action of AgNPs on Microorganisms: 5A photomicrograph showing freely distributed K .pneumoniae population. 5B photomicrograph showing E.coli cells after treating with nanoparticles. 5C photomicrograph showing a mass agglutinated but alive Vibrio alginolyticus bacteria. 5D photomicrograph showing scattered P.shigellioids bacterial aggregation. 5E photomicrograph showing P. aeruginosa aggregation process with silver nanoparticles addition. The Figure 5 represent to the Gram of bacterial strains to show the mechanical action of silver nanoparticles that bind on the bacterial cell wall. Gram staining of AgNPs treated bacteria showed the disintegration of cellular organisation in bacteria. The bacterial cells were ruptured after binding with AgNPs. The study indicated that the green synthesized AgNPs interfere with the molecular built up of bacterial cell wall. The antibacterial activity of the synthesized AgNPs showed varying degree of inhibition in relation to bacterial species and concentration. The optical density value revealed that 15µl dose of AgNPs is the MIC for V. alginolyticus, K.pneumoniae, P.aeroginosa, B.sustillus and P.shigelloids.
For the bacteria V.harveyi, S.paratyphi,
V.mimicus and V.alginolyticus the MIC was 25µl. Silver nanoparticles were found to be less active in killing S.aureus and E.coli. Okafor et al [17] had also reported that the antibacterial sensitivity of AgNPs on the Gram
+ve
S.aureus was greatly lower than that of the Gram-ve
E.coli due to the presence of thick peptidoglycan layer that protects against toxins and chemicals. But in the present investigation both the Gram positive S.aureus and Gram 12
negative E.coli exhibited low sensitivity. So the mechanism of the silver nanoparticles on bacteria is still unclear. The antibacterial activity was examined by comparing colonyforming capability of different bacteria treated by various concentration of AgNPs. Antibacterial activity is studied qualitatively by agar medium by well diffusion and quantitatively interms of the MIC. A lower MIC (OD value) corresponds to higher antibacterial effectiveness. As given in Bar diagram 1 AgNPs could effectively kill bacteria in a concentration-dependent manner. Luo et al., [18] had reported that the nanoparticles induce oxidative stress to bacteria and induce ROS production. Further, under normal circumstances cells are able to defend themselves against ROS damage with antioxidant enzymes. But when the nanoparticles are inside the cell, the nanoparticles could restrain antioxidative enzymes to inhibit the capability of removing ROS. Priester et al., [19] also reported that the nanoparticles break the balance of oxidant/antioxidant and generate the accumulation of ROS in bacteria. It has also been reported that when silver nanoparticles are attached to the surface of cell membrane, the respiratory function and permeability of the bacterial cell become unstable. Other studies suggest that when bacteria are treated with silver ions, DNA tends to lose its ability to replicate. However exact mechanism of AgNPs on different bacterial cells needs a further study.
4. Conclusion: In the present study silver nanoparticles were synthesized using the extracts of inflorescence of the Cocos nucifera. The green synthesized silver nanoparticles were characterized using UV-visible Spectroscopy, FT-IR Spectroscopy and TEM analysis. The phytochemical study of selected fraction of the inflorescence extract showed in the presence of reducing sugar. So the reducing sugar was found to be major agent for silver nanoparticles synthesis. The synthesized silver nanoparticles were tested against ten bacterial strains among these the V. alginolyticus, K.pneumoniae, P.aeroginosa, B.sustillus and P.shigelloids were highly sensing to synthesized silver nanoparticles. Microscopical images of Gram strains of bacterias show that the mechanical action of silver nanoparticles that bind on the bacterial cell wall.
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J. Ramyadevi, K. Jeyasubramanian, A. Marikani, G. Rajakumar, A.A. Rahuman. Mater. Lett. 71 (2012) 114.
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[2]
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D. Jain, H. Kumar Daima, S. Kachhwaha, S.L. Kothari. Digest Journal of Nanomaterials and Biostructures. 4 (2009) 557.
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V.K. Sharma, R.A. Yngard, Y. Lin. Adv. Colloid Interface Sci. 145 (2009) 83.
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D.K. Bozanic, B. Dimetrijevic, N. Bibic, A.S. Luyt, V. Djokovic. Carbohydr. Polym. 83 (2011) 883.
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L.R. Jaidev, G. Narasimha. Colloids Surf., B. 81 (2010) 430.
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Green synthesis of silver nanoparticles from the ethyl acetate: methanol extract of the inflorescence of Cocos nucifera (Family: Arecaceae) for enhanced antibacterial activity R.Mariselvam1, A.J.A.Ranjitsingh1*, A.Usha Raja Nanthini1, K.Kalirajan2, C.Padmalatha3 and P.Mosae Selvakumar4 1 2
Department of Zoology, Sri Paramakalyani College, Alwarkurichi, Tamilnadu, India
Department of Chemistry, Sri Paramakalyani College, Alwarkurichi, Tamilnadu, India 3
The Principal, M.V.M. Govt. College for women, Dindigul, Tamilnadu, India.
4
Department of Chemistry, Karunya University, Coimbatore, Tamilnadu, India * Corresponding Author
Email: [email protected]
Preparation of AgNPs using the selected fraction of the ethyl acetate – methanol
extracts of the inflorescence of Cocos nucifera. Synthesis of silver nanoparticles by using plant extract as reducing agent. The green synthesized AgNPs were characterized by using UV/Visible spectroscopy, FTIR and TEM study. The Green synthesized nanoparticles exhibited a good antibacterial activity against selected bacterial human pathogens.
The mechanism of action of AgNPs on bacterial strain was analysed by using Gram straining procedure.
15
Green synthesis of silver nanoparticles from the ethyl acetate: methanol extract of the inflorescence of Cocos nucifera (Family: Arecaceae) for enhanced antibacterial activity R.Mariselvam1, A.J.A.Ranjitsingh1*, A.Usha Raja Nanthini1, K.Kalirajan2, C.Padmalatha3 and P.Mosae Selvakumar4 1 2
Department of Zoology, Sri Paramakalyani College, Alwarkurichi, Tamilnadu, India
Department of Chemistry, Sri Paramakalyani College, Alwarkurichi, Tamilnadu, India 3
The Principal, M.V.M. Govt. College for women, Dindigul, Tamilnadu, India.
4
Department of Chemistry, Karunya University, Coimbatore, Tamilnadu, India * Corresponding Author
Email: [email protected]
16
