Green Synthesis And Characterization Of Silver Nano Particles From Leaf Aqueous Extract Of Aloe vera (L.) Burm. And Its Anti Microbial Activity

Dr. S. Ananthalakshmi1, R.Kavitha2 ,S.Kalaivani2 , J. Emili Saranya2

1. Research Advisor, 2. Research Scholar, Department of Chemistry,

Urumu Dhanalakshmi College, Tiruchirappalli-19, Tamilnadu, India.

drlakshmichem@gmail.com , kalaipriya01@gmail.com.

ABSTRACT

Silver nano particles were synthesized using extract of Aloe vera (L.) Burm. which has been proven active against micro-organisms. Characterization was done by using Ultra-Visible (UV- Vis) spectrophotometer, Scanning Electron Microscopy (SEM), FT-IR and antimicrobial activity. SEM showed the formation of silver nano particles with an average size of 400 nm. It can be concluded that the gel of Aloe vera (L.) Burm can be good source for synthesis of nano particles of better antimicrobial activity against microorganisms. The important outcome found in the study is the development of value added products front medicinal plant Aloe vera (L.) Burm for biomedical and nano particles based industries.

KEYWORDS: Silver Nano Particles, Green Synthesis, Aloe vera (L.) Burm, Anti microbial Activity.

INTRODUCTION

Nanotechnology can be termed as the fabrication, characterization, exploration and application of nanosized (1-100 nm) materials for the development of science. It deals with the study of extremely minute structures and the prefix “nano” is a Greek word which means “dwarf or miniature”. Nanotechnology provides the ability to engineer the properties of materials by controlling their size, and this has been driven research towards a multitude of potential uses for nanomaterials. Nanotechnology has been spread to number of areas including biomedical services, cosmetics, drug gene delivery, environmental health, food, health care, catalysis, mechanics, non linear optical devices, optics, photo-electrochemical application, single electron transistors, and space industries.

The Nanoparticles (NPs) received a particular attention for their positive impact in improving many sectors of economy, including consumer products, pharmaceutics, cosmetics, transportation, energy and agriculture etc., and are being increasingly produced for a wide range of new applications within industry are emerging rapidly. A very interesting application of NPs in the scope of life sciences is their use as ‘smart’ delivery system. Metal NPs are of great scientific interest as they bridge the gap between the bulk and atomic structures. NPs have unique physicochemical properties, like., high surface area, high reactivity, tunable pore size and particle morphology. Recent advances in nanotechnology include the incorporation of metallic NPs into diverse industrial, medical, and household products.

Silver is of great choice in the field of biological systems, living organisms and medicine among the various noble metals. Silver has been not only proven as an effective tool for retarding and preventing the bacterial infections but also they are found to exhibit wound healing activity. Colloidal silver nanoparticles (AgNPs) exhibits distinct properties such as catalytic, antibacterial, good conductivity and chemical stability. The investigations on AgNPs have attained significance due to their use in opto-electronics systems. Anti-microbial activity and silver-embedded fabrics used in sporting equipment. However, still there is a need for economical, commercially viable as well as environmentally clean route of synthesis for AgNPs.

A number of physical and chemical approaches are explored for the preparation of nanoparticles. Physical method involves laser ablation and evaporation condensation methods whereas in chemical method utilizes chemical reductants (NaBH4, ethanol, ethylene glycol etc.), aerosol technique, electrochemical or sonochemical deposition, photochemical reduction and laser irradiation technique. Although in the chemical synthesis of NPs, generation of hazardous by products is highlighted as the environmental contaminants as well as involvement of certain chemicals is expensive and may lead to the presence of noxious chemical species tangled on the surface of NPs, which may have adversarial effect on environment.

Recent developments in plant tissue culture techniques in fabrication of NPs have shown promising results to improve the productivity to many folds. As plants potentially eliminate the environmental issues by making the NPs more biocompatible, it is necessary to increase the efficiency of the locally available and unexplored plants resources for the green synthesis of AgNPs and clarifies the possible mechanism involved in synthesis, is still infancy. The silver nanoparticles have wide spread antimicrobial resistance in biological process [1-11].

Chemicals used for nanoparticles synthesis and stabilization are toxic and lead to non-eco-friendly by products. Thus, there is an increasing demand for green nanotechnology [12]. Many biological approaches for both extracellular and intracellular nanoparticles synthesis have been reported till date using microorganisms including bacteria, fungi and plant materials [13, 14].

In the recent days, silver nanoparticle have been synthesized from the naturally occurring sources and their products like green tea (Camellia sinensis), Neem (Azadirachtaindica), leguminous shrub (Sesbaniadrummondii), various leaf broth, natural rubber, starch, lemongrass leaves extract Aloe vera plant extract etc., [15].

Aloe vera is a shrubby, perennial succulent plant of Liliaceae family having turgid leaves joined at the stem in a rosette pattern. Plant is also characterized by stemless large, thick, fleshy leaves having a sharp apex and a spiny margin [16]. Aloe vera has been shown to have anti-inflammatory, immuno stimulatory and cell growth stimulatory activities [17-19]. Activity against a variety of infectious agents in terms of anti-viral, anti-fungal and anti-bacterial has been also reported [20-22]. Since early times, Aloe vera gel has been used for the treatment of several skin cuts and burn abnormalities too [23].

Nano crystalline silver particles have found tremendous applications in the field of biomedical and this was the main route to carried out our present study. Antimicrobial properties of silver nanoparticles caused the use of the nano metal in different field of medicine, various industry, and health. Hence we aimed to; Synthesize silver nanoparticles, characterize them by the UV visible spectrum, FT-IR, Scanning Electron Microscopy techniques and to study the antimicrobial activity of the synthesised compounds. Due to the development of new material for biomedical and nanoparticles based industries, plant mediated synthesis of silver nanoparticles have more advantageous and shows best antimicrobial activities, we aimed to study anti-microbial activities of the synthesized Silver nanoparticles using Aloe Vera (L.) Burm. extract.

METHODS AND MATERIALS

Preparation of Aloe vera (L.) Burm. extract

The collected Aloe vera (L.) Burm. plant was washed thoroughly with running tap water for 5 minutes to remove dust and then it was washed with deionized water. Aloe vera (L.) Burm. gel was pulped out from the Aloe vera (L.) Burm. plant and washed for 5 minutes with deionized water. These gel was crushed properly and filtrated using filter paper (pore size 25. M). The filtered Aloe vera (L.) Burm. extract was collected and stored for the preparation of silver nanoparticles.

Preparation of silver nanoparticles

Aloe vera (L.) Burm. extract and silver nitrate solution in a specified quantitative were used to prepare silver nanoparticles and the colour was noted. Initially 5ml of Aloe vera (L.) Burm. extract was taken in a 50 ml beaker and 10 ml of 0.1N silver nitrate solution was slowly added with constant stirring using mechanical stirrer and we’re heated approximately to 90 ˚C for 45 minutes. The brownish black solution formed were collected and cooled for 20 minutes. Under the solution there was the formation of brownish black precipitate obtained which was a silver nanoparticles and the solution was decayed slowly and the residue were kept to dry without disturbing.

The supernatant liquid was centrifuged at 10000 rpm for 30 minutes to isolate the silver nanoparticles from plant materials and other compound present in the solution. The residue was collected, washed three times with deionized water and dried and the residues were used for the further analysis. The Table (I and II) indicates the different ml used to prepare the silver nanoparticles.

Table – I Equal quantity of Aloe vera (L.) Burm. and 0.1N AgNO3 solution studied.

0.1N AgNO3 (ml)

Aloe vera (L.) Burm. extract (ml)

10

10

15

10

20

10

Table – II Variable quantity of Aloe vera (L.) Burm. and 0.1N AgNO3 solution studied.

0.1N AgNO3 (ml)

Aloe vera (L.) Burm. extract (ml)

10

5

10

10

10

15

Fig. 1. Photograph of the plant Aloe vera (L.) Burm.

The above same procedure was followed for the preparation of silver nanoparticles for different ratio mixtures and the colour of the solution were noted. The dried silver nanoparticles were characterized by UV, FT-IR and SEM. UV-Visible spectrometry was used to identify, characterize and analyse the silver absorption. Scanning electron microscopy (SEM) was used to find out the surface and morphological characterisation at nanometer of Synthesised Ag nanoparticles. Fourier transformed Infrared spectroscopy (FT-IR) was used to identify the organic functional group and determined which of them are attached to Ag nanoparticle’s surface. And finally the antimicrobial activities of silver nanoparticles was studied.

MICRO ORGANISMS AND CULTURE MEDIA

Bacterial cultures such as, Staphylococcus aureus, Bacillus subtilis, E.coli, Psudomonas ,A.niger and Candida albicans were obtained from Eumic Analytical Lab and Research Institute, Tiruchirappalli. Bacterial strains were maintained on Nutrient agar slants (Hi media) at 4˚C.

INOCULUM PREPARATION

Bacterial cultures were subcultured in liquid medium (Nutrient broth) at 37˚C for 8hrs and further used for the test (105-106 CFU/ml). These suspensions were prepared immediately before the test was carried out.

PREPARATION OF CULTURE MEDIA

NUTRIENT AGAR MEDIUM

Nutrient agar medium is one of the most commonly used medium for several routine bacteriological purposes:

Ingredients Grams/Litre

Peptone:5gm

Beef extract:3gm

Agar:15gm

Sodium chloride:5gm

Yeast extract:1.5gm

pH:7.0

After adding all the ingredients into the distilled water it was boiled and dissolved the medium completely and sterilized by autoclaving at 15 psi pressure (121˚C) for 15 minutes.

NUTRIENT BROTH

The nutrient broth was prepared by the same composition without agar. After adding all the ingredients into the distilled water it was boiled to dissolve the medium completely and sterilized by autoclaving at 15 psi pressure (121˚C) for 15 minutes.

PREPARATION OF PLANT MATERIAL

Leaves, of the plant materials taken for this study were shade dried individually at room temperature and then powdered by using electric, blender. About 10gm of fresh plant materials (Leaves) were extracted with 100ml of distilled water 90:10. They were kept for seven days at room temperature (31˚C) for complete extraction. After seven days the extracts were filtered through What man no.1 filter paper. This extract was collected and kept in refrigerator.

ASSAY OF ANTIMICROBIAL ACTIVITY

MICROBIAL INOCULUM PREPARATION

The nutrient broth were prepared, then identified bacterial colonies were inoculated into the broth culture were used for antimicrobial activity.

KIRBY BAUER AGAR WELL DIFFUSION ASSAY

The nutrient agar medium was prepared and sterilized by autoclaving at 121˚C 15 psi pressure for 15 minutes then aseptically poured the medium into the sterile petriplates and allowed to solidify the Bacterial broth culture was swabbed on each petriplates using a sterile buds. Then wells were made by well cutter. The organic solvent extracts of leaves were added to each well aseptically.

This procedure was repeated for each Petri plates then the petriplates were incubated at 37˚C for 24 hrs. After incubation the plates were observed for the zone of inhibition.

RESULTS AND DISCUSSION

The present investigation entitled “Green synthesis of silver nanoparticles using Aloe vera (L.) Burm. extract and its characterization and antimicrobial activity” was conducted to study the green synthesis of AgNPs by reduction of aqueous silver ions using selected plant extract and the effect of different reaction conditions were studied. The outcomes during the course of investigation have been portrayed in different Tables and Figures and are described Effect of different physical appearance on the green synthesis of silver nanoparticles

Initially Aloe vera (L.) Burm. plant were screened for the green synthesis of AgNPs. The synthesis of silver nanoparticles was monitored by colour change of the plant extract after the bio reduction of silver nitrate. The formation of silver nanoparticles was confirmed by changing in the solution colour from pale yellow to dark brown.

Fig.2 Pale yellow colour of the Aloe vera (L.) Burm. extract

Fig.3 50 ml beaker shows the colour of the Aloe vera (L.) Burm extract after mixed with 0.1N silver nitrate solution and heated standard flask shows the colour of 0.1N silver nitrate solution.

Fig.4 Colour of the solution after mixing of 0.1N silver nitrate solution and Aloevera (L.) Burm. extract before heating and stirrin g

Fig.5. Dark brownish colour solution of silver nanoparticles obtained after the heating along with mechanical stirrer

Fig.6. Silver nanoparticles after dried

UV-VISIBLE spectrum of silver nitrate solution in Aloe vera (L.) Burm. extract

UV-VISIBLE spectrum of reaction medium confirmed the presence of Ag nanoparticles. The characteristic absorption peaks of 0.1N silver nitrate solution in Aloe vera (L.) Burm. recorded which indicates the absorption of silver.

To confirm the formation and stability of synthesis nanoparticle were assayed by UV-Viable spectrophotometer, Fig.7 shows the UV-Visible spectra of AgNO3 (10-3M). As shown in figure 7, UV –vis spectra showed that in the range of low amounts of the extract the absorption spectra exhibit a gradual increase of the absorbance accompanied with a shift in the λmax of SPR band absorption peak from 250 to 280 nm. Further increasing the concentration of plant extract with constant amount of AgNO3, the λmax was shifted to longer wavelengths. The inset UV-vis spectrum was related to the Ag ions before the bio-reduction event, in which an absorption band appeared at 300 nm. The appearance of the band at 410 nm, along with absence of 300 nm absorption band indicate of the successful synthesis of AgNPs under experimental conditions. Such a characteristic Surface Plasmoon Resanance (SPR) peak for AgNPs has been reported to predominantly appear in the range of 300-500 nm.

In fig 7 the absorption peak of 300 nm shows the presence of metal Ag. In fig 8 there is no any absorption peak noted in the UV- visible spectrum of Aloe vera (L.) Burm. which indicates the absence of any metal atom in the extract.

But, in the fig 9 of UV-visible spectrum of Aloe vera (L.) Burm. and AgNO3 solution, the absorption peak is shifted to 410 nm with a broad absorption peak confirms the formation of nanoparticle in the Aloe vera (L.) Burm. AgNO3 solutions.

Fig.7. UV-Visible spectrum of 0.1N silver nitrate solution

Fig.8.. UV-Visible spectrum of Aloe vera (L.) Burm.

Fig.9. UV-VISIBLE spectrum of silver nitrate solution in Aloe vera (L.) Burm.

FT-IR ANALYSIS

FT-IR spectrum was used to identify the possible functional group of biomolecules in the plant extract that might be responsible for the bio-reduction and coating of AgNPs. FT-IR measurements were carried out to identify the biomolecules for capping and efficient stabilization of the metal nanoparticles synthesized.

The FT-IR spectrum of silver nanoparticles (Figure 10) showed the band between 3417cm-1corresponds to O-H group in the biomolecules. The IR bands at 2926 and 2855.21 cm-1 due to C-H stretching vibration modes in hydrocarbon chains. In the case of AgNPs, a large shift in the absorbance peak with decreased band intensity was observed at 1384.63cm-1.

The spectra also illustrate prominent shift in the wave numbers corresponding to amide (1652.5 – 1600 cm-1), and the peak at 1628.33 cm-1 indicates the presence of free amino group which may be interacted with AgNPs surface maintain the stability of nanoparticles. The FT-IR Spectrum in Figure.10 indicates the capping of the nanoparticles with the extract constituents.

Fig.10. FT-IR of green synthesized silver Nano particle.

SEM analysis

SEM image shows the morphological character, size and surface of the AgNPs synthesized by the extract of Aloe vera (L.) Burm. under optimized physical conditions. SEM microscopy which revealed that the AgNPs are around 200 nm in size with the mixture of many shapes i.e. triangle, rhombus, and spherical are clearly observed and spherical are predominant. SEM determination showed the formation of AgNPs, which were well dispersed and the aggregation of the particles could be seen. The corresponding SEM micrographs being obtained at an accelerating voltage of 10 kv at various magnifications. At such magnification, the green synthesized silver nanoparticles showed rough areas on which micro pores and macro pores were clearly identifiable. In the image, the particle sizes range from 89.06 nm to 89.22 nm is obtained. The Figures (11 & 12) show that the particles are highly crystalline in nature.

Fig.11 Fig.12

Fig.11 & 12. SEM image of AgNP under different magnification and obtained from the extract of Aloe vera (L.) Burm.

Antimicrobial activities of silver nanoparticles

The green synthesized silver nanoparticles are tested for its antimicrobial activity towards the micro organism. Silver nanoparticles were divided and prepared in four different concentration of 25µl, 50µl, 75µl, 100µl and these are correlated with the controlled medium. The synthesized silver nanoparticles is active against the gram positive bacteria, gram negative bacteria and fungal which is listed in Table III.

Antimicrobial activity assay of the synthesized AgNps, nanoparticles  was studied against the growth of Gram Positive bacteria (Bacillus subtiles and  Staphylococcus aureus), Gram negative bacteria (E. coli , and Pseudomonas aeruginosa) and Fungi (Candida albicansand A.niger) using the standard agar well diffusion technique. The results are illustrated in Fig.13 -18 and Table III.

The mean zone of inhibition produced by the AgNPs against tested bacterial and fungal strains ranged from 7 mm to 16 mm under the control of Gentamicin antibiotic disc. The organisms used are very sensitive to standard antibiotic Gentamicin and have registered the zone of inhibition 15, 25, 20, 15, 15 and 15 mm respectively (Table -III and figures 13-18). The green synthesized silver nanoparticles has produced zone inhibition range from16-23 (mm/ml) against Bacillus subtilis, 18 -24 (mm/ml) against Staphylococcus aureus, 20 – 28 (mm/ml) against E.coli 18 -24 (mm/ml) against Pseudomonas, 22 – 30 (mm/ml) Candida albicans, 22- 32 (mm/ml) against A.niger.

The zone of inhibition values show that antimicrobial activity of the green synthesised silver nanoparticles is in a dose dependent manner. This may be due to the presence of antimicrobial compound in the nano particles. Thus, this result shows that Silver nanoparticles could also be useful as the antibiotics.

Table III Antimicrobial evaluation data of the Silver nanoparticles.

SAMPLE

DMSO Extract 100 µl added and Zone of inhibition (mm/ml)

25 µl

50 µl

75 µl

100 µl

Control

Bacillus subtilis

16

18

20

23

15

Staphylococcus aureus

18

20

22

24

25

E.coli

20

22

25

28

20

Pseudomonas

18

20

21

24

15

Candida albicans

22

24

27

30

15

A.niger

22

24

28

32

15

CONTROL: Gentamicin antibiotic disc

The above characterization of green synthesized silver nanoparticles has shows the antimicrobial activities which can be applicable in the field of biomedical and nanotechnology industries. The following images show the antimicrobial activities towards the microorganisms.

Fig.13. Zone inhibition of the green synthesized silver nanoparticles against Bacillus subtilis

Fig.14. Zone inhibition of the green synthesized silver nanoparticles against Staphylococcus aureus

Fig.15. Zone inhibition of the green synthesized silver nanoparticles against E.coli.

Fig.15. Zone inhibition of the green synthesized silver nanoparticles against E.coli.

Fig.17. Zone inhibition of the green synthesized silver nanoparticles against Candida albicans

Fig.18. Zone inhibition of the green synthesized silver nanoparticles against A.niger.

CONCLUSION

In the present study we focused on green synthesis of silver nanoparticles using Aloe vera ( L.) Burm. extract. Further, these synthesized silver nanoparticles are characterised using UV- Visible technique. The morphology and particle size are determined by SEM analysis. The primary confirmatory for the silver nanoparticles was colour change in UV- Visible. Absorption spectra of silver nanoparticles showed the peak shifted to approximately 410 nm with a broad absorption peak. SEM analysis confirms that the particles are in the range from 89.05 nm –89.22 nm. FT-IR analysis also been discussed. Based on the measured zone of inhibition values it is observed that the silver nanoparticles shows the antimicrobial activity. Therefore, the green synthesized silver nanoparticles can be promising candidate for pharmaceutical, biomedical and environmental applications.

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FTIR-Nano Powder-

NameDescription

4000 400350030002500200015001000500

100

0

10

20

30

40

50

60

70

80

90

cm-1

%T

1384.63cm-1

3417.14cm-1

1628.33cm-1 1101.48cm-1

1030.46cm-1

2926.16cm-1

1193.85cm-1 618.71cm-1

2855.21cm-1

824.29cm-1

2395.93cm-1

2426.26cm-1

910.30cm-12092.04cm-1