We use cookies to ensure our website works properly and to personalise your experience. Cookies policy
1,2,3 Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India
1Department of Botany, Government College for Men(A), Kadapa, Andhra Pradesh, India
This study explores the green synthesis of gold nanoparticles using leaf extracts of E.lushingtonii and its characterization using Uv-vis, FT-IR, DLS, Zeta potential, XRD, SEM and TEM. Green synthesized AuNPs exhibited Uv-vis absorbance at 538nm wave length. FT-IR spectroscopic studies the obtained peaks at 3450 cm-1 and 1650 cm-1 indicating the functional groups (OH- phenols) and (N-H-proteins/amides) are mainly responsible for capping and stabilisation of synthesised AuNPs. The surface charge of the AuNPs was determined to be -28.6 mV. Crystallographic planes were observed by XRD and crystalline nature was validated by SAED analysis. TEM high resolution microscopic studies reveal that the nanoparticles are spherical in shape with sizes ranging from 7 to 25 nm. The efficacy of AuNPs on inhibition of bacterial and fungal growth was determined using disc diffusion method on selected strains. The findings indicated that plant extract and AuNPs both possess antimicrobial properties. From the results, the green synthesized gold nanoparticles are effective bactericidal agents, which they can be used in biomedical applications.
Nanotechnology have become a remarkable expansion in recent times. There is an incredible development in nanoparticle applications for scientific applications in various fields, covering a wide range of disciplines such as electronics, biomedical, biosensing and therapeutics. Nanoparticles (NPs) are considered as high-performance structures due to their small size and high volume ratio, they have distinct physical and chemical properties than their macro form (1). Among the various metal nanoparticles, Au nanoparticles (NPs) in particular, have received tremendous attention. AuNPs have high potential due to their biocompatibility, optical, photocatalytic, antimicrobial, and cytotoxic properties (2). AuNPs possess large surface areas that can be conveniently functionalized with various biomolecules by means of Au-thiolate chemistry, facilitating the attachment of different moieties, such as antibodies, peptides, and biocompatible polymers with good biocompatibility and targeting capability (3). Their unique optical properties, high chemical stability, good biocompatibility, and easy functionalization make them promising candidates for a variety of biomedical applications, including bioimaging, biosensing, drug delivery (4), cancer diagnosis, and therapeutics (5).
Nanomaterials can be synthesized using various physical, chemical, and biological methods. Even though the Chemical and physical methods are beneficial for the synthesis of metal nanoparticles but have certain disadvantages such as hazardous by products during synthesis, low biocompatibility, and involves high cost. (6, 7). Biological methods employs the use of biological organisms such as bacteria, fungi, algae, and plants, as reducing and stabilizing agents in the synthesis process. In contrast to physical and chemical methods the biological method is considered as green synthesis which can be considered as sustainable, eco-friendly, low cost and safe method for the synthesis of metal nanoparticles (8, 9). But, a significant drawback of microbe-mediated synthesis is that it is not industrially feasible due to its lab maintenance. Plant extracts possess rich phytochemical constituents (bioactive compounds) that are useful as both reducing and stabilizing agents which results in effective and rapid synthesis of nanoparticles and achieves a greater yield (10). The bioactive compounds in medicinal plants substantially influencing the shape, size, and other key features of nanoparticles (11). Hence, plant-based biosynthesis of nanomaterials is considered as the best and the most acceptable method due to enhanced stability and greater biocompatibility (12).
E.lushingtonii is an endemic and threatened (vulnerable) plant species naturally growing in open deciduous forests of southern peninsular India. Ethanobotanically this plant is used as antidote for and snake bite and scorpion sting (13). Till today no work on synthesis of nanoparticles is found on E. lushingtonii. Hence, an attempt has been made to synthesise the AuNPs using leaf extract of E.lushingtonii including its characterization and evaluation of their antimicrobial properties in the present study.
MATERIALS AND METHODS:
Plant material collection: Eriolaena lushingtonii leaves were collected from the field, washed with distilled water and shade dried at room temperature, around 10 days and the dried leaf material pulverised using an electric blender to obtain a fine powder.
Plant Extract preparation: 25g of fine leaf powder was extracted with 100ml of Milli-Q water in a boiling water bath for 1 hour. Extract was filtered with Whatman no.1 filter paper and filtrate was collected and stored at room temperature for green synthesis of AuNPs.
Synthesis of Gold Nanoparticles (AuPs): Tetrachloroaurate salt (HAuCl4, 99.98%) was obtained from Sigma-Aldrich and used as gold precursor. Using deionized water 1 mM tetra chloroaurate(HAuCl4) solution was prepared. To the 95 mL of 1 mM aqueous tetra chloroaurate (HAuCl4) solution, 5 mL of leaf extract was added with the ratio of 20:1, the sample was kept at room temperature, until the colour of aqueous solution changed from red to purple colour ( 14, 15)
Characterizations Techniques: The characterization of the synthesized gold nanoparticles (AuNPs) was performed using several analytical techniques. UV-Spectro UV-20280 Double beam 1200 1/mm spectrometer (Shimadzu, UV-1800 UV-VIS Spectrometer) was employed to monitor the reduction of the gold solution to nanoparticles with in the wavelength range between 450 to 700nm. Fourier-Transform Infrared Spectroscopy (FT-IR) analysis were performed using an Alpha BRUKER Transmission Spectrometer by KBr pellet method and scanned in the range of 400 to 4000 cm-1. Analysis of FT-IR was used to recognize the functional groups of the bioactive components involved in the synthesis of gold nanoparticles. The dried sample of AuNPs was examined for the structure and composition using powder X-ray diffraction spectroscopy. The data was recorded using PANalyticXPert Pro (λ = 0.15406 nm) at 45 kV and 20 mA. The dried sample was scanned in the range of 2θ = 10–80° with 2°/min. Structural, elemental composition, and particle size of the AuNPs were elucidated by energy-dispersive X-ray spectroscopy SEM-EDAX: Jeol 6390LA/ OXFORD XMXN, HRTEM: Jeol /JEM 2100. Size and shape of gold nanoparticles were characterized through SEM. The particle and surface morphology of AuNPs were measured with Transmission Electron Microscopy (TEM), by using HF-3300 advanced 300 kV TEM/STEM from Hitachi to determine the size and shape of AuNPs.
Evaluation of antimicrobial activity
Synthesised AuNPs was analysed for antimicrobial activity against three gram positive bacterial strains like Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212 and three Gram negative bacterial strains like Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 43816, Pseudomonas aeruginosa ATCC 15442. Antifungal activity was tested on four fungal strains Aspergillus flavus ATCC 9643, Aspergillus flavus ATCC,16404 Candida albicans ATCC 10231 and Trichoderma harzianum ATCC 20476.
Microbial stains procured from Dept. of Microbiology, Sri Venkateswara University, Tirupati. Disc diffusion assay method was followed using standard protocol (Cruickshank 1986). For this 20 µl of 100 µg/ml concentration of plant extract, AuNPs, Sterptomycin/Flucanazole and 1 mM concentration of Chloroauric acid (HAuCl4) solution were applied on separate filter paper discs (Whatman No. 1 filter paper with 6 mm diameter) and allowed to dry before being placed on the agar medium. Triplicates of each concentration were tested and incubated at 370C for 24 h. in Incubation Chamber. Diameter of the zones was measured in centimetres (cm) with the help of scale and the results were tabulated. Values are expressed as mean ±SE.
Fig.1. a, Eriolaena lushingtonii-Habitat b. E. lushingtonii-twig
Fig.2. a. Plant extract before and after synthesis b. Surface plasmon resonance analysis of synthesised AuNPs with UV–Vis spectroscopy shows a typical broad peak at 538 nm
Fig.3. FT-IR spectrum of synthesised AuNPs -broad peaks at 3450 cm-1 and 1650 cm-1
Fig.4. Particle size (DLS) and Zeta potential value of synthesized AuNPs
b. EDAX Analysis of synthesised AuNPs shows 0.86% weight percentage of Au metal
Table-1: EDAX Analysis
|
Element |
Wt% |
Atomic % |
|
C |
42.27 |
60.86 |
|
O |
22.21 |
22.18 |
|
Mg |
0.24 |
0.16 |
|
Al |
0.23 |
0.14 |
|
Cl |
7.15 |
3.22 |
|
Ca |
6.31 |
2.58 |
|
Au |
20.59 |
10.86 |
Fig.6. 50 nm and 20nm resolution studies and of synthesised AuNPs with TEM analysis show size range from 7 to 25 nm with spherical-shaped particles and SAED pattern shows crystallographic nature of nanoparticles
Fig.7. XRD spectra for EL-Leaf- AuNPs
Fig.9. Antifungal activity of green synthesised gold nanoparticles (AuNPs) from Leaf extract of E.lushingtonii
Table-2: Antimicrobial activity of EL-Leaf-AuNPs
|
Name of the organism |
PE |
AuCl2 |
AuNPs |
Standard Streptomycin/Flucanozole |
|
8.7±0.15 |
- |
7.8±0.12 |
19.8±0.11 |
|
|
E.faecalis |
9.5±0.08 |
- |
8.7±0.18 |
21.5 ±0.18 |
|
9.3 ±0.13 |
- |
8.1±0.19 |
18.2±0.32 |
|
|
12.5±0.24 |
9.1±0.21 |
10.4±0.24 |
23.5±0.26 |
|
|
10.8±0.21 |
8.4±0.13 |
8.7±0.18 |
22.7±0.10 |
|
|
11.2±0.11 |
8.9±0.32 |
9.1±0.10 |
21.8±0.21 |
|
|
8.9 ± 0.18 |
- |
7.8±0.15 |
10.2 ±0.21 |
|
|
A.niger |
9.3 ±0.52 |
- |
8.5±0.36 |
10.9±0.69 |
|
A.flavus |
9.8±1.17 |
- |
8.7±0.18 |
11.2±0.45 |
|
T.harzianum |
9.1±0.45 |
- |
8.4±0.64 |
11.9±0.38 |
Fig.10. Graph showing antimicrobial activity of different extracts
RESULTS AND DISCUSSION:
Synthesis and Characterization of AuNPs: The synthesis of gold nanoparticles were confirmed with a colour change pattern of plant extract from pale green to purple immediately after the addition of plant extract to the tetrachloroaurate (HAuCl4) solution. Au3+ ions have been successfully reduced to Au0 (metallic gold) by the hydroxyl or amino groups of phytochemicals of plant extract. UV-Vis Spectroscopic studies was conducted to identify the characteristic surface plasmon resonance peak at 538 nm, confirming the formation of AuNPs. Similar peak at 538 nm with Uv-Vis at was observed during confirmation of gold nanoparticles by Das et al.,2024 using Jamun leaf extracts, peaks at 534nm and 562nm was observed by Arif etal., 2020 using Uncaria gambir and Ghramh et al., 2019 using Euphorbia pepulus leaf extract (16,17,18). Peaks at 500–550 nm is characteristic of the surface plasmon resonance (SPR) of gold nanoparticles (20). The results of the FTIR spectroscopy indicates that the broad peaks obtained at 3450 cm-1 and 1650 cm-1 were assigned for O–H bond of phenols and N–H bond of primary amines, respectively (Fig.3). This suggests that the hydroxyl groups of phenols and amide groups of proteins forming a layer on the surface of nanoparticles, act as capping agents. Following the confirmation of synthesis of AuNPs, the colloidal solution was centrifuged at 12,000 rpm for 20 min together with 3–4 distilled water washes to remove the unattached compounds. The centrifuged ELL-AuNPs were kept in an oven for drying and the dried powder is used for further characterization studies.
SEM, TEM and XRD Analysis:
SEM analysis at 1µm resolution studies show that the particles appear irregular and nearly spherical (Fig.5a). The surface of the particles appear rough and uneven. The EDAX analysis of synthesised sample shows 20.59% weight percentage of Au metal along with 42.27% of carbon, 22.21% of oxygen, 0.24% of magnesium, 0.23% of aluminium, 7.15% chlorine, and 6.31% of calcium (Table-1; Fig.5b.).TEM analysis studies provided the morphology of gold nanoparticles with high resolution. 50nm and 20nm resolution studies of TEM image analysis of synthesized AuNPs show that the distribution of particles are spherical and uniform.
The crystalline nature and phase purity of the synthesized gold nanoparticles (AuNPs) were evaluated using Selected Area Electron Diffraction (SAED). The obtained diffraction pattern exhibits well-defined, concentric polycrystalline rings, characteristic of a highly crystalline Face-Centered Cubic (FCC) metallic lattice. The SAED analysis confirms that the nanoparticles possess an FCC crystal structure. The indexing of the (311) reflection serves as a primary indicator of the gold phase, providing a structural fingerprint that complements the morphological data obtained from bright-field TEM imaging. Powder X-ray diffraction pattern in Fig. 7. shows that the Au-NPs synthesized is in crystalline structure. The spectrum gives an intense peak at 2θ = 38.47°, 44.84°, 66.05°, and 78.00° which correspond to the (111), (200), (220), and (311) plane proving the structure of Au-NPs to be face center cubic (fcc). The crystallinity of Au-NPs is pure by comparing its XRD pattern with the database; JCPDS file number 00-004-0784 (21).
Antimicrobial actvity:
Antimicrobial activity was done following the Disc diffusion method and the results were tabulated in Table-2 . Among the tested bacterial pathogens, E.coli exhibited more susceptibility to both leaf extract (12.5mm) and AuNPs (10.4 mm). It was followed by Pseudomoas aeruginosa (11.2mm for leaf extract and 9.1mm for AuNPs) and the least by Bacillus subtilis (8.7 mm for leaf extract and 7.8mm for AuNPs). Gram-negative bacteria are more susceptible to both plant extract and AUNPs than Gram-positive bacteria. Similar findings on gram negative bacterial inhibition was observed with PG-AuNPs by Sathiyaraj et al., 2021 (24). This might be attributed due to the differences in their cell wall thickness. Gram negative bacteria, AuNPs easily penetrate through the cell membrane and causes damages to the cell (22, 23). The antibacterial property of gold NPs was achieved in two processes. They inhibited the metabolic process by changing the membrane potential and lowering adenosine triphosphate (ATP) synthase activity. Second, they rejected the ribosome's subunit for tRNA binding, effectively dismantling its biological process. Both leaf extract and AuNPs showed the bactericidal property against the tested bacterial strains in a concentration dependent manner. Among the four fungal strains Aspergillus flavus (9.8 mm for leaf extract and 8.7mm for AuNPs and A.niger (9.5 for leaf extract and 8.4mm for AuNPs) are susceptible. Least by Candida albicans (8.9mm for leaf extract and 7.8 mm for AuNPs). The antimicrobial efficiency of leaf extract was more than to AuNPs.
CONCLUSION
This study successfully demonstrated the synthesis and characterization of gold nanoparticles using leaf extract of E.lushingtonii. The biosynthesized AuNPs are spherical and the size ranges from 7-25 nm. Both plant extracts and synthesized nanoparticles possess significant antimicrobial properties. The AuNPs showed a higher and moderate antibacterial activity against gram negative and gram-positive bacteria respectively. The biocompatibility of biosynthesised AuNPs have potential applications in drug delivery system.
REFERENCES
Pasupuleti Sivaramakrishna, Sade Ankanna, Nataru Savithramma, Green synthesis, Characterization and Antimicrobial properties of Gold Nanoparticles using Leaf extract of Eriolaena lushintonii Dunn, Int. J. of Pharm. Sci., 2026, Vol 4, Issue 5, 5237-5246, https://doi.org/10.5281/zenodo.20309254
10.5281/zenodo.20309254