Mohammad Akhtar Rasool*1, Dr. Girendra Kumar Gautam2, Dr. Durga Prasad Panda3,
Dimak Chand Sahu4
1Research Scholar, Department of Pharmacy, Bhagwant University, Ajmer (RJ), India.
2Director, Shri Ram College of Pharmacy, Muzaffarnagar (UP), India.
3Research Supervisor, J. K. College of Pharmacy, Bilaspur (CG), India.
4Associate Professor, J. K. College of Pharmacy, Bilaspur (CG), India.
The synthesis of nanoparticles using plant extracts has gained considerable attention in the field of nanotechnology since it is easy, simple, and cost effective and does not make use of toxic chemicals. In this work, gold (Au) nanoparticles were synthesized using the Aconitum heterophyllum leaf extract. Nanoparticles (NPs) prepared with plant extract are more stable, suitable and faster therapeutic efficacy against fungal infection. Moreover, the preparations of gold nanoparticles with plant extract are more uniform in shape and size. The metal nanoparticles were characterized by UV-visible spectroscopy, Fourier transform infrared spectroscopy, zeta potential, XRD, AFM, TEM, STEM, EDX analysis. The Minimum inhibitory concentration of the nanoparticles on various microorganisms (S.aureus, E.coli, P.auruginosa, B.subtilis and C.albicans) was determined along with its anti-biofilm and photocatalytic activity. The gold nanoparticles exhibited better and enhanced activity in inhibiting the microorganisms. And the photocatalytic activity was found to be better in the gold nanoparticles.
A variety of plant species are well known for their therapeutic, anti-inflammatory as well as antimicrobial properties. Over a period of time, the different parts of plants have been studied to identify their uses and have been effectively used to treat microbial infections. A number of disease causing microorganisms are havoc over the world population due to their increased resistance against the market available drugs as well as the increase in the number of stronger and more potent microbial strains. Microorganisms such as Escherichia coli, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa and Bacillus subtilis are capable of causing a multitude of diseases ranging from mild infections to life threatening diseases.
Nanoparticles have gained considerable attention in the field of nanoscience and nanotechnology mainly due to their size, structure and functions which provides a high surface area as compared to the bulk materials. They have been employed in various fields such as in drug delivery, biosensors, therapeutics etc. Many nanoparticles are known to have antimicrobial properties and can be manipulated according to the desired application. Thus, they can provide a means for developing new drugs with enhanced activity and effectivity.
A large number of chemical and physical methods of nanoparticle synthesis have already been used to successfully produce nanoparticles with good antimicrobial properties. However, these methods have certain drawbacks such as release of toxic chemicals, expensive machinery, complicated techniques etc. These issues can be sorted by the use of biosynthesis methods of nanoparticle production which can either employ plants or microorganisms in the synthesis process, plants being more preferable due to the unpredictable nature of microorganisms. Aconitum heterophyllum has shown strong antimicrobial as well as antifungal properties. They can be used in the treatment of a multitude of skin infections such as dry skin, itching, rashes as well can fungal infections such as candidiasis etc. With the help of such plants, we can help impart important characters to the nanoparticles to improve their performance. In this study the gold nanoparticles were synthesized using the leaf extract of Aconitum heterophyllum plant. The nanoparticles were then characterized by using techniques such as UV-visible spectroscopy, Fourier transform infrared sprectroscopy, zeta potential, XRD, AFM, TEM, STEM and EDX analysis. These methods helped to identify the functional groups donated by the plant extract to the synthesized nanoparticles as well as study their shape, size, structure and behavior. The nanoparticles were then tested for antimicrobial activity against a range of microorganisms and their minimum inhibitory concentration was identified. Their activity against the biofilm formed from the bacteria, Staphylococcus aureus was also identified. The nanoparticles were also tested for photocatalytic degradation properties.
MATERIALS AND METHODS:
Chemicals and Plant Sample:
The reagents used were of analytical grade obtained from Merck (Mumbai, India). The chemicals used for the synthesis were of analytical grade. We purchased Chloroauric acid [HAuCl4] from Merck (India), Ativisha leaf extract was prepared in distilled water. All glass wares are properly rinsed with chromic acid followed by distilled water and dried. Biological reduction of silver nitrate for its nanocomposite by Ativisha leaf extract is carried out at room temperature.
Preparation of plant extract:
The collected leaves of Aconitum heterophyllum were washed with deionized water and dried. 4g of fresh leaves were crushed using a mortar and pestle and mixed with 200ml of deionized water. The extract was filtered using Whatman filter paper. After filtration, equal amount of ethanol was added to precipitate the mucilage present in the extract. The extract was centrifuged at 7000rpm for 10mins to make it mucilage free. The supernatant was collected and stored at 4°C.
Synthesis of gold nanoparticles:
Chloroauric acid [HAuCl4], 0.5mM was mixed with the plant extract of Aconitum heterophyllum leaves (40mg/ml). The final reaction mixture was made up to 1ml and incubated in water bath at 70°C for 10mins. The formation of Au nanoparticles was observed with a color change from yellow to pink. The mixture was centrifuged at 12000rpm for 20mins to separate the nanoparticles which were then dried to obtain their powdered form. Various parameters such as change in the concentration of plant extract, Chloroauric acid etc. were changed to obtain maximum and optimum output.
An anti-microbial assay was performed to test the activity of the AuNPs against S.aureus, E.coli, P.aeruginosa, B.subtilis and C.albicans. Mueller Hinton broth was used for the bacteria and sabouraud dextrose broth was used for the fungus. Various concentrations of the nanoparticles were added to the media along with inoculums prepared with the optical density adjusted to a 0.5 McFarland standard in a 1ml reaction mixture.
Biofilms of S.aureus were formed on a 96-well microtiter plate. Gold nanoparticles along with 100µl of inoculum in Tryptic soy broth were separately seeded in wells and incubated at 37°C for 24 h. A control was simultaneously performed with the absence of nanoparticles. The well contents were discharged and the wells were washed with deionized water. The MTT reduction assay was performed to evaluate viability of the biofilms. The MTT solution (50µl, 2mg/ml in PBS) was added to the wells and incubated for 2h. The MTT solution was removed after staining and 100µl of DMSO was added to dissolve the MTT formazan product. The DMSO solution was then transferred to another plate and the optical density was measured using a microplate reader at 570nm.
Photocatalytic activity of Au nanoparticles
The photocatalytic activity of the AuNPs was studied by degradation of the dye, methylene blue under sunlight irradiation. 1ml of nanoparticle solution (1mg/ml) and 100µl of methylene blue dye solution (1mg/ml) was made upto 10ml with deionized water. A control was prepared and kept under similar conditions. The suspension was then put under sunlight irradiation under constant stirring. At intervals of every 1 h, 500µl of the suspension was taken and centrifuged for 15 min at 5000 rpm. The supernatant was then scanned at the wavelengths from 350 to 850nm using a Microplate reader to study the degradation of dye in the presence of nanoparticles.
RESULTS AND DISCUSSION
Synthesis of gold nanoparticles with change in conc. of Chloroauric acid:
Various concentrations of Chloroauric acid (0.1-1mM) were mixed with the plant extract of Aconitum heterophyllum and incubated at 70oC. The best peak was obtained at the 0.5mM. Therefore, it was chosen for further studies.
Graph 1: PE (100mg/ml) with varying conc. of Chloroauric acid (0.1-1mM)
Synthesis of gold nanoparticles with change in conc. of plant extract:
Various concentrations of plant extract (10-100mg/ml) were mixed with Chloroauric acid (0.5mM) and incubated at 70oC. The best peak was obtained at the 100mg/ml but the desired color indicating the size and formation of the nanoparticles was at the concentration of 40mg/ml. Therefore, it was chosen for further studies.
Graph 2: Chloroauric acid (0.5mM) with varying concentration of plant concentration (10-100mg/ml)
The formation of gold nanoparticles was monitored using a Microplate reader to obtain the UV-vis spectra. The nanoparticle solution was scanned between the wavelengths of 300 to 700. The peak for gold nanoparticles was found to be at 544nm and 553nm respectively.
Graph 3: UV-vis spectroscopy of AuNPs
Fourier Transform Infrared Spectra:
The FTIR spectra can help in study the functional groups present in the synthesized nanoparticles. Since the nanoparticles were synthesized from the Aconitum heterophyllum leaf extract, its functional group get attached to the nanoparticles and impart additional characteristics. The region below 1500 cm-1 is known as the fingerprint region which is due to the bending vibrations of the molecule. The band noticed between 3271 to 3266 cm-1may be assigned to O-H stretching. The peak between 1710 and 1680 cm-1 may indicate a stretching of C=O bonds.
Fig. 1: FTIR spectra of plant extract GNPs
Atomic Force Microscopy:
AFM was carried out for gold nanoparticles to study the surface topology using the MNOVA software. It was found that the nanoparticles were spherical in shape. The average size of the gold NPs was found to be <50 nm.
Fig. 2: AFM of Gold NPS
XRD pattern was carried out to understand the crystalline nature of the nanoparticles. The peaks were identified which corresponded to the mentioned planes. This showed that the nanoparticles are crystalline and face-centered cubic in nature.
Fig. 3: XRD of AuNPs
The zeta potential is an indicator of the stability and surface charge of the colloidal dispersions. It was carried out using a zeta particle size analyser the values of zeta potential for AuNPs were found to be -18.2 and -23.9. The negative values indicate moderate stability.
Fig. 4: Zeta potential of AuNPs
Transmission Electron Microscopy:
TEM analysis can be used to confirm the formation of AuNPs and show their shape and size. It was carried out using a cryo-transmission electron microscope (Thermo scientific, Talos). Fig 5 shows the Au nanoparticles which are of spherical as well as triangular shape. The nanoparticles were of <20nm in size.
Fig. 5: TEM analysis of AuNPs
Anti-Microbial Activity of Nanoparticles:
The Minimum inhibitory concentration (MIC) of Au NPs was found for the microorganisms, E.coli and S.aureus. It was found to be 0.45mg/ml and 0.16mg/ml for E.coli and S.aureus respectively.
Table 1: MIC of AuNPs
The growth curves were obtained for the selected microorganisms in the presence and absence of the AuNPs. Effective inhibition in the growth of the microorganisms in the presence of gold nanoparticles was seen which is due to their antimicrobial activity. The nanoparticles reduced the exponential growth phase of the microorganisms and also resulted in an early death. The inhibition activity of the gold nanoparticles for the concentrations taken was found to be effective against E.coli and S.aureus.
The growth curve of E.coli in presence and absence of gold NPs (0.45 mg/ml) over a period of 24 h was generated. The growth of the microorganism was much lesser in the presence of AuNPs and the lag phase was achieved much faster between 10 to 15 mins.
Graph 4: Growth curve of E.coli in presence and absence of gold NPs
The growth curve of S.aureus in presence and absence of gold NPs (0.16mg/ml) over a period of 24 h was obtained. The growth of the microorganism was much lesser in the presence of AuNPs and death of the organism occurred at 10mins.
Graph 5: Growth curve of S.aureus in presence and absence of AuNPs
The anti-biofilm activity of the synthesised gold nanoparticles was studied on S.aureus. It can be seen that there is growth of the S.aureus biofilm is absence of any inhibiting agents. In the fig 17 and 18 which consist of the slides with the AuNPs, It can be seen that there is inhibition in the growth of the biofilm to a great extent. The activity of the AuNPs in inhibiting the biofilm formation was much more ability due to the additional biofilm inhibiting ability of the silver nanoparticles.
Graph 6: Results of biofilm assay
The nanoparticles were synthesized from the plants extract of Aconitum heterophyllum the synthesized nanoparticles were then characterized to identify their properties. The nanoparticles were found to contain functional groups obtained from plant leaves. The gold nanoparticles exhibited effective anti-microbial action against E.Coli, S.aureus, B.Subtilis, P. aeruginosa and C.albicans, anti-biofilm activity on the biofilm formed from S.aureus. The anti-microbial and anti-biofilm activity of the AuNPs was found to be best due to the additional properties of the gold nanoparticles present in the formulation.
1. R. Dabur et al, “Antimicrobial activity of some indian medicinal plant” AFR.J. Tradit, 2007; vol.4: app. 313-318.
2. S. S. Shankar, A. Rai, A. Ahmad, and M. Sastry, “Rapid synthesis of Au , Ag , and bimetallic Au core – Ag shell nanoparticles using Neem ( Azadirachta indica ) leaf broth,” 2004; 275: pp. 496–502.
3. M. M. Kumari, J. Jacob, and D. Philip, “Spectrochimica Acta Part A : Molecular and Biomolecular Spectroscopy Green synthesis and applications of Au – Ag bimetallic nanoparticles,” Spectrochim. ACTA PART A Mol. Biomol. Spectrosc., 2015; 137: pp. 185–192.
4. M. Yasir, J. Singh, M. K. Tripathi, P. Singh, and R. Shrivastava, “Green synthesis of silver nanoparticles using leaf extract of common arrowhead houseplant and its anticandidal activity,” Pharmacogn. Mag., 2018; 13(52): pp. 840–844,.
5. M. Hamelian, S. Hemmati, K. Varmira, and H. Veisi, “Green synthesis, antibacterial, antioxidant and cytotoxic effect of gold nanoparticles using Pistacia Atlantica extract,” J. Taiwan Inst. Chem. Eng., 2018; vol. 0: pp. 1–10.
6. L. Z. S. Brambilla, E. H. Endo, D. A. G. Cortez, and B. P. Dias, “Anti-biofilm activity against Staphylococcus aureus MRSA and MSSA of neolignans and extract of Piper regnellii,” Rev. Bras. Farmacogn., 2017; 27(1): pp. 112–117.
7. K. Gopinath et al., “Green synthesis of silver, gold and silver/gold bimetallic nanoparticles using the Gloriosa superba leaf extract and their antibacterial and antibiofilm activities,” Microb. Pathog., 2016.
8. E. R. León, E. L. Rodríguez, C. R. Beas, G. Plascencia-villa, R. Alfonso, and I. Palomares, “Study of Methylene Blue Degradation by Gold Nanoparticles Synthesized within Natural Zeolites,” vol. 2016, 2016.
9. T. Mazhar, V. Shrivastava, and R. S. Tomar, “Green Synthesis of Bimetallic Nanoparticles and its Applications : A Review,” 2017; 9(2): pp. 102–110.
10. B. Syed et al., “Journal of King Saud University-Science Phyto-biologic bimetallic nanoparticles bearing antibacterial activity against human pathogens,” J. King Saud Univ. - Sci., 2018; pp. 1–6.
11. L. Lu, G. Burkey, I. Halaciuga, and D. V Goia, “Journal of Colloid and Interface Science Core – shell gold / silver nanoparticles : Synthesis and optical properties,” 2013; vol. 392: pp. 90–95,.
12. S. S. Shankar, A. Rai, A. Ahmad, and M. Sastry, “Rapid synthesis of Au , Ag , and bimetallic Au core – Ag shell nanoparticles using Neem (Azadirachta indica) leaf broth,” 2004; vol. 275: pp. 496–502,.
13. G. Sharma et al., “Journal of King Saud University – Science Novel development of nanoparticles to bimetallic nanoparticles and their composites : A review,” J. King Saud Univ. - Sci., 2017.
Received on 18.10.2021 Modified on 07.01.2022
Accepted on 14.02.2022 © RJPT All right reserved