Curcumin Mediated Gold Nanoparticles and Analysis of its Antioxidant, Anti-inflammatory, Antimicrobial Activity Against Oral Pathogens

ABSTRACT

Objective:

To green synthesise gold nanoparticles using curcumin and to analyse its antioxidant, anti-inflammatory, and antimicrobial activity among oral pathogens.

Material and Methods:

Biosynthesised Curcumin Gold nanoparticles (CuAuNP) were evaluated by UV-visible spectrophotometer (UV-Vis), Transmission Electron Microscopy (TEM), and evaluation of antioxidant, anti-inflammatory and antibacterial activity against oral pathogens.

Results:

Synthesized CuAuNP were characterized using UV-visible spectrophotometry and showed peak absorption at 530nm. CuAuNp showed a 90.3% maximum scavenging ability of DPPH at a concentration of 50 μg/mL. CuAuNP exhibited 79.6 % of the highest anti-inflammatory activity at 50μg/mL than the standard drug diclofenac. TEM image clearly showed uniformly dispersed spherical-shaped gold nanoparticles with a size of about 20 nm. The biosynthesized nanoparticle was tested for its antimicrobial effect, and it showed a potent effect against S. aureus, E. faecalis, and C. albicans at 100µg/ mL. Enterococcus faecalis has a maximum zone of inhibition of 14 mm at 100µg/ mL of CuAuNp. Among gram-positive bacteria, a maximum zone of inhibition of 12 mm at 100µg/ mL was seen in S. aureus compared to S mutans. Candida albicans showed a maximum zone of inhibition of 18 mm at 25 μg/mL of CuAuNp.

Conclusion:

Curcumin-mediated gold nanoparticles with 20 nm size were effective and had strong antioxidant and anti-inflammatory activity at 50µg/ mL, antimicrobial action inhibiting microbes at 100µg/mL concentration that can be used in treating various Oral mucosal lesions.

Keywords:
Metal Nanoparticles; Curcumin; Anti-Infective Agents

Introduction

Conventional Drug delivery has a low profile in biodistribution, targeting, aqueous solubility, therapeutic index, and low bioavailability. These limitations are currently solved by nanotherapeutics that have promising effects [¹]. The renowned visionary talk given by the American physicist Richard Feynman, "There's plenty of room at the bottom" in 1959, is accepted to have given an applied birth to the area of nanotechnology [²]. Nanotechnology tends to have an innovative progression that prompts material organization at the nanometer scale (one billionth of a meter). In recent years, the biosynthesis of metallic nanoparticles has become a significant focus in nanotechnology for its medical applications [³].

In the previous two decades, noble metal nanoparticles (MNPs), particularly gold nanoparticles (AuNPs), have progressively accomplished extraordinary consideration from the scientific community because of their remarkable natural, physical, synthetic, and optical properties [⁴]. This unique property of gold with chemical inertness, resistance to surface oxidation, and less cytotoxicity make them get prioritised for nanotechnologies and applications [⁵]. They have discovered numerous applications in different regions in biotechnology [⁶], drug delivery [⁷], biosensing, and catalysis [⁸].

Properly functionalised gold nanoparticles act as a drug reservoir for small molecules with prolonged blood presence. The tunability of AuNPs allows complete control of surface properties for targeting and sustained release of bioactive molecules [⁹].

Depending on the reducing agents, nanoparticles can be synthesised by physicochemical and biological approaches. The physicochemical methods are generally costly and tedious. However, the biological methods use enzymes or secondary metabolites from plants from microorganisms, fungi, and polyphenolic-rich compounds to overcome the drawbacks of the physicochemical approach [¹⁰].

Green chemistry uses naturally occurring functionally rich biomaterials such as plant extract, which could be an alternative for producing nanoparticles in eco-friendly methods. Curcumin is one of the most useful plant-based biomaterials, safe with minimal toxicity from turmeric. It has the potential for green synthesis due to its polyphenol trigger during the reduction process [¹¹]. Its curative aspects include antioxidant, anticancer, anti-inflammatory, antimicrobial, and radioprotective properties [¹²]. Curcumin is effective against proinflammatory cytokines, cyclooxygenase, and Prostaglandin E. in wound healing [¹³].

Two main steps of nanoparticle synthesis are reducing and stabilising agents. Using a reducing agent causes electrons from curcumin to reduce metal from M^x+ to M^o, transforming from bulk metal to its electrical state. Using stabilising agent stabilises nanoparticles and protects them from aggregation. Repulsive force controls the size and shape of nanoparticles [¹⁴]. Despite its promising medicinal properties, curcumin shows poor bioavailability with undetectable concentrations in blood and extra-intestinal tissues. These limitations are due to low absorption, chemical instability, fast metabolism, and high systemic elimination [¹⁵]. Our previous research concentrated on the synthesis of novel turmeric gold nanoparticles [⁵] and curcumin-mediated silver nanoparticles [¹⁶] for its application in oral mucosal lesions. In this present study, curcumin was used as a reducing and stabilizing agent to synthesize gold nanoparticles. This study aimed to green synthesise gold nanoparticles using curcumin, characterized by UV-Visible spectrophotometry, TEM analysis, and evaluation of its antioxidant, anti-inflammatory, and antimicrobial activity against four oral pathogens.

Material and Methods

Materials

Curcumin, Gold chloride, DPPH(2,2diphenyl-1-picrylhydrazyl), Ascorbic Acid, and Bacterial media were purchased from (HiMedia Laboratories, Mumbai, India).

Synthesis of Curcumin Mediated Gold Nanoparticles

0.1g of curcumin was dissolved in 5 mL dimethyl sulphoxide, and 45 mL of distilled water was added. 1mM of gold (III) chloride trihydrate (HAuCl4.3H₂O) was measured and dissolved in 75 mL of distilled water, and 25 mL of prepared curcumin extract was added. The reaction mixture was stirred for 48 hours at 800 rpm in a magnetic stirrer. The synthesized gold nanoparticles were separated by centrifugation at 8000rpm for 10 minutes. Colour change was visually observed every 1 hour. The obtained pellets were purified by washing them with ethanol and water 2-3 times. The purified pellet was stored in the refrigerator for further use.

Characterization of Gold Nanoparticles

The green synthesized nanoparticles were characterized for their optical properties using a UV-visible double-beam spectrophotometer. The morphological characteristics, such as the size and shape of the nanoparticles, were analysed using transmission electron spectroscopy (TEM). The functional groups were responsible for synthesising curcumin-mediated gold nanoparticles were characterised using Fourier Transform infrared spectroscopy.

Antioxidant Activity

The antioxidant potential of the CuAuNP was determined using a DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. 1ml of CuAuNP with different concentration (10μL, 20μL, 30μL, 40μL, 50μL) of solutions were added to 1ml DPPH (80μg/mL) 0.2mm solution in methanol (0.1g/L) and incubated at 30 minutes in room temperature. Methanolic-coloured DPPH is reduced to a non-coloured solution. The reduction in absorbance was measured at 517nm. The standard solution was the ascorbic acid. Inhibition percentage was calculated using the absorbance of sample solution: (Absorbance of control-Absorbance of sample/ Absorbance of control) x100.

The absorbance of DPPH and methanol is the absorbance of control, and the absorbance of DPPH and sample extract is the absorbance of the sample.

Anti-inflammatory Activity

Anti-inflammatory activity was performed by Albumin denaturation assay. Different concentrations of CuAuNP of 10μL, 20μL, 30μL, 40μL, and 50μL respectively, were added to 2ml of 1% aqueous Bovine Serum Albumin (BSA) mixed with 400μL of methanolic extract, pH of the reaction mixture was 6.8 and 20 min incubation was done and heated at 57° C for 20 min in a water bath. The mixture was cooled, and absorbance was observed at 660nm. BSA mixture with 30% methanol solution is controlled. Different concentration of diclofenac sodium is standard. The experiment was repeated thrice.

Percentage inhibition is: =(Absorbance of control-Absorbance of sample)/ Absorbance of control × 100.

Antimicrobial Activity

The antimicrobial activity was done by using the agar well diffusion technique. 10µL of fresh microbial cultures such as Streptococcus mutans, Staphylococcus aureus, Enterococcus faecalis, and Candida albicans were inoculated in sterile Hi-Veg broth medium and incubated for 18 hours in an orbital shaker at 120-150rpm. Mueller Hinton agar was prepared (For Candida albicans, Rose Bengal Agar was used). The antimicrobial activity was done to analyze the efficacy of Cucumber-intervened gold nanoparticles against oral pathogens at different concentrations. The oral pathogens were swabbed on the surface of each sterile MHA plate (For Candida albicans RBA plates). A gel puncher was used to cut four wells into each plate. The first three wells were loaded with three different concentrations (25μL, 50μL,100μL) of biosynthesized gold nanoparticles. A standard antibiotic (Amoxicillin) was loaded in the fourth wall. The plates were incubated at 37 °C for 24 hours (Candida albicans-48 hours of incubation). After incubation, the plates were observed and measured for a zone of inhibition around each well.

Results

Visual Observation

The initial colour of the gold chloride solution was pale yellow, and after adding curcumin extract, it quickly changed to dark yellow within 15 minutes at 38 °C. After 2 hours of synthesis, the curcumin-mediated gold nanoparticles showed dark greenish-yellow colour. This colour change indicates the ability of curcumin extract to reduce, which leads to the reduction of gold chloride to gold nanoparticles. The colour change process of curcumin-mediated gold nanoparticles was evident in (Figures 1A, B, C, D) which showed Curcumin with DMSO extract, Gold (III) chloride trihydrate Solution, Curcumin Gold Chloride Mixture, and Curcumin Gold Chloride nanoparticle.

Figure 1
Colour change process of curcumin mediated gold nanoparticles A. Curcumin with DMSO extract B. Gold (III) chloride trihydrate Solution C. Curcumin Gold Chloride Mixture D. Curcumin Gold Chloride nanoparticle.

UV-vis Spectroscopy

Green synthesised Cu Au Np was analysed by Ultraviolet (UV)-visible spectrophotometer (ELICO SL201 UV-V is spectrophotometer). Blank was the control solution, and the nanoparticle solution was simultaneously scanned from 400-650nm. The absorption spectra of biosynthesized gold nanoparticles showed their maximum absorption peak at 530 nm (Figure 2). The absorption peak at 530nm is due to the excitation of surface plasmon resonance, which further confirmed the presence of reduced gold nanoparticles by curcumin extract, ascertaining the synthesis of CuAuNP. They were monitored at different intervals of 1, 2, 6, and 24 hours. Further purification was carried out by centrifugation at 6,500rpm for 15 min, and the nanoparticles were collected in the form of pellets which were further used for analytical characterisation.

Figure 2
UV-visible spectra of curcumin-mediated gold nanoparticles.

Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) (Make; PHILIPS Model; CM 200) was assisted in analysing the shape and size of the synthesized gold nanoparticles (CuAuNP), which was depicted in Figures 3 and 4. TEM images are obtained with various magnification ranges by a high-resolution megapixel camera. CuAuNp aqueous solution was deposited on a carbon-coated copper grid and was analysed. The shape of the nanoparticles was found to be spherical and uniformly dispersed in nature, with a size of about 20nm. They were spherical shaped with smooth edges and were well dispersed. AuNps, which are well dispersed, are surrounded by curcumin extract that serves as a capping agent.

Figure 3
TEM images of curcumin-mediated gold nanoparticles.

Figure 4
The bar diagram shows the antioxidant activity of CuAuNPs compared with standard: DPPH free radical scavenging assay.

Antioxidant Activity

DPPH free radical scavenging potential of various concentrations of green synthesized AuNP is shown in Figure 4. The antioxidant potential of CuAuNp was performed using free radical DPPH. Methanolic violet-coloured DPPH is reduced to a yellow or non-coloured solution by hydrogen or electron. Antioxidants are bio compounds in plant extract with functional groups assigned on AuNp, which reacts with free oxygen radicals and reduces DPPH. Biosynthesised AuNP exhibited 90.3% maximum inhibitory activity of DPPH radical at the highest concentration of 50μg/ml. Dose-dependent antioxidant activity was described as comparable with the DPPH scavenging potential of ascorbic acid (Standard).

Anti-Inflammatory Activity

Different concentrations of methanolic CuAuNp extract exhibited inhibition of protein denaturation (Figure 5). CuAuNp showed protein denaturation inhibitory activity of 41.7%, 57.5%, 67.6%, 77.4%, and 79.6%, respectively, comparable with commercially available synthetic, anti-inflammatory drug Diclofenac. Maximum protective activity and highest inhibition of CuAuNp was 80 % at a concentration of 50μg/mL. When the extract concentration increased, there was a dependent rise in anti-inflammatory activity comparable with the standard.

Figure 5
The bar diagram shows the anti-inflammatory activity of CuAuNPs compared with the standard bovine serum albumin assay.

Antimicrobial Activity

The antimicrobial effect of biosynthesized gold nanoparticles was tested against four oral pathogens: S. aureus, S. mutans, E. faecalis, and C. albicans. They were two gram-positive, one gram-negative, and one fungal pathogen. Wells of 5 mm were loaded with different concentrations of 25 μL, 50 μL, and 100 μL of CuAuNP. Results demonstrated that the gold nanoparticles, when compared with the standard (Amoxicillin, C. albicans-Fluconazole) group, hold an average antimicrobial effect against all four pathogens, which increased its effect dose-dependently, as depicted in Figures 6 and 7. A maximum zone of inhibition was observed in S. aureus and E.faecalis at 100μg/ mL and C. albicans at 25μg/ mL.

Figure 6
Antimicrobial activity of Curcumin mediated gold nanoparticles against (a) Candida albicans, b) Enterococcus faecalis, c) Streptococcus mutans, and d) Staphylococcus aureus.

Figure 7
The bar diagram shows the zone of inhibition in mm for different concentrations of curcumin-mediated gold nanoparticles against oral pathogens.

Discussion

Phytomedicine is developing today for treating various oral diseases [¹⁷]. The most important advantage is that there are less severe side effects, and it is also effective in curing diseases. Nanomedicine is also a blooming field that helps treat chronic diseases [¹⁸]. Among various nanomaterials, gold nanoparticles have unique physicochemical properties and are an efficient drug in Nanomedicine. An essential property of them is they accurately target cells. Several drugs are coated with gold nanoparticles to improve efficacy [¹⁹].

Curcumin is one of the most useful phytochemicals derived from "turmeric" and has the potential for the green synthesis of Au NPs. During the reduction process, Polyphenol in turmeric may trigger the synthesis of Au NPs [²⁰]. Time consumption, cost, and low curcumin solubility are the drawbacks of physicochemical methods [²¹]. Eco-friendly, cost-effective, green methods are warranted in the synthesis of Gold Nanoparticles, wherein in our study, gold (III) chloride trihydrate is used as a Gold precursor, and curcumin is used as a reducing and capping agent.

Visual observation was used as an initial identification tool to confirm the synthesis of nanoparticles [²²]. Colour change from yellow to ruby red showed the synthesis of gold nanoparticles synthesised from Curcuma wenyujin [²³]. In our study, the initial color of the gold chloride solution was pale yellow, which changed to dark yellow within 15 min and dark greenish yellow, which indicated the reducing ability of curcumin extract that led to the reduction from gold chloride to gold nanoparticles. Similarly, Brazilian red propolis mediated the green synthesis of gold nanoparticles, which was observed by a colour change from pale yellow to dark red [²⁴]. The different colors of AuNPs, from light pink to dark red, depend on these nanoparticles' size, shape, and structural characteristics [²⁵].

UV-visible spectrophotometry was used to detect the synthesis of nanoparticles at various time intervals [¹⁴]. Existing works also reported obtaining the absorption peak at 530nm for gold nanoparticles synthesized by using Brazilian red propolis extract, which was similar to our study where gold nanoparticles showed their maximum absorption peak at 530 nm due to the excitation of surface plasmon resonance that confirmed the presence of reduced gold nanoparticles by curcumin extract. In a curcumin and potassium carbonate study, curcumin was used as a reducing agent, where Au³⁺ was easily reduced to Au⁰. A minor peak was obtained at 425nm, which was reduced considerably, and a major peak was obtained at 525nm, which increased gradually after 4 hours. This change was due to the reaction of curcumin in solution with a reduction of Au³⁺ to Au⁰ [²⁶].

Previous research works reported that the TEM results of synthesized gold nanoparticles from Curcuma wenyujin extract showed a nanoparticle size around 20nm [²³].

Antimicrobial application is due to ultrasmall size and shape as small as 250 times than bacteria, causing an electrostatic interaction between Au from the nanoparticles and negative charge on the cell wall of microbes resulting in distortion such as permeability, osmolarity, electron transport leading to cell death [²⁷]. Small nanoparticles have a great bactericidal effect by binding with a larger bacterial cell membrane surface area. There are several theories. One among them is nanoparticles, which have a strong predilection for reacting with sulfhydryl and phosphorous groups on the cell walls, thus causing significant damage resulting in the release of bacterial cell contents [²⁸]. Another hypothesis is that Au NPs enter the bacterial cell membrane, thus attaching to NADH dehydrogenase, generating highly reactive oxygen species and exhausting the ATP, thus interrupting the respiratory chain. These radicals interact with intercellular constituents and DNA, destroying microbes [²⁹]. Higher concentrations of Nps interact with cytoplasmic organelles and bacterial nucleic acid [³⁰].

Bacterial cell wall composition shows the difference in antimicrobial activity. AuNPs show superior activity against Gram-negative bacteria than Gram-positive bacteria. A thick peptidoglycan layer of polysaccharide chain crosslinked by short peptides causes a hard, rigid structure for NPs to penetrate. Similarly, in our study, there was minimal antimicrobial activity against gram-positive microbes such as Streptococci mutans, and average antimicrobial activity was against gram-positive S. aureus, gram-negative E. faecalis 100µg/Ml, fungal pathogen C. albicans at 25 µg/ mL which increased its effect in a dose-dependent manner. Enterococcus faecalis has a maximum zone of inhibition of 14 mm at 100µg/ mL of Cu Au Np. Among gram-positive bacteria, the maximum zone of inhibition of 12 mm at 100µg/ mL was seen in S. aureus compared to S mutans.

In contrast, Nirmala Grace and Padalia [³¹] enhanced nanoparticle antimicrobial activity by having surface modification by coating with aminoglycoside antibiotics, thus increasing gram-positive bacterial activity [³¹, ³²]. Similarly, Wang et al. [³³] revealed gentamycin-resistant E coli in their study. The drug promotes NP dissolution and increases Ag ion concentration, causing bacterial growth inhibition and cell death [³³]. In accordance with our study, Thangamani and Bhuvaneshwari [³⁴], Au NPs synthesised using Simarouba glauca leaf extract NP size decreased with various morphological variations such as a prism, spherical like particles and found antimicrobial activity against S. aureus, S. mutans, B. subtilis, E. coli, Proteus vulgaris, and K. pneumonia. Nanoparticles with sizes ranging from 6-71 nm had superior antibacterial properties against E. coli, P. aeruginosa, and K. pneumonia with 200 μg/mL completely inhibited microbial growth [³⁴]. Similar to our study, Cu Au Nps is effective against Gram-negative microbes such as E. coli at a low concentration of 100 μg/mL. Antimicrobial activity can be used to fight and control fungal pathogens. Balasubramanian et al. [³⁵] synthesised Au NPs from the Jasminum auriculatum leaf extract, which showed an antifungal effect against Aspergillus fumigatus. Similarly, our study with a minimal concentration of 100 μg/mL was effective against Candida albicans with the maximum zone of inhibition of 18 mm seen at 25 μg/mL of Cu Au Np.

Conclusion

Curcumin is an antioxidant-rich natural polyphenol that is highly used for human consumption and well-being. In this study, curcumin was intervened with gold nanoparticles observed by a colour change to dark greenish-yellow. The synthesised nanoparticle was characterised by a UV-Visible spectrophotometer, which showed a surface plasmon peak at 530nm with TEM showing spherically shaped at 20 nm, confirming the presence of curcumin. The antimicrobial activity of the nanoparticle showed its resistance against C. albicans at 25µg/ mL, S. aureus, and E. faecalis at 100µg/ mL. The biosynthesized nanoparticle can be used as an effective medicine in treating oral mucosal lesions in the future.

Data Availability

The data used to support the findings of this study can be made available upon request to the corresponding author.

Acknowledgements

We thank Nanobiomedicine Lab, Saveetha Dental College for providing insight and expertise that greatly assisted this research.

• Financial Support
  None.

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Edited by

Academic Editor: Wilton Wilney Nascimento Padilha

Publication Dates

• Publication in this collection
  27 Nov 2023
• Date of issue
  2023

History

• Received
  04 May 2022
• Reviewed
  15 Nov 2022
• Accepted
  02 Jan 2023