- Academic Editor
†These authors contributed equally.
Background: Methods like the bio-synthesis of silver nanoparticles (Ag NPs) using plant extracts have become promising due to their eco-friendly approach. The study aimed to examine the utilization of Garcinia gummi-gutta fruit phytochemicals as agents in the biosynthesis of Ag NPs, evaluation of the antimicrobial, antioxidant, and anti-cancerous properties, as well as the photocatalytic ability of bio-synthesized Ag NPs against Crystal Violet (CV), a triphenylmethane dye. Methods: The characterization of the physical properties of the Ag NPs synthesized via the green route was done using UV–Vis spectrophotometry (UV–Vis), X-ray Diffraction (XRD), Fourier Transform Infrared Spectrophotometry (FTIR), Scanning Electron Microscopy (SEM), Zeta potential analysis, and Transmission Electron Microscopy (TEM). The dye degradation efficiency of CV was determined using synthesized Ag NPs under UV light by analyzing the absorption maximum at 579 nm. The antimicrobial efficacy of Ag NPs against E. coli, S. aureus, Candida tropicalis, and Candida albicans was examined using the broth dilution method. The antioxidant and anti-cancer properties of the synthesized Ag NPs were assessed using the DPPH and MTT assays. Results: The UV analysis revealed that the peak of synthesized Ag NPs was 442 nm. Data from FTIR, XRD, Zeta potential, SEM, and TEM analysis confirmed the formation of nanoparticles. The SEM and TEM analysis identified the presence of spherical nanoparticles with an average size of 29.12 nm and 24.18 nm, respectively. Maximum dye degradation efficiency of CV was observed at 90.08% after 320 min without any silver leaching, confirming the photocatalytic activity of Ag NPs. The bio-efficiency of the treatment was assessed using the Allium cepa root growth inhibition test, toxicity analysis on Vigna radiata, and Brine shrimp lethality assay. Conclusions: The findings revealed the environmentally friendly nature of green Ag NPs over physical/chemically synthesized Ag NPs. The synthesized Ag NPs can effectively be used in biomedical and photocatalytic applications.
Nanotechnology is gaining attention in the fields of material science, biomedicine, and water treatment due to its unique structural and biomedical characteristics, contributing to its widespread applications in different fields of science. The nano-size and composition of these materials influence their electronic, optical, catalytic, and magnetic properties. Metal nanoparticles can be used as potential antimicrobial, antioxidant, anti-inflammatory, anti-cancerous, and photocatalytic agents due to their unique properties [1, 2]. Bio-synthesized nanoparticles especially green nanoparticles are the focus of the field due to their non-toxic and eco-friendly nature as compared to the nanoparticles synthesized by chemical and biological methods. The potential applications of green synthesized NPs in the fields of medicine and environmental remediation have been explored by researchers [3, 4, 5].
Green-synthesized metal nanoparticles have the potential to be employed as antimicrobial agents against bacteria and fungi. Silver nanoparticles (Ag NPs) synthesized using Alocasia indica leaf extract were reported to have significant antimicrobial activity (99% inhibition) against S. aureus bacteria [6]. The study explored the applicability of green Ag NPs in dental implants by investigating the anti-biofilm activity of the NPs [6]. Similarly, various studies have investigated the potential applicability of green synthesized NPs as antimicrobial agents, as well as for biomedical purposes [7, 8]. The nanostructure and increased surface area of these materials enable easier penetration of these particles into the microbial cells. Green Ag NPs are significantly effective against multi-drug-resistant bacteria [9].
Ag NPs synthesized from plant extracts can also be used as antioxidant agents due to their potential ability to scavenge free radicals. This property of the green nanoparticles can be effectively used in pharmacognostic applications in modern medicine [10]. The cytotoxic properties of these nanoparticles render them suitable anticancer agents in cancer therapy. Researchers have explored the potential applications of biosynthesized NPs in the treatment of renal cell carcinoma [4]. Biogenic Ag NPs studies revealed improved activity of silver nanoparticles on various cancer cell lines [2].
The modern era of industrialization and urbanization has increased water
pollution and one of the major contributors to this pollution is the discharge of
dye effluents into water bodies. Crystal Violet (CV) (C
Garcinia gummi-gutta, also known as Malabar tamarind, is a member of the Clusiaceae family that is frequently utilized in culinary applications in Western Africa and South East Asia. The fruit contains different phytochemicals such as maleic acid, hydroxy citric acid, tartaric acid, citric acid, phosphoric acid, vitamin C, tartaric acid, and many phenolic compounds. The fruit extracts are used in natural weightless products. It is also used in the treatment of diarrhea, rheumatoid conditions, ulcers, bowel complaints, inflammation, and more [18].
The interest in fruit extract stems from its unexplored applications in various fields such as sustainable nanotechnology and various bio-medicinal applications despite their widespread use in culinary purposes and their easy availability. This knowledge gap generates an opportunity to explore its untapped potential and contribute to the existing scientific understanding. Even though Garcinia gummi-gutta fruit extract (GFE) was reported in the synthesis of biogenic Au nanocrystals, its potential in the bioreduction of Ag NPs has received less attention [18, 19]. Here, we aim to investigate the possibility of ecologically friendly AgNP synthesis using Garcinia gummi-gutta fruit extract.
The antibacterial, anti-cancerous, and antioxidant activities of Ag NPs synthesized from Garcinia gummi-gutta fruit extract, as well as their photocatalytic potentials have not yet been investigated. Examining these characteristics can offer useful information on the prospective uses of the synthesized Ag NPs in several industries, including biotechnology, medicine, and environmental remediation.
In this study, Ag NPs were synthesized from silver nitrate using GFE via green synthesis, with plant phytochemicals acting as reducing and capping agents. The synthesized GFE Ag NPs were employed as an antibacterial, antioxidant, anti-cancer, and nano-catalyst in the degradation of CV dye. Utilizing LC-MS analysis, the degraded products were investigated and examined in more detail. We further investigated the bio efficiency of the treatment by testing the toxicity of CV and NP-treated solutions using actual environmental samples.
All chemicals were procured from Hi-media Laboratories Pvt. Ltd. India. Fresh fruits of Garcinia gummi-gutta (L.) were procured from fields in Kottayam, India. The dust and impurities were removed by thorough washing. The fruit pericarp and seeds were separated and dried. The dried fruit pericarp was stored at room temperature for further use.
Approximately 10 g of Garcinia gummi-gutta dry fruits were suspended in
100 mL of distilled water and subjected to a 1-hour reflux extraction at 40
°C [20]. The extract was filtered through Whatman No.1 and used
for the synthesis of Ag NPs. For the synthesis of Ag NPs, 40 mL 0.01 M AgNO
Absorption spectra of the GFE Ag NPs were recorded using UV-vis absorption
spectroscopic analysis (200-800 nm) (Shimadzu UV-1800ENG240V UV
spectrophotometer). FTIR spectra of the plant extract and the GFE Ag NPs were
studied using a Shimizu IR sprit with reflectance QATR-S spectrophotometer at a
scanning range of 500–4000 cm
The antibacterial potential of GFE Ag NPs was investigated by determining the
Minimum Inhibitory Concentration (MIC) of the NPs against bacterial and fungal
strains. The MIC of the GFE Ag NPs was obtained using the broth dilution method
validated by Clinical Laboratory Standards Institute. The bacterial strains
E. coli (ATCC 10536) and S. aureus (ATCC 25923), and the fungal
strains Candida tropicalis (ATCC 10231) and Candida albicans
(ATCC 90028) were procured from Microbial Type Culture Collection and Gene Bank
(MTCC), Institute of Microbial Technology, IMTECH, Chandigarh, India. The active
bacterial and fungal cultures were grown in nutrient broth (NB) and Potato
Dextrose Broth (PDB), respectively, for 24 hours at 37 °C. The desired
concentrations of GFE Ag NPs were prepared by suspending the NPs in sterile
distilled water and by sequential dilutions from 300 to 10 µg
The antioxidant capabilities of the synthesized GFE Ag NPs were evaluated using
the previously described method with minor changes [1]. For the study, various
concentrations of the NPs (10–60 µg
Where A
The in vitro cytotoxic activity of GFE Ag NPs was analyzed using HEP-G2
cell lines procured from ATCC. The trypsinized monolayer cell culture was
adjusted to a cell count of 1.0
The concentration of the Ag NPs required to inhibit 50% (IC
The photocatalytic degradation ability of Ag NPs was investigated in an aqueous CV solution (0.1 mM) and UV light was used as the light source. NP solution (0.25 mg/mL) was prepared by suspending the synthesized Ag NPs in distilled water by sonicating the solution. CV solution (10 mL) was combined with 20 mL of Ag NPs. The control was a CV solution devoid of NP. The solutions were exposed to UV light for 320 minutes while being constantly stirred. The reaction process was monitored using a UV-vis spectrophotometer by measuring dye absorbance at 10, 20, 40, 80, 160, and 320 min intervals [27]. Degradation efficiency was calculated using the equation.
Where C
To analyze the degraded products of CV, GFE-NP-treated solution at 320 min was subjected to LC-MS analysis using a Shimadzu LCMS8040 model equipped with a Triple quadrupole analyzer and SPD40- UV-vis detector.
Eco-toxicity of the treatment was analyzed by monitoring the toxicity of the untreated CV solution. The reduction in the toxicity of treated dye effluents was also assessed by investigating the Allium cepa root growth inhibition test, Vigna radiata toxicity study, and by performing the Brine shrimp death experiment. Clean bulbs were grown in distilled water for 72 hours before being subjected to synthesized NP solutions (0.25 mg/mL), test samples (0.1 mM CV solution), and Ag NPs-treated CV dye solutions for 48 hours for the Allium cepa root growth inhibition test. As a control, distilled water was used. The root length of the onion bulbs in each solution was measured after exposure, and the percentage of growth inhibition was calculated [28, 29]. The seeds of Vigna radiata were immersed in distilled water overnight for germination. Each sprout was exposed to test samples including synthesized Ag NPs solutions, CV solution (0.1 mM), and Ag NPs-treated dye solutions. As a control, a seedling was subjected to distilled water. Each sample set was treated with 10 mL sample solutions per day, and the experiment was carried out for 3–4 days. On the 4th day, the Vigna radiata seedlings were harvested and the length of the leaf and stem root, and the total length of the seedling were measured, subsequently, the percentage of leaf, stem, and root growth inhibition was calculated [27, 28]. The brine shrimp lethality experiment was used to examine the treatment efficacy as a quick technique for analyzing the cytotoxic effects of synthesized Ag NPs, and treated and untreated dye solutions. The Brine prawn, Artemia salina (L), was nurtured in the same manner as previously described [30]. After 48 hours, matured nauplii were used for analysis. For the toxicity analysis, 5 mL of artificial seawater mixed with 5 mL of a test solution (synthesized Ag NPs, untreated or treated CV dye) was collected in test tubes and 10 mL of artificial seawater was kept as the control for the assay. A total of 30 matured nauplii were introduced to each of the samples (both the test samples and the control samples). For 24 hours, the setup was left uncovered under a constant light source. The number of surviving shrimps and dead nauplii was counted after 24 hours of exposure, and the percentage of mortality was computed [29].
The presence of phytochemicals such as flavonoids, alkaloids, and many other phytochemicals in Garcinia gummi-gutta extracts has been observed in phytochemical investigations [31]. During the reduction reaction, these phytochemicals act as reducing and capping agents in the synthesis of Ag NPs. The initial yellow color in the reaction mixture turned brownish-black, indicating the synthesis of Ag NPs in the mixture. The presence of a large number of phytochemicals in the plant extract that acted as reducing and capping agents in the production of Ag NPs was confirmed by the color change observed within 20 minutes of the reaction. After 30 minutes of reaction, the color of the reaction mixture changed to brownish black, indicating that the Ag NPs in the reaction mixture had been fully reduced. The reaction mixture was further examined to confirm the production of Ag NPs (Fig. 1). Similar observation was seen using banana peel extracts [21].
Schematic representation of the green synthesis of Ag NPs.
(A) Dried Garcinia gummi-gutta fruit pericarp. (B) Reflux extract of
Garcinia gummi-gutta fruits. (C) AgNO
UV-vis spectrophotometry was used to confirm the production of Ag NPs in the reaction mixture after the GFE Ag NPs were synthesized. In the UV-vis absorption spectra of pure Ag NPs, a surface plasmon resonance (SPR) peak at 442 nm was identified, confirming its formation (Fig. 2A). The GFE and green synthesized Ag NPs were analyzed using FTIR to identify potential biomolecules involved in Ag NP synthesis. The acquired spectrum revealed several distinct peaks that confirm and support the generation of Ag NPs and the role of plant phytochemicals in Ag NP synthesis.
(A) Spectrum showing the SPR peak, and (B) XRD pattern of Ag NPs synthesized from GFE. SPR, Surface Plasmon Resonance; GFE, Garcinia gummi-gutta fruit extract; XRD, X-ray Diffraction.
The resulting FTIR spectra of GFE showed peaks at 2921.61 (cm
Fourier Transform Infrared Spectrophotometry (FTIR) of the Garcinia gummi-gutta fruits extract and the synthesized Ag NPs.
The crystalline phase of the synthesized GFE Ag NPs was obtained by X-ray
Diffraction (XRD) analysis. XRD spectrum of the synthesized Ag NPs revealed the
characteristic peaks of Ag NPs with 2-theta values, 38.06
The synthesized Ag NPs were subjected to Dynamic Light Scattering (DLS) analysis
to monitor the fluctuations in the light intensity of the synthesized NPs [36].
The study found particles of two distinct sizes, which could be attributed to the
agglomeration of the synthesized NPs (Fig. 4A). The synthesized Ag NPs had an
average particle size of 98.52 nm. The charge distribution of the synthesized Ag
NPs was confirmed by zeta potential analysis. The observed zeta potential value
of the synthesized Ag NPs (–39.4
Dynamic Light Scattering (DLS) analysis of the green synthesized Ag NPs. (A) Particle size analysis of the green synthesized Ag NPs. (B) Zeta potential analysis of the green synthesized Ag NPs.
Field emission scanning electron microscopy (FE-SEM) (A), High-resolution transmission electron microscopy (HRTEM) images (B) with Selected area electron diffraction (SAED) patterns (C) of green synthesized Ag NPs.
The TEM analysis of the synthesized GFE Ag NPs was performed to better analyze the surface characteristics and the crystalline nature of the NPs (Fig. 5B). TEM scans revealed spherical-shaped particles averaging 24.18 nm in size. The SAED patterns of the GFE Ag NPs revealed concentric rings, confirming the crystalline structure of the NPs. According to XRD examination, the d-spacing values of SAED patterns were 0.244, 0.208, 0.146, and 0.123 nm, corresponding to the [111], [200], [220], and [311] planes of Face centered cubic planes, respectively (Fig. 5C) [2]. EDS tests were used to determine the elemental composition of the GFE Ag NPs, revealing very intense silver peaks followed by carbon and oxygen (Fig. 6). The presence of carbon and oxygen can be attributed to bioactive substances such as flavonoids, phenols, and alkaloids that function as capping agents on the surface of biogenic Ag NPs [2].
The Energy Dispersive X-ray (EDX) spectrum of the synthesized Ag NPs.
To evaluate the antimicrobial properties of the synthesized GFE Ag NPs against
bacterial strains, E. coli (ATCC 10536) and S. aureus (ATCC
25923), and the fungal strains, Candida tropicalis (ATCC
10231) and Candida albicans (ATCC90028), the MIC
analysis was performed with GFE Ag NPs at varying concentrations (20–100
µg
Microorganism | Microbial strains | IC |
MIC (µg |
Percentage of inhibition at MIC (%) |
Bacteria | E. coli | 30.26 | 100 | 96.69 |
S. aureus | 50.84 | 100 | 96.59 | |
Fungi | Candida albicans | 87.16 | 100 | 94.05 |
Candida tropicalis | 50.79 | 100 | 95.11 |
MIC, Minimum Inhibitory Concentration.
Antioxidants are substances that may scavenge free radicals, thus protecting
cells from harmful effects of reactive oxygen species created in cells.
Antioxidants are important characteristics that maintain the balance between
oxidants and antioxidants and thereby control the defense mechanism of the body
[37]. DPPH is a free radical with a purple color, possessing absorption maxima at
517 nm and becomes yellow color upon neutralization of the free radicals. The
antioxidant Ag NPs donate electrons or protons to the DPPH radicals, reducing
those radicals that subsequently leads to a reduction in the color intensity, and
thereby a reduced absorbance. Varying concentration of ascorbic acid (2–12
µg
The GFE Ag NPs were tested for anti-cancer activity against HEP-G2 hepatic
cancer cell lines. The cytotoxic activity of GFE Ag NPs at various concentrations
(10–100 M) was tested in vitro using the MTT assay (Fig. 7). Higher
cytotoxic activity of HEP-G2 cell lines was observed with increasing
concentrations of GFE Ag NPs (Fig. 7E). The IC
Cytotoxic potential of synthesized Ag NPs on HEP-G2 cell lines. (A) HEP-G2 control cell lines (B), (C), (D) HEP-G2 cell lines treated with synthesized Ag NPs (100 µM). (E) Graphical representation of reduction in cell viability of HEP-G2 cell lines with different concentrations of Ag NPs.
The photocatalytic performance of the synthesized Ag NPs was investigated by using the synthesized Ag NPs to degrade CV dye. The absorption maxima of CV were recorded at 579 nm using the UV-vis Spectrophotometer. The absorption spectra revealed decreasing peaks at various time intervals (Table 2). After 320 minutes, the percentage degradation efficiency was calculated to be 90.08% of UV irradiation (Figs. 8,9). Smaller-sized NPs due to their larger surface area significantly increase the rate of photocatalytic degradation. Since it may increase the number of the coordinate atoms, mediating adsorbed dyes could enhance photocatalytic degradation [40, 41].
Time interval | Absorbance at 579 nm |
control | 0.452 |
00 min | 0.197 |
10 min | 0.167 |
20 min | 0.155 |
40 min | 0.128 |
80 min | 0.127 |
160 min | 0.080 |
320 min | 0.043 |
Photocatalytic degradation of Crystal Violet using green synthesized Ag NPs.
Image depicting the degradation of Crystal Violet at different time intervals.
The effectiveness of CV degradation is determined by the identification of the degraded products, which provides a better understanding of the photocatalytic degradative mechanism. In the present study, degraded products generated during the photocatalytic degradation reaction was examined using LC-MS analysis of the NP-treated CV solution at the point of completion of the reaction. The resulting chromatogram showed a notable intensity difference between the control and the treated CV solution, indicating that CV was effectively degraded (Fig. 10A). Seven separate m/z peaks were identified (Fig. 10B). These peaks were, in order, 372.3, 358.3, 344.2, 330.3, 316.4, 302.2, and 288.1. The components m/z 372.3 represent the parent molecules that are CV in their ionised form. And the remaining components m/z 358.2, 344.2, 335.2, 330.20, 316.4, and 295.2 were parent CV, mono-, di-, tri-, tetra-, penta-, and hexa-N-de-methylated intermediates, respectively (Fig. 11). Similar results were observed by He et al. [42] in a microwave-assisted catalytic degradation of CV by nickel dioxide nanosuspensions.
LC-MS analysis of the degraded products. (A) chromatogram comparison of control CV and the NP-treated dye solution. (B) Mass spectra of N-demethylated products of CV from the CV degradation.
UV-mediated photocatalytic degradation mechanism of CV by GFE Ag NPs.
The N-demethylation of CV occurred gradually, as observed according to the degraded products identified by LC-MS analysis. The presence of some isomeric intermediates was identified from the LC-MS spectra. There were two isomeric intermediates for the di-N-de-methylated product, three for the tri-N-de-methylated product, and two for the tetra-N-de-methylated product (Fig. 11). De-methylation and the destruction of the conjugated ring structures are the two main ways that CV degrades [42]. The photocatalytic activity of the green synthesized GFE-Ag NPs was used in the present study to describe the degradation of CV through de-methylation of the dye molecules.
Wastewater containing dye effluents released into land or water bodies can have significant effects on soil fertility and organisms at different tropic levels. Hence the release of treated (degradation products) and untreated dyes should be monitored to reduce toxicicity or making it non-toxic for living organisms. An Allium cepa root growth inhibition test was carried out to analyze the bio-efficiency of the treatment. Onions treated with CV dye before the treatment with NPs have been shown to exhibit 52.70% inhibition on onion root growth. This was reduced to 22.00% for the samples that were exposed to dye solutions treated with Ag NPs synthesized from GFE (Table 3). Similarly, the effect of treated and untreated solutions dye solutions on V. radiata was monitored for the growth inhibition % of the leaf, stem, and root of the green gram sprouts. CV solution before treatment with NPs retarded the growth of the green gram sprouts, and the samples exposed to AgNP-treated solutions have shown normal growth similar to the control samples. The Brine shrimp (Artemia salina) toxicity analysis was carried out based on the mortality % of the dye solutions before and after treatment with NPs. All the Brine shrimp nauplii exposed to untreated CV solutions died within 24 h of incubation, indicating the severe toxicity of CV on brine shrimp growth. The mortality rate of the brine shrimp nauplii exposed to dye solutions that were treated with green synthesized Ag NPs was reduced to 20–25%, depicting the efficient degradation of dye compounds in the solution by the green synthesized Ag NPs (Table 3).
Conditions | Allium cepa toxicity | Vigna radiata toxicity | Brine shrimp lethality | |||
Root growth inhibition (%) | Leaf toxicity (%) | Stem toxicity (%) | Root toxicity (%) | Total growth inhibition (%) | Mortality (%) | |
Distilled water | 0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
2.67 |
CV control | 52.70 |
24.33 |
38.93 |
46.50 |
22.89 |
100.00 |
Synthesized Ag NP | 18.71 |
7.07 |
7.84 |
24.81 |
9.66 |
4.11 |
Test - treated | 22.00 |
8.08 |
7.33 |
32.51 |
10.59 |
22.69 |
The GFE Ag NPs were characterized using several spectroscopic and microscopic investigations. The SEM examination revealed spherical-shaped NPs with an average size of 29.12 nm. Similar results were observed during TEM analysis. The GFE Ag NPs were proven to have good antimicrobial, antioxidant, and anti-cancerous activities. This study investigated the photocatalytic capability of Ag NPs in the breakdown of CV dye. Phytotoxicity studies of the Ag NP-treated products revealed significant toxicity reduction of dye and its degraded products. The current study uses renewable and eco-friendly methods for Ag NP synthesis and its biological and catalytic properties. This methodology can be used alone or used with a hybrid system for the efficient removal of dye molecules from textile effluents.
The data presented in this study are available on request from the corresponding authors.
This research article was produced through collaboration between the authors. Conceptualization, VAA, BB, and JKS; Writing the original manuscript, JTK, and BB; Methodology, data curation, and formal analysis, JTK, AM, KRRR, MP, AMA, J-TC; Review and editing, BB, VAA, AMA, KRRR, J-TC; Interpretation, and review/revision, AM, VAA, KRRR, and JKS. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
Not Applicable.
All the authors are thankful to their respective Universities and Institutes for their support. The authors are thankful to Common Instrumentation Lab, Materials Research Lab-Department of Physics, CHRIST (Deemed to be University) for providing the instrumentation facilities and Major Research Project (MRP DSC-1830, MRP DC-1934), Centre for Research, CHRIST (Deemed to be University) for providing the DLS analysis facilities. We also thank the Department of Physics, Cochin University of Science and Technology for providing the SEM analysis facilities. The authors acknowledge the Central Research Facilities, Centre for Nano and Soft Matter Sciences, Bengaluru for providing TEM facilities and this work was funded by the Researchers Supporting Project Number (Project Number: RSP2023R261) King Saud University, Riyadh, Saudi Arabia.
This research received no external funding.
Given the role as Guest Editor and Editorial Member, Jen-Tsung Chen had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Graham Pawelec. The authors declare no conflict of interest.
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