Published at : 10 Jul 2024
Volume : IJtech
Vol 15, No 4 (2024)
DOI : https://doi.org/10.14716/ijtech.v15i4.6147
Harits Atika Ariyanta | 1. Department of Pharmacy, Faculty of Health and Pharmacy, Universitas Gunadarma, Depok 16424, Indonesia 2. Research Center for Biomass and Bioproducts, National Research and Innovation Agency (BRIN) |
Hotlina Nainggolan | Department of Pharmacy, Faculty of Health and Pharmacy, Universitas Gunadarma, Depok 16424, Indonesia |
Siti Aysah Denti | Department of Pharmacy, Faculty of Health and Pharmacy, Universitas Gunadarma, Depok 16424, Indonesia |
Yega Segara M | Department of Pharmacy, Faculty of Health and Pharmacy, Universitas Gunadarma, Depok 16424, Indonesia |
Cahya Mukti Setiyanto | Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok 16424, Indonesia |
Indila Mayrosa | Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok 16424, Indonesia |
Widya Fatriasari | 1. Research Center for Biomass and Bioproducts, National Research and Innovation Agency (BRIN), Jl Raya Bogor KM 46 Cibinong 16911, Indonesia 2. Research Collaboration Center for Biomass-Based Nano C |
Yoki Yulizar | Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok 16424, Indonesia |
Tribidasari A. Ivandini | Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok 16424, Indonesia |
We
have successfully developed a direct, one-step phyto-synthesis of stable black
gold nanostructure (AuNS) using garlic extract (GE). The presence of abundant S
in GE has a high affinity for Au, forming strong Au-S bonds to induce the
growth of anisotropic crystals on Au and continue to form an extensive network
of nanowires resembling a dendrimer. The synthesized garlic-induced stable black AuNS was observed by a
color change from brick-red to black through the addition of GE at various
concentrations and confirmed by the UV–Vis spectrophotometer, particle size
analyzer (PSA), transmission electron microscope (TEM), Fourier transform
infrared (FTIR), and x-ray diffraction (XRD) spectrophotometer. In addition,
the antibacterial, antioxidant, and cytotoxic properties of the black AuNS were
studied. Black-AuNs showed no antibacterial activity on gram-negative bacteria
(Escherichia coli) and two gram-positive bacteria (Staphylococcus aureus and Bacillus subtilisin) but had very high antioxidant
activity (99.68% DPPH inhibition). The cytotoxic effect of black-AuNS was
studied in normal cells and showed that the inhibition in both Vero and Chang
cells was dose-dependent. These findings offer new opportunities to develop a
“green” superstructure of black AuNS for other biomedical applications.
Garlic; Green-Synthesis; Gold Nanoparticles; Nanodendrite; Nanowire
Nanotechnology
has become a very broad research topic in recent years, and it has piqued the
interest of many researchers
Black
AuNS is a potential super particle that has been reported to have broadband
absorption properties throughout the visible and infrared domains. Their
unique morphology is driven by the anisotropic growth of small nanoparticles,
leading to the self-assembly of an extensive network of nanowires that
resembles a dendrimer. Indeed, the unique properties of black AuNS are suitable
for applications such as photothermal therapy, surface-enhanced spectroscopy,
or solar energy conversion. Several successful fabrication methods of black
AuNS have been reported. Aqueous black colloids of gold nanostructured were
synthesized with the simple addition of NaBH4 to HAuCl4
at a very precise concentration in ice temperature. Unique reticular gold
nanostructure produced in this approach exhibits high conductivity and low
reflection. However, black colloids produced are only stable at room
temperature for up to one week before forming a black precipitate
Generally,
the gold nanoparticles (AuNP) green synthesis method substitutes secondary
metabolites from plant extracts for strong reducing agents such as NaBH4
to reduce Au3+ to Au0 and produce conventional spherical
particles. However, in this study, NaBH4 was used as an Au3+ reducing
agent to produce Au0 crystal seed, and garlic extract was used to
drive further anisotropic Au0 crystal growth. Garlic (Allium sativum L.) is
recognized for its high organosulfur (S) content, with 3% allicin being a
predominant form (Gutiérrez-del-Río, Fernández, and
Lombó, 2018), and its wide availability. The abundance of S in garlic
extract (GE) results in a strong Au-S interaction, which facilitates the
anisotropic growth of Au0 crystals
2.1. 2.1. Materials
and Characterization
White
garlic was purchased from a traditional market located in Depok, Jawa Barat,
Indonesia. Tetrachloroauric(III) acid trihydrate 99% (HAuCl4.3H2O),
ethanol, nutrient agar, barium chloride dihydrate (BaCl2.2H2O),
sodium chloride (NaCl), sulfuric acid (H2SO4),
2,2-diphenyl-1-Picrylhydrazyl (DPPH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT), fetal bovine
serum (FBS) and dulbecco’s modified eagle medium (DMEM) were purchased from
Sigma Aldrich. Vero (ATCC CCL-81) and
Chang cell (ATCC
CCL-13) were obtained from the
Primate Anima Study Center of Bogor Agricultural Institute.
Material characterization was carried out using
several analytical instruments, including an IR Prestige-21 Shimadzu Fourier
Transform Infra Red (FTIR), Shimadzu 2600 UV-Vis spectrophotometer, Malvern Zetasizer Nano ZSP Particle Size
Analyzer (PSA), Hitachi H9500 Transmission Electron Microscope
(TEM), and Miniflex 600-Rigaku X-Ray
Analytical Instrument X-Ray Diffraction (XRD).
2.2.
Preparation of Garlic Extract
To prepare the garlic
extract (GE), the salting out extraction (SOE) method was used with slight modifications
based on previous studies (Ariyanta, Ivandini, and
Yulizar, 2021a). Initially, 100 grams of washed and dried garlic was
blended with 20 mL of water, and the resulting garlic juice was sonicated for
20 minutes with 300 mL of ethanol p.a. The resulting mixture was then filtered,
and the filtrate was taken to the SOE system, which consisted of a mixture of
excess ammonium sulfate and ethanol. Following the SOE process, an organic
phase containing allicin was obtained, and the GE was stored in a freezer until
it was used for synthesis.
2.3.
Synthesis of Garlic-Induced Black-AuNS
Garlic-induced black AuNS was
performed by adding 35 µL of HAuCl4 5 x
10-3 M to 12.5 mL of distilled water with
the help of a magnetic stirrer. The resulting solution was added to 35 µL
of GE with varying concentrations of 1, 5, 10, and 100% and 62µL of fresh NaBH4 0.35M to produce AuNS-1, AuNS-5, AuNS-10, and
AuNS-100, respectively. The mixture was stirred for 10 minutes until a color
change occurred and was further characterized using a UV-Vis spectrophotometer,
FTIR, XRD, PSA, and TEM. In comparison, AuNP
synthesis was carried out using the same procedure with 5% GE but without the
addition of NaBH4 and was carried out for 2 hours under UV lamp
irradiation.
2.4.
Antibacterial
Activity Assay
The
well diffusion method was used in this study to test the antibacterial activity
of AuNS against three bacterial strains: Staphylococcus
aureus, Escherichia coli, and Bacillus subtilis.
To standardize the bacterial suspension, the McFarland turbidity standard of
0.5 was used. The NA media was sterilized and then cultured for bacteria, and a
perforator no 5 was used to perforate the media to form wells. Each well was
filled with 50 µL of AuNS-1, AuNS-5, AuNS-10, AuNS-100, AuNP, negative control,
or positive control (chloramphenicol). The petri dishes were incubated at 37°C
for 24 hours, and the bacterial inhibition zone was observed and measured using
a calliper.
2.5.
DPPH Radical Scavenging Assay
The antioxidant activity of
the AuNPs synthesized was determined using the DPPH free radical scavenging
assay. About 2 mL of freshly
prepared DPPH solution (0.004% w/v) was mixed with 100 mg/mL of AuNS using a vortex and kept in the dark
at room temperature for 1 h. The absorbance of the control and treated DPPH was
measured at 517 nm, and the inhibition percentage was calculated using equation
1:
Where
2.6.
MTT Assay
In-vitro cytotoxic analysis was done to analyze the effect of AuNS on normal cell inhibition using MTT. This method was carried out by growing Vero (ATCC CCL-81) and Chang (ATCC CCL-13) cells with a concentration of 5000 cells in 100µl DMEM supplemented with Fetal Bovine Serum (FBS) of 5%, Penicillin of 100U/mL and streptomycin 100ug/mL. The sample of AuNS with a different concentration was added after the cells were 50% confluent (24 h). On day 3, the MTT test was performed by adding 10µl MTT of 5mg/mL per well and incubating them at 37ºC for 4 h. The media was removed after incubation, and ethanol was added to dissolve the formazan crystals. The absorbance value was measured at a wavelength of 595 nm, and the inhibition percentage was calculated using equation 2:
3.1.
Characterization of Garlic-Induced
Black AuNS
The
GE obtained through the SOE method was characterized using FTIR to analyze
their functional groups before being used for AuNS synthesis. The GE FTIR
spectra in Figure 1 shows the absorption at a wavenumber of 3000-3300, 1610,
1450, 1130, 937, and 580 cm-1 indicating the vibration of O-H
stretching, C=C stretching, -O-H bending in carboxylic, S=O sulfoxide, C–H
deformation in =CH2, and C-H bending in alkynes, respectively. These
FTIR spectra are in agreement with our previous study showing the presence of
allicin in GE
The
synthesis of AuNS was performed by reducing Au3+ from HAuCl4
with NaBH4 to produce Au0 crystal seed. Next, GE was
added to induce anisotropic growth. Organic molecules tend to selectively bind
to one face of the nanocrystal, causing it to extend asymmetrically (Stanca et al., 2015). Given the high
affinity of Au-S, allicin present in GE could strongly bind to the surface of
the growing crystal, resulting in anisotropic growth
Figure 2a shows the UV-Vis
absorbance of AuNS in different concentrations of the added GE. A sharp
absorption peak at a wavelength of 520 nm occurs when 1% GE is added, which is
associated with the LSPR of spherical AuNP and gradually decreases with
increasing concentration due to a color change of the mixture caused by a
structural change. According to the UV-Vis spectrophotometry results, adding 1%
GE visually produces a brick red color, which darkens with the addition of 5%
GE, then turns dark purple with the addition of 10% GE and finally black with
the addition of 100% GE. Green synthesis of AuNP without NaBH4 under
UV lamp irradiation was performed as a comparison. The colloid produced was
pink in color and had poor stability (less than 7 days), as indicated by the
formation of a black precipitate. In contrast, AuNS-100 showed very good
stability until the 35th day of observation, as seen from the insignificant
shift in wavelength and decrease in absorbance (Figure 2b). For 35 days, the
black color obtained on AuNS-100 remained stable, with no precipitate forming.
At the appropriate concentration, GE can also act as a capping agent, influencing
the stability of AuNS. This can be seen in their FTIR spectra, which show the
wavenumber shift of GE before and after being used for AuNS synthesis (Figure
1).
Figure 1 The FTIR spectra of (GE) and AuNS-100
Furthermore,
the zeta potential value of AuNS shifted to the right from -68 mV/s, -48 mV/s,
-36 mV/s to -33 mV/s with the addition of 1, 5, 10, and 100 % GE, respectively
(Figure 3a). Some reports mention that nanoparticles with a zeta potential
close to +/- 30 mVs are considered as a stable colloidal suspension system that
prevents nanoparticle aggregation
Figure 3 (a) Zeta potential and (b) Size distribution curves of various AuNS
Figure 4 TEM image of
(a) AuNP, (b) AuNS-1, (c) AuNS-5, (d) AuNS-10, and (e) AuNS-100
The TEM image in Figure 4 confirms the results of the previous
analysis. Figure 4a depicts the morphology of AuNP synthesized without NaBH4
under UV lamp irradiation. The resulting particle size appears to be larger
than AuNS in diameter. The addition of 1% GE resulted in small spherical gold
particles with diameters ranging from 3 to 7 nm and a red brick colloid (Figure
4b). When the GE concentration was raised to 5%, the small spherical gold
particles became closer together and coalesced into a larger size of 10-15 nm
(Figure 4c). This was continued until the addition of 10% GE caused spheres to
assemble and fuse to form short dendrite structures (Figure 4d). Finally,
Figure 4e shows the formation of an extensive network of dendrite structures at
100% GE increments.
Based on the results
obtained, the growth mechanism of the synthesized garlic-induced black AuNS can
be proposed. Initially, the small gold particles resulting from the reduction
of Au3+ to Au0 by NaBH4 undergo self-assembly
to form a short dendrite structure. The growth then continues due to the strong
binding affinity of allicin to Au, leading to the formation of an extensive
dendrimer (Figure 5). It is worth noting that when the volume of 100% GE was
increased under the same synthesis conditions, a black precipitate with a clear
supernatant gradually formed (data not shown).
Figure 5 The proposed
growth mechanism of garlic-induced black AuNS
XRD
spectrometry was utilized to confirm the crystal structure of the black AuNS.
XRD pattern of Figure 6 identifies peaks for face-centered cubic (fcc)
polycrystalline gold at of 38.53o, 44.63o, 64.66o,
and 78.75o, corresponding to (111), (200), (220) and (311) planes,
which match with JCPDS Au number 04.0748. No other peaks of crystallographic
impurities were found to confirm the purity of the gold crystallite.
Figure 6 X-Ray diffraction spectrum of black AuNS-100 |
3.2. Antimicrobial Properties of Garlic-Induced
Black AuNS
In this work,
antimicrobial activity towards Escherichia coli, Staphylococcus
aureus, and Bacillus subtilis showed negative results for AuNP,
AuNS-1, AuNS-5, AuNS-10, dan AuNS 100. This is in accordance with previous
studies that reported that AuNS did not have antibacterial activity on both
gram-positive and gram-negative bacteria. The antimicrobial activity of
co-existing chemicals involved during the synthesis that was not completely
removed from the surface of the AuNS could explain why they appear to have
antimicrobial activity
3.3. Antioxidant Properties of Garlic-Induced
Black AuNS
Oxidative stress is caused
by an imbalance between antioxidants and free radicals, which can be harmful to
human health. Antioxidant compounds play an important role in inhibiting the
production and neutralizing free radicals
Figure 7 a) DPPH free
radical scavenging activity of 100mg/mL GE,
AuNP, and various AuNS towards 0.004%
(w/v) DPPH solution; b) In vitro cytotoxicity of AuNP and AuNS-100
against Vero and Chang cells |
The antioxidant activity
of AuNS and AuNP, as shown in Figure 7a, was compared to that of GE in this
study. At the same volume of particle addition to DPPH, black AuNS-100 was able
to inhibit DPPH by 99.68%. Meanwhile, AuNP was only able to inhibit DPPH by
63.94%. The ability of these different antioxidants depends on the chemical
composition, particle size, and surface layer of AuNPs, resulting in different
physical and chemical properties, including their interactions to neutralize
free radicals
3.4. Cytotoxic Properties of Garlic-Induced
Black AuNS
The
cytotoxic effects of AuNP are commonly evaluated using the MTT assay method on
both cancerous and normal cells. Studies indicate that it has a cytotoxic
effect on cells in vitro
At
a concentration of 30 mg/mL, black AuNS-100
inhibited normal cells (Vero and Chang cells) with a low percentage of
inhibition. The inhibition of black AuNS-100 was dose-dependent in both Vero
and Chang cells. Whereas the percentage of inhibition against vero cells in
AuNP was already high (83.68%) at 30 mg/mL, the
percentage of inhibition against chang cells was nearly the same as in black
AuNS-100 (Figure 7b).
The
mechanism of AuNS in inhibiting cells is due to their ability to penetrate
cells, releasing large amounts of ions that cause toxicity, and apoptosis
occurs
In summary, we successfully developed a one-step
“green” synthesis of black-AuNS using GE. The presence of S in GE promotes the
formation of strong Au-S interaction that induces the anisotropic growth of the
AuNS. Visually, there was a color change during the synthesis process from
brick-red to black through the addition of GE in various concentrations. The
black color obtained on AuNS-100 has excellent stability, with morphology in
the form of a dense network of branched particles mimicking dendrimers.
Furthermore, the antibacterial, antioxidant, and cytotoxicity properties of
black AuNS were investigated. Black-AuNS had very high antioxidant activity but
no antibacterial activity against gram-negative bacteria (Escherichia coli)
and two gram-positive bacteria (Staphylococcus aureus and Bacillus
subtilis). The cytotoxic effect of black-AuNS on normal cells was also
evaluated, and it was discovered that the inhibition was dose-dependent in both
Vero and Chang cells. These findings open up new avenues for developing a
"green" superstructure of black AuNS for other biomedical applications.
This
work was funded by Hibah Penelitian Dasar Kompetitif Nasional (PDKN) 2022, No.
033/E5/PG.02.00/2022 from the Ministry of Education, Culture, Research and
Technology of Indonesia through the Higher Education Service Institute of
Region III (LLDIKTI III) and the Directorate of Research and Community service,
Universitas Gunadarma.
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