Highly Antibacterial Activity Against Methicillin-resistant Staphylococcus aureus of Silver Nanoparticles Synthesized by Citrus maxima Peel Extract

doi:10.21203/rs.3.rs-160930/v1

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Highly Antibacterial Activity Against Methicillin-resistant Staphylococcus aureus of Silver Nanoparticles Synthesized by Citrus maxima Peel Extract

https://doi.org/10.21203/rs.3.rs-160930/v1

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A cost-effective and green technique was performed for the synthesis of silver nanoparticles (AgNPs) from a plant resource using Citrus maxima peel (CMP) extract as a reducing agent. The formation of AgNPs was confirmed by UV-Vis Spectroscopy at the wavelength range of 400−500 nm. The optimized conditions for the AgNPs synthesis using CMP extract as a reducing agent were determined. At these conditions, the X-ray diffraction (XRD) and the high-resolution transmission electron microscopy (HRTEM) results revealed the face-centered cubic structure of AgNPs had a highly crystalline with the particle size in a range of 10−20 nm. The Fourier transform infrared spectroscopy (FT-IR) demonstrated the presence of flavonoid, terpenoid, phenolic, and glycosides in phytochemical compositions of CMP extract which can act as the reducing agents for AgNPs formation. The antibacterial effect of the AgNPs was evaluated against Methicillin-resistant Staphylococcus aureus (MRSA) by implementing the minimum inhibitory concentration (MIC), minimum batericidal concentration (MBC), and the zone of inhibition tests. The AgNPs exhibited effective antibacterial activity against bacteria with an average diameter of inhibition zones of 11.7 mm, the MIC of 8.27 µg/mL, and the MBC of the 16.54 µg/mL.

Organic Chemistry

Polymer Science

Sunlight irradiation

silver nanoparticles

antibacterial

Citrus maxima peel

Improper use of antibiotics promotes the growth of resistant bacteria. The prevalence of multidrug-resistant infections has become a severe challenge in clinical practice. Bacteria such as S. aureus, B. cereus, P. aeruginosa, E. coli, etc. are becoming increasingly difficult to treat with conventional antibiotics [1]. Resistance can be transmitted between bacteria via plasmids. Unfortunately, resistant bacteria emerged shortly after the clinical use of new synthetic, synthetic antibiotics. Bacteria possess several universal mechanisms to fight different types of antibiotics. These mechanisms include producing antibiotic-specific degradation enzymes, altering membrane permeability, mutating/modifying efficient sites, and adjusting the translation system. Therefore, the urgent need is to develop biocides with alternative mechanisms. Silver nanoparticles (AgNPs) are useful in inhibiting drug-resistant bacteria [2,3]. It seems that the bacteria have a low resistance to AgNPs, supporting their use as a suitable biocide. An earlier study showed that AgNPs penetrated bacterial cells. The results showed that AgNPs could interact directly with cellular macromolecules. However, the bactericidal mechanism of AgNPs is not precise, with some controversial theories as follows. The first mechanism [4]: Oxidized AgNPs, releases free silver ions from the nanoparticle surface to be toxic to bacteria. However, characters containing fixed AgNPs exhibited better antimicrobial performance than silver ion coated surfaces, suggesting that AgNPs and Ag⁺ have different bactericidal pathways. The second mechanism [5]: AgNPs break down the membrane/cell wall and thus inhibit aerobic respiration, damage deoxyribonucleic acid (DNA), disturb biosynthesis, and protein folding. The last mechanism [6]: Reactive oxygen species (ROS) generated by the AgNPs stimulate the light and then kill the bacteria. However, some studies find that AgNPs are in vitro antioxidants. In recent years, the exploitation of green technology to synthesize AgNPs has gained remarkable achievements. Various claims show that the AgNPs biosynthesis process focuses on a number of plant sources, in which the extraction of plant parts such as leaves [2,7], root [2], seed [8], stem and flower [9], and fruit [10] have been used effectively. Consequently, the synthesis of the AgNPs using plant extracts offers advancements over other methods with more effective applications, especially in antibacterial activities. In addition, the formation of AgNPs on supports, typically SiO₂, with the aim of enhancing dispersion also provides high antibacterial performance [11].

Citrus maxima as a popular fruit is widely grown in Southeast Asia, especially in Vietnam. In many countries, in huge quantities, a Citrus maxima peel has always been considered a biomass waste. It has been reported that some phenolic acids, coumarin flavonoids, rutin, and terpenoids are abundant in the peel extract of Citrus maxima [12,13]. These compounds are considered natural antioxidants. Besides, they can also be used as an effective reducing agent to produce nanosilver. Therefore, AgNPs obtained from this process are more biocompatible and suitable for biomedical and industrial applications. This study conducted a synthesis of AgNPs at room temperature under sunlight irradiation, based on reducing AgNO₃ solution using Citrus maxima peel extract (collected from Ben Tre province, Vietnam). Furthermore, we studied their antimicrobial properties against Methicillin-resistant Staphylococcus aureus with the standardized inoculum of bacteria of 1.5´10⁷ CFU.mL^-¹. Fabrication, characterization, and antibacterial activity testing of AgNPs using CMP extract are illustrated in Fig. 1 (more detailed, in Supplementary Information).

AgNPs formation was shown at room temperature under the irradiation of sunlight. The surface plasmon band of AgNPs demonstrated it observed in the wavelength region of 400-450 nm (Figure S1a). The intensity of this absorption increases as the concentration of AgNO₃ rises from 0.75 to 1.25 mM. It shows an increase in the concentration of AgNPs, along with an increase in the concentration of AgNO₃. However, AgNPs formation decreased at higher AgNO₃ concentrations (1.5 mM). Agglutination of small particles leading to a decrease in the concentration of AgNPs (lower absorption intensity) took place in this case. With the ratio of AgNO₃/CMP extract of 19/1, the formation of AgNPs was almost negligible (Figure S1b). Increase the ratio of AgNO₃/CMP extracts up to 16/4; the formation rate of AgNPs increases significantly. It is explained that the high concentration of biological molecules in the CMP extract has effectively reduced Ag⁺ ions to Ag^o [14]. AgNPs formation rate almost unchanged when increasing the ratio of AgNO₃/ CMP extract up to 15/5. Besides, it also has a red-shift of the absorption band, leading to an increase in the size of AgNPs. The effect of reaction time on the formation of AgNPs was also investigated. The AgNPs formation was observed at 120 minutes (Figure S1c). AgNPs formation intensity increased and maintained after 330 minutes. From the obtained results, the optimized conditions for the synthesis of AgNPs using CMP extract as a reducing agent were determined involving the presence of the light illumination, the volume ratio of AgNO₃ solution/CMP extract of 18/2, the AgNO₃ concentration of 1.25 mM and the reaction time of 330 minutes.

X-ray diffraction analysis (XRD) of AgNPs synthesized at the optimized conditions (Fig.S3) showed Several Bragg reflections with 2θ of 38.5, 46.7, 64.5, and 77.3° recorded corresponding to (111), (200), (220) and (311) lattice planes, characterizing of the face-centered cubic structure of AgNPs (JCPDS card No. 89-3722). A few intense with unassigned peaks in AgNPs structure's vicinage at 2θ = 28.2, 32.5, 55.5, and 56.9° exhibited bio-organic phase crystals [24] in the CMP extract, which was useful for the reduction of Ag⁺ ions. The average crystal size of AgNPs was estimated as 10.4 nm following the Debye–Scherrer equation.

FTIR spectra of CMP and AgNPs are recorded to find possible functional groups responsible for AgNPs biosynthesis (Fig.S4). The results showed that the biological molecules of CMP stick to the surface of the AgNPs solution. The peak at 3415 cm^-¹ can be attributed to the O-H elongation oscillation of phenol groups on flavonoid rings [15], Absorption bands at 2930 and 1620 cm^-¹ are estimated to be due to prolonged fluctuation of C-H and C=C, [15], respectively. The peak at 1375 cm^-¹ is assumed to be due to bending vibrations in the plane of δ (O-H). The peak at 1745 cm^-¹ could be a sign of the C=O group. Furthermore, the peaks at 1055 and 620 cm^-1 can be attributed to the oscillation of ν(C-O) and δ(C-H), respectively. It reported that flavonoids such as naringenin, hesperidin, and naringin are the main compounds found in CMP extract [12], which reduces Ag⁺ and adsorption on the AgNPs surface to enhance their stability. Besides, other bioactive compounds such as phenolic acids, limonoids, coumarin, quercetin, rutin, and carotenoids also play an essential role in preparing AgNPs. In comparison with the pure CMP extract, insignificant differences of absorption bands with weaker signals were observed in AgNPs solution, attributed to a decrease of the biomolecules' concentration (involving the reduction of Ag⁺ ions to Ag⁰ [16]).

The HRTEM analysis showed the nanoparticles were spherical in shapes with a diameter range of 10–20 nm (Fig.S5). The HRTEM image demonstrated that the AgNPs had three planes of structured AgNPs, including the (111) and (200) planes with the d-spacings of 0.27 and 0.24 nm, respectively. The EDS spectrum of AgNPs delivers high intense major peaks of elemental Ag with an atomic percentage of 70.8%, which was typical for the absorption of metallic silver because of surface plasmon resonance (Fig. S6). The appearance of the weaker signals of O (11.2 wt.%), C (15.4 wt.%), and Cl (1.6 wt.%) may be owing to the presence of bio-molecules that are bound to the surface of AgNPs. Besides, the carbon composition also is existed by the carbon tape covered the sample. The zeta potential in the double electrical layer surrounding AgNPs is −17.7 mV (Figure S7a). The result confirmed that the AgNPs possess high stability in the aqueous solution. The size distribution showed that AgNPs with a diameter of 12.2 nm had the highest appearance (Fig. S7b). This result is completely consistent with XRD and HRTEM analysis. The use of CMP extract as reducing and stabilizing agents permitted to obtain the spherical nanoparticles had small and uniform size at room temperature with sunlight irradiation assistance (Table 1).

Table 1. Synthesis of AgNPs using the leaf extract of different plants.

Plants	Plant’s part	Shapes	Size (nm)	References
Alternanthera dentate	Leaf	Spherical	50–100	[17]
Tea extract	Leaf	Spherical	20–90	[18]
Pistacia atlantica	Seed	Spherical	10–50	[19]
Acalypha indica	Leaf	Spherical	20–30	[20]
Citrus sinensis	Peel	Spherical	10–35	[21]
Vitis vinifera	Fruit	Spherical	30–40	[22]
Eclipta prostrate	Leaf	Triangles, pentagons, hexagons	35–60	[23]
Centella asiatica	Leaf	Spherical	30–50	[24]
Carica papaya	Leaf	Spherical	25–50	[25]
Nelumbo nucifera	Leaf	Spherical, triangular	25–80	[26]
Citrus maxima	Peel	Spherical	10-20	This work

Fig.S8 showed the inhibition zone of the synthesized AgNPs sample against Methicillin-resistant Staphylococcus aureus (MRSA) bacteria. The average inhibition zone diameter was 11.7 nm. The antibacterial activities of the obtained AgNPs were further determined by their corresponding minimum inhibitory concentration (MIC) and minimum batericidal concentration (MBC) shown in the Fig.S9. For MIC analysis, it was observed that the exponential phase of bacteria delayed in the presence of AgNPs and this phenomenon was more obvious with the increase of AgNPs concentration (Fig.S9a). The AgNPs synthesized at the optimized condition could delay the exponential phase of bacteria. The AgNPs could completely inhibit the MRSA bacteria at MIC of 8.27 µg/mL (N/16). For MBC analysis, the MBC value was found to be 16.54 μg/mL (N/8).

In conclusion, a cost-effective, fast, eco-friendly, and convenient green route for synthesizing AgNPs using CMP extract as a reducing agent at ambient temperature was proposed. The AgNPs synthesized at the optimized conditions were formed as highly crystallized spherical particles with a size range of 10-20 nm. The obtained AgNPs showed efficient antibacterial activity against Methicillin-resistant Staphylococcus aureus. Hence, the use of a green method for synthesizing AgNPs in this work may suggest using such nanoparticles in the inhibition of future antibiotic-resistant bacteria.

Funding: Not applicable.

Competing Interests: The authors declare that they have no conflict of interest.

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Highly Antibacterial Activity Against Methicillin-resistant Staphylococcus aureus of Silver Nanoparticles Synthesized by Citrus maxima Peel Extract

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