Green-synthesized silver nanoparticles of Cinnamomum zeylanicum and their biological activities

Asia Noureen; Faisal Ahmad; Farhan Younas; Gezahign Fentahun Wondmie; Samir Ibenmoussa; Gamal A. Shazly; Mohammed Bourhia

doi:10.1515/chem-2024-0128

Article Open Access

Green-synthesized silver nanoparticles of Cinnamomum zeylanicum and their biological activities

Asia Noureen , Faisal Ahmad , Farhan Younas , Gezahign Fentahun Wondmie , Samir Ibenmoussa , Gamal A. Shazly and Mohammed Bourhia

Published/Copyright: March 3, 2025

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From the journal Open Chemistry Volume 23 Issue 1

Abstract

Several drugs cure pathogenic microorganisms; however, they all have cost, toxicity, and pathogenic resistance. The present study was designed to evaluate the antibacterial, antifungal, and antileishmanial efficacy, as well as the toxicity profile, of nanoparticles (NPs). Synthesized silver nanoparticles (AgNPs) of Cinnamomum zeylanicum (C. zeylanicum) bark extract was characterized using several techniques were UV-visible spectroscopy verified NPs production. The absorption peak of C. zeylanicum bark extract was 418 nm. Fourier transform infrared spectroscopy used to identified the functional groups in plant extract responsible for reducing AgNO₃ to AgNPs. Scanning electron microscopy showed AgNPs morphology. The synthesized NPs were spherical, rectangular, and oval. The synthesized NPs were analyzed for phase distribution, crystallinity, and purity using X-ray diffraction. C. zeylanicum bark extract AgNPs possess crystal cubic structure, and the Debye-Scherrer equation determined the average particle size of 10.508 nm from full width at half-maximum of the diffraction peaks. In this study, an environmentally friendly synthesis of AgNPs from C. zeylanicum bark extract was tested for antibacterial, antifungal, and antileishmanial efficacy against E. coli, S. aureus, Trichoderma harzianum, and Leishmania tropica (KWH23) strains. Fresh human blood cells were also tested. Green synthesized NPs were effective against pathogenic bacteria. Low dose of AgNPs can be used to reduce toxicity.

Keywords: Cinnamomum zeylanicum bark extract; silver nanoparticles; antibacterial activity; antifungal activity; antileishmanial activity; cytotoxic effect

1 Introduction

Nanotechnology is evergreen research due to its non-toxicity and higher significance than chemical synthesis. The green synthesis of NPs has become an emerging area of research in the twenty-first century due to its non toxic and environment friendly processes. “Nanotechnology is the science to control matter at the molecular level [1]. Nanotechnology is a reliable and environment-friendly process for the synthesis of not developing nano-scale particles ranging from 1 to 100 nm [2]. Biosynthesis gives progression over chemical and physical strategy, but high temperature and toxic chemical is not required for their synthesis [3]. Although silver nanoparticles (AgNPs) have a tremendous role in various antibacterial applications, silver metal’s interaction with microbes is not fully understood yet. Therefore, it has been hypothetically approved that AgNPs caused cell inhibition, cell transduction, and cell lysis, respectively [4]. Plants are often a source of easy and cost-effective medication, whether in pure energetic principle agents or traditional preparations [5,6]. Employing plant extracts to produce NPs by green synthesis has many advantages such as they are quite safe, easily available, and have a wide diversity of metabolites that might support reduction reactions [7]. Due to its potential stability, the synthesis procedure of metal NPs, specifically AgNPs using plant extract, has become a major concern for scientists. It has an outstanding application in biological imaging, drug delivery, gene silencing, antimicrobial, and chemical sensing [8]. Due to the increasing complexities in our natural ecosystems due to man’s interference and global environmental changes, pathogens are becoming more resistant and complex due to frequent mutations, resulting in extreme consequences like treatment failure. Natural products were discovered alternate to synthetic antibacterial agents [9]. Due to their high penetration ability and functional strategies, NPs are opening new promising ways for medical applications and other industries [10].

The bark of Cinnamomum zeylanicum (C. zeylanicum) is widely used in fragrance production. It is primarily used as a flavoring agent in a wide range of used extensively in preparing foods like sweets, spicy candies, chocolate tea, hot cocoa, and liqueurs. It acts as a volatile oil in medicine preparation and is also used to treat colds. Furthermore, it is affective for digestive problems and used for diarrhea treatment. The bark of C. zeylanicum has a highly anti-oxidant reaction [11]. C. zeylanicum plant plays a pivotal role in imparting distinctive flavor due to the presence of characteristic phytochemical compounds, and their important oils and related ingredients likewise play a significant role in antimicrobial, antidiabetic, and antifungal activities [12,13,14]. Cinnamon contains different kinds of sticky compounds, like cinnamaldehyde, cinnamate, cinnamic acid, and several essential oils for living organisms [15]. According to Vangalapati et al. [16], the bark of C. zeylanicum contains 65–80% cinnamaldehyde and 5.00–10.00% eugenol.

2 Experimental details

2.1 Collection of plant and extraction

The bark of C. zeylanicum was obtained from a local market in Islamabad, Pakistan, and washed two times with tap water and once with distilled water. The bark was dried. After drying, the bark was crushed into fine powder.

2.2 Green synthesis of NPs

C. zeylanicum bark powder was weighed. In a conical flask, 50 mL of distilled de-ionized water was taken, in which 5 g C. zeylanicum bark powder was dissolved. The flask was kept on the hot plate for 30 min at 60°C and in a shaking bath to get the homogenous mixture. The flask was kept at 40°C in shaking incubator for 48 h. Through Whatman filter paper, the plant extract was filtered (Filter paper with pore size 90 nm). AgNo₃ solution of six different molarities, 15, 10, 7.5, 5, 2.5, and 1 mM, were used for reactions. The plant extract was added to the salt solution in a 1:2 volume ratio and incubated at various pH levels for a period. Visual observation of the color change from pale yellow to dark brown proved the reduction of the salt solution into silver ions, whereas the control color remained constant. At the end of the reaction, the colloidal solution was centrifuged. For 10 min at 10,000 rpm, the reaction mixture was centrifuged three times. The variations in experimental samples revealed the synthesis of AgNPs [17].

2.3 Optimization of AgNPs

The size and form of NPs were controlled, and the synthesis of NPs was increased by optimizing several factors.

2.4 Concentrations of plant extracts and silver nitrate solution

Different concentrations were examined to identify the optimal concentrations of plant extracts and salt. The final concentrations of plant extract were 0.25, 1.25, 2.5, 5, and 7.5 μg/mL. The silver salt solution was utilized in six concentrations (15, 10, 7.5, 5, 2.5 mM, and 1 mM). The solution were kept in dark at room temperature for reaction.

2.5 Conditions optimization

Following the method reported by Mittal et al. [18], the optimal pH for biological production of AgNPs was found. The AgNPs were produced at a pH range of 0–10. A 0.1 M sodium hydroxide solution was used to alter the pH of the reaction mixture. In a static incubator (JSSI-300CL, Korea), AgNPs were synthesized at various temperatures (25, 35, 45, and 60°C). The reaction mixture was stored in dark test tubes. The reaction time, or how long the silver salt interacts with the plant extract, determines the size of the NPs produced. AgNP synthesis was evaluated at various reaction times (30 min, 1, 2, 3, 4, and 24 h). In contrast, the other parameters remained constant. The reaction mixture was kept in dark flasks while the other parameters remained constant.

3 Characterization of AgNPs

3.1 UV-visible spectroscopy

Silver ions’ bio-reduction in solution was observed using UV-visible spectrum (PerkinElmer Lambda 950UV/V spectrophotometer, UK) with a resolution of 1 nM between 200 and 700 nm wavelengths.

3.2 Fourier transform infrared spectroscopy (FTIR)

For reducing AgNO₃ to Ag Ions, various functional groups in the C. zeylanicum bark extract were identified using FTIR. FTIR (Perkin-Elmer spectrum 100, United States) of AgNPs were recorded between 1,000 and 4,000 cm⁻¹.

3.3 X-ray diffraction (XRD) analysis

XRD (Rigaku Geiger Flex D-max III/c diffractometer USA) examination was used to measure the phase purity, grain size, and crystallinity of NPs. To undertake an XRD spectroscopic investigation, the material was first lyophilized. Double-sided sticky tape was used to adhere AgNPs to silica supports. The analysis was carried out utilizing a (Rigaku Geiger Flex D-max III/c diffractometer, USA) with a copper ray tube operating at 30 kV and 20 mA over a range of 2 from 30 to 70°C.

3.4 Scanning electron microscopy (SEM)

The shape, size, and microstructure of the NPs were characterized by a SEM (JSM 6390LV, JOEL, USA).

3.5 Antibacterial activity

AgNPs from C. zeylanicum bark extract was tested for antibacterial efficacy against gram-negative and gram-positive microorganisms. After 24 h of incubation on the Muller Hinton Agar plate, 0.5 McFarland standard (1 × 10⁸ CFU/mL) of fresh bacteria culture was introduced. In the wells, different concentrations of C. zeylanicum BE AgNPs (100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μg/mL) were serially administered. Pen-strep (0.5%) and water were positive and negative controls, respectively. At 37°C, agar plates were incubated for 24 h. The zone of inhibition was assessed after 24 h of incubation. The tests were performed in triplicate. The minimum inhibitory concentration (MIC) values were determined using the broth microdilution method [19]. At a final concentration of 100 μg/mL, NPs were used. NPs solutions (10 μL) were added in each microtiter plate well, and 180 μL nutrient broth medium was added in the remaining wells. Each well-received 10 μL of inoculum. The negative control was water, while the treated group was broth and inoculum containing standard medicine (pen-strep). The microtiter plates were incubated at 37°C for 24 h. Three tests were conducted on each NPs. An ELISA reader, the microtiter plate’s absorbance at 620 nm was measured.

3.6 Antifungal activity

The antifungal activity of NPs was tested on one fungal strain (Trichoderma harzianum). The approach of Jenny et al. [20] was modified to test the antifungal activity of biologically produced NPs. The 20 mL potato dextrose agar (PDA) autoclaved solution was poured into sterilized Petri plates, and the fungal solution’s turbidity was adjusted with 0.5 McFarland standard (1 × 108 CFU/mL). After adjusting the turbidity, add 20 μL of fungal solution into sterilized PDA Petri plates. In the wells, different concentrations of C. zeylanicum AgNPs (100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μg/mL) were serially administered. Amphotericin B was utilized as a positive control, and water was used as a negative control. At 37°C, PDA plates were incubated for 24 h. The zone of inhibition was assessed after 24 h of incubation. The experiments were carried out in triplicate. For each NP exhibiting antifungal activity against test pathogens, the MIC was assessed spectrophotometrically. The broth micro-dilution method (Sharma et al. [21]) was used to determine the MIC values. The final concentrations of NPs tested were (100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μg/mL). In each well, 180 µL nutrient broth medium was added. A micropipette was then used to perform two-fold serial dilutions so that each well received 10 μL of a successively decreasing concentration of NPs solution. After that, 10 μL of inoculum was administered to each well. As a positive control, broth containing the standard drug (Amphotericin B 0.05%) was employed, while water was used as a negative control. For 24 h, the microtiter plates were incubated at 37°C. Each NP was evaluated three times. Using an ELISA reader and a microtiter plate, absorbance was measured at 520 nm.

3.7 Antileishmanial activity

3.7.1 Anti-promastigote assay

The antileishmanial activity of biologically synthesized NPs was evaluated in vitro against L. tropica promastigote culture. L. tropica promastigotes were incubated at 24°C temperature in media containing 25 mM 4-(2-hydroxyethyl)-1-piper-azi-neethanesulfonic acid (HEPES) buffer, sodium bicarbonate, 10% heat-inactivated fetal bovine serum (FBS), and 2% pen-strep antibiotic. 1 × 10⁶ cells/mL of the promastigotes form of L. tropica from the logarithmic growth phase were introduced into each well of RPMI 1640 containing 10% FBS. Subsequently, L. tropica incubated in media for 48 hours as positive control group, water (negative control), and various concentrations of NPs (100, 50, 25, 12, 6, 3, and 0.7 μg/mL). The reference drug tartar emetic (TE) was then serially administered in each well of the plate. The growth of the cells was monitored in a microplate reader by taking the optical density (OD) of the cells after 48 h of incubation at 24°C. The antileishmanial activity was measured using in vitro reactions, which were done in triplicate, and the results were expressed as IC₅₀/72 h.

3.7.2 Anti-amastigote assay

Amastigotes grow outside the living cells and are axenic amastigotes [22]. They set guidelines for cultivating axenic amestigotes. The algorithmic growth phase of axenic amastigotes was also harvested and cultured in RPMI 1640 media, and pH of 5.5 was adjusted for their growth. At 33°C, the cells were incubated for 7 days in humified CO₂ (5%). By changing the temperature and PH, promastigotes transformed into axenic amastigotes. In each well of the 96-well microtiter plate (106 cells/mL), RPMI media containing a culture of axenic-amastigotes were added. The different concentrations of NPs (100, 25, 15, 12.5, 6.5, 3.0, 1.5, 0.75 μg/mL) water and (TE reference drug) were added to each well and incubated at 25°C ± 1°C for 72 h. All the experiments were performed in triplicates. A microplate reader was used for the OD of the cells. IC₅₀ value of the biologically synthesized NPs showing anti-amastigote activity was calculated by graph pad prism.

3.7.3 Cytotoxicity of NPs on human blood cells

A cytotoxic assay was performed to analyze the cytotoxicity of biologically synthesized AgNPs on fresh human blood cells. The fresh human blood was collected from healthy volunteers in a vacuum cleaner. Using phosphate buffer (PBS), wash the human blood cells three times and centrifuge at 3,000 rpm for 2 min. After centrifugation, the erythrocytes were separated from the blood and settled at the bottom of the Eppendorf tube. The blood cell suspension was serially incubated with biologically synthesized AgNPs at 37°C for 3 h. The reaction mixture was centrifuged at 6,000 rpm after incubation for 10 min. Using a UV-visible spectrophotometer (T80+, PG instruments), the released hemoglobin was monitored at 576 nm. Experiments were performed in triplicates. The red blood cells lysed with Triton X-100 (0.1%) were used as a positive control, and the cell suspensions (red blood) in PBS were used as a negative control. Percentage hemolysis was calculated by using the following formula:

% Hemolysis = (OD at 576 in AgNPs solution – OD at 576 nm in PBS) ̸OD at 576 nm in 0.1% Triton X-100 – OD at 576 nm in PBS) × 100.

4 Results

4.1 Green synthesis of AgNPs

When C. zeylanicum bark extract was mixed with silver nitrate salt solution, the color changed from yellowish to dark brown, indicating the formation of AgNPs.

4.2 Conditions optimization

4.2.1 Concentrations of plant extracts, silver nitrate solution

Several concentrations of the plant extract (0.25, 1.25, 2.5, 5, and 7.5 mg/mL) were used from the stock solution. The ideal plant extract concentration was 200 μL and concentration of silver nitrate salt solution was 10 mM, silver nitrate was used at concentrations ranging from 0.25 to 15 mM. As the concentration of AgNO₃ raised, the synthesis of AgNPs increased. pH has a significant impact on AgNPs synthesis. At various pH levels, in the range of 2–14, the production of AgNPs was optimized; however, neutral pH, i.e., pH 7, was the best. At this pH, UV-visible spectroscopy showed a strong peak, indicating that AgNPs were synthesized and were identical in size. Synthesis of AgNPs were optimized at different temperatures, but 40°C was an ideal temperature. AgNPs can be synthesized at low temperatures (below 25°C ), and high temperatures (above 60°C); however, room temperature (40°C) was shown to be the best temperature because tiny, spherical AgNPs were produced at 40°C temperature and demonstrated single plasmon resonance in UV-visible spectroscopy. The reaction time, or how long the silver salt interacts with the plant extract, determines the size of the NPs produced. Even though AgNPs were produced at all reaction times, the yields varied. As the silver salt concentration and reaction time were increased, the yield of AgNPs increased, along with a change in the surface plasmon band wavelength to a higher range, indicating AgNPs agglomeration. The best time to synthesize AgNPs in the dark was 12 h, while other parameters remained constant, resulting in a higher yield of AgNPs with a single and short wavelength peak in UV-visible spectroscopy.

4.3 UV-visible spectroscopy

The absorbance spectrum of biologically synthesized AgNPs of C. zeylanicum bark extract (Figure 1) shows sharp absorbance at (418 nm).

Figure 1

UV of AgNps. C. zeylanicum synthesis of AgNPs was confirmed by UV-Vis spectroscopy technique. UV-Vis spectra of biologically synthesized AgNps of C. zeylanicum bark extract showed sharp absorbance at 418 nm.

4.4 FT-IR

The potential biomolecules are responsible for encapsulating the reduced AgNPs made from bark extract and reducing silver ions. The prominent peaks of the AgNPs’ FTIR spectrum (Figure 2) were noticed at 2996.92, 1775.19, 1380.8, 1245.0, 1048.2, and 749.77 cm⁻¹. The peak at 1775.9 is attributed to the vibration stretching of aldehyde carbonyl group (C═O) and the strong (C═O) starching representing a high concentration of cinnamaldehyde and aldehydes in C. zeylanicum bark extract. The peak was shifted to 1775.19 in AgNPs C. zeylanicum bark extract, showing the formation of AgNPs. The peak at 2996.92 was assigned for (═C–H) stretching, peak 1,380 showed C–H bending of alkane, 1,245 was the C–O–C bond of aromatic acid ester, and C–OH groups of phenolic compounds, peak 1,048 was assigned for –CH₃ bending, and 749 represent ═CH of the benzene ring.

Figure 2

FTIR of AgNPs of C. zeylanicum showed the identification of functional groups. The stretching and vibrations of chemicals such as phenol, flavonoids, terpenoids, and proteins in the plant extract were responsible for stabilizing and capping of AgNPs.

4.5 XRD analysis

An XRD analysis was carried out to determine the crystalline structure of AgNPs in C. zeylanicum bark extract. The peaks of different intensity were obtained at 2θ range of 30–70°. Three prominent diffraction peaks were observed at angles of 38.10°, 44.60°, and 64.67° (2θ), corresponding to the 111, 200, and 220 Bragg reflections, respectively (Figure 3). The XRD peaks’ distinct line widening reveals that the synthesized material consists of particles in the nanoscale range. AgNps have a cubic crystal shape (CC). The results of AgNPs are similar to the international pattern of the joint committee on powder diffraction data (JCPDS card no 00-004-0783) (Swanson, Tatge., 195356). We identified the peaks’ intensity, position, and width using this XRD patterns analysis and their full width at half-maximum (FWHM) data. The diffraction peaks have been indexed as crystalline cubic structures. The average particle size of AgNPs was calculated from the FWHM of the diffraction peaks using the Debye-Scherrer equation [23].

D = k λ / B cos θ ,

where D is the nanoparticle’s diameter, λ is its wavelength, β is its FWHM, and Ө is its diffraction angle. Calculations were carried out to determine the size of AgNPs based on several refraction peaks. The findings revealed that the average size of AgNPs of C. zeylanicum bark extract was 10.508 nm.

$Figure 3 XRD AgNPs of C. zeylanicum bark extract. The diffraction peaks of AgNPs of C. zeylanicum bark extract has been indexed as crystalline cubic structures. The average particle size of AgNps was calculated from the FWHM of the diffraction peaks using the Debye-Scherrer equation, and the average size was 10.508 nm.$

Figure 3

XRD AgNPs of C. zeylanicum bark extract. The diffraction peaks of AgNPs of C. zeylanicum bark extract has been indexed as crystalline cubic structures. The average particle size of AgNps was calculated from the FWHM of the diffraction peaks using the Debye-Scherrer equation, and the average size was 10.508 nm.

4.6 SEM

SEM images of AgNPs of C. zeylanicum bark extract (Figure 4) were observed, and it was confirmed that the diameter of NPs was below 100 nm at a magnification of ×40,000. Particles were observed to be spherical. The average size of AgNPs of C. zeylanicum bark extract was 53–66 nm.

Figure 4

FESEM images of AgNPs of C. zeylanicum bark extract. (a) and (b) showed SEM images of AgNPs C. zeylanicum bark extract. The biologically synthesized AgNPs have a high density and are predominantly spherical.

4.7 Antibacterial activity

The antibacterial activity results revealed that the biologically synthesized AgNPs were active against Gram-negative bacteria (E. coli) and Gram-positive bacteria (S. aureus). (Figure 5) shows the zones of inhibition of NPs (100, 50, 25, 12.5, 6.25, 3, 1.5, 0.75 μg/mL). Negative control (H₂O) and positive control (pen-strep) were used against gram negative bacteria (E. coli). AgNPs of C. zeylanicum bark extract showed the highest activity against E. coli (17.66 ± 0.57 mm) at 100 µg/mL. The lowest antibacterial activity was reported against E. coli (3.3 ± 2.8 mm) at 3 µg/mL concentration. The zone of inhibition of negative control (water) was 0 ± 0 mm and positive control (pen-strep) was 22 ± 0 mm. The AgNPs of C. zeylanicum bark extract at concentrations of 1.5 and 0.75 µg/mL showed no activity. The MIC (Figure 6) of C. zeylanicum bark extract against E. coli was 6.25 µg/mL with an IC₅₀ value of 5.1576. AgNPs of C. zeylanicum bark extract (Figure 7) showed the highest activity against S. aureus (19.66 ± 0.57 mm) at 100 µg/mL. The lowest antibacterial activity was reported against S. aureus (12.66 ± 0.57 mm) at 3 µg/mL concentration. The zone of inhibition of negative control was 0 ± 0 mm and positive control was 23 ± 0 mm. The AgNPs of C. zeylanicum bark extract at concentrations of 1.5 and 0.75 µg/mL showed no activity. The MIC (Figure 8) of C. zeylanicum bark extract against S. aureus was 2.5 µg/mL with an IC₅₀ value of 3.735.

Figure 5

Graphical presentation of antibacterial activity against gram-negative bacterial strain of E. coli (ATCC # 8739). AgNPs showed the highest antibacterial activity (17.66 ± 0.57 mm) at 100 µg/mL and the lowest (3.3 ± 2.8 mm) at a concentration of 3 µg/mL.

Figure 6

IC₅₀ of AgNPs of C. zeylanicum bark extract against E. coli (ATCC # 8739).

Figure 7

Graphical presentation of antibacterial activity against gram-positive bacterial strain S. aureus (ATCC# 6538). AgNPs of C. zeylanicum bark extract showed the highest activity against S. aureus (19.66 ± 0.57 mm) at 100 µg/mL and lowest activity (12.66 ± 0.57 mm) at a concentration of 3 µg/mL.

Figure 8

IC₅₀ of AgNPs of C. zeylanicum bark extract against S. aureus (ATCC# 6538).

4.8 Antifungal activity

AgNPs of C. zeylanicum bark extract (Figure 9) showed the highest activity against Trichoderma harzianum (11.33 ± 1.52 mm) at 100 µg/mL. The lowest antibacterial activity was reported against Trichoderma harzianum (7 ± 1 mm) at 3 µg/mL concentration. The zone of inhibition of negative control was 0 ± 0 mm and positive control was 14.33 ± 0.57 mm. At concentrations of 1.5 and 0.75 µg/mL, AgNPs of C. zeylanicum bark extract showed no activity. The MIC (Figure 10) of C. zeylanicum bark extract was 3 µg/mL, and the IC₅₀ value was 2.488.

Figure 9

Graphical presentation of Antifungal activity of AgNPs of C. zeylanicum bark extract. AgNPs showed the highest activity against Trichoderma harzianum (11.33 ± 1.52 mm) at 100 µg/mL. The lowest antifungal activity (7 ± 1 mm) was reported at 3 µg/mL concentration.

Figure 10

IC₅₀ of AgNPs C. zeylanicum bark extract against fungal strain Trichoderma harzianum.

4.9 Antileishmanial activity

4.9.1 Anti-promastigote assay

Antileishmanial efficacy of biologically synthesized AgNPs was examined for the strain of Leishmania tropica KWH23. The effect of AgNPs on metabolic activity (Figure 11) of promastigote against eight different concentrations (100, 50, 25, 12.5, 6.25, 3, 1.5, 0.75 μg/mL) was determined. At 72 h after incubation, the survival of promastigotes cells was evaluated in both the control and test groups. After 72 h of incubation, the metabolic activity of promastigotes decreased with the increase in the concentration of AgNPs. Compared to the control group, the metabolic activity of Leishmania tropica promastigotes was positively affected at each concentration of AgNPs. The IC₅₀ value of AgNPs of C. zeylanicum bark extract was 2.664 μg/mL. The results indicated that the increased concentration of AgNps decreased the cell viability of Leishmania tropica. The highest concentration of AgNPs was 100 μg/mL, showing maximum effects on the metabolic activity of promastigotes.

Figure 11

Inhibitory concentration (IC₅₀) of promastigotes of L. tropica at different concentrations of AgNPs of C. zeylanicum bark extract.

4.9.2 Anti-amastigote assay

The algorithmic growth phase of axenic amastigotes was also harvested and cultured in RPMI 1640, and PH 5.5 was adjusted for their growth. At 33°C, the cells were incubated for 7 days in humified CO₂ (5%). By changing the temperature and pH, promastigotes transformed into axenic amastigotes. In each well of the 96-well microtiter plate (106 cells/mL), RPMI media containing a culture of axenic-amastigotes were added. The different concentrations of biologically synthesized NPs (100, 25, 15, 12.5, 6.5, 3.0, 1.5, 0.75 μg/mL) along with water (negative control) and (TE positive control) were added to each well and incubated at 25°C ± 1°C for 72 h. The effects of AgNPs of C. zeylanicum bark extract on Leishmania tropica (axenic amastigotes) metabolic activity were also examined. Effects of various concentrations (Figure 12) of AgNPs (100, 50, 25, 12.5, 6.25, 3, 1.5, 0.75 μg/mL) on Leishmania tropica showed that the metabolic activity of leishmanial cells consistently decreased by increasing the concentration of AgNPs. The metabolic activity of Leishmania tropica amastigotes was positively affected at each concentration of AgNps compared to the control group. The IC₅₀ value of AgNPs of C. zeylanicum was 1.123 μg/mL.

Figure 12

Inhibitory concentration (IC₅₀) of anti-amastigotes of L. tropica at different concentrations of AgNps of C. zeylanicum bark extract.

4.9.3 Cytotoxicity of NPs on human blood cells (hemolytic activity)

Hemolytic activity was performed on fresh human blood cells. It is important to synthesized less toxic and more effective NPs to cure pathogenic diseases. It was necessary to evaluate the biocompatibility of these NPs on normal human cells to ensure their non-toxic usage. In this study, the biologically synthesized NPs were used at different concentrations and evaluated for their cytotoxicity against human macrophages. It was clear from (Figure 13) that all the NPs were non-biocompatible at 200 μg/mL. AgNPs of C. zeylanicum showed hemolytic activity (<80%) at 200 μg/mL concentration and (<30%) at a concentration of 12.5 μg/mL. The results indicated that the AgNPs were non-biocompatible at higher concentrations and biocompatible at lower concentrations when compared with 0.5% Triton X-100 (as positive control).

Figure 13

Cytotoxicity of C. zeylanicum bark extract in normal cell (hemolytic assay).

5 Discussion

According to Hyllested et al. [24], in the synthesis of AgNPs, color change is a key indicator. According to other investigations, the color change from yellowish brown to dark brown indicates the presence of colloidal AgNPs [25,26].

According to Otunola et al. [27], the optimization of all parameters (plant extract concentrations, silver nitrate solution, pH, temperature, and time) is essential for the synthesis of AgNPs of the desired size and shape, as well as the prevention of NP agglomeration. Thus, at close to ambient temperature, neutral pH, and a 12-h reaction period, energy-efficient AgNPs were synthesized in a solution containing 1 mM silver salt. For the analysis of NPs, UV-vis spectroscopy is a beneficial approach. UV spectroscopy was used to characterize the NPs. The plant extract chemicals converted AgNO₃ to Ag⁺ ions in the aqueous solution. The reduction of Ag ions to AgNPs has been attributed to the activation of plasmon vibrations of AgNPs in the solution, which was verified by UV-vis spectra. The surface plasmon resonance (SPR) for colloidal silver has been reported to range from 320–390 nm to 420–450 nm [28]. The UV absorption of plant extract AgNPs suggests that bioactive substances such as proteins stabilized and hindered the transition of electrons from AgNPs’ surface. The colloidal silver SPR reveals that only AgNPs were present in the dispersion. FTIR has proven to be a valuable approach for characterizing and identifying functional groups. According to Sasidharan et al. [29], the FTIR spectrum of pure chemicals was so distinctive that it resembled a molecular fingerprint. The study discovered stretching vibrations of substances, including phenol, flavonoids, terpenoids, and proteins, proving that these molecules are responsible for AgNP stabilizing and capping [19]. According to the AgNPs data [30,31], polyhydroxy phenolics and flavonoids in the plant extract were the key biomolecules that can decrease silver ions and convert them to AgNPs.

Peak intensity, position, and width were identified using XRD patterns analysis, and FWHM values were also included. The diffraction peaks of AgNPs were exhibiting a crystalline cubic structure. The high degree of crystallinity of the AgNPs was reflected in the peak intensity. The diffraction peaks, on the other hand, were broad, indicating a tiny crystallite size. The same results were concluded by Wani et al. [32]. The existence of metal particles of nano-size was confirmed by SEM examination of AgNPs. They were spherical, with a diameter of less than 100 nm. The antimicrobial activities of biologically produced AgNPs demonstrated they were active against microbes. It was obvious that as the concentration of AgNPs of C. zeylanicum bark extract increases, the inhibitory activity also increases. The significant surface area to volume ratio of AgNPs resulted in increased antimicrobial activity. Combining biomolecules with AgNPs gave AgNps a higher activity than an aqueous extract because silver nitrate contains the ionic form of silver (Ag⁺), which has more activity than extract [33]. Previous research has also shown that AgNps from plant extracts have potent antimicrobial properties [34]. Producing less hazardous and more effective agents is critical to cure pathogenic diseases. Cytotoxicity is a critical test that must be completed first. AgNPs have the potential to be an effective treatment for deadly diseases. The results indicate that the AgNPs of C. zeylanicum bark extract was non-biocompatible at higher concentration and biocompatible at lower concentration when compared with 0.5% Triton X-100 (as positive control). According to the findings, C. zeylanicum bark extract AgNPs can be an efficient antimicrobial agent with minimal toxicity at low doses.

6 Conclusion

We effectively synthesized and characterized AgNPs from C. zeylanicum bark extract using a variety of approaches. The AgNPs had a cubic crystal shape and a size of 10.508 nm. AgNPs have also shown antibacterial, antifungal, and antileishmanial activity against various pathogenic pathogens. AgNPs were discovered to be non-toxic to both human cells and the environment. As a result, we conclude that AgNPs derived from C. zeylanicum bark extract are a good option for developing innovative and environmentally friendly antibacterial agents. The study results suggest that the AgNPs synthesized from C. zeylanicum bark extract exhibited non-biocompatibility at higher concentrations and biocompatibility at lower concentrations than 0.5% Triton X-100 (used as a positive control). The AgNPs also demonstrated antimicrobial activity against various pathogenic microorganisms with minimal toxicity at low doses. Therefore, the study concludes that AgNPs synthesized from C. zeylanicum bark extract have the potential to be used as an effective and eco-friendly antimicrobial agent.

Acknowledgements

The authors extend their appreciation to the Researchers Supporting Project number (RSPD2025R1118), King Saud University, Riyadh, Saudi Arabia.

Funding information: The research was financially supported through Researchers Supporting Project Number (RSPD2025R1118), King Saud University, Riyadh, Saudi Arabia.
Author contributions: Conceptualization, writing the original draft, performed experiments, formal analysis, investigations, data curation: Asia Noureen, Faisal Ahmad, Farhan Younas, and Gezahign Fentahun Wondmie. Investigations, resources, project administration, reviewing, and editing: Samir Ibenmoussa, Gamal A. Shazly, Mohammed Bourhia, and Arshad Islam.
Conflict of interest: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Ethical approval: Ethical Approval is not applicable to this article.
Data availability statement: Data will be available upon request from the corresponding author.

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Received: 2024-03-05

Revised: 2024-11-02

Accepted: 2024-12-11

Published Online: 2025-03-03

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/chem-2024-0128

Keywords for this article

Cinnamomum zeylanicum bark extract; silver nanoparticles; antibacterial activity; antifungal activity; antileishmanial activity; cytotoxic effect

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