Green fabrication of silver nanoparticles using leaf extract of tropical vine Momordica charantia: spectral characterization and in vitro cytotoxicity evaluation on human breast cancer cells

ABSTRACT

The use of environmental materials such as plant extract for the synthesis of silver nanoparticles (AgNPs) offers numerous benefits of eco-friendliness and compatibility for pharmaceutical and other biomedical Applications. In this study, we used Momordica charantia leaf extract as a reducing agent for the synthesis of AgNPs. Characterization of the AgNPs was done by UV-Visible Spectroscopy, FTIR, SEM, TEM and EDX. Furthermore, we established the in vitro anticancer efficacy of these nanoparticles against the human breast cancer cell line MCF-7 using MTT assay, nuclear morphology testing, and staining. In addition to the above, the anti-bacterial effect was also tested against a range of bacterial isolates using well-diffusion method. These AgNPs also demonstrated impressive results when tested for their free-radical scavenging efficiency on the MCF-7 breast cancer cell line, indicating future potential for their use in medicine. These findings indicated the possible applicability of biosynthesized AgNPs derived from Momordica charantia L.

KEYWORDS:

• Momordica charantia L
• silver nanoparticles
• antioxidant
• cytotoxic effect
• breast cancer
• fluorescent staining

Introduction

Nanoparticles play an important role in pharmaceutical industrial, biomedical and biotechnological applications. Nanotechnology is rapidly growing, impacting many aspects of human existence and generating interest in biological sciences, agriculture, biomedical, food industry, textiles, environmental ecosystems, etc [1]. Of the several green synthesis methodologies, plant extract-mediated green synthesis has additional practical uses. Plant-mediated green synthesis is quicker than traditional biosynthesis methods because raw materials are readily available, the process is inexpensive, and it is sustainable. Plant materials such as leaves, roots, shoots, flowers, fruit pulp, bark, and seeds have been successfully exploited for efficient nanoparticle biosynthesis. Many plants have also been reported to be used in the green production of metal nanoparticles. As a result, nanoparticle manufacturing is becoming highly significant. Silver is the metal most commonly used in the treatment of disinfection, mental illness, nicotine addiction, and infectious disorders, such as gonorrhoea and syphilis [2]. They also exhibit chemical characteristics such as stability and conductivity, along with biological characteristics including antibacterial, antiviral, antifungal, and anti-inflammatory activities [3]. Several studies have demonstrated the potential antibacterial effects of silver nanoparticles (AgNPs) in particular. A study by Saxena et al. (2010) shows results of the antibacterial activity of silver nanoparticles, extracted from Onion (Allium cepa), on E.coli and S. typhimurium [4]. Other researchers carried out novel works on silver nanoparticles to further support its antibacterial activity [5,6]. In addition to the antibacterial activities Majeed et al. (2016) also portray the potential anticancer activities, of silver nanoparticles, against the MCF-7 breast cancer cell line, when tested in vitro [6]. Testing has been done on the anticancer properties of silver nanoparticles made from plant aqueous extract. The study by Rahaman et al. (2015) showed that the IC₅₀ dose of silver nanoparticles leads to an increase in intracellular reactive oxygen species and significantly diminished mitochondrial membrane potential, indicating the effective involvement of apoptosis in cell death when tested in Jurkat cells in vitro [7–9].

Cancer is a dangerous illness that is one of the leading causes of mortality, globally. External and internal factors can contribute to the onset of the illness. Despite significant progress in improving chemotherapeutic anticancer drugs, the majority of them have severe hazardous side effects. Several anticancer medicines have limited therapeutic effects due to their lack of selectivity towards cancer cells [10]. Breast cancer is the second most common cancer among Indian women, and it is more common in urban than rural areas and among women who are of greater socioeconomic classes. There are thought to be over 100,000 new cases of breast cancer diagnosed each year in India [11]. According to a recent assessment by the Indian Council of Medical Research, there will be 106,124 new cases of breast cancer in India in 2015 and 123,634 by 2020 [12].

A tropical and subtropical vine belonging to the Cucurbitaceae family called Momordica charantia is widely grown in Asia, Africa, and the Caribbean for its extremely bitter edible fruit. The jagged margins of the leaves, which give the impression that they have bit, are what gave rise to the Latin word Momordica, which means ‘to bite’. The bitter gourd (common name for M. charantia) has effective trypanocidal and antifungal properties. Furthermore, this species exhibits antiviral, anti-HIV and anti-tumour properties. Since current anti-diabetic drugs/candidates such as fenofibrate, rosiglitazone and thiazolidinediones, and GW50156 suffer from some shortcomings, M. charantia fruit in itself, seems to be a promising candidate as it is effective in abolishing the four main symptoms of diabetes. M. charantia also contains lectins (a category of proteins) that display anti-HIV and anti-tumour activities. In the study carried out by Agrawal & Beohar (2010) on chemopreventive and anticarcinogenic effects of Momordica charantia extracts, it was shown that the animals (C57 Bl mice with melanoma tumour model) treated with extracts of Momordica had an increased life span compared to the mice in the control group, hence proving it’s ant-carcinogenic effects. Thus, it is safe to say that M. charantia is proven effectively in portraying anti-bacterial, immune-stimulating, anti-leukaemic, antiprotozoal, antiviral, antifungal, antidiabetic, and anticancer characteristics [13–17].

Materials and methods

Collection of plant extract

The leaves of Momordica charantia L. were collected from the medicinal market in the Coimbatore district, Tamil Nadu, India and authenticated by the Research Officer, Siddha Central Research Institute, Government of India.

Preparation of momordica charantia L leaf extract

Previous research on leaf extracts of M. charantia shows the use of dried leaves for the preparation of aqueous extract using distilled water or alcohol, depending upon the study. For instance, a study by Costa et al. (2010) on the antimicrobial activities of M. charantia extracts suggests the preparation of leaf extract by rota evaporation with ethanol [18]. Umukuro and Ashorobi (2006) prepared the leaf extract by drying the leaves in the oven and then using distilled water to soak the dried leaves. This filtrate from this solution was evaporated to get a sticky residue [19].

About 10 g of fresh leaves of Momordica charantia L were taken and washed thoroughly with distilled water to remove dust particles. These washed leaves were cut into very small pieces and boiled in 100 mL of distilled water for an hour in a round-bottom flask with a condenser. A similar approach of preparation, using fresh leaves, was carried out by Krithiga & Briget (2015) during their experiment to assess the antimicrobial activity of AgNPs from the M. charantia leaf extract. The leaf extract was filtered using Whatman No.1 filter paper to Obtain the pure leaf extract [20,21].

Phytochemical study

The prepared extracts were subjected to phytochemical tests for plant Secondary metabolites; tannins, saponins, steroids, alkaloids and glycosides. The presence of these metabolites was determined by various methods and tests. The presence of tannins was recorded using a ferric chloride test. Carbohydrates and amino acids were determined using Benedicts and Ninhydrin reagents respectively. A foam test was performed to detect the presence of saponins in the extract. Hager’s reagent and alkaline tests were performed to prove the presence of corresponding alkaloids and flavonoids [22–27].

Evaluation of the antioxidant potential of aqueous extract of momordica charantia L

Antioxidants are substances that can prevent or slow damage to cells caused by free radicals. A free radical is a molecule or molecular fragment that contains one or more unpaired electrons in its outermost orbital. It causes damage to various tissues by the action of new reactive oxygen species in a chain reaction [28]. Further studies for determining phenolic contents of this Cucurbitaceae species, such as the one carried out by Kubola & Siriamornpun (2008) suggest the usage of hydroxy free radical scavenging assay, where the reaction mixture was prepared using ferrous sulphate, EDTA, phosphate buffer and various other supporting constituents and its absorbance measured at 520 nm. The inhibition rate was calculated as the hydroxy radical scavenging activity [29]. It is seen that DPPH and FRAP assays are most commonly used to analyse the antioxidant potential of M. charantia, as also used in a study by Fongmoon (2013) to scrutinize the antioxidant activity of bitter melon from Thailand [30].

Total reducing power assay

1.0 ml of extract solution at different concentrations (20–100 μl) was mixed with 2.5 ml of 0.2 mol/L phosphate buffer (pH 6.5) and 2.5 ml 1% potassium ferric cyanide solution. Then, the mixture was incubated at 50°C for 20 min. At the end of the incubation, 2.5 ml of 10% trichloroacetic acid was added to the mixture and centrifuged at 3000 rpm for 10 min. The upper layer of solution was collected and mixed with 2.5 ml of distilled water and 05 ml of 0.1% ferric chloride solution. The absorbance was measured at 700 nm against a blank [31].

Hydroxyl radical scavenging activity

The scavenging activity of sample extracts on hydroxyl radical was measured according to the method of Klein et al. (1999), Various concentrations (20–100 µg) of extracts were added with 1.0 ml of iron-EDTA solution, 0.5 ml of EDTA solution (0.0186), and 1.0 ml of DMSO (0.85% DMSO (v/v) in 0.1 M phosphate buffer, pH 7.4) sequentially. The reaction was initiated by adding 0.5 ml of ascorbic acid (0.22%) and incubating at 80–90°C for 15 min in a water bath. After incubation, the reaction was terminated by the addition of 1.0 ml of ice-cold TCA (17.5% w/v). Three ml of Nash reagent was added and left at room temperature for 15 min. The reaction mixture without a sample was used as a control. The intensity of the colour formed was measured spectrophotometrically at 412 nm against a reagent blank [32].

Biosynthesis of silver nanoparticle

Five millilitres of the fresh leaf extract of Momordica charantia L was dropped into 45 mL of AgNO3 (1 mM) solution with constant stirring on a magnetic stirrer at 25 + 2°C [33–35]. The yellow colour of the solution started to change gradually and turned reddish brown within 1 h. It was considered a visual sign of the growth of AgNPs. After the completion of the reaction, the solution was centrifuged at 8000 rpm for 40 min at 20°C and repeatedly washed three times. It was collected and further centrifuged at 14,000 rpm with double-distilled water for 20 min. Finally, it was dehydrated by ethanol, dried using a desiccator, and stored for the necessary characterization and biological studies [36].

Characterization of AgNPs

The absorption spectra of synthesised AgNPs were recorded by using UV-visible spectroscopy from 300 to 600 nm (BioTek, Synergy LX multimode reader) at the intervals of 15 min, 50 min, 18 h, and 48 h by taking distilled water as blank. This method was also carried out in the study carried out by Elumalai et al. (2010) to characterise silver nanoparticles from Euphorbia hirta, where the absorption spectrum was measured at 5 h after diluting a small aliquot of their reaction medium into distilled water. Later the UV-visible spectrograph was recorded as a function of time [5]. Another study by Mala et al. (2017) to compare the AgNPs synthesised from Coccinia grandis and M. charantia, also used UV-visual spectroscopy at a different wavelength of 200–800 nm. The graph was plotted against wavelength on the X-axis and absorbance on the Y-axis. The absorbance peaks were obtained between 435 and 445 nm [37]. The study taken by Rashid et al. (2017) on the characterization of phytoconstituents and evaluation of the antimicrobial activity of silver-extract nanoparticles synthesised from Momordica charantia fruit extract, suggested the usage of FTIR spectra in the range of 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹ [38]. In the study done by David et al. (2014) to characterise AgNPs from M. charantia, FTIR measurements were performed on a Thermo Scientific Nicolet iS5 instrument in the diffuse reflectance mode at a resolution of 4 cm⁻¹ in KBr pellets [39]. Similar to previous studies, the FTIR spectra of the solid leaf extract of Momordica charantia and AgNPs were taken by a Shimadzu IRTracer-100 from 4000 to 400 cm⁻¹. It helped to detect the functional groups in the extract and the AgNPs that might be accountable for synthesising the nanoparticles.

Energy dispersive analysis X-ray (EDX) spectrometer works using the photon nature of light. In the X-ray range, the energy of a single photon will be enough to produce a measurable pulse of X-ray radiation. A semiconductor material will be used to detect the X-ray along with processing electronics to analyse the spectrum [40]. EDX spectrum for the biosynthesised silver nanoparticles was obtained from the LEO-1530 instrument coupled with an EDX detector. The air-dried AgNPs were placed on the stub using carbon adhesive that was fixed before being gold-sputtered coated. The analysis of AgNP size distribution, shape and size was performed using transmission electron microscopy (TEM) and high-resolution scanning electron microscopy (HR-SEM) (CARL ZEISS) [41–43].

Anti-bacterial effects of AgNPs synthesized from Momordica charantia L

The test organisms used were clinical isolates viz., Staphylococcus aureus, Streptococcus pyogenes E.coli and Klebsiella pneumoniae which were obtained from the Department of Microbiology, Coimbatore Medical College and Hospital (CMHC), Coimbatore. The bacterial and the fungal cultures were maintained on nutrient agar medium and potato dextrose agar (PDA) medium respectively, unlike in the research by Qamar et al. (2020) where Sabouraud dextrose agar culture media plates were prepared and inoculated with the test species [44].

The samples were tested by the well diffusion method. Different concentration of the AgNPs (10 μg/ml to 30 μg/ml) was prepared by reconstituting with dimethylsulfoxide (DMSO). The test microorganisms were seeded into the respective medium by spread plate method 10 μl (10 cells/ml) with the 24 h cultures of bacteria growth in nutrient broth. After solidification, the filter paper wells (5 mm in diameter) impregnated with the extracts were placed on test organism-seeded plates. Amoxicillin (10 μg) was used as a standard for antibacterial tests. The antibacterial assay plates were incubated at 37°C for 24 h. The diameters of the inhibition zones were measured in mm [45,46].

In vitro cytotoxic effect of AgNPs synthesized from Momordica charantia L

Breast cancer (MCF-7) was procured from the National Center for Cell Sciences (NCCS), Pune, India. The selected cancer cells were maintained in Dulbecco’s modified eagles’ medium (DMEM) supplemented with 2 mM l-glutamine and balanced salt solution (BSS) adjusted to contain 1.5 g/L Na₂CO₃, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM l-glutamine, 1.5 g/L glucose, 10 mM (4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid) (HEPES) and 10% foetal bovine serum (GIBCO, U.S.A.). Penicillin and streptomycin (100 IU/100 µg) were adjusted to 1 mL/L. The cells were maintained at 37°C with 5% CO₂ in a humidified CO₂ atmosphere. The selected cells that were grown on coverslips (1 × 105 cells/coverslip) were incubated with AgNPs at different concentrations, and they were then fixed in ethanol: acetic acid solution (3:1, v/v). The coverslips were gently mounted on glass slides for the morphometric analysis. Three monolayers per experimental group were micro-graphed. The morphological changes of the cells were analysed using Nikon (Japan) bright field inverted light microscopy at 10× magnification. The inhibitory concentration (IC₅₀) value was evaluated using an MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The cells were grown (1 × 104 cells/well) in a 96-well plate for 48 h into 80% confluence. The medium was replaced with fresh medium containing diluted AgNPs samples in various concentrations, and the cells were further incubated for 48 h. The culture medium was removed, and 100 µL of the MTT [3-(4,5-dimethylthiozol-2-yl)-3,5 diphenyl tetrazolium bromide] (Hi-Media) solution was added to each well and incubated at 37°C for 4 h. After removal of the supernatant, 50 µL of DMSO was added to each of the wells and incubated for 10 min to solubilise the formazan crystals. The optical density was measured at 620 nm in an ELISA multi-well plate reader (Thermo Multiskan EX, U.S.A.) [47]. The OD value was used to calculate the percentage of viability using the following formula:

% of viability = [OD value of experimental sample/OD value of experimental control]×100

Fluorescent staining

Approximately 1 µL of a dye mixture (100 mg/mL acridine orange (AO) and 100 mg/mL ethidium bromide (EtBr) in distilled water) was mixed with 0.9 ml of cell suspension (1 × 105 cells/mL) on clean microscope coverslips. The pretreated cancer cells were collected, washed with phosphate-buffered saline (PBS) (pH 7.2) and stained with 10 µL of AO/EtBr. After incubation for 2 min, the cells were washed twice with PBS (5 min each) and visualised under a fluorescence microscope (Nikon Eclipse, Inc, Japan) at 400× magnification with an excitation filter at 580 nm.

Similarly, the cells were placed on a glass coverslip in a 6-well plate and treated with complex for 24 h. The fixed cells were permeabilized with 0.2% triton X-100 (50 μl) for 10 min at room temperature and incubated for 3 min with 10 μl of DAPI by placing a coverslip over the cells to enable uniform spreading of the stain. The cells were observed under a (Nikon Eclipse, Inc, Japan) fluorescent microscope.

Statistical analysis

Analytical measurements were conducted in triplicate, and the resulting experimental data are presented as mean ± standard deviation (SD). Statistical analyses were performed using Origin software (version 7.0383; OriginLab Corporation, Northampton, MA 01060, U.S.A.). The statistical significance of the data was assessed through two-sample independent t-tests and one-way ANOVA using GraphPad InStat 3 (San Diego, CA, U.S.A.), with a significance limit set at a 5% probability level.

Results and discussion

Phytochemical analysis of momordica charantia L.

Phytochemical investigations were conducted on the leaves of Momordica charantia L. using established methods to assess the presence of secondary metabolites. The outcomes of the phytochemical analysis revealed the existence of carbohydrates, amino acids, saponins, tannins, alkaloids, flavonoids, glycosides, and steroids in these leaves. Notably, previous studies examining the phytochemical composition of M. charantia L. leaves, employing similar testing methods outlined in Section 2.3, reported the presence of all the aforementioned components, as presented in Table 1. Additionally, these earlier research findings identified the occurrence of cardiac glycosides and steroids alongside the previously mentioned compounds. This collective data underscores the consistent presence of a range of vital secondary metabolites in Momordica charantia L. leaves, signifying their potential significance in various pharmacological and therapeutic applications. The confirmation of cardiac glycosides and steroids in the present study further enriches our understanding of the phytochemical profile of these leaves, offering new avenues for future research and exploration of their potential health benefits [26].

Table 1. Phytochemical screening of Momordica charantia L.

Phytochemical Component	Name of the test	Results
Carbohydrates	Benedicts test	+
Amino acids	Ninhydrin test	+
Saponin	Foam test	++
Alkaloids	Hager’s test	+
Tannins	Ferric chloride test	+
Flavonoids	Alkaline test	+

‘+’ Present in Trace Amount, ‘++’ Present in higher amounts, ‘-’ Not Present.

Evaluation of the antioxidant potential of aqueous extract of momordica charantia L

The aqueous extracts exhibited notable antioxidant potential, as evident from the results of the total reducing power assay and hydroxy radical scavenging activity. An observation aligns with findings from prior research on various plant species, which also indicate that antioxidants play a role in reducing the Fe³⁺/ferricyanide complex to its ferrous form. The experiment was done in triplicate and the mean ± SD values were recorded (n = 3). In particular, the aqueous extract of Momordica charantia L. demonstrated a significant increase in reducing power, with absorbance values rising from 0.09 ± 0.010 at a concentration of 25 μg/ml to a substantial 1.09 ± 0.037 at 125 μg/ml. The IC₅₀ value for this extract was determined to be 35 µg/ml, underscoring its potent antioxidant properties. The scavenging of hydroxy radicals by these extracts is likely attributable to their phenolic compounds, which possess the ability to donate electrons to hydrogen peroxide, effectively neutralising it into water. This hydroxy radical scavenging activity exhibited a dose-dependent pattern, with values increasing from 0.03 at 25 µg/ml to 0.09 at a concentration of 125 µg/ml. The IC₅₀ value for this activity was determined to be 89 µg/ml, highlighting the extract’s efficacy in combating hydroxy radicals. These results emphasise the antioxidant capabilities of the aqueous extracts and suggest their potential use in mitigating oxidative stress and related health conditions.

Total reducing power assay

The reducing power of the samples was found to be increased with the increase in concentrations of the samples. The experiment was done in triplicate and the mean ± SD values were recorded (n = 3). The reducing power of the aqueous extract of Momordica charantia L. was observed to be increased from the absorbance of 0.09 ± 0.010 at 25 μg/ml to 1.09 ± 0.037 at 125 μg/ml (Figure 1). The standard antioxidant ascorbic acid showed its maximum activity in the absorbance of 1.61 ± 0.014 µg/ml at a concentration of 125 µg/ml and the results indicated that the standard exhibited its maximum activity when compared to the aqueous extract of Momordica charantia L. The effect of reducing power was increased in a dose-dependent manner. IC₅₀ value was found to be 35 µg/ml. The results from previous research, on various species, also support the hypothesis in the presence of antioxidants that caused the reduction of Fe³⁺/ferricyanide complex to the ferrous form [48].

Figure 1. Reducing power assay of fruit extract of Momordica charantia L.

Hydroxyl radical scavenging activity

The scavenging of hydroxy radicals by extracts could be attributed to their phenolics, which can donate electrons to hydrogen peroxide, thus neutralising it to water [49]. The experiment was done in triplicate and the mean ± SD values were recorded (n = 3). The scavenging activity of the aqueous extract of Momordica charanita L. against hydroxyl radicals was noticed to be increased from 0.03 ± 0.016 at 25 µg/ml to 1.24 ± 0.095 at a concentration of 125 µg/ml scavenging increased at a dose-dependent manner (Figure 2). The standard antioxidant ascorbic acid showed its maximum activity in the absorbance of 1.69 ± 0.047 at a concentration of 125 µg/ml the standard exhibited its maximum activity when compared to the aqueous extract of Momordica charanita L. IC₅₀ value from the hydroxy radical scavenging activity was found to be 89 µg/ml.

Figure 2. Hydroxyl radical scavenging of fruit extract of Momordica charantia L.

Biosynthesis of silver nanoparticles

The silver nanoparticles were successfully synthesised using fruit extract of M. charantia L. plant. The visible observation confirms the synthesis of silver nanoparticles. Initially, the fruit extract was yellow (Figure 3). The colour gradually changed to reddish brown on the addition of silver nitrate to the extract. This indicates the formation of silver nitrate in the reaction mixture [5]. The colour changes were observed after 2 h of incubation.

Figure 3. Synthesis of nanoparticles (a) Plant extract (b) Silver nitrate (c) Synthesized AgNPs solution.

Characterization of AgNPs

Furthermore, silver nanoparticles were biosynthesised using leaf extracts, and there in vitro cytotoxic effects on the breast cancer cell line MCF-7 were investigated. When the hue of the synthesis solution gradually changed from yellow to reddish brown, silver nanoparticle formation was established. EDX analysis, FTIR spectroscopy, and UV-visible spectroscopy were used to characterise the produced nanoparticles. The SPR bands with a centre at 411 nm in the absorbance versus wavelength graph produced from the UV spectrum provide evidence that AgNPs were synthesised in the solution. The 3298, 2919, 1643, 1401, 1326, 1193, 1030, 537, and 467 cm-1 FTIR peaks were found. The existence of silver nanoparticles was confirmed by an optical band peak around 3 KeV in the EDX measurement. These results were supported by various previous research carried out on M. charantia leaves and fruits [39.40].

UV-Visible spectrum

The synthesis of AgNPs in the solution is confirmed by the SPR bands with a centre at 411 nm (Figure 4). Surface Plasmon Resonance, a size-dependent quantum mechanical process, is what causes the peak to occur (SPR). When the valence electrons’ De-Broglie wavelength matches or falls below the particle size, this effect becomes significant [5,37].

Figure 4. UV-visible spectroscopy analysis of AgNPs synthesized extract of Momordica charantia.

FTIR spectroscopy

The FTIR characterization is used to find the molecules and their functional group present. The FTIR spectra revealed the presence of different functional groups like carboxylic acids, amides, amines, phenols, alkanes, esters, alkyl halides and ethers (Figure 5). This functional group plays a very important role in these silver nanoparticle’s synthesis. In the previous research by Mala et al. (2017) on fruit and leaf extract of M. charantia, the FTIR peaks for the leaf extracts were obtained at ∼3298, ∼2919, ∼1643, ∼1401, ∼1326, ∼1193, ∼1030, ∼537 and ∼467 cm⁻¹ [37]. To further support the current results, research carried out by David et al. (2014) revealed prominent peaks for AgNPs in FTIR 1020, 1384, 1630 and 3424 cm⁻¹ due to C – N stretching (due to the presence of aliphatic amines) and a particular shift was observed from 3445 to 3424 cm⁻¹ that may indicate the involvement of OH functional group in the reduction of Ag⁺ ions [39].

Figure 5. The FTIR analysis of aqueous fruit extract of Momordica charantia L. Ag nanoparticles.

Energy Dispersive Spectroscopy (EDX)

Energy dispersive spectroscopy works using the particle nature of light. In the X-ray range of wavelength, the energy of photons (light particles) is just enough to produce a measurable, pulsating X-ray. The JEOL JSM 7600F was used to conduct the EDX analysis. The energy dispersive analysis by X-ray spectrum of the simple Momordica charantia L leaf extract is shown in Figure 6, and it can be seen that, in addition to the oxygen present from the bioorganic component of the extract, the extract contains several other inorganic ions. There is confidence that silver has been properly identified because identification lines for the main emission energies of silver (Ag) are displayed and they correlate with peaks in the spectrum. The obtained silver nanoparticles displayed an optical band peak around 3 KeV, thus confirming its presence. Similar results were produced with EDAX analysis by Gandhiraj et al. (2018) [40].

Figure 6. EDX pattern of Ag nanoparticles.

SEM and TEM imaging

AgNP were made as a thin film on a carbon plate and was allowed to air out on a hot plate at 100°C for 2 h, and then it was gold-coated. A scanning electron microscope was employed to look at the exterior morphology and characteristics of the surface of AgNPs. Visual representations of the size and morphology of synthesised M. charantia AgNPs revealed by SEM micrographs are shown in Figure 7. The SEM images also illustrates that the particles were not homogeneous in terms of shape and size. All potential spherical and irregular AgNPs geometries were visible under the micrographs. The common AgNP grain size was determined to be between 10 and 40 nm. Due to the large surface area, particles appear to cluster slightly, resulting in the production of medium-sized particles [39,50]. A TEM study was carried out to more precisely determine the crystalline properties and the size of the generated nanoparticles. The Ag TEM photos, which revealed that the particles are primarily spherical as seen in Figure 8, are consistent with the SEM data. In this image, the bulk of the Ag NPs are portrayed as spherical, with average particle sizes of 40 nm. The SEM and XRD analyses, which depict the morphological structure of the nanoparticles, agree with the TEM results.

Figure 7. SEM image of AgNPs synthesized from M. charantia.

Figure 8. TEM image of AgNPs synthesized from M. charantia.

Anti-bacterial effects of AgNPs synthesized from Momordica charantia L.

The antibacterial efficacy was evaluated using the well-diffusion technique, and it was noted that the most substantial zone of inhibition, measured at 30 µg, was observed for the following bacterial strains: Staphylococcus aureus, Streptococcus pyogenes, E. coli, and Klebsiella pneumoniae (Figure 9). Notably, Staphylococcus aureus exhibited the highest degree of inhibition. This experiment was meticulously conducted in triplicates (n = 3) and the mean ± SD values of the inhibition zone were recorded in Table 2, emphasising the rigour of the results. Furthermore, the antibacterial impact varies among the different bacterial species tested, with Staphylococcus aureus and Streptococcus pyogenes exhibiting the most pronounced response to the treatment. Moreover, the introduction of AgNPs extract into the growth medium at a concentration of 30 µg/ml notably reduced the Minimum Inhibitory Concentration (MIC) for the inoculated bacterial species. In a related study conducted by Coutinho et al. (2010), the MIC for Staphylococcus aureus was initially determined to be 1024 µg/ml. However, the incorporation of the AgNPs led to a remarkable reduction in its MIC to 32 µg/ml. These findings underscore the potential of the silver nanoparticles obtained Momordica charantia fruit extract as a potent antibacterial agent, particularly in the case of Staphylococcus aureus, and contribute to our understanding of its antimicrobial properties [51]. A similar result was observed by Abalaka et al. (2010) in their study to test the antibacterial activity of M. charantia whole plant, where the MIC of Staphylococcus aureus was shown to be 40 µg/ml [52].

Figure 9. Inhibition of bacterial growth by well-diffusion method in (a) Staphylococcus aureus; (b) Streptococcus pyogenes; (c) E. coli; (d) Klebsiella pneumoniae.

Table 2. Antibacterial activity of AgNPs synthesised from Momordica charantia L. against pathogenic microorganisms.

Microorganisms	Zone of inhibition in various NP concentration (in mm)
	Standard	10µg/ml	20µg/ml	30µg/ml
Staphylococcus aureus	15 ± 0.26	-	-	10 ± 0.15
Streptococcus pyogenes	18 ± 0.19	-	-	16 ± 0.09
E. coli	10 ± 0.12	-	-	8 ± 0.17
Klebsiella pneumoniae	12 ± 0.22	-	`-	11 ± 0.36

In vitro cytotoxic effect of Momordica charantia L

The MTT assay serves as a valuable tool for assessing cellular metabolic activity, providing insights into cell viability, proliferation, and cytotoxicity. The presented graph illustrates the variations in the percentage of cell viability in response to treatment with Momordica charantia L. silver nanoparticle extract compared to untreated cells. The experiment was done in triplicate and the mean ± SD values were recorded (n = 3). When subjected to different concentrations (5, 10, 25, 50, and 100 µg/ml) of Momordica charantia L. silver nanoparticles, a significant inhibitory effect on MCF-7 cells is observed (Figure 10) Doxorubicin (Dox) is used as the standard drug which is compared with the activity of nanoparticles on breast cancer cells (Figure 11). The yellow arrows show the normal shape of MCF-7 cell and the red arrows represents the apoptotic cells. Notably, the IC₅₀ value of Momordica charantia derived AgNPs, which represents the concentration at which 50% of the cells are inhibited, is determined to be 25 ± 0.2. This outcome underscores the potential of Momordica charantia L. silver nanoparticles as an agent with cytotoxic properties against MCF-7 cells. The concentration-dependent response suggests that as the concentration of nanoparticles increases, the inhibitory effect on cell viability becomes more pronounced. This finding has important implications for potential applications in cancer treatment and warrants further investigation to understand the mechanisms underlying this cytotoxicity and its specific relevance in cancer therapy.

Figure 10. Momordica charantia L. silver nanoparticles treated MCF-7 breast cancer cell line.

Figure 11. Effect of Momordica charantia L. silver nanoparticles against MCF-7 cancer cell line.

AO/EB staining

The study utilised acridine orange (AO), a cell-permeable dye that can penetrate both live and dead cells. In live cells, AO emitted a strong and uniform green fluorescence, while in dead cells; it displayed green or orange fluorescence, indicating the occurrence of apoptosis or necrosis. In contrast, ethidium bromide (EB) entered cells with compromised membrane integrity, resulting in an orange or intense red fluorescence. The combined AO/EB staining method served to distinguish between normal, apoptotic, and necrotic cells. The dual AO/EB fluorescent staining assay was employed to detect morphological changes in cell membranes associated with apoptosis following treatment with chemopreventive agents. When applied to MCF-7 cells treated with the extract, this staining assay not only revealed viable cells but also demonstrated significant morphological alterations (Figure 12). These changes included cell shrinkage, chromatin condensation, and inter-nucleosomal fragmentation, indicative of the impact of the treatment on cell morphology. This analysis highlights the potential influence of the tested extract on cell structure and suggests its relevance in the context of apoptosis-related research and chemopreventive investigations.

Figure 12. Ao/etbr staining of aqueous extract of Momordica charantia L. silver nanoparticles against MCF-7 cancer cell.

DAPI STAINING (4’,6-diamidino-2-phenylindole)

According to the results from the MTT assay, an additional investigation using DAPI staining was conducted to explore the potential induction of apoptosis by Momordica charantia L. Silver nanoparticles extract. The treatment of cells with varying concentrations (10, 25, and 50 µg/ml) of the extract led to discernible nuclear morphological alterations in comparison to normal cells, suggesting the initiation of apoptosis. The MTT assay on MCF-7 cells after treatment with various concentrations of synthesised AgNPs by Momordica charantia L. is found in Table 3. Notable changes in cell morphology included cellular shrinkage, fragmentation of nuclei into distinct pieces, and the generation of cells with diverse sizes in the treated cell population. The observed morphological variations indicated a concentration-dependent occurrence of apoptosis, with higher concentrations of Momordica charantia L. silver nanoparticles extract associated with more pronounced changes (Figure 13). The data is presented as the mean ± standard deviation (SD) derived from three independent experiments within each group (Figure 14). These findings underscore the potential of the extract to induce apoptosis in a dose-dependent manner, revealing its promising role in influencing cellular behaviour.

Figure 13. DAPI staining of aqueous extract of Momordica charantia L. silver nanoparticles against MCF-7 cancer cell.

Figure 14. Cytotoxic effect of AgNPs based on Momordica charantia L. against MCF-7 cell line.

Table 3. Effects of varying concentrations of AgNPs on the MCF-7 cell line using the MTT assay.

Concentration (µg/ml)	MCF-7 Cell Line
	% Cell viability	% Cell inhibition
10	78.68 ± 0.03	21.32 ± 0.03
25	66.27 ± 0.3	33.64 ± 0.3
50	45.71 ± 0.04	54.27 ± 0.04
Control	100	0

Conclusion

This research introduces a safe and effective method for the synthesis of silver nanoparticles (AgNPs) using an aqueous extract of Momordica charantia L. Various analytical techniques, including UV-Vis, FTIR, SEM, TEM and EDAX spectroscopy, were employed to comprehensively analyse these green-synthesised nanoparticles. The FTIR results suggested that the phytoconstituents present in Momordica charantia L. were responsible for reducing silver ions and capping the AgNPs. Furthermore, EDAX analysis confirmed the crystalline nature of the AgNPs, with silver being the dominant element at 3 KeV. The AgNPs demonstrated significant antibacterial and antioxidant properties, with the highest efficacy recorded at the highest concentration of 100 µg/L. They exhibited substantial antimicrobial activity against both gram-positive and gram-negative bacteria. Notably, these green-synthesised AgNPs displayed a potent inhibitory effect on the growth of breast cancer MCF-7 cells, indicating their potential as a promising candidate for further research in antibacterial, antioxidant, and in vitro cytotoxic studies on MCF-7 cell lines. The study also observed that higher concentrations of copper nanoparticles from Momordica charantia L. induced more significant morphological alterations, suggesting concentration-dependent apoptosis. This study underscores the environmentally friendly aspect of AgNPs production using an aqueous extract of Momordica charantia L., emphasising its potential as a life-saving agent in anticancer therapies. Further investigations are warranted to explore the potential applications of AgNPs in the treatment of metastatic breast cancer.

Acknowledgments

Authors are thankful to the Researchers Supporting Project number (RSPD2024R728), King Saud University, Riyadh, Saudi Arabia.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.