Silver nanoparticles green-synthesized ethanol–water extract of Cleistocalyx operculatus buds and their antimicrobial activities

In the current study, using Cleistocalyx operculatus bud extracts to synthesize silver nanoparticles is considered a novel, eco-friendly, and low-priced process that advances chemical and physical methods. C. operculatus bud extracts played a dual part in reducing and stabilizing silver nanoparticles. The formation of silver nanoparticles was recognized on UV–Vis spectra at an absorption maximum of 432 nm. Transmission electron microscopy images detected the quasi-spherical nanoparticle shape with an average size of 26.2 nm corresponding with water extract and 27.5 nm synthesized by ethanol–water extract. The obtained silver nanoparticles had moderate stability with zeta potential ranging from − 15.4 to − 38.4 mV. The phase purity of the bio-synthesized materials was confirmed by an X-ray diffraction pattern. The Fourier transform infrared analysis demonstrated hydroxyl groups acting as stabilizing agents in the extract. Silver nanoparticles synthesized from ethanol–water extract exhibited higher antibacterial and anticancer activity than nanomaterials prepared with water extract. Positive-gram bacteria (Bacillus subtilis, Lactobacillus fermentum, and Staphylococcus aureus, negative-gram bacteria (Escherichia coli, Pseudomonas aeruginosa, and Salmonella enterica), and cancer cell lines (lung cancer A549, hepatic cancer Hep-G2, epidermal carcinoma KB, and breast cancer MCF-7) were both inhibited by the nanoparticles synthesized with ethanol–water extract.


Introduction
Nowadays, nanoscience is a speedily evolving field that has contributed to producing a wide range of various synthesized metal nanoparticles.Metal nanoparticles are widely applied in medicine, environmental remediation, and drug delivery.For instance, gold nanoparticles were recognized as safe nanocarriers widely used in drug delivery (Yang et al. 2022); palladium nanoparticles were not only applied in catalysis to replace platinum nanoparticles but also were materials to inhibit bacterial/cancer cells (Joudeh et al. 2022).In comparison, copper nanoparticles were used as pesticides and fertilizers in agricultural activities (Hemmati et al. 2020;Bakshi et al. 2021).Due to bactericidal and fungicidal activity, silver has been widely researched for synthesizing at the nanoscale and controlling microbial proliferation.Generally, physical and chemical approaches were used to produce silver nanoparticles (AgNPs).Pure phase and simple operation are considered outstanding advantages of these methods.However, the high cost, energy consumption, and hazardous effects on the environment are considered the primary disadvantages (Ijaz et al. 2020).Recently, the biogenic synthesized AgNPs have become essential for nanoparticle formation with a simple, rapid, and eco-friendly route.Plant-based AgNPs were formed with phytochemicals in the extracts, including pectin, vitamins, phenolics, and flavonoids, as reductants and stabilizers.Nagar recognized that biomolecules such as terpenoids and flavanones were responsible for capping and stabilizing AgNPs.In an aqueous medium, the AgNPs synthesized at 30 °C exhibited high degradation activity for acid orange 10 and acid orange 52 by peroxomonosulphate (Nagar et al. 2019b).Tyagi used Tagetes erecta leaf-enriched ascorbic acid and polyphenol compounds to produce AgNPs under sunlight irradiation for 30 min (Tyagi et al. 2021).Pradeep reported that AgNPs biosynthesis using Hypericum perforatum containing phenolic acids and flavonoids acted as efficient silver-reducing and capping agents after 5 days of reaction (Pradeep et al. 2022).Similarly, phenolics and flavonoids in the Linacanthus nutans leaf and stem were suggested as the main phytochemicals responsible for reducing Ag + ions.Nevertheless, the presence of flavonoids and pectin in Citrus maxima peel extract and sunlight assisted in forming and stabilizing AgNPs (Nguyen 2020).Consequently, using plant extracts for reducing Ag + provided an environmentally low-priced technology for high-scale production.
Bacteria are considered a significant threat facing medical remediation since the beginning of antibiotic-resistant bacterial due to their evolution.Drug resistance began as a complicated problem caused by the over-treatment of antibiotics and drugs in curing infectious diseases (Holmes et al. 2016).Therefore, developing innovative drugs for treating microbial pathogens is an urgent solution.The freshly evolving nanotechnology accelerated the production of metal nanoparticles, especially AgNPs, with high antibacterial activity and nonhazardous to humans at low concentrations (Xu et al. 2021).When used in low dosages, the non-toxicity of the bio-synthesized AgNPs grows to be an interesting issue for medical biotechnology.AgNPs may be a substitute for antibiotic drugs presenting better impacts on multidrugresistant bacteria.Due to the high surface volume ratio and binding activity, nanoparticles were used as an anticancer drug (Morais et al. 2020).Banana leaf-mediated AgNPs synthesis has high potential in vitro cytotoxicity investigation in malignant cancerous cells such as the lung cancer cell line A549 and breast cancer cell line MCF-7 (Raghavendra et al. 2021).AgNPs synthesized by Mangifera indica seed extract get a high cytotoxic effect against cervical cancer cell line HeLa and breast cancer cell line MCF-7; less cytotoxic effect on regular fibroblast cell line (Donga et al. 2021).Green synthesized AgNPs using Heliotropium bacciferum extract exhibit 50% cell inhibition of breast cancer cell line MCF-7 at 5.44 µg/mL AgNPs and colorectal carcinoma cell line HCT-116 at 9.54 µg/mL AgNPs (Khan et al. 2021).Comparably, AgNPs formed by Zingiber officinale leaf also showed effective anticancer against pancreatic cancer cell lines (PANC-1, AsPC-1, and MIA PaCa-2) without cytotoxicity activity against normal cell line HUVEC (Wang et al. 2021).
Cleistocalyx operculatus (C.operculatus), an edible plant, is typically distributed in Southeast Asia and contains high amounts of phenolic compounds.The primary phytochemical leaf and bud extract of C. operculatus were terpenoids, flavonoids, and phloroglucinols.C. operculatus leaves and flower buds were utilized for inflammatory conditions, such as bruises, acne, and skin ulcers (Pham et al. 2020).Although C. operculatus extract contained possible reducing chemicals, the use of C. operculatus extract in the AgNPs synthesis has rarely been reported.For example, C. operculatus leaf extract was also employed as a reducing agent for synthesizing AgNPs decorated on graphene surfaces (Thi et al. 2022) and reducing Ag + ions to AgNPs on the TiO 2 surface (Nguyen et al. 2022).The presence of phytochemical compounds (carbohydrates, phenolic acids, flavonoids, amino acids, and proteins) in the extract affects the formation of AgNPs (Mittal et al. 2015;Patil et al. 2017).Several factors directly affect extraction, such as extraction time, extraction temperature, and solvent.Among the organic solvents commonly used to extract different parts of plants (leaves, flowers, seeds, and peels), ethanol is considered more common and less toxic than methanol, acetone, and hexane.Also, the phytochemicals present in the plant were better soluble in ethanol than in water (Yoswathana et al. 2013;Mokrani et al. 2016;Jeyaraj et al. 2021;Romano et al. 2022).
This work concentrated on the plant extract-synthesized AgNPs from C. operculatus buds as a reducing and stabilizing agent.The influence of ethanol-water solvent used as extraction solvent on the AgNPs formation was also evaluated.Furthermore, the antimicrobial activities of synthesized AgNPs using water extract and ethanol-water extract were compared on positive-gram bacteria (Bacillus subtilis, Lactobacillus fermentum, and Staphylococcus aureus, negativegram bacteria (Escherichia coli, Pseudomonas aeruginosa, and Salmonella enterica), and cancer cell lines (lung cancer A549, hepatic cancer Hep-G2, epidermal carcinoma KB, and breast cancer MCF-7).

Materials
The precursor for the AgNPs formation was silver nitrate (AgNO 3 , Merck), while double-distilled water/ethanol-water was considered a solvent for extracting.Cleistocalyx operculatus buds (C.operculatus) were collected from a local grocery store.The extract was prepared using C. operculatus buds extracted in 100 mL double-distilled water/ethanol-water at T °C for t min.The heterogeneous solutions were refrigerated to room temperature, purified with Whatman filter paper, and centrifuged at 6800 rpm for 60 min to eliminate unsolvable solids.The resulting filtrate was stored in an amber-colored glass container in the dark, labeled as Ext-W (extraction in water) and Ext-E (extraction in ethanol-water), and stored at 5 °C for the AgNPs synthesis.The obtained extract played both reductants and stabilizers for AgNPs synthesis.

Extract of C. operculatus buds assisted in the AgNPs formation
The extract of C. operculatus buds was used as a green, reducing, and stabilizing agent to synthesize AgNPs.Therefore, parameters, including the concentration of C. operculatus buds, extraction temperature, and time that affected the phytochemicals of the extract, were studied in detail.Silver nitrate (0.0085 g) was soluted into 50 mL of the extract to maintain an Ag + concentration of 1 mM.The synthesized solutions were prepared in an amber-colored glass container to avoid reducing Ag + ions by light irradiation and continuously stirred at room temperature.The pale yellow solutions were converted from yellow to red-orange with a gradually increasing reaction time, indicating that AgNPs formed (Soto et al. 2019).Investigating the effect of numerous parameters on the AgNPs formation, several experiments were carried out with varying concentrations of C. operculatus buds (2.5, 5.0, 7.5, 10.0, and 12.5 g/L), extraction temperatures (30, 40, 50, 60, and 70 °C), and extraction time (10, 20, 30, 40, and 50 min).AgNPs samples using water extract were denoted as AgNPs-W, while AgNPs-E samples were obtained from the ethanol-water extract.

Measurements
UV-visible absorption spectra (UV-vis) of the bio-synthesized AgNPs were recorded on a Jasco model V730 spectrophotometer in the 300-700 nm range.X-Ray diffraction (XRD) was analyzed on a Bruker D2 Phaser instrument with CuK α excitation (λ = 1.5418Å) operating at 40 kV and 40 mA.Zeta potential measurements of the synthesized solutions were carried out using a Zetasizer SZ-100 (Horiba, Japan) at 25 °C.Transmission electron microscopy (TEM) images of the prepared AgNPs were obtained using a JEOL model JEM-1400 instrument operating at 120 kV.The particle-size distribution of the produced AgNPs was measured by ImageJ software of TEM images and estimated Origin 9.0 with the Gauss method.Nicolet 6700 Fourier transform infrared (FTIR) instrument was used to verify the presence of functional groups stabilized by the as-prepared AgNPs.The efficiency synthesis was determined by converted Ag + concentration using the inductively coupled plasma optical emission spectroscopy (ICP-OES).

Analysis of phenolic components in the extract
The presence of phenolic in the C. operculatus bud extracts was quantified by the Folin-Ciocalteu method with alteration (Chandra et al. 2014).The samples containing 250 µL of extract, 250 µL of water, and 250 µL of Folin-Ciocalteu phenol reagent were prepared, respectively.After stirring and maintaining room temperature for 5 min, 250 µL of 10% w/w Na 2 CO 3 solution was introduced, followed by heating up at 50 °C for 20 min.The absorbance of the analyzed sample was determined on UV-vis spectra at a wavelength of 765 nm.The water-soluble and ethanol-water-soluble phenolic content was shown in mg gallic acid equivalents (GAE) per g of C. operculatus buds.Each test was accomplished thrice for repetitions; Excel software calculated means and standard deviations.

Antibacterial activity
The reference bacterial strains used for the antibacterial assay were positive grams: Bacillus subtilis ATCC 6633 (B.subtilis), Lactobacillus fermentum N4 (L.fermentum), Staphylococcus aureus ATCC 13709 (S. aureus) and negative grams: Escherichia coli ATCC 25922 (E.coli), Pseudomonas aeruginosa ATCC 15442 (P.aeruginosa), and Salmonella enterica ATCC 35664 (S. enterica) using the well-diffusion method.Tryptic soy broth and tryptic soy agar were employed for bacteria.The pathogenic cultures were subcultured and incubated at 37 °C for 24 h to attain 5 × 10 5 CFU/mL using MacFarland's standard.Ampicillin and cefotaxime were used to control positive-gram and negative-gram bacteria, respectively.96-well microplates evaluated the 50% inhibition concentration IC 50 of the synthesized AgNPs and the absorbance was quantified at 630 nm.

Antiproliferative activity on cancer cell lines
Various categories of cancer cell lines, such as lung cancer (A549), hepatic cancer (Hep-G2), epidermal carcinoma (KB), and breast cancer (MCF-7), were assessed for the proliferative effect of the synthesized AgNPs.Dulbecco's modified Eagle's medium (DMEM), complemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, was accustomed to subculture the cell lines in an incubator with 5% CO 2 .Cells were stained with trypan blue and living cells were counted on a hemocytometer.The density of cancer cells was calibrated to 3 × 10 4 CFU/well and the suspension was seeded into a 96-well plate for 72 h.
A549, Hep-G2, KB, and MCF-7 cell lines were tested with varied concentrations: 0.12, 0.47, 1.88, and 7.50 pM for bio-synthesized AgNPs-W while concentrations of AgNPs-EW were 0.15, 0.59, 2.38, and 9.50 pM.Dimethyl sulfoxide (DMSO) was used as the solvent to dilute the concentrations.After treatment, the cells were incubated for 72 h with 5% CO 2 .After that, 100 mg/mL of the MTT reagent was added, and the mixture was incubated at 37 °C for 4 h.The generated formazan was solubilized by adding 100 μL of DMSO.In this experiment, Ellipticine was used as a control sample.The absorbance was quantitatively measured at 540 nm on a 96-well plate.
The percentage of the bacteria/cell growth inhibition was calculated as follows (Kamiloglu et al. 2020) where I is the percentage of cell growth inhibition.A sample, A blank , and A control are the absorbance of the tested, blank, and control samples, respectively.
Each test in the present investigation was performed thrice for repetitions.Means and standard deviations were calculated using GraphPad Prism software.

Synthesizing AgNPs with water extract of C. operculatus buds (AgNPs-W)
To investigate the reducing/stabilizing role of C. operculatus buds extract, several samples such as 1 mM AgNO 3 solution (Ag), water extract of C. operculatus buds (Ext), and AgNO 3 dissolved with water extract of C. operculatus buds (Ag-Ext) were prepared.After stirring at ambient temperature for 24 h, all as-prepared samples were recorded from 300 to 700 nm on a UV-vis spectrophotometer.The AgNPs biofabrication could be determined by a chromatic transformation of the reaction substrate since the color conversion from yellowish to brown-orange indicated a reduction of Ag + ion to Ag° (Fig. 1).The formation of AgNPs surface plasmon at 432 nm with an intensity of 0.299 on Ag-Ext.These results agreed well with the results in the literature that indicated the characteristic peak at 434 nm belongs to the AgNPs formation (Erdogan et al. 2019).Adsorption peak was absent in the case of C. operculatus buds extract (Ext) and AgNO 3 solution (Ag).The bioreduction of AgNPs involved (1) phytochemicals such as alkaloids, phenolics, terpenoids, flavonoids, amino acids, and alcohol (Shaikh et al. 2021).Therefore, the current result may be interpreted based on the reducing effect of phenolics, quantified to 2.43 ± 0.12 mg GAE/g for the 7.5 g/L of C. operculatus bud water extract.
The concentration of C. operculatus bud extract is a vital parameter affecting the AgNPs formation.To optimize a suitable concentration of C. operculatus bud extract, the influence of C. operculatus bud extract on the AgNPs synthesis was studied in a range of 2.5-12.5 g/L.The UV-vis spectra of the obtained with several C. operculatus bud concentrations are illustrated in Fig. 2a.The absorbance intensity enhanced and shifted to a lower wavelength as C. operculatus buds increased from 2.5 to 12.5 g/L.The lack of reducing/stabilizing agents was at low extract concentrations, leading to little AgNPs formation and aggregation.In contrast, more AgNPs were formed in the presence of numerous reductants, and the formed AgNPs were also well stabilized by stabilizers at high extract concentrations (Mishra et al. 2020).Therefore, the low absorbance intensity of AgNPs peak was observed at lower than 5.0 g/L of C. operculatus bud extract.In comparison, the high values reached the range concentrations of C. operculatus bud extract from 7.5 to 12.5 g/L.However, there was a slight rise in intensity when the concentration of C. operculatus buds shifted from 10 to 12.5 g/L.Regarding the particle growth, it can be seen that the nano-size of the synthesized materials gradually increased in the presence of more concentration of C. operculatus buds (2.5-12.5 g/L).Similar results are reported by Nagar et al. using Azadirachta indica leaf extract for synthesizing AgNPs: a weak absorption peak was obtained at a low percentage (5%) due to the insufficient reducing agents for transferring Ag + ions.As the percentage of leaf increased up to 10%, the intensity of the absorption peak and size nanoparticles also increased owing to more reductants generated in the extract and the AgNPs agglomeration began to occur (Nagar et al. 2019a).Based on the results in Fig. 2a, 10 g/L of C. operculatus buds was selected as an optimal value in additional tests.
Figure 2b shows the UV-vis spectra of the obtained AgNPs at various extraction temperatures (30-70 °C) for 30 min.At the same time, the concentration of the C. operculatus bud extract was kept constant at 10 g/L and reaction mixtures were conducted at ambient temperature for 24 h.It was observed that 60-70 °C was a beneficial temperature for preparing extract with an AgNPs absorbance of 0.470 compared to different temperatures.Generally, the solubility of the total phenolics increased as the extraction temperature increased, resulting in more phenolic contents being extracted.Therefore, numerous reductants and stabilizers were produced at 60-70 °C, leading to high absorbance for samples synthesized with C. operculatus bud extracted.
The results were similar to those reported in the literature, which showed that higher than 50 °C was enough temperature to extract total phenolic contents (Liaudanskas et al. 2017;Zhong et al. 2019).A similar trend with the effect of C. operculatus bud concentration observed at numerous extraction temperatures is that the particle size increased with increasing extraction temperature.At the extraction temperature of 70 °C, agglomeration started to occur in the prepared solution because of more phytochemicals, leading to an insignificant difference in the AgNPs absorbance between the samples prepared at 60 and 70 °C.Thus, the suitable temperature for extracting C. operculatus bud to synthesize AgNPs was 60 °C in the present study.
Another factor affecting the extraction efficiency of phenolics was the extraction time, so the extraction duration's influence on the AgNPs formation was also studied in the range of 10-50 min.A considerable increase in the absorbance of the bio-synthesized samples was observed by prolonging the extraction duration in a range of 10-50 min (Fig. 2c).It means increasing the extraction time from 10 to 50 min resulted in forming more phytochemicals to produce a higher quantity of AgNPs.This trend was similar to a study by Vishwasrao on the Kalipatti sapota fruit for synthesizing AgNPs (Vishwasrao et al. 2019).However, the particle size was overgrown with high phenolics resulting in large particles and an insignificant increase in the absorbance of bio-synthesized AgNPs for 40 min compared to 50 min.To create AgNPs efficiently, the appropriate duration selected for extracting was 40 min in the present work.Based on the obtained results from Fig. 2, it can be concluded that the optimal parameters were carefully chosen for synthesizing AgNPs-W as 10.0 g/L of C. operculatus buds extracted at 60 °C for 40 min.

Synthesizing AgNPs with ethanol-water extract of C. operculatus buds (AgNPs-E)
López-Miranda extracted Sargassum in three solvents: water, ethanol, and ethanol-water for synthesizing AgNPs.The obtained indicated that ethanol-water was more favorable for the AgNPs formation than water and the nonpresence of AgNPs used ethanol extract.According to López-Miranda, nanoparticles obtained from the ethanol-water extract had a three-time higher concentration than the water extract due to a higher content of reducing and stabilizing agents (López-Miranda et al. 2021).In another study by Rahman, the investigation of AgNPs synthesis from ethanol-water extracts using Phyllanthus niruri showed that the presence of ethanol increased the solubility of phytochemicals in the extract.However, a large amount of ethanol was disadvantageous for forming AgNPs (Rahman et al. 2022).The AgNPs-E formation dependence on ethanol-water extracts was demonstrated in Fig. 3a.There was a similarity to the observations of López-Miranda and Rahman with this present work.The presence of ethanol produced more phenolics in the extract (Fig. 3b).When the phenolic content exceeded, it was unfavorable for forming AgNPs due to the competition of the complexation process between Ag + ions and phenolics.The highest AgNPs concentration at 15% ethanol-water (v/v) corresponded to 2.34 mg GAE/g, the AgNPs concentration decreased at 20% ethanol-water in the presence of phenolics of 3.15 mg EAG/g, and the absence of AgNPs was recognized on a sample containing 100% ethanol-water (4.62 mg EAG/g of phenolics).The obtained results proposed that 15% ethanol-water was a suitable solvent for AgNPs formation from C. operculatus bud.The AgNPs formed from 1 mM AgNO 3 and 10.0 g/L of C. operculatus bud 15% ethanol-water were denoted as AgNPs-E.
The AgNPs formed by reductants in the extract of C. operculatus buds are illustrated in Fig. 4. According to Fig. 4 Mechanism for reducing Ag + ions to AgNPs using extract of C. operculatus buds Bawazeer et al., hydroxyl groups in phytochemicals reduced Ag + ions to Ag° and stabilized the formed Ag° (Bawazeer et al. 2021).Quercetin was characterized as a typical substance for the main phytochemical components of C. operculatus buds.Firstly, two equivalents of Ag + displaced H + in ortho-hydroquinone moiety to create a silver complex.The silver complex generates Ag° and releases free orthoquinone moiety in the following step.Finally, these produced quinones are responsible for stabilizing the formed Ag° (Shujahadeen et al. 2019).
The synthesis efficiency of AgNPs using water and ethanol-water extract was determined at the optimal conditions following formula (2) and the calculated values were presented in Table 1.The green-synthesized AgNPs-W and AgNPs-E were centrifuged at 6800 rpm for 6 h to remove AgNPs.The obtained solutions were used to determine the concentration of Ag + ions unconverted to AgNPs by ICP-OES.The synthesis efficiency for AgNPs-W was 59.5% and 75.6% was obtained from AgNPs-E green synthesis.From Table 1, more reducing agents are produced for reducing Ag + ions to AgNPs in the ethanol-water extract, improving the synthesis efficiency contrasted with water extract.In the synthesis reactions of silver nanoparticles using plant extracts, the presence of reducing agents is always more abundant than that of Ag + ions because the excess of reductants will stabilize the forming nanoparticles, enhancing the stability and reducing agglomeration of the nanoparticles (Raja et al. 2017;Jha et al. 2018).The presence of residue reductants on the surface of AgNPs in the present work is recognized from the FTIR spectra and shown in Fig. 5b.Therefore, the yield of AgNPs green synthesis was calculated according to the amount of Ag + ions as the following formula where H is the efficient synthesis of AgNPs; C 0 and C are Ag + concentrations in the precursor and the bio-synthesized AgNPs solution, respectively.

Characteristics of AgNPs-E/AgNPs-W and their antibacterial/anticancer activity
XRD patterns studied the structures of AgNPs-W and AgNPs-E.The XRD pattern with 2θ values extending in a range of 10°-80° symbolized four peaks involving diffraction from the ( 111), ( 200), ( 220), and ( 311) planes (Fig. 5a).Diffraction of the (111) plane on samples: AgNPs-W and AgNPs-E was also greatest.The peaks were indexed based on silver's face-centered cubic (FCC) lattice and were completely consistent with the standard data JCPDS No 04-0783.The average crystallite size was assessed from the FWHM of the diffraction peak by applying the Debye-Scherrer formula where K represents Scherrer's constant (K = 0.94), D signifies crystallite size, β expressed in radians is the full width at half maximum (FWHM) of the peak, and θ denotes the diffraction peak position.The FWHM was estimated from the Gaussian function fitted with four peaks (111), ( 200), (220), and (311) by the software Origin 9.0.The average crystallite size ranged from the highest for AgNPs-W (16.4 nm) to the lowest for AgNPs-E (12.8 nm) in Table 2.
Figure 5b shows the FTIR spectra of the purified AgNPs and the extract of C. operculatus buds by different solvents.The purified nanoparticles exhibited absorption peaks at 1077, 1385, 1636, 2926, and 3447 cm −1 due to C-O-C symmetrical stretching, cyclic C-C=C, OH bending of water, C-H stretching, and O-H stretching, respectively (Elhawary et al. 2020).The peaks with slight shifts were  After AgNPs formation, there was also a decline in peak intensity compared with the extract.From the FTIR spectra, it may be inferred that the phytoconstituents in the extract were in charge of the reduction.Further, the extracts formed a layer on the surface of the obtained AgNPs that prevented agglomeration (Abbai et al. 2016).
SEM images in Fig. 6 indicated that the silver nanoparticles were spherical with particle size < 50 nm.According to Jyoti et al., the maximum absorbance was a wavelength of 410-450 nm, corresponding to the spherical nanoparticles (Jyoti et al. 2016).Furthermore, there was clustering from small-sized particles to generate larger-sized nanoparticles.TEM images detailly observed the shapes and sizes of the synthesized AgNPs in various synthetic solvents compared to SEM images.The typical TEM of water-synthesized AgNPs-W and ethanol-water-synthesized AgNPs-E are shown in Fig. 7a and b.The TEM images showed a more uniform formation of spherical on AgNPs-E than AgNPs-W.The average particle size was 27.5 nm for AgNPs-W and 26.2 nm for AgNPs-E.The particle-size distribution of the synthesized solutions is also represented in Fig. 7c and d.The particle size of the AgNPs-W sample ranged from 8 to  54 nm, while that of the AgNPs-E sample occupied sizes of 10 to 38 nm.AgNPs obtained from water and ethanol-water extracts were both negatively charged (Fig. 8).The zeta potential of AgNPs-W and AgNPs-E was − 38.4 and − 15.4 eV, respectively.The presence of a stabilizing agent was considered a determinant of a material's surface charge.For example, starch formed the surface of AgNPs charge-neutral (Abbaszadegan et al. 2015), chitosan created a positively charged surface (Dara et al. 2020), and phenolics conferred a negative charge on the composites (Jayeoye et al. 2022).The zeta potential of AgNPs-W differed from AgNPs-E due to phytochemicals in the extracts.In addition, more phenolics are participating in reducing Ag + to Ag 0 on AgNPs-E compared to AgNPs-W through the reaction efficiency (Table 1), leading to fewer phenolics as stabilizing agents for AgNPs-E than AgNPs-W.Therefore, the zeta potential of AgNPs-E lowered charge-negative than AgNPs-W.
AgNPs were highly antimicrobial against various microorganisms, including bacteria, fungi, and cells (Rathi Sre et al. 2015) (Braithwaite et al. 2014).In the present work, the as-prepared AgNPs-W and AgNPs-E were tested for inhibitory activity against human pathogenic microorganisms by measuring the half-maximal inhibitory concentration (IC 50 ).Inhibitory efficiency and IC 50 values of AgNPs-W and AgNPs-E are represented in Table 3 and Fig The results demonstrated that the AgNPs-E exhibited promising antibacterial activity against the clinical pathogens compared to AgNPs-W.
Compared with the antibacterial activity, the inhibitory activity against cancer cells of AgNPs-W and AgNPs-E was high, with the inhibitory efficiency reaching > 90%.Evaluating the antiproliferative effects of the plant-assisted AgNPs in detail, numerous concentrations of nanoparticles were introduced to cancer cell lines, such as A549, Hep-G2, KB, and MCF-7.The results showed a continuous decrease in cell growth with an accelerating concentration of AgNPs (Fig. 10).Table 3 shows that the bio-synthesized AgNPs-W were more toxic toward MCF-7 than the cells (A549, Hep-G2, and KB).In contrast, AgNPs-E exhibited significant anticancer activity against the liver cell line Hep-G2.There were numerous studies using plant extracts for synthesizing AgNPs.However, C. operculatus bud extract has not yet been investigated for forming AgNPs and the use of ethanol-water as a solvent in the AgNPs synthesis is still limited.Table 4 compared the results in the present work with the literature that used ethanol-water as a solvent for extraction.The ethanol concentration used to extract C. operculatus in the present work was much lower than in other studies.Moreover, our study has spherical particles with an average size of 28 nm, similar to the results of AgNPs, which used the ethanol-water extract of Murraya paniculata leaf, Sargassum, and Phyllanthus niruri.Predominantly, the synthesis efficiency of AgNPs (75.6%) using the ethanol-water extract of C. operculatus was determined.The synthesized AgNPs displayed inhibitory activity against some cancer cell lines such as KB, Hep-G, A549, and MCF7.

Conclusions
The synthesis of AgNPs using C. operculatus buds by a green route had several advantages, such as an economical, efficient, and eco-friendly advantage of locally available and easy-to-find raw plants.The plant-mediated AgNPs synthesis is bio-compatible for a wide-ranging selection of biomedical applications.An ethanol-water solvent is used more effectively for extracting reducing and stabilizing agents present in the extract of C. operculatus buds than an aqueous solvent.The obtained AgNPs are the negatively charged sphere in an average of 26.2 nm for AgNPs-W and 27.5 nm for AgNPs-E.The synthesis efficiency of AgNPs-E (75.6%) was higher than that of AgNPs-W (59.5%).Furthermore, the fabricated AgNPs exhibited high bacteria and antiproliferative effects on various human cancer

Fig. 2
Fig. 2 AgNPs-W formation dependence on parameters: a concentration of C. operculatus buds, b extraction temperature, and c extraction time.Experimental conditions: 1 mM AgNO 3 , C. operculatus buds of 2.5-12.5 g/L extracted at 60 °C for 30 min, and reactions occurred at ambient temperature for 24 h.Experimental conditions: 1 mM AgNO 3 , C. operculatus buds of 10.0 g/L extracted at 30-70 °C for 30 min, and reactions occurred at ambient temperature for 24 h.Experimental conditions: 1 mM AgNO 3 , C. operculatus buds of 10.0 g/L extracted at 60 °C for 10-50 min, and reactions occurred at ambient temperature for 24 h

Fig. 3
Fig.3AgNPs-E formation dependence on ethanol-water extracts (EtOH).Experimental conditions: 1 mM AgNO 3 , 10.0 g/L of C. operculatus bud ethanol-water extracted at 60 °C for 40 min, and reactions occurred at ambient temperature for 24 h

Fig. 5
Fig. 5 XRD patterns (a) and FTIR spectra (b) of the synthesized AgNPs: the water extract of C. operculatus buds (Ext-W), the ethanol-water extract of C. operculatus buds (Ext-E)

Fig. 9
Fig. 9 Antibacterial activity of the green-synthesized AgNPs-W (a) and AgNPs-E (b) at the numerous AgNPs concentrations

Table 1
Ag + concentration in the precursor and bio-synthesized solution

Table 2
Phase composition and crystallite size of the bio-synthesized AgNPs-W and AgNPs-E

Table 3
IC 50 of the greensynthesized AgNPs on bacteria and cancer cells *Positive-gram bacteria: Ampicillin, negative-gram bacteria: Cefotaxime, and cancer cells: Ellipticine

Table 4
Evaluation of the results in the present research with the recent literature using ethanol-water extract for AgNPs synthesis