Barriers and facilitators to hospital pharmacists’ engagement in medication safety activities: a qualitative study using the theoretical domains framework Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-153 Page 140 Silver nanoparticle biogenically synthesised by Psychotria malayana Jack: Physicochemical, cytotoxic and antimicrobial characterisations Nur Afifah Mohd Zulkafly1, Deny Susanti2, Tengku Karmila Tengku Mohd. Kamil3 and Muhammad Taher1* ABSTRACT Introduction: Silver nanoparticles are targeted for antimicrobial and cytotoxic properties to combat antimicrobial resistance and chemoresistance. Green synthesis of silver nanoparticle method is widely used because it is environmental-friendly using biological substances as reducing and stabilising agents. Psychotria malayana Jack is rich with a wide range of phytochemicals that able to synthesise silver nanoparticle. Methods: The leaves of P. malayana Jack was extracted with ethanol-water solvent via ultrasound assisted extraction and the extract was analysed using liquid chromatography- mass spectrometry (LC-MS). The extract was then added to silver nitrate solution for 24 hours. The formation of AgNPs-PM was analysed using UV-visible spectrophotometry, scanning electron microscopy, zeta particle size and zeta potential analysis. The synthesised AgNPs-PM were tested for their cytotoxicity on human colorectal adenocarcinoma cells (Caco-2) and human epithelial breast adenocarcinoma cells (MCF-7) using 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) colourimetric assay. For antibacterial activity, the nanoparticles were tested on Gram-negative Escherichia coli and Pseudomonas aeruginosa and Gram-positive Bacillus subtilis and Staphylococcus aureus using disc diffusion method. Results: AgNPs-PM were successfully synthesised using P. malayana Jack extract. LC-MS analysis showed the presence of flavonoids, amino acids and heterocyclic compounds . An attempt in cytotoxic activity test showed that at concentrations between 12.5 µg/ml to 400 µg/ml of AgNPs-PM, no cytotoxic activity was observed. Whereas, in antibacterial assay, 2 mg/ml AgNPs-PM tested on the bacterial strains showed weak inhibition on their growth. Conclusion: AgNPs-PM has been successfully synthesised and characterised. However, the AgNPs-PM possess low bioactivities of cytotoxic and antibacterial activities. ARTICLE HISTORY: Received: 3 July 2023 Accepted: 25 July 2023 Published: 31 July 2023 KEYWORDS: Silver nanoparticles, P. malayana Jack, flavonoids, cytotoxic, antimicrobial HOW TO CITE THIS ARTICLE: Mohd Zulkafly N. A., Susanti D., Tengku Mohd Kamil T. K. & Taher M. (2023). Silver nanoparticle biogenically synthesised by Psychotria malayana Jack: Physicochemical characterisations, cytotoxic and antimicrobial activities. Journal of Pharmacy, 3(2)., 140-153 doi: 10.31436/jop.v3i2.244 Authors’ Affiliation: 1 Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University Malaysia, Jalan Sultan Ahmad Shah, Bandar Indera Mahkota, Pahang, Malaysia. 2 Department of Chemistry, Faculty of Science, International Islamic University Malaysia, Jalan Sultan Ahmad Shah, Bandar Indera Mahkota, Pahang, Malaysia 3Department of Pharmacy Practice, Faculty of Pharmacy, International Islamic University Malaysia, Jalan Sultan Ahmad Shah, Bandar Indera Mahkota, Pahang, Malaysia. *Corresponding author: Email address: mtaher@iium.edu.my Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-152 Page 141 Introduction In the last few decades, research on nanotechnology is being widely adapted worldwide. Nanoparticles refer to particles within the size range of 1 nm to 100 nm and they have many potential applications in various areas such as biotechnology, medicine, pharmaceutics, industries, biology and agriculture (Song & Kim, 2009; Susanti, Haris, Taher, & Khotib, 2022). In medical study, nanoparticles assist in more rapid diagnosing of diseases and more efficient disease treatments because functional molecules can be attached selectively to the metallic nanoparticles such as silver, platinum and gold, which allow the transportation of the molecules to the target site under the influence of magnetic field (Ahmed et al., 2022; Muhamad, Ab.Rahim, Wan Omar, & Nik Mohamed Kamal, 2022). In other field such as nanotechnology, metallic nanoparticles are being widely utilised due to their high reactivity and high surface area to volume ration (Muhamad et al., 2022; Susanti et al., 2022). Silver nanoparticles (AgNPs) are gaining attention among other metallic nanoparticles as they have remarkable biological and physicochemical properties due to their distinct surface plasmon resonance (Muhamad et al., 2022). This surface plasmon resonance refers to electronic oscillations of the conduction electrons on nanoparticle’s surfaces due to the interaction with the electromagnetic radiation (Eze, Tola, Nwabor, & Jayeoye, 2019). In synthesising the metallic nanoparticles such as AgNPs, a few methods have been introduced: physical, chemical and biological (Susanti et al., 2022). Physical method renders a few disadvantages which include small number of AgNPs yield, a long completion period of the whole process and the utilisation of high energy which can cause a release of excessive heat to the surroundings. With chemical method, the production yield may be high, but the production of the toxic by-products is a big downturn of this process (Muhamad et al., 2022). Due to disadvantages presented via physical and chemical methods, the synthesis of nanoparticles using biological method such as bacteria, fungi and plant mediated synthesis is now being widely adapted (Susanti et al., 2022). From the economical factor, green synthesis is cost-effective as the biological component of the biological agents which include bacteria, fungi, yeast, plant, viruses and by-products of these agents can act as the reducing agents (Alarjani, Huessien, Rasheed, & Kalaiyarasi, 2022; Susanti et al., 2022). They also function as capping agents which are crucial for the stability and biocompatibility of nanoparticles (Susanti et al., 2022). Figure 1: The leaves and fruit bunch of P. malayana Jack. The mature plant possesses dark-green leaves with a fruit bunch (Figure 1). The phytochemicals presence in the leaves extract of P. malayana Jack, or also known as “meroyan sakat” or “salung” in Malaysia can be utilised as the reducing and stabilising agents for the synthesising process of AgNPs in this study. Plants from genus Psychotria are rich with alkaloids as their major compounds such as indole, quinoline, isoquinoline, monoterpene indole, flavonoids, cyclic peptides, terpenoids and coumarins (Calixto et al., 2016). Among these phytochemicals, flavonoids are well known for its role as reducing and capping agent, as a replacement for the use of toxic chemical products (Ahmad et al., 2022). In this study, the leaves extract of P. malayana Jack was utilised to produce AgNPs. Other advantages of using green synthesis method include environmentally friendly as toxic chemicals are not being used with no application of high pressure, temperature, and energy. It is also stated that the use of plant mediated synthesis in nanoparticles production is simple as it can be produced in a single step at a room temperature, easy to manage commercial-scale processes and highly stable in storage (Alarjani et al., 2022; Susanti et al., 2022). Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-152 Page 142 Studies reported that the emergence of multidrug- resistant bacterial strains due to misuse of antibiotics has become a major concern to the human health all over the world (Murray et al., 2022; Wang, Hu, & Shao, 2017). A predictive statistical models stated that the deaths associated with antimicrobial resistance in the year 2019 was estimated to be 4.95 million deaths (Murray et al., 2022). It was proven in many previous literatures that silver ions and silver-modified inorganic materials such as AgNPs exhibit multiple antimicrobial activity. This includes their ability to interfere with the bacterial DNA activity and generation of free radicals which induce the reactive oxygen species leading to bacterial cell death (Ahmad et al., 2022). These mechanisms are different to most antibiotics which commonly work by targeting the cell wall synthesis, translation machinery and DNA replication machinery. These unique properties of AgNPs make them a comparable choice to antibiotic in the treatment of microbial infections as antibiotic’s resistance mechanisms are not applicable to them (Ahmad et al., 2022; Susanti et al., 2022). Aside from that, reports state that AgNPs also demonstrate antitumourigenic activity on tumour models (Muhamad et al., 2022). Based on the research, AgNPs which is concentration dependent can induce apoptosis or the programmed cell death in in vitro studies. Furthermore, AgNPs can also induce the alterations in the cell morphology, reduce cell viability and its metabolic activity and causing an increase in oxidative stress which lead to mitochondrial damage. This then leads to significant DNA damage and cell death. Thus, there is a potential usage of the AgNPs in cancer treatment (Zhang, Ma, Gu, Huang, & Zhang, 2020). Therefore, this study was done to investigate the antimicrobial activity and cytotoxicity of AgNPs-PM for their potential application in human use. Methodology 1. Plant sample collection and extraction Fresh leaves of P. malayana Jack, voucher specimen (PIIUM 0008-1) was collected at Kuantan, Pahang, Malaysia. The leaves were air dried at a controlled temperature drier (40 °C) for three days. The leaves were ground mechanically into fine powder using a mechanical grinder. 50g of the leaves powder were mixed with 500 ml ethanol-water (Ethanol 95%, GENE Chemicals) (80:20) in a 500 ml Erlenmeyer flask. The extraction was done via ultrasound-assisted extraction method at a temperature of 48 °C for 40 minutes using a probe sonicator (Qsonica Ultrasonic Sonicator Converter Model CL-334). The leaves extract was then filtered using filter papers (NICE Qualitative 102) and stored in a refrigerator at 4 °C (Bimakr, Ganjloo, Zarringhalami, & Ansarian, 2017). 2. Liquid Chromatography Mass Spectrometry Quadrupole of Flight (LC-MS-QTOF) Analysis Sample preparation: P. malayana Jack leaves extract was dried via rotary evaporator (IKA HB 10 basic) at a speed of 130 rpm and a temperature of 50°C. The dried extract was reconstituted with methanol to a final concentration of 10 mg/ml, then it was diluted to a concentration of 1 mg/ml with methanol. Prior to analysis via LC-MS-QTOF (6520 Agilent Techologies, SA, USA), the extract was filtered using a 0.22 µm pore of PVDF membrane size syringe filter. LC-MS method: The chromatographic separation was operated using Agilent ZORBAX Eclipse Plus C18 Rapid Resolution HT (2.1 x 100 mm) 1.8 µm with (A) 0.1% formic acid in distilled water and (B) 0.1% formic acid in acetonitrile for positive mode. The gradient elution programme was 0.00 – 18.00 min, 5 – 95% (B); 18 – 23 minutes; 95% (B); 23.0 minutes; 5% (B). It was run for 30 minutes. Prior to new injection, re-equilibration of LC condition was conducted for 2 minutes. The sample injection volume and the flow rate of mobile phase was set at 2 µl and 0.25 ml/min, respectively. The mass spectrometer was operated in positive electrospray ionisation (ESI) mode with optimum gas temperature at 325°C, gas flow at 11 L/min and nebuliser at 35 psi. Data analysis: The chromatographic profiles were analysed using Agilent Mass Hunter Qualitative Analysis B.05.00 software (Agilent Technologies, Santa Clara, CA, USA) based on the accurate mass data identified and the predicted compounds were annotated using METLIN database (Al-Abd et al., 2015). 3. Preparation of silver nitrate (𝑨𝑨𝑨𝑨𝑵𝑵𝑵𝑵𝟑𝟑) solution 1 mM and 5 mM of AgNO3 solutions were prepared by dissolving 0.0153g and 0.0764g of AgNO3 powder (EMSURE, Macedonia) into 90 ml deionised water, respectively. 4. Synthesis of silver nitrate (𝑨𝑨𝑨𝑨𝑵𝑵𝑵𝑵𝟑𝟑) solution 10 ml of P. malayana Jack leaves extract was added slowly into a 90 ml AgNO3 solution of two different concentrations (1 mM and 5 mM) under continuous stirring (100 rpm) using a magnetic stirrer to ensure the 1:9 ratio of the plant extract to AgNO3 solution. The synthesis process was continued for 24 hours. After 24 hours, the synthesised solution was centrifuged at 7500 rpm for 15 minutes at 4 °C to purify it (Supra 22K, Korea). The resulting pellet was resuspended with a small amount of deionised water and was dried in an oven at 40 °C. The resulting powder of AgNPs-PM was left at room temperature for future use. Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-152 Page 143 5. Characterization of silver nanoparticles (AgNPs-PM) The characterisation of the synthesised AgNPs-PM was carried out by Ultraviolet and Visible spectrophotometer (UV-Vis), scanning electron microscopy (SEM), zetasizer and zeta potential analysis. UV-Vis Spectroscopy analysis: The green synthesised AgNPs-PM were sampled at 1, 2, 4, 8 and 24 hours for analysis via UV-Visible double beam spectrophotometer (SHIMADZU UV-1800, Japan). Deionised water was used as a blank and reference solution. The spectrum was then recorded in the scanning range of 350 nm to 800 nm. Morphological analysis: The image of the biosynthesised AgNPs-PM, the nanoparticles were analysed under Zeiss EVO-50X Scanning Electron Microscopy (SEM) instrument. Then, on a non-conduction carbon tape that functions as the stabiliser, AgNPs-PM powder was prepared by simply sprinkling 2 mg of the powder sample on the tape on the sample holder. After being fixed, SEM analysis was performed and the AgNPs-PM powder was analysed at room temperature. Zetasizer and Zeta Potential analysis: The analysis sample was an aqueous solution of biosynthesised AgNPs-PM that was placed inside a Malvern Zetasizer instrument. The instrument then identified the electrical potential of ions surrounding a particle at its border as well as ions adsorbed in the diffuse layer at 25oC that was run 12 times. 6. Cytotoxic assay Cell culture: Human colorectal adenocarcinoma cells (Caco-2) and human epithelial breast adenocarcinoma cells (MCF-7) were cultured at 37℃ in a 5% CO2 incubator (Thermo Scientific Heraeus BB15) in Eagle’s minimal essential medium, EMEM (ATCC 30-2003, Manassas, VA) supplemented with 10% fetal bovine serum, FBS (Gibco, Brazil) and 1% (v/v) penicillin- streptomycin (Nacalai Tesque, INC., Kyoto, Japan). After the cells reached confluency at 80%, trypsinisation process was applied to detach and subculture the cells. The cells were then seeded into a 96-well plate at a density of 15,000 cells per well. Cell viability assay: The evaluation of the cytotoxic activity of the synthesised AgNPs was done via 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colourimetric assay. The seeded cells were treated with various concentrations (400 µg/ml, 200 µg/ml, 100 µg/ml, 50 µg/ml, 25 µ/ml and 12.5 µg/ml) of AgNPs-PM and an anticancer drug Tamoxifen as positive control. The cells were incubated for 24 hours. After 24 hours, the cells were treated with 20 µg of MTT (5 mg/ml) and re-incubated for 30 minutes at 37℃. The formed crystals of formazan were dissolved using 200 µL of dimethyl sulfoxide, DMSO (EMPLURA, USA) and re- incubated for another 30 minutes at 37℃. Using a microplate reader, the difference in colour intensities (absorbance) was recorded at 630 nm. 7. Antimicrobial assay Two Gram positive bacteria, Bacillus subtilis and Staphylococcus aureus and two Gram negative bacteria, Escherichia coli and Pseudomonas aeruginosa were cultured in nutrient broth medium. Then, they were placed in the incubator for 18 hours at 37 ± 1℃. Disc diffusion assay was employed to screen for antimicrobial activity of AgNPs-PM. The grown microbes were sub-cultured on petri dishes and the discs were treated with 10 µl of 2 mg/ml AgNPs-PM, 10% P. malayana plant extract, 2 mg/ml AgNO3 solutions and deionised water (negative control). Amoxicillin discs were used as the positive control. Then, the petri dishes were put in a CO2 incubator (Binder) at 37oC for 24 hours. The zone of inhibition around the discs was measured in mm and was compared with the negative control. 8. Statistical study Statistical analysis was done via Statistical Package for Social Sciences (SPSS). The tests were conducted in sets of three and the data were reported as mean ± standard deviation (SD) using one-way ANOVA test. The individual correlations were obtained via Duncan’s technique. If P < 0.05, the value will be considered as significantly different (Anbumani et al., 2022). Results and Discussion 1. LC-MS-QTOF analysis of P. malayana Jack leaves extract The preliminary compound identification was performed using LC-MS-QTOF analysis by comparing the m/z spectra belonging to each compound to the mass spectra database of METLIN (Figure 2). The analysis provided that there was a total of 80 compounds detected. Information on the chemical composition of the 10 major compounds from the analysis such as name, molecular formula, m/z, mass and classification of each compound were listed in Table 1. Among the compounds analysed were categorised as phospholipid, steroid, ketone, amino acids and flavonoids (flavans, falavanols and leucoanthocyanidin) (Table 1). Although, there were some unknown compounds as the data of the compounds are not available in the METLIN database. The available literatures studying genus Psychotria reported that among chemical compounds found were indole alkaloids, cyclic peptides or cyclotides, quinoline and isoquinoline alkaloids, flavonoids, terpenoids, coumarins and tannins (Calixto et al., 2016). This is in line with the findings from LC-MS analysis of P. malayana Jack (Figure 2 and Table 1). Many literatures also reported that among the phytochemicals that were responsible for synthesis of AgNPs were flavonoids, amino acids, tannins, polyphenols, sterols, heterocyclic compounds, triterpenoids, terpenoids, alkaloid, etc. due their ability to Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-152 Page 144 act as the reducing, capping and stabilising agents (Ahmad et al., 2022; Alarjani et al., 2022; Nadaf et al., 2022; Patil & Kim, 2017). Thus, attributable to the biological activity of P. malayana Jack, it can be utilised in the synthesis of AgNPs-PM. 2. Green synthesis of silver nanoparticles, AgNPs-PM For the synthesis, 1 mM and 5 mM AgNO3 solutions were added to the plant extract and changes were observed every 1, 2, 6, 8 and 24-hours (Figure 3A-3B). The changes in colour intensity when synthesising nanoparticles with 1 mM concentration were minimal, but darkest after 24 hours incubation. When the process was done with 5 mM, the colour of the solutions turned from green to dark brown after 1 hour incubation. As cited in many literatures, the formation of dark brown solution which is due to the excitation of AgNPs’ surface plasmon resonance confirms the successful synthesis of AgNPs (Ahmad et al., 2022). The yield of the AgNPs-PM was weighed after high-speed centrifugation process. The findings provided that 1 mM yielded the lowest and 5 mM the most (Figure 3C). The development of AgNPs-PM synthesised using 5 mM AgNO3 solution was further observed using other characterisation methods. 2.1 Proposed mechanism for synthesis of AgNPs A general mechanism for the formation of AgNPs include three main phases which are activation phase, growth phase and termination phase (Makarov et al., 2014). During the activation phase, there will be reduction of metal ions and nucleation of the reduced metal atoms. Phytochemicals such as flavonoids in its keto form will convert into enol form. This will in turn release reactive hydrogen. However, due to the presence of two hydroxyl groups on the same carbon, the enol form is deemed as unstable, and thus, will convert back to its keto form. At this stage, the liberated reactive hydrogen causes the reduction of metal ions to metal atom, specifically Ag+ to Ag0, which then combine with each other forming small AgNPs. In addition to flavonoids, tannins also act as reducing agents (Ahmad et al., 2022). In the growth phase, the small adjacent AgNPs coalesce spontaneously into larger particles. This process continues until the particles assume a stable shape and size. Finally, in the termination phase, AgNPs will acquire the most favourable conformation due to the influence of the phytochemicals that function as stabilising agents (Makarov et al., 2014). Figure 2: LC-MS analysis of P. malayana Jack. The sample was reconstituted with methanol to the final concentration of 10 mg/mL, then diluted to the concentration of 1 mg/mL with methanol. The sample was then filtered using a 0.22 μm pore size syringe filter before analysis. Chromatographic separation was performed at 40°C using Agilent ZORBAX Eclipse Plus C18 Rapid Resolution HT (2.1 x 100 mm) 1.8 μm with (A) 0.1% formic acid in dH20 and (B) 0.1% formic acid in acetonitrile for positive mode. Mass spectrometer was operated in positive electrospray ionisation (ESI) mode with optimum gas temperature at 325°C, gas flow at 11 L/min and nebuliser at 35 psi, respectively. Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-152 Page 145 Table 1: 10 major compounds of P. malayana Jack analysed by LC-MS-QTOF analysis No Compound name Molecular formula m/z Mass (g/mol) Classification 1 LysoPE(0:0/18:4(6Z,9Z,12Z,15Z)) C23H40NO7P 491.286 473.252 Lipid (Phospholipid) 2 Eplerenone C24H30O6 415.2116 414.2043 Lipid (Steroid) 3 4-(o-Carboxybenzamido) glutaramic acids C13H14N2O6 295.0926 294.085 - 4 N-Cyclohexanecarbonyloentadecyl amine C22H43NO 338.3421 337.3347 Ketone 5 2-Amino-3-methyl-1-butanol C5H13NO 104.1069 103.0997 Amino acids 6 Unknown - 533.3315 532.3242 - 7 Oritin-4beta-ol C15H14O6 291.0862 290.079 Flavonoids (Flavans, Flavanols and Leucoanthocyanidin) 8 Unknown - 393.2862 375.2525 - 9 Unknown - 568.4265 567.4192 - 10 Purine C5H4N4 121.0509 120.0473 Heterocyclic aromatic organic compound Figure 3: AgNPs-PM incubated at 1, 2, 6, 8 and 24hours incubation, respectively using (A) 1 mM AgNO3 solution (B) 5 mM AgNO3 solution (C) Yield of AgNPs-PM obtained from two different molarity of AgNO3 Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-152 Page 146 3. Characterisation of silver nanoparticles, AgNPs-PM 3.1 UV-Visible spectrophotometer analysis The formation of the synthesised AgNPs-PM was confirmed via UV-Visible spectroscopy analysis by measurement of surface plasmon resonance (Patra & Baek, 2014). UV-Vis absorption spectra of P. malayana Jack leaves extract showed the hypsochromic and hyperchromic shift of the UV-Visible spectra of the leaves extract, and AgNPs-PM at different incubation time (Figure 4). In this study, the collected absorption spectra within 350 nm to 800 nm showed maximum absorption at 412.0 nm (1 hr), 416.1 nm (2 hr), 422.2 nm (6 hr), 437.2 nm (8 hr) and 449.0 nm (24 hr). It was also observed that the absorption and intensity of the peak increased as incubation time increased. The broad peak in the range of 550 nm to 650 nm were attributed to the presence of flavonoids with aromatic benzene conjugated at C-2 and C-3. Another broad band that was centred at 520 nm suggested a conjugated benzene with electron donating group such as NH and OH, also suggesting flavonoids (Ahmad et al., 2022). The data from LC-MS-QTOF analysis provided that one of the major phytoconstituents of P. malayana Jack extract was flavonoids. The observations on this analysis provided that flavonoids were the major constituent of P. malayana Jack that was responsible for the reduction and stabilisation of AgNPs- PM (Patil & Kim, 2017). The observation on the AgNPs- PM bands from this analysis provided that the hypochromic shift moved towards lower wavelength. These new maximum absorption peaks of AgNPs range from 410 nm to 450 nm represent the surface plasmon resonance of AgNPs-PM. . Figure 4: UV-Vis absorption spectra of (A) the green synthesized AgNPs-PM after incubation at 1, 2, 6, 8 and 24 hours (B) P. malayana Jack leaves extract measured between 350-800 nm. An arrow indicated a specific band for silver nanoparticles. Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-152 Page 147 3.2. Scanning electron microscopy (SEM) analysis The morphological characteristics of the synthesised AgNPs-PM such as size and shape were directly viewed using SEM via electron scanning. These characteristics were associated with the toxicity, drug and tissue targeting, drug release and the biological fate of AgNPs- PM (Patra & Baek, 2014). The SEM image indicated that the AgNPs-PM formed were mostly aggregated, which is attributed to the function of the phytochemicals of P. malayana Jack leaves extract (Figure 5). The formed AgNPs-PM were having a hexagonal cluster and the particle sizes of AgNPs-PM analysed ranged from 75 nm to 145 nm, which was magnified under 4000x. Some factors could have an influence in the formation of the size and shape of AgNPs-PM such as temperature of the reaction medium, time of the synthesising reaction, exposure to light and the storage conditions (Patra & Baek, 2014; Ye et al., 2022). Aggregation of nanoparticles may occur during the storage, in which they might shrink or grow, thus affecting their potential activities. However, the information provided by SEM about the size distribution and the true population average is limited (Patra & Baek, 2014). Figure 5: SEM analysis of AgNPs-PM. Characteristic morphology of silver nanoparticles produced in this study. 3.3 Zeta particle size and zeta potential analysis Dynamic Light Scattering (DLS) analysis provided information on the particle hydrodynamic size, zeta potential and polydispersity index (PDI) of AgNPs-PM (Suriyakala et al., 2022). In this study, the average size distribution of the synthesised AgNPs-PM as analysed via zetasizer nanomachine was 110.1 ± 64.66 nm (Table 2). Particle size and their morphology are the most important parameters in determining their properties. It had been proven in many studies that nanoparticles are more efficient in delivering drugs than microparticles due to nanoparticles having larger surface areas, thus more drug interactions can be observed (Patra & Baek, 2014). Many literatures accepted the descriptive size of nanoparticles to be between 1 nm to 100 nm (Susanti et al., 2022). In pharmaceutical field, those particles having a diameter of 10 nm to 1000 nm are also regarded as nanoparticles (Mazayen, Ghoneim, Elbatanony, Basalious, & Bendas, 2022). Zeta potential measurement can provide predictive data on the surface charge, which affects the storage stability of the colloidal dispersion (Patil & Kim, 2017; Suriyakala et al., 2022). The maximum zeta potential value of the formed AgNPs-PM (Table 2) was approximately -117 ± 15.2 mV. This high negative value indicated that there were electrostatic repulsion between the synthesised AgNPs-PM, making them stable. The negative value also indicated that there were presence of negatively charged functional groups from the P. malayana Jack leaves extract (Suriyakala et al., 2022). Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-152 Page 148 In addition, zeta potential at ± 35 mV suggested a formation of stable particles (Buszewski et al., 2018). Previous findings also stated that in order to avoid particle aggregation and ensuring its stability, either a high positive or a high negative value of zeta potential must be achieved (Patra & Baek, 2014). Table 2. Average size distribution and zeta potential of AgNPs-PM analysed using zetasizer instrument. Particle size analysis Particle size average (d.nm) 110.1 ± 64.66 PdI 0.287 Zeta potential analysis Zeta potential (mV) -117 ± 15.2 Zeta deviation 18.2 Conductivity (mS/cm) 0.551 4. Cytotoxic Assay Cancer is one of the most leading death-causing diseases and 1 out of 3 people has the possibility to get cancer (Alyami, Alyami, & Almeer, 2022; Zhang et al., 2020). Different types of cancer therapies being offered include chemotherapy, surgery, radiation, hormonal, targeted and immunotherapy. However, the therapies are challenging due to induction of enormous side effects, which include neuropathies, alopecia and gastrointestinal and skin disorders. In addition, high rate of recurrence and multi-drug resistance against common chemotherapeutic drugs are other factors that limit the therapeutic efficacy (Alyami et al., 2022; Gavas, Quazi, & Karpiński, 2021). Thus, to avoid the systemic side effects and drug resistance, many researchers are focusing on the development of nanomaterials as an alternative formulation that can specifically target tumour cells (Zhang et al., 2020). Cytotoxic assay was used to measure the ability of tested compounds in killing cell lines (Shelembe, Mahlangeni, & Moodley, 2022). In this study, the cytotoxic effect of AgNPs-PM at different concentrations of 400 µg/ml, 200 µg/ml, 100 µg/ml, 50 µg/ml, 25 µg/ml and 12.5 µg/ml was investigated against Caco-2 and MCF-7 cell lines using MTT cell viability assay. The formation of purple formazan crystals is dependent on NADPH and oxidoreductase enzymes of the cancer cells. Thus, the intensity of purple colour is directly proportional to the cell viability (Shelembe et al., 2022). The absorbance of the dissolved formazan crystals was measured via microplate reader at 570 nm. The number of viable cells is proportional to the absorbance and percentage viability (%) was calculated using the following formula: % Cell viability = Asample Auntreated x 100 (Eq. 1) Many studies from in vitro assays reported that AgNPs possess cytotoxic activity on several human cell lines, which include human peripheral blood mononuclear cells, human bronchial epithelial cells, red blood cells, liver cells (HepG2), human colorectal cells (Caco-2) and human epithelial breast cells (MCF-7) (Liao, Li, & Tjong, 2019; van der Zande et al., 2016). In this study, the percentage viability of Caco-2 and MCF- 7 cell lines was reduced after interaction against various concentrations of AgNPs-PM (Figure 6). In Caco-2 cells, after incubation with 12.5 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml and 400 µg/ml, the cell viability calculated was 89.2%, 84.6%, 82.2%, 88.6%, 80.18% and 81.675, respectively. However, previous study reported that at a concentration of 5 µg/ml of AgNPs, 50% of cell will be inhibited after exposure for 48 hours (Zein, Alghoraibi, Soukkarieh, Salman, & Alahmad, 2020). In MCF-7 cells, the cell viability calculated against the increasing concentration of AgNPs-PM was 97.5%, 96.7%, 90.2%, 95.14%, 90.97% and 89.7%, respectively. Whereas the IC50 of AgNPs against MCF-7 in a study conducted by Fard, Tafvizi, and Torbati (2018) was found to be 9.85 µg/ml. Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-152 Page 149 Figure 6: Percentage viability of AgNPs-PM on Caco-2 and MCF-7 against various concentrations ranging from 12.5 µg/ml to 400 µg/ml of AgNPs-PM However, the findings from this study showed that the cytotoxicity of AgNPs-PM on Caco-2 cells and MCF-7 were both insignificant. It was also expected that the percentage viability of the two cell lines to decrease with increasing concentration of AgNPs-PM as AgNPs exhibit concentration-dependent cell death (Alyami et al., 2022; Zhang et al., 2020). However, in this study, the trend fluctuated. In addition to that, the standard errors of this assay were high, indicating that the results were not reliable, thus, the percent viability might not reflect a true value of the overall cytotoxic assay. This less reliable result might be due to variable density of cell in each well, unsuitable incubation time of cells in MTT, the wavelength at which the optical density was measured, and the type of culture media used (Ghasemi, Turnbull, Sebastian, & Kempson, 2021). In this assay, it was also observed that the percentage inhibition of AgNPs-PM against Caco-2 cells were more prominent than MCF-7 cells. This finding might be attributed to the higher sensitivity of Caco-2 cells to AgNPs-PM than MCF-7 cells (van der Zande et al., 2016). 5. Antibacterial Assay Many pathogenic bacteria are showing resistance to various antibiotics. To address this issue, new antibiotics are necessary (Ahmad et al., 2022). AgNPs were proven to possess antimicrobial properties, making them suitable alternatives to antibiotics (Nguyen et al., 2021). This study investigated the activity of AgNPs- PM, AgNO3 solutions and P. malayana Jack leaves extract on four bacterial strains via disc diffusion method. The tested bacteria were B. subtilis, E. coli, P. aeruginosa and S. aureus. In this study, the negative control discs were treated with deionised water, and the positive control discs were treated with antibiotic amoxicillin. For the sample discs, they were treated with 10% P. malayana plant extract, 2 mg/ml AgNPs-PM and 2 mg/ml AgNO3 solutions 2 mg/ml AgNPs-PM and 2 mg/ml AgNO3 solutions both showed antimicrobial activity on the microbial growth against all the tested bacterial strains (Figure 7). At a same concentration of 2 mg/ml, AgNPs-PM exhibited good inhibition on the growth of P. aeruginosa, followed by S. aureus, B. subtilis and E. coli at 3.00 ± 0.17 mm, 2.00 ± 0.28 mm, 1.75 ± 0.14 mm and 1.25 ± 0.26 mm, respectively. The activity of 2 mg/ml AgNO3 solutions were almost comparable to AgNPs- PM, but with a larger zone of inhibition than AgNPs- PM. The zone of inhibition caused by AgNO3 solutions was 2.00 ± 0.35 mm, 2.50 ± 0.41 mm, 4.00 ± 0.00 mm and 3.50 ± 0.22 mm on P. aeruginosa, followed by S. aureus, B. subtilis and E. coli, respectively. This is in line with the previous publications stating that silver ions and silver-modified materials like AgNPs possess multiple antimicrobial activity such as induction of reactive oxygen species (Ahmad et al., 2022). Since the activity of AgNPs-PM was both greatest on P. aeruginosa and least on E. coli, it is unknown 0 20 40 60 80 100 12.5 25 50 100 200 400 Pe rc en ta ge v ia bi lit y (% ) Concentration of AgNPs (ug/ CaCO2 MCF7 Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-152 Page 150 whether AgNPs work better on Gram-negative bacteria or Gram-positive bacteria. In addition, the true mechanism of how AgNPs exhibit antimicrobial activity is still unclear (Ahmad et al., 2022). The proposed mechanisms are grouped into three main actions which are induction of oxidative stress, release of metal ion and non-oxidative mechanism. These actions can either act independently or simultaneously. This can lead to denaturation of the proteins and leaking of the cell contents (Goyal, Verma, Kharewal, Gahlaut, & Hooda, 2022). However, when the bacterial strains were tested against the 10% P. malayana plant extract, no zone of inhibition was seen and measured, indicating that the extract does not possess antimicrobial activity. From this assay, it can be concluded that the antimicrobial activity of AgNPs-PM was caused by the synthesised AgNPs solely (Nguyen et al., 2021). There is no significant difference of the antibacterial activity between the samples (AgNPs-PM and AgNO3 solutions) and Amoxicillin against B. subtilis and E. coli. However, testing on P. aeruginosa and S. aureus showed that there is a large significant difference observed between the samples (AgNPs-PM and AgNO3 solutions) and Amoxicillin. . Figure 7: Zone of inhibition (mm) of four bacterial strains against AgNPs-PM, AgNO3, P. malayana Jack extract. The test was conducted by disc diffusion method using amoxicillin disc as a positive control and deionised water as negative control. Conclusion The study was conducted by synthesising AgNPs-PM from P. malayana leaves extract via biological route which is known as green synthesis method. The major phytochemicals of P. malayana Jack as analysed by LC- MS-QTOF, namely flavonoids, amino acids and heterocyclic aromatic organic compound which were possible for the formation of AgNPs-PM as they act as the reducing and stabilising agents for the process. Characterisations were done on the synthesised AgNPs- PM to study their characteristics as the nature of the AgNPs play an important role in their application. The investigations proposed that the formed AgNPs-PM were having hexagonal cluster with the size of around 75 nm to 145 nm as viewed under electron microscope. Another analysis also confirmed that the size distribution of AgNPs-PM was 110.1 ± 66.64 nm with a zeta potential of approximately -117 ± 15.2 mV, indicating that stable AgNPs-PM were successfully synthesised using green synthesis method. Their cytotoxicity was also determined via MTT colourimetric assay and it was found out that at concentrations of 12.5 µg/ml, 25 µg/ml, 50 µg/ml¸ 100 µg/ml, 200 µg/ml and 400 µg/ml of AgNPs-PM, the cell viability of both Caco-2 and MCF-7 cell lines were over 80%. This finding indicated that the percentage inhibition was low and insignificant. However, in this study, a few confounding factors may have an effect on the result, thus, making it unreliable. AgNPs-PM were also studied for antimicrobial activity against B. subtilis, E. coli, P. aeruginosa and S. aureus using disc diffusion method, and 0 2 4 6 8 10 12 14 B. subtilis E. coli P. aeruginosa S. aureus Zo ne o f i nh ib iti on (m m ) Bacterial strains AgNPs-PM AgNO3 solution P. malayana Jack extract Amoxicillin Deionised water Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-152 Page 151 it was observed that AgNPs-PM inhibited the growth of the four bacterial strains, with the highest inhibition on P. aeruginosa and the lowest inhibited was E. coli. Thus, the biogenic synthesis of AgNPs-PM might be a promising process for the production of other metallic nanoparticles which could have potential applications in various fields. However, the improvement on this study for cytotoxic and antimicrobial assays can be made by adjusting the size and formulation of nanoparticles. Author contributions MT and DS design the study. MT, DS, TK supervise the works. NA conduct the research and collect the data. NA wrote the manuscript. MT review the manuscript. All authors have read the manuscript. Acknowledgements The research was supported by Department of Pharmaceutical Technology, Kulliyyah of Pharmacy, International Islamic University Malaysia. Conflict of Interest The authors declare that there is no conflict of interest in the writing of this manuscript. References Ahmad, N., Fozia, Jabeen, M., Haq, Z. U., Ahmad, I., Wahab, A., … Khan, M. Y. (2022). Green Fabrication of Silver Nanoparticles using Euphorbia serpens Kunth Aqueous Extract, Their Characterization, and Investigation of Its in Vitro Antioxidative, Antimicrobial, Insecticidal, and Cytotoxic Activities. BioMed Research International, 2022. https://doi.org/10.1155/2022/5562849 Ahmed, O., Sibuyi, N. R. S., Fadaka, A. O., Madiehe, M. A., Maboza, E., Meyer, M., & Geerts, G. (2022, February 1). Plant Extract-Synthesized Silver Nanoparticles for Application in Dental Therapy. Pharmaceutics, Vol. 14. MDPI. https://doi.org/10.3390/pharmaceutics14020380 Al-Abd, N. M., Mohamed Nor, Z., Mansor, M., Azhar, F., Hasan, M. S., & Kassim, M. (2015). Antioxidant, antibacterial activity, and phytochemical characterization of Melaleuca cajuputi extract. BMC Complementary and Alternative Medicine, 15(1). https://doi.org/10.1186/s12906-015-0914- y Alarjani, K. M., Hussein, D., Rasheed, R. A., & Kalaiyarasi, M. (2022). Green synthesis of silver nanoparticles by Pisum sativum L. (pea) pod against multidrug resistant foodborne pathogens. Journal of King Saud University - Science, 34(3). https://doi.org/10.1016/j.jksus.2022.101897 Alyami, N. M., Alyami, H. M., & Almeer, R. (2022). Using green biosynthesized kaempferol-coated sliver nanoparticles to inhibit cancer cells growth: an in vitro study using hepatocellular carcinoma (HepG2). Cancer Nanotechnology, 13(1). https://doi.org/10.1186/s12645-022- 00132-z Anbumani, D., Dhandapani, K. vizhi, Manoharan, J., Babujanarthanam, R., Bashir, A. K. H., Muthusamy, K., … Kanimozhi, K. (2022). Green synthesis and antimicrobial efficacy of titanium dioxide nanoparticles using Luffa acutangula leaf extract. Journal of King Saud University - Science, 34(3). https://doi.org/10.1016/j.jksus.2022.101896 Bimakr, M., Ganjloo, A., Zarringhalami, S., & Ansarian, E. (2017). Ultrasound-assisted extraction of bioactive compounds from Malva sylvestris leaves and its comparison with agitated bed extraction technique. Food Science and Biotechnology, 26(6), 1481–1490. https://doi.org/10.1007/s10068-017-0229-5 Buszewski, B., Railean-Plugaru, V., Pomastowski, P., Rafińska, K., Szultka-Mlynska, M., Golinska, P., … Dahm, H. (2018). Antimicrobial activity of biosilver nanoparticles produced by a novel Streptacidiphilus durhamensis strain. Journal of Microbiology, Immunology and Infection, 51(1), 45–54. https://doi.org/10.1016/j.jmii.2016.03.002 Calixto, N. O., Pinto, M. E. F., Ramalho, S. D., Burger, M. C. M., Bobey, A. F., Young, M. C. M., … Pinto, A. C. (2016, August 1). The genus psychotria: Phytochemistry, chemotaxonomy, ethnopharmacology and biological properties. Journal of the Brazilian Chemical Society, Vol. 27, pp. 1355–1378. Sociedade Brasileira de Quimica. https://doi.org/10.5935/0103- 5053.20160149 Eze, F. N., Tola, A. J., Nwabor, O. F., & Jayeoye, T. J. (2019). Centella asiatica phenolic extract- mediated bio-fabrication of silver nanoparticles: Characterization, reduction of industrially relevant dyes in water and antimicrobial activities against foodborne pathogens. RSC Advances, 9(65), 37957–37970. https://doi.org/10.1039/c9ra08618h Fard, S. E., Tafvizi, F., & Torbati, M. B. (2018). Silver nanoparticles biosynthesised using Centella asiatica leaf extract: apoptosis induction in MCF‐ 7 breast cancer cell line. IET Nanobiotechnology, 12(7), 994–1002. https://doi.org/10.1049/iet- nbt.2018.5069 Gavas, S., Quazi, S., & Karpiński, T. M. (2021). Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-152 Page 152 Nanoparticles for Cancer Therapy: Current Progress and Challenges. Nanoscale Research Letters, 16, 173. https://doi.org/10.1186/s11671- 021-03628-6 Ghasemi, M., Turnbull, T., Sebastian, S., & Kempson, I. (2021). The mtt assay: Utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis. International Journal of Molecular Sciences, 22(23). https://doi.org/10.3390/ijms222312827 Goyal, B., Verma, N., Kharewal, T., Gahlaut, A., & Hooda, V. (2022). Structural effects of nanoparticles on their antibacterial activity against multi-drug resistance. Inorganic and Nano-Metal Chemistry. Taylor and Francis Ltd. https://doi.org/10.1080/24701556.2021.2025103 Liao, C., Li, Y., & Tjong, S. C. (2019). Bactericidal and Cytotoxic Properties of Silver Nanoparticles. International Journal of Molecular Sciences, 20(2), 449–449. Makarov, V. V, Love, A. J., Sinitsyna, O. V, Makarova, S. S., Yaminsky, I. V, Taliansky, M. E., & Kalinina, N. O. (2014). ‘Green’ Nanotechnologies: Synthesis of Metal Nanoparticles Using Plants (Vol. 6). Mazayen, Z. M., Ghoneim, A. M., Elbatanony, R. S., Basalious, E. B., & Bendas, E. R. (2022). Pharmaceutical nanotechnology: from the bench to the market. Future Journal of Pharmaceutical Sciences, 8(1). https://doi.org/10.1186/s43094- 022-00400-0 Muhamad, M., Ab. Rahim, N., Wan Omar, W. A., & Nik Mohamed Kamal, N. N. S. (2022). Cytotoxicity and Genotoxicity of Biogenic Silver Nanoparticles in A549 and BEAS-2B Cell Lines. Bioinorganic Chemistry and Applications, 2022, 1–22. https://doi.org/10.1155/2022/8546079 Murray, C. J., Ikuta, K. S., Sharara, F., Swetschinski, L., Robles Aguilar, G., Gray, A., … Naghavi, M. (2022). Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet, 399(10325), 629–655. https://doi.org/10.1016/S0140-6736(21)02724-0 Nadaf, S. J., Jadhav, N. R., Naikwadi, H. S., Savekar, P. L., Sapkal, I. D., Kambli, M. M., & Desai, I. A. (2022, November 1). Green synthesis of gold and silver nanoparticles: Updates on research, patents, and future prospects. OpenNano, Vol. 8. Elsevier Inc. https://doi.org/10.1016/j.onano.2022.100076 Nguyen, N. H., Nhi, T. T. Y., Van Nhi, N. T., Cuc, T. T. T., Tuan, P. M., & Nguyen, D. H. (2021). Comparative Study of the Silver Nanoparticle Synthesis Ability and Antibacterial Activity of the Piper Betle L. And Piper Sarmentosum Roxb. Extracts. Journal of Nanomaterials, 2021. https://doi.org/10.1155/2021/5518389 Nipun, T. S., Khatib, A., Ahmed, Q. U., Nasir, M. H. M., Supandi, F., Taher, M., & Saiman, M. Z. (2021). Preliminary phytochemical screening, in vitro antidiabetic, antioxidant activities, and toxicity of leaf extracts of Psychotria malayana Jack. Plants, 10(12). https://doi.org/10.3390/plants10122688 Nipun, T. S., Khatib, A., Ahmed, Q. U., Redzwan, I. E., Ibrahim, Z., Khan, A. Y. F., … El-Seedi, H. R. (2020). Alpha-Glucosidase inhibitory effect of psychotria malayana Jack Leaf: A rapid analysis using infrared fingerprinting. Molecules, 25(18). https://doi.org/10.3390/molecules25184161 Patil, M. P., & Kim, G. Do. (2017, January 1). Eco- friendly approach for nanoparticles synthesis and mechanism behind antibacterial activity of silver and anticancer activity of gold nanoparticles. Applied Microbiology and Biotechnology, Vol. 101, pp. 79–92. Springer Verlag. https://doi.org/10.1007/s00253-016-8012-8 Patra, J. K., & Baek, K. H. (2014). Green Nanobiotechnology: Factors Affecting Synthesis and Characterization Techniques. Journal of Nanomaterials, Vol. 2014. Hindawi Limited. https://doi.org/10.1155/2014/417305 Shelembe, B., Mahlangeni, N., & Moodley, R. (2022). Biosynthesis and bioactivities of metal nanoparticles mediated by Helichrysum aureonitens. Journal of Analytical Science and Technology, 13(1). https://doi.org/10.1186/s40543-022-00316-7 Song, J. Y., & Kim, B. S. (2009). Rapid biological synthesis of silver nanoparticles using plant leaf extracts. Bioprocess and Biosystems Engineering, 32(1), 79–84. https://doi.org/10.1007/s00449-008-0224-6 Suriyakala, G., Sathiyaraj, S., Babujanarthanam, R., Alarjani, K. M., Hussein, D. S., Rasheed, R. A., & Kanimozhi, K. (2022). Green synthesis of gold nanoparticles using Jatropha integerrima Jacq. flower extract and their antibacterial activity. Journal of King Saud University - Science, 34(3). https://doi.org/10.1016/j.jksus.2022.101830 Susanti, D., Haris, M. S., Taher, M., & Khotib, J. (2022, June 2). Natural Products-Based Metallic Nanoparticles as Antimicrobial Agents. Frontiers in Pharmacology, Vol. 13. Frontiers Media S.A. https://doi.org/10.3389/fphar.2022.895616 van der Zande, M., Undas, A. K., Kramer, E., Monopoli, M. P., Peters, R. J., Garry, D., … Bouwmeester, H. (2016). Different responses of Caco-2 and MCF-7 cells to silver nanoparticles are based on highly similar mechanisms of action. Nanotoxicology, 10(10), 1431–1441. https://doi.org/10.1080/17435390.2016.1225132 Mohd Zulkafly et al. (2023) Journal of Pharmacy, 3(2), 140-152 Page 153 Wang, L., Hu, C., & Shao, L. (2017, February 14). The antimicrobial activity of nanoparticles: Present situation and prospects for the future. International Journal of Nanomedicine, Vol. 12, pp. 1227–1249. Dove Medical Press Ltd. https://doi.org/10.2147/IJN.S121956 Ye, L., Cao, Z., Liu, X., Cui, Z., Li, Z., Liang, Y., … Wu, S. (2022, May 25). Noble metal-based nanomaterials as antibacterial agents. Journal of Alloys and Compounds, Vol. 904. Elsevier Ltd. https://doi.org/10.1016/j.jallcom.2022.164091 Zein, R., Alghoraibi, I., Soukkarieh, Ch., Salman, A., & Alahmad, A. (n.d.). In-vitro anticancer activity against Caco-2 cell line of colloidal nano silver synthesized using aqueous extract of Eucalyptus Camaldulensis leaves. Zhang, D., Ma, X. L., Gu, Y., Huang, H., & Zhang, G. W. (2020, October 29). Green Synthesis of Metallic Nanoparticles and Their Potential Applications to Treat Cancer. Frontiers in Chemistry, Vol. 8. Frontiers Media S.A. https://doi.org/10.3389/fchem.2020.00799 Introduction In the last few decades, research on nanotechnology is being widely adapted worldwide. Nanoparticles refer to particles within the size range of 1 nm to 100 nm and they have many potential applications in various areas such as biotechnology, medicine,... Silver nanoparticles (AgNPs) are gaining attention among other metallic nanoparticles as they have remarkable biological and physicochemical properties due to their distinct surface plasmon resonance (Muhamad et al., 2022). This surface plasmon resona... Due to disadvantages presented via physical and chemical methods, the synthesis of nanoparticles using biological method such as bacteria, fungi and plant mediated synthesis is now being widely adapted (Susanti et al., 2022). From the economical facto... Figure 1: The leaves and fruit bunch of P. malayana Jack. The mature plant possesses dark-green leaves with a fruit bunch (Figure 1). The phytochemicals presence in the leaves extract of P. malayana Jack, or also known as “meroyan sakat” or “salung” in Malaysia can be utilised as the reducing and stabilising... Studies reported that the emergence of multidrug-resistant bacterial strains due to misuse of antibiotics has become a major concern to the human health all over the world (Murray et al., 2022; Wang, Hu, & Shao, 2017). A predictive statistical models ... Aside from that, reports state that AgNPs also demonstrate antitumourigenic activity on tumour models (Muhamad et al., 2022). Based on the research, AgNPs which is concentration dependent can induce apoptosis or the programmed cell death in in vitro s... Methodology 1. Plant sample collection and extraction Fresh leaves of P. malayana Jack, voucher specimen (PIIUM 0008-1) was collected at Kuantan, Pahang, Malaysia. The leaves were air dried at a controlled temperature drier (40 C) for three days. The leaves were ground mechanically into fine powder usi... 2. Liquid Chromatography Mass Spectrometry Quadrupole of Flight (LC-MS-QTOF) Analysis Sample preparation: P. malayana Jack leaves extract was dried via rotary evaporator (IKA HB 10 basic) at a speed of 130 rpm and a temperature of 50 C. The dried extract was reconstituted with methanol to a final concentration of 10 mg/ml, then it was ... 3. Preparation of silver nitrate (𝑨𝒈,𝑵𝑶-𝟑.) solution 1 mM and 5 mM of Ag,NO-3. solutions were prepared by dissolving 0.0153g and 0.0764g of Ag,NO-3. powder (EMSURE, Macedonia) into 90 ml deionised water, respectively. 4. Synthesis of silver nitrate (𝑨𝒈,𝑵𝑶-𝟑.) solution 10 ml of P. malayana Jack leaves extract was added slowly into a 90 ml Ag,NO-3. solution of two different concentrations (1 mM and 5 mM) under continuous stirring (100 rpm) using a magnetic stirrer to ensure the 1:9 ratio of the plant extract to Ag,NO... 5. Characterization of silver nanoparticles (AgNPs-PM) The characterisation of the synthesised AgNPs-PM was carried out by Ultraviolet and Visible spectrophotometer (UV-Vis), scanning electron microscopy (SEM), zetasizer and zeta potential analysis. UV-Vis Spectroscopy analysis: The green synthesised AgNPs-PM were sampled at 1, 2, 4, 8 and 24 hours for analysis via UV-Visible double beam spectrophotometer (SHIMADZU UV-1800, Japan). Deionised water was used as a blank and reference solution. The s... Morphological analysis: The image of the biosynthesised AgNPs-PM, the nanoparticles were analysed under Zeiss EVO-50X Scanning Electron Microscopy (SEM) instrument. Then, on a non-conduction carbon tape that functions as the stabiliser, AgNPs-PM powde... Zetasizer and Zeta Potential analysis: The analysis sample was an aqueous solution of biosynthesised AgNPs-PM that was placed inside a Malvern Zetasizer instrument. The instrument then identified the electrical potential of ions surrounding a particle... 6. Cytotoxic assay Cell culture: Human colorectal adenocarcinoma cells (Caco-2) and human epithelial breast adenocarcinoma cells (MCF-7) were cultured at 37℃ in a 5% ,CO-2. incubator (Thermo Scientific Heraeus BB15) in Eagle’s minimal essential medium, EMEM (ATCC 30-200... 7. Antimicrobial assay Two Gram positive bacteria, Bacillus subtilis and Staphylococcus aureus and two Gram negative bacteria, Escherichia coli and Pseudomonas aeruginosa were cultured in nutrient broth medium. Then, they were placed in the incubator for 18 hours at 37 ± 1℃... 8. Statistical study Statistical analysis was done via Statistical Package for Social Sciences (SPSS). The tests were conducted in sets of three and the data were reported as mean ± standard deviation (SD) using one-way ANOVA test. The individual correlations were obtaine... Results and Discussion 1. LC-MS-QTOF analysis of P. malayana Jack leaves extract The preliminary compound identification was performed using LC-MS-QTOF analysis by comparing the m/z spectra belonging to each compound to the mass spectra database of METLIN (Figure 2). The analysis provided that there was a total of 80 compounds det... The available literatures studying genus Psychotria reported that among chemical compounds found were indole alkaloids, cyclic peptides or cyclotides, quinoline and isoquinoline alkaloids, flavonoids, terpenoids, coumarins and tannins (Calixto et al.,... 2. Green synthesis of silver nanoparticles, AgNPs-PM For the synthesis, 1 mM and 5 mM Ag,NO-3. solutions were added to the plant extract and changes were observed every 1, 2, 6, 8 and 24-hours (Figure 3A-3B). The changes in colour intensity when synthesising nanoparticles with 1 mM concentration were mi... 2.1 Proposed mechanism for synthesis of AgNPs A general mechanism for the formation of AgNPs include three main phases which are activation phase, growth phase and termination phase (Makarov et al., 2014). During the activation phase, there will be reduction of metal ions and nucleation of the re... Figure 2: LC-MS analysis of P. malayana Jack. The sample was reconstituted with methanol to the final concentration of 10 mg/mL, then diluted to the concentration of 1 mg/mL with methanol. The sample was then filtered using a 0.22 μm pore size syringe... Table 1: 10 major compounds of P. malayana Jack analysed by LC-MS-QTOF analysis Figure 3: AgNPs-PM incubated at 1, 2, 6, 8 and 24hours incubation, respectively using (A) 1 mM AgNO3 solution (B) 5 mM AgNO3 solution (C) Yield of AgNPs-PM obtained from two different molarity of AgNO3 3. Characterisation of silver nanoparticles, AgNPs-PM 3.1 UV-Visible spectrophotometer analysis The formation of the synthesised AgNPs-PM was confirmed via UV-Visible spectroscopy analysis by measurement of surface plasmon resonance (Patra & Baek, 2014). UV-Vis absorption spectra of P. malayana Jack leaves extract showed the hypsochromic and hyp... . Figure 4: UV-Vis absorption spectra of (A) the green synthesized AgNPs-PM after incubation at 1, 2, 6, 8 and 24 hours (B) P. malayana Jack leaves extract measured between 350-800 nm. An arrow indicated a specific band for silver nanoparticles. 3.2. Scanning electron microscopy (SEM) analysis The morphological characteristics of the synthesised AgNPs-PM such as size and shape were directly viewed using SEM via electron scanning. These characteristics were associated with the toxicity, drug and tissue targeting, drug release and the biologi... 3.3 Zeta particle size and zeta potential analysis Dynamic Light Scattering (DLS) analysis provided information on the particle hydrodynamic size, zeta potential and polydispersity index (PDI) of AgNPs-PM (Suriyakala et al., 2022). In this study, the average size distribution of the synthesised AgNPs-... Zeta potential measurement can provide predictive data on the surface charge, which affects the storage stability of the colloidal dispersion (Patil & Kim, 2017; Suriyakala et al., 2022). The maximum zeta potential value of the formed AgNPs-PM (Table ... Table 2. Average size distribution and zeta potential of AgNPs-PM analysed using zetasizer instrument. Many studies from in vitro assays reported that AgNPs possess cytotoxic activity on several human cell lines, which include human peripheral blood mononuclear cells, human bronchial epithelial cells, red blood cells, liver cells (HepG2), human colorec... However, the findings from this study showed that the cytotoxicity of AgNPs-PM on Caco-2 cells and MCF-7 were both insignificant. It was also expected that the percentage viability of the two cell lines to decrease with increasing concentration of AgN... 5. Antibacterial Assay Many pathogenic bacteria are showing resistance to various antibiotics. To address this issue, new antibiotics are necessary (Ahmad et al., 2022). AgNPs were proven to possess antimicrobial properties, making them suitable alternatives to antibiotics ... 2 mg/ml AgNPs-PM and 2 mg/ml Ag,NO-3. solutions both showed antimicrobial activity on the microbial growth against all the tested bacterial strains (Figure 7). At a same concentration of 2 mg/ml, AgNPs-PM exhibited good inhibition on the growth of P. ... Since the activity of AgNPs-PM was both greatest on P. aeruginosa and least on E. coli, it is unknown whether AgNPs work better on Gram-negative bacteria or Gram-positive bacteria. In addition, the true mechanism of how AgNPs exhibit antimicrobial act... . Figure 7: Zone of inhibition (mm) of four bacterial strains against AgNPs-PM, AgNO3, P. malayana Jack extract. The test was conducted by disc diffusion method using amoxicillin disc as a positive control and deionised water as negative control. Conclusion The study was conducted by synthesising AgNPs-PM from P. malayana leaves extract via biological route which is known as green synthesis method. The major phytochemicals of P. malayana Jack as analysed by LC-MS-QTOF, namely flavonoids, amino acids and ... Author contributions MT and DS design the study. MT, DS, TK supervise the works. NA conduct the research and collect the data. NA wrote the manuscript. MT review the manuscript. All authors have read the manuscript. Acknowledgements The research was supported by Department of Pharmaceutical Technology, Kulliyyah of Pharmacy, International Islamic University Malaysia. Conflict of Interest The authors declare that there is no conflict of interest in the writing of this manuscript. References