Biogenic Metal Transformations from Plectranthus amboinicus: Complexes, Nanoparticles, and Therapeutic Applications

Introduction

Copper, zinc, and manganese are essential trace elements with diverse biological roles, including enzymatic activity, antioxidant functions, and maintenance of bone and skin health.^1–3 Metal complexes and nanoparticles of these elements have garnered significant interest for their therapeutic applications, including their anti-inflammatory and antibacterial properties^4–6 for applications in biosensors and drug delivery systems.^7–9 The biological activity of metal-based materials often surpasses that of their individual components, making them promising candidates for medical applications.^10,11

Flavonoids are widely distributed in medicinal plants and are potent antioxidants with remarkable metal-binding capacity. Their proficiency in coordinating metal ions often enhances biological activity,^12,13 leading to improved therapeutic effects. Plectranthus amboinicus, a medicinal plant traditionally used across tropical regions,^14,15 contains rich deposits of polyphenolic compounds. These compounds not only serve as excellent ligands for metal complexation but also act as biological reducing agents in the creation of nanoparticles.^16,17

The study explores a unique approach that leverages P. amboinicus extract to synthesise both metal complexes and oxide nanoparticles. This dual-pathway method demonstrates how plant metabolites can serve different roles, such as coordinating ligands for complex formation and reducing agents for nanoparticle synthesis. This approach addresses the growing need for sustainable material synthesis methods while providing products suitable for biological applications.

Experimental

Materials and Chemicals

All reagents, mineral salts, and solvents were of analytical grade, procured from reputable suppliers such as Merck, BDH, and Aldrich. Solutions were prepared using ultrapure water (resistivity >18.2 MΩ·cm).

Plant Material Collection and Extraction

Leaves of P. amboinicus were collected from Yafae and Yemen, and their identity was confirmed by Professor Dr. Abdul-Nasir Al-Gefri (voucher specimen number PA-2020-01). The plant material was thoroughly rinsed with tap water, followed by sterilised water, then air-dried naturally over a period of four weeks. The dried leaves were ground into a fine powder using a mechanical grinder.

For extraction, 200 g of the powdered plant material was macerated in 2000 mL of 80% methanol at room temperature (~25°C) for 48 hours with intermittent stirring. The mixture was filtered through Whatman No. 1 filter paper, and the filtrate was concentrated under reduced pressure at 60°C using a rotary evaporator (Heidolph, model R-200). The resultant extract was stored at 4°C for subsequent use.¹⁸

Preparation of the n-Butanol Fraction

The segmentation process, adapted from an established method and illustrated in Figure 1, involved macerating 10 g of dried, ground plant material in 100 mL of distilled water for 20 hours, followed by four filtrations. The aqueous phase was first extracted three times with chloroform to remove sugars and monoglycosides, then concentrated. Subsequent extraction with ethyl acetate (three times) targeted phenolic compounds, with the ethyl acetate fractions concentrated. Finally, the remaining aqueous phase was extracted four times with n-butanol to recover additional phenolics, and concentrated. Multiple n-butanol extractions ensured maximum recovery.¹⁹

Figure 1 Polyphenolic extraction process.

Analysis of n-Butanol Fraction Extract by LC-MS/MS

The polyphenolic constituents of the n-butanol extract were analysed using an Orbitrap Elite LC-MS/MS system (Thermo Fisher Scientific) fitted with a C18 column (2.1 × 50 mm, 1.7 μm).

• Injection volume: 3 μL
• Mobile phase: Solvent A (0.1% formic acid in water), Solvent B (acetonitrile)
• Gradient programme: from 5% B to 95% B over 20 minutes
• Flow rate: 0.3 mL/min
• Ionisation mode: Electrospray ionisation (ESI) in negative mode
• MS parameters: capillary voltage 3.0 kV, sheath gas at 40 psi, auxiliary gas at 10 psi, spray temperature 350°C.

Compounds were identified by comparing fragmentation patterns, retention times, and spectral data with authentic standards and spectral databases such as MassBank and METLIN. Fragmentation analysis focused on characteristic ions of flavonoids and phenolic compounds, aiding in structural confirmation.

Synthesis Methods for Metal-Based Materials

Two distinct synthetic routes have been developed to obtain different metal-based materials from P. amboinicus: metal complexes through coordination chemistry, and metal oxide nanoparticles through green synthesis. Each approach utilises specific extraction methods and reaction conditions to obtain the desired products.

Synthesis of Metal Complexes

Following the methodology described by Bamigboi et al,²⁰ n-butanol plant extract (0.15 g) was combined with metal salts (0.27 g), including CuSO₄·5H₂O, ZnSO₄·7H₂O, and Mn(CH₃COO)₂·4H₂O, in methanol (10 mL). NaOH was used to bring the solution pH to between 9 and 10, followed by a 2-hour incubation at 60 °C. The resulting precipitate was collected upon cooling, purified by ethanol recrystallisation, and stored in sealed containers.

Synthesis of Oxide Nanoparticles

Metal oxide nanoparticles were synthesised based on the procedure outlined by Singh et al²¹ with certain adjustments. The process involved mixing methanolic extract (0.2 g) with 80% methanol (20 mL) and metal salts (0.1 g each of CuSO₄·5H₂O, ZnSO₄·7H₂O, and Mn(CH₃COO)₂·4H₂O). NaOH (1 M) was used to bring the pH of the solution to 9, followed by 2 hours of magnetic stirring at 50 °C. Nanoparticle formation is indicated by immediate colour changes. The products were isolated by centrifugation (5000 rpm, 20 min), rinsed with distilled water, followed by drying at 80 °C for a duration of 3 hours.

Characterisation of Metal Complexes and Metal Oxide Nanoparticles

Spectroscopic Analysis Using UV-Vis

Absorption spectra were recorded using a SCINCO S-3100 UV-Vis spectrophotometer (scanning range 200–800 nm). Samples were prepared by diluting in a mixture of DMSO and methanol (2:1 v/v). Spectra were obtained at room temperature using quartz cuvettes with 1 cm path length.

FT-IR Spectroscopy

FTIR spectra were collected with a PerkinElmer FT-IR spectrometer (model C116189) (range 4000–400 cm⁻¹). Samples were prepared as KBr pellets or as thin films on the ATR accessory. Spectral resolution was set to 4 cm⁻¹, with an average of 32 scans.

X-Ray Diffraction

XRD analyses were performed on a Bruker D8 Advance diffractometer (Cu Kα, λ = 1.5406 Å) operating at 40 kV and 45 mA. Data were collected over a 2θ range of 5–100°, with a step size of 0.02°, and a scan rate of 1° per minute. Crystallite sizes were estimated using the Scherrer equation.

¹H NMR Spectroscopy

¹H NMR spectra were acquired on a Bruker Avance Neo 500 MHz spectrometer at 25°C, using DMSO-d₆ as the solvent. Chemical shifts (δ) are reported in parts per million (ppm) relative to residual solvent signals.

FESEM-EDX Analysis

Morphological and elemental characterisation was performed using a Carl Zeiss Supra 55 scanning electron microscopy (FESEM) system equipped with energy-dispersive X-ray (EDX) capability. This instrument, with a resolution of 1 nm, enabled detailed analysis of the nanostructure morphology while simultaneously providing elemental composition data through EDX spectroscopy.

Antimicrobial Screening

Flavonoid extracts, their metal complexes, the methanolic plant extract, and nanoparticle oxides were tested against four bacteria—Staphylococcus aureus (NCTC 12493), Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922)—and Candida albicans, sourced from Aden Central Laboratories.

Antimicrobial activity was assessed via agar diffusion²² at concentrations of 40, 80, 120, and 160 mg/mL. Antibiotics—clindamycin (2 µg), ofloxacin (5 µg), ampicillin (10 µg)—served as positive controls, with DMSO as negative control.

Antioxidant Tests

Samples were dissolved in DMSO (10 mg/mL) and tested across various concentrations, compared to ascorbic acid.²³

DPPH Assay

Using 517 nm spectrophotometry, samples (25–150 μg/mL) were evaluated for free radical scavenging.²⁴ The percentage inhibition was calculated as:

where As is the absorbance of the sample, and Ac is the absorbance of the control (only DPPH solution).

In addition, the IC50 was estimated graphically.

ABTS Assay

ABTS radical cation, prepared by reacting ABTS with potassium persulfate, was diluted to 0.70 ± 0.02 at 734 nm.²⁵ Sample solutions (diluted appropriately) were mixed with ABTS, and absorbance was measured after 6 minutes to assess antioxidant activity.

Anticancer Assay

Cytotoxicity was evaluated against AML (THP-1) and Calu-3 lung cancer cells using WST-1 and SRB assays, respectively.

Cell Culture

THP-1 cells were maintained in RPMI; Calu-3 in DMEM, both supplemented with antibiotics and serum, incubated at 37°C in 5% CO₂.

Cytotoxicity Assays

THP-1 viability was assessed with WST-1; 3×10³ cells per well, treated with test compounds for 48 hours, then measured at 450 nm.

Calu-3 viability was measured via SRB; 5×10³ cells per well, treated for 72 hours, fixed with TCA, stained with SRB, and read at 540 nm. Cisplatin served as positive control.^26,27

Flow Cytometry

Post-treatment, cells (10⁵) were stained with Annexin V-FITC and PI, incubated in the dark, and analysed using flow cytometry. Data acquisition involved 12,000 events per sample, with analysis of apoptotic and necrotic populations performed via quadrag.^28,29

Statistical Analysis

All experiments were performed in triplicate (n = 3), and the data are presented as mean ± standard deviation. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test, with p < 0.05 considered statistically significant. Size distribution analysis was performed on a minimum of 200 particles per sample.

Results and Discussion

Preparation of Metal Complexes and Metal Oxide Nanoparticles

The synthesis of metal complexes and metal oxide nanoparticles was achieved through environmentally friendly methods using P. amboinicus extract. The formation of these materials was evidenced by distinct colour changes during the synthesis process.³⁰ As shown in Figure 2, the reactions between copper (II) ions and polyphenol extract produced green complexes (50 ± 1.2 nm), whereas their interaction with methanolic extract yielded blackish-brown nanoparticles (39 ± 1.6 nm).³¹ The zinc-based materials exhibit different colourations, with yellow complex (43 ± 2.1 nm) and white nanoparticles (38 ± 1.3 nm).³² In contrast, both manganese complex (60 ± 1.2 nm) and nanoparticles (55 ± 1.2 nm) exhibit a dark brown colouration.^33,34 These colour variations result from specific interactions between the plant phytochemicals and metal ions during the formation process.

Figure 2 Construction of metal complexes and metal oxide nanoparticles.

Mechanisms of Metal Complex and Nanoparticle Formation Using Plectranthus Amboinicus Extracts

Fabrication of metal complexes and nanoparticles employing P. amboinicus extracts involves distinct pathways governed by the phytochemical composition and reaction conditions of the extract (Table S1). The following sections in detail:

Metal Complex Formation

1. Ligand coordination: Flavonoids isolated from the n-butanol fraction (eg, kaempferol and apigenin) act as polydentate ligands. The hydroxyl (-OH) and carbonyl (C=O) groups coordinate with the metal ions (Cu²⁺, Zn²⁺, and Mn²⁺) to form stable complexes:

This chelation is favoured by the deprotonation of phenolic -OH groups at alkaline pH (9–10).

2. Hydroxide incorporation: The addition of NaOH introduces hydroxide ions (OH⁻), further stabilising the complexes by occupying vacant coordination sites:

This step is crucial for achieving octahedral geometries, as evidenced by the UV-Vis spectra (eg Jahn-Teller distortion in Cu²⁺ complex at 530 nm).

3. Precipitation and crystallisation: Upon cooling, the complexes precipitated owing to their reduced solubility. Recrystallisation with ethanol removed the unreacted ions, yielding pure complexes with distinct colours (green for Cu, yellow for Zn, and brown for Mn).

Metal Oxide Nanoparticle Formation

1. Reduction of metal ions: Polyphenols and flavonoids in the crude methanolic extract acted as reducing agents. For example, genistein donates electrons to reduce Cu²⁺ into Cu⁰.

2. Nucleation and growth: Reduced metal atoms aggregate to form nuclei, which then grow into nanoparticles. The reaction pH (9.0) and temperature (50 °C) optimised particle size control.

Immediate colour changes (eg black-brown for CuO nanoparticles) indicate the formation of nuclei.

Spherical morphologies (observed via FESEM) are a result of isotropic growth.

3. Stabilisation by Phytochemicals: Residual flavonoids and proteins are adsorbed onto nanoparticle surfaces, preventing aggregation. FTIR peaks at 593 cm⁻¹ (M-O bonds) and EDX metal-to-oxygen ratios (~1:1) confirm oxide formation.

Analytical Validation

UV-Vis spectroscopy: Complexes exhibit ligand-to-metal charge transfer bands (eg, Cu²⁺ complexes at 340 nm). Nanoparticles show characteristic absorption bands in the UV-Vis region (eg, ZnO at 350 nm) attributed to band gap transitions.

FT-IR spectroscopy: Shifts from 1629 cm⁻¹ (C=O in complexes) to 1558 cm⁻¹ (M-O in nanoparticles) confirm distinct bonding environments.

X-Ray diffraction (XRD): Sharp peaks in complexes (eg Zn at 2θ = 22°) versus broader peaks in nanoparticles (eg MnO at 2θ = 29°) reflect differences in crystallinity.

This dual-pathway mechanism highlights the versatility of P. amboinicus in synthesising both coordination compounds and nanoparticles, which are suitable for medical uses like antimicrobial agents or drug transport mechanisms.

Analysis of n-Butanol Fraction Extract by LC-MS/MS

LC-MS/MS analysis of polyphenolic compounds (Figure 3) identified several biologically significant flavonoids (Table S2) (Figure 4), including kaempferol, apigenin, naringin, and genistein. These compounds are recognised for their antioxidant properties and therapeutic potential in the treatment of Alzheimer’s and other neurodegenerative diseases.^35,36 P. amboinicus contains coumarins and flavonoid glycosides, which represent a valuable source of these typically expensive and hard-to-obtain compounds. These flavonoids have diverse biological activities, including anticancer properties through tumour growth inhibition, cardioprotective effects,^37,38 and antibacterial and anti-inflammatory properties,^39,40 thus supporting traditional medicinal applications. The identified flavonoids exhibit strong metal-binding capabilities through their hydroxyl or carbonyl groups, forming complexes that often exhibit enhanced antioxidant properties compared with free flavonoids.¹⁰

Figure 3 LC-MS/MS of n-butanol fraction extract.

Figure 4 Some flavonoids detected by LC-MS/MS analysis.

UV-Visible Spectroscopic Analysis

The UV-vis spectrum of P. amboinicus n-butanol extract exhibited characteristic flavonoid absorption peaks at 230 and 290 nm, attributed to aromatic π–π * transitions (Figure 5a), whereas the methanolic extract showed a broad absorption band spanning 200–390 nm (Figure 5b). The copper complexes showed shifts to 220, 340, and 530 nm (Figure 5a),^41,42 with a 530 nm band suggesting a distorted octahedral structure due to the Jahn-Teller effect, whereas the copper oxide nanoparticles exhibited a single peak at 250 nm (Figure 5b).^43,44 Zinc complexes demonstrate absorption peaks at 260 and 340 nm, reflecting ligand-based transitions, whereas zinc oxide nanoparticles show a characteristic peak at 350 nm.^45,46 The manganese complexes exhibited shifted peaks at 250 and 420 nm, with a weak 420 nm band indicating octahedral geometry for the high-spin Mn²⁺ complexes, and the corresponding oxide nanoparticles displayed peaks at 270 and 330 nm.^47,48

Figure 5 UV‒vis absorption spectra of the polyphenolic extract and the metal complexes (a), methanolic extract, and metal oxide nanoparticles (b).

All the metal complexes retained shifted phenolic compound absorption peaks, whereas the metal oxide nanoparticle spectra showed distinct patterns without methanolic extract absorption features. This spectral evidence suggests the role of the methanolic extract as a reducing agent for the formation of metal oxide nanoparticles.

Fourier Transform Infrared Spectroscopy (FT-IR)

The FT-IR spectra revealed distinct structural characteristics of the synthesised materials (Figure 6a and b). The n-butanol extract exhibited characteristic bands at 3421 cm⁻¹ (O-H stretching), 2924 cm⁻¹ (C-H stretching), 1629 cm⁻¹ (C=O stretching), and 1384 cm⁻¹ (C-O stretching).^49,50

Figure 6 FT-IR spectra of the polyphenolic extract and metal-complex: (a) methanolic extract and (b) metal oxide nanoparticle.

Metal Complexes (Figure 6a):

• Copper complex showed shifts to 3398 cm⁻¹ (O-H), 1558 cm⁻¹ (C=O), and new bands at 593 cm⁻¹ (Cu-O)
• Zinc complex displayed bands at 3412 cm⁻¹ (O-H), 1562 cm⁻¹ (C=O), and 545 cm⁻¹ (Zn-O)
• Manganese complex exhibited shifts to 3405 cm⁻¹ (O-H), 1565 cm⁻¹ (C=O), and 528 cm⁻¹ (Mn-O)

Metal Oxide Nanoparticles (Figure 6b):

• CuO nanoparticles: characteristic peaks at 535 cm⁻¹ and 590 cm⁻¹. This is consistent with what is stated in the literature.⁵¹
• ZnO nanoparticles: strong band at 450–500 cm⁻¹
• MnO nanoparticles: distinctive peaks at 520 cm⁻¹ and 580 cm⁻¹

These spectral changes confirm successful complex formation and nanoparticle synthesis.^52,53

XRD Analysis

X-ray diffraction demonstrated the successful creation of both metal complexes and nanoparticles in the powder samples. The diffraction patterns exhibit distinctive characteristics for each material.

Methanolic and Polyphenol Extracts

• Methanolic extract (Figure 7a): minimal peaks at 2θ = 25°, 28°, 30°, and 45°
• Polyphenol mixture (Figure 7b): crystalline properties with peaks at 2θ = 21°,29°, 32°, 39°, 40°, and 43°

Figure 7 XRD for: (a) methanolic extract, (b) polyphenol extract, (c) copper complex, (d) copper oxide nanoparticles (CuONPs), (e) zinc complex, (f) zinc oxide nanoparticles (ZnONPs), (g) manganese complex, and (h) manganese oxide nanoparticles (MnONPs).

Copper-Based Materials

• Copper complex (50 ± 1.2 nm) (Figure 7c): distinctive peaks at 2θ = 26°, 37°, 39°, 55°, 59, 63°, and 70°.
• Copper oxide nanoparticles (39 ± 1.6 nm) (Figure 7d): peaks at 2θ = 18°, 20°, 22°, 24°, 29°, 30°, 32°, 35°, 42°, 53°, and 60°, aligning with reference data⁵⁴

Zinc-Based Materials

• Zinc complex (43 ± 2.1 nm) (Figure 7e): peaks at 2θ = 23°, 25°, 28°, 31°, 33°, 39°, 40°, 45°, and 56°.
• ZnO nanoparticles (38 ± 1.3 nm) (Figure 7f): peaks at 2θ = 29°, 30°, 32°, 34°, 47°, 52°, 60°, and 68°, consistent with literature findings.^55,56

Manganese-Based Materials

• Mn complex (60 ± 1.2 nm) (Figure 7g): absorption peaks at 2θ = 30°, 36°, 39°, 40°, 42°.

The manganese oxide nanoparticles (55 ± 1.2 nm) (Figure 7h) showed peaks at 2θ = 19°, 29°, 35°, 41°, 45°, and 50°. The more pronounced and intense peaks in the zinc complex suggest their enhanced crystalline nature compared to that of the ZnO nanoparticles. Plant-synthesised MnO nanoparticles typically exhibit a weakly crystalline nature.⁵⁷ The crystallite sizes were determined using the Debye-Scherrer equation. Research indicates that plants of the Lamiaceae family can produce nanoparticles of varying sizes and shapes, which are influenced by plant extract concentration.⁵⁸

¹H NMR Analysis

¹H NMR spectroscopic analysis was conducted using the samples dissolved in DMSO-d⁶. The methanolic extract (Figure 8a) showed less intense peaks in the aromatic region, whereas the polyphenol mixture spectrum (Figure 8b) exhibited signals at 6–8 and 9–13 ppm, corresponding to the benzene ring protons of phenolic compounds.^59,60 Signals around 6.5 ppm indicated mono- and di-substituted phenyl rings in the plant extracts, whereas peaks at 9–13 ppm confirmed the presence of flavonoids in the polyphenol mixture. The signal at 1.21 ppm was assigned to methyl protons (-CH₃), and the signals between 3.2–3.8 ppm corresponded to methylene (-CH₂-) and methoxy (O-CH₃) protons, respectively.

Figure 8 Continued.

Figure 8 Continued.

Figure 8 Continued.

Figure 8 1H NMR for: (a) methanolic extract, (b) polyphenol extract, (c) copper complex, and (d) copper oxide nanoparticles (CuONPs), (e) zinc complex, and (f) zinc oxide nanoparticles (ZnONPs), (g) manganese complex, and (h) manganese oxide nanoparticles (MnONPs).

The metal complexes displayed distinct peak shifts at 6–13 ppm, as evidenced by the copper complex (Figure 8c), while copper oxide nanoparticles (CuONPs) (Figure 8d) showed no signals in the aromatic region. Similarly, the zinc complex (Figure 8e) exhibits characteristic peaks, whereas the zinc oxide nanoparticles (ZnONPs) (Figure 8f) lack aromatic signals. Manganese complex (Figure 8g) showed a broad peak, indicating high spin characteristics,^61,62 whereas manganese oxide nanoparticles (MnONPs) (Figure 8h) displayed no aromatic region.

All metal oxide nanoparticles spectra confirmed the absence of phenolic compounds, with signals at 2.50 ppm and 3.12–3.39 ppm attributed to DMSO-d⁶ solvent and water, respectively.⁶³ The ¹H NMR data demonstrate clear spectral differences between the metal complexes and their corresponding metal-oxide nanoparticles.

EDX Analysis

The elemental makeup of the sample was analyzed with Energy-dispersive X-ray spectroscopy (EDX) for the metallic complexes and metal oxide nanoparticles synthesised using Plectranthus amboinicus. EDX (Table S3) of the copper (Figure 9a), zinc (Figure 9b), and manganese complex (Figure 9c) revealed varying elemental compositions. The copper complex showed carbon (46.21 wt%), oxygen (33.58 wt%), and copper (20.21 wt%).

Figure 9 EDX for: (a) copper complex, (b) zinc complex and (c) manganese complex.

As shown in Table S4 and Figure 10, the analyses of the copper oxide nanoparticles (Figure 10a), zinc oxide nanoparticles (Figure 10b), and manganese oxide nanoparticles (Figure 10c) demonstrate different compositions. CuONPs contained oxygen (33.04 wt%), copper (43.34 wt%), and carbon (22.95 wt%). A strong characteristic peak at 8 keV indicated metallic copper in the CuONPs, consistent with previous studies,^51,64 whereas the copper complex showed a weaker peak at this position. The oxygen peak appeared at 0.5 keV in the metal oxide nanoparticles and at 0.2 keV in the complexes, confirming distinct formation pathways.

Figure 10 EDX for: (a) copper oxide nanoparticles (CuONPs), (b) zinc oxide nanoparticles (ZnONPs), and (c) manganese oxide nanoparticles (MnONPs).

The zinc complex displayed peaks similar to those of copper complex for oxygen and carbon, with distinctive zinc peaks at 1.5 and 8.8 keV. ZnONPs showed a higher zinc content than their complex,⁶⁵ whereas zinc complex contained more carbon owing to their structural composition.

Manganese oxide nanoparticles exhibited less pronounced carbon peaks but sharper manganese peaks than their complex, with a higher oxygen content in the nanoparticles. The carbon signal at 0.4 keV in metal oxide nanoparticles may originate from the sample carrier, atmospheric carbon species, or residual plant extract.^66,67

According to EDX data, carbon was the dominant element in the complexes, whereas the metal oxide nanoparticles showed the highest peaks for metal and oxygen, confirming their formation. A metal-to-oxygen ratio (M: O) of approximately 1:1 in all nanoparticles indicated the formation of CuO, ZnO, and MnO.

Spectrum processing identified the following deletable peaks: copper complex (1.600, 2.077, 3.340, 11.089 keV), CuONPs (1.257–3.591 keV), zinc complex (1.269–4.335 keV), ZnONPs (1.260–3.600 keV), manganese complex (1.493–3.708 keV), and MnONPs (1.259–3.168 keV).

FESEM Analysis

Surface morphology analysis using scanning electron microscopy (FESEM) revealed distinct characteristics for each compound (Figure 11a–h):

Figure 11 FESEM analysis; (a) Copper complex, (b) Copper oxide nanoparticles (CuONPs), (c) zinc complex, (d) zinc oxide nanoparticles (ZnONPs), (e) manganese complex, and (f) manganese oxide nanoparticles (MnONPs).

Copper-Based Materials

• Copper complex (50 ± 1.2 nm) (Figure 11a): flat sheet-like structures with agglomerates, attributed to the preparation method and pH conditions^68,69
• Copper oxide nanoparticles (39 ± 1.6 nm) (Figure 11b): uniform spherical shapes with even distribution^70,71

Zinc-Based Materials

• Zinc complex (43 ± 2.1 nm) (Figure 11c): ellipsoidal morphology with consistent size distribution⁷²
• ZnO nanoparticles (38 ± 1.3 nm) (Figure 11d): Hierarchical flower-like structures with distinct petals⁷³

Manganese-Based Materials

• Mn complex (60 ± 1.2 nm) (Figure 11e and f): sheet-like structures with a layered arrangement
• Manganese oxide nanoparticles (55 ± 1.2 nm) (Figure 11g and h): spherical with minimal agglomeration⁷⁴

The FESEM micrographs confirmed the uniform particle distribution and distinct morphological features of each compound. These size distributions, particularly those spanning 10 nm to 50 nm, are deemed ideal for medical applications owing to their unique therapeutic properties.⁷⁵ Higher magnification images (Figure 11f and h) further revealed the detailed surface characteristics and crystalline nature of the synthesised materials.

Antimicrobial Screening

The metal complexes demonstrated greater antimicrobial activity than the metal oxide nanoparticles (Figure 12). The flavonoid extract was more effective than the methanolic extract, with copper complexes showing the highest activity—25.47 cm against Staphylococcus aureus, 24 cm against Klebsiella pneumoniae and E. coli, and 26.17 cm against Pseudomonas aeruginosa (Table S5)—surpassing previous reports.⁷⁶ Zinc complexes displayed similar activity to copper against E. coli (22 cm), while manganese complexes exhibited antimicrobial effects, though less potent than copper-zinc complexes. All complexes outperformed the flavonoid extract, aligning with existing literature.⁷⁷ They also inhibited Candida albicans, albeit less effectively. The methanolic extract showed the weakest activity. Copper and zinc oxide nanoparticles demonstrated higher antimicrobial efficacy than manganese oxide, with copper complexes outperforming antibiotics against all microbes except Candida albicans.⁷⁸

Figure 12 Antimicrobial activity of metal complexes and metal oxide nanoparticles.

Antioxidant Results

Copper oxide nanoparticles exhibited the strongest antioxidant activity in both DPPH and ABTS assays. The scavenging capacity followed the order: copper oxide nanoparticles > copper complexes > zinc complexes > zinc oxide nanoparticles > manganese oxide nanoparticles > manganese complexes > flavonoid extract > methanolic extract (Table S6). The IC₅₀ values for radical scavenging of these nanoparticles were higher than in previous studies.^79,80 In the DPPH test, IC₅₀ values were 28.32 μg/mL for copper complexes and 17.12 μg/mL for copper oxide nanoparticles; zinc complexes and zinc oxide nanoparticles showed IC₅₀ values of 37.46 and 32.97 μg/mL, respectively (Figure 13). In the ABTS assay (Figure 14), IC₅₀ values were 11.03 μg/mL for copper nanoparticles and 22.5 μg/mL for their complexes, exceeding previous findings,⁸¹ with ascorbic acid at 10.65 μg/mL (Table S6). Manganese complexes and manganese oxide nanoparticles showed IC₅₀ values of 55.04 and 46.83 μg/mL (DPPH), and 49.53 and 43.67 μg/mL (ABTS). The methanolic extract was the least effective antioxidant.

Figure 13 DPPH scavenging capacity of metal complexes and metal oxide nanoparticles.

Figure 14 ABTS scavenging capacity of ascorbic acid, metal complexes, and metal oxide nanoparticles.

Anticancer Results

The WST-1 assay on THP-1 leukaemia cells and the SRB assay on Calu-3 lung cancer cells (Figures 15, 16 and Table S7) demonstrated significant anticancer activity, with copper oxide nanoparticles showing the highest efficacy, followed by zinc oxide nanoparticles. Copper oxide nanoparticles exhibited IC₅₀ values of 13.63 μg/mL (THP-1) and 26.67 μg/mL (Calu-3), surpassing previous reports.^82,83 Zinc oxide nanoparticles had IC₅₀ values of 37.23 μg/mL and 58.42 μg/mL, respectively, also higher than prior data.⁸⁴ Manganese oxide nanoparticles showed IC₅₀s of 60.8 μg/mL and 80.46 μg/mL. The methanolic extract was less active. Although metal complexes displayed anticancer effects, they were less effective than nanoparticles, likely due to the superior tissue penetration of nanoscale materials. The order of anticancer activity was: copper complexes > zinc complexes > manganese complexes.

Figure 15 IC50 values metal complexes and metal oxide nanoparticlesin THP-1 cells.

Figure 16 IC50 of metal complexes and metal oxide nanoparticlesin Calu-3 cells.

Flow Cytometry Results

Flow cytometry revealed that all studied metal complexes and metal oxide nanoparticles induced apoptosis in Calu-3 cells (Figure 17A–C). Nanoparticles caused greater apoptosis than complexes, consistent with their higher cytotoxicity, likely due to their size and composition. The flavonoid extract induced more apoptosis than the methanolic extract. Over time, apoptosis increased: late apoptosis (Q2) rose after 72 hours, while early apoptosis (Q4) peaked within the first 24 hours. Early apoptosis (Q4) indicates initial cell death with membrane integrity; late apoptosis (Q2) reflects advanced cell death with membrane perforation; necrosis (Q1) involves non-apoptotic cell death, and healthy cells are represented in Q3. The increase in apoptosis over time is consistent with previous studies.⁸⁵ Copper oxide nanoparticles induced the highest late apoptosis (28.6%) after 72 hours, with total apoptosis reaching 39%. Zinc and manganese oxide nanoparticles showed lower rates, at 26.06% and 23.93%. Metal complexes exhibited higher apoptosis than the metal oxides, with total rates of 31.72%. The apoptotic effects of these materials enhance their therapeutic potential, as apoptosis analysis provides insight into cell death stages and drug efficacy.⁸⁶

Figure 17 Continued.

Figure 17 Continued.

Figure 17 Mechanism of cell death in Calu-3 lung cancer cell line after treatment with metal complexes and metal oxide nanoparticles, (A) After 24 hours, (B) After 48 hours, (C) After 72 hours.

Conclusion

This study presents an eco-friendly method for synthesising metal-based materials using Plectranthus amboinicus extracts. The dual-pathway synthesis successfully produced well-defined metal complexes and oxide nanoparticles, with sizes of approximately 50 nm (copper), 43 nm (zinc), 60 nm (manganese), and their corresponding oxides—39 nm, 38 nm, and 55 nm respectively. Spectroscopic and microscopic analyses confirmed the formation of sheet-like complexes and spherical, crystalline nanoparticles, as evidenced by XRD patterns.

The controlled synthesis and distinctive properties of these materials underpin their significant biological activity, often surpassing standard antibiotics in antimicrobial tests. Their notable antioxidant capacity was linked to their ability to inhibit cancer cell growth in leukaemia and lung cancer models. Both complexes and nanoparticles induced apoptosis, further enhancing their therapeutic potential.

The utilisation of plant metabolites as natural reducing and stabilising agents underscores the potential for sustainable and eco-friendly biomedical material production. Looking ahead, further research is warranted to evaluate the biocompatibility and toxicity profiles of these materials, alongside surface functionalisation strategies to optimise their therapeutic efficacy. Developing scalable manufacturing protocols and exploring their applications against resistant microbes, as well as elucidating drug delivery mechanisms, are essential steps towards translating these materials from laboratory research to clinical use.

This green synthesis approach marks an important stride towards sustainable nanomedicine production, fostering advances across multidisciplinary biomedical research and opening new avenues for the development of novel, eco-friendly therapeutic agents.