Sample collection and preparation

Plectranthus amboinicus leaves were collected from the Maejo area, San Sai District, Chiang Mai Province, Thailand. The leaves were washed with distilled water and dried in a hot air oven at 60 °C for 24 hours. Once dried, they were ground into a fine powder. Three separate 20-gram portions of the powdered leaves were weighed and placed into round-bottom flasks for extraction.

Microwave-assisted extraction procedure

Bioactive compounds were extracted using microwave-assisted extraction (MAE) at a power of 450 watts, using 200 mL of 50% ethanol as the solvent. The extraction was conducted at three different time intervals: 10, 15, and 20 minutes. ¹¹ The extracts were filtered using Whatman No.1 filter paper, and the solvents were partially evaporated under reduced pressure using a rotary evaporator. Extracts were then stored at −40 °C for further analysis.

Drying methods and yield determination

The filtered extracts were subjected to two drying techniques: freeze-drying (PF) and tray-drying (PT). The percentage yield (% yield) was calculated using the formula: % Yield = ( Weight of Dried Extract / Weight of Starting Material ) × 100

Caffeic acid quantification by HPLC

Extracts from MAE (PT10, PT15, PT20, PF10, PF15, PF20) were analyzed for caffeic acid content. Each 0.05 g sample was dissolved in ethanol and diluted to 50 mL. Solutions were filtered through a 0.45 μm membrane filter and placed into 2 mL vials. High-performance liquid chromatography (HPLC) was used for quantification. Conditions are listed in Table 1.

Total Phenolic Content (TPC) determination

TPC was measured using the Folin–Ciocalteu method on PF10, PF15, and PF20 samples. Extracts (200 μL) were mixed with 1,000 μL of Folin–Ciocalteu reagent and 800 μL of 7.5% sodium carbonate. After incubation at room temperature for 60 minutes, absorbance was measured at 765 nm. A gallic acid standard curve (16–250 mg/L) was used to calculate phenolic content, reported as mg gallic acid equivalents per gram (mg GAE/g extract). ^{12, 13}

Antioxidant activity (DPPH assay)

The antioxidant activity of PF10, PF15, and PF20 was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method. ¹⁴ Various concentrations of extract (0.125–4 mg/mL) and L-ascorbic acid (12–400 μg/mL) were tested. After mixing 50 μL of each sample with 100 μL DPPH (2,000 μM), the reaction was incubated in darkness for 30 minutes. Absorbance was read at 517 nm.

Scavenging activity (%) was calculated as: % DPPH = [ ( A _ control − A _ sample ) / A _ control ] × 100

The IC ₅₀ value, indicating the concentration required to scavenge 50% of DPPH radicals, was used to compare antioxidant potential.

Antimicrobial activity

Bacterial preparation

PF15 was tested against Staphylococcus aureus (ATCC 25932), Staphylococcus epidermidis (ATCC 14990), and Cutibacterium acnes (ATCC 6919). Bacteria were cultured in nutrient broth at 37 °C for 12–18 hours and adjusted to a 0.5 McFarland standard, giving an approximate cell density of 1.5 × 10 ⁸ CFU/mL.

Extract preparation for antimicrobial testing

A 1,000 mg/mL stock solution of PF15 was prepared in sterile distilled water, filtered through a 0.2 μm membrane and used for antimicrobial testing.

Disc diffusion assay

Sterile discs were loaded with 10 μL of extract, dried, and placed on agar plates seeded with test bacteria. Sterile water and ampicillin (10 μg/disc) served as negative and positive controls. Plates were incubated at 37 °C for 24 hours and inhibition zones were measured. ¹⁵

MIC determination (Broth microdilution)

PF15 was serially diluted in 96-well plates (1,000–0.48 mg/mL). Wells were inoculated with bacteria, and plates incubated at 37 °C for 24 hours. The MIC (Minimal Inhibitory Concentration ) was the lowest concentration with no visible bacterial growth. ¹⁵

MBC determination (Drop plate method)

Aliquots from each MIC well were plated on nutrient agar. After incubation, the MBC (Minimal Bactericidal Concentration was the lowest concentration at which no bacterial colonies formed. ¹⁵

Anti-inflammatory activity assay

THP-1 Cell culture and differentiation

THP-1 monocytes (ATCC TIB-202) were cultured in RPMI-1640 medium with 10% FBS and 1% antibiotics. Differentiation into macrophages was induced using 50 ng/mL PMA for 48 hours, followed by 24 hours in fresh medium. ¹⁶

Cell viability assay

Differentiated cells were treated with PF15 extract (0–200 μg/mL) and 0.5 μg/mL LPS (lipopolysaccharides) for 24 hours. PrestoBlue ^® reagent was added, and absorbance was measured at 570 nm to assess cell viability. ¹⁷

Cytokine measurement by ELISA

Cells were treated with PF15 (12, 25, and 50 μg/mL) along with LPS. Supernatants were collected after 24 hours and analyzed for TNF-α, IL-1β, IL-6, and IL-10 using ELISA kits (Thermo Fisher). Results represent means ± standard deviation (SD) from triplicate experiments.

Cream Formulation with PF15

An anti-acne cream was formulated using the MIC concentration of PF15. The aqueous phase containing PF15 was heated to 70 °C, combined with the oil phase, and emulsified for 30 minutes. The cream was cooled to 35–40 °C, mixed until homogeneous, and stored for further analysis.

Stability and safety evaluation

Accelerated stability testing

The cream underwent 6 thermal cycles (4 °C for 24 h and 45 °C for 24 h) to assess changes in pH, odor, color, texture, and phase separation. ¹⁸

Antimicrobial activity of the final product

Using the disc diffusion method, antimicrobial activity was evaluated against S. aureus, S. epidermidis, and C. acnes. Cream without extract served as a negative control.

Microbial contamination testing

Aerobic Plate Count (APC)

Cream samples were diluted and plated on plate count agar. Colonies were counted after 24 hours at 37 °C, reported as CFU/g.

Yeast and mold contamination

Samples were plated on Dichloran Rose Bengal Chloramphenicol agar after serial dilution. Colonies were counted after 24 hours to determine CFU/g.

Escherichia coli detection

Diluted samples were cultured in Lauryl Tryptose broth and incubated. Gas formation indicated presumptive E. coli. Confirmation was done using EC Broth and MPN methodology.

Staphylococcus aureus detection

Samples were diluted and plated on Mannitol Salt agar. Colony morphology and color change were used for identification. CFU/g was reported. ¹⁹

Salmonella spp. detection

Samples were pre-enriched in Tryptone Soya broth, streaked onto SS Agar, and incubated for 24 hours. Growth consistent with Salmonella spp. was recorded as “present” or “absent” in 25 g of sample. ²⁰

All results are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA, followed by Duncan’s Multiple Range Test. A p-value < 0.05 was considered significant. Data analysis was conducted using GraphPad Prism version 9.0.0.121.

Microwave-assisted extraction (MAE) using 50% ethanol for 10, 15, and 20 minutes, followed by either tray drying (PT10, PT15, PT20) or freeze-drying (PF10, PF15, PF20), yielded extracts with distinct physical characteristics and varying efficiencies. Tray-dried extracts produced yields of 22.4, 23.5, and 22.5 g, respectively, demonstrating consistent extraction efficiency across the time intervals. These values are in alignment with the findings of, ²¹ which reported diminishing returns with extended extraction beyond optimal durations. The resulting tray-dried powders were dark green in color, indicating good retention of chlorophyll, flavonoids, and phenolic compounds ( Figure 1). Conversely, freeze-dried extracts yielded slightly lower quantities—19.8, 21.8, and 21.0 g for PF10, PF15, and PF20, respectively—yet retained a fine, homogeneous powder with vibrant green coloration. Despite lower yields, freeze-drying was effective in preserving thermolabile bioactives and improving powder texture. These visual differences in texture and color between drying methods ( Figure 2) highlight the critical influence of post-extraction drying on the final product characteristics, with freeze-drying offering superior preservation at the expense of yield. ²²

Tray-Dried Extracts (PT)

High-performance liquid chromatography (HPLC) analysis confirmed the presence of caffeic acid in all tray-dried extracts. The retention times observed for PT10, PT15, and PT20 were 13.94, 12.19, and 13.92 minutes, respectively, with corresponding concentrations of 1.04, 1.07, and 1.03 mg/g ( Figure 3b-d). These results reflect consistent caffeic acid retention across varying extraction times, with PT15 slightly outperforming others. However, the overall concentration remained modest, suggesting that tray drying may limit phenolic preservation.

Freeze-dried extracts (PF)

Caffeic acid content in freeze-dried extracts was significantly higher: 3.15, 3.33, and 3.29 mg/g for PF10, PF15, and PF20, respectively ( Figure 4b-d). Retention times were consistent with the standard, confirming accurate identification. PF15 exhibited the highest caffeic acid content, indicating that 15 minutes of MAE under freeze-drying conditions is optimal for phenolic preservation. The increased levels of caffeic acid in PF samples may be attributed to the lower thermal stress during drying, which minimizes degradation of sensitive compounds. ²¹

The comparative analysis ( Figure 5) clearly illustrates that freeze-drying consistently outperformed tray drying in preserving caffeic acid content across all extraction durations. This emphasizes the role of gentle drying techniques in maintaining the chemical integrity of phytochemicals, supporting the use of freeze-drying for applications targeting high bioactive potency.

The TPC was highest in PF15, recorded at 70.46 ± 0.49 mg GAE/g extract, followed by PF10 and PF20. The differences were statistically significant ( p < 0.05), indicating that extraction time had a measurable impact on phenolic recovery ( Figure 6). Notably, the 15-minute extraction (PF15) demonstrated optimal balance between yield and stability, aligning with earlier research indicating that mid-range MAE durations improve phenolic extraction without inducing degradation. ^{23, 21} These results collectively underscore the effectiveness of microwave-assisted extraction at 15 minutes in conjunction with freeze-drying as the most favorable combination for preserving caffeic acid and phenolic content in P. amboinicus leaf extracts. The application of this optimized method is particularly advantageous in the development of high-performance cosmeceutical products where antioxidant activity is desired.

Antioxidant activity

The Plectranthus amboinicus leaf extract obtained under optimized conditions (PF15) demonstrated significant antioxidant activity, as assessed using the DPPH radical scavenging assay. The extract exhibited a markedly low IC ₅₀ value, indicating a strong ability to neutralize free radicals. Among the three extraction durations tested—10, 15, and 20 minutes—the PF15 extract showed the most potent antioxidant effect, with a statistically significant difference in IC ₅₀ values compared to PF10 and PF20 ( p < 0.05) ( Figure 7). These findings align with the work of, ²⁴ who reported the antioxidant potential of P. amboinicus. The enhanced activity at 15 minutes suggests that this extraction time enables maximal release and preservation of antioxidant phenolic compounds, including caffeic acid and other flavonoids. This result underscores the importance of extraction time optimization in maximizing the functional properties of herbal extracts. Such antioxidant efficacy supports the potential use of P. amboinicus extract in cosmetic formulations targeting oxidative stress-related skin issues, such as aging and environmental damage.

Antimicrobial activity of PF15

Inhibition against skin pathogens

The antimicrobial potential of PF15 was evaluated using the paper disc diffusion method. At a concentration of 1,000 mg/mL, PF15 exhibited clear zones of inhibition against S. aureus, S. epidermidis, and C. acnes. The diameter of the inhibition zones was significantly larger than those of the negative control (distilled water), indicating strong antibacterial efficacy ( Table 2). These results support the growing evidence that P. amboinicus possesses natural antimicrobial compounds effective against common skin pathogens. Its ability to inhibit C. acnes, a key contributor to acne development, reinforces its potential application in dermatological formulations such as anti-acne creams and antimicrobial cosmetics. ²³

Minimum Inhibitory and Bactericidal Concentrations (MIC & MBC)

The MIC and MBC values of the PF15 extract were determined using a broth microdilution assay. Results indicated that the MIC values against S. aureus, S. epidermidis, and C. acnes were 7.81, 3.91, and 3.91 mg/mL, respectively, while the corresponding MBC values were 31.25, 15.63, and 15.63 mg/mL ( Table 3). These data demonstrate that PF15 not only inhibits bacterial growth but also exhibits bactericidal effects at relatively low concentrations, particularly against C. acnes, a clinically relevant acne pathogen. The observed antibacterial potency is consistent with prior reports on the antimicrobial activities of P. amboinicus. ^{21, 25, 26} These findings provide strong support for the use of this plant extract in the formulation of herbal-based skin care products designed to prevent or treat bacterial skin infections.

Anti-inflammatory activity of PF15

Effect on THP-1 cell viability

The cytotoxicity of PF15 was assessed in human THP-1 monocytes using the PrestoBlue ^® viability assay. As illustrated in Figure 8, PF15 at concentrations ranging from 6 to 50 μg/mL did not significantly reduce cell viability compared to the untreated control group. Cell viability remained above 80% at all tested concentrations, indicating that PF15 was non-cytotoxic under the experimental conditions. A statistically significant increase in viability was observed at certain concentrations ( p < 0.01 and p < 0.001), suggesting a potential dose-dependent proliferative or protective effect on THP-1 cells.

Modulation of cytokine secretion in LPS-stimulated THP-1 macrophages

To evaluate the anti-inflammatory potential of PF15, the levels of pro- and anti-inflammatory cytokines were quantified in lipopolysaccharide (LPS)-stimulated THP-1 macrophages following treatment with PF15 at concentrations of 12, 25, and 50 μg/mL. LPS stimulation significantly increased the secretion of TNF-α, IL-1β, and IL-6 compared to the untreated control ( ^## p < 0.001). However, treatment with PF15 significantly reduced the levels of these pro-inflammatory cytokines in a dose-dependent manner (* p < 0.05 and ** p < 0.001), as shown in Figure 9. In contrast, the level of IL-10, a key anti-inflammatory cytokine, was significantly upregulated upon PF15 treatment, suggesting an immunomodulatory shift favoring resolution of inflammation. These results demonstrate that PF15 effectively suppresses pro-inflammatory signaling while enhancing anti-inflammatory responses in macrophages.

The IL-6/IL-10 ratio serves as a reliable biomarker for evaluating the balance between pro- and anti-inflammatory responses. As depicted in Figure 10, LPS stimulation significantly elevated the IL-6/IL-10 ratio, indicating a dominant pro-inflammatory state. Treatment with PF15 significantly reduced this ratio in a dose-dependent manner, suggesting a shift toward an anti-inflammatory phenotype and restoration of immune homeostasis.

The results of this study provide robust evidence that P. amboinicus leaf extract (PF15) exhibits significant anti-inflammatory activity through multiple mechanisms. The extract not only suppressed key pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) but also enhanced the production of IL-10, a cytokine critical for immune regulation and inflammation resolution. These findings are consistent with previous studies on plant-derived bioactive compounds exhibiting immunomodulatory effects in macrophage models. ^{27, 28} The observed inhibition of TNF-α and IL-6 aligns with established mechanisms of anti-inflammatory plant compounds that interfere with Toll-like receptor (TLR)-mediated signaling pathways. ²⁹ Suppression of IL-6 is particularly noteworthy, as its overexpression is implicated in the pathogenesis of chronic inflammatory diseases. ³⁰ The upregulation of IL-10 further supports the role of PF15 in promoting an anti-inflammatory environment, a mechanism also observed in other herbal interventions. ^{31, 32} Importantly, the decrease in the IL-6/IL-10 ratio suggests a rebalancing of immune responses, indicative of improved inflammatory resolution. ³³ Similar regulatory effects have been reported in studies involving flavonoids and polyphenols, ^{16, 17} highlighting the potential of PF15 as a natural anti-inflammatory agent.

Prototype cream formulation and evaluation

A topical cream was formulated using PF15 and compared against a control formulation lacking the extract ( Figure 11). The extract-based cream displayed a light green color, smooth consistency, and favorable spreadability. It emitted a mild, herbal-minty scent, characteristic of the extract, and exhibited no signs of phase separation, sedimentation, or instability ( Table 4). The measured pH of 5.5 aligns with the optimal range for topical applications and skin compatibility. ³⁴ These physical and organoleptic properties indicate the successful incorporation of P. amboinicus extract into a stable cosmetic matrix with potential applications in acne treatment and skin care product development. ³⁵

Accelerated stability assessment of PF15 cream

The PF15 cream underwent six thermal cycles (alternating storage at 4 ± 1 °C and 45 ± 1 °C) to simulate long-term environmental stress. The cream maintained a consistent texture, color, and fragrance across all cycles, with no evidence of phase separation or degradation. The pH remained stable at 5.5 throughout the testing period, confirming the formulation’s stability under extreme storage conditions. ^{36– 38}

Antibacterial efficacy of formulated PF15 cream

Following accelerated stability testing, the PF15 cream retained its antibacterial activity against S. aureus, S. epidermidis, and C. acnes. The inhibition zones measured 11± 1.00 mm, 12± 0.82 mm, and 12± 0.82 mm (Mean ± SD, n = 3) respectively ( Figure 12), confirming that the antimicrobial properties of the extract were not compromised during storage. These results support its efficacy as a topical agent targeting acne-associated pathogens. ^{39, 40}

Microbial contamination assessment

Microbiological testing of the PF15 cream was conducted in compliance with the Thai Cosmetics standard and ⁴¹ The cream was assessed for aerobic plate count (APC), yeast and mold contamination, and the presence of Escherichia coli, Staphylococcus aureus, and Salmonella spp. All results were within the acceptable limits, confirming the microbiological safety of the final product ( Table 5).

These findings confirm that the PF15 cream meets safety standards for cosmetic use, is free from pathogenic microorganisms, and is suitable for topical applications. Safety evaluations based on FDA guidelines—including cytotoxicity and microbial contamination assessments—indicate that the formulation is safe for consumer use. This aligns with previous safety studies of P. amboinicus in cosmetic applications. ⁴² The successful formulation of a stable, effective, and safe cream containing P. amboinicus leaf extract (PF15) demonstrates the practical potential of this plant as a bioactive ingredient in dermatological and cosmeceutical products. The extract retained its biological properties post-formulation and post-stability testing, making it a promising candidate for anti-acne, antimicrobial, and anti-inflammatory skincare applications.