Chiang Mai Journal of Science

Comparative Effects of Solvent Polarity on Phenolic and Flavonoid Contents, Antioxidant Activity, and Cytotoxicity of Litsea elliptica Leaf Extracts

1. INTRODUCTION

The Lauraceae family is an economically important group of flowering plants widely distributed throughout tropical and subtropical regions, particularly in Asia. Members of this family are extensively utilized as sources of medicines, spices, essential oils, fragrances, and timber. The genus Litsea comprises numerous evergreen trees and shrubs distributed across Asia, North and South America, and other tropical regions [1,2]. Species of Litsea have long been used in traditional medicine for the treatment of gastrointestinal disorders, diabetes, respiratory diseases, inflammation, and traumatic injuries [3–5]. Recent pharmacological studies have demonstrated that extracts and isolated compounds from Litsea species possess diverse biological activities, including antioxidant, antimicrobial, anti-inflammatory, anticancer, antidiabetic, analgesic, cardioprotective, and wound-healing effects. These activities are largely attributed to the presence of phenolic compounds, flavonoids, alkaloids, terpenoids, and essential oils [6–8].

Litsea elliptica Blume (synonym Litsea petiolata Hook.f.) is a tropical tree species native to Southeast Asia and commonly found in Thailand and Peninsular Malaysia. In Thailand, it is locally known as “Thummong” or “Tham Mung” and is traditionally consumed as a culinary herb and flavoring agent [9]. The leaves are widely used in local cuisine because their characteristic aroma resembles that of the giant water bug (Lethocerus indicus) [10, 11]. In addition to its culinary applications, L. elliptica has long been used in traditional medicine for the treatment of stomach ulcers, fever, headaches, and other ailments. Previous studies have reported that crushed leaves are applied to relieve headaches, while the leaves have also been used as an herbal remedy for stomach ulcers and fever. Furthermore, methanolic leaf extracts have been shown to inhibit the growth of Helicobacter pylori, supporting its traditional use in gastrointestinal disorders. Chemopreventive activity associated with a reduced occurrence of stomach cancer in Thailand and significant antimutagenic activity have also been reported [12, 13]. Phytochemical investigations have revealed that L. elliptica contains a diverse range of bioactive constituents, including phenolic compounds, flavonoids, alkaloids, terpenoids, and volatile compounds. Extracts and essential oils obtained from different plant parts have demonstrated antioxidant, antimicrobial, antifungal, insecticidal, mosquito-repellent, and cytotoxic activities. In particular, methanolic extracts of L. elliptica leaves exhibited strong antioxidant activity and moderate cytotoxicity against A549 human lung carcinoma cells, while phytochemical profiling identified compounds such as phytol, tocopherols, fatty acid derivatives, and phenolic constituents that may contribute to these biological effects [9, 14]. Similar antioxidant and antiproliferative activities have also been reported in other Litsea species and members of the Lauraceae family, suggesting that this plant group represents a promising source of bioactive compounds with pharmaceutical potential [15,16]. In addition to its biological activities, the safety profile of L. elliptica has also been investigated. Previous toxicological studies demonstrated that oral administration of L. elliptica essential oil produced no significant adverse effects in experimental animals and was considered safe under the tested conditions. These findings support the continued investigation of L. elliptica as a potential source of natural therapeutic agents.

Despite increasing evidence supporting the pharmacological potential of Litsea elliptica, previous studies have primarily focused on essential oils, methanolic extracts, volatile constituents, or specific plant parts. Moreover, earlier investigations mainly examined phytochemical composition, antioxidant activity, or cytotoxicity separately, with cytotoxic evaluations limited to A549 human lung carcinoma cells. Consequently, the influence of extraction solvent polarity on phytochemical recovery and the resulting antioxidant and cytotoxic activities of L. elliptica leaf extracts remains poorly understood. Furthermore, the cytotoxic potential of L. elliptica leaf extracts against human colorectal cancer cells has not been previously investigated. Water, methanol, and ethanol were selected as extraction solvents because they possess distinct polarity indices (9.0, 5.1, and 4.3, respectively), enabling evaluation of the influence of solvent polarity on the extraction of phytochemicals with different chemical characteristics. Since solvent polarity strongly affects the solubility and extraction efficiency of phenolics, flavonoids, alkaloids, terpenoids, and other secondary metabolites, variations in extraction solvent are expected to influence both phytochemical composition and biological activity [17, 18]. To date, a systematic comparison of aqueous, ethanolic, and methanolic extracts of L. elliptica leaves has not been reported. Furthermore, these findings suggest that L. elliptica may possess bioactive constituents relevant to gastrointestinal health, warranting further investigation into its potential against gastrointestinal-derived malignancies [12, 13]. Therefore, HCT116 human colorectal carcinoma cells were selected as a biologically relevant model to evaluate the cytotoxic potential of L. elliptica leaf extracts. Colorectal cancer is among the most prevalent malignancies worldwide and remains a leading cause of cancer-related mortality. Despite substantial advances in surgery, chemotherapy, radiotherapy, and targeted therapies, treatment resistance, tumor heterogeneity, and treatment-associated toxicities continue to limit therapeutic success. Consequently, natural products remain an important source of bioactive compounds for anticancer drug discovery.

Therefore, the present study aimed to evaluate the effects of solvent polarity on extraction yield, total phenolic content, total flavonoid content, antioxidant activity, and cytotoxic activity of L. elliptica leaf extracts obtained using water, ethanol, and methanol. Antioxidant capacity was assessed using DPPH and ABTS radical scavenging assays, while cytotoxic activity was evaluated against HCT116 human colorectal carcinoma cells and Vero normal cells. By integrating phytochemical, antioxidant, and cytotoxic evaluations, this study provides new insights into the influence of solvent polarity on the biological activities of L. elliptica leaf extracts and their potential application as natural antioxidant and anticancer agents.

2. MATERIALS AND METHODS

2.1 Chemicals, Reagents, and Standards
All solvents, reagents, and standards used were of analytical grade. The chemicals used in this study were distilled water, ethanol, methanol (all ≥99% purity for solvents), 1,1-diphenyl-2-1 picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), Trolox, gallic acid, quercetin, aluminum chloride hexahydrate, sodium carbonate (Na₂CO₃), Folin-Ciocalteu reagent, potassium persulfate, and other compounds. Additionally, materials including Dulbecco’s modified Eagle’s medium with high glucose (DMEM), penicillin/streptomycin solution (100×), fetal bovine serum (FBS), and 0.25% trypsin-EDTA (1×) were procured from GIBCO (Barcelona, Spain). Neutral red (NR) and all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Merck® (Darmstadt, Germany) unless otherwise specified.

2.2 Sample Collection and Preparation
Fresh samples of fully expanded leaves of Litsea elliptica Blume were collected from Khamong Subdistrict, Tha Mai District, Chanthaburi Province, Thailand (12.6858° N, 101.9650° E), during February–March 2023. Plant identification was confirmed by Assoc. Prof. Sutharat Khonkayan (Plant Taxonomist, Program of Industrial Biology, Roi-Et Rajabhat University), and a voucher specimen (RBFL-SK143) at the Herbarium of the Faculty of Liberal Arts and Science, Roi-Et Rajabhat University, Thailand.

The collected leaves were thoroughly washed with tap water to remove adhering debris, rinsed with distilled water, and dried in a hot-air oven at 50 °C for 48 h. The dried leaves were then ground into a fine powder using a laboratory blender. For ethanolic and methanolic extraction, 20 g of leaf powder was macerated separately in 100 mL of solvent in a stoppered 250 mL Erlenmeyer flask for 72 h at room temperature with occasional shaking. The extracts were filtered through Whatman No. 1 filter paper, and the residues were re-extracted twice under the same conditions. The filtrates from each extraction were combined. For aqueous extraction, 20 g of leaf powder was extracted with 100 mL of distilled water by decoction at 80 °C for 1 h. The extraction procedure was repeated three times, and the combined extracts were filtered through Whatman No. 1 filter paper. The combined filtrates were concentrated under reduced pressure using a rotary evaporator (Solventvap, Linbel, Zhengzhou, China) to obtain crude extracts. The extracts were stored at −20 °C in tightly sealed containers protected from light until analysis.

2.3 Total Phenolic Content (TPC) Determination
The TPC was quantified using an adapted Folin-Ciocalteu method [19]. The diluted extract (20 µL) was mixed with a solution of 7.5% Na₂CO₃ (80 µL) and 10% Folin-Ciocalteu’s phenol reagent (100 µL). Three replicates of each sample were used for the experiment, which was conducted in 96-well microplates and incubated for 30 minutes at about 25°C in the dark. The microplate reader (M965 Plus, Metertech Inc., Taipei, Taiwan) was used to measure the absorbance at 750 nm. Gallic acid was employed for the calibration curve, and the gallic acid dilution series (10-500 µg/mL) was used to express the data as mg GAE/g extract.

2.4 Total Flavonoid Content (TFC) Determination
The total flavonoid content (TFC) was determined using the aluminum chloride colorimetric method, adapted from Vongsak et al. [19]. Quercetin was used as the reference standard. Briefly, 100 µL of plant extract solution (100 µg/mL) or quercetin standard (1–100 µg/mL) was transferred into a 96-well microplate. Subsequently, 100 µL of 2% (w/v) aluminum chloride (AlCl₃) solution was added to each well and mixed thoroughly. The reaction mixture was incubated for 30 min at room temperature under dark conditions. After incubation, the absorbance was measured at 405 nm using a microplate reader. A calibration curve was constructed using quercetin, and the total flavonoid content was expressed as milligrams of quercetin equivalents per gram of extract (mg QE/g extract). All measurements were carried out in triplicate, and the results were reported as mean values.

2.5 DPPH Radical Scavenging Assay
The antioxidant activity of Litsea elliptica leaf extracts was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazine) radical scavenging assay, with IC₅₀ values reported as the primary outcome, following the method developed by Yen et al. [20]. A 0.1 mM DPPH solution was freshly prepared in methanol. Extract solutions were prepared over a broad concentration range (1–1000 µg/mL) to ensure adequate coverage of the full dose–response profile. In a 96-well microplate, 20 µL of extract solution was mixed with 180 µL of DPPH solution and incubated for 30 min at room temperature in the dark. Absorbance was measured at 517 nm using a microplate reader. Methanol mixed with DPPH solution served as the negative control, while Trolox was used as a positive control. The percentage of DPPH radical scavenging activity was calculated according to the equation:

% inhibition = [(A_control − A_sample) / A_control] × 100

where A_control is the mixture of methanol/ethanol/ water and DPPH solution and A_sample is the absorbance in the presence of extract and DPPH solution. Dose–response curves were constructed by plotting percentage the DPPH scavenging activity against extract concentration, and IC₅₀ values were determined by linear regression analysis of the linear portion of the concentration–inhibition curves. All experiments were performed in triplicate, and results were expressed as mean IC₅₀ ± SD.

2.6 ABTS Radical Scavenging Assay
ABTS radical scavenging activity was evaluated using IC₅₀ values as the primary outcome, following the method described by Mareček et al. [21]. The ABTS•⁺ radical cation was generated by reacting 7 mM ABTS with 2.45 mM potassium persulfate, and the mixture was incubated in the dark at room temperature for 12–16 h. Prior to analysis, the ABTS•⁺ solution was diluted with methanol to achieve an absorbance of 0.70 ± 0.02 at 734 nm. Extract solutions were prepared within a concentration range of 1–100 µg/mL to ensure reliable absorbance measurements, as higher concentrations resulted in near-complete radical scavenging and signal saturation approaching the lower detection limit at 734 nm. In a 96-well microplate, 50 µL of extract solution at the designated concentrations was mixed with 100 µL of the diluted ABTS•⁺ solution and incubated for 15 min at room temperature in the dark. Absorbance was measured at 734 nm using a microplate reader. Methanol mixed with ABTS•⁺ solution served as the negative control, while Trolox was used as the positive control. The percentage of ABTS radical scavenging activity was calculated according to the equation:

% inhibition = [(A_control − A_sample) / A_control] × 100

where A_control is absorbance of a mixtures of ABTS•⁺ solution with blank solvents and and A_sample is absorbance of the mixture of sample extract/standard Trolox and ABTS•⁺ solution. Dose–response curves were constructed by plotting percentage inhibition against extract concentration, and IC₅₀ values (µg/mL), defined as the concentration required to inhibit 50% of ABTS•⁺ radicals, were determined by linear regression analysis of the linear portion of the concentration–inhibition curves. All experiments were performed in triplicate, and results were expressed as mean IC₅₀ ± SD.

2.7 Cell Cytotoxicity Assay Using the Neutral Red Uptake Assay
Human colorectal carcinoma HCT116 cells and African green monkey kidney Vero cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/mL penicillin–streptomycin (Gibco, USA). Cells were maintained at 37°C in a humidified incubator containing 5% CO₂. Cell cytotoxicity was evaluated using the Neutral Red uptake assay, as previously described Pocasap et al. [22]. Briefly, HCT116 and Vero cells were seeded into 96-well plates at a density of 5 × 10⁴ cells/well and incubated overnight to allow cell attachment prior to treatment. The cells were then treated with different concentrations of L. elliptica leaf extracts (10–1000 µg/mL) or cisplatin (10–400 µg/mL) for 24 h. After treatment, the culture medium was removed and replaced with neutral red solution (50 µg/mL), followed by incubation for 2 h. The cells were washed once with phosphate-buffered saline (PBS), and the incorporated dye was extracted using acidified propan-2-ol. Absorbance was measured at 537 nm with a reference wavelength of 650 nm using an EnSight multimode plate reader (PerkinElmer, MA, USA). Cisplatin was used as a positive control, and untreated cells served as the negative control. Cell inhibition (%) was calculated as:

Cell inhibition (%) = ((ODcontrol - ODsample)/ODcontrol) x 100

The half-maximal inhibitory concentration (IC₅₀) was calculated from dose–response curves generated by plotting percentage cell inhibition against sample concentration. The selectivity index (SI) was calculated as follows:

SI = IC₅₀ (normal cells) / IC₅₀ (cancer cells)

2.8 Statistical Analysis
All experiments were performed in triplicate, and the results were expressed as the mean ± standard deviation. The IC₅₀ values for the DPPH and ABTS assays were ascertained via linear regression analysis of the linear portion of the concentration–inhibition curves, employing the regression equation and the coefficient of determination (r²). A one-way analysis of variance (ANOVA), succeeded by Tukey’s HSD post hoc test, was utilized to compare mean values across the different extracts, with statistical significance defined at p < 0.05. Furthermore, Pearson’s correlation coefficients were calculated to evaluate the relationships among total phenolic content, total flavonoid content, and antioxidant activity. All statistical analysis were statistically analyzed using the Statistical Package for the Social Sciences (SPSS, version 24).

3. RESULTS AND DISCUSSION

3.1 Extraction Yield
The extraction yield of L. elliptica leaves varied according to the solvent used. The aqueous extract produced the highest yield (27.10% of dry leaf weight), followed by the ethanolic (20.59%) and methanolic (18.50%) extracts. These differences reflect the influence of solvent polarity on extraction efficiency and the solubility of plant constituents. Water, ethanol, and methanol differ in polarity and therefore extract different groups of phytochemicals. The higher polarity of water facilitates the extraction of polar constituents, resulting in a greater overall extraction yield. Similar solvent-dependent extraction patterns have been reported for other Litsea species and medicinal plants [18, 23]. These findings demonstrate the importance of solvent selection in determining phytochemical recovery and the resulting biological activities of plant extracts.

3.2 Quantification of Total Phenolic and Total Flavonoid Contents
The total flavonoid content (TFC) of Litsea elliptica leaf extracts was determined using the aluminium chloride colorimetric assay and expressed as quercetin equivalents (QE). As shown in Table 1 and Figure 1, flavonoid content varied significantly among the extracts, indicating a marked influence of solvent polarity on flavonoid recovery. The ethanolic extract exhibited the highest TFC (126.8 ± 0.8 mg QE/g extract), followed closely by the methanolic extract (124.3 ± 0.9 mg QE/g extract), whereas the aqueous extract contained significantly lower levels (111.3 ± 1.9 mg QE/g extract). No significant difference was observed between the ethanolic and methanolic extracts (p > 0.05), although both contained significantly higher flavonoid concentrations than the aqueous extract.

The relatively high flavonoid contents obtained in all extracts indicate that L. elliptica leaves are a rich source of flavonoid compounds. Flavonoids are widely recognized for their antioxidant, anti-inflammatory, antimicrobial, and anticancer properties [24, 25]. The superior extraction efficiency of ethanol and methanol is consistent with the moderately polar nature of flavonoids, which are generally more soluble in aqueous organic solvents than in water alone. Similar solvent-dependent extraction patterns have been reported for other medicinal plants and members of the Litsea genus [26].

Total phenolic content (TPC) was determined using the Folin–Ciocalteu assay and expressed as gallic acid equivalents (GAE). Significant differences in phenolic content were observed among the extraction solvents (Table 1 and Figure 1). The ethanolic extract exhibited the highest TPC (322.9 ± 3.7 mg GAE/g extract), followed by the methanolic extract (274.8 ± 10.8 mg GAE/g extract), while the aqueous extract contained substantially lower phenolic levels (106.7 ± 7.6 mg GAE/g extract). These findings indicate that ethanol was the most effective solvent for extracting phenolic constituents from L. elliptica leaves.

Comparison with other Litsea species further highlights the phytochemical richness of L. elliptica. Methanolic extracts of L. polyantha were reported to contain 511.47 ± 22.30 mg GAE/g extract and 230.79 ± 5.44 mg QE/g extract of total phenolics and flavonoids, respectively [26]. Although direct comparisons should be interpreted cautiously because of differences in plant material, extraction procedures, and analytical methods, the relatively high phenolic and flavonoid contents observed in the present study indicate that L. elliptica is a valuable source of bioactive phytochemicals. Notably, ethanol extraction yielded the highest phenolic recovery, supporting its suitability for the development of antioxidant-rich food, nutraceutical, and pharmaceutical products.

3.3 Antioxidant Activity of L. elliptica Leaf Extracts
The DPPH and ABTS assays are based on different radical species and reaction mechanisms, thereby providing complementary information regarding antioxidant activity. DPPH primarily evaluates the scavenging capacity of lipophilic antioxidants, whereas ABTS is applicable to both hydrophilic and lipophilic antioxidant systems [21, 27]. The use of both assays therefore enabled a more comprehensive assessment of the antioxidant potential of L. elliptica leaf extracts.

As shown in Figure 2, all extracts exhibited concentration-dependent radical scavenging activity in both assays, although antioxidant potency varied markedly according to the extraction solvent. Based on IC₅₀ values (Table 2), antioxidant activity followed the order: ethanol extract > methanol extract > aqueous extract. In the DPPH assay, the ethanolic and methanolic extracts exhibited strong antioxidant activity, with IC₅₀ values of 14.9 ± 0.5 and 15.1 ± 1.1 µg/mL, respectively, whereas the aqueous extract showed considerably weaker activity (IC₅₀ = 585.5 ± 10.2 µg/mL). Similarly, in the ABTS assay, the ethanolic and methanolic extracts demonstrated potent radical scavenging activity with IC₅₀ values of 2.6 ± 0.1 and 2.9 ± 0.1 µg/mL, respectively, both of which were lower than that of Trolox (IC₅₀ = 5.7 ± 0.1 µg/mL). In contrast, the aqueous extract exhibited only moderate activity (IC₅₀ = 83.2 ± 0.2 µg/mL). According to the classification proposed by Moga et al. [21, 28], the ethanolic and methanolic extracts can be considered highly active antioxidants, whereas the aqueous extract ranged from moderate to weak depending on the assay employed.

Previous studies have also demonstrated the antioxidant potential of L. elliptica. Goh et al. [9] reported ABTS IC₅₀ values of 116.24 ± 3.47 and 326.28 ± 4.72 µg/mL for methanolic extracts of young and mixed L. elliptica leaves, respectively. The methanolic extract evaluated in the present study exhibited a substantially lower ABTS IC₅₀ value (2.9 ± 0.1 µg/mL), suggesting strong radical-scavenging activity. Variations among studies may be attributed to differences in geographical origin, plant developmental stage, environmental conditions, and extraction methodology. While Goh et al. investigated young and mixed leaves collected in Brunei Darussalam using Soxhlet extraction, the present study examined fully expanded leaves collected from eastern Thailand and extracted by maceration. Despite these methodological differences, both studies consistently indicate that L. elliptica leaves possess considerable antioxidant potential.

3.4 Correlation Between Total Phenolic Content, Total Flavonoid Content, and Antioxidant Capacity
Pearson correlation analysis was utilized to assess the impact of phenolic and flavonoid components on the antioxidant capacity of L. elliptica leaf extracts. This method allowed for an investigation of the relationships among total phenolic content (TPC), total flavonoid content (TFC), and the IC₅₀ values obtained from the DPPH and ABTS assays (see Table 3). A strong positive correlation was observed between the IC₅₀ values obtained from the DPPH and ABTS assays (r = 1.000, p < 0.001), indicating good agreement between the two methods in evaluating the radical scavenging capacity of the extracts.

Furthermore, TPC was strongly positively correlated with TFC (r = 0.983, p < 0.001), indicating that extracts with higher phenolic contents also tended to contain higher flavonoid levels. This finding suggests that flavonoids may contribute substantially to the overall phenolic content of the extracts. In addition, both TPC and TFC exhibited strong negative correlations with DPPH and ABTS IC₅₀ values (r = −0.972 to −0.979, p < 0.001), indicating that extracts containing higher levels of phenolic and flavonoid compounds generally possessed greater antioxidant activity.

3.5 L. elliptica Leaf Extracts Exhibited Cytotoxicity Against HCT116 and Vero Cell Lines
The cytotoxic effects of L. elliptica leaf extracts were evaluated against HCT116 human colorectal carcinoma cells and Vero (African green monkey kidney) cells, which served as a normal cell model for assessing extract safety. All extracts exhibited dose-dependent cytotoxic activity against HCT116 cells (Figure 3).

Interestingly, the cytotoxicity of L. elliptica leaf extracts against HCT116 cells did not parallel their antioxidant activity, total phenolic content (TPC), or total flavonoid content (TFC). Although the ethanolic extract exhibited the highest TPC, TFC, and antioxidant activity, it showed lower cytotoxicity against HCT116 cells than the methanolic and aqueous extracts. A similar observation was reported by Goh et al. [9], who demonstrated that extracts with higher antioxidant activity were not necessarily the most cytotoxic toward A549 lung carcinoma cells. These findings suggest that the phytochemicals responsible for antioxidant activity may differ from those mediating cytotoxic effects.

Previous phytochemical investigations of L. elliptica have identified diverse classes of bioactive compounds, including flavonoid glycosides (quercitrin and isoquercitrin), alkaloids (isoboldine and reticuline), nucleosides (uridine), and steroid glycosides [33]. Therefore, the selective cytotoxicity observed in the aqueous extract may be attributed to polar water-soluble constituents that were not adequately represented by the total phenolic and total flavonoid measurements. In addition, aqueous extraction may preferentially enrich such polar bioactive compounds while reducing the co-extraction of less polar constituents associated with toxicity toward normal cells [34-35]. This interpretation is consistent with the markedly lower toxicity of the aqueous extract toward Vero cells and its substantially higher selectivity index (SI = 8.42) compared with the methanolic extract, ethanolic extract, and cisplatin.

Although phenolic compounds and flavonoids may contribute to cancer cell inhibition through redox-modulating mechanisms, other classes of phytochemicals may play a more important role in mediating selective cytotoxicity [36-37]. The primary objective of the present study was to evaluate the biological activity and preliminary safety of L. elliptica leaf extracts rather than to identify the individual bioactive compounds responsible for the observed effects. Therefore, detailed chemical characterization using advanced analytical techniques such as HPLC, LC-MS, or GC-MS was beyond the scope of the current work and remains an important area for future investigation.

The pronounced activity of the aqueous extract against HCT116 cells, together with its low toxicity toward normal cells, highlights its potential as a source of selective anticancer compounds. To our knowledge, this is the first report demonstrating selective cytotoxicity of an aqueous extract of L. elliptica against HCT116 colorectal cancer cells. Further studies are currently planned to investigate the mechanisms underlying this activity, particularly intracellular reactive oxygen species (ROS) generation and apoptosis-related pathways. In addition, HPLC, LC-MS/MS-based phytochemical profiling and bioassay-guided fractionation will be performed to identify the major active constituents responsible for the observed selective cytotoxicity.

4. CONCLUSIONS

The present study demonstrates that solvent polarity significantly influences the extraction yield, phytochemical composition, antioxidant activity, and cytotoxic effects of Litsea elliptica Blume (syn. Litsea petiolata Hook.f.) leaf extracts. Although the aqueous extract produced the highest extraction yield, ethanol and methanol were more effective in extracting phenolic and flavonoid compounds. The ethanol extract exhibited the highest total phenolic and flavonoid contents and consequently showed the strongest antioxidant activity in both DPPH and ABTS assays. Correlation analysis revealed strong inverse relationships between phenolic and flavonoid contents and antioxidant IC₅₀ values, confirming the important contribution of these compounds to the antioxidant capacity of the extracts. Cytotoxicity evaluation against HCT116 colorectal cancer cells revealed that the methanolic and aqueous extracts exhibited comparable inhibitory activities. Notably, the aqueous extract demonstrated the highest selectivity toward HCT116 cells (SI = 8.42), indicating substantially lower toxicity toward normal Vero cells compared with the other extracts and the positive control, cisplatin. These findings suggest that L. elliptica leaves contain bioactive constituents with both antioxidant and selective anticancer potential.

Overall, L. elliptica leaves represent a promising natural source of antioxidant and anticancer compounds. However, further studies involving phytochemical profiling, bioassay-guided isolation of active constituents, mechanistic investigations, and in vivo evaluation are required to confirm the therapeutic potential of the observed biological activities.

ACKNOWLEDGEMENTS

The authors of the research paper express their sincere gratitude to the Faculty of Pharmacy, Nakhon Ratchasima College, Nakhon Ratchasima province for providing all the necessary support for this work. We extend our gratitude to Assist. Prof. Dr. Nichakan Peerakham and Assist. Prof. Suthharat Khonkayan for her support in plant identification and collection.

AUTHOR CONTRIBUTIONS

Kwanchayanawish Machana, Sirirat Phakpaknam and Anusorn Thampitak: Conceptualization; Methodology; Formal analysis, Conceptualization, and Writing –original draft. Pattarakorn Sirikul and Pornpimol Namwichaisirikul: Data curation and Formal analysis. Atcharee Chatsuwannakij and Chonlada Jadprakob: Visualization; Formal analysis and Investigation. Nitcha Prapruttipong and Wuttipat Khamphiranon: Supervision, Software and Validation. Kanokwan Waiyanont, Natcha Namsiripongpun and Atthasit Saengthong: Writing - Reviewing and Editing, Project administration. Natthida Weerapreeyakul: Conceptualization, Methodology, Cell Culture Assay, Formal Analysis. All authors have read and agreed to the published version of the manuscript.

CONFLICT OF INTEREST STATEMENT

The authors are required to declare whether or not they hold any conflicting interests.

DECLARATION OF USE OF GENERATIVE AI

The authors acknowledge using generative AI tools, like OpenAI's ChatGPT, solely for language editing and improvement of clarity. The tool was not used to generate or modify experimental data, results, or citations. All content was carefully reviewed and approved by the authors, who take full responsibility for the final manuscript.

FUNDING

This research was financially supported by Nakhonratchasima College, Thailand.

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