Tropical Journal of Natural Product Research
Tropical Journal of Natural Product Research (TJNPR) is the official scientific publication of the Natural Product Research group, University of Benin; and it is available in both printed and electronic formats. The journal is published monthly. Tropical Journal of Natural Product Research (TJNPR) is a peer-reviewed, open access online international journal which publishes original research papers. The journal welcomes submission from scholars and experts for possible publication from all over the world. The scope of the journal includes: Pharmaceutical research, chemistry and biochemistry of naturally occurring compounds, biological evaluation of crude extracts, ethnomedicine, traditional and complementary medicine, ethnopharmacology, biomedical research, Biotechnology, Evaluation of natural substances of land and sea and of plants, microbes and animals, pharmacognosy, bioavailability, clinical, pharmacological, toxicological studies and pharmacokinetics of phytochemicals, Isolation and characterization of compounds, structure elucidation, synthesis and experimental biosynthesis of natural Product as well as developments of methods in these areas are welcomed in the journal. Finally, research papers in fields on the chemistry-biology boundary, papers discussing the screening of natural Product for biological activity against different health targets are also encouraged.
University of Benin and Natural Product Research Group www.uniben.edu
ERRATUM: Impact of some Bioactive Compounds of Flavonoid-Rich Fraction of Monodora tenuifolia, Benth on Glucose Concentration and Certain Proteins Associated with Glucose Metabolism using Molecular Docking Approach
Hyperglycemia is a hallmark of diabetes mellitus, known to be a metabolic disorder caused by excess blood glucose due to lack of insulin secretion, or cellular insensitivity to insulin or damage to pancreatic β-cell. This study focused on the potential of flavonoid-rich fraction of M. tenuifolia seeds (FRFMTS) to reducing plasma glucose concentration in diabetic rats and determine the molecular interaction of some phytochemicals in FRRMTS against certain proteins associated with diabetes mellitus. The plant sample was pulverized and extracted at 1g in 5 ml of 80% v/v ethanol to obtain hydro-ethanol extract subjected to solvent-solvent fractionation to obtain the FRFMTS used for HPLC screening. Seven (7) groups of male Wistar rats containing six (6) rats each were used. The groups were normal control, 25 mg/kg FRFMTS, 50 mg/kg FRFMTS, diabetic control, diabetic rats + 25 mg/kg FRFMTS, diabetic rats + 50 mg/kg FRFMTS and diabetic rats + metformin (6.67 mg/kg). A glucose oxidase kit was used to determine blood glucose concentrations, while the molecular interactions with some target proteins were docked using the extra precision (XP) algorithm. The results revealed a significant increase in the plasma glucose of diabetic control group in comparison to normal control group. Diabetic rats treated with 25 mg/kg FRFMTS, 50 mg/kg FRFMTS and metformin showed that glucose concentrations significantly reduced compared to diabetic control group. The molecular docking model revealed highest binding affinity of quercetin to acetyl-CoA carboxylase, glucose-1-transporter, glycogen synthase kinase 3β, glucagon-like peptide 1 and insulin receptor kinase. This study suggests that FRFMTS possesses glucose lowering ability and quercetin present in FRFMTS binds effectively to proteins linked to diabetes and could elicit antihyperglycemic effect.
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Ethnobotanical and Ethnopharmacological Study of Medicinal Plants Used for Respiratory Diseases in the Sefrou Region, Morocco
The wide variety of flora in the Sefrou region indicates the great potential of traditional knowledge. Despite being a perfect place to research aromatic and medicinal plants, this area has not yet received much attention. In Morocco, respiratory disorders represent a major public health concern. Our study aims to investigate the traditional knowledge of the Sefrou population regarding the use of medicinal plants to treat respiratory ailments. The survey included 199 individuals from various age groups, gender, study level, family situation, income, and source of information. Data was gathered and analyzed to determine parameters, including fidelity level and family importance value. The floristic analysis of this study identified 38 plant species belonging to 19 families. The two dominant families were Lamiaceae (12 species) and Asteraceae (4 species). The data shows the frequency of Mentha pulegium L., Artemisia herba alba Asso, and Origanum compactum Benth with RFC values of 59.4%, 49.7%, and 35.5%, respectively. The infusion was the most common preparation method. According to our results, the leaf was the most commonly used part to treat respiratory diseases (PPV=0.397), and Tuberculosis was the most treated category with an ICF=0.97. The current survey indicates that, despite the advancement of modern medicine, the indigenous people of the Sefrou region mostly utilize medicinal plants to treat ailments. It is also necessary to carry out laboratory studies to extract and determine these plants phytochemical constituent, and identify their toxicological effects.
GC-MS Analysis, Determination of Total Flavonoid Content, Sun Protection Factor, and In Vitro Anti-inflammatory Activity of Ethanol Extract of Kokang (Lepisanthes amoena) Leaves
Lepisanthes amoena, locally known as kokang, is an indigenous plant from Kalimantan. The plant is traditionally used by the Dayak and Kutai tribes in East Kalimantan (Indonesia) for the treatment of skin inflammation. This study aimed to determine the phytochemical constituents, total flavonoid content, sun protecting factor (SPF), and anti-inflammatory activity of the ethanol extract of L. amoena leaves. Powdered L. amoena leaves were extracted by maceration in 96% ethanol. The chemical constituents of the extract were identified by gas chromatography-mass spectrometry (GC-MS). Total flavonoid content was determined using colorimetric method. The determination of sun protecting factor (SPF) was carried out using a UV-Vis spectrophotometer at a wavelength range of 290-320 nm. Anti-inflammatory activity was tested using the protein denaturation inhibition assay. GC-MS analysis identified five key volatile constituents, with Benzyl (1,2,3-thiadiazol-4-yl) carbamate (29.37%) identified as the major compound. The total flavonoid content of ethanol extract of L. amoena leaves was 7.105 ± 0.065% w/w QE. Results of the SPF determination indicate that L. amoena leaf extract has "ultra" UV-protection activity at 850–950 ppm with SPF values ranging from 16.351 to 20.543. The ethanol extract of L. amoena leaves exhibited strong anti-inflammatory activity, with IC₅₀ value of 18.933 ppm in the protein denaturation inhibition assay, which was significantly lower than that of diclofenac sodium (IC₅₀ = 23.006 ppm). These findings suggest that L. amoena leaf extract holds potential as a natural source of anti-inflammatory and photoprotective agents.
Innovative Formulation of Solid Lipid Nanoparticle Loaded with Carrot Seed Essential Oil for Potential Antioxidant Activity and Sun Protection
The discovery of natural sun protection and antioxidant agents is vital, but the low bioavailability of these ingredients often limits their use. This study aimed to formulate and characterize a serum containing SLN-CSEO-APs (solid lipid nanoparticle-carrot seed essential oil) with anti-aging and photoprotective properties to evaluate its antioxidant and sun protection effects. SLN was prepared using hot homogenization, heating lipids above their melting point, and dissolving the emulsifier in water. The physical characteristics, stability, antioxidant activity, and sun protection efficacy of the serum were evaluated. Antioxidant activity was assessed using DPPH and ABTS methods, while sun protection was measured by ultraviolet B (UVB) and ultraviolet A (UVA) protection. SLN-CSEO-APs serum showed a spherical morphology with a particle size of 257.12 nm ± 28.82, a polydispersity index of 0.50 PDI ± 0.00, and a zeta potential of -23.19 mV ± 0.81. The serum hydrogel had a pH within the physiological range for skin, with optimal viscosity, spreadability, and adhesion. Over time, serum stability decreased, as indicated by the reduction in all parameters. The serum demonstrated strong antioxidant potential, with IC50 values for ABTS and the positive control closely matched. Antioxidant activity was moderate, with values of 0.157 mg AAE/g dw for DPPH and 0.644 mg TE/g dw for ABTS. UVB protection was medium (13.549 ± 0.827), and UVA showed PA++ (4.202 ± 0.040). SLN-CSEO-APs serum provides significant antioxidant and sun protection, making it a promising skincare ingredient.
Formulation Development and Quality Evaluation of a Topical Cream Containing a Polyphenol-Rich Extract from Spilanthes acmella Murr.
This study aimed to develop and evaluate a stable oil-in-water (O/W) topical cream incorporating a polyphenol-rich extract from Spilanthes acmella Murr. (S. acmella), a plant recognized for its antioxidant potential. The extract, obtained via reflux extraction with 80% ethanol, demonstrated notable free radical scavenging activity in the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, exhibiting an IC₅₀ (Inhibitory concentration 50) value of 563.26 µg/mL. The cream was prepared using the hot emulsification method. Formulation variables, including oil phase concentration and composition (olive oil and cetyl alcohol), the emulsifier system (Tween 80/Span 80 blend), stirring duration, and humectant type, were systematically optimized to achieve the desired physicochemical stability and aesthetic properties. The optimized formulation contained 10% S. acmella extract, a 15% oil phase, a 10% emulsifier mix, and 10% glycerin. Three pilot batches were produced, all yielding a smooth, homogeneous, grayish-green cream with a pH of 5.69 ± 0.20, which is consistent with the physiological pH of human skin. The cream demonstrated good stability under stress conditions, excellent spreadability (29.87 ± 0.33 cm²), and a consistent total polyphenol content of 56.82 ± 0.69 mg gallic acid equivalents per gram. Microbial limit tests confirmed the product’s microbiological safety, and in vivo dermal irritation tests in rabbits (ISO 10993-10) showed no signs of skin irritation. Overall, the formulated cream was found to be stable, safe, and rich in antioxidant compounds, suggesting its potential as a promising dermo-cosmetic product.
Ethanol Extract of Cosmos caudatus Attenuates Oxidative Stress and Inflammation in a Testosterone-Induced Benign Prostatic Hyperplasia Rat Model via Reduction of Malondialdehyde, Interleukin-6, and Tumor-Infiltrating Lymphocytes Activity
Benign Prostatic Hyperplasia (BPH) is a common age-related urological condition driven by chronic inflammation and oxidative stress, prompting the search for safer phytotherapeutic alternatives. This study evaluated the in vivo efficacy of ethanolic extract of Cosmos caudatus leaves in modulating the oxidative-inflammatory axis underlying testosterone-induced benign prostatic hyperplasia. Blood samples (0.5–1 mL) were collected from the retro-orbital plexus at baseline (T0), post-induction (T1), and post-treatment (T2) of thirty male Rattus norvegicus obtained from the Laboratory Animal Unit, Universitas Sebelas Maret, were allocated into five groups (n=6): Normal, Control, Positive Control receiving Finasteride, and treatment groups administered C. caudatus extract at 125 mg/kg or 250 mg/kg. BPH was induced using testosterone propionate (3 mg/kg/day, s.c.) for 28 days, followed by a 28-day treatment period. Serum malondialdehyde, interleukin-6, stromal tumor-infiltrating lymphocytes, prostate weight, and prostate index were evaluated. Testosterone induction significantly increased oxidative stress, systemic inflammation, and stromal lymphocytic infiltration, together with notable prostate enlargement (p<0.05). Treatment with both doses of C. caudatus extract markedly reduced malondialdehyde and interleukin-6 levels, suppressed tumor-infiltrating lymphocytes infiltration, and decreased prostate weight and prostate index compared with the untreated benign prostatic hyperplasia group (p<0.05), showing therapeutic effects comparable to Finasteride. These findings demonstrate that C. caudatus effectively attenuates benign prostatic hyperplasia progression through strong antioxidant and anti-inflammatory mechanisms, indicating its potential as a promising phytotherapeutic candidate targeting the oxidative-inflammatory axis in benign prostatic hyperplasia.
Cytotoxic Activity Against Breast Cancer of Ethyl Acetate Fraction of the Fruiting Bodies Ganoderma multiplicatum through Inducing Apoptosis
Breast cancer remains the most frequently diagnosed malignancy among women worldwide, underscoring the need for safer and more effective therapeutic agents, particularly those derived from natural medicinal resources. This study evaluated the cytotoxic and apoptosis-inducing activities of the ethyl acetate fraction (GMEA) obtained from the 96% ethanol extract of Ganoderma multiplicatum (GME) against human breast cancer MCF-7 cells. Triterpenoid quantification using the vanillin assay revealed enrichment of these compounds in GMEA (5.84 ± 1.41 mg/g) compared with GME (4.83 ± 0.40 mg/g). GMEA significantly reduced MCF-7 cell viability in a dose-dependent manner, with an IC50 value of 147.69 µg/mL after 48 h. Flow cytometry demonstrated marked increases in both early (0.8% -16.1%) and late apoptosis (0.7% - 2.8%). Importantly, GMEA exhibited minimal cytotoxicity toward human fibroblasts at concentrations ≤ 400 µg/mL, suggesting selective activity against cancer cells. Collectively, these findings indicate that triterpenoid enriched fraction from Ganoderma multiplicatum exert cytotoxic effects through apoptosis induction and highlight this species as a promising and underexplored source of natural anticancer agents.
Traditional Wisdom on Cultivated Edible Plants: Ethnobotany of the Kui Ethnic Group in Sangkha District, Surin Province, Northeastern Thailand
This study explores the ethnobotanical knowledge of cultivated edible plants among the Kui community in Sangkha District, Surin Province, Northeastern Thailand, emphasising their cultural, nutritional, and medicinal roles. A total of 77 species from 44 families were documented, with Fabaceae and Zingiberaceae being the most represented. Introduced species (62.34%) were more common than native species (37.66%), reflecting historical crop dissemination and cultural adoption, while native species continue to play important roles in cultural identity and ecosystem resilience. Herbs and trees were the predominant growth forms, supporting both immediate and long-term subsistence needs. Analyses of use value (UV), fidelity level (FL), and informant consensus factor (Fic) highlighted key species central to local livelihoods and healthcare, including Citrus hystrix DC., Morinda citrifolia L., Oryza sativa L., and Piper sarmentosum Roxb., demonstrating multifunctionality in both culinary and medicinal contexts. Leaves, rhizomes, and inflorescences were the most commonly used plant parts, suggesting deliberate selection for nutritional and pharmacological benefits. These cultivated edible plants not only sustain traditional food practices but also support livelihoods, income generation, and intergenerational knowledge transfer. The findings provide a basis for future phytochemical, nutritional, socio-cultural, and agroecological studies aimed at enhancing biodiversity conservation, food security, and sustainable use of ethnobotanical resources.
Comparative Chemotype Characterization and Antibacterial Activity of Essential Oils Obtained from the Leaves of Three Litsea species from Vietnam
The genus Litsea (family Lauraceae) is well known for its essential oils, which are traditionally valued for their richness in monoterpenes and sesquiterpenes—compounds with notable antibacterial and antioxidant properties. This study analyzed the chemical composition and antibacterial effects of essential oils obtained from the leaves of three Litsea species: Litsea cubeba (PH1), Litsea glutinosa (PH2), and Litsea verticillata (PH3). Essential oils were obtained through hydrodistillation and analyzed using gas chromatography–mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID). The results showed significant chemical differences among the three species. PH1 oil displayed a typical profile dominated by 1,8-cineole (22.60 %), sabinene (17.12 %), α-pinene (6.21 %), and β-pinene (5.01 %). In contrast, PH2 was dominated by β-elemene (12.78 %), β-caryophyllene (10.13 %), and linalool (9.60 %), while PH3 contained a high amount of β-caryophyllene (19.75 %), β-caryophyllene epoxide (15.05 %), α-caryophyllene (8.50 %), and much lower levels of 1,8-cineole (2.05 %). The antibacterial activity of the oils was evaluated against four bacterial strains: Escherichia coli, Salmonella typhi, Staphylococcus aureus, and Pseudomonas aeruginosa, using the agar well diffusion method. All essential oils demonstrated inhibitory activity, with the strongest effects (inhibition zone up to 20.1 ± 0.4 at 60 % against S. aureus) exhibited by PH1, which is rich in monoterpenes. These findings, therefore, highlight the chemical and biological diversity among Litsea species and emphasize their potential as sources of natural antibacterial agents for pharmaceutical and cosmetic applications.
Computational Insights into the Interaction of Silver Nanoparticles from Mimosa pudica linnaeus with Tumor Necrosis Factor-α and Heat Shock Protein 60: An In-Silico Approach
Mimosa pudica linnaeus has been recognised for its diverse range of bioactive compounds, with antibacterial, anti-inflammatory, hepatoprotective, and antiparasitic effects. This study aims to investigate the inhibitory potential of bioactive compounds from Mimosa pudica linnaeus against Blastocystis sp., TNF-α, and HSP60 using an in-silico method. The analysis involved predicting biological activity using PASS, evaluating drug-likeness according to Lipinski’s Rule of Five, and assessing the ADME properties and toxicity profiles of Mimosa pudica linnaeus phytochemicals. The PASS analysis showed that Apigenin, p-Hydroxybenzoic acid, Quercetin, and Naringenin had the highest probability of activity across various categories. Among the 18 compounds analysed, 13 met all Lipinski’s criteria, while 17 showed high gastrointestinal absorption. However, 9 compounds exhibited high toxicity effects. Molecular docking analysis revealed that Jasmonic acid, Naringenin, and Monoamidomalonic acid exhibited lower binding affinities than metronidazole (a standard drug) against Blastocystis sp. Luteolin and Naringenin also showed potential as competitive inhibitors of TNF-α. However, 4-(2-phenylethyl) phenol and Fisetin showed the lowest binding affinity scores of -7.47 and -7.75 kcal/mol, respectively, against HSP60. Molecular docking results showed that the ligands interacted with the target proteins via hydrophobic and hydrogen-bond interactions, suggesting the formation of stable protein-ligand complexes. These results show that bioactive compounds of Mimosa pudica linnaeus have potential as therapeutic candidates with promising anti-inflammatory properties.
The Therapeutic Effect of Bidens Pilosa as Anti-Colon Cancer in Alcohol- Induced Rats: In Vivo and In Silico Method
Bidens pilosa leaves ethanol extract (EBP) has been evaluated for its anti-inflammatory, anti-mycobacterium, and immunomodulatory properties. However, the potential of EBP against colon injury in alcoholic rats remains elusive. In this regard, the potential EBP was investigated for its anti-cancer effect in alcohol-induced colon injury. The EBP with three different doses was treated for six weeks in the alcohol animals. Blood level and colon tissues were examined in alcohol-induced colon. The modern pharmacological analysis was conducted to prove the potential of EBP against colon carcinogenesis. The results defined that alcohol administration significantly disrupted the goblet and crypt cells in the colon tissue. Alcohol decreased the blood parameters; notably, an MCV level of 52.68 ± 1.35 is lower than the control group at 55.5 ± 4.43 (P < 0.05). In the alcohol model, EBP supplementation (500 and 750 mg/kg) improved the colon histological damage and restored the blood parameters. The bioinformatic studies exhibited that the EBP bioactive compounds regulated the signaling pathway of main targets, notably CASP3, EGFR, CCL2, and STAT3, in the alcohol-induced colon carcinogenesis. These results are considered to be the underlying reference to investigate the potential EBP against alcohol-induced colorectal injury.
ERRATUM: Synergistic Inhibition of Chronic Myeloid Leukemia: Combining Imatinib and Naringin to Overcome Drug Resistance
Chronic Myeloid Leukemia (CML) treatment with Imatinib often encounters the challenge of drug resistance, limiting its long-term efficacy. This study aimed to evaluate the cytotoxic effects and underlying mechanisms of Naringin, both alone and in combination with Imatinib, on Imatinib-sensitive (K562-S) and Imatinib-resistant (K562-R) CML cell lines. Imatinib resistance in K562 cells was induced through a stepwise exposure to the drug. Cytotoxicity was assessed using the MTT assay, while flow cytometry was employed to determine the expression levels of P-glycoprotein (P-gp), extracellular signal-regulated kinase (ERK), and protein kinase B (AKT). Molecular docking studies were conducted to explore potential interaction mechanisms. The combination treatment significantly reduced cell viability and downregulated P-gp, ERK, and AKT expression in both cell lines, indicating a synergistic effect. These findings suggest that Naringin can potentiate the therapeutic efficacy of Imatinib, offering a promising strategy to overcome drug resistance and improve clinical outcomes in CML management.
Characterization and Temperature-Dependent Adsorption Potential of Hydrothermally Synthesized Manganese Oxide Nanoparticles
Water pollution usually results from discharge of untreated or partially treated human and industrial waste into water bodies, and nanomaterials are increasingly being recognized as a sustainable alternative remediation approach. The aim of the present study is to determine the structural properties and possible adsorption property of hydrothermally synthesized MnO2 nanoparticles. The synthesized nanoparticles were characterized using X-ray diffraction, scanning electron microscopy, energy-dispersive X-ray spectroscopy, Brunauer-Emmett-Teller surface area analysis, Fourier-transform infrared spectroscopy and UV-Visible spectroscopy. The nanoparticles primarily exhibited small crystallite sizes, structural disorder, and mostly amorphous material. Manganese was dominant in the nanoparticles as evidenced by surface shape and chemical composition but diminished with rising temperature. The surface area peaked at 110oC and decreased at higher temperatures possibly due to pore collapse and sintering effect, suggesting that annealing temperature had a significant impact on the mesoporous structure. FTIR and UV-Visible data suggest the presence of surface hydroxyl groups evidenced by moisture absorption, an O-Mn-O stretching that promotes hydrogen bonding and bandgap energies that allow for possible visible-light photocatalysis. Incorporating these results with existing research, the adsorption mechanism is expected to include electrostatic interactions controlled by surface charge, chemisorption via redox-active Mn sites, cation exchange regulated by potassium intercalation, and synergistic physisorption. The study also suggests that controlled thermal treatment enhanced adsorption performance as nanoparticles annealed at 90-110°C are expected to exhibit optimal adsorption qualities. Overall, findings from this study offer a starting point for producing efficient MnO2-based adsorbents capable of removing organic pollutants and heavy metals in aquatic environment.
A Randomized-Controlled Trial Study of Telecare-Based Interprofessional Collaboration: A New Strategy in Tuberculosis Treatment
To provide optimal treatment for tuberculosis (TB), an interprofessional team is required, but most hospitals cannot provide such standards. A potential alternative method for better medication adherence is telecare. This study aims to develop a telecare service based on interprofessional collaboration to elevate medication adherence and enhance therapy outcomes for individuals with tuberculosis. The research was a single-blind randomized control trial. Data collection was carried out at three points: baseline, one month, and three months post-intervention. The primary outcome measured was the bacterial conversion, while secondary outcomes included medication adherence and the quality of TB patients’ lives. Using an individually randomized sample, 100 patients (50 patients per group) were selected. The instruments used included the medication adherence rating scale-5, knowledge questionnaire, and St. George's Respiratory Questionnaire. Data were analyzed with Mann-Whitney test. A significant difference was observed in the knowledge scores between the control and experimental groups, indicated by a p-value of 0.007 (p < 0.05). The experimental group showed a higher median score (76.92) compared to the control group (61.54). Significant differences were also observed in the medication (p = 0.008, p < 0.05). In the experimental group, 50 patients demonstrated higher medication adherence (median score 25.00). Regarding patients’ quality of life, a significant difference was observed between the two groups. The control experimental group had better quality of life. Overall, the study concluded that telecare-based interprofessional collaboration had a positive impact on improving knowledge, medication adherence, quality of life in TB patients and bacterial conversion.
ERRATUM: Aqueous Extract Of Ampelopsis Cantoniensis With The Addition Of Curcuma Longa And Taraxacum Officinale: LD50 On Zebrafish (Danio Rerio) Embryos And Nanoformulations For Bacteria Inhibition
Herbal extracts are promising sources of bioactive compounds with potential antimicrobial and biomedical applications. However, their safety and bioavailability often limit their practical use. This study aimed to evaluate the acute toxicity and antibacterial activity of an aqueous extract of Ampelopsis cantoniensis combined with Curcuma longa and Taraxacum officinale (Am-CT extract), and to enhance its performance through nanoformulation. The Am-CT extract, containing 29.69% dihydromyricetin, was tested for toxicity in zebrafish embryos at concentrations ranging from 1.875 to 15 g L⁻¹. The results indicated that the extract was safe, with an LC₅₀ value of 7.81 g L⁻¹, showed weak teratogenicity (EC₅₀ = 7.422 g L⁻¹), and had a therapeutic index (TI) of 1.025. Three Am-CT nanoformulations were prepared using chitosan (CS) and carboxymethyl cellulose (CMC) and characterized by FESEM, DLS, and FTIR analyses. The nanoformulation with a CMC/CS ratio of 1/2 exhibited particle sizes of 80–100 nm and demonstrated stronger antibacterial activity against Escherichia coli and Staphylococcus aureus than the crude extract. Both the extract and its nanoformulation were more cytocompatible than dihydromyricetin, confirming their potential as safe and effective antibacterial agents.
ERRATUM: Formulation and Characterization of Nanostructured Lipid Carriers Composed of Solid Lipids (Cetyl Palmitate and Stearic Acid) and Liquid Lipid (Nyamplung Oil [Calophyllum inophyllum L.])
Nanostructured lipid carriers (NLCs) are lipid-based drug delivery systems made from a mix of liquid and solid lipids, stabilized by surfactants. Nyamplung (Calophyllum inophyllum L) oil, rich in oleic acid (48.49%), has shown in recent studies to enhance wound healing and dermatological performance when used in formulations like bigels and hydrogels. The present study aimed to formulate and characterize NLC system composed of solid lipids (cetyl palmitate and stearic acid) and a liquid lipid (nyamplung oil) for effective drug delivery. Six different NLC formulations (I-VI) were prepared by varying the concentrations of cetyl palmitate, stearic acid, and nyamplung oil. The NLC formulations were characterized by assessing their pH, viscosity, particle size, and polydispersity index (PDI). The particle size analysis revealed that the majority of formulations had particle sizes below 1000 nm, which is consistent with the nanoscale range needed for NLC systems, while the viscosity values confirmed good consistency and homogeneity. Uniform particle production and stability were suggested by the monodisperse distributions shown by the PDI values. For all evaluations, the physical properties of Formulations I–III generally satisfied the required standards. Meanwhile Formula VI's particle size exceeded 1000 nm, indicating a less stable dispersion at that lipid ratio. Formulations IV–VI also showed acceptable physicochemical stability. Notably, the use of nyamplung oil contributed additional benefits, such as antioxidant and anti-inflammatory properties, enhancing the overall therapeutic potential. The study highlights the use of locally available natural ingredients to innovate lipid-based delivery systems, offering promising applications in cosmeceutical and pharmaceutical formulations.
Phytochemical Profiling and Bioactivity Evaluation of Perilla Species from Thai Nguyen Province, Vietnam
Perilla frutescens, belonging to the Lamiaceae family, serves as a traditional medicinal herb in East Asia, offering benefits such as anti-inflammatory, antimicrobial, and antitumor effects. This study aimed to compare the phytochemical composition and biological activities of two varieties- P. frutescens var. crispa forma and P. frutescens var. frutescens—cultivated in Thai Nguyen province, northern Vietnam. Leaf samples were dried and extracted with ethanol using maceration, followed by GC-MS characterization and in vitro assessments of antimicrobial and antitumor effects. GC–MS profiling revealed 39 bioactive compounds, with Perilla ketone dominating in var. frutescens (69.41%) and 1-cyclohexene-1-carboxaldehyde, 4-(1-methylethenyl)- (61.48%) in var. crispa forma. Var. crispa forma exhibited a more diverse composition, particularly of sesquiterpenes such as caryophyllene and nerolidol. Biological assays demonstrated concentration-dependent antimicrobial effects against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus flavus, Aspergillus brasiliensis, and Candida albicans. Notably, the extract of var. crispa forma showed superior inhibitory zones and surpassed the antifungal standard amphotericin B against C. albicans. Cytotoxicity testing on AGS gastric cancer cells using the MTT assay showed potent anticancer activity, with var. crispa forma achieving an IC₅₀ of 10.43 µg/mL, compared to 15.83 µg/mL for var. frutescens. These findings suggest that ethanol extracts of P. frutescens, particularly var. crispa forma, possess strong antimicrobial and anticancer potentials.
The Employment of FTIR Spectroscopy and Multivariate Data Analysis for Authentication Study of Sacha Inchi Seed Oil
Sacha inchi (Plukenetia volubilis) is recognized as one of the world’s most versatile plants, with nearly all parts suitable for use as food ingredients. Sacha inchi seed oil (SIO) commands a premium price in the online market, approximately tenfold higher than conventional vegetable oils such as palm oil (PO), making it a prime target for economically motivated adulteration. This study aimed to establish a robust analytical approach for the discrimination and quantification of PO and soybean oil (SBO) adulterants in authentic SIO using Fourier Transform Infrared (FTIR) spectroscopy coupled with chemometrics. FTIR spectral data were acquired for pure SIO, PO, SBO, and their mixtures across the mid-infrared range (4000–650 cm⁻¹). Principal component analysis (PCA) of full-spectrum absorbance values revealed spectral proximity between SIO and SBO, indicating potential challenges in visual differentiation. Principal component regression (PCR) applied to selected spectral regions (3010–2830 and 1800–700 cm⁻¹) yielded the highest calibration and prediction performance, with coefficients of determination (R²) values of 0.9991 and minimal root mean square error of calibration (RMSEC = 0.620) and validation (RMSEP = 0.658). The developed models demonstrated high accuracy and precision, validating the reliability of FTIR-chemometric integration for authenticating SIO and detecting adulteration with lower-cost oils. This approach offers a rapid, non-destructive, and cost-effective solution for quality control in functional oil authentication.
ERRATUM: Antimicrobial Activity of Tripitsajuk Crude Extract against Escherichia coli and Pseudomonas aeruginosa
The escalating prevalence of antibiotic-resistant gram-negative bacteria necessitates urgent exploration of alternative therapeutic approaches. Tripitsajuk (TPJ), a traditional Thai medicinal formulation containing Myristica fragrans (nutmeg), Oenanthe javanica (water dropwort), and Syzygium aromaticum (clove), has shown promising antimicrobial properties against gram-positive organisms, yet its efficacy against gram-negative pathogens remains uninvestigated. This study evaluated the antimicrobial potential of Tripitsajuk crude extracts against clinically significant gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Plant materials were extracted using two solvent systems: 40% ethanol (TPJHE40) and absolute ethanol (TPJE) through seven-day maceration. Antimicrobial assessment employed the agar well diffusion technique with extract concentrations ranging from 12.5 to 100 mg/mL. Gentamicin served as the positive control, while 10% dimethyl sulfoxide functioned as the negative control. Statistical analysis was performed using one-way analysis of variance followed by Tukey's post-hoc test with significance established at P < 0.05. TPJE extraction yielded 11.228% compared to 9.903% for TPJHE40. Only TPJE exhibited antimicrobial activity against the two tested organisms. Against E. coli, TPJE showed concentration-dependent inhibition from 12.5 mg/mL (12.00±0.00 mm) to 100 mg/mL (17.11±0.11 mm). P. aeruginosa demonstrated greater resistance, responding only to 100 mg/mL (11.44±0.29 mm). TPJHE40 extract failed to produce measurable inhibition zones at any concentration tested. The absolute ethanol extract of Tripitsajuk demonstrated significant antimicrobial efficacy against gram-negative bacteria at high concentrations, with E. coli displaying higher susceptibility compared to P. aeruginosa. These findings establish Tripitsajuk as a promising candidate for development into adjunctive or alternative antimicrobial therapeutics, particularly for managing drug-resistant gram-negative bacterial infections.
Dietary Zingiber officinale (Ginger) Supplementation Ameliorates Lead Carbonate-Induced Hepato-Renal Toxicity and Inflammation in Female Wistar Rats
In low and middle-income countries, lead contamination remains a significant public health threat due to weak regulatory enforcement. Lead exposure induces oxidative stress and systemic inflammation, leading to liver and kidney dysfunction. This study aimed to investigate the ameliorative effects of dietary ginger (Zingiber officinale) supplementation against lead-induced damage in Wistar rats. Female Wistar rats were divided into four groups (n = 6): Control (A); lead carbonate (30 mg/kg/day, oral) (B); lead + 1% ginger diet (C); and lead + 5% ginger diet (D) for 28 days. Serum levels of creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), total antioxidant capacity (TAC), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and liver histology were assessed. Lead exposure significantly (p < 0.05) elevated markers of hepato-renal injury (creatinine, AST, and ALT), increased the oxidative stress marker (MDA), and raised pro-inflammatory cytokines (TNF-α, IL-6). Conversely, it depleted the total antioxidant capacity (TAC) and the anti-inflammatory cytokine IL-10, while inducing severe liver histopathological damage compared to the control group (Group A). Dietary ginger supplementation dose-dependently attenuated these lead-induced alterations. Notably, the 5% ginger diet (Group D) significantly (p < 0.05) reduced all markers of organ damage and inflammation, decreased MDA, and increased both TAC and IL-10 levels compared to Group B. In conclusion, dietary ginger, particularly at a 5% supplementation level, demonstrates significant protective effects against lead-induced hepato-renal toxicity, oxidative stress, and inflammation in rats, highlighting its potential as a natural hepato-renal protective agent.
Potential Cytotoxic Isolates of Spigelia anthelmia Linn. against T47D and WiDr Cancer Cells
Natural products continue to provide valuable leads for anticancer drug discovery, yet limited studies have addressed the bioactive constituents of Spigelia anthelmia Linn. This research aimed to isolate and characterize cytotoxic metabolites of S. anthelmia using a bioassay-guided isolation approach. Sequential extraction, vacuum liquid chromatography (VLC), and preparative thin-layer chromatography (PTLC) yielded a semi-purified fraction designated as Isolate A. Cytotoxicity was evaluated against T47D breast cancer and WiDr colon cancer cells using the MTT assay. Isolate A significantly reduced cell viability in a dose-dependent manner (ANOVA, p < 0.05), with IC₅₀ values of 166.92 ± 5.10 µg/mL (T47D) and 245.24 ± 6.25 µg/mL (WiDr), whereas doxorubicin exhibited IC₅₀ values of 40.05 ± 2.30 µg/mL and 3.57 ± 0.45 µg/mL, respectively. Flow cytometry with Annexin V–FITC/PI staining revealed a visible shift of cell populations toward early and late apoptotic quadrants, indicating apoptosis as the predominant mechanism of cell death. Spectroscopic characterization by FTIR, LC–MS, and NMR suggested that Isolate A is a phenolic/alkaloid-type compound (m/z 218 [M+H]⁺) containing hydroxyl, carbonyl, and heteroaromatic groups. Collectively, these findings demonstrate that S. anthelmia contains apoptosis-inducing metabolites with measurable cytotoxicity, supporting its potential as a natural source of anticancer leads and warranting further purification and mechanistic investigation. To our knowledge, this is the first study to combine bioassay-guided isolation, apoptosis profiling, and spectroscopic characterisation of S. anthelmia metabolites in T47D and WiDr cancer cells.
Ethanol Extract of Hymenocardia acida Tul (Euphorbiaceae) Ameliorates Pentylenetetrazole-Induced Seizures and Neuroinflammation in Mice
Epilepsy is a non-communicable neurological condition with recurrent seizures resulting from uncontrolled neuronal firing as its significant symptoms. Since the practice of traditional medicine and the use of herbal products is common among middle- to low-income countries, it has become imperative to investigate the probable benefit of these products in the treatment of epilepsy. This study evaluated the possible neuroprotective role of Hymenocardia acida leaf extract against the neuronal damage in a mouse model of chemically-induced seizures using pentylenetetrazole (PTZ). Fifty (50) mice were divided into five groups (n=10) comprising a normal control group, which received vehicle, a PTZ-only group (35 mg/kg, i.p), a 50 mg/kg H. acida group, a 100 mg/kg H. acida group and a positive control group, which received 300 mg/kg diazepam. A total of ten (10) PTZ injections were administered to mice in groups 3-5 on alternate days, one hour after treatments. The resulting seizures were graded by severity. The brain tissues were harvested and used for the determination of pro-inflammatory cytokine expression. Data was analysed using one-way ANOVA followed by Tukey’s test at p<0.05 significance. The results showed that the ethanolic extract of H. acida (EEHA) significantly reduced the severity of PTZ-induced seizures as from day 5. The PTZ-induced overexpression of pro-inflammatory cytokines was significantly ameliorated by both doses of EEHA. In conclusion, the neuroprotective effect of EEHA may be due to its anti-inflammatory ability, as evidenced in the amelioration of pro-inflammatory cytokine expression.
Post-germination Application of Trichoderma asperellum for the Biocontrol of Macrophomina phaseolina in Cowpea
Cowpea (Vigna unguiculata (L.) Walp.) serves as a food source for humans and forage for animals. However, its production is affected by disease-causing fungi, of which Macrophomina phaseolina is a significant pathogen. Trichoderma was employed as a biofungicide to manage the disease in the screenhouse. Three strains of Trichoderma asperellum were isolated from the soil. The fungal spore suspensions of the Trichoderma strains were prepared, formulated into seven different treatment combinations, and applied to the cowpea potted soil five days after the germination of the cowpea to investigate their biocontrol ability on M. phaseolina and assess their effects on cowpea growth. The experiment's results showed that cowpea plants treated with T. asperellum differed significantly in plant height, stem girth, and leaf number compared to those treated with M. phaseolina alone (p < 0.05). Trt3 (54.6815 cm), Trt1 (54.0125 cm), and Trt5 (52.9375 cm) gave a higher plant height than in control 1 (M. phaseolina-treated cowpea - 44.9667 cm). Also, Trt7 (0.5413) and Trt3 (0.5258) gave a higher stem girth than in control 1 (M. phaseolina-treated cowpea - 0.3333 cm), while Trt6 (20.292) gave a higher leaf number than in control 1 (M. phaseolina-treated cowpea - 8.833). Additionally, Trt3 and Trt7 exhibited disease incidences of 22% and 67%, respectively, compared to control 1, which had a 100% incidence. Meanwhile, Trt7 showed 8% disease severity, compared to control 1, which had 100%. Therefore, post-germination Trichoderma application proved to be an effective strategy for controlling M. phaseolina, and it also has the potential to enhance cowpea biomass for sustainable food security.
Performance of Ipomoea batatas L. Pigment as a pH Strip and Acid–Base Titration Indicator
Purple sweet potato tubers (PSPT) are rich in anthocyanins, pigments that exhibit visible colour changes across varying pH levels. This study aims to evaluate the performance of PSPT pigment (PSPTP) as both a pH strip and a titration indicator for strong acid–strong base (SASB), strong acid–weak base (SAWB), weak acid–strong base (WASB), and weak acid–weak base (WAWB) titrations. PSPTP was extracted from dried tubers using medical-grade (MG) and analytical-grade (AG) ethanol, resulting in PSPTP-MG and PSPTP-AG, respectively. Strips were fabricated by immobilizing the extracts on cellulose paper, producing PSPTPIP-MG and PSPTPIP-AG. The strips were unchanged at pH 1–7, pale blue at pH 8–9, green at pH 10–12, and yellow at pH 13–14. PSPTPIP-MG was stable for 14 days, while PSPTPIP-AG lasted 21 days; both gradually faded but retained acceptable stability for up to 180 days. They demonstrated excellent sensitivity and produced clear results when applied to household chemicals. As titration indicators, PSPTP-MG and PSPTP-AG showed good precision (CV 0.09%–1.17%) across all titration types but were accurate only for SASB and SAWB titrations (recovery 102.4%–102.5%). In the analysis of pharmaceutical samples, SASB and SAWB titrations yielded acid concentrations of 0.042 M for PSPTP-MG and PSPTP-AG, and 0.041 M for phenolphthalein (PP) in unspiked samples, while spiked samples yielded 0.165 M with all three indicators. Overall, this study demonstrates that PSPTP performs effective as both a pH strip and a titration indicator, particularly for SASB and SAWB titrations.
Phytochemical Composition, Antioxidant, and Acetylcholinesterase Inhibitory Activities of Pluchea pteropoda Extract
Pluchea pteropoda (Asteraceae) has been traditionally employed in folk medicine; however, its phytochemical characteristics and biological activities remain insufficiently defined. This study aimed to elucidate the phytochemical composition, antioxidant properties, and acetylcholinesterase (AChE) inhibitory potential of leaf and root extracts prepared using 45% and 70% ethanol. Four extracts of 45% ethanol extract of leaves (L45), 70% ethanol extract of leaves (L70), 45% ethanol extract of roots (R45), and 70% ethanol extract of roots (R70) were obtained and subjected to qualitative phytochemical screening, quantitative determination of total phenolic and flavonoid contents, DPPH radical scavenging and FRAP reducing power assays, as well as AChE inhibition assessment via Ellman’s method. Data were analyzed using regression-based IC50 estimation and reported as the mean of triplicate measurements. Leaf extracts exhibited substantially higher phenolic (up to 52.89 mg GAE/g) and flavonoid contents (20.62 mg QE/g) compared to root extracts. Antioxidant performance varied according to solvent strength: L45 demonstrated the most potent DPPH scavenging activity (IC50 = 194.8 µg/mL), whereas L70 showed superior reducing capability. AChE inhibitory activity followed the order L70 > L45 > R70 > R45, although L70 remained approximately 16 times less potent than galantamine. Collectively, the findings indicate that P. pteropoda possesses noteworthy antioxidant potential and moderate AChE inhibition, suggesting its promise as a natural source for the development of phytotherapies targeting oxidative stress and neurodegenerative disorders.
Rice Bran Dextrose Agar as a Cost-Effective Alternative to Standard Fungal Culture Media
Microorganisms require carbohydrate-rich media for growth. Rice bran, a carbohydrate-dense by-product, offers a cost-effective alternative to synthetic media like Sabouraud dextrose agar (SDA) and potato dextrose agar (PDA), making it a promising ingredient for mushroom cultivation in laboratories. This research evaluated the performance of rice bran dextrose agar (RBDA) as a culture medium compared to standard SDA and PDA media for fungal growth. Rice bran dextrose agar was formulated (A1, A2, A3, B1, B2, B3, C2, and C3) and optimized by varying dextrose and agar concentrations to support fungal growth. Aspergillus niger, Candida albicans, and Aspergillus fumigatus were tested, and media performance was assessed by measuring colony diameters on day five. A statistical analysis was conducted to compare the fungal growth on RBDA with that on standard media, SDA, and PDA. On day five, the Candida albicans colony on RBDA (C1) showed a significantly larger diameter (16.46 mm, p < 0.05) compared to other RBDA formulations and SDA. For Aspergillus niger, no significant difference was observed (p > 0.05), but RBDA C1 (75.1 mm) showed better growth than the control (SDA), although lower than A2 (76.5 mm) and C3 (79.9 mm). Aspergillus fumigatus grown on RBDA (pH 5.91) showed significantly better growth (28.55 mm, p < 0.05) than SDA and PDA. The study’s findings indicated that RBDA medium demonstrated effective performance in supporting the growth of Candida albicans, Aspergillus niger, and Aspergillus fumigatus, suggesting that rice bran is a viable natural alternative to synthetic mushroom culture media.
Induction of JUN and FOS Expression by Gallic Acid Derivatives in MCF-7 Breast Cancer Cells: An in Silico and RT-PCR Study
Breast cancer remains the leading malignancy worldwide and continues to cause high morbidity and mortality. Although chemotherapy, hormonal therapy, immunotherapy, and targeted treatments are available, these therapeutic options frequently induce adverse effects and contribute to resistance, limiting long-term outcomes. Gallic acid and its derivatives have been widely reported to exert cytotoxic and pro-apoptotic effects on various cancer cell lines, including MCF7 breast cancer cells. However, the underlying molecular mechanisms, particularly those involving apoptosis-related gene modulation, have not been fully elucidated. This study investigated the expression changes of key apoptosis-related genes in MCF7 cells following treatment with two gallic acid derivatives, N-octyl gallamide and N-tert-butyl gallamide, using integrated in silico and in vitro approaches. Differentially expressed genes (DEGs) were identified from the GSE158788 dataset using GEO2R. Protein–protein interaction networks were constructed using Cytoscape, and KEGG pathway enrichment was performed to determine relevant signaling pathways. JUN and FOS, identified as hub genes, were validated using quantitative RT-PCR in MCF7 cells treated with IC₅₀ and 2×IC₅₀ concentrations of the test compounds. DEG and PPI analyses identified JUN and FOS as key hub genes associated with gallic acid–mediated apoptosis. RT-PCR results demonstrated that N-octyl gallamide significantly upregulated JUN (ΔΔCTs 1.25 ± 0.251; p < 0.05) and FOS (1.82 ± 0.691; p < 0.05), exceeding the effects of tamoxifen. In contrast, N-tert-butyl gallamide did not significantly alter JUN or FOS expression (ΔΔCTs 0.76 ± 0.053; p > 0.05). N-octyl gallamide exhibits strong potential as an anticancer agent through JUN and FOS activation in MCF7 cells. These findings highlight its promise for further development as an alternative or adjuvant breast cancer therapy.
VEGF and Macrophage Expression in Pulp Inflammation Following Administration of a Combination of Tricalcium Silicate Cement and Galam (Melaleuca cajuputi var. cumingiana) Leaf Extract: In Vivo and In Silico Studies
The use of natural compounds with anti-inflammatory and regenerative effects for wound healing has been found beneficial. Galam (Melaleuca cajuputi var. cumingiana) is a typical Indonesian medicinal plant with anti-inflammatory activity. This study aimed to evaluate the combined effect of galam leaf extract and tricalcium silicate cement on vascular endothelial growth factor (VEGF) and macrophage expression in pulpitis rat model. Twenty-four male Wistar rats were divided into six groups: Groups 1 and 2 - received a combination of galam leaf extract and tricalcium silicate cement for 3 and 7 days, respectively; Groups 3 and 4 - received tricalcium silicate cement alone for 3 and 7 days, respectively; Groups 5 and 6 - received 40% propylene glycol for 3 and 7 days, respectively. After the treatment periods, macrophage and VEGF expressions were assessed by histological and immunohistochemical analyses. The binding interactions of phytoconstituents of galam leaf with VEGF protein was predicted through molecular docking simulations. Results showed a significant increase in VEGF expression and a reduction in pulp inflammation in the combination groups compared to the controls. Macrophage numbers increased on day 3, indicating immune activation, and decreased by the 7th day, suggesting inflammation resolution. The compound dammarane-3,12,25-triol present in galam leaf demonstrated strong binding affinity to VEGF in silico, suggesting potential angiogenic effect. These findings indicate that the combination of galam leaf extract and tricalcium silicate cement enhanced pulp healing by reducing inflammation, regulating macrophage activity, and increasing VEGF expression in pulpitis rat model.
Assessment of Essential Oil Yield from Artemisia herba-alba and the Endemic Artemisia mesatlantica in the Middle Atlas of Morocco: A Step Toward Sustainable Valorization
Species of the genus Artemisia play a central role in Moroccan traditional pharmacopoeia and aromatic plant exploitation; however, several endemic taxa remain poorly documented in terms of their physicochemical and productive potential. This study presents a comparative evaluation of Artemisia herba-alba and the endemic Artemisia mesatlantica collected from the Middle Atlas region, with a specific focus on moisture content and essential oil yield under standardized post-harvest and extraction conditions. Aerial parts were harvested in April following AFNOR (NF T 75-402) standards, dried using both solar and controlled-temperature oven methods, and subjected to hydrodistillation using a Clevenger-type apparatus. The two species exhibited marked quantitative differences in water content. A. herba-alba showed a low average moisture content of 8.3% (range: 4.63–16.12%), whereas A. mesatlantica displayed significantly higher values averaging 23.5% (range: 20.71–25.81%), reflecting contrasting tissue hydration and water-retention capacities. Essential oil yields were low for both taxa, reaching 0.14 ± 0.01% for A. herba-alba and 0.16 ± 0.01% for A. mesatlantica on a dry-mass basis. Despite the modest absolute yields, A. mesatlantica consistently exhibited slightly higher values, even in the context of elevated moisture levels, suggesting potential anatomical or physiological specificities influencing volatile compound production and storage. These results emphasize the strong sensitivity of Artemisia essential oil production to ecological context, phenological stage, and post-harvest handling. Furthermore, they provide valuable baseline data for the future phytochemical characterization, bioactivity assessment, and sustainable valorization of the endemic A. mesatlantica within Moroccan medicinal and aromatic plant sectors.
Response Surface Methodology-Driven Optimization of HPLC for the Determination of Caffeine, Chlorogenic Acid, and Caffeic Acid in Coffee
Most studies on coffee bioactive compounds have focused on single targets and relied on trial-and-error optimization. This study aims to develop a high-performance liquid chromatographic (HPLC) method optimized by response surface methodology using a central composite design (RSM-CCD), and a design of experiments (DoE) approach for the simultaneous quantification of caffeine (CAF), chlorogenic acid (CGA), and caffeic acid (CA) in roasted coffee samples. Separation was performed on a Luna C18 (2) column (250 × 4.6 mm; 5 μm). Three factors – methanol ratio, flow rate, and methanol gradient were investigated and optimized through 17 CCD runs, evaluating retention time, peak area, resolution, tailing factor, and theoretical plates. The optimum condition of 31.6% methanol, 0.81 mL/min flow rate, and 1%/min gradient enabled the simultaneous elution and separation of CGA, CAF, and CA at a retention time of 6.325, 7.444, and 9.326 minutes, respectively, and over a 12-minute chromatographic runtime. Validation according to International Council for Harmonization (ICH) Q2 (R1) guidelines confirmed excellent linearity (R² ≥ 0.998), LOD/LOQ (ppm) of 0.1718/0.5207 (CGA), 0.1773/0.5373 (CAF), and 0.0813/0.2464 (CA), with precision below 2% and recovery between 94.05% and 112.42%. Robusta levels (mg/g) were CGA (10.083 ± 0.229), CAF (18.838 ± 0.238), and CA (2.067 ± 0.044) while Arabica levels (mg/g) were CGA (16.641 ± 0.211), CAF (9.916 ± 0.157), and CA (2.178 ± 0.041). The RSM-driven HPLC successfully demonstrated efficient factor interaction modeling and robustness improvement. Consequently, the developed DoE-based method provides a rapid, selective, and reliable method for coffee quality evaluation and authenticity assessment.