Phytochemical profile and biological activities of Kerianthera preclara, Kutchubaea insignis, Pagamea duckei, Pagamea macrophylla, and Rudgea lanceifolia (Rubiaceae)

1 Introduction

The Amazon rainforest harbors one of the largest reservoirs of plant biodiversity in the world. However, the chemical composition and biological potential of many of its species remain poorly investigated. In this context, the screening of crude plant extracts represents an important strategy for identifying promising plants for further phytochemical studies. When combined with biological assays, this approach provides preliminary information on bioactive potential and supports future investigations involving bioassay-guided fractionation, isolation, and structural characterization of active metabolites (Atanasov et al., 2021; Suffredini et al., 2023; Miranda et al., 2024; Savickiene and Raudone, 2024).

Among Amazonian plant families, Rubiaceae stands out because of its remarkable chemical diversity and its ability to produce a wide variety of secondary metabolites, including alkaloids, iridoids, saponins, coumarins, anthraquinones, terpenoids, and phenolic compounds. Many of these metabolites have been associated with antibacterial, antifungal, antiviral, antiparasitic, cytotoxic, antitumor, and insecticidal activities, highlighting the family as a source of natural products with pharmacological and agricultural relevance. Moreover, the occurrence of alkaloids such as quinine and emetine underscores the therapeutic importance of Rubiaceae and reinforces the value of the chemical and biological investigation of its underexplored species (Di Giorgio et al., 2006; Martins and Nunez, 2015; Habtemariam, 2018; Hussain et al., 2019; Wang et al., 2020; Crispim et al., 2025; De Souza et al., 2025; Debasish et al., 2025; Franco et al., 2025).

From a chemosystematics perspective, Rubiaceae exhibits distinct patterns of secondary metabolite distribution among its subfamilies. Species of Ixoroideae are predominantly characterized by iridoids and triterpenes, whereas Cinchonoideae is recognized for monoterpene indole alkaloids and related alkaloids. Members of Rubioideae commonly accumulate alkaloids and anthraquinones. This is particularly evident for species of the genera Kerianthera, Kutchubaea, Pagamea, and Rudgea, whose chemistry has received little attention despite their occurrence in one of the most biodiverse ecosystems on the planet (Martins and Nunez, 2015; Sousa and Chaves, 2019; Cole et al., 2024; Santivañez and Angolini, 2025).

Despite the recognized chemical and pharmacological potential of the Rubiaceae family, many of its Amazonian genera remain understudied. Based on the literature, no phytochemical studies have been found for the genera Kerianthera and Kutchubaea. Within the genus Pagamea, only Pagamea guianensis Aubl. and Pagamea coriacea Spruce ex Benth. have been evaluated for their biological activities (De Smet, 1985; Shepard, 2004; Roumy et al., 2007; Suffredini et al., 2007), while P. duckei and P. macrophylla have not been subjected to any phytochemical or biological studies. Similarly, the genus Rudgea has been studied in only a few species, in which alkaloids, triterpenes, saponins, and anthraquinones have been described, along with antifungal and cytotoxic activities. Therefore, there are no studies on Rudgea lanceifolia Salisb (Young et al., 1998; Lopes et al., 1999; Alves et al., 2004; Oliveira et al., 2007; Schinnerl et al., 2012). These gaps highlight the need to expand research on Amazonian species of the Rubiaceae family, contributing to our understanding of their chemical diversity and the biological potential of their secondary metabolites.

Limited phytochemical knowledge of these genera restricts the assessment of their biological potential. Since Rubiaceae produces secondary metabolites with pharmacological and agricultural relevance, phytochemical and biological screening of underexplored species is an effective strategy for identifying new bioactive compounds and selecting promising species for detailed chemical investigation.

The biological evaluation of plant extracts is an important strategy for identifying species with promising pharmacological and agricultural potential. Antimicrobial activity is particularly relevant in view of the increasing emergence of antimicrobial resistance, which has intensified the search for new bioactive natural products (Atanasov et al., 2021; Jouogo et al., 2022; Palanisamy et al., 2024; WHO, 2025). On the other hand, plant extracts may exhibit antioxidant activity through mechanisms such as free radical scavenging and the reduction of pro-oxidant metal ions, thereby protecting biological systems against oxidative damage. Consequently, chemical assays such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and ferric reducing power are widely employed for the preliminary evaluation of the antioxidant potential of natural products (Apak et al., 2016; Phuyal et al., 2020; Cosme et al., 2020; Bibi et al., 2022; Yamauchi et al., 2024; Lisjak et al., 2026). In addition, the Artemia salina lethality assay provides a simple and reliable approach for preliminary toxicity screening; extracts that exhibit high toxicity against A. salina may have cytotoxic potential, but this would require isolating these compounds and testing them on cell lines (tumoral or healthy) (Meyer et al., 1982; Krishnaraju et al., 2005; Ismail et al., 2025), whereas insecticidal evaluation against Sitophilus zeamais, one of the major pests of stored grains, contributes to the identification of plant-derived compounds with potential applications in sustainable pest management (Prior et al., 2005; Suleiman et al., 2015; Isman, 2020; Akbar et al., 2022; Jiménez-Galindo et al., 2023; Lv et al., 2023).

Based on the scarce phytochemical and pharmacological information available, Kerianthera preclara J.H.Kirkbr., Kutchubaea insignis Fisch. ex DC., Pagamea duckei, Pagamea macrophylla, and R. lanceifolia were selected for investigation. To the best of our knowledge, this is the first study to provide a comparative phytochemical characterization and biological evaluation of these Amazonian Rubiaceae species. The phytochemical profiles of the hexane and methanolic extracts were characterized by thin-layer chromatography (TLC) and proton nuclear magnetic resonance (¹H NMR), while their antibacterial, antioxidant, insecticidal, and general toxicity activities were assessed using preliminary bioassays. These findings contribute new phytochemical and biological data for previously unexplored species and provide a foundation for future bioassay-guided isolation and characterization of bioactive natural products.

2 Materials and methods

2.1 General experimental procedures

The plant material was dried in a forced-air oven (TE-394/2, Tecnal, Piracicaba, Brazil). Crude extracts were prepared using commercial-grade solvents purchased from Química Araguaya (São Paulo, Brazil), followed by extraction in an ultrasonic bath (USC-2800, Unique, Indaiatuba, Brazil; 40 kHz) and solvent removal under reduced pressure using a rotary evaporator (model 550, Fisatom, São Paulo, Brazil).

Phytochemical analyses of all extracts and fractions were performed by comparative thin-layer chromatography (TLC) on silica gel 60 F₂₅₄ aluminum-backed plates (Sigma-Aldrich, St. Louis, MO, United States). Chromatograms were examined under ultraviolet light and developed using appropriate visualization reagents for the detection of different classes of secondary metabolites. The reagents included anisaldehyde–sulfuric acid, ceric sulfate [Ce(SO₄)₂], iodine vapor, ferric chloride (10% ethanolic solution), potassium hydroxide (KOH), diphenylborinyloxyethylamine/polyethylene glycol (NP/PEG; 1% methanolic solution of diphenylborinyloxyethylamine followed by 5% ethanolic polyethylene glycol 4,000), and Dragendorff’s reagent.

Anisaldehyde–sulfuric acid, ceric sulfate, and NP/PEG were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Analytical-grade iodine was obtained from CRQ Produtos Químicos Ltda (Diadema, Brazil), ferric chloride from Vetec Química Fina Ltda (Rio de Janeiro, Brazil), potassium hydroxide from Merck KGaA (Darmstadt, Germany), and methanol and absolute ethanol from CAQ–Casa da Química Indústria e Comércio Ltda (Diadema, Brazil). Dragendorff’s reagent was prepared from analytical-grade basic bismuth subnitrate and ACS-grade potassium iodide purchased from Dinâmica Química Contemporânea Ltda (Indaiatuba, Brazil).

¹H Nuclear Magnetic Resonance (NMR) spectra were acquired on a Bruker Fourier 300 Ultrashield spectrometer (Bruker BioSpin, Rheinstetten, Germany) operating at 300 MHz. Deuterated chloroform (CDCl₃; Cambridge Isotope Laboratories, Tewksbury, MA, United States) and deuterated dimethyl sulfoxide (DMSO-d _*6*; Sigma-Aldrich, St. Louis, MO, United States) were used as NMR solvents.

For the antibacterial assays, 2,3,5-triphenyltetrazolium chloride (TTC) and analytical-grade dimethyl sulfoxide (DMSO) were obtained from Nova Biotecnologia (São Paulo, Brazil). Absorbance measurements were performed using a Multiskan™ GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States).

Antioxidant assays were carried out using a Cirrus 80ST UV–Vis spectrophotometer (Femto, São Paulo, Brazil). Contact toxicity assays against S. zeamais employed analytical-grade acetone obtained from Neon Comercial Reagentes Analíticos Ltda (Suzano, Brazil).

2.2 Plant material and extraction

Branches and leaves of Kerianthera preclara J.H.Kirkbr. were collected at the Bosque da Ciência on Campus I of the National Institute of Amazonian Research (INPA), while samples of Kutchubaea insignis Fisch. ex DC. (branches, leaves, and fruits), Pagamea macrophylla Spruce ex Benth. (branches and leaves), and Rudgea lanceifolia Salisb. (branches and leaves) were obtained from the Adolpho Ducke Forest Reserve. Additionally, two collections of Pagamea duckei Standl. were collected at the Campina/Campinarana Biological Reserve from individuals under different sunlight exposure conditions: one from a shaded environment (PD 1), from which branches and leaves were collected, and another collected in a sunny environment (PD 2), from which the inflorescences were also collected. All collections were made in Manaus, Amazonas, Brazil, and the respective herbarium specimens are deposited at the INPA Herbarium. Information regarding the geographic coordinates, voucher numbers, and records in the National Genetic Heritage Management System (SisGen) for the six samples studied is presented below: K. preclara (voucher number 299253; 3°05′49″ S, 59°59′13″ W; SisGen A56DEC4), K. insignis (voucher number 299251; 2°33′48″ S, 59°34′32″ W; SisGen A9FE558), P. macrophylla (voucher number 255657; 2°57′06″ S, 59°57′27″ W; SisGen A4C0796), R. lanceifolia (voucher number 299252; 2°34′13″ S, 59°34′25″ W; SisGen AA318E2), P. duckei (PD 1) from a shaded environment (voucher number 299249; 2°21′07″ S, 60°00′56″ W; SisGen A4C0796) and P. duckei (PD 2) from a sunny environment (voucher number 299250; 2°21′09″ S, 60°09′00″ W; SisGen A4C0796). The plant material was first dried in an oven at 35 °C, ground in a hammer mill and a knife mill, and subjected to sequential extraction in order of increasing polarity using hexane (Hex) and methanol (MeOH) as solvents. For each stage, a ratio of 1 g of plant material to 3 mL of solvent was used. Extraction was assisted by an ultrasonic bath (40 kHz) for 20 minutes, repeated at least four times (1 g/ 3 mL w/v, four times). Finally, the extracts obtained were concentrated in a rotary evaporator at 55 rpm. In total, 14 hexane extracts and 14 methanol extracts were obtained.

2.3 Comparative thin-layer chromatography analysis of the crude extracts

For TLC analysis, all extracts were standardized to a concentration of 10 mg/mL. Chromatographic plates were initially examined under ultraviolet (UV) light at 254 and 365 nm and by physical visualization (Collins et al., 2006). The plates were subsequently treated with specific visualization reagents to detect different classes of secondary metabolites. Anisaldehyde–sulfuric acid reagent and iodine vapor were used as universal reagents (Wagner and Bladt, 1996); ceric sulfate [Ce(SO₄)₂] for terpenes (Chaves, 1997); ferric chloride (FeCl₃) and diphenylborinyloxyethylamine/polyethylene glycol (NP/PEG) for phenolic compounds (Matos, 2009; Wagner and Bladt, 1996); potassium hydroxide (KOH) for quinones (Wagner and Bladt, 1996); and Dragendorff’s reagent for nitrogen-containing compounds, particularly alkaloids (Matos, 2009).

2.4 ¹H NMR analysis of the crude extracts

¹H NMR spectra were acquired at 300 MHz for all extracts. For sample preparation, 10 mg of each extract was dissolved in 500 µL of deuterated solvent, using CDCl₃ for the hexane extracts and DMSO-d _*6* for the methanolic extracts. Spectra were acquired under standardized instrumental conditions within each extract group. For the hexane extracts, 64 scans (NS) were collected using a receiver gain (RG) of 7.42029, whereas the methanolic extracts were acquired with 64 scans and an RG of 32.252.

2.5 Artemia salina lethality assay

The toxicity of all hexane and methanolic extracts was evaluated using the A. salina lethality assay according to the method described by Meyer et al. (1982). Briefly, 10 mg of A. salina cysts were incubated in 3.8% (w/v) artificial seawater under continuous aeration and fluorescent light at 25 °C for 48 h to obtain freshly hatched nauplii.

Ten nauplii were transferred to each well of a 24-well microplate containing the test solutions. The extracts were evaluated at concentrations ranging from 32.5 to 1,000 μg/mL. Potassium dichromate (0.1%) and 5% dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. All treatments were performed in triplicate, and nauplii mortality was recorded after 24 h of exposure.

The median lethal concentration (LC₅₀) was determined for each extract, and toxicity was classified according to the criteria proposed by Nguta et al. (2011).

2.6 Antibacterial activity

Antibacterial activity was evaluated using the broth microdilution method according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2015), with minor modifications. The extracts were tested against Aeromonas hydrophila (CBAM 0162), Acinetobacter baumannii (CBAM 0664), Citrobacter freundii (CBAM 0702), Escherichia coli (CBAM 0006), Enterobacter cloacae (CBAM 0200), Klebsiella pneumoniae (CBAM 0051), Morganella morganii (CBAM 0138), Pseudomonas aeruginosa (CBAM 0519), Serratia marcescens (CBAM 0069), Staphylococcus aureus (CBAM 0026), and Staphylococcus epidermidis (CBAM 0613). All strains were obtained from the Culture Collection of Amazonian Microorganisms (CBAM) at the Instituto Leônidas e Maria Deane–Fiocruz Amazônia (ILMD-Fiocruz, Manaus, Brazil).

Bacterial strains were reactivated in Mueller-Hinton broth for 16–24 h at 37 °C. The inoculum density was adjusted to the 0.5 McFarland standard and diluted 1:20 to obtain a final concentration of approximately 5 × 10⁴ colony-forming units (CFU) per well.

Hexane and methanolic extracts were dissolved in 5% dimethyl sulfoxide (DMSO) to prepare stock solutions (10 mg/mL). All extracts were initially screened at a final concentration of 1,000 μg/mL. Extracts that inhibited bacterial growth by ≥ 70% were selected for determination of the minimum inhibitory concentration (MIC) by two-fold serial microdilution (1,000–31.25 μg/mL).

Oxytetracycline (125 μg/mL) was used as the positive control, whereas Mueller-Hinton broth containing 5% DMSO served as the negative control. Optical density was measured at 625 nm immediately after plate preparation and again after 24 h of incubation at 37 °C. Bacterial viability was subsequently assessed by adding 40 µL of 2,3,5-triphenyltetrazolium chloride (TTC). All experiments were performed in triplicate. Statistical analyses were performed using GraphPad Prism version (v8.0.2). Data were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparisons test to compare each treatment group with the negative control. Differences were considered statistically significant at p < 0.05, with a 95% confidence interval.

2.7 Antioxidant activity

The antioxidant activity of the methanolic extracts was evaluated according to the method described by Novaes (2008) using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay and the Fe³⁺/1,10-phenanthroline reducing assay. Only methanolic extracts were evaluated. Each extract (5 mg) was dissolved in methanol and the volume adjusted to 10 mL, yielding a final concentration of 500 μg/mL.

Ascorbic acid (AA) was used as the reference antioxidant at concentrations ranging from 90 to 720 μg/mL. Aliquots of 10 µL from each standard solution were used to construct calibration curves for both assays. Calibration curves were prepared prior to sample analysis to verify assay linearity (R² > 0.95) and to allow quantitative comparison between the extracts and the reference antioxidant. All procedures were carried out under reduced light to minimize photooxidation.

For the DPPH assay, a 28 μg/mL DPPH solution was prepared by dissolving the reagent in 1 mL of dichloromethane (CH₂Cl₂) and diluting to 100 mL with methanol. The reaction mixture consisted of 990 µL of DPPH solution and 10 µL of extract solution. After incubation for 30 min at room temperature, absorbance was measured at 517 nm.

The Fe³⁺/1,10-phenanthroline assay was performed using a 0.25% 1,10-phenanthroline solution prepared in sodium acetate buffer (pH 4.5). The ferric solution was prepared from ammonium ferric sulfate dissolved in 1% hydrochloric acid. The reaction mixture contained 980 µL of the phenanthroline solution, 10 µL of the ferric solution, and 10 µL of the extract solution. After 60 min of incubation, absorbance was recorded at 515 nm. All assays were performed in triplicate.

Antioxidant activity was expressed as ascorbic acid equivalents using the corresponding calibration curves, according to Martins et al. (2014). Extracts with equivalence values approaching 1.0 were considered to exhibit antioxidant activity comparable to that of ascorbic acid. The equivalence was calculated using the mass of the extract (5 µg in 10 µL) inside the oxidant solution, the variation in absorbance (ABS), and the linearity coefficient of AA (LCoe) and angular coefficient of AA (ACoe) of the ascorbic acid curve, as illustrated by the following equation:

E q u i v a l e n c e = 5 A B S c o n t r o l − A B S e x t r a c t − ACoe / LCoe

2.8 Insecticidal activity against Sitophilus zeamais

Contact toxicity against S. zeamais (maize weevil) was evaluated according to the method described by De Lira et al. (2015). Initially, a screening assay was performed using the methanolic extracts of branches and leaves from K. preclara, K. insignis, P. duckei (PD 1 and PD 2), P. macrophylla, and R. lanceifolia. All extracts were tested at a dose of 4 µg/insect. Extracts that caused ≥50% mortality and exhibited sufficient solubility in acetone were selected for concentration-response bioassays to determine the LC₅₀ and the concentration required to kill 90% of the insects (LC₉₀).

Selected extracts were dissolved in acetone to obtain doses of 2.5, 5.0, 7.5, 10.0, 15.0, and 20.0 µg/insect. A 0.5 µL aliquot of each solution was topically applied to the dorsal thorax of each insect. Insects in the negative control group received 0.5 µL of acetone only. For each treatment, 20 unsexed adults were maintained in plastic containers (4.0 cm in diameter × 6.0 cm in height) under darkness at 28 C ± 2 °C. Mortality was assessed after 7 days. All bioassays were performed in quadruplicate.

LC₅₀ and LC₉₀ values, together with their respective 95% confidence intervals, were estimated by probit analysis using ProbitCalc software (version 1.1.0).

3 Results and discussion

As the first comparative phytochemical and biological investigation of these five Amazonian Rubiaceae species, this study aimed to provide a preliminary characterization of their chemical profiles and evaluate their antibacterial, antioxidant, insecticidal, and general toxicity activities in order to identify species with promising bioactive potential. The integration of phytochemical screening with complementary biological assays enabled the selection of priority species for future bioassay-guided isolation and pharmacological investigation. The experimental workflow and the main biological findings are summarized in Figure 1.

3.1 Thin-layer chromatography profile of the extracts

Thin-layer chromatography (TLC) provided a preliminary overview of the chemical composition of the extracts (Figure 2). In the hexane extracts, anisaldehyde-sulfuric acid revealed predominantly lilac and pink spots, whereas Ce(SO₄)₂ produced brown and light lilac bands, indicating the predominance of terpenoid constituents. This finding agrees with the low polarity of hexane, which preferentially extracts lipophilic metabolites such as terpenes, steroids, and fatty acids (Gonzalez-Hernandez et al., 2024). Under UV light (365 nm), blue fluorescent bands were observed in the branches extracts of Pagamea duckei (PD 1 and PD 2), Pagamea macrophylla, Rudgea lanceifolia, and Kerianthera preclara (extracts 1, 3, 9, 11, and 13), suggesting the occurrence of compounds with similar chromatographic behavior and chromophoric groups. In contrast, UV light at 254 nm and the remaining detection reagents produced no characteristic bands indicative of alkaloids or phenolic compounds. Because TLC provides only a preliminary phytochemical profile, definitive compound identification will require complementary analytical approaches, including mass spectrometry (LC-MS/MS) and the isolation of individual metabolites followed by NMR-based structural elucidation.

Under ultraviolet light (254 and 365 nm), the methanolic extracts displayed chromatographic bands characteristic of compounds containing chromophoric groups associated with aromatic structures or conjugated double bonds (Figure 3). Except for the extracts of Kutchubaea insignis (7 and 8), Rudgea lanceifolia (12), and Kerianthera preclara (14), all extracts exhibited blue fluorescence at 365 nm.

Development with NP/PEG enhanced the fluorescence intensity, particularly in the methanolic extracts of Pagamea duckei (2, 4, and 5), suggesting the occurrence of phenolic compounds, especially glycosylated flavonoids. Yellow fluorescent bands remained visible at the application site after derivatization. These observations were supported by FeCl₃, which produced the characteristic black spots coloration of phenolic compounds. Enhanced blue fluorescence was also observed in the methanolic branches extracts of P. duckei (PD 1 and PD 2) and in the leaves extract of K. insignis. In K. preclara, fluorescence enhancement was restricted to compounds retained near the application site, exhibiting distinct retention factor (Rf) values from those observed in the other species.

Oliveira et al. (2007) reported the occurrence of anthraquinones and naphthoquinones in cell cultures of Rudgea jasminoides, supporting the chemotaxonomic relevance of these metabolites within Rubioideae. Likewise, the methanolic extracts of P. duckei (1 and 3) produced pink bands after application of KOH, suggesting the occurrence of quinones, particularly anthraquinones.

The methanolic extracts of K. insignis developed with anisaldehyde-sulfuric acid and Ce(SO₄)₂ exhibited characteristic blue bands together with dark bands under ultraviolet light at 254 nm, suggesting the presence of iridoids. Acidic derivatization reagents promote the hydrolysis of iridoid glycosides, producing characteristic blue-colored derivatives (Zgórka, 2008). In addition, blue bands were observed in all extracts, particularly near the application site, indicating the possible occurrence of glycosylated iridoids. These findings agree with the recognized chemotaxonomic importance of iridoids in the Ixoroideae subfamily (Sousa and Chaves, 2019). Similar compounds have been reported for Duroia saccifera and isolated from other species of the genus, including Duroia hirsuta and Duroia macrophylla (Page et al., 1994; López-Vázquez, 2019; Zanca, 2020).

Among the investigated species, only the methanolic branches and leaves extracts of K. preclara (13 and 14) produced orange bands after derivatization with Dragendorff’s reagent, indicating the presence of nitrogen-containing metabolites, most likely alkaloids. Alkaloids are among the major chemotaxonomic markers of the Cinchonoideae subfamily and have been widely reported in several genera of this lineage (Parveen et al., 2024; De Souza et al., 2025). Likewise, alkaloids have previously been isolated from species of Isertia, a genus belonging to the same subfamily as Kerianthera (Bohrmann et al., 1969; Lau-Cam and Tashiro, 1971; Esterl et al., 1983).

Overall, the chromatographic profiles were consistent with the chemotaxonomic patterns described for Rubiaceae, suggesting the occurrence of phenolic compounds, quinones, iridoids, and alkaloids in the investigated species. Nevertheless, these findings should be regarded as preliminary, and confirmation of the proposed metabolites will require complementary analyses, including LC-MS/MS, compound isolation, and multidimensional NMR spectroscopy.

3.2 Phytochemical profiling of the crude extracts by ¹H NMR

The ¹H NMR spectra of the hexane extracts were dominated by resonances characteristic of lipophilic metabolites, particularly terpenoids, fatty acids, and phytosterols (Figure 4). Intense signals between δ_H 0.60 and 1.80 corresponded to methyl groups typical of terpenoid structures, whereas the prominent resonance at δ_H 1.25 was attributed to the aliphatic chains of fatty acids. Signals between δ_H 1.80 and 2.50 were assigned to methylene and methine protons, while resonances in the δ_H 3.00–4.00 region suggested the presence of methoxylated constituents.

The occurrence of phytosterols was supported by the characteristic olefinic resonance at δ_H 5.35 (H-6), together with methyl signals at δ_H 0.70 and 0.71 and the low-intensity multiplet at δ_H 3.50 assigned to H-3. Additional olefinic signals at δ_H 5.12 further supported the occurrence of stigmasterol. Overall, these spectral features are consistent with the presence of β-sitosterol and stigmasterol, two phytosterols commonly reported in nonpolar plant extracts (Sobolev et al., 2005; Gnocchi et al., 2021).

Only weak resonances were observed in the aromatic region (δ_H 6.00 to 8.00), indicating that aromatic metabolites occur in relatively low abundance in the hexane extracts. Nevertheless, extracts of Pagamea duckei and Kutchubaea insignis displayed more evident signals in this region, suggesting the presence of aromatic or conjugated olefinic compounds. In addition, the branches extracts of P. duckei, Pagamea macrophylla, and Kerianthera preclara exhibited downfield resonances at δ_H 12.29, 11.45, and 10.48, respectively, consistent with strongly deshielded protons, such as hydrogen-bonded hydroxyl groups or carboxylic acid hydrogens.

The ¹H NMR spectra of the methanolic extracts (Figure 5) exhibited resonances characteristic of terpenoid, glycosylated, and aromatic metabolites. Signals between δ_H 0.60 and 1.00 were assigned to methyl groups typical of terpenoid skeletons, whereas the intense resonance at δ_H 1.25 corresponded to methylene groups of long aliphatic chains, characteristic of fatty acids. These signals were particularly intense in the methanolic extracts of Kerianthera preclara and in the leaves and inflorescence extracts of Pagamea duckei, corroborating the chromatographic profiles obtained after derivatization with anisaldehyde-sulfuric acid and Ce(SO₄)₂.

Resonances between δ_H 2.50 and 5.00 were attributed to methylene, methine, and methoxyl protons. Signals between δ_H 3.00 and 4.50 are characteristic of oxygenated carbons and include anomeric, oxymethine, and oxymethylene hydrogens commonly associated with glycosylated metabolites, such as iridoid glycosides and phenolic glycosides (Heitzman et al., 2005; Lee et al., 2025).

The methanolic extracts of Kutchubaea insignis exhibited a characteristic doublet at δ_H 6.00 together with a broad singlet at δ_H 7.50, corresponding to the H-6 and H-3 olefinic protons of iridoids containing a C-3/C-4 enol ether system (Es-Safi et al., 2006; Dinda et al., 2007; Wang et al., 2011). Similar singlets around δ_H 7.50 were also detected in the branches extract of Rudgea lanceifolia and in both methanolic branches and leaves extracts of K. preclara. Combined with the TLC profiles, these spectral features are consistent with the occurrence of iridoid-type metabolites.

The methanolic branches and leaves extracts of K. preclara also displayed olefinic resonances together with low-intensity signals between δ_H 9.00 and 10.00, corresponding to highly deshielded proton environments. Although these resonances, detected together with the positive reaction observed with Dragendorff’s reagent, are consistent with the occurrence of nitrogen-containing metabolites, putatively assigned to alkaloids. Previous reports of alkaloids in several Rubiaceae species support this interpretation (Berger et al., 2022; De Souza et al., 2025; Santivañez and Angolini, 2025).

The ¹H NMR spectra of P. duckei exhibited aromatic resonances between δ_H 6.00 and 8.00, with signal intensity varying according to collection conditions. While the branches extracts showed similar profiles, leaves extracts collected from a sunny area (PD 2) displayed more intense aromatic resonances than those collected from a shaded area (PD 1). This pattern agrees with previous reports demonstrating that increased ultraviolet radiation stimulates the accumulation of phenolic compounds involved in photoprotection and antioxidant defense (Del Valle et al., 2020; Lan et al., 2024). These observations are consistent with the TLC profiles, which also suggested the occurrence of phenolic constituents in this species.

Overall, the TLC profiles and ¹H NMR spectra revealed chromatographic and spectroscopic features consistent with the occurrence of terpenoids, glycosylated metabolites, iridoid-type metabolites, phenolic compounds, quinones, and nitrogen-containing metabolites in the investigated species. Among the evaluated taxa, P. duckei exhibited the strongest evidence for phenolic constituents, whereas K. preclara and K. insignis showed spectral features compatible with iridoid and alkaloid-type metabolites. However, these phytochemical assignments should be regarded as putative, as they are based on TLC and ¹H NMR analyses of crude extracts (Wolfender et al., 2019). Definitive identification of these metabolites will require complementary analytical approaches, including LC-MS/MS, isolation of individual compounds, and structural elucidation by one- and two-dimensional NMR spectroscopy.

3.3 Toxicity of the extracts against Artemia salina

Among the hexane extracts, only the extract obtained from the branches of Rudgea lanceifolia exhibited toxicity against Artemia salina, causing 56.6% nauplii mortality at 1,000 μg/mL. Among the methanolic extracts, only the extract from the branches of Kerianthera preclara remained active at 250 μg/mL, producing mortality rates of 43.3%, 46.6%, and 70.0% at 250, 500, and 1,000 μg/mL, respectively. The methanolic leaves extract of R. lanceifolia caused 40.0% mortality at 1,000 μg/mL. In Pagamea duckei, methanolic leaves extracts from both collections (PD 1 and PD 2) resulted in 36.6% mortality at the same concentration, whereas the branches extract from PD 2 exhibited higher activity, with mortality rates of 46.6% and 66.6% at 500 and 1,000 μg/mL, respectively.

According to Nguta et al. (2011), extracts with LC₅₀ values below 100 μg/mL are considered highly toxic and may represent promising sources of compounds with cytotoxic potential. Although LC₅₀ values were not determined for all active extracts, the concentration-dependent mortality observed for the methanolic extract from the branches of K. preclara indicates the presence of bioactive constituents worthy of further investigation.

The phytochemical profile of K. preclara, obtained by TLC and ¹H NMR analyses, suggested the occurrence of nitrogen-containing metabolites, terpenoids, and glycosylated compounds, which may contribute to the observed toxicity. However, the relationship between the biological activity and these putative metabolite classes remains hypothetical and should be confirmed through compound isolation and structural characterization.

Comparable findings have been reported for Isertia hypoleuca, another member of the tribe Isertieae, whose extracts exhibited pronounced toxicity against Artemia spp (LC₅₀ < 10 μg/mL) (Gupta et al., 1996). Since Rubiaceae is recognized as a rich source of alkaloids, terpenoids, and glycosylated metabolites (Volobuff et al., 2019; De Moura et al., 2021), the toxicity observed for K. preclara is consistent with the chemical diversity reported for the family. Nevertheless, the metabolites responsible for this activity remain unknown, highlighting the need for bioassay-guided fractionation, compound isolation, and structural elucidation.

3.4 Antibacterial activity

The antibacterial screening revealed that only the hexane extracts of R. lanceifolia showed inhibition above 50%, against Citrobacter freundii (61.74%) and Morganella morganii (69.75%) (Table 1). In contrast, the methanolic extracts of P. duckei and K. preclara exhibited the most relevant antibacterial responses among the evaluated species.

TABLE 1. Percentage of antibacterial inhibition of the extracts from the species evaluated.

The extracts were evaluated at 1,000 μg/mL. Data expressed as inhibition percentage (%) compared to negative control. (−) samples that showed no activity, with inhibition values lower than 20%.

p < 0.001.

p < 0.01.

p < 0.5, (n = 3). Acinetobacter baumannii (AB), Aeromonas hydrophila (AH), Citrobacter freundii (CF), Escherichia coli (EC), Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Serratia marcescens (SM), Enterobacter cloacae (ECL), Klebsiella pneumoniae (KP), Morganella morganii (MM), and Staphylococcus epidermidis (STE), Bra (branches), Lea (leaves), Inf (inflorescences), Fru (fruits), PD, 1 (collection in a shaded area) and PD, 2 (collection in a sunny area).

The methanolic extract from the branches of P. duckei (PD1) showed inhibition against C. freundii (73.86%) and Escherichia coli (71.77%), whereas the leaves extract inhibited Serratia marcescens (75.85%). The leaves extract from plants collected from a sunny area (PD 2) showed activity against Aeromonas hydrophila (68.62%), C. freundii (75.28%), and Staphylococcus epidermidis (53.81%), while the inflorescence extract inhibited S. marcescens (50.94%). The distinct antibacterial profiles observed between PD 1 and PD 2 suggest that environmental conditions may influence the production or accumulation of bioactive metabolites in P. duckei.

The methanolic extract from the branches of K. preclara showed the highest inhibition against E. coli (100%), whereas the leaves extract exhibited strong activity against Klebsiella pneumoniae (95.27%). Extracts showing inhibition above 70% in the initial screening were subsequently evaluated by minimum inhibitory concentration (MIC) assays (Table 2). However, none of the extracts completely inhibited bacterial growth at the highest tested concentration (500 μg/mL), resulting in MIC values above this concentration. These findings indicate that the antibacterial effects observed in the preliminary screening may depend on extract concentration and suggest that the active constituents may occur at relatively low abundance within the crude extracts.

TABLE 2. Minimum inhibitory concentration (MIC) of methanolic extracts from Amazonian Rubiaceae species against bacterial strains.

Data expressed as inhibition percentage (%) compared to negative control. (−) samples that showed no activity, with inhibition values lower than 20%.

p < 0.001.

p < 0.01.

p < 0.5, (n = 3). Extracts from leaves (Lea), branches (Bra) of the species: Kerianthera preclara e Pagamea duckei in a shaded area (PD, 1) and a sunny area (PD, 2).

Several representatives of this family have demonstrated antibacterial activity against both Gram-positive and Gram-negative bacteria, with phenolic compounds and alkaloids frequently associated with these effects (Castro et al., 2016; Liu et al., 2016; Chassagne et al., 2021; Jouogo et al., 2022; Layug et al., 2025). Therefore, the antibacterial activity observed for P. duckei and K. preclara may be related to the occurrence of bioactive metabolites within these chemical classes; however, this hypothesis requires confirmation through targeted chemical analyses and isolation studies.

All evaluated extracts showed limited activity against Pseudomonas aeruginosa and Acinetobacter baumannii. This reduced susceptibility has also been reported for other plant-derived extracts and may be associated with the intrinsic resistance mechanisms of these species, including low outer membrane permeability, efficient efflux systems, and enhanced detoxification capacity, which reduce the effectiveness of both conventional antibiotics and plant-derived bioactive compounds (Li et al., 2015; Breijyeh et al., 2020; Jubair et al., 2021).

To our knowledge, this study represents the first report of antibacterial activity for K. preclara, P. duckei, and R. lanceifolia. Although the observed effects are preliminary, these findings expand the biological knowledge of poorly investigated Amazonian Rubiaceae species and provide a basis for future studies focused on bioassay-guided fractionation, isolation, and structural characterization of the metabolites responsible for the observed activity.

3.5 Antioxidant activity

Among the methanolic extracts evaluated, only those from P. duckei exhibited antioxidant activity in the tested assays, with the branches extract from plants collected from a sunny area (PD 2) showing the strongest response in both DPPH (1.6 equivalents of ascorbic acid) and Fe³⁺/phenanthroline (2.8 equivalents) assays (Table 3). The higher antioxidant capacity observed in PD 2 may be associated with differences in the accumulation of phenolic metabolites, as suggested by the TLC profiles and ¹H NMR spectra, which indicated a greater abundance of aromatic compounds in this sample. Similar patterns have been reported by Jadidi et al. (2023), who demonstrated that increased exposure to photosynthetically active radiation and UV-B can stimulate the accumulation of phenolic and flavonoid compounds, resulting in enhanced antioxidant responses in plants.

TABLE 3. Antioxidant potential of methanolic extracts determined by DPPH and Fe³⁺/phenanthroline assays and expressed as ascorbic acid equivalents.

The antioxidant activity was interpreted according to the scale proposed by Martins et al. (2014), in which antioxidant equivalence values lower than 1.0 indicate very active samples, values between 1.1 and 2.0 indicate active samples, values between 2.1 and 3.0 indicate moderate antioxidant activity, and values higher than 3.1 indicate inactive samples (n = 3). Bra (branches), Lea (leaves), Inf (inflorescences), Fru (fruits), PD, 1 (collection in a shaded area) and PD, 2 (collection in a sunny area).

Phenolic metabolites are widely recognized for their antioxidant properties due to their ability to scavenge reactive species and chelate transition metals, as well as their involvement in plant defense mechanisms, adaptation to environmental stress, and pigmentation processes (Takó et al., 2020). In Rubiaceae species, antioxidant responses evaluated by the DPPH assay have also been associated with the occurrence of phenolic constituents, including compounds such as p-coumaric acid (Duarte et al., 2025; Meha and Sudhir, 2025).

However, it is important to consider that chemical antioxidant assays provide a preliminary estimation of the reducing capacity and radical-scavenging potential of plant extracts. Although these methods are useful for screening purposes, they evaluate specific chemical properties and do not necessarily reflect antioxidant effects under biological conditions. Therefore, complementary approaches, including cellular models and other biological systems, are required to confirm the biological relevance of the observed antioxidant responses (Apak et al., 2016; Yamauchi et al., 2024; Lisjak et al., 2026).

To our knowledge, this is the first report describing the antioxidant potential of P. duckei and comparing samples collected under contrasting light conditions. The antioxidant responses observed for this species indicate the presence of metabolites with reducing and radical-scavenging properties, highlighting P. duckei as a promising candidate for further phytochemical investigations. Nevertheless, the compounds responsible for this activity remain to be identified through complementary chemical analyses, isolation, and structural characterization.

3.6 Insecticidal activity against Sitophilus zeamais

The evaluated extracts showed different levels of contact toxicity against Sitophilus zeamais (Table 4). Among the tested species, the methanolic extract from Pagamea macrophylla leaves exhibited the strongest insecticidal effect, with the lowest lethal concentrations (LC₅₀ = 3.82 ± 1.11 µg/insect and LC₉₀ = 15.73 ± 1.13 µg/insect), indicating high activity at low doses. In contrast, the methanolic leaf extract of R. lanceifolia showed the lowest toxicity, requiring higher concentrations to achieve mortality (LC₅₀ = 90.83 µg/insect and LC₉₀ = 420.60 µg/insect). Differences among plant organs were also observed for K. preclara, in which the leaf extract showed greater toxicity (LC₅₀ = 8.94 µg/insect and LC₉₀ = 58.82 µg/insect) compared with the branch extract (LC₅₀ = 12.30 µg/insect and LC₉₀ = 150.30 µg/insect). The methanolic extract from P. duckei branches also showed relevant insecticidal activity, with LC₅₀ and LC₉₀ values of 11.46 and 55.83 µg/insect, respectively.

TABLE 4. Median lethal concentration values (LC₅₀ and LC₉₀) and Probit analysis parameters for methanolic extracts active against Sitophilus zeamais.

number of insects used in the test.

degrees of freedom.

lethal concentration and confidence interval.

calculations using ProbitCalc software (v1.1.0).

minimum and maximum lethal concentrations estimated by the statistic, (n = 4). PD, 1 (collection in a shaded area).

The pronounced toxicity of P. macrophylla leaf extract suggests that this organ may accumulate higher levels or a more diverse profile of metabolites with insecticidal potential. Similarly, the differences observed between leaves and branches of K. preclara indicate that metabolite distribution among plant tissues may influence biological activity, reflecting variations in chemical defense strategies (Yang et al., 2020; Gajger and Dar, 2021; Qasim et al., 2024).

The insecticidal responses observed in this study are consistent with the preliminary chemical profiles obtained by TLC and ¹H NMR analyses, which suggested the presence of metabolite classes such as terpenoids, fatty acids, phenolic compounds, and nitrogen-containing compounds. Several studies have demonstrated that terpenoids and fatty acid derivatives can exert insecticidal effects through multiple mechanisms, including disruption of respiratory processes, penetration through the insect cuticle, and neurotoxic effects (Isman, 2006; De Lira et al., 2015; Rosa et al., 2020; De Pimentel et al., 2023; Wanna et al., 2025). Likewise, nitrogen-containing metabolites, particularly alkaloids, have been associated with interference in nervous system function and cellular processes in insects (Gajger and Dar, 2021). However, the specific compounds responsible for the activity observed in the present study remain to be determined through isolation and structural characterization.

To our knowledge, this is the first report describing the insecticidal potential of P. duckei, P. macrophylla, and K. preclara against S. zeamais. The results highlight these Amazonian Rubiaceae species as promising sources of metabolites with insecticidal properties and provide a foundation for future investigations involving bioassay-guided fractionation, isolation, and identification of the compounds responsible for the observed effects.

4 Conclusion

This study provides a preliminary phytochemical and biological characterization of five poorly investigated Amazonian Rubiaceae species. The results revealed distinct chemical profiles and biological activities, with K. preclara showing antibacterial activity and preliminary evidence of nitrogen-containing metabolites, P. duckei exhibiting antioxidant and antibacterial potential associated with phenolic-like metabolites, and P. macrophylla demonstrating promising insecticidal activity against S. zeamais. TLC and ¹H NMR analyses suggested the putative occurrence of terpenoids, iridoids, phenolic compounds, glycosylated metabolites, and nitrogen-containing compounds. However, their definitive identification requires further studies involving advanced chemical analyses, isolation, and structural elucidation. To our knowledge, this is the first integrated phytochemical and biological assessment of these Amazonian Rubiaceae species, providing a foundation for future investigations aimed at discovering bioactive natural products.

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Footnotes

Edited by: Carmen Lucia Cardoso, University of São Paulo, Brazil

Reviewed by: Minhajur Rahman, University of Chittagong, Bangladesh

Mohd Shahanbaj Khan, B. S. Abdur Rahman Crescent Institute of Science and Technology, India