Comprehensive Chemical Characterization and Bioactivity Assessment of Anacardium occidentale Leaf Extract

Abstract

Anacardium occidentale Linn. is a tropical fruit tree of global significance, native to South America and widely cultivated worldwide. This study aimed to annotate metabolites, isolate and characterize agathisflavone, develop and validate a high-performance liquid chromatography with diode-array detection (HPLC-DAD) method for its quantification, and evaluate the antimicrobial and cytotoxic activities of a standardized leaf extract. Metabolite profiling was performed using ultra-performance liquid chromatography coupled to a quadrupole/time-of-flight tandem mass spectrometer (UPLC-QTOF-MS/MS) and molecular networking based on the Global Natural Products Social Molecular Networking (GNPS) platform. A total of 25 compounds were annotated in the extract, predominantly gallic acid derivatives and flavonoids, and fractionation furnished agathisflavone. The validated method showed satisfactory validation parameters, and the agathisflavone content was determined to be 6.7 mg g^–1. The extract was active against Candida tropicalis (minimum inhibitory concentration, MIC = 62.5 μg mL^-1). However, extract concentrations above 100 μg mL^-1 reduced normal human lung fibroblast (GM07492-A) cell viability (half-maximal inhibitory concentration, IC₅₀ = 83.38 μg mL^-1, SI = 1.33 (selectivity index)), indicating low selectivity. These results provide data for future studies on the quality control and standardization of this herbal medicine, suggest its antifungal potential, and indicate that it exhibits moderate cytotoxicity.

Keywords:
Anacardiaceae; biflavonoid; cashew; gallic acid; GNPS

Introduction

Anacardium occidentale Linn. (Anacardiaceae), commonly known as cashew tree, is a tropical fruit tree of global economic and ethnobotanical significance. The cashew tree is native to South America, particularly Brazil, but it has been cultivated worldwide. The largest cashew nut producers are Southern and Southeastern Asia and sub-Saharan Africa.¹

Anacardium occidentale has been registered in the Brazilian Pharmacopoeia, and its bark has been used to relieve mild non-infectious diarrhea.²,³ Besides the bark, folk medicine has traditionally used different parts of this plant, including the leaves, to treat infections, inflammation, and gastrointestinal disorders.⁴,⁵ Additionally, plant leaves represent an abundant and renewable plant material that is often regarded as an agricultural by-product, which makes them attractive for developing standardized phytomedicines and applications that add value to crop species.⁶

Previous studies⁵,⁷-¹⁵ have demonstrated that A. occidentale leaves are rich in phenolic acids, gallotannins, and flavonoids, and that these leaves exhibit antioxidant, antimicrobial, and cytotoxic activities in various models. These classes of secondary metabolites have been associated with antimicrobial effects in medicinal and crop plants and investigated as alternative or complementary agents against fungal pathogens.⁶,¹⁶ Nevertheless, most reports on A. occidentale leaves have relied on non-standardized extracts, partial chemical characterization, or isolated endpoints, which has limited their translational utility.

Fungal infections caused by Candida species are therapeutically challenging and underlie infections that range from superficial mucocutaneous candidiasis to severe invasive and systemic diseases. These infections affect immunocompromised individuals and hospitalized patients and are associated with high morbidity and mortality rates.¹⁷ Furthermore, the increasing prevalence of non-albicans Candida species is concerning because many of these species exhibit reduced susceptibility to commonly prescribed antifungal drugs. In this context, C. tropicalis has emerged as a clinically relevant and highly virulent pathogen, underscoring the need for new antifungal agents with defined chemical composition and favorable safety profile.¹⁶,¹⁸

Among the flavonoids reported in natural products, agathisflavone, a biflavonoid that has been detected in A. occidentale¹⁹ and other taxa,²⁰ has emerged as a promising chemical marker. However, its quantitative determination in cashew leaf preparations remains insufficiently explored. In addition to this limitation, studies that correlate defined chemical markers with biological activity and safety assessment are scarce.

Given the consolidated but heterogeneous evidence regarding the phenolic profile and biological activities of A. occidentale leaves, this study aimed to profile the ethanolic leaf extract using ultra-performance liquid chromatography coupled with quadrupole/time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) and molecular networking based on the Global Natural Products Social Molecular Networking (GNPS); to isolate agathisflavone and confirm its structure; to develop and validate a simple high-performance liquid chromatography with diode-array detection (HPLC-DAD) method for agathisflavone quantification; and to evaluate the antifungal activity of the agathisflavone-standardized leaf extract against C. tropicalis and its cytotoxicity toward normal human lung fibroblasts.

Experimental

Chemicals

Solvents were of analytical (Synth, Diadema, Brazil) or HPLC (Merck, Darmstadt, Germany) grade. Ultrapure water was obtained from a Simplicity water system (Millipore, Bedford, USA). Deuterated methanol and deuterated dimethyl sulfoxide were used as nuclear magnetic resonance (NMR) solvents (Sigma-Aldrich, USA).

Plant material and extract preparation

A. occidentale leaves were collected in São Carlos, São Paulo, Brazil (22°1’34”S, 47°52’26”W), in April 2013 (first harvest) and April 2024 (second harvest). The specimen was authenticated by Valéria Maria M. Gimenez, and a voucher (SPFR 16254) was deposited in the Herbarium of the Department of Biology, Laboratory of Plant Systematics, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo. Collection and genetic heritage access licenses were Nos. 42929-3 and SisGen A1484F8, respectively.

After drying and grinding, 52 g of leaves were extracted with ethanol (3 × 200 mL) in an ultrasonic bath for 30 min, in triplicate. After filtration and solvent evaporation, a total of 8.4 g of A. occidentale leaf crude extract (AOE) was obtained from the pooled ethanolic macerates of the first harvest, corresponding to a yield of 16.2% (m/m) relative to the dried leaves. The same batch of AOE was used for UPLC-QTOF-MS/MS analyses and bioassays.

Chemical analysis of the extract by UPLC-QTOF-MS/MS

For UPLC-QTOF-MS/MS analyses, AOE was dissolved in methanol at 1 mg mL^-1, and 0.6 µL of this solution was injected into the chromatographic column. The analyses were performed on an ACQUITY UPLC System coupled to a Waters Xevo G2-XS QToF mass spectrometer (Waters, USA). An ACQUITY UPLC HSS T3 C18 column (1.8 μm, 100 × 2.1 mm, Waters Corporation, Ireland) was used to perform the chromatographic separation at 40 °C. The mobile phases consisted of eluents A (water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid) at 0.5 mL min^-1. Gradient elution was set as follows: 5-100% B from 0 to 10 min, 100% B from 10 to 14 min, and return to the initial condition after 15.0 min, with equilibration for 5 min.

Mass spectra were acquired by using ESI (electrospray ionization) in the negative mode for m/z ranging from 100 to 1500 Da. The equipment was operated in the data-dependent acquisition (DDA) mode. Each MS scan lasted 0.1 s and was followed by an MS² scan of the most intense ions. For MS² fragmentation, a low-collision energy ramp from 10 to 40 eV and a high-collision energy ramp from 50 to 80 eV were used to acquire data in the centroid format. The equipment was operated in the high-resolution mode at 6.0 GHz and for 76.0 μs; leucine-enkephalin was employed as standard. The spectrometer parameters were capillary voltage of 2.5 kV, cone voltage of 40 V, source temperature of 120 °C, desolvation temperature of 350 °C, cone gas flow of 50 L h^-1, and desolvation gas flow of 800 L h^-1.

Metabolite annotation

Metabolites were annotated through MS² analysis by using the GNPS web platform, manual interpretation with the MZmine 3 software (version 3.4.27; MZmine Development Team, Münster, Germany),²¹ and searches for potential metabolite candidates based on compounds that have been isolated from plants belonging to the family Anacardiaceae, genus Anacardium, and A. occidentale. Additionally, metabolites were annotated on the basis of mass accuracy (with a maximum error of 5 ppm), isotopic pattern distribution obtained from MS¹ (to generate molecular formulas), and adduct formation.

Metabolites were also annotated on the basis of the confidence criteria proposed for HRMS (high-resolution mass spectrometry) based identification. GNPS library matches were supported by accurate mass (≤ 5 ppm), isotope pattern, and MS/MS fragmentation (level 2), while tentative structures based on literature and diagnostic fragments were classified as putative candidates (level 3).²²

The LC-MS/MS data file was converted to the mzXML format by using MSConvert version 3.0 (ProteoWizard, Palo Alto, USA). Then, the converted file was uploaded and analyzed at the GNPS platform by using WinSCP version 6.5.5 (Martin Prikryl, Prague, Czech Republic).

The molecular network was generated by using the online workflow with the following parameters: the precursor ion mass and MS/MS fragment tolerances were set to 0.5 and 0.05 Da, respectively; the cosine score was set above 0.65; and at least five common fragment ions were required per MS/MS spectrum. Library annotations were obtained by comparing the MS/MS spectra and GNPS spectral libraries; at least five matching fragment ions and a cosine score above 0.65 were required.²³ The resulting network was visualized using Cytoscape version 3.10.4 (The Cytoscape Consortium, San Diego, USA). The broad precursor and fragment ion mass tolerances were selected to ensure that spectral coverage was comprehensive and compatible with public spectral libraries. However, all the proposed annotations reported herein were subjected to stringent high-resolution validation criteria that were compatible with QTOF-MS/MS data using the MZmine 3 software. Final acceptance required precursor ion mass errors within a narrow ppm range (≤ 5 ppm), consistent isotope patterns, and the presence of diagnostic fragment ions characteristic of the respective compound classes, as supported by high-resolution MS/MS spectra and literature data.

Agathisflavone isolation

AOE (4.7 g) was dissolved in 250 mL of methanol/water (8:2 v/v) and subjected to liquid-liquid partitioning. The dissolved sample was extracted with hexane and ethyl acetate. The separated phases were concentrated in a rotary evaporator, to yield the hexane (HF, 0.74 g), EtOAc (EF, 0.87 g), and hydromethanol (HMF, 3.04 g) fractions.

EF (0.87 g) was subjected to solid-phase extraction (SPE, silica ODS, 230-400 mesh-ASTM, Sigma-Aldrich) by using methanol/water as the mobile phase. This yielded four subfractions: EF-1 (methanol/water, 30:70 v/v), EF-2 (methanol/water, 50:50 v/v), EF-3 (100% methanol), and EF-4 (100% methanol). The presence of agathisflavone was confirmed in EF-2, which was purified on a Sephadex LH-20 (GE-Healthcare) column (570 × 20 mm) and eluted with methanol. Agathisflavone was isolated from subfractions 17-20 (6 mg, 98% purity, as determined by HPLC-DAD), and its chemical structure was assigned by comparing the ¹H and ¹³C NMR data with published data. The spectra were acquired on a Bruker Avance 400 and DRX 500 spectrometers and are provided in the Supplementary Information (SI) section. NMR data were processed using SpinWorks version 4.0 (K. Marat, University of Manitoba, Winnipeg, Canada).

Development and validation of a method for agathisflavone determination

A Shimadzu LC-20AD (Shimadzu, Kyoto, Japan) system was used to develop and validate the method. This system was equipped with a pump (LC-20AD), a degasser (model DGU-20A3), a DAD detector (SPD-M20A), an autosampler (SIL-20AHT), an oven (CTO-20A), a control module (CBM-20 A), and Software LCsolution.

A stock standard agathisflavone solution (1.0 mg mL^-1) was prepared by dissolving an accurately weighed amount of the compound in methanol. Five working standard solutions (2, 5, 10, 25 and 50 µg mL^-1) were obtained by serial dilution.

To quantify agathisflavone, extracts were prepared by weighing 3 g of dried and ground leaves from the second harvest and extracting them with 30 mL of methanol in an ultrasonic bath for 10 min. The solutions were filtered, and the solvent was evaporated in a fume hood. Sample solutions (0.5 mg mL^-1) were prepared by dissolving an accurately weighed amount of extract in 20 mL of methanol. For agathisflavone quantification, AOE (3.18 mg mL^-1) was prepared by dissolving an accurately weighed amount of AOE (127 mg) in 20 mL of methanol, which was followed by sequential dilution with methanol.

All the solutions were filtered through a 0.45-µm membrane filter before being analyzed by chromatography. The working standard agathisflavone solutions and A. occidentale sample solutions (20 μL) were injected into a Luna C18 column (250 × 4.6 mm, 5 μm; Phenomenex) maintained at 40 °C. The mobile phase consisted of acetonitrile (solvent B) and water containing 2% acetic acid (solvent A); the following gradient program was applied: 2% to 100% B in 30 min, 100% B for 5 min, return to 5% B in 3 min, and equilibration for 15 min. The flow rate was maintained at 1.0 mL min^-1. To record the chromatogram, the DAD detector was set at 270 nm.

The analytical method was validated for specificity, linearity, sensitivity, accuracy, and precision.²⁴,²⁵ Specificity was demonstrated by analyte purity across the agathisflavone peak in extract samples. Linearity between the peak area and concentration was evaluated by using calibration curves obtained with five standard concentrations, in triplicate. Data were analyzed by linear regression; Excel 2013 (Microsoft Corporation, Redmond, USA) was employed. The limits of detection (LOD) and quantification (LOQ) were determined from the calibration curve by using the equations LOD = 3.3σ/S and LOQ = 10σ/S, where σ represents the standard deviation of the blank, and S is the slope of the calibration curve. Ten replicate measurements of the blank were used for the calculations.

Recovery was assessed by using leaf crude extract spiked with agathisflavone at 5 µg mL^-1 (n = 9) or not. Peak areas were measured, and recovery percentages were calculated. Method precision was evaluated by repeatability, by using the recovery data, and the relative standard deviation (RSD) was determined.

Antifungal activity

The AOE (obtained from the first harvest) antifungal activity was evaluated against Candida tropicalis (ATCC 13903) and Fusarium oxysporum (F4, obtained from the institutional fungal culture collection);²⁶ the broth microdilution method was employed for yeasts and filamentous fungi.²⁷,²⁸ C. tropicalis was cultured on CHROMagar and Sabouraud dextrose agar at 30 °C for 48 h, while F. oxysporum was grown on potato dextrose agar for five days, to obtain conidia. The inocula were adjusted to 0.5-2.5 × 10³ colony-forming units (CFU) per milliliter (mL) for C. tropicalis and 1 × 10⁴ conidia mL^-1 for F. oxysporum. AOE was dissolved in 2% dimethyl sulfoxide (DMSO) and diluted in Roswell Park Memorial Institute (RPMI) 1640 (2000-3.9 µg mL^-1). Microplates were inoculated and incubated at 37 °C for 48 h (C. tropicalis) and 35 °C for 72-96 h (F. oxysporum). The controls included a sterility control (2% DMSO), an antifungal agent (amphotericin B, tested from 0.315 to 16 µg mL^-1), and reference strains, namely Candida krusei (ATCC 6258) and Candida parapsilosis (ATCC 22019) for yeasts and Aspergillus flavus (ATCC 204304) for filamentous fungi. Aqueous resazurin solution (0.001%) was added to visualize yeast viability. The minimum inhibitory concentration (MIC) was defined as the lowest concentration with blue coloration (C. tropicalis) or absence of visible growth (F. oxysporum). All the tests were performed in triplicate.

Antibacterial activity

The AOE antibacterial activity (AOE obtained from the first harvest) was determined against Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (clinical isolate).

The bacteria were grown in Brain Heart Infusion agar for 24 h, and Mueller-Hinton broth was used for the assay. The inoculum was adjusted to 5 × 10⁵ CFU mL^-1.²⁹ The samples were dissolved in 2% (v/v) DMSO and diluted from 2000 to 3.90 µg mL^-1 in culture medium. The suspensions were pipetted into plates containing sample dilutions and incubated at 35 °C for 24 h. Wells containing only medium served as sterility controls, and wells with medium and inoculum served as growth controls. Escherichia coli (ATCC 25922) was used as reference strain, and ampicillin was employed as reference drug over a concentration range of 0.125 to 256 µg mL^-1. Aqueous resazurin solution (0.001%) was added to indicate bacterial viability. MIC was defined as the lowest concentration with blue coloration. All the tests were performed in triplicate.

Cytotoxicity assay

Normal human lung fibroblast (GM07492A) cells were used to assess the AOE cytotoxic potential by using the XTT colorimetric assay kit (Roche Diagnostics). Briefly, GM07492A cells (10⁴ cells per well) were seeded in 96-well microplates with 100 µL of HAM F10/DMEM medium containing AOE (19.5-2500 µg mL^-1) prepared in 0.1% DMSO. After incubation at 37 °C for 24 h, the medium was removed, and the cells were washed with 100 µL of PBS (phosphate-buffered saline). Then, 100 µL of phenol red-free HAM-F10 medium and 25 µL of XTT reagent were added per well. The plates were incubated at 37 °C for 17 h. Absorbance was measured at 450 nm on a microplate reader with a reference wavelength of 620 nm. Cytotoxicity was expressed as the concentration that inhibited cell growth by 50% (IC₅₀). The experiments were performed in triplicate. The AOE selectivity index (SI) was calculated as SI = IC₅₀/MIC. Statistical analyses were performed by using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test to compare treatment groups. Results are reported as mean ± standard deviation, with statistical significance set at α = 0.05.

Results and Discussion

Chemical composition

The GNPS database enabled annotation of 15 metabolites through spectral library searches of the molecular and fragment ions (Figure 1a). GNPS molecular networking provided additional structural evidence, which supported metabolite annotation and allowing visualization of metabolite families, including flavonols (Figure 1b) and gallic acid derivatives (Figure 1c), as well as their potential biological relevance to the extract.

Figure 1.
Molecular network obtained from UPLC-QTOF-MS/MS data acquired in negative ionization mode from Anacardium occidentale leaf extract. (a) Complete feature-based molecular network generated by GNPS and visualized in Cytoscape. Red nodes represent metabolites annotated by spectral library matching. Nodes associated with blank samples were removed from the final network. Expanded view of the cluster containing annotated (b) flavonol and (c) gallic acid derivatives.

Additionally, 10 compounds were annotated on the basis of prior knowledge of the species chemistry and fragmentation patterns (Figure 2, Table 1, and in the SI section Figures S1-S35).

Figure 2.
Metabolites tentatively annotated in A. occidentale crude extract: melibiose (1), shikimic acid (2), galloyl-glucose (3), gallic acid (4), protocatechuic acid hexoside (5), galloyl shikimic acid (6), epigallocatechin (7), procyanidin B1 (8), catechin (9), gallic acid methyl ether (10), 1,3,6-tri-O-galloyl-glucose (11), myricetin hexoside (12), 1,2,3,6-tetra-O-galloyl-glucose (13), ethyl gallate (14), quercetin-3-O-galactoside (15), quercetin galloyl hexoside (16), quercetin-3-O-xyloside (17), kaempferol-3-O-glucoside (18), cyanidin-3-O-(2’’-galloyl)-galactoside (19), quercetin-3-O-rhamnoside (20), kaempferol-3-O-arabinoside (21), quercetin galloyl pentoside (22), 3’’-galloyl-quercitrin (23), quercetin (24), and agathisflavone (25).

Table 1.
Metabolites tentatively annotated in the Anacardium occidentale leaf crude extract (AOE)

tR / min	Identification	Identification confidence levela	Molecular formula	Adduct ion	Calculated mass (m/z)	Experimental mass (m/z)	Mass error / ppm	Fragment ions	Ref.
0.53	1	2	C12H22O11	[M + FA – H]–	387.1139	387.1141	0.5	341; 221; 179; 119; 101	GNPS
0.58	2	3	C7H10O5	[M – H]–	173.0450	173.0448	–1.2	155; 137; 111	7
0.84	3	3	C13H16O10	[M – H]–	331.0665	331.0653	–3.6	211; 169; 151; 125; 123	7
1.12	4	3	C7H6O5	[M – H]–	169.0137	169.0129	–4.7	125	7
1.59	5	3	C13H16O9	[M – H]–	315.0716	315.0713	–1.0	153; 108	30
1.73	6	3	C14H14O9	[M – H]–	325.0560	325.0546	–4.3	169; 125	7
1.81	7	2	C15H14O7	[M – H]–	305.0661	305.0647	–4.6	167; 137; 125	GNPS, 7
1.86	N.a.b	3	C18H24O13	[M – H]–	447.1139	447.1135	–0.9	305; 152; 108	–
1.94	N.a.b	3	C7H6O4	[M – H]–	153.0188	153.0186	–1.3	109	–
2.09	N.a.	5	–	–	–	451.2182	0.7	401; 337; 272	–
2.24	8	2	C30H26O12	[M – H]–	577.1346	577.1353	1.2	407; 289; 245; 125	GNPS, 7, 31
2.48	9	2	C15H14O6	[M – H]–	289.0712	289.0704	–2.8	245; 203	GNPS, 32
2.53	10	3	C8H8O5	[M – H]–	183.0293	183.0290	–1.6	168; 124	33
2.69	N.a.c	3	C20H20O13	[M – H]–	467.0826	467.0827	0.2	423; 315; 169	–
2.71	11	2	C27H24O18	[M – H]–	635.0884	635.0889	0.8	557; 465; 313; 169	GNPS, 7
2.92	N.a.	5	–	–	435.2230	435.2219	–2.5	382; 295; 232	–
3.04	12	2	C21H20O13	[M – H]–	479.0826	479.0824	–0.4	317; 316; 287; 271; 214	GNPS, 7
3.14	13	2	C34H28O22	[M – H]–	787.0994	787.0999	0.6	617; 465; 313; 169	GNPS, 7
3.21	N.a.c	3	C22H26O13	[M – H]–	497.1295	497.1305	2.0	435; 313; 236; 169; 124	–
3.27	14	3	C9H10O5	[M – H]–	197.0450	197.0446	–2.0	169; 124	35
3.32	15	2	C21H20O12	[M – H]–	463.0877	463.0855	-4.8	301; 300; 271; 151; 107	GNPS, 7
3.35	N.a.	5	–	–	–	469.0503	–	169; 125	–
3.40	16	2	C28H24O16	[M – H]–	615.0986	615.0991	0.8	463; 301; 151	GNPS, 7
3.50	17	2	C20H18O11	[M – H]–	433.0771	433.0763	–1.8	300; 301; 271	GNPS, 7
3.55	18	2	C21H20O11	[M – H]–	447.0927	447.0936	2.0	285; 284; 255; 227	GNPS, 7
3.61	19	3	C28H24O15	[M – H]–	599.1037	599.1050	2.2	335; 285; 169	7
3.68	20	3	C21H20O11	[M – H]–	447.0927	447.0929	0.4	301; 300; 271; 255	7
3.82	21	2	C20H18O10	[M – H]–	417.0822	417.0816	–1.4	284; 255; 227	GNPS
3.98	N.a.d	3	C21H20O10	[M – H]–	431.0978	431.0968	–2.3	358; 284	–
4.06	22	3	C27H22O15	[M – H]–	585.0880	585.0862	–3.1	301; 151	7
4.17	23	2	C28H24O15	[M – H]–	599.1037	599.1037	0.0	301; 151	GNPS
4.36	N.a.d	3	C27H22O14	[M – H]–	569.0931	569.0934	0.5	285	–
4.47	N.a.d	3	C28H24O14	[M – H]–	583.1088	583.1103	2.6	285	–
4.53	24	2	C15H10O7	[M – H]–	301.0348	301.0345	–1.0	178; 151	GNPS
5.30	25	1	C30H18O10	[M – H]–	537.0822	537.0833	2.1	443; 417; 399; 375	GNPS, 31

The compound annotations were categorized according to confidence levels. Most features were assigned as level 2 (i.e., they matched the GNPS database) and a few remaining features as level 3 (tentative candidate). Agathisflavone isolation and structural confirmation (level 1) reinforced that this compound is a relevant quality-control marker for A. occidentale. This systematic classification made the chemical assignments more robust and addressed a common limitation of untargeted metabolomics studies.²²

The annotated metabolites included gallic acid derivatives and flavonoids, which confirmed previous reports on the A. occidentale chemical profile. ⁷,¹²,³⁰-³⁵

Gallic acid derivatives

Six compounds were tentatively annotated (3, 6, 10, 11, 13, and 14) as gallic acid derivatives, mainly gallic acid esters. Three gallotannin were annotated as monogalloylglucose (3), as trigalloyl-glucose (11), and as tetragalloyl‐glucose (13). The MS/MS spectra of compounds 11 and 13 showed characteristic fragment ions that corresponded to consecutive elimination of a gallic acid (170 Da) and a galloyl (152 Da) moiety. Compounds 3, 11, and 13 presented fragment ions at m/z 169 and 125, which were associated with glucose (162 Da) and CO₂ (44 Da) loss. Compound 4 produced an [M – H]^– ion at m/z 169.0137, annotated as gallic acid, and at m/z 125, associated with CO₂ loss. Among the gallic acid derivatives, loss of a methyl (15 Da) and an ethyl (28 Da) group from the molecular ion supported the presence of gallic acid methyl ether (10) and gallic acid ethyl ether (14), respectively, while the fragment ion at m/z 124 in the MS/MS spectra of compounds 10 and 14 originated from subsequent decarboxylation. The MS/MS spectrum of galloyl shikimic acid (6) showed a characteristic fragment ion at m/z 169, which corresponded to a deprotonated gallic acid moiety and represented neutral loss of 156 Da due to ester bond cleavage. An additional fragment ion at m/z 125, generated by decarboxylation of the gallic acid fragment, supported the presence of a galloyl group.

Flavan-3-ol derivatives

The MS/MS spectrum of epigallocatechin (7) in the negative ionization mode showed extensive fragmentation. Such fragmentation yielded an intense product ion at m/z 204, attributed to C-ring retro-Diels-Alder (RDA) cleavage, and diagnostic phenolic fragments at m/z 137 and 125. An additional fragment ion was observed at m/z 167, associated with the trihydroxylated B-ring. This fragmentation pattern is consistent with the pattern reported for flavan-3-ol and reflects a high degree of epigallocatechin hydroxylation, thereby supporting its tentative annotation. The MS/MS spectrum of catechin (9) showed characteristic flavan-3-ol fragmentation, including a product ion at m/z 245 (neutral loss of 44 Da) from the precursor ion. Additional fragment ions at m/z 205 and 203 arose from heterocyclic C-ring cleavage.

Flavonols

The data showed the presence of one flavonol (24) and nine glycosylated flavonols (12, 15, 16-18, and 20-23). The glycosylated flavonols underwent losses that corresponded to the sugar moiety, which generated the aglycone ion (quercetin: m/z 301, kaempferol: m/z 285, and myricetin: m/z 317) that subsequently fragmented according to the typical flavonol pattern. Additionally, there was characteristic MS/MS fragmentation in the negative ionization mode including C-ring RDA cleavage, to yield diagnostic product ions associated with the aglycone A-ring (m/z 179 and 151).

Miscellaneous compounds

Among the annotated compounds, melibiose (1) was tentatively annotated based on fragment ions arising from glycosidic bond cleavage, generating product ions corresponding to monosaccharide units (m/z 179). The fragment ion at m/z 111 in the MS/MS spectrum of shikimic acid (2) was considered the main diagnostic fragment, as a result of CO₂ (44 Da) and H₂O (18 Da) losses. The MS/MS spectrum of protocatechuic acid hexoside (5) showed a characteristic neutral loss of 162 Da. This corresponded to hexose cleavage and yielded a diagnostic fragment at m/z 153, assigned to deprotonated protocatechuic acid. In the negative ionization mode, the MS/MS spectrum of procyanidin B1 (8) exhibited characteristic fragmentation dominated by quinone methide cleavage, which yielded the monomeric catechin/epicatechin ion at m/z 289. The MS/MS spectrum of cyanidin-3-O-(2”-galloyl)-galactoside (19) in the negative ionization mode showed a diagnostic product ion at m/z 285, which corresponded to deprotonated cyanidin arising from glycosidic bond cleavage (neutral losses of 162 and 152 Da). The presence of the galloyl-related fragment at m/z 169 further supported the tentative annotation of a galloylated cyanidin galactoside. In the negative ionization mode, agathisflavone (25) exhibited characteristic MS/MS fragmentation, including an ion at m/z 417 arising from partial cleavage of one flavone unit via C-ring fragmentation.

Chromatographic analysis and agathisflavone isolation

Figure 3 shows the base peak chromatogram. The most intense ions were detected at m/z 387.1141 (t_R 0.53 min), 173.0448 (t_R 0.58 min), 183.0290 (t_R 2.53 min), 469.0503 (t_R 3.35 min), and 537.0833 (t_R 5.30 min), which corresponded to compounds 1, 2, and 10; an unannotated compound; and compound 25, respectively. Agathisflavone was detected in the extract and isolated for use as a reference standard during HPLC-DAD method development and validation. Agathisflavone has been detected in A. occidentale¹⁹ and reported as a relevant bioactive biflavonoid,²⁰ but its use as a reference standard in A. occidentale remains limited.

Figure 3.
UPLC-QTOF-MS/MS base peak intensity chromatograms obtained in negative ion mode for A. occidentale crude extract (AOE).

Using liquid-liquid partition, solid phase extraction, and Sephadex LH-20 column chromatography, agathisflavone (25) was isolated from the leaf crude ethanol extract derived from the first harvest. The NMR data (¹H and ¹³C, Figure S36 and S37 in the SI section) were consistent with literature values reported for agathisflavone, thereby confirming the identity of compound 25.³⁶ HPLC-DAD analysis at 270 nm indicated a purity of 98% for this compound (Supplementary chromatogram, Figure S38), which showed that it is a suitable chemical marker for quality control.

Method development and validation

The analytical conditions were established to determine agathisflavone on the basis of a trial-and-error approach, by using A. occidentale leaf crude extract derived from the second harvest. Under the established analytical conditions, the peak corresponding to agathisflavone was detected (t_R 19.66 min) in the crude extract, confirming the presence of the compound of interest (Figure 4a). The similarity between the UV spectra of the agathisflavone standard and of the corresponding peak in the crude extract analysis reinforced the analyte identity (Figures 4d and 4f). The selectivity was confirmed by using a diode array detector, which showed a peak purity of 0.999.

Figura 4.
Chromatograms in the UV region (270 nm) of (a) OEA derived from the second harvest, (b) OEA derived from the first harvest, and (c) agathisflavone standard. UV-vis spectra of (d) the peak at t_R = 19.66 min, (e) the peak at t_R = 19.78 min, and (f) agathisflavone standard. Analytical conditions: acetonitrile/water + 2% acetic acid linear gradient (2 to 100% acetonitrile in 30 min, and 100% acetonitrile for 10 min). The flow rate was 1.0 mL min^-1, the oven temperature was 40 °C and Phenomenex Luna column.

The area of the peak obtained at λ = 270 nm was used to construct the agathisflavone calibration curve. The method was linear at agathisflavone concentrations ranging from 2 to 50 µg mL^-1; RSD was lower than 5% for triplicate analyses. The final calibration curve was described by the equation y = 42244 x − 65031 (r² = 0.9934). The slope was 42244 (95% CI: 35924–48564), and the intercept was −65031 (95% CI: −(226260–96198)). The LOD and LOQ were 0.48 and 1.59 µg mL^-1, respectively.

Recovery and repeatability tests at 5 µg mL^-1 showed 108.9% recovery with an RSD of 3.24%. The agathisflavone content was 16.4 mg g^-1 of extract derived from the second harvest, as determined from the external calibration curve. The agathisflavone content was 6.7 mg g^-1 of extract derived from the first harvest (Figures 4b and 4e). Chemical variability between the extracts derived from the first and second harvests was expected due to natural environmental and physiological factors. Despite this limitation, it was possible to detect and quantify agathisflavone in both cases. The measured agathisflavone concentration indicated that this compound is not merely a trace constituent and may play a meaningful role in the biological profile of the extract.

Developing and validating an HPLC-DAD method to quantify agathisflavone constitutes a significant methodological contribution of this study. While earlier reports have focused on qualitative profiling,⁷,³⁷ a validated quantitative method allows the agathisflavone content in crude extracts to be reliably determined, which is essential for standardization, reproducibility, and future pharmacological evaluations.

Antimicrobial and cytotoxic activities

Table 2 lists the MIC results obtained for AOE derived from the first harvest against S. aureus, P. aeruginosa, C. tropicalis, and F. oxysporum. According to the criteria adopted for plant extracts (MIC ≤ 100 µg mL^-1: strong activity; MIC between 100 and 500 µg mL^-1: moderate activity; MIC between 500 and 1000 µg mL^-1: weak activity),³⁸ AOE showed strong activity against C. tropicalis (MIC = 62.5 µg mL^-1), moderate activity against S. aureus and P. aeruginosa (MIC = 500 µg mL^-1), and weak activity against F. oxysporum (MIC =1000 µg mL^-1). Although AOE had a higher MIC than the reference drugs, the AOE antifungal effect against C. tropicalis supports its ethnomedicinal use and calls for its further bioassay-guided fractionation to identify its active constituent.

Table 2.
Minimum inhibitory concentration (MIC) determined for Anacardium occidentale extract

Microorganism	MIC / (µg mL-1)
	AOEa	Ampicillin	Amphotericin
Staphylococcus aureus (ATCC 25923)	500	2	n.t.
Pseudomonas aeruginosa (clinical isolate)	500	> 256	n.t.
Candida tropicalis (ATCC 13903)	62.5	n.t.	0.5
Fusarium oxysporum (sequenced environmental isolate - F4).	1000	n.t.	4.0

• ^a
  AOE: Anacardium occidentale crude extract; n.t.: not tested.

The quality control MIC of amphotericin B against C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 (1.0 and 0.25 µg mL^-1, respectively) was within the CLSI recommended ranges.²⁷ Against A. flavus ATCC 204304, the MIC of amphotericin B was 1.0 µg mL^-1,²⁸ and against E. coli ATCC 25922, the reference drug ampicillin showed MIC of 0.25 µg mL^-1. P. aeruginosa is intrinsically resistant to ampicillin, so the lack of activity observed for this drug against this strain is expected and does not indicate experimental failure.

The obtained MIC for AOE against C. tropicalis was lower than the MIC previously reported for other plant extracts against this species. A study⁵ reported that a leaf extract did not produce an inhibition halo against C. tropicalis. In contrast, an extract obtained by using 80% methanol at 60 °C provided MIC of 500 µg mL^-1 against C. albicans,⁸ which is higher than MIC observed in the present study.

Additionally, MIC of 62.5 µg mL^-1 has been reported for a leaf extract against C. albicans,³⁹ which suggested that extraction conditions, chemical composition, or synergistic interactions among metabolites may influence the extract antifungal potency. Although gallotannins and flavonoids,⁹,⁴⁰ metabolite classes with documented antifungal properties, were present in the extract and possibly contributed to the observed activity, direct evidence remains limited. Future studies evaluating the MICs of purified agathisflavone would help to clarify the relative contributions of individual metabolites. Additionally, it should be acknowledged that gallotannins often dominate the antifungal activity of crude plant extracts.¹⁶ In contrast to the pronounced activity against C. tropicalis, AOE weakly inhibited F. oxysporum, which suggested that its antifungal effect was more specific on yeasts than on filamentous fungi.

Several of the annotated compounds, including gallic acid (4), galloyl-glucose derivatives (3, 4, 5, 11, and 13), catechin (9), epigallocatechin (7), and procyanidin B1 (8), are widely recognized for their antimicrobial properties, and their detection reinforced that phenolic compounds contribute to the A. occidentale biological activities. The occurrence of multiple galloyl-glucoses and flavonoid glycosides in AOE further highlighted a feature that is associated with antifungal and antibacterial effects.⁶,¹⁶

Figure 5 shows the mean results of three independent cell viability assays, with bars indicating the standard error of the mean. AOE obtained from the first harvest reduced GM07492A cell viability in a concentration-dependent manner, and IC₅₀ was 83.38 ± 2.93 µg mL^-1. Given that AOE had MIC of 62.5 µg mL^-1 against C. tropicalis, the calculated SI was 1.33. Therefore, selectivity for fungal cells over normal fibroblasts was limited, so further fractionation must be conducted, and individual compounds must be tested.

Figura 5.
Cell viability of normal human lung fibroblast (GM07492A) cells after treatment with AOE derived from the first harvest. Negative control (no treatment); solvent control (1% DMSO); positive control (25% DMSO). Statistical analyses were performed by using one-way ANOVA, followed by Tukey’s multiple comparisons test to compare treatment groups. Values are the mean ± standard deviation.*Significantly different from the negative group (p < 0.05).

Conclusions

This study presents a comprehensive metabolite profiling of A. occidentale leaves based on UPLC-HRMS/MS and GNPS-based molecular networking. The profiling revealed a diverse composition comprised mainly of gallic acid, flavan-3-ol, and flavonol derivatives. These findings are consistent with previous reports on the polyphenol-rich nature of A. occidentale and support its ethnobotanical and pharmacological relevance. In addition, a HPLC-DAD method to quantify agathisflavone was validated, representing an important advance compared to earlier qualitative studies.

A limitation of the present study is that it focused on a single collection site, which may not fully capture the chemical variability associated with environmental, seasonal, or genetic factors. Future studies should focus on isolating the identified metabolites and evaluating their biological activity, as well as on assessing chemical variability across different plant materials and growing conditions. Ongoing efforts are being directed toward obtaining a greater amount of purified agathisflavone to perform MIC assays and to clarify how it contributes to the observed antifungal activity.

Supplementary Information

Supplementary information (MS/MS and NMR data) is available free of charge at http://jbcs.sbq.org.br, as PDF file.

Supplementary PDF

Supplementary PDF

Data Availability Statement

The mass spectrometry data supporting the findings of this study have been deposited in the MassIVE repository under accession number MSV000100342. The datasets generated during the validation study have been deposited in Zenodo (DOI: 10.5281/zenodo.18084435). Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors acknowledge financial support from CNPq No. 306106/2024-8; CAPES No. 001; and FAPESP Nos. 2016/10313-4 and 2024/00915-3. The use of ChatGPT (OpenAI) for the initial generation of the graphical abstract is also acknowledged. The final image was manually modified and refined using Paint 3D.

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Edited by

• Editor handled this article:
  João Henrique Ghilardi Lago (Associate)

Publication Dates

• Publication in this collection
  03 Aug 2026
• Date of issue
  2026

History

• Accepted
  28 Apr 2026