Chemical Profiling by UHPLC–MS/MS and In Vitro Antioxidant, Antimicrobial, and Cytotoxic Properties of Lepidoceras peruvianum Kuijt Leaves and Fruits

ABSTRACT

Rationale

Lepidoceras peruvianum Kuijt is an understudied hemiparasitic species endemic to the Peruvian Andes that lacks comprehensive phytochemical characterization. Investigating its metabolome is important for understanding its chemotaxonomic relevance and potential as a source of bioactive compounds.

Methods

Leaves and fruits of L. peruvianum were subjected to untargeted metabolomic profiling using high‐resolution UHPLC–ESI‐Orbitrap‐MS/MS in positive and negative ionization modes. Metabolites were annotated based on accurate mass measurements, isotopic patterns, collision‐induced dissociation (CID) fragmentation data, and spectral matching with the Global Natural Products Social Molecular Networking (GNPS), METLIN, and MassBank databases. Structural elucidation employed diagnostic fragmentation pathways, including retro‐Diels–Alder (RDA), heterocyclic ring fission (HRF), quinone methide (QM), and benzofuran‐forming (BFF) cleavages. Antioxidant activity was evaluated using DPPH●, ABTS●+, and FRAP assays, while antimicrobial activity and toxicity were assessed through antibacterial testing and the Artemia salina lethality assay.

Results

Leaves contained 34 metabolites, mainly flavan‐3‐ols, proanthocyanidins, flavonols, lignans, and isoquinoline alkaloids, whereas fruits were characterized by anthocyanins, organic acids, and polar lipids. Total phenolic content was higher in leaves (292.4 mg GAE/g) than in fruits (216.8 mg GAE/g), corresponding to stronger antioxidant activity. Leaves extracts exhibited greater antibacterial activity against Staphylococcus aureus , while fruit extracts showed stronger effects against Gram‐negative bacteria. LC₅₀ values in the A. salina assay were 181.8 μg/mL for leaves and 475.9 μg/mL for fruits.

Conclusions

This study provides the first comprehensive metabolomic characterization of L. peruvianum, revealing organ‐specific chemical diversity and notable bioactive properties. The findings demonstrate the utility of HRMS/MS fragmentation analysis for metabolite annotation and identify this endemic species as a promising source of bioactive natural products.

1. Introduction

Ultra‐high‐performance liquid chromatography coupled with mass spectrometry (UHPLC–MS/MS) has transformed metabolomic analysis by enabling the simultaneous detection and structural characterization of metabolites in complex matrices, such as food and medicinal plants [1, 2, 3, 4, 5]. The integration of high‐resolution mass spectrometry (HRMS) and tandem mass spectrometry (MS/MS) enhances compound annotation by allowing precise identification of secondary metabolites—including isomers [6], oligomers, and polymers [7]—based on retention time, accurate mass measurements, and fragmentation pattern analysis [8]. The use of both positive and negative electrospray ionization modes ensures broader metabolite coverage and improved sensitivity and fragmentation consistency [5, 9, 10]. Additionally, spectral matching against databases such as the Global Natural Product Social Molecular Networking (GNPS), the National Institute of Standards and Technology (NIST14), and the MassBank of North America strengthens the reliability of compound annotation [5].

The systematic phytochemical characterization of underexplored endemic species is particularly relevant given the growing interest in natural sources of redox‐active metabolites. Oxidative stress is implicated in the pathogenesis of multiple chronic conditions, and plant‐derived phenolic compounds are widely recognized for their capacity to modulate reactive oxygen species (ROS) through radical scavenging, metal chelation, and redox signaling regulation [11, 12]. Therefore, identifying novel phenolic‐rich species remains a strategic priority in antioxidant research.

Lepidoceras peruvianum Kuijt (Eremolepidaceae) is a hemiparasitic species endemic to Peru, distributed between 3500 and 3600 m above sea level in the Mesoandean region between Pampa and Mantaro basins. Together with Lepidoceras chilense, it represents one of only two species within the genus Lepidoceras Hooker F. [13]. Initially collected by Weberbauer in 1910 in Totorobamba (Huamanga Province, Ayacucho, Peru) and later formally described by Kuijt in 1985, it has been recognized as a species known only from its type collection [13, 14]. Due to its restricted distribution, lack of conservation measures, and anthropogenic pressures such as firewood extraction, its population may be at risk. Recent rediscovery in rocky outcrops of the Huaraca Stone Forest (Vinchos District) confirmed its parasitic association with Berberis flexuosa, traditional topical use for inflammatory conditions, and production of edible fruits [15].

Preliminary phytochemical screening has revealed the presence of triterpenes and/or steroids, quinones, flavonoids, cardiotonic compounds, phenols and/or tannins, saponins, and coumarins in leaves, while fruits contain flavonoids, phenolic compounds, anthocyanins, and triterpenes/steroids. Initial in vitro evaluations also suggested antioxidant potential. As a hemiparasitic plant, L. peruvianum can partially fix carbon autotrophically while absorbing inorganic nutrients from its host [16]. Although parasitic plants may negatively impact host species, several taxa exhibit recognized medicinal properties, including applications in dermatological and metabolic disorders [17].

Peru harbors remarkable botanical endemism, particularly within the Andean region, with approximately 5509 endemic plant taxa (27.9% of its flora) [14]. This high biodiversity underscores the importance of chemically characterizing endemic species to support conservation and potential sustainable utilization.

Antioxidant capacity is commonly assessed using complementary colorimetric assays, such as 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH●), 2,2′‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS●+), or ferric reducing antioxidant power (FRAP), which enable comparative evaluation of radical‐scavenging and reducing properties [11, 12]. These activities are frequently correlated with total phenolic content [2], reinforcing the biological relevance of phenolic metabolites.

Bioprospecting efforts have identified plant‐derived flavonoids, terpenoids, and alkaloids with antimicrobial effects mediated through membrane disruption, enzyme inhibition, and oxidative imbalance in microbial cells [18, 19, 20, 21]. Additionally, the Artemia salina lethality assay provides a rapid, cost‐effective preliminary screening method for assessing cytotoxicity and the biological activity of extracts [22, 23].

Given the lack of comprehensive phytochemical and bioactivity studies on L. peruvianum Kuijt, we hypothesized that this endemic species contains structurally diverse phenolic metabolites that confer measurable antioxidant and antimicrobial activities. Therefore, this study aimed to (i) perform untargeted metabolomic profiling of leaves and fruit extracts using UHPLC–MS/MS, (ii) quantify total phenolic content, (iii) evaluate in vitro antioxidant capacity using DPPH•, ABTS•+, and FRAP assays, (iv) assess antibacterial activity, and (v) determine preliminary cytotoxicity using A. salina bioassay. This integrative approach provides the first comprehensive chemical and functional characterization of this Andean hemiparasitic species.

2. Materials and Methods

2.1. Plant Material

Leaves and fruits of L. peruvianum Kuijt were collected from rocky outcrops in the Bosque de Piedras de Huaraca, located in the community of Huaraca, Anchacchuasi (Vinchos District), Huamanga Province, Ayacucho, Peru (13°18′58.9″S; 74°26′55.8″W; 3627 m a.s.l.).

Taxonomic identification was carried out at the Herbarium San Marcos, Museo de Historia Natural, Universidad Nacional Mayor de San Marcos (Lima, Peru). A voucher specimen was deposited under accession number 001‐2024‐USM‐MHN (identification record 329‐USM‐MHN‐2023).

2.2. UHPLC–ESI‐MS/MS Metabolomic Profiling

Leaves and fruits were freeze‐dried using a lyophilizer (Labotec, South Africa) at −42°C and 0.013 mbar for 72 h. The dried material was ground in a porcelain mortar and stored in hermetically sealed aluminum containers, protected from light and humidity until analysis.

For metabolomic extraction, 30 mg of lyophilized powder was transferred to a 10mL volumetric flask and extracted with methanol/acetonitrile/water (5:3:2, v/v/v). The mixture was sonicated (150 W, 0.7 A) for 25 min at room temperature. Extracts were filtered through a 0.22μm polyvinylidene fluoride membrane filter and stored at −20°C until LC–MS/MS analysis. All extractions were performed in triplicate.

Chromatographic separation was carried out on a Dionex UltiMate 3000 UHPLC system coupled to a Q Exactive Plus Hybrid Quadrupole‐Orbitrap mass spectrometer (Thermo Fisher Scientific GmbH, Germany) equipped with a heated electrospray ionization (HESI) source. Separation was achieved using a Luna Omega C18 column (100 Å, 150 mm × 2.1 mm, 1.6 μm; Phenomenex, USA). The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The gradient elution was performed as follows: 90% A at 0–1 min, 70% A at 5–18 min, 0% A at 20–22 min, and re‐equilibration to 90% A until 30 min. The flow rate was maintained at 0.3 mL min⁻¹; the injection volume was 2 μL, and the column temperature was set at 40°C.

Mass spectra were acquired in both negative and positive ionization modes over a scan range of 120–1500 m/z. Full MS scans were recorded at a resolution of 35 000 (at m/z 200) with an automatic gain control target of 1 × 10⁶ and maximum ion injection time of 100 ms. Data‐dependent MS/MS spectra were obtained at a resolution of 17 500 with an automatic gain control target of 2 × 10⁵ and a maximum ion injection time of 80 ms. The electrospray ionization (negative/positive) voltage was set at 2.5/3.0. The capillary temperature was 250°C, the auxiliary gas heater temperature was 400°C, the sheath gas flow rate was 50 arbitrary units, the sweep gas flow rate was 1 arbitrary unit, and the S‐lens RF level was set at 100. Normalized collision energy values 30 and 40 were used in negative mode and 20 and 40 in positive mode. Compound annotation was based on accurate mass measurements (mass error ≤5 ppm), isotopic distributions, and MS/MS fragmentation pattern comparisons against mzCloud, METLIN, and Mass Bank databases, and identification confidence was assigned according to the Metabolomics Standards Initiative (MSI) criteria.

2.3. Total Phenolic and Flavonoids Assay

Hydroethanolic extracts (70% ethanol) of L. peruvianum Kuijt leaves and fruits were obtained by percolation at room temperature. The extracts were filtered and concentrated under reduced pressure using an R‐3000 rotary evaporator (BÜCHI Labortechnik AG, Switzerland) at 40°C. The concentrated extracts were subsequently dried using a Mini Spray Dryer B‐290 (BÜCHI Labortechnik AG) operating at an inlet temperature of 120°C, a feed flow rate of 2 mL min⁻¹, and 100% aspiration [15]. All determinations were performed in triplicate.

2.3.1. Total Phenolic Content

Total phenolic content was determined using Folin–Ciocalteu colorimetric method [24]. Briefly, 100 μL of extract solution (320 μg mL⁻¹ in methanol) was mixed with 500 μL of diluted Folin–Ciocalteu reagent (1:10) and 400 μL of 7.5% sodium carbonate solution. After incubation for 30 min at room temperature in the dark, absorbance was measured at 765 nm using a GENESYS 150 UV–Vis spectrophotometer (Thermo Fisher Scientific, USA). Gallic acid was used to construct the calibration curve (y = bx + a; R ² ≥ 0.99), and results were expressed as milligrams of gallic acid equivalents per gram of dry extract (mg GAE/g). All measurements were performed in triplicate.

2.3.2. Total Flavonoid Content

Total flavonoid content was determined using the aluminum trichloride colorimetric method [12]. A methanolic extract solution was prepared at a concentration of 800 μg mL⁻¹. Briefly, 2 mL of the extract solution was mixed with 0.5 mL of 2% aluminum chloride solution, and the mixture was diluted to 5 mL with methanol. The reaction mixture was incubated at room temperature for 30 min, and absorbance was measured at 415 nm using a GENESYS 150 UV–Vis spectrophotometer (Thermo Fisher Scientific, USA). A calibration curve was constructed using rutin as the reference standard (y = bx + a; R ² ≥ 0.99). The results were expressed as milligrams of rutin equivalents per gram of dry extract (mg RUE/g). All measurements were performed in triplicate.

2.4. Anthocyanin Quantification

Lyophilized fruit material (15 g) was macerated in 200 mL of 70% ethanol acidified with citric acid (pH 3.3) for 24 h at room temperature. The macerate was filtered and concentrated under reduced pressure using an R‐3000 rotary evaporator (BÜCHI Labortechnik AG) at 40°C. The concentrated extract was subsequently freeze‐dried using a lyophilizer (Labotec) at −42°C and 0.013 mbar for 48 h.

Total anthocyanin content was determined using the pH‐differential method [25]. The dry anthocyanin extract was dissolved in 70% ethanol to obtain a stock solution of 10 mg mL⁻¹. This solution was further diluted to 1 mg mL⁻¹ in buffer solutions at pH 1.0 and 4.5. Absorbance was measured at 520 and 720 nm, with the corresponding buffer used as the blank. All measurements were performed in triplicate. Total anthocyanin content was calculated according to Equation (1).

$$\begin{array}{c}\text{Total anthocyanin}\left(\mathrm{mg}/100\mathrm{g}\right)\\ =\left\{\left[\left(A^{*}\times \mathrm{MW}\right)/\left(\mathrm{\varepsilon }\times l\right)\right]\times \left[\mathrm{DF}\right]\times \left[\left({V\mathrm{s}}_{\mathrm{mL}}\right)/{W\mathrm{s}}_{\mathrm{g}}\right]\right\}\times 100\end{array}$$ (1)

where A* represents the absorbance difference calculated as (A ₅₂₀ − A ₇₂₀) at pH 4.5; MW, molecular weight for cyanidin‐3‐glycoside (449.2 g mol⁻¹); DF, dilution factor; ɛ, molar extinction coefficient for cyanidin‐3‐glycoside (26 900 L mol⁻¹ cm⁻¹); l, path length (1 cm); Vs, sample volume (mL); Ws, sample weight (g).

2.5. Antioxidant Capacity Assays

2.5.1. DPPH Radical Scavenging Assay

The antioxidant capacity was evaluated using the DPPH• scavenging method described by Brand‐Williams et al. [26], as modified by Sousa et al. [27]. Briefly, dry extracts from leaves and fruits were dissolved in methanol to obtain concentrations ranging from 25 to 250 μg mL⁻¹. An aliquot of 300 μL of each concentration was mixed with 2.7 mL of DPPH• methanolic solution (40 μg mL⁻¹). The reaction mixture was incubated for 30 min at room temperature in the dark, and absorbance was measured at 515 nm using methanol as a blank.

Antioxidant activity was calculated according to Equation (2):

$$\text{Antioxidant activity}\left(\%\right)=\left[\left({\mathrm{A}}_{\mathrm{c}}-\left({\mathrm{A}}_{\mathrm{s}}-{\mathrm{A}}_{\mathrm{b}}\right)\right)/\left({\mathrm{A}}_{\mathrm{c}}\right)\right]\times 100$$ (2)

where A _c represents the absorbance of the control (300 μL of methanol and 2.7 mL of DPPH● solution); A _s, corresponds to the absorbance of the sample or Trolox solution mixed with DPPH●, and A _b, absorbance blank (300 μL of extract dilution or Trolox + 2.7 mL of methanol).

Trolox was used as a positive control, and antioxidant activities of extracts and Trolox were compared at a concentration of 100 μg mL⁻¹. The median effective concentration (EC₅₀) was determined by plotting the percentage of remaining DPPH• against extract concentration and fitting a nonlinear regression model. A calibration curve of DPPH• (1–40 μg mL⁻¹) in methanol was used to determine the residual radical concentration. Data analysis was performed using OriginPro version 8. All measurements were carried out in triplicate.

2.5.2. ABTS•⁺ Scavenging Assay

ABTS•⁺ assay was performed according to the method of Rivas et al. [12]. ABTS•⁺ was generated by mixing equal volumes of 7 mM ABTS and 2.45 mM potassium persulfate solutions, then allowing the mixture to react for 16 h at room temperature in the dark. Prior to analysis, the ABTS•⁺ solution was diluted with 96% ethanol to obtain an absorbance of 0.70 ± 0.02 at 734 nm. A stock solution of each extract (500 μg mL⁻¹) was prepared in 96% ethanol and further diluted to obtain concentrations ranging from 25 to 250 μg mL⁻¹. An aliquot of 20 μL of each dilution was mixed with 980 μL of ABTS•⁺ solution and incubated for 7 min at room temperature. Absorbance was measured at 734 nm using 96% ethanol as a blank.

The percentage of antioxidant activity was calculated according to Equation (2), where A _c represents the absorbance of the control (20 μL of 96% ethanol + 980 μL of ABTS●⁺ solution); A _s, corresponds to the absorbance of the extract or Trolox solution with ABTS●⁺ solution. Trolox was used as a reference antioxidant under the same experimental conditions. The EC₅₀ value was calculated from the dose–response curve obtained by plotting antioxidant activity percentage against extract concentration using nonlinear regression analysis in OriginPro version 8. All assays were performed in triplicate.

2.5.3. Determination of Antioxidant Capacity by the FRAP Method

The FRAP assay was performed according to Rivas et al. [12], with slight modifications. The FRAP reagent was freshly prepared by mixing 25 mL of 0.3 mM acetate buffer (pH 3.6) with 2.5 mL of 10 mM 2,4,6‐tripyridyl‐s‐triazine (TPTZ) solution prepared in 40 mM hydrochloric acid and 2.5 mL of 20 mM ferric chloride hexahydrate. The reagent was incubated in a water bath at 37°C prior to use. A stock solution of the extract (500 μg mL⁻¹) was prepared in 96% ethanol, and working solutions were obtained by serial dilution to yield final concentrations ranging from 25 to 250 μg mL⁻¹. For the assay, 20 μL of each concentration was mixed with 980 μL of FRAP reagent and incubated in the dark at room temperature for 30 min. Absorbance was measured at 593 nm using a UV–Vis spectrophotometer, with 96% ethanol as the blank.

Antioxidant activity was calculated as a percentage of ferric reducing capacity using Equation (2), where A _c corresponds to the absorbance of the control (20 μL of 96% ethanol plus 980 μL of FRAP reagent), A _s represents the absorbance of the sample or Trolox standard, and A _b corresponds to the blank absorbance. Trolox was used as a reference antioxidant under identical experimental conditions. Results were expressed as a percentage of antioxidant activity, as described in Section 2.5.2.

2.6. Antibacterial Activity

2.6.1. Modified Agar Well Diffusion Method

Antibacterial activity was evaluated using a modified agar well diffusion method [1, 28]. Reference strains were reactivated on tryptic soy agar (TSA) and incubated at 35°C ± 2°C for 18–24 h. A microbial suspension was prepared by adding sterile saline solution (0.85%, w/v) to the fresh culture and vortexing to homogenize. The turbidity was adjusted to 0.5 McFarland standard (approximately 1.5 × 10⁸ CFU mL⁻¹), confirmed spectrophotometrically.

An aliquot of 100 μL of the standardized inoculum was added to 25 mL of molten Mueller–Hinton agar (MHA) maintained at 40°C, gently homogenized, and poured into sterile Petri dishes. After solidification, wells of 6 mm diameter were aseptically made using a sterile cork‐borer, and 100 μL of the test solution was dispensed into each well. Plates were allowed to prediffuse at room temperature for 15 min and subsequently incubated at 35°C ± 2°C for 24 h. For antifungal evaluation against Candida albicans , Sabouraud dextrose agar (SDA) was used as the culture medium. Antimicrobial activity was determined by measuring the diameter of the inhibition zones (mm) using a digital Vernier caliper. Each plate contained five wells for the test sample and one well for the positive control [1, 8]. All assays were performed in triplicate.

2.6.2. Determination of Minimum Inhibitory Concentration

Following inoculation, the microplates were allowed to stand at room temperature for 15 min to ensure adequate interaction between the extract and the microbial suspension. Subsequently, plates were incubated at 35°C ± 2°C for 18–24 h under aerobic conditions. The distribution of treatments within the microplate is presented in Supplementary Table S1.

For extracts exhibiting intrinsic coloration or potential interference with resazurin visualization, minimum inhibitory concentration (MIC) determination is performed by comparing the color of inoculated test wells with their corresponding extract control wells (without inoculum). MIC was defined as the lowest concentration at which no visible color change was observed relative to the blank control, indicating complete inhibition of microbial metabolic activity. For the reference antibiotic, MIC was defined as the lowest concentration that prevented the color transition of resazurin from blue to pink, reflecting bacterial metabolic reduction of the indicator [1, 29, 30]. Antibacterial activity was evaluated against Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 6538, and Bacillus spizizenii ATCC 6633. Antifungal activity was assessed against Candida albicans ATCC 10231.

2.7. Preliminary Toxicity Assay Using A. salina

The preliminary toxicity of the dry leaves and fruit extracts of L. peruvianum Kuijt was evaluated using the A. salina lethality bioassay, following previously described procedures [24, 25]. Briefly, 50 mg of A. salina cysts were incubated in 350 mL of artificial seawater (3.8 g/100 mL) in an Erlenmeyer flask under continuous aeration and artificial illumination at room temperature (20°C–25°C) for 24 h to obtain free‐swimming nauplii. A stock solution of the dry extract (10 000 μg mL⁻¹) was prepared by dissolving 20 mg of extract in 0.5 mL of dimethyl sulfoxide and 1.5 mL of distilled water. Serial dilutions were subsequently prepared in artificial seawater to obtain final concentrations of 1000, 500, 100, and 10 μg mL⁻¹ in a final volume of 5 mL. Ten active nauplii were transferred into each vial containing the different extract concentrations, and assays were conducted in triplicate. A drop of yeast suspension (3 mg of dry yeast in 5 mL of seawater) was added to each vial as a food source. After 24 h of exposure at room temperature, the number of surviving nauplii was recorded. The median lethal concentration (LC₅₀) was calculated using Probit analysis as previously described [31, 32].

2.8. Data Analysis

Data distribution was assessed using the Shapiro–Wilk test to evaluate normality, while homogeneity of variances was verified using Levene's test.

Comparisons between two independent groups were conducted using Student's t‐test. For multiple group comparisons, one‐way analysis of variance (ANOVA) was applied, followed by Tukey's post hoc multiple comparison test when significant differences were detected. Statistical significance was established at 95% confidence level (p < 0.05).

All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) software, version 29.0 (IBM Corp., Armonk, NY, USA).

3. Results and Discussion

This pioneering study employed UHPLC–MS/MS to characterize the metabolite profile of L. peruvianum Kuijt, revealing distinct phytochemical compositions between leaves and fruits (Figure 1). A total of 34 metabolites were detected in leaves, including 16 flavonoids (flavanols, proanthocyanidins, flavonols, flavanones, and dihydrochalcones), 5 alkaloids, 4 lignans, and minor constituents such as organic acids and polar lipids. In contrast, fruits exhibited a differentiated metabolic pattern characterized by the presence of anthocyanins (absent in leaves) and lack of alkaloids. Flavonoids remained the dominant class in fruits, including anthocyanins, flavanols, proanthocyanidins, flavonols, and flavanones. Notably, fruits showed a higher relative abundance of organic acids (17.39% vs. 5.88% in leaves) and polar lipids (17.39% vs. 2.94% in leaves), whereas flavan‐3‐ols and proanthocyanidins were more abundant in leaves (23.53%) than in fruits (13.04%). Organic acids in fruits may contribute to the regulation of acidity, osmotic balance, and stress adaptation [33]. Additional metabolites identified in both organs included lignans, gingerols, sugars, nucleosides, and amino acids/peptides (Table 1). Table 2A and 2B summarize the compounds tentatively identified in leaves and fruits, respectively. These findings represent the first comprehensive metabolomic characterization of L. peruvianum and highlight its chemical diversity and potential bioactive relevance.

FIGURE 1.

Total‐ion chromatograms of L. peruvianum Kuijt leaves and fruits analyzed in negative (upper) and positive (lower) ESI modes. Abbreviations: NL, normalization level; FTMS, Fourier transform mass spectrometry; ESI, electrospray ionization.

TABLE 1.

Bioactive compounds analyzed from L. peruvianum Kuijt leaves and fruits.

Type of metabolite	Leaves (%)	Fruits (%)
Amino acids and peptides	5.88	4.35
Sugars	5.88	4.35
Organic acid	5.88	17.39
Nucleosides	2.94	4.35
Flavan‐3‐ol and proanthocyanidins	23.53	13.04
Flavonol	8.82	17.34
Flavanone	11.76	4.35
Dihydrochalcone	2.94	0.00
Anthocyanins	0.00	8.70
Lignans	11.76	4.35
Alkaloids	14.71	0.00
Alkyl phenols	2.94	4.35
Lipids	2.94	17.39

TABLE 2A.

Chemical compounds identified from L. peruvianum Kuijt leaves by UHPLC–MS/MS.

No.	RT (min)	Adduct	MS (m/z) ion	Mass error (ppm)	MS2 ions (m/z)	Molecular formula	Annotation [reference] (class)
1	1.64	[M + H]+	175.1191	0.9	60.0564, 70.0658, 112.0872, 116.0709, 130.0975, 158.0924	C6H14N4O2	Arginine (amino acid)
2	1.8	[M − H]−	181.0712	2.68	163.0604, 131.0341, 119.0340, 101.0234, 89.0233, 85.0284, 71.0227, 59.0127	C6H14O6	Mannitol (sugar)
3	1.81	[M − H]−	341.1089	1.57	179.0555, 161.0447, 143.0341, 119.0340, 113.0235, 101.0234, 89.0233, 71.0127, 59.0128	C12H22O11	Sucrose (sugar)
4	2.05	[M + H]+	308.0911	0.15	291.0644, 245.0591, 233.0590, 179.0484, 162.0219, 144.0114, 130.0500, 116.0168, 84.0449, 76.0222	C10H17N3O6S	Glutathione (peptide)
5	2.07	[M − H]−	133.0134	1.51	115.0027, 89.0233, 72.9920, 71.0127	C4H6O5	Malic acid (organic acid)
6	2.14	[M + H]+	268.1042	0.48	136.0619, 115.0392, 85.0289, 73.0291, 57.0342	C10H13N5O4	Adenosine (nucleoside)
7	2.19	[M − H]−	133.0134	1.73	173.0085, 129.0182, 111.0078, 87.0077, 85.0284	C6H8O7	Citrate (organic acid)
8	3.57	[M − H]−	577.1357	2.92	407.0771, 339.0874, 289.0716, 245.0816, 205.0500, 161.0236, 125.0235	C30H26O12	B‐type procyanidin dimer (isomer 1) [34, 35, 36] (proanthocyanidin)
		[M + H]+	579.1501	0.77	427.1026, 409.0917, 301.0705, 287.0549, 275.0549, 259.0603, 247.0602, 191.0340, 163.0390, 139.0390, 127.0391
9	5.69	[M − H]−	577.1354	2.3	407.0769, 339.0876, 289.0716, 245.0815, 205.0502, 175.0391, 161.0236, 137.0235, 125.0235	C30H26O12	B‐type procyanidin dimer (isomer 2) [34, 35, 36] (proanthocyanidin)
		[M + H]+	579.1501	0.77	427.1023, 409.0916, 301.0705, 287.0549, 275.0548, 259.0600, 247.0601, 233.0444, 191.0338, 163.0390, 139.0390, 127.0391
10	6.9	[M − H]−	577.135	1.66	407.0769, 339.0869, 289.0869, 245.0815, 203.0705, 179.0340, 161.0235, 137.0235, 125.0234	C30H26O12	B‐type procyanidin dimer (isomer 3) [34, 35, 36] (proanthocyanidin)
		[M + H]+	579.1496	0.18	427.1021, 409.0916, 301.0706, 287.0548, 275.0547, 259.0600, 247.0600, 233.0444, 191.0338, 163.0389, 139.0390, 127.0391
11	7.6	[M − H]−	289.0717	3.44	245.0815, 221.0813, 203.0706, 187.0397, 179.0393, 161.0600, 151.0392, 137.0235, 125.0234, 109.0284	C15H14O6	Catechin [34, 37, 38, 39] (flavan‐3‐ol)
		[M + H]+	291.0862	0.46	273.0756, 249.0761, 207.0652, 165.0547, 147.04440, 139.0390, 123.443
12	7.94	[M − H]−	865.1984	1.14	525.0712, 407.0771, 289.0714, 243.0295, 217.0503, 161.0236, 125.0234	C45H38O18	B‐type procyanidin trimer (isomer 1) [34, 35, 36] (proanthocyanidin)
		[M + H]+	867.2135	0.45	697.1540, 579.1490, 437.0866, 407.0760, 397.0916, 289.0706, 275.0548, 245.044, 163.0389, 139.0390, 127.0391
13	8.57	[M − H]−	577.135	1.66	407.0767, 339.0871, 289.0714, 245.0815, 203.0708, 179.0340, 161.0235, 137.0234, 125.0234	C30H26O12	B‐type procyanidin dimer (isomer 4) [34, 35, 36] (proanthocyanidin)
		[M + H]+	579.1498	0.24	427.1023, 409.0915, 301.0706, 291.0862, 271.0599, 259.0601, 247.0601, 233.0444, 191.0340, 163.0389, 139.0390, 127.0391
14	9.38	[M − H]−	289.0715	9.38	245.0814, 221.0814, 203.0706, 187.0392, 179.0341, 165.0184, 151.0391, 137.0233, 125.0223, 109.0284	C15H14O6	Epicatechin [34, 35, 40] (flavan‐3‐ol)
		[M + H]+	291.0863	0.05	273.0755, 249.0755, 207.0651, 165.0546, 147.0440, 139.0390, 123.0443
15	9.75	[M − H]−	865.1975	0.08	525.0814, 407.0766, 289.0713, 243.0294, 217.0497, 175.0392, 161.023, 125.0234	C45H38O18	B‐type procyanidin trimer (isomer 2) [34, 35] (proanthocyanidin)
		[M + H]+	867.2135	0.47	297.1546, 579.1491, 437.0866, 427.1022, 289.0706, 289.0706, 271.0601, 247.0601, 163.0390, 139.0390, 127.0391
16	10.12	[M + FA‐H]−	787.2662	0.92	417.1552, 402.1310, 387.1080, 181.0499, 166.0267, 96.4146	C34H46O18	Syringaresinol diglucoside (lignan)
17	10.71	[M − H]−	609.1457	1.12	300.0273, 271.0244, 255.0295, 178.9978, 151.0028	C27H30O16	Quercetin‐3‐O‐rutinoside (rutin) [39, 40] (flavonol glycoside)
		[M + H]+	611.1604	0.43	465.1023, 303.0498, 129.0547, 85.0289, 71.0498
18	10.89	[M − H]−	449.1086	1.72	287.0558, 193.0136, 175.0029, 151.0028, 135.0442, 125.0231	C21H22O11	Eriodictyol hexoside (isomer 1) (flavanone)
19	11.06	[M − H]−	463.0881	2.15	300.0272, 271.0244, 255.0296, 178.9980, 151.0027	C21H20O12	Quercetin hexoside (flavonol glycoside)
20	11.22	[M − H]−	593.1512	1.95	327.0504, 285.0399, 255.0296, 229.0506, 227.0342, 178.9976, 151.0029	C27H30O15	Kaempferol‐3‐O‐rutinoside [37, 41] (flavonol glycoside)
		[M + H]+	595.1664	1.06	449.1090, 287.0550, 129.0547, 83.0289, 85.0289, 71.0498
21	11.5	[M − H]−	579.2084	1.99	417.1550, 402.1311, 387.1082, 181.0498, 166.0263	C28H36O13	Syringaresinol 4′‐glucoside (lignan)
22	11.76	[M − H]−	433.1138	1.96	313.0715, 271.0609, 177.0186, 151.0027, 145.0285, 119.0492, 93.0334	C21H22O10	Naringenin‐7‐O‐glucoside (flavanone)
23	11.85	[M − H]−	449.1089	2.45	329.0506, 287.0555, 259.0616, 181.0135, 166.9977,151.0027, 135.0441	C21H22O11	Eriodictyol hexoside (isomer 2) [40] (flavanone)
24	11.87	[M]+	354.1335	0.34	336.1227, 275.0699, 247.0752, 206.0811, 189.0783, 188.0706, 165.0546, 149.0597	C20H20NO5	Protopine [42, 43] (alkaloid)
25	11.96	[M − H]−	435.1292	2.25	273.0765, 229.0865, 179.0341, 167.0341, 125.0233, 123.0440, 123.0440, 119.0493, 93.0334	C21H24O10	Phloretin‐2′‐O‐hexoside (dihydrochalcone)
26	12.23	[M − H]−	805.292	0.81	595.2179, 580.1922, 565.1799, 550.1837, 417.1550, 402.1310, 387.1503, 359.1128, 225.0762, 210.0526, 195.0655, 181.0497, 166.0263	C39H50O18	Buddlenol D 4‐O‐glucopyranoside (lignan)
27	12.27	[M + H]+	370.165	0.3	352.1543, 337.1543, 321.1090, 306.0878, 306.0878, 290.0937, 206.0812, 188.0707, 181.0859, 165.0911, 149.0598	C21H24NO5	Allocryptopine (alkaloid)
28	12.47	[M + H]+	338.1388	0.22	323.1151, 322.1074, 308.0917, 294.1123, 279.0898	C20H19NO4	Dihydroberberine [44] (alkaloid)
29	13.27	[M]+	352.1544	0.33	337.1303, 336.1230, 322.1073, 308.1280, 294.1128	C21H22NO4	Palmatine [43, 45] (alkaloid)
30	13.39	[M]+	336.123	0.22	321.0993, 320.0917, 306.0761, 304.0966, 292.0967	C20H18NO4	Berberine [35, 42] (alkaloid)
31	13.79	[M − H]−	643.2401	2.38	595.2179, 580.1944, 565.1671, 550.1837, 417.1557, 402.1315, 387.1563, 359.1144, 225.0762, 210.0527, 195.0656, 180.0420, 165.0184	C33H40O13	Buddlenol D 4‐O‐glucopyranoside (lignan)
32	14.13	[M − H]−	271.0611	3.8	227.0707, 177.0185, 165.0186, 151.0028, 119.0492, 107.0128, 119.0492, 107.0128, 93.0335, 83.0127, 65.0021	C15H12O5	Naringenin [38, 39] (flavanone)
		[M + H]+	273.0757	0.04	171.0289, 153.0183, 147.0441, 123.0442, 119.0494
33	16.74	[M − H]−	293.1758	3.77	236.1049, 221.1540, 207.1387, 192.1149, 127.0758, 71.0127	C17H26O4	6‐Gingerol (β‐hydroxy ketone)
34	18.54	[M + HCOO‐H]−	559.3123	1.84	277.2170, 253.0926, 161.0446, 119.0339, 101.0233, 89.0232	C27H46O9	9,12,15‐Octadecatrienoic acid hexoside (lipid)

Note: Putative identification by spectral similarity using the following databases: GNPS, NIST14, and MassBank of North America.

TABLE 2B.

Chemical identification of L. peruvianum Kuijt fruits by UHPLC–MS/MS.

No.	RT (min)	Adduct	MS ion (m/z)	Mass error (ppm)	MS2 ions (m/z)	Molecular formula	Annotation [reference] (class)
1	1.57	[M + H]+	175.1191	0.6	60.0564, 70.0658, 112.0872, 16.0709, 160.0975, 158.0924	C6H14N4O2	Arginine (amino acid)
2	1.71	[M − H]−	181.0708	0.91	163.0602, 131.0338, 119.0338, 101.0232, 89.0232, 85.0282, 71.0126, 59.0126	C6H14O6	Mannitol (sugar)
3	1.75	[M − H]−	195.0502	1.34	177.0396, 159.0289, 129.0182, 99.0075, 89.0075, 75.0075, 59.0127	C6H12O7	Gluconic acid (organic acid)
4	1.78	[M − H]−	135.0288	−0.07	117.0183, 89.0232, 75.0075, 72.9919, 59.0127	C4H8O5	Threonic acid (organic acid)
5	1.89	[M − H]−	175.0239	1.29	146.9601, 115.0025, 87.0075, 71.0126, 59.0127	C6H8O6	Ascorbic acid (organic acid)
6	1.95	[M − H]−	133.0132	0.68	115.0025, 89.0232, 72.9919, 71.0126	C4H6O5	Malic acid (organic acid)
7	2.03	[M + H]+	268.1039	−0.45	136.0619, 115.0392, 85.0289, 85.0289, 73.0291, 57.0342	C10H13N5O4	Adenosine (nucleoside)
8	2.2	[M − H]−	593.1514	2.25	284.0321, 147.0076, 125.0233	C27H31O15	Cyanidin‐3‐O‐rutinoside (isomer 1) [39, 53] (anthocyanin)
		[M + H]+	595.1658	0.14	449.1073, 287.0549
9	6	[M − H]−	577.1346	0.91	407.0766, 339.0867, 289.0714, 245.0815, 203.0708, 161.0235, 137.0233, 125.0233	C30H26O12	B‐type procyanidin dimer [35, 36, 37, 39, 40] (proanthocyanidins)
		[M + H]+	579.1496	−0.18	427.1021, 409.0916, 301.0706, 301.0706, 287.0548, 275.0547, 259.0600, 247.0600, 233.0444, 191.0338, 163.0389, 139.0390, 127.0391
10	6.8	[M − H]−	289.0715	2.89	245.0815, 221.0813, 203.0706, 187.0393, 179.0342, 165.0185, 165.0185, 151.0391, 137.0235, 125.0234, 109.0284	C15H14O6	Catechin [34, 35, 37, 39] (Flavan‐3‐ol)
		[M + H]+	291.0864	0.29	273.0773, 249.0759, 207.0653, 165.0547, 165.0547, 1147.0441, 139.0390, 123.0443
11	7.6	[M + H]+	595.1658	0.14	449.1073, 287.0549	C27H31O15	Cyanidin‐3‐O‐rutinoside (isomer 2) [53] (anthocyanin)
12	8.97	[M − H]−	289.0717	3.55	245.0815, 221.0811, 203.0706, 187.0392, 179.0341, 165.0183, 151.0391, 137.0234, 125.0234, 109.0284	C15H14O6	Epicatechin (flavan‐3‐ol)
		[M + H]+	291.0865	0.05	273.0762, 249.763, 207.0653, 165.0546, 147.0441, 139.0390, 123.0443
13	10.42	[M − H]−	609.1459	1.53	300.0275, 271.0249, 255.0292, 178.9979, 151.0028	C27H30O16	Quercetin‐3‐O‐rutinoside [39] (flavonol glicoside)
		[M + H]+	611.1604	−0.43	465.1016, 303.0498, 129.0547, 85.0289, 71.0498
14	10.76	[M − H]−	463.0883	2.55	300.0273, 271.0246, 255.0295, 178.9977, 151.0029	C21H20O12	Isoquercetin (flavonol)
15	10.96	[M − H]−	593.1516	2.45	285.0401, 255.0296, 229.0505, 151.0029	C27H30O15	Kaempferol‐3‐O‐hexosil‐3″‐deoxyhexoside [39] (flavonol glicoside)
16	11.23	[M − H]−	579.2087	2.63	417.1552, 402.1306, 387.1079, 181.0499, 166.0263	C28H36O13	Syringaresinol 4′‐glucoside (lignan)
17	11.95	[M − H]−	303.0511	3.83	275.0558, 259.0605, 241.0500, 217.0501, 199.0393, 175.0393, 153.0393, 125.0234	C15H12O7	Taxifolin (flavonol)
18	13.9	[M − H]−	327.2177	3.48	291.1959, 229.1442, 211.1335, 183.1384, 171.1020, 97.0648, 85.0284	C18H32O5	9,12,13‐Trihydroxy‐10,15‐octadecadienoic acid (lipid)
19	14.08	[M − H]−	271.0612	4.13	227.0706, 177.0185, 151.0028, 119.0492, 107.0128, 93.0335	C15H12O5	Naringenin (flavanone)
20	14.4	[M − H]−	329.2334	3.4	229.1441, 211.1334, 183.1384, 171.1020, 139.1119, 127.1119, 99.0805	C18H34O5	9,12,13‐Trihydroxy‐10‐octadecenoic acid (lipid)
21	16.49	[M − H]−	293.1758	3.46	236.1049, 221.1540, 207.1382, 192.1152, 127.0754, 71.0127	C17H26O4	6‐Gingerol (β‐hydroxy ketone)
22	18.34	[M + COOH‐H]−	559.3123	1.84	413.0988, 277.2170, 253.0925, 161.0442, 101.0235, 89.0230	C27H46O9	9,12,15‐Octadecatrienoic acid hexoside (lipid)
23	18.63	[M − H]−	452.278	1.96	255.2326, 296.0373, 140.0108, 78.9579	C21H44NO7P	1‐Palmitoyl‐2‐hydroxy‐sn‐glycero‐3‐ phosphoethanolamine (lipid)

Note: Putative identification by spectral similarity using the following databases: GNPS, NIST14, and MassBank of North America.

As a hemiparasitic species, L. peruvianum retains photosynthetic capacity while extracting water and nutrients from host plants through specialized haustorial connections. These structures establish functional links with the host xylem, enabling passive transfer of water and dissolved solutes driven by water potential gradients. Hemiparasites are known to acquire not only water and mineral nutrients but also organic carbon sources [46, 47], nitrogen‐containing compounds such as amino acids and organic acids [41], and signaling molecules including phytohormones, messenger RNAs, transcription factors, small interfering RNAs, and even viral particles [48]. Maintenance of a negative water potential is essential for this resource‐acquisition strategy, often achieved by accumulating osmotically active solutes such as mannitol [46, 47, 49], which was particularly abundant in leaves. This physiological strategy reflects a dynamic balance between autotrophic metabolism and host dependence, integrating endogenously synthesized and host‐derived metabolites to optimize survival.

Among the identified metabolites, adenosine was detected in both leaves and fruits as a protonated ion [M + H]⁺ at m/z 288, eluting at approximately 2 min under the applied chromatographic conditions. The MS/MS fragmentation spectra of the precursor ion exhibited highly consistent profiles in both matrices, thereby confirming the identity of adenosine across leaves and fruits in both cases. Extracellular adenosine has been associated with activation of plant defense signaling pathways and stress responses [50]. Therefore, its presence in L. peruvianum may be related to adaptive and defensive mechanisms, potentially linked to its hemiparasitic lifestyle and environmental interactions.

The metabolomic analysis also revealed the presence of polar lipids in both leaves and fruits of L. peruvianum. All lipidic compounds were detected in negative ionization mode, showing well‐defined precursor ions and characteristic MS/MS fragmentation patterns that enabled their tentative identification (Table 2A and 2B). In leaves, the lipid fraction was mainly represented by lysophosphatidylethanolamine derivatives, whereas fruits contained both lysophosphatidylethanolamine and oxylipins. These lipid classes are recognized as bioactive signaling molecules that mediate molecular interactions between plants and pathogens. Notably, the oxylipins detected in fruits, including 9,12,13‐trihydroxy‐10,15‐octadecadienoic acid and 9,12,13‐trihydroxy‐10‐octadecenoic acid, have been associated with the induction of plant defense gene expression and antimicrobial responses [51]. Likewise, lysophospholipids, such as lysophosphatidylcholine, are involved in plant innate immunity, contributing to defense activation and the regulation of fruit ripening processes, which may explain their higher relative abundance in fruits [52].

UHPLC–MS/MS analysis provided key structural information based on retention time, accurate precursor ion mass, and diagnostic fragmentation profiles. Collision‐induced dissociation (CID) generated specific fragmentation patterns that served as molecular fingerprints for compound annotation. The main fragmentation mechanisms observed included retro‐Diels–Alder (RDA) cleavage, heterocyclic ring fission (HRF), quinone methide fission (QM), and benzofuran‐forming (BFF) fission [7, 34, 40, 42, 52]. These fragmentation pathways were clearly identified in the MS² spectra obtained for L. peruvianum extracts and were particularly informative for the structural characterization of flavonoids, lignans, alkaloids, and alkyl phenols.

A total of 16 flavonoids were tentatively identified in leaves, predominantly flavonols and proanthocyanidins. In fruits, 10 flavonoids were detected, with anthocyanins and flavonols representing the most prominent subclasses (Table 2A and 2B). This differential distribution suggests organ‐specific biosynthetic regulation and may contribute to the distinct biological properties observed between leaves and fruits.

Within the flavan‐3‐ol group and their oligomeric derivatives (proanthocyanidins), two monomeric units—catechin and epicatechin—were identified. In negative ion mode, catechin is detected as a deprotonated molecule [M − H]⁻ at m/z 289.0717. The fragment at m/z 137 is attributed to HRF, whereas the fragment at m/z 125 results from a RDA cleavage [34, 37]. The fragment at m/z 137 is attributed to HRF. The ion observed at m/z 125 results from RDA cleavage of the C‐ring [35].

In positive ion mode, catechin is observed as a protonated molecule [M + H]⁺ at m/z 291.0862. The fragment ion at m/z 273 corresponds to the loss of a water molecule, while the ion at m/z 249 arises from ethanol elimination. The fragment at m/z 165 is generated through HRF, involving the elimination of catechin's A ring. The ion at m/z 147 may originate either from further water loss of the m/z 165 fragment or from HRF of the m/z 273 ion (−126 Da). The fragment at m/z 139 is attributed to RDA cleavage [7]. Additionally, the ion m/z 123, previously reported by Li and Deinzer [7], has been associated with a Benzofuran‐Forming Fission (BFF) fragmentation pathway. Our observations indicate that the fragmentation path in positive mode is more straightforward to interpret (Figure 2A).

FIGURE 2.

MS/MS fragmentation pathways of polyphenols and alkaloids from L. peruvianum. (A) Principal fragmentation pathways of catechin under positive ionization mode. (B) Fragmentation pathways of B‐type procyanidin dimer under negative ionization mode. (C) Fragmentation pathways of B‐type procyanidin trimer under negative ionization mode. (D) Fragmentation pathway of quercetin‐3‐O‐rutinoside [M + H]⁺, showing the sequential loss of rhamnosyl and glucosyl units to yield the aglycone quercetin. (E) ESI–MS/MS spectrum of the protopine alkaloid showing the molecular ion [M]⁺ at m/z 354 and characteristic RDA fragmentation pathway.

Epicatechin, an isomer of catechin, exhibits the same precursor ions in both ionization modes: [M + H]⁺ at m/z 291.0863 in positive mode and [M − H]⁻ at m/z 289.0715 in negative mode. Their fragmentation patterns are highly similar (Tables 2A and 2B), making retention time analysis essential for differential identification. Both catechin and epicatechin were detected in leaves and fruits.

Proanthocyanidins, which are oligomeric flavan‐3‐ols [7], were particularly abundant in leaves. Six type B proanthocyanidins were tentatively identified in leaves extracts (Table 2A). Four isomers of proanthocyanidin B dimers (isomers 1–4) were identified. These isomers display highly similar precursor ions and fragmentation profiles, with differentiation achieved through retention times of 3.57, 5.69, 6.90, and 8.57 min, respectively. Each isomer generates in negative ion mode a deprotonated molecule [M − H]⁻ at m/z 577 and in positive ion mode a protonated molecule [M + H]⁺ at m/z 579. In negative ion mode, the fragment at m/z 289 corresponds to catechin/epicatechin monomer, produced via QM fission mechanism [34, 35, 40], confirming that the monomeric units of these dimers consist of catechin and/or epicatechin (Figure 2A).

Two type B proanthocyanidin trimers (isomers 1 and 2) were also identified in leaves. As structural isomers, they shared identical precursor ions and fragmentation profiles in both ionization modes, with differentiation based on retention times of 7.94 and 9.75 min, respectively. In negative ion mode, both isomers of proanthocyanidin trimers exhibit precursor ions as deprotonated molecules [M − H]⁻ at m/z 865 (Table 2A). The fragment ion at m/z 407 is generated via a RDA cleavage of the C ring, accompanied by water elimination. Additionally, the fragment at m/z 289, corresponding to a catechin/epicatechin monomer, is produced through the QM fission (QM cleavage) [34, 42]. In positive ion mode, the protonated molecule [M + H]⁺ at m/z 867 is observed, together with the fragment ion at m/z 579, which results from QM fission with the elimination of one catechin/epicatechin residue [7].

In fruits, only one type B proanthocyanidin dimer has been detected. The ions observed were the protonated molecule [M + H]⁺ at m/z 579 in positive ion mode and the deprotonated molecule [M − H]⁻ at m/z 577 in negative ion mode, both exhibiting fragmentation profiles consistent with the isomers identified in leaves (Figure 2C). However, the distinct retention time of 6 min suggests that this dimer may represent a different isomer from those detected in leaves (Table 2B).

Proanthocyanidins are among the most abundant polyphenols biosynthesized by plants and play crucial roles in defense against abiotic and biotic stress, as well as in the regulation of seed germination and dormancy [40]. In addition to their ecological function, these compounds are widely recognized for their antioxidant, anti‐inflammatory, anticancer, neuroprotective, and cardioprotective properties [7, 40], which may contribute to the biological activities observed in L. peruvianum Kuijt.

Three flavonols (compounds 17, 19, and 20; Table 2A) were identified in leaves and detected in both ion modes. Their MS/MS fragmentation patterns featured sequential losses of sugar moieties, facilitating clear identification of the aglycone in positive mode.

Regarding compound 17 (Table 2A), the precursor ion detected as a protonated molecule [M + H]⁺ at m/z 611 yields the fragment ion m/z 465, consistent with the loss of one rhamnosyl residue [M + H‐146]⁺. Fragment at m/z 303 corresponds to the quercetin aglycone, resulting from the combined loss of one rhamnosyl and one hexosyl residues [M + H‐308]⁺, thereby confirming the identification of this component as quercetin‐3‐O‐rutinoside (Figure 2C).

Compound 19 (Table 2A) was observed as a deprotonated molecule [M − H]⁻, with an exact mass of 463.0881. The fragmentation profile reveals a prominent product ion at m/z 300, corresponding to the quercetin aglycone. This fragmentation occurs due to the loss of a hexosyl residue and an additional hydrogen [M − 2H‐162]⁻. This fragmentation profile supported its tentative identification as quercetin hexoside.

Concerning compound 20 (Table 2A), the precursor ion detected as a protonated molecule [M + H]⁺ at m/z 595 produces a fragment ion at m/z 449, corresponding to the loss of a rhamnosyl residue [M + H‐146]⁺. The fragment ion at m/z 287 is identified as the kaempferol aglycone, resulting from the combined loss of rhamnosyl and hexosyl residues [M + H‐308]⁺, thereby confirming the compound as kaempferol‐3‐O‐rutinoside. This fragmentation pathway is also observed in negative ion mode, where the kaempferol aglycone [M − H‐308]⁻ corresponds to ion m/z 285 [54].

From fruits, four flavonols were detected in both positive and negative ionization modes. Their fragmentation pattern involves the sequential loss of sugar residues, with the aglycone clearly observed, particularly in positive ionization mode. Compound 13 (Table 2B) exhibits the precursor ion detected as a protonated molecule [M + H]⁺ at m/z 611, corresponding to quercetin‐3‐O‐rutinoside. The fragment ion at m/z 303, derived from the precursor ion at m/z 611, corresponds to the quercetin aglycone formed by the loss of one rhamnosyl and one glucosyl residue. Compound 14 shows a precursor ion observed as a deprotonated molecule [M − H]⁻ at m/z 463, generating a fragment ion at m/z 151 through RDA cleavage [M − H‐glucose‐C₈H₈O₂]⁻ [55].

In negative mode, the precursor ion [M − H]⁻ corresponding to m/z 593 for compound 15 (Table 2B) yields the fragment ion m/z 285, corresponding to the aglycone kaempferol, resulting from the loss of one hexose and one deoxyhexoside residue [M − H‐308]⁻. This component was identified as kaempferol‐3‐O‐hexosil‐3″‐deoxyhexoside. The precursor ion [M − H]⁻ at m/z 303 has been identified as dihydroflavonol taxifolin.

Leaves extracts revealed four flavanones: two isomers of eriodictyol hexoside, naringenin, and naringenin‐7‐O‐glucoside. The fragmentation profiles of these compounds involved the characteristic loss of sugar residues in glycosylated forms, with the aglycone ions readily distinguishable. In fruits extracts, only naringenin was detected among flavanones.

Flavanones are recognized as bioactive compounds with significant pharmacological potential. Eriodictyol has been reported to regulate the transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2), a central mediator of antioxidant defense pathways [56], thereby contributing to antioxidant, neuroprotective, cardioprotective, and hepatoprotective effects, as well as improved insulin sensitivity [57]. Naringenin has been associated with inhibition of prostate and breast cancer metastasis, modulation of immune responses, and improvement of insulin resistance [38].

A dihydrochalcone was identified in leaves extracts, exhibiting a precursor ion in negative mode at m/z 435. The MS/MS spectrum of this ion reveals the fragment ion at m/z 273 corresponding to the aglycone phloretin, resulting from glycosidic cleavage. Based on this fragmentation behavior, the compound was identified as phloretin‐2′‐O‐hexoside [54].

Fruits extracts were distinguished by the presence of anthocyanins, detected as two isomeric forms (Table 2B). The characteristic fragmentation pattern of anthocyanins involved sequential loss of sugar residues, most clearly observed in positive mode. In negative mode, fragmentation typically included the loss of a sugar moiety and hydrogen [M − 2H‐sugar] [10, 40]. The precursor ion detected as a protonated molecule [M + H]⁺ at m/z 595 (compound 8; Table 2B) yields a fragment ion at m/z 287, corresponding to cyanidin aglycone, formed by the loss of one hexosyl and one rhamnosyl residue. Consequently, compound 8 has been identified as cyanidin‐3‐O‐rutinoside (isomer 1). In negative ion mode, the precursor ion observed as a deprotonated molecule [M − H]⁻ at m/z 593 produces a fragment ion at m/z 284, corresponding to the cyanidin aglycone. This fragment results from the loss of hexosyl and rhamnosyl residues together with one hydrogen [M − 2H‐308]⁻.

Compounds 8 and 11 shared identical precursor ions and fragmentation profiles in positive ionization mode, confirming their isomeric nature. Therefore, differentiation between these anthocyanins was achieved by retention time analysis, with retention times of 2.2 and 7.6 min, respectively (Table 2B).

Anthocyanins are bioactive natural pigments widely recognized for their antioxidant, antimicrobial, antitumor, immunomodulatory, neuroprotective, and antidiabetic properties. Their biological effects are mediated through multiple molecular mechanisms, including modulation of the cyclooxygenase pathway, free radical scavenging, regulation of inflammatory cytokine signaling, and interaction with mitogen‐activated protein kinase (MAPK) pathways [58]. The presence of anthocyanins exclusively in fruits extracts of L. peruvianum may therefore contribute significantly to their potential antioxidant and antimicrobial activities.

Four lignans—syringaresinol diglucoside, syringaresinol 4′‐glucoside, buddlenol‐D 4‐O‐glucopyranoside, and buddlenol‐D—were identified in leaf extracts, whereas fruits contained syringaresinol 4′‐glucoside (Table 2A and 2B). These compounds were detected exclusively in negative ionization mode, and their fragmentation pathway indicates the potential loss of sugar residues.

The phenolic compound 6‐gingerol was detected in both leaves and fruits in negative ionization mode. This bioactive molecule has been reported to possess antioxidant and anti‐inflammatory properties [59] and has been associated with therapeutic effects against hyperglycemia and renal and cardiovascular complications.

Leaf extracts were further characterized by the presence of five isoquinoline alkaloids that were identified in leaves. Among them, two possess a protopine nucleus (protopine and allocryptopine), two contain a protoberberine nucleus (palmatine and berberine), and one features a dihydroberberine nucleus.

Due to the presence of nitrogen atoms in the alkaloid structure, these compounds readily undergo protonation, allowing their detection in positive ion mode predominantly as protonated molecules [M + 1]⁺. In some cases, radical cations [M]⁺ may also be observed (Table 2A). The fragmentation pathway varies according to the molecular framework of each alkaloid. Alkaloids featuring a protopine or tetrahydroberberine nucleus typically exhibit fragmentation pathways dominated by RDA cleavage, whereas those with a protoberberine nucleus are characterized by sequential elimination of methyl radicals and carbon monoxide losses, rather than RDA processes [42].

The precursor ion observed as a radical cation [M]⁺ at m/z 354.1335 produces diagnostic fragment ions at m/z 206 and m/z 149 (Table 2A), consistent with RDA cleavage [42], thereby confirming its identification as protopine. This isoquinoline alkaloid is reported to exhibit a broad spectrum of biological activities, including antibacterial, antiviral, antifungal, antispasmodic, anti‐inflammatory, neuroprotective, and antithrombotic effects, as well as modulatory influence on cardiovascular system [60] (Figure 2E).

The precursor ion detected as a protonated molecule [M + H]⁺ at m/z 370.165 generates fragment ions at m/z 206 and m/z 165, both arising from RDA cleavage. In addition, the fragment ion at m/z 188 results from the elimination of water from the ion at m/z 206 [61], confirming the compound as allocryptopine.

The precursor ion observed as a protonated molecule [M + H]⁺ at m/z 338.1388 has been identified as a dihydroberberine, a hydrogenated derivative of berberine. This alkaloid exhibits anti‐inflammatory activity by modulating cellular signaling pathways, including the MAPK cascade, nuclear factor kappa B, interleukins, tumor necrosis factor alpha, and nitric oxide production. Furthermore, previous studies highlighted its therapeutic potential in the management of atherosclerosis, diabetes and obesity [44].

In positive ionization mode, palmatine was detected as a radical cation [M]⁺ at m/z 352.1544. This protoberberine alkaloid has demonstrated notable antitumor potential against breast carcinoma [62] and prostate cancer, and also exhibits antioxidant, anti‐inflammatory, neuroprotective, antibacterial, and antiviral activities, in addition to regulating lipid metabolism [63]. The precursor ion observed as a radical cation [M]⁺ at m/z 326.123 undergoes sequential eliminations of methyl groups and carbon monoxide, yielding fragment ions at m/z 321, 320, 304, and 306 [35, 42]. This fragmentation pattern is characteristic of alkaloids with a protoberberine nucleus, confirming its identification as berberine. Previous studies have reported a wide range of bioactive properties for berberine, including anti‐inflammatory, anticancer, antidiabetic, and analgesic effects [36, 44, 64]. Moreover, it has been shown to exert antidepressant, cardioprotective, antioxidant, and neuroprotective activities [65] as well as antimicrobial and anti‐human immunodeficiency virus effects [66], highlighting its therapeutic potential in neurodegenerative diseases such as Alzheimer's [36, 37].

Isoquinoline alkaloids such as berberine have previously been reported in species of the genus Berberis, which serve as hosts for Lepidoceras. The detection of multiple isoquinoline alkaloids in the hemiparasitic plant L. peruvianum Kuijt therefore suggests a potential translocation process from its host, B. flexuosa. Previous studies have demonstrated that parasitic and hemiparasitic plants can sequester alkaloids from their hosts as defensive metabolites via bidirectional translocation. For instance, isoquinoline alkaloids from Berberis montana have been shown to be transferred to the hemiparasite Tristerix verticillatus [67]. Such transfer of defense metabolites may allow hemiparasites to exploit host‐derived compounds to enhance their own defense mechanisms. Furthermore, given the increasing evidence of functional horizontal gene transfer in parasitic plants, further investigation of the interaction between B. flexuosa and L. peruvianum is warranted to determine whether the presence of berberine‐type alkaloids arises from metabolite translocation or from horizontal gene transfer mechanisms.

Using UHPLC–ESI‐MS/MS analysis, a wide range of bioactive metabolites was identified in the hemiparasitic species L. peruvianum. Many of these metabolites are known to participate in cellular signaling pathways and to act as regulatory molecules involved in the development or prevention of diseases such as cancer. To our knowledge, this study represents the first comprehensive phytochemical screening of L. peruvianum, providing a foundation for future pharmacological and biochemical studies aimed at exploring the biological potential of this species.

The quantification of total phenolic content using Folin–Ciocalteu method revealed that leaves extracts contained significantly higher levels of phenolic compounds than fruits extracts. Total phenolic content reached 292.4 ± 0.49 mg GAE g⁻¹ in leaves, whereas fruits showed 216.8 ± 0.91 mg GAE g⁻¹ (p < 0.05; Table 3). These findings are consistent with those reported by Aronés Jara et al. [15], although the values remain lower than those reported for other medicinal plants. For example, Sousa et al. [27] evaluated five medicinal species—Terminalia brasiliensis, Terminalia fagifolia, Copernicia cerifera , Cenostigma macrophyllum var. acuminata, and Qualea grandiflora—and reported total phenolic contents reaching up to 763.63 ± 2.5 mg GAE g⁻¹. Similarly, total flavonoid content in L. peruvianum leaves was 44.0 ± 1.1 mg RUE g⁻¹, which is lower than values reported for other plant extracts such as Myrtus communis , where flavonoid contents range between 77.30 ± 0.80 and 129.96 ± 2.29 mg RUE g⁻¹ [68].

TABLE 3.

Total phenolic content from L. peruvianum Kuijt leaves and fruits.

Plant part	Total phenols (mg GAE/g)	Total flavonoids (mg RUE/g)	Anthocyanins (mg/100 g)
Leaves	292.4 ± 0.49*	44.0 ± 1.09	—
Fruits	216.8 ± 0.91*	—	1263.1 ± 14.27

Note: Mean of three determinations: mg GAE/g, milligrams equivalent to gallic acid per gram of extract (y = 0.01x + 0.0262; r = 0.999); mg RUE/g, milligrams equivalent to rutin per gram of extract; mg/100 g (y = 0.0259x + 0.0044; r = 0.998), milligrams of anthocyanin expressed as cyanidin‐3‐glycoside per 100 g of extract.

^*Two‐tailed Student's t test at 95% confidence interval.

The fruits of L. peruvianum also presented measurable anthocyanin content; however, these levels were lower than those reported in other species collected from the same region, including Gaultheria glomerata, Gaultheria myrsinoides, and Berberis lutea [15].

The antioxidant potential of L. peruvianum leaves and fruits was evaluated using three in vitro assays: DPPH•, ABTS•⁺, and FRAP. Antioxidant activity was expressed both as percentage inhibition (Figure 3) and as EC₅₀ values (Figure 3), which represent the concentration required to achieve 50% of maximal antioxidant activity. Lower EC₅₀ values indicate greater antioxidant potential. Trolox was used as the reference antioxidant.

FIGURE 3.

In vitro antioxidant activity percentage from L. peruvianum Kuijt leaves and fruits compared to Trolox. EC₅₀, median effective concentration: curve of DPPH^● (y = 0.02764x + 0.0086; r = 0.9999); the averages are based on three repetitions and include the standard error of the mean (±). Levene statistics (p = 0.001); ANOVA (α = 0.05); Tukey test (α = 0.05): the superscript letters (a, b, and c) are homogeneous subsets.

The results demonstrated that leaves extracts exhibited higher antioxidant activity than fruits extracts across all three assays, although both remained statistically lower than Trolox (p < 0.05). Likewise, the EC₅₀ values obtained for leaves were lower than those for fruits but still significantly higher than those observed for Trolox (p < 0.05). These results confirm the strong antioxidant activity of Trolox and demonstrate the antioxidant potential of L. peruvianum (Figure 3). Notably, the antioxidant capacity observed in this species was higher than that reported for other plants collected from the Huaraca Stone Forest in Peru using similar analytical methods [23]. Figure 3 illustrates the three most representative concentrations (100, 150, and 200 μg mL⁻¹) selected from three homogeneous subsets, whereas Figure 3 shows the EC₅₀ values of leaves and fruit extracts compared with Trolox. The reported values correspond to the mean of three replicates and include the standard error of the mean (±). Homogeneity of variance was evaluated using Levene's test (p = 0.001), and differences among groups were analyzed using ANOVA followed by Tukey's multiple comparison test (α = 0.05), where different letters (a, b, and c) indicate statistically homogeneous subsets.

The compounds identified in leaves extracts (Table 2A) include several metabolites previously reported to possess antioxidant activity. The antioxidant capacity of these molecules is largely related to their chemical structure, which enables them to donate electrons and neutralize free radicals, thereby preventing oxidative damage to cellular components [69]. Among these compounds, glutathione plays a fundamental role as an intracellular antioxidant that protects cells against oxidative stress [63, 70]. Flavonoids such as catechin and epicatechin, commonly found in green tea and other plant‐derived foods, are well known for their strong antioxidant activity [67]. Likewise, quercetin‐3‐O‐rutinoside, quercetin hexoside, and kaempferol‐3‐O‐rutinoside are flavonoids recognized for their ability to protect cells from oxidative damage [71]. Procyanidin dimers and trimers (type B), belonging to the flavonoid family, have also been reported to exhibit significant antioxidant properties [72]. In addition, lignans such as syringaresinol diglucoside and syringaresinol 4′‐glucoside have been reported to be effective antioxidant compounds [73]. Other identified flavonoids, including naringenin, naringenin‐7‐O‐glucoside [70], and eriodictyol hexoside [74], have similarly been associated with antioxidant activity.

In the A. salina bioassay control group, no mortality was observed, confirming that the diluent used in the experiment was not toxic to the nauplii. The median lethal concentration (LC₅₀) of L. peruvianum extracts was calculated by fitting a logarithmic curve to the number of dead nauplii according to the Probit method [31, 32]. The reported values correspond to the mean of three replicates, and the associated standard error indicates the precision of the measurements.

Preliminary toxicity tests indicated that L. peruvianum exhibits moderate toxicity. The LC₅₀ values obtained for leaves extracts were significantly lower than those for fruits extracts, which may be attributed to the higher abundance of isoquinoline alkaloids detected in leaves (Table 4). Plants frequently produce secondary metabolites such as alkaloids and polyphenols as chemical defense mechanisms against herbivores and pathogens. Alkaloids may exert toxic effects by inhibiting enzymes, disrupting neural signaling, or interfering with DNA and protein synthesis [75, 76]. Polyphenolic compounds, including catechins and proanthocyanidins, may also contribute to toxicity, either directly or by forming more reactive compounds during oxidation [76]. In addition, these molecules can bind to proteins, altering their structure and biological function [72].

TABLE 4.

Toxicity bioassay on A. salina of L. peruvianum Kuijt.

Plant part	LC50 (μg mL−1)	Category
Leaves	181.8 ± 1.14	Moderately toxic
Fruits	475.9 ± 1.10	Moderately toxic

Abbreviations: LC₅₀, median lethal concentration; relatively innocuous, >1500 μg mL⁻¹; practically not toxic, 1000–1500 μg mL⁻¹; slightly toxic, 500–1000 μg mL⁻¹; moderately toxic, 100–500 μg mL⁻¹; highly toxic, 10–100 μg mL⁻¹; extremely toxic, 1–10 μg mL⁻¹.

The antimicrobial evaluation revealed that methanolic extracts of L. peruvianum leaves and fruits exhibit antibacterial activity against several tested bacterial strains, but no inhibitory effect against yeast. Overall, fruit extracts showed greater antibacterial activity than leaves extracts, suggesting a higher concentration or more effective combination of antibacterial compounds in fruits (Table 5).

TABLE 5.

Antibacterial activity of the methanolic extract from leaves and fruits of L. peruvianum Kuijt.

	Leaves		Fruits		Standard
Microorganism	Inhibition zone* (mm)	MIC (mg/mL)	Inhibition zone* (mm)	MIC (mg/mL)	Inhibition zone (mm)	MIC (μg/mL)
E. coli	11.8 ± 0.01c	100.20	12.5 ± 0.01c	12.52	21.5 ± 0.14b	0.50
P. aeruginosa	11.4 ± 0.20c	100.20	12.7 ± 0.01d	25.06	13.5 ± 0.25c	0.50
S. typhimurium	10.4 ± 0,02b	100.20	11.0 ± 0.01c	12.52	18.2 ± 0.69a	0.50
S. aureus	13.7 ± 0.02a	6.27	10.5 ± 0.01a	25.00	16.5 ± 0.10ª	32.25
B. spizizenii	10.8 ± 0.01b	6.27	15.3 ± 0.05b	12.50	20.5 ± 0.45b	8.00
C. albicans	0.0 ± 0.00	N.R.	0.0 ± 0.00	N.R.	17.6 ± 0.77	N.R.

Note: The mean and standard error of the mean are shown for five replicates. Standard skewness coefficient between −2 and 2. Levene's test (p value > 0.05). ANOVA (p value < 0.05). Tukey's test (superscripts indicate homogeneous subsets). N.R.: not reported. Ciprofloxacin was used as the positive control for bacteria, while clotrimazole was used for C. albicans .

Both leaves and fruits extracts inhibited bacterial growth, with inhibition zones ranging from 10.4 to 15.3 mm. As expected, these values were lower than those observed for the positive control (ciprofloxacin), a broad‐spectrum antibiotic with high antibacterial efficacy. Gram‐negative bacteria, including E. coli , P. aeruginosa , and S. typhimurium were inhibited by both extracts. However, the fruits extract consistently produced larger inhibition zones than the leaves extract. This trend was particularly evident for E. coli and S. typhimurium , where inhibition zones of 12.5 and 11.0 mm were recorded for fruit extracts, compared with 11.8 and 10.4 mm for leaf extracts (Table 5; Figure 4).

FIGURE 4.

Inhibition zones of the methanolic extract from L. peruvianum Kuijt leaves and fruits against Gram‐positive and Gram‐negative bacteria, and a yeast. (A) E. coli ATCC 8739; (B) P. aeruginosa ATCC 9027; (C) S. typhimurium ATCC 14028; (D) S. aureus ATCC 6538; (E) B. spizizenii ATCC 6633; (F) C. albicans ATCC 10231. Ciprofloxacin (CP) was used as the positive control for the bacteria, while clotrimazole (Cl) was used for C. albicans; 1, 2, 3, 4, and 5 represent the replicates.

Inhibition of the Gram‐positive strains S. aureus and B. spizizenii yielded notable results. In the case of S. aureus , the leaves extracts proved more effective, producing an inhibition zone of 13.7 mm compared with 10.5 mm for the fruits extract, with a MIC of 6.27 mg mL⁻¹. Conversely, B. spizizenii showed greater susceptibility to the fruits extract, which produced an inhibition zone of 15.3 mm. However, MIC value for the fruits extract (12.50 mg mL⁻¹) was higher than that observed for the leaves extract (6.27 mg mL⁻¹; Table 5; Figure 5).

FIGURE 5.

MIC of the methanolic leaves and fruits extracts from L. peruvianum Kuijt against Gram‐positive and Gram‐negative bacteria. MIC, minimum inhibitory concentration; SC, sterility control (Mueller–Hinton broth with resazurin, without inoculum); GC, growth control (inoculum plus sterile Mueller–Hinton broth). MIC test for C1–C10 and A–C: dilutions of the extract plus inoculum and resazurin. Extract control for C1–C10 and D–F: dilutions of the extract plus Mueller–Hinton broth and resazurin (no inoculum). Standard control for C1–C10 and G–H: dilutions of ciprofloxacin standard. ●, indicates the MIC for L. peruvianum. ►, indicates the MIC for ciprofloxacin.

These findings suggest that the chemical composition of the extracts differs between leaves and fruits, thereby influencing their antimicrobial activity. Differences in the concentration and composition of flavonoids, tannins, and other secondary metabolites may account for the observed variation in antibacterial effects.

The MIC assays (Figure 5) confirmed that both leaves and fruits extracts exhibit antimicrobial activity, although relatively high concentrations were required when compared with conventional antibiotics. The MIC values for E. coli , P. aeruginosa , and S. typhimurium ranged from 10.4 to 12.7 mg mL⁻¹, considerably higher than the MIC for ciprofloxacin (0.5 μg mL⁻¹), indicating lower antibacterial potency. In contrast, S. aureus and B. spizizenii were more sensitive to extracts, with MIC values of 6.27 mg mL⁻¹ for the leaves extracts and 25.00 and 12.50 mg mL⁻¹ for the fruits extracts, respectively. These results suggest that the leaves extracts may exert a more selective inhibitory effect against Gram‐positive bacteria.

No antifungal activity against C. albicans was detected, as no inhibition zones were observed in any of the tested samples. These findings suggest that the compounds present in L. peruvianum either lack significant antifungal activity against this yeast or are present at concentrations insufficient to produce measurable inhibitory effects.

The antimicrobial activity observed in the leaves and fruits extracts can be largely attributed to the presence of several bioactive metabolites identified in the chemical profile, particularly polar lipids. Notably, the fruits extracts exhibited greater antimicrobial efficacy, which may be associated with the presence of two lipid classes: oxylipins and lysophosphatidylethanolamines. These compounds are highly bioactive and play important roles in plant defense mechanisms against pathogens and parasites. Oxylipins, for example, have been reported to exhibit antimicrobial activity at concentrations of approximately 100 mM against Gram‐negative phytopathogenic bacteria such as Pseudomonas syringae , Xanthomonas campestris , and Erwinia carotovora [51].

This observation supports the greater efficacy of the fruits extracts against Gram‐negative bacteria such as E. coli, P. aeruginosa , and S. typhimurium , which showed MIC values of 12.5, 25.1, and 12.5 mg mL⁻¹, respectively. These values indicate greater antibacterial activity than the leaves extracts, which contain only lysophosphatidylethanolamines and exhibited an MIC of 100.2 mg mL⁻¹ against all three bacterial strains.

Both extracts also demonstrated activity against Gram‐positive bacteria, including S. aureus and B. spizizenii. For the fruits extracts, MIC values were 25.0 and 12.5 mg mL⁻¹, respectively, whereas the leaves extracts showed an MIC value of 62.7 mg mL⁻¹ for both strains.

In addition to lipids, the presence of flavonoids—classified as polyphenolic compounds—may also contribute significantly to the antimicrobial activity. These molecules can interact with bacterial cell membranes, compromising membrane integrity and interfering with the synthesis of amino acids required for microbial growth [72]. Such mechanisms could explain the inhibitory activity observed against both Gram‐positive and Gram‐negative bacteria. The strongest responses were observed against S. aureus and B. spizizenii, both Gram‐positive strains, with MIC values as low as 6.27 mg mL⁻¹ in some assays.

Regarding Gram‐negative bacteria, the fruits extracts exhibited greater activity (MIC values of 25.0 and 12.5 mg mL⁻¹) compared with the leaves extracts (MIC value of 100.2 mg mL⁻¹). This difference may be associated with higher concentrations of various organic acids in the fruit extracts of L. peruvianum. Among these, malic acid and ascorbic acid are particularly notable, as they have been associated with antimicrobial activity through the downregulation of genes involved in bacterial motility (such as flhA, flaA, and flgE) and outer membrane proteins such as BabA, thereby interfering with signal transduction, adhesion, and motility processes [77].

Overall, the results indicate that methanolic extracts of L. peruvianum exhibit moderate antibacterial activity against several Gram‐positive and Gram‐negative bacterial strains, with greater efficacy observed in fruits extracts. However, the relatively high MIC values compared with conventional antibiotics suggest that the antibacterial potency of these extracts is limited. Furthermore, the absence of antifungal activity against C. albicans suggests that their antimicrobial potential may be primarily restricted to bacterial pathogens.

Future research should focus on the fractionation and isolation of the bioactive compounds responsible for the observed antibacterial activity, as well as on evaluating possible synergistic effects between these compounds and commercial antibiotics in order to enhance antimicrobial efficacy.

Finally, the results from the A. salina bioassay provide useful preliminary information on the potential toxicity of the extracts. Nevertheless, it is important to note that the toxic effects observed in A. salina cannot be directly extrapolated to humans or other aquatic organisms. Consequently, these findings should be considered as an initial screening step that highlights the need for more comprehensive and specific toxicity studies.

4. Conclusion

An untargeted UHPLC–MS/MS analysis identified a diverse phytochemical profile in the leaves and fruits of L. peruvianum Kuijt, including several phenolic compounds associated with antioxidant activity. The hemiparasitic nature of this species may contribute to the diversity of metabolites detected, potentially reflecting metabolic interactions with its host plants.

The antioxidant assays demonstrated that leaves extracts exhibited higher antioxidant capacity than fruits extracts, although both plant organs showed measurable antioxidant potential. In addition, methanolic extracts of L. peruvianum showed moderate antibacterial activity against both Gram‐positive and Gram‐negative bacteria, with slightly greater efficacy in the fruits extracts.

The toxicity evaluation using A. salina bioassay revealed moderate toxicity, indicating the need for further toxicological studies with more specific biological models to better assess safety and potential biomedical applications. Overall, these findings highlight L. peruvianum as a promising natural source of bioactive compounds with antioxidant and antibacterial properties. Furthermore, characterizing its phytochemical profile contributes to scientific knowledge of this hemiparasitic species and underscores the importance of conserving the biodiversity of Andean ecosystems where it occurs.

Author Contributions

Marco Rolando Aronés Jara: conceptualization, methodology, investigation, funding acquisition, supervision, resources, project administration. Kirianova Godoy Bautista: methodology, investigation. Freshsia Ingrid Ortiz Pérez: software, data curation. Edgar Cárdenas Landeo: formal analysis, data curation. Edith Eveling Conislla Cáceres: investigation, validation. Hugo Roberto Luna Molero: validation, visualization. Juan Clímaco Paniagua Segovia: investigation, methodology, formal analysis. Jaeson Santos Calla Choque: writing – review and editing, validation, visualization. Anas Rashid: validation, visualization, writing – review and editing, conceptualization, project administration, supervision. María Segunda Aurora Prado: conceptualization, validation, visualization, writing – review and editing, project administration, supervision.

Conflicts of Interest

The authors declare no conflicts of interest.

Institutional Review Board Statement

This study was approved by Universidad Nacional de San Cristóbal de Huamanga, Ayacucho, Perú (391‐2014‐UNSCH‐COG‐R).

Supporting information

Table S1: Distribution of extract and ciprofloxacin dilutions in the microplates.

Data Availability Statement

The data that supports the results and findings of this study are available from the corresponding authors upon request.