Isolation and Tentative Characterization of a Genistein-like Compound from the Mistletoe Dendrophthoe pentandra (L.) Miq.

Abstract

Dendrophthoe pentandra (L.) Miq., a hemiparasitic mistletoe widely used in Indonesian traditional medicine, is abundant in flavonoids; nevertheless, many of these compounds are inadequately characterized beyond the well-known flavonols. This study aimed to isolate and characterize a putative genistein-like compound, metabolomics standard initiative level 2–3, from the ethyl acetate fraction of leaves using a multi-instrument analytical approach. Reverse phase high performance liquid chromatography analysis revealed prominent peaks with retention times comparable to those of a genistein reference in both analytical and preparative conditions. The Fourier transform infrared spectroscopy spectrum of the isolate exhibited functional groups characteristic of an isoflavone framework, including phenolic O–H stretching, conjugated C=O carbonyl bands, and aromatic C=C and C–O vibrations. High-resolution liquid chromatography–electrospray ionization–tandem mass spectrometry quadrupole time-of-flight analysis detected a deprotonated molecular ion [M–H]^- at m/z 269.0449, along with diagnostic fragment ions consistent with retro-Diels–Alder cleavage commonly observed in isoflavone aglycones. Although the analysis remained semi-quantitative and lacked orthogonal confirmation (e.g., nuclear magnetic resonance and peak-purity assessment), the combined analytical evidence was consistent with a tentative genistein-like annotation. Semi-quantitative recalculation indicated an estimated concentration of 0.5267 µg/mL in the injected ethyl acetate fraction, corresponding to an approximate relative abundance of 2.63% within the analyzed fraction. Because this value was below the estimated limit of quantification, the result should be interpreted as a relatively low-abundance estimate rather than a validated absolute quantitative measurement. These findings provide preliminary evidence supporting the possible occurrence of a genistein-like compound within the Loranthaceae family, although definitive structural confirmation requires further investigation.

Keywords

genistein-like compound isoflavone LC–ESI–MS/MS QToF RP-HPLC phytochemical analysis

Introduction

Duku mistletoe, Dendrophthoe pentandra (L.) Miq. is a hemiparasitic species of the Loranthaceae family that colonizes Lansium domesticum (Corrêa) Kosterm. It is widely distributed across Southeast Asia and holds a long-standing history of use in Indonesian traditional medicine to treat various conditions, including inflammation, hypertension, metabolic and reproductive disorders, and infectious diseases.¹ Previous studies have linked these therapeutic uses to the plant’s rich phenolic metabolites, particularly flavonoids, and related polyphenolic compounds with antioxidant and pharmacological activities.^2,3 Despite growing interest in pharmacology, the phytochemical documentation of this species remains inconsistent, with a predominant focus on common flavonols.

Previous phytochemical investigations have consistently identified several flavonoid and flavonol-related constituents in D. pentandra (L.) Miq., including quercetin derivatives and related phenolic compounds.^3–5 Yet, this recurring emphasis on flavonols has overshadowed the broader chemical diversity expected within Loranthaceae, leaving significant gaps in the characterization of other flavonoid subclasses. Recent advances in plant metabolomics and chemotaxonomic profiling have highlighted that plant secondary metabolite diversity is often underestimated. This is particularly evident in non-model and parasitic plant species, where unique biosynthetic adaptations may occur. Isoflavones, in particular, have never been comprehensively reported in the family, despite their relevance to plant defense and adaptive physiology. This absence is particularly noteworthy because isoflavones such as genistein and daidzein are predominantly associated with Fabaceae and other leguminous taxa, where they are considered characteristic phytoestrogenic metabolites.^6,7 Genistein (Figure 1), a well-known isoflavone, is commonly reported in soybeans, legumes, alfalfa (Medicago sativa) sprouts, and red clover, which are recognized as major dietary sources of phytoestrogenic isoflavones.^8–11 Recent phytochemical and metabolomic studies have emphasized that isoflavonoids are rarely detected outside Fabaceae and are only occasionally reported in non-leguminous taxa, highlighting their chemotaxonomic significance.^6,12 Consequently, their occurrence in other plant lineages has attracted growing scientific attention. Thus, their putative occurrence in Loranthaceae represents both a chemotaxonomic anomaly and raises intriguing biosynthetic questions.

Figure 1.

Structure of Genistein (5,7-dihydroxy-3-[4-hydroxyphenyl]-chromen-4-one).¹⁰

The chemotaxonomy of D. pentandra (L.) Miq. has traditionally relied on flavonol profiles, leaving its entire metabolic repertoire inadequately elucidated. Reports on isoflavonoids outside of Fabaceae are rarely found, and the absence of confirmed isoflavones in Loranthaceae highlights a clear gap in phytochemical knowledge. This gap has gained new relevance following Lazuardi et al.¹³ who detected mass spectral features in the ethyl acetate fraction of D. pentandra (L.) Miq. with molecular weights similar to those of genistein. Although these findings were preliminary and based solely on mass spectral similarity, they represent an important early indication that isoflavone-like metabolites may exist in this species.

From a biosynthetic perspective, the potential occurrence of isoflavone-like compounds in a non-Fabaceae plant is unexpected, as the canonical isoflavone biosynthetic pathway is largely restricted to legumes. In legumes, isoflavone biosynthesis is typically mediated by isoflavone synthase (IFS), a cytochrome P450 enzyme (CYP93C) responsible for the aryl migration step that distinguishes isoflavonoids from other flavonoid subclasses.^14,15 Recent reviews have further emphasized that isoflavonoid metabolism is highly lineage-dependent and remains predominantly associated with leguminous plants.¹⁶ Nevertheless, several studies have demonstrated the production of genistein and daidzein in non-leguminous dicot and monocot tissues, including experimentally transformed plant systems, indicating that isoflavonoid biosynthetic capacity may extend beyond traditional Fabaceae lineages.¹⁷ If verified, these findings may expand current understanding of isoflavone biosynthesis beyond the Fabaceae, where these pathways are most frequently reported.¹⁴

Despite these initial indications, the evidence’s tentative nature necessitates more focused, targeted isolation and characterization efforts. Therefore, this study aimed to tentatively isolate and characterize isoflavone-like compounds from the ethyl acetate fraction of D. pentandra (L.) Miq. leaves. This objective was designed to further evaluate previously reported MS-based indications while addressing an important phytochemical gap in the current literature. In addition, the study sought to obtain an initial semi-quantitative estimate of this compound, which gave context for its relative abundance within the plant matrix. This approach aligns with metabolomics standards for compound annotation, metabolomics standard initiative (MSI) level 2–3, which uses multiple complementary analytical techniques to support tentative identification in the absence of full structural confirmation.

In pursuing this goal, chromatographic and spectrometric techniques were employed as complementary analytical tools for preliminary structural interpretation. Given the expected low-abundance of isoflavones in this species, a multi-instrumental approach was employed to provide cumulative evidence for tentative identification, acknowledging that definitive structural confirmation via nuclear magnetic resonance (NMR) was beyond the scope of this initial investigation. Within this framework, semi-quantitative parameters were incorporated to support comparative interpretation of chromatographic responses rather than absolute quantification. Even at the tentative annotation level (MSI level 2–3), these findings provide a basis for future studies involving structural elucidation, extensive metabolomic mapping, and biological activity assessment.

Materials and Methods

Plant Materials and Authentication

Fresh leaves of D. pentandra (L.) Miq. were collected in September 2024 from the host plant (L. domesticum) (Corrêa) Kosterm in Banuayu Village, Muara Enim, South Sumatra, Indonesia. Subsequently, a certified curator at the Herbarium Bogoriense, Indonesia, determined that the voucher specimen (ELSA-185402), reviewed on October 31, 2024, was authentic with certificate No. B-3868. The samples were shade-dried, subsequently pulverized with an HR3000 grinder (Hangzhou Feiru, China), and stored for extraction.

Materials and Instruments

Pro-analytical grade chemical solvents were used, including methanol, n-hexane, and ethyl acetate (Emsure^→, Merck, Darmstadt, Germany; Cat. No. 1.06009, 1.04367, and 1.09623, respectively)–methanol, water, and acetonitrile for chromatography (LiChrosolv^→, Merck, Darmstadt, Germany; Cat. No. 1.06018, 1.15333, and 1.00029, respectively). The genistein primary reference material (GPRM) with CAS No. 446-72-0, purity >97% (92136-10MG, Supelco, Sigma-Aldrich) was used as the analytical standard.

The instruments used in this study included a rotary evaporator (Eyela N-1200B, Tokyo Rikakikai Co., Japan), a sonicator (Branson 8800 Ultrasonic Cleaner, USA), an high performance liquid chromatography (HPLC) system (Agilent 1260 Infinity II, Agilent Technology Inc., USA), a chromatography column (Pyrex, Japan), an Fourier transform infrared spectroscopy (FTIR) spectrometer (IRPrestige-21, Shimadzu, Japan), a liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) quadrupole time-of-flight (QToF) system (Waters Xevo™ G3 QToF, Waters Corporation, USA), a Poroshell 120 EC-C18 analytical column (4.6 × 100 mm, 2.7 µm; Agilent Technologies, Cat. No. 695975-902), an Agilent Prep-C18 column (250 × 10 mm, 5 µm; Cat. No. 440905-802), and an Acquity UPLC BEH C18 column (2.1 × 150 mm, 1.7 µm; Waters, Cat. No. 186002347).

Maceration, Extraction, and Fractionation

Simplicia of D. pentandra (L.) Miq. leaves (450 g) were subjected to cold maceration with analytical methanol (1:10, w/v) for 72 hours, with stirring every 12 hours, filtered, and concentrated using an Eyela N-1200B evaporator at 15 rpm and 40 °C, followed by drying under nitrogen flow. Purification of the crude extract was performed by sequential liquid–liquid partitioning with n-hexane and ethyl acetate to obtain fractions enriched in semi-polar phenolic constituents. Crude extract (3 g per batch) was added to 10 mL of warm water and sonicated until homogeneous. This partitioning procedure was repeated in multiple batches to process a total of 30 g of crude extract for yield determination. The mixture was triturated with n-hexane seven times (7 × 50 mL), followed by trituration with ethyl acetate three times (3 × 50 mL). Each fraction resulting from the partition is collected separately and dried using the same evaporation method. The ethyl acetate fraction, which is commonly enriched with semi-polar flavonoid-related compounds in D. pentandra (L.) Miq. was selected for HPLC analysis.^3,5

Sample and Standard Preparation for HPLC Analysis

A GPRM was dissolved in HPLC-grade methanol to prepare a 100 μg/mL stock solution. From this, five standard concentrations were prepared for calibration: 0.1, 0.5, 1, 2, and 4 μg/mL, for retention time (RT) comparison, and semi-quantitative analysis. The ethyl acetate fraction of D. pentandra (L.) Miq. was prepared in HPLC-grade methanol at two concentrations: 20 μg/mL for analytical purposes and 1 mg/mL for preparative isolation. All solutions were sonicated and vortexed, then filtered through a 0.45 μm PTFE (25 mm), transferred to autosampler vials, and subsequently analyzed chromatographically under identical conditions for both samples and standards.

RP-HPLC Conditions for Analysis

Reverse phase (RP)-HPLC analysis was performed using modified reversed-phase chromatographic conditions adapted from previously published analytical methods.^18,19 The analysis was performed using an Agilent 1260 Infinity II system equipped with a binary pump, autosampler, diode array detector (DAD), fraction collector, and Agilent OpenLab CDS software. A Poroshell 120 EC-C18 analytical column was employed for qualitative and semi-quantitative profiling, while an Agilent Prep-C18 preparative column was used for purification and isolation. The mobile phase consists of Eluent A (water) and Eluent B (methanol) for chromatography, delivered isocratically at a 30:70 (v/v) ratio. The flow rate was set to 0.5 mL/min for the analytical column and 2.0 mL/min for the preparative column, with injection volumes of 20 and 100 µL, and total run times of 18 and 30 min, respectively. The column temperature was maintained at 25 °C. Initial wavelength scanning was performed over 220–270 nm, after which 260 nm was selected as the monitoring wavelength based on the target compound’s highest peak area. All injections were performed in triplicate to ensure chromatographic reproducibility. RT and peak area were consistent across replicate injections, indicating good analytical repeatability and reproducible peak profiles under identical conditions. The separation conditions were selected to achieve stable, reproducible retention of the isoflavone compounds. System suitability parameters, including RT, peak area, peak height, 50% peak width, symmetry factor, selectivity, and theoretical plate number (N), were obtained from both sample and standard chromatograms. These parameters were monitored to assess chromatographic performance and injection-to-injection consistency throughout the analytical sequence. A fraction collector was used to collect the isolated subfraction with RT identical to the reference standard, which was subsequently dried for further analysis.

HPLC Validation

Basic validation parameters were assessed to support semi-quantitative RP-HPLC analysis. Linearity is evaluated using a calibration curve prepared over a range of standard concentrations (0.1–4 μg/mL), and the correlation coefficient (R²) is used to assess the linearity of the detector response. Precision was evaluated via triplicate injections of the standard, and the relative standard deviation (RSD) of RT and peak area was calculated to estimate repeatability. Injection-to-injection consistency was further assessed by examining the variation in chromatographic peak characteristics across replicate runs, including RT shifts and peak area fluctuations. The limits of detection (LOD) and limit of quantification (LOQ) were estimated following the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) regression-based approach, using the standard deviation (SD) of the calibration curve residuals and the slope of the regression line. LOD and LOQ were calculated using the equations: LOD = 3.3 × (SD/slope) and LOQ = 10 × (SD/slope).²⁰ These validation parameters were considered sufficient for exploratory semi-quantitative interpretation, although the method was not intended to represent a fully validated quantitative analytical procedure.

FTIR Analysis

Isolated subfraction from D. pentandra (L.) Miq. leaves extract was characterized by FTIR spectrophotometry using an InfraRed Prestige-21 with the KBr pellet transmission technique. Dried samples were finely ground and mixed with FTIR-grade KBr at a 1:200 (w/w) ratio, then compressed into 4-mm transparent pellets using a Mini Hand Press (MHP-1, Shimadzu). FTIR spectra were acquired between 4,000 and 500 cm⁻¹ and with a 20 s scan time and 4 cm⁻¹ resolution. The GPRM pellets were prepared using the same procedure to ensure analytical consistency–spectrum analysis of the isolate based on peak values in the infrared region. Functional group assignment focused on diagnostic isoflavone absorption bands, including phenolic O–H stretching, conjugated carbonyl C=O stretching, aromatic C=C vibrations, and C–O–C/C–O (phenolic/ether) stretching.^19,21,22

LC–ESI–MS/MS QToF Analysis

The isolated subfraction from the D. pentandra (L.) Miq. leaves was characterized using high-resolution LC– ESI–MS/MS QToF, following previously reported phenolic profiling approaches with modifications suitable for flavonoid analysis.^23,24 Chromatographic analysis was performed on an Acquity UPLC system coupled to a Waters Xevo™ G3 QToF mass spectrometer equipped with an electrospray ionization (ESI) interface. Separation of compounds was detected using an Acquity UPLC^→ BEH C18 reversed-phase column (2.1 × 150 mm, 1.7 μm). The column temperature was maintained at 40 °C, while the sample temperature was set to 15 °C, with an injection volume of 1 μL. The mobile phase consisted of two eluents: Eluent A (5 mM ammonium acetate) and Eluent B (acetonitrile). Gradient elution was applied at a flow rate of 0.3 mL/min with a total run time of 16 min, increasing Eluent B from an initial 5%–90% over 10 min; holding at 90% for 3 min, and then returning to the initial condition. Mass spectrometric detection was conducted in negative ESI mode over an m/z range of 50–1,500. The capillary voltage was set to 2.5 kV, and the cone voltage to 40 V. Source and desolvation temperatures were maintained at 100 °C and 250 °C, respectively. Tandem mass spectrometry (MS/MS) fragmentation was performed using a collision energy ramping from 6 to 40 eV to generate diagnostic ion products. LockSpray correction was applied at a flow rate of 10 μL/min to ensure mass accuracy, and data acquisition and processing were performed using MassLynx™ v4.2 software. Reference MS/MS spectra of the standard compound were obtained by direct injection into the ESI–QToF system in negative ion continuum mode and used for comparison with sample-derived precursor ions.

Data Interpretation and Semi-quantification

Data interpretation was conducted using a multi-level analytical approach. Chromatographic identity was assessed by comparing RT and peak characteristics between the isolated compound and GPRM, following established phytochemical identification guidelines.^25,26 Functional group analysis was performed by comparing FTIR absorption bands of the isolate with those of a characteristic isoflavone.²¹ Accurate mass measurements and MS/MS fragmentation patterns were interpreted by comparison with previously reported fragmentation behavior of isoflavone, including retro-Diels–Alder (RDA) cleavage pathways.^23,27

Compound identification was assigned as a putative annotation in accordance with the MSI level 2–3, based on spectral similarity without full structural confirmation. Semi-quantitative estimation of the putative genistein-like compound in the ethyl acetate fraction of D. pentandra (L.) Miq. was performed using external calibration by linear regression of the GPRM peak areas over a concentration range of 0.1–4 μg/mL, consistent with the validated RP-HPLC analytical conditions.

Ethical Statement

Plant material was collected from cultivated host plants under routine agricultural management practices. D. pentandra (L.) Miq. is not classified as an endangered or protected species in Indonesia (Minister of Environment and Forestry Regulation No. P.106/2018). No specific permits are required for sampling, as the material was obtained outside of protected conservation areas. The collection of plant materials complies with applicable national regulations and internationally recognized biodiversity conservation guidelines.

Results and Discussion

Extraction Yield of D. pentandra (L.) Miq. Leaves

Cold maceration of 450 g of dry leaves yielded 35.52 g of crude extract (7.89% w/w). A total of 30 g of crude extract was subjected to partitioning through repeated batch processing (3 g per batch), yielding n-hexane (1.25 g, 4.16%), ethyl acetate (4.55 g, 15.16%), and methanolic residue (21.22 g, 70.74%) fraction (Table 1), with yields expressed relative to the total processed extract.

Table 1.

Fraction Yield Obtained from Liquid–Liquid Partitioning of Crude Extract of D. pentandra (L.) Miq. Leaves.

Fraction Name	Weight (g)	Yield (% w/w)
n-hexane	1.25	4.16
Ethyl acetate	4.55	15.16
Methanol extract residue	21.22	70.74
Total recovered	27.02	90.06

Note: Partitioning was performed in repeated batches (3 g per batch; total 30 g processed). Yields are expressed relative to the total crude extract. Minor mass differences reflect cumulative recovery and handling losses.

The solvent partitioning results revealed that the methanolic residue represented the largest recovered fraction of the crude extract (70.74% w/w), while the ethyl acetate fraction constituted a substantial semi-polar fraction (15.16% w/w). The recovery of material in the ethyl acetate fraction suggests the presence of semi-polar constituents, including flavonoids, glycosylated flavonoid derivatives, and other phenolic compounds previously reported in D. pentandra leaves.^3,4 The enrichment of phenolic constituents in this fraction further supports the phytochemical potential of D. pentandra (L.) Miq. as a source of bioactive metabolites.⁵ Hemiparasitic plants are known to accumulate flavonoids as defense and physiological regulatory molecules, which may contribute to the phytochemical profile of D. pentandra (L.) Miq.^3,28

HPLC Isolation and Semi-quantification

The RP-HPLC analysis of the ethyl acetate fraction revealed five distinct peaks with RTs of 2.40, 2.95, 4.29, 5.21, and 8.46 min (Figure 2A). The peak eluting at 4.29 min (peak No. 4) was selected as a primary putative target peak due to its proximity to the GPRM peak at 4.15 min (Figure 2B). The preparative RP-HPLC purification suggests this chromatographic agreement. The target subfraction consistently eluted at 14.54 min, which closely aligns with the RT of GPRM at 14.57 min. Additional chromatographic parameters—namely, peak area, peak height, peak width at 50%, symmetry, and selectivity factor (α > 1)—showed comparable chromatographic behavior between the isolate and the reference substance (Table 2). Semi-quantitative analysis using external calibration with GPRM yielded a linear response over the evaluated concentration range (0.10–4.00 µg/mL), with a regression equation of y = 101.44x – 3.4755 (R² = 0.9976) (Figure 3). Recalculating using the regression equation yielded an estimated concentration of 0.5267 µg/mL in the injected ethyl acetate fraction (20 µg/mL). This corresponded to an approximate relative abundance of 2.63% within the analyzed fraction (Table 3). Using the ICH regression-based approach, the estimated LOD and LOQ were 0.31 and 0.95 µg/mL, respectively. Because the calculated concentration was below the estimated LOQ, the result should be interpreted as a low-abundance estimation rather than a validated absolute quantitative measurement.

Figure 2.

RP-HPLC Analysis: (A) Chromatogram of the Ethyl Acetate Fraction of Dendrophthoe pentandra (L.) Miq. Leaves at 20 µg/mL, Exhibiting a Distinct Peak at RT 4.29 Min (Peak No. 4) Identified as a Putative Genistein-like Compound; (B) Chromatogram of the GPRM at 2 µg/mL Exhibiting a Central Peak at RT of 4.15 Min.

Table 2.

Comparison of the Central Peak Chromatographic Parameters of the Ethyl Acetate Fraction of Dendrophthoe pentandra (L.) Miq. Leave Extract and GPRM Using a Preparative RP-HPLC System at λ 260 nm.

Parameters	Ethyl Acetate Fraction	Genistein PRM	Description
RT (min)	14.54	14.57	Difference 0.03 min.
Peak area (mAU.min)	34,756.64	39,018.34	The fraction peak area is approximately 89% of the standard’s peak area.
Peak height (mAU)	1,144.27	1,289.53	The fraction peak height is approximately 88% of the standard peak height.
Peak width 50% (min)	0.4724	0.4722	The fraction peaks are identical (with a difference of 0.0002).
Symmetry factor	0.80	0.84	The symmetry factor is identical (difference 0.04).
Selectivity (α)	1.12	1.20	The α value is α > 1.

Figure 3.

Calibration Curve of GPRM (0.1–4 µg/mL) for Semi-quantifying Estimation of Putative Genistein-like Compound.

Table 3.

Estimating Putative Genistein-like Compound Levels in the Ethyl Acetate Fraction and the Plant Dendrophthoe pentandra (L.) Miq.

Parameter	Value
Sample analyzed	Ethyl acetate fraction
Injection concentration	20 µg/mL
Target peak RT	4.29 min
Mean peak area	49.9533
Calibration equation	y = 101.44x − 3.4755
Calculated concentration in the injected solution	0.5267 µg/mL
Relative proportion in the injected fraction	2.63%
LOD	0.31 µg/mL
LOQ	0.95 µg/mL
Analytical interpretation	Detected above LOD but below LOQ; estimated as a low-abundance constituent*

Note: *The estimated value should be interpreted as a low-abundance constituent rather than a reliable quantitative measurement because the calculated concentration was below the LOQ.

Repeated injections showed RSD values of 0.27%–0.91% for peak area and 0.03%–0.11% for RT, indicating good chromatographic repeatability under the applied analytical conditions. It is important to note that no peak-purity assessment (e.g., DAD-based spectral analysis) was performed for the selected chromatographic peak. Therefore, the possibility of co-elution cannot be excluded, and the observed chromatographic signal may represent more than one co-eluting component. In addition, a detailed UV spectral comparison was not included, representing an additional limitation of the chromatographic assessment. This limitation should be considered when interpreting RT similarity, as compounds with similar polarity may produce overlapping responses.

RP-HPLC analysis of the ethyl acetate fraction yielded a prominent peak (peak #4) at RT ~4.29 min (Figure 2A), which corresponds to the RT of the primary reference material genistein (GPRM) at RT ~4.15 min. Selecting a single dominant peak for analysis may introduce selection bias; however, this approach was justified by reproducible retention behavior and consistent chromatographic profiles across replicate injections. Similarly, on a preparative C18 column, the isolate eluted at 14.54 min compared to 14.57 min for the reference genistein standard. This high chromatographic agreement indicates that the isolated compound has very similar polarity and interactions to genistein. Several chromatographic parameters (RT, peak width, symmetry factor α > 1, etc.) were comparable between the isolate and the GPRM, further supporting their similarity. However, RT alone is not a reliable means of identifying a chemical. Under the same conditions, different phenolic compounds could elute at the same time or have the same RT. It should be noted that isoflavones and flavonols sometimes exhibit overlapping RT ranges; therefore, retention similarities, while encouraging, are only preliminary indications. In the absence of peak-purity confirmation, co-elution cannot be excluded and may influence the interpretation of the chromatographic similarity observed between the isolate and reference standard.

In the current study, the retention agreement observed at the analytical and preparative scales supports the tentative classification of the isolate as an isoflavone, in contrast to typical flavonol compounds such as quercetin previously reported in D. pentandra (L.) Miq. The RT differences between the isolate and GPRM were relatively small (~0.14 min and ~0.03 min in analytical and preparative RP-HPLC, respectively). Low RT RSD values across replicate injections further supported chromatographic reproducibility under the applied analytical conditions.^23,25 Repeated injections also produce low RSD values for both peak area and RT, supporting good chromatographic consistency across replicate injections under the applied analytical conditions. The peak additionally showed satisfactory symmetry and resolution from adjacent peaks, indicating effective chromatographic separation of the detected component.^18,26 However, it should be noted that no specific peak-purity tests (e.g., diode array spectral analysis) were performed on this HPLC peak. Therefore, the presence of undetected impurities that co-eluted cannot be completely ruled out.

Semi-quantitative recalculation using external calibration with GPRM yielded an estimated concentration of 0.5267 µg/mL in the injected ethyl acetate fraction, corresponding to an approximate relative abundance of 2.63% within the analyzed fraction. This result suggests that the detected compound is a low-abundance constituent of the ethyl acetate fraction. However, because the calculated concentration remained below the estimated LOQ (0.95 µg/mL), the result should be interpreted cautiously as a relative abundance estimation rather than a validated absolute quantitative measurement.

Nevertheless, the calibration model showed good linearity (R² = 0.9976), while replicate injections showed satisfactory chromatographic consistency.^25,26 Although exploratory in nature, these findings support the tentative presence of low-abundance genistein-like compounds within the analyzed fraction when interpreted together with the chromatographic and spectrometric evidence.^19,23,27 In summary, the RP-HPLC data indicate the presence of a putative genistein-like compound in the extract. However, additional spectroscopic evidence is still required to verify its identity because retention behavior alone is not chemically definitive.

FTIR Spectral Analysis

FTIR analysis served as a complementary structural characterization tool, supporting RP-HPLC findings and providing additional evidence before LC–ESI–MS/MS analysis. The FTIR spectra of the isolated subfraction and GPRM revealed highly comparable spectral features characteristic of an isoflavone-like framework. The isolate showed a broad phenolic O–H stretching band at ~3,277 cm⁻¹, a conjugated carbonyl (C=O) absorption at ~1,641 cm⁻¹, aromatic C=C stretching ring vibrations around ~1,505 cm⁻¹, and C–O/C–O–C stretching bands in the ~1,183–1,091 cm⁻¹ range (Figure 4). These spectral features are generally consistent with oxygenated flavonoid or an isoflavone-type structure, although they do not provide definitive structural identification. To facilitate comparison, the principal absorption bands of the isolate and the corresponding features of the GPRM spectra assignments are summarized in Table 4.

Figure 4.

Overlay FTIR Spectra of the GPRM (Red) and the Isolate sub-fraction (Black) from Dendrophthoe pentandra (L.) Miq. by Transmission Pellet KBr, Illustrating Comparable Specific Functional Group Absorptions at Wavenumbers 3,277 cm⁻¹ (O–H), 1,641 cm⁻¹ (C=O), 1,505 cm⁻¹ (C=C), and 1,183–1,091 cm⁻¹ (C–O/C–O–C).

Table 4.

Assignments of Functional Groups Corresponding to Significant FTIR Absorption Peaks for the GPRM and the Isolate Subfraction from Dendrophthoe pentandra (L.) Miq. Assigns a Specific Functional Group to the Putative Isoflavone-like Aglycones.

Functional Groups	PRM of Genistein		Isolate Subfraction		Assignment
Functional Groups	Peak (cm⁻¹)	Intensity	Peak (cm⁻¹)	Intensity	Assignment
O–H stretching	3,412.22 3,119.03	31.95 29.26	3,277.20	24.20	Phenolic hydroxyl (H-bonded)
C=O (conjugated carbonyl)	1,643.42	22.06	1,641.59	21.64	Isoflavone 4-oxo group
C=C aromatic	1,507.43	21.94	1,505.22	21.44	Aromatic skeletal ring vibrations
C–O/C–O–C	1,193.02 1,044.50	25.19 47.61	1,183.38 1,091.76	20.34 23.26	Phenolic/ether stretching

The broad O–H absorption band suggests the presence of multiple hydrogen-bonded hydroxyl groups, as expected for polyhydroxylated flavonoids and consistent with the isoflavone chromophore.^25,29 Similarly, the absorption bands at ~1,641 and ~1,643 cm⁻¹ in the isolate and GPRM, respectively, are compatible with a conjugated C=O group commonly associated with the 4-oxo-benzopyranone scaffold of isoflavones along with characteristic phenolic O–H and aromatic C=C vibrations. The slightly lower C=O stretching frequency compared with non-conjugated carbonyls (>1,700 cm⁻¹) supports conjugation with an aromatic system characteristic of flavonoid-type compounds.^21,30 The aromatic C=C stretching band around ~1,505 cm⁻¹ supports the presence of a conjugated aromatic ring system. In the fingerprint region (<1,200 cm⁻¹), bands near ~1,183 cm⁻¹ and ~1,091 cm⁻¹ may be assigned to C–O/C–O–C ether or aryl stretching vibrations, which are compatible with oxygenated flavonoid-type structures. Minor differences between the isolate and the reference spectrum, including slight peak shifts or broadening, may reflect matrix effects, hydrogen bonding, or minor impurities. Therefore, the FTIR data support, but do not independently confirm, the assignment of the isolate as an isoflavone.³⁰

Overall, the FTIR profile of the isolate was consistent with a polyphenolic flavonoid-like framework comparable to that of the GPRM. However, FTIR analysis alone cannot reliably distinguish closely related flavonoid or isoflavonoid isomers, particularly those with similar hydroxylation patterns that may produce comparable infrared spectra. Accordingly, the FTIR findings in this study were interpreted solely as supportive evidence of a general isoflavone-like structural framework rather than as definitive structural confirmation.³¹ Given these inherent limitations, high-resolution LC–ESI–MS/MS analysis was subsequently employed to further assess molecular identity based on accurate mass and diagnostic fragmentation behavior.

High-resolution MS/MS Characterization

High-resolution LC–ESI–MS/MS QToF analysis provided the most discriminative analytical evidence supporting the tentative identification of the isolated molecule as a genistein-like isoflavone. The LC–MS chromatogram of the subfraction isolate showed a prominent peak at RT 5.36 ± 0.06 min under UPLC conditions (Figure 5A), with no significant signal in the blank, indicating that the observed peak originated from the extract sample rather than analytical contaminants or instrumental artifacts.

Figure 5.

(A) LC–MS Spectra ToF MSE (50–1,500) 6eV ESI: Overlay Spectra of a Blank Methanol and a Subfraction Isolate of Dendrophthoe pentandra (L.) Miq., Indicating that All Peaks Originate from the Sample and No Significant Signals Were Observed in the Blank; and (B) Spectra MS Showing the Peak of the Isoflavone-like Aglycone at RT 5.36 min.

The negative ion mass spectrum showed that a deprotonated molecular ion [M–H]⁻ at m/z 269.0449 was observed at both low and high collision energies (Figure 6B), and closely matched the reference standard signal at m/z 269.0492 (Figure 6A). The observed mass is in good agreement with the theoretical exact mass of genistein (C₁₅H₁₀O₅; calculated [M–H]⁻ = 269.0453), with a mass deviation of approximately 0.0004 Da (~1.5 ppm). This value falls within the mass accuracy range commonly applied to metabolomic annotation workflows.^24,27,31 This high mass accuracy is consistent with a genistein-like molecular formula, although it does not conclusively establish definitive structure identity.^24,32 Additionally, a direct comparison with the genistein reference material under identical UPLC–MS conditions demonstrated closely comparable retention behavior, which further supports the tentative assignment of the detected compound.^31,33

Figure 6.

MS/MS Spectra QToF MSE (50–1,500) 6eV ESI: (A) Direct Injection Continuum Spectra of GPRM; (B) High- and Low-energy Spectra of the Ethyl Acetate Subfraction of Dendrophthoe pentandra (L.) Miq. (Negative Ion Mode).

The MS/MS fragmentation pattern of the precursor ion at m/z 269 further supports the classification of the isolate as an isoflavone-type compound. Collision-induced dissociation produces a set of characteristic product ions, including a prominent fragment at m/z 133, together with ions at m/z 181 and m/z 113. The observed fragment ions at m/z 133 and m/z 181 are consistent with diagnostic RDA cleavage of the heterocyclic C-ring in genistein and related isoflavone aglycone, producing characteristic A- and B-ring fragments that are widely reported under negative ESI–MS/MS conditions.^19,23,27 Among these products, the ion at m/z 133 is considered particularly informative for genistein-related structures and is frequently reported as a diagnostic fragment arising from RDA cleavage pathways in isoflavones.^19,23 In contrast, flavonols generally exhibit distinct fragmentation patterns and typically do not produce a dominant m/z 133 ion under comparable analytical conditions.^23,27

In the context of D. pentandra (L.) Miq, this distinction is particularly relevant, as previous phytochemical investigations have primarily reported flavonols such as quercetin and morin.⁵ Overall, these observations support the tentative assignment of the isolate to an isoflavone-like aglycone. However, the fragmentation patterns should be interpreted as supportive rather than definitive, as structurally related flavonoids may exhibit partially overlapping fragmentation behavior.

Integrative Discussion

Collectively, the combined chromatographic, spectroscopic, and mass spectrometric evidence provides a coherent basis for the tentative identification of the isolated compound. RP-HPLC analysis revealed a dominant semi-polar constituent with retention behavior comparable to that of genistein reference. However, RT alone cannot serve as a definitive structural criterion due to the potential for co-elution among phenolic compounds. FTIR spectroscopy further supported this interpretation by indicating a conjugated carbonyl and polyphenolic framework consistent with an isoflavone-like structure. However, its ability to distinguish closely related isomeric structures remains limited. In contrast, high-resolution LC–ESI–MS/MS offered the most discriminative information through accurate mass determination and diagnostic fragmentation patterns, supporting a tentative identification in accordance with MSI level 2–3 criteria.

The possible occurrence of a putative genistein-like compound in D. pentandra (L.) Miq. may contribute to expanding the current understanding of phytochemical diversity of the Loranthaceae and suggests that isoflavonoid-like metabolites may occur beyond the Fabaceae. From a chemotaxonomic perspective, this observation is noteworthy because isoflavones are predominantly associated with Fabaceae taxa and are only occasionally reported in non-leguminous plant lineages.^6,7 Accordingly, the present findings may indicate a broader distribution of flavonoid biosynthetic capacity than previously recognized.

This observation also raises important questions regarding the underlying biosynthetic mechanisms. In legumes, isoflavone biosynthesis is typically mediated by IFS, a CYP93C pathway commonly associated with Fabaceae taxa.^14,15 If verified, these findings may expand current knowledge of isoflavonoid biosynthesis beyond the Fabaceae, where these pathways are most frequently described. The presence of a genistein-like compound in a non-Fabaceae species may reflect alternative biosynthetic routes, convergent evolution, or possible metabolic interactions between the hemiparasitic plant and its host. Recent advances in isoflavonoid biosynthesis research suggest that specialized metabolic pathways may be more evolutionarily dynamic than previously recognized.¹⁶ Consistent with this view, experimental evidence has further shown that isoflavonoid production can occur beyond traditional legume lineages under specific genetic or physiological contexts.¹⁷

Compared with well-established sources of genistein, such as soybeans and other legumes, the relative abundance estimated in this study is low, suggesting that the compound is a minor constituent of the plant matrix. Similar low-abundance detections have occasionally been reported in exploratory phytochemical studies of non-leguminous species, where these metabolites are often present at trace levels and require sensitive analytical methods. Accordingly, the present findings should be interpreted within the context of exploratory phytochemical research, in which tentative metabolite annotations serve as a basis for targeted follow-up rather than definitive structural confirmation. To the best of our knowledge, this study provides the first tentative evidence for a putative genistein-like compound in this plant lineage.

Study Limitations

This study provides a tentative characterization of a genistein-like compound using a multi-instrumental analytical approach; however, several limitations should be acknowledged. The absence of peak-purity assessment (e.g., DAD-based analysis) means that co-elution cannot be completely excluded, and the observed chromatographic signal may represent more than one co-eluting component. In addition, the proposed compound annotation relies primarily on chromatographic behavior, FTIR spectroscopy, and high-resolution MS/MS data. Although complementary, these techniques are not sufficient for definitive structural elucidation. The absence of NMR analysis further limits structural confirmation, and the current metabolite annotation should therefore be regarded with MSI level 2–3. Moreover, the reported concentration was derived from semi-quantitative external calibration and should be interpreted only as an approximate relative abundance estimate. Collectively, these considerations indicate that the present findings should be interpreted as exploratory phytochemical evidence and require further confirmation through orthogonal analytical techniques.

Conclusion

This study reports the tentative isolation and characterization of a putative genistein-like compound from the ethyl acetate fraction of D. pentandra (L.) Miq. leaves. The assignment was corroborated by convergent chromatographic, spectroscopic, and high-resolution mass spectrometric evidence consistent with MSI level 2–3 annotation. The detected compound appeared to be a low-abundance constituent of the ethyl acetate fraction, and its relative abundance should be interpreted cautiously because the calculated concentration was below the estimated LOQ. These findings provide preliminary evidence for the presence of isoflavone-like metabolites within the Loranthaceae family and may help expand the current understanding of phytochemical diversity in this lineage.

Future studies should focus on comprehensive structural confirmation, bioactivity evaluation, and broader metabolomic investigation across diverse host-parasite systems to better understand the biosynthetic and chemotaxonomic significance of these compounds.

Footnotes

Acknowledgements

The author acknowledges the Directorate of Research and Community Service, the Directorate General of Research and Development, the Ministry of Higher Education, Science, and Technology of the Republic of Indonesia, for the research grant. The author expresses appreciation to the Rector, the Dean of the Faculty of Veterinary Medicine, and the Coordinator of the Doctoral Program in Veterinary Medicine at Airlangga University for their support in providing facilities, laboratories, and unlimited library access, which enabled the completion of this work.

Authors’ Contribution

All authors made substantial contributions to conception and design, acquisition of data, or analysis, and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agreed to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors’ requirements/guidelines.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Data Availability Statement

All the data is available with the authors and shall be provided upon request.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

This study does not involve animal experiments or human subjects.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The research is funded by the Ministry of Higher Education, Science, and Technology, Republic of Indonesia with project No. 2394/2025.

Informed Consent

Not applicable.

Publisher’s Note:

All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, editors, or reviewers. This journal remains neutral concerning jurisdictional claims in published institutional affiliation.

Use of Artificial Intelligence-assisted Tools:

The authors declare that they have not used artificial intelligence (AI) tools for writing and editing of the manuscript, and no images were manipulated using AI.

Abbreviations Used

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Isolation and Tentative Characterization of a Genistein-like Compound from the Mistletoe Dendrophthoe pentandra (L.) Miq.

Abstract

Keywords

Introduction

Structure of Genistein (5,7-dihydroxy-3-[4-hydroxyphenyl]-chromen-4-one). 10

Materials and Methods

Plant Materials and Authentication

Materials and Instruments

Maceration, Extraction, and Fractionation

Sample and Standard Preparation for HPLC Analysis

RP-HPLC Conditions for Analysis

HPLC Validation

FTIR Analysis

LC–ESI–MS/MS QToF Analysis

Data Interpretation and Semi-quantification

Ethical Statement

Results and Discussion

Extraction Yield of D. pentandra (L.) Miq. Leaves

HPLC Isolation and Semi-quantification

Calibration Curve of GPRM (0.1–4 µg/mL) for Semi-quantifying Estimation of Putative Genistein-like Compound.

FTIR Spectral Analysis

High-resolution MS/MS Characterization

MS/MS Spectra QToF MSE (50–1,500) 6eV ESI: (A) Direct Injection Continuum Spectra of GPRM; (B) High- and Low-energy Spectra of the Ethyl Acetate Subfraction of Dendrophthoe pentandra (L.) Miq. (Negative Ion Mode).

Integrative Discussion

Study Limitations

Conclusion

Footnotes

Acknowledgements

Authors’ Contribution

Consent to Participate

Consent for Publication

Data Availability Statement

Declaration of Conflicting Interests

Ethical Approval

Funding

Informed Consent

Publisher’s Note:

Use of Artificial Intelligence-assisted Tools:

Abbreviations Used

References

Structure of Genistein (5,7-dihydroxy-3-[4-hydroxyphenyl]-chromen-4-one).¹⁰