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

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.

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.

OPEN IN VIEWER

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.

OPEN IN VIEWER 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.

OPEN IN VIEWER

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.

OPEN IN VIEWER Figure 3.

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

OPEN IN VIEWER

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.

OPEN IN VIEWER 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).

OPEN IN VIEWER

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
	Peak (cm−1)	Intensity	Peak (cm−1)	Intensity
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.

OPEN IN VIEWER 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

OPEN IN VIEWER 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.