Introduction

The Neolitsea genus, belonging to the Lauraceae family, comprises woody perennial shrubs and trees found in evergreen forests. It encompasses 85 species distributed throughout tropical and subtropical regions of Southeast Asia. Economically, Neolitsea species are rich sources of medicine, timber, spices, and perfumes1. Apart from serving as a reservoir of valuable resources, the genus serves its good reputation in folk medicines. Neolitsea species plants have been used in traditional medicine for the treatment of various illnesses including furuncle, carbuncle, edema, fractures, eruptions on fingers, and rheumatic arthralgia and especially, the bark and leaves of N. cassia were used for the treatment of fractures1. Ethanolic extracts of leaves of N. sericea var. aurata showed the presence of 14 new alkaloids using the HPLC-SPE-NMR technique2 and 37 known alkaloids were reported from this genus3. The genus is rich in alkaloids, mainly isoquinoline, and aporphine4, sesquiterpenes, triterpenes, and steroids3.

Neolitsea pallens (D. Don) Momiyama & H. Hara (Family: Lauraceae), commonly known as Pale Litsea, is an evergreen tree. In India, it is mainly distributed in the states of Jammu & Kashmir, Himachal Pradesh, and Uttarakhand, thriving at altitudes ranging from 1500-3000 m5. N. pallens holds significant ethnobotanical value, being utilized for various purposes. The leaves and bark serve as spices, and the freshly pressed juice of the plant is esteemed for its efficacy as a hair tonic6. Additionally, the local inhabitants in Himachal Pradesh collect its stems or leaves for fuel in household food preparation7. Despite its diverse uses, the chemistry of N. pallens remains largely unexplored. Previous investigations focused on the steam-distilled volatile oils composition of its leaves, bark, and fruits using GC and GC-MS8. The leaf oil was rich in sesquiterpenoids with furanogermenone (30.6%), β-caryophyllene (19.3%) and germacrene D (12.7%) as major constituents. The bark oil was dominated by oxygenated sesquiterpenoids represented by furanogermenone (59.1%), germacrone (9.3%), 10-epi-γ-eudesmol (7.8%) and curcumenol (5.3%). Furanogermenone (54.8%), trans-β-ocimene (8.8%), sabinene (6.4%) and germacrene D (4.0%) constituted the major proportion of the fruit oil. Furanogermenone constituted the highest proportion in all three oils. However, the non- volatile chemical constituents present in the leaves have remained unknown until now.

Given that leaves are often the primary site for biosynthesis and accumulation of secondary metabolites, including bioactive compounds like phenolic compounds and terpenoids, studying the leaves of N. pallens is essential for understanding their chemical composition. Additionally, leaves are renewable and readily accessible plant parts, making them suitable for phytochemical studies aimed at identifying bioactive compounds for pharmaceutical purposes. Phenolics, which are known for their bioactive properties and associated health benefits, have not been previously reported in the Neolitsea genus9. Therefore, the objective of this study was to comprehensively investigate the chemistry of N. pallens leaves, with a focus on phenolic compounds. To achieve this objective, 25% aqueous methanol extract of the leaves was analysed by ultra performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS), a state of art technique in plant metabolomics.

Material and methods

Chemicals: Methanol, chloroform, and n-hexane (LR grade) were purchased from Merck (India). Methanol and water (LC–MS grade) were purchased from J.T. Baker (Center Valley, PA, USA) and Carlo Erba (Val de Reuil, France), respectively. Formic acid was obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France) and fresh distilled water was prepared in the laboratory using a distillation unit.

Collection of plant material and its extraction

Plant sample: The fresh leaves of N. pallens (20 accessions) were collected from their natural habitat in Majhrana, Himachal Pradesh, India (Fig. 1), at an altitude of 2316 m (31°12.014″ N and 77°18.288″ E) in the month of March, 2021. The plant collection adhered to all relevant guidelines and the permission was obtained from the Divisional Forest Officer, Shimla, Himachal Pradesh, India. The plant specimen (flowering stage) was authenticated at Systematic Botany Discipline of Forest Botany Division of the ICFRE-Forest Research Institute (FRI) Dehradun. The voucher specimen AN: 172,813 was also deposited in the herbarium of the Systematic Botany Discipline.

Figure 1
figure 1

Neolitsea pallens growing in Majhrana, Himachal Pradesh, India.

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Extraction of the leaves and sample preparation: The leaves collected from 20 accessions were pooled, and made into a composite sample which was lyophilized at − 40 °C and milled into a powder using a mixer grinder. The leaves were extracted sequentially using n-hexane, chloroform, and 25% aqueous methanol, respectively. Primarily, 5 g of powdered leaves were extracted with 50 mL of n-hexane using ultrasonication for 1 h then the supernatant was filtered through Whatman no.1 filter paper. The residue was re-extracted two more times with fresh solvent. The filtrates were combined and passed through charcoal black for removal of chlorophyll from the extract and concentrated in a vacuum using a rotary evaporator (Buchi, Switzerland) at the temperature 40 °C (279.42 mmHg). The residue was subsequently subjected with chloroform followed by 25% aqueous methanol three times and the liquid extracts, after treatment with charcoal black for removal of chlorophyll, were concentrated in vacuum using a rotary evaporator at the temperatures 42 °C (400 mmHg) and 50 °C (406 mmHg), respectively. All the extracts were collected individually and stored at 2–4 °C for their chemical examinations. The yield (%) of 25% aqueous methanol extract was found to be 15.85 ± 1.26 (Mean ± S.D). A fresh solution (100 ppm) of the extract was prepared in methanol and filtered through a 0.22-μm polyvinylidene difluoride (PVDF) membrane (MILLEX GV filter unit) and transferred into a UPLC autosampler vial prior to LC–MS analysis.

UPLC-Q-TOF-MS/MS analysis of 25% aqueous methanol extract: UPLC-QTOF-MS/MS analysis of 25% aqueous methanol extract was conducted using an Agilent 6546 system, a Quadrupole time-of-flight (QTOF) mass spectrometer coupled with an Agilent 1290 Infinity II UPLC system via Dual AJS (ESI) electrospray ionization Source (Agilent Technologies, Santa Clara, CA, 95,051, US). The Agilent 1290 UPLC system consisted of a quaternary pump (G1311A), an online vacuum degasser (G1322A), an auto sampler (G1329A), and a diode array detector (G1315D). Chromatographic separation of the extract was achieved using an Agilent ZORBAX RRHD Eclipse Plus reversed phase C18 column (2.1 × 100 mm, 1.8 μm). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 100% methanol (solvent B), with a flow rate of 0.4 mL/min under the following gradient elution program: 5% B at 0–2 min, 5–20% B at 2–6 min, 20–45% B at 6–18 min, 45–95% B at 18–25 min, and a post time of 3 min, resulting in a total run time of 28 min. The sample injection volume was 5 μL, and the column temperature was set at 35 °C. Mass spectrometric analysis was carried out on an Agilent 6520 QTOF mass spectrometer in Negative ESI mode. The resolving power of the QTOF analyzer was set above 10,000 (FWHM, full width at half maximum), and spectra were acquired within a mass range of m/z 100–1700. Nitrogen gas was used for nebulizing, drying in the ionization source, and also for collision in the CID (Collision-induced Dissociation) cell. The sheath gas temperature was maintained at 350 °C, with a flow rate of 11 L/min, and the fragmentor voltage was set to 100 V. The capillary voltage was adjusted to 3500 V, the nebulizer pressure to 35 psi, and the drying gas flow rate to 10 L/min. Collision energy values for MS/MS experiments were fixed at 10 eV, 20 eV, and 40 eV for all the selected masses. The total number of injections was 7, consisting of 3 blank injections followed by 3 sample injections for MS analysis, and then 1 sample injection for Auto MS/MS analysis. Data analysis was performed using Agilent Mass Hunter Workstation Data Acquisition software version B.03.01 (version 10.0 Qualitative Analysis, Agilent Technologies, Santa Clara, CA, 95051, USA), which generated the molecular formula with a mass accuracy limit of 5 ppm (related to the contribution to mass accuracy, isotope abundance, and isotope). To obtain chemical structure information, the following databases were consulted: PubChem (https://pubchem.ncbi.nlm.nih.gov), ChemSpider (http://www.chemspider.com/), Food Database (http://foodb.ca/), Mass Bank (http://www.massbank.jp/), and Human Metabolome Database (https://hmdb.ca/).

Results

In the present investigation, the metabolite profiling of the 25% aqueous methanolic extract of N. pallens leaves was elucidated by employing reverse phase UPLC-QTOF-MS and tandem mass spectrometry (MS/MS) under negative ionization mode. Figure 2 depicts the total ion chromatogram of the chemical compounds and the assigned peak numbers correspond to the elution order of the compounds as enumerated in Table 1. A total of 56 chemical compounds were tentatively identified by examination of their MS/MS fragmentation patterns and comparison with the chemical structures of previously reported compounds. Comprehensive MS data for the identified compounds are succinctly presented in Table 1, encompassing retention time (rt), compound class, experimental m/z, experimental mass, theoretical mass, mass error in parts per million (ppm), molecular formula, and MS/MS fragment ions by considering the registered mass spectra fragmentation patterns in databases, as well as the observed fragmentation patterns in the current study and previously reported data in the literature. The identified compounds were systematically categorized into distinct classes, comprising four organic acids, five phenolic acids and derivatives, six tannins, two phenylpropanoid glycosides, four lignan glycosides, thirty-one flavonoids, and four miscellaneous compounds. The comprehensive characterization of all 56 compounds in N. pallens leaves was conducted for the first time through the analysis of mass spectrometry fragmentation patterns of the compounds utilizing UPLC-Q-TOF–MS/MS technique.

Figure 2
figure 2

Total ion chromatogram of 25% aqueous methanol extract of Neolitsea pallens leaves using negative ionization mode in UPLC-QTOF-MS/MS technique.

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Table 1 Retention time and mass spectroscopic characteristic data of constituents identified in 25% aqueous methanol extract of Neolitsea pallens leaves using negative ionization mode in UPLC-QTOF-MS/MS technique.
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Discussion

Organic acids. Four compounds were tentatively identified as organic acids. Compound 1 was detected at a retention time (rt) of 0.755 min, appearing as an [M-H]− ion at m/z 191.0197. The molecular formula was assigned as C6H8O7 using Electrospray Ionization High-Resolution Mass Spectrometry (ESI-HRMS). Tandem mass spectral analysis revealed fragment ions at m/z 173 [M-H-H2O]−, 155 [M-H-2H2O]−, 111 [M-H-2H2O-CO2]−, and 85 [M-H-2H2O-CO2-C2H2]−. Integrating the mass spectrometry data and interpreting the MS/MS fragmentation pattern (Scheme 1 [supporting information]), this compound was identified as citric acid10.

Compound 2, eluting at a retention time of 0.833 min, was detected as an [M-H]− ion at m/z 133.014. The ascribed molecular formula, C4H6O5 was established through precise mass determination. Upon fragmentation, it yielded product ions at m/z 115 [M-H-18Da]− and m/z 71 [M-H-18-44Da]−, indicative of the sequential loss of a water molecule (−H2O) followed by the elimination of a carbon dioxide molecule (−CO2) from the precursor ion. Accordingly, through comprehensive analysis of the acquired MS/MS data and cross-referencing with published literature, this compound was identified as malic acid11. The elucidated fragmentation pattern of this compound is depicted in scheme 2 (supporting information).

Compound 3 (rt 1.240 min) was detected as an [M-H]− ion at m/z 203.0192 with a molecular formula C7H8O7 and it was tentatively identified as daucic acid based on its fragmentation pattern (Scheme 3 [supporting information]) and online database (PubChem and FooDB). The MS/MS spectra exhibited distinctive product ions at m/z 184 [M-H-18Da]−, m/z 140 [M-H-18-44Da]−, and m/z 97 [M-H-18-44-44Da]−, attributed to the sequential loss of H2O, H2O + CO2, and H2O + 2CO2 from the parent ion. Notably, the fragment ion at m/z 97 [M-H-H2O-2CO2]− emerged as the base peak in the spectrum.

Compound 4 (rt 1.371 min) displayed a [M-H]− molecular ion at m/z 117.0191and tentatively identified as succinic acid (C4H6O4) through MS spectral comparison with published data12. The MS/MS spectra revealed distinct fragment ions at m/z 99 [M-H-18Da]− and m/z 73 [M-H-44Da]−, indicative of sequential losses of H2O and CO2, respectively (Scheme-4 [supporting information]).

Phenolic acids and derivatives. Five compounds were identified within this class, comprising two phenolic acids and three derivatives of phenolic acids. Compound 5 (rt 2.450 min) displayed a [M-H]− precursor ion at m/z 191.0191, with a molecular formula C7H12O6. The MS/MS fragment ions at m/z 173, m/z 129, and m/z 111 corresponded to neutral losses of H2O, H2O + CO2, and 2H2O + CO2, respectively. The current MS/MS data, along with reported literature13,14,15, led to the identification of compound 5 as quinic acid. The proposed fragmentation pattern is illustrated in scheme 5 (supporting information). Compound 8 (rt 6.893 min) displayed an [M-H]− ion at m/z 457.1344, with the molecular formula C20H26O12 determined by ESI-HRMS. The MS/MS spectra revealed product ions at m/z 163 and 119. A characteristic fragment ion at m/z 163 [M-H-C11H18O9]−, representing the loss of deoxyribosyl-rhamnosyl (−294 Da) moiety, further fragmented into m/z 119 by the loss of a CO2 molecule (Scheme 8 [supporting information]). Considering the current MS/MS data and previously reported literature, this compound was identified as cis-p-coumaric acid 4-[apiosyl-(1- > 2)-hexoside]16,17,18,19. Compound 9 (rt 7.158 min) with the molecular formula C16H18O9, exhibited a precursor ion [M-H]− at m/z 353.0872. Upon fragmentation, it yielded a distinct and intense fragment ion at m/z 191 [M-H-C9H6O3]− resulting from the neutral loss of the p-coumaric acid moiety (−162 Da). This fragment was identified as quinic acid [Scheme 9 [supporting information]. Consequently, the compound (9) was identified as chlorogenic acid, supported by the comprehensive analysis of MS/MS data and corroborated by findings from previous reports14,20,21. Compounds 12 (rt: 7.966 min) and 16 (rt: 9.426 min) were detected as [M-H]− ions at m/z 385.1133, and their molecular formula was determined to be C17H22O10 through HRMS analysis. Both compounds were recognized as isomers, featuring similar fragment ions at m/z 223, 179, 208, 164, and 149, corresponding to common neutral losses. Notably, the MS/MS analysis revealed fragment ions at m/z 223 [M-H-163Da]−, attributed to the loss of the sugar moiety. Subsequent fragmentation produced daughter ions at m/z 208, 179, 164, and 149 through the loss of neutral molecules CH3, CO2, CO2 + CH3, and CO2 + 2CH3, respectively (Scheme 12 and Scheme 16 [supporting information]). The collective evidence from MS/MS data and previously reported literature22, allowed the confident identification of compounds 12 and 16 as isomers of 1-O-sinapoyl glucose.

Tannins. Six compounds were identified in this class, including one hydrolyzable tannin and five condensed tannins, commonly referred to as proanthocyanidins. Compound 6, eluted at a retention time of 2.633 min, exhibited a molecular ion [M-H]− at m/z 331.0664, corresponding to the molecular formula C13H16O10. The MS/MS spectra unveiled distinct product ions at m/z 168 [M-H-164Da]− and m/z 125 [M-H-C6H12O5-44Da]−. The characteristic fragment ion was observed at m/z 168 due to loss of rhamnose [-C6H12O5] and it was identified as gallic acid (Scheme 6 [supporting information]). Consequently, based on the acquired MS/MS data and corroborating literature23,24, Compound 6 was identified as a 3-glucogallic acid. Compounds 10 (rt: 7.220 min) and 28 (rt: 11.950 min) were detected as [M-H]− ions at m/z 577.1345, featuring the molecular formula C30H26O12. They were tentatively identified as proanthocyanidin dimers based on previously reported literature25,26,27,28. The MS/MS spectra of both compounds exhibited similar fragment ions at m/z 425, 289, 451, 299, 125, and 407, generated through a specific fragmentation reaction outlined in Scheme 10 (supporting information). The product ion at m/z 425 [M-H-152Da]− was produced by retro-Diels–Alder (RDA) fission, further fragmented into 407 [M-H-152-18Da]− due to the neutral loss of an H2O molecule. Additionally, the fragment ion at m/z 289 [M-H-288Da]− resulted from quinone methide (QM) fission, while ions at m/z 125 [M-H-126Da]− and 451 [M-H-126Da]− were formed by heterocyclic ring fission from the parent ion. According to published reports25,26,27,28, the current MS/MS data, and a significant difference in retention time, compounds 10 and 28 were tentatively identified as isomers of procyanidin B5 and procyanidin B8, respectively (Scheme 10 [supporting information]).

Compounds 11 (rt: 7.364 min) and 24 (rt: 11.398 min) were detected with a precursor [M-H]− ion at m/z 863.182, and they were assigned the molecular formula C45H36O18 based on accurate mass measurements. Both compounds were identified as isomers of proanthocyanidin trimers, exhibiting similar product ions at m/z 711 [M-H-152Da]−, indicative of a loss of 152 Da through RDA fission. In the MS/MS spectra of compound 24, product ion at m/z 737 was observed via heterocyclic ring fission, and ions at m/z 575 and 287 resulted from QM fission, respectively. Compound 11 exhibited product ion at m/z 411, possibly through QM fission followed by RDA fission. The proposed fragmentation pathway is illustrated in Schemes 11 and 24 (supporting information). Based on the current MS/MS data and information from previously reported literature25,29,30,31,32,33,34,35, these two compounds were tentatively identified as isomers of procyanidin type 1A1B.

Compound 23 (rt: 11.258 min) was detected as an [M-H]− ion at m/z 575.1193, and its molecular formula was assigned as C30H24O12 based on accurate mass measurements. This compound was identified as a proanthocyanidin dimer, relying on previously reported literature25. The tandem mass spectra produced fragment ions at m/z 423, 449, 285, 289, and 125. These fragment ions were produced through specific fragmentation pathways (Scheme 22 [supporting information]). The fragment ion at m/z 423 [M-H-152Da]− was formed through RDA fission and m/z 285 was formed due to QM fission. Additionally, the product ions at m/z 449 [M-H-126Da]− and 125 [M-H-150Da]− were produced through heterocyclic ring fusion. Based on the current MS/MS data and information from previously reported literature, this compound could be identified as proanthocyanidin A2.

Lignan glycoside. Three compounds, 17 (rt: 9.648 min), 18 (rt: 9.751 min), and 29 (rt: 12.789 min), were detected as [M-H]− ions at m/z 521.202, with a molecular formula of C26H34O11. These compounds, were identified as isomers with a common characteristic peak at m/z 359 [M-H-162Da]−, resulting from the loss of a sugar moiety (-C6H10O5). The fragment ion peak at m/z 329 [M-H-162-31Da]− was commonly observed in compounds 18 and 29 due to the loss of a methoxy group (–OCH3) from m/z 359. Additionally, the daughter ion at m/z 341 [M-H-162-18Da]− was observed in compound 29 due to the loss of a neutral water molecule (-H2O) from m/z 359. In contrast, fragment ions at m/z 344 [M-H-162–15Da]− and 313 [M-H-162-15-31Da]− were observed in compound 17, indicating the loss of a methyl group (–CH3) followed by a methoxy group (–CH3O) from m/z 359. The fragmentation patterns of these three compounds were presented in Schemes 17, 18, and 29 (supporting information). According to published reports36 and the current MS/MS data, these three compounds were identified as isomers of isolariciresinol-O-glucoside, respectively.

Compound 21 (rt: 10.898 min) was observed as an [M-H]− ion at m/z 581.2231 with molecular formula (C28H38O13). In the MS/MS spectra, a prominent characteristic fragment ion at m/z 419 [M-H-162Da]− was observed, resulting from the loss of the rhamnose moiety (−C6H10O5). Upon further fragmentation, the ensuing fragment ions were observed at m/z 404 [M-H-162–15Da]− and m/z 373 [M-H-162–15-31Da]−, indicative of sequential losses of a methyl group in the radical form (−CH3˙) followed by a methoxy group (-CH3O˙), respectively (Scheme 20 in the Supporting Information). Considering the current MS/MS data and relevant literature37, compound 21 was tentatively identified as lyoniresinol 9-glucoside.

Flavonoids. Flavonoids constitute a diverse and prevalent class of polyphenolic compounds. Chemically, they consist of a 15-carbon skeleton comprising two aromatic rings (A and B) linked by a three-carbon bridge (C). This class represents a paramount group of phenolic compounds, notably abundant in 25% aqueous methanol extract of N. pallens leaves. Within this classification, a total of 31 compounds were identified and characterized within the sub-class of flavonols. Particularly, compounds 19, 20, 22, 32, 36, 30, 31, 33, 35, 37, 38, 41, 43, 42, 47, and 52 were identified as quercetin-based derivatives (Fig. 3A), while compounds 25, 26, 34, 40, 45, 48, 54, 39, 44, 46, 49, 50, 51, 53, and 55 were determined to be kaempferol-based derivatives (Fig. 3B).

Figure 3
figure 3

(A): Proposed Fragmentation Pathways for Quercetin-Based Derivatives. (B): Proposed Fragmentation Pathways for Kaempferol-Based Derivatives.

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Four compounds, 19 (rt 10.133 min), 20 (10.199 min), 32 (13.82 min), and 36 (14.479 min), were identified as [M-H]− ions at m/z 609.14, sharing the identical molecular formula C27H30O16. These compounds were characterized as isomers, featuring a common characteristic product ion at m/z 300 [M-H-309Da]−, corresponding to quercetin [M-H]− unit, potentially arising from the loss of a sugar moiety such as glucosyl-rhamnosyl (-C12H21O9). Moreover, in compound 20, an additional product ion at m/z 462 [M-H-147Da]− was observed, indicative of the loss of rhamnosyl moiety (-C6H11O4). Through the integration of the present MS/MS data and insights from documented literature38, these compounds were identified as isomers of rutin (quercetin-O-rhamnoside-hexoside). The fragmentation patterns of these compounds were presented in Scheme 36 [supporting information].

Compound 22 (rt 10.91) exhibited an [M-H]− ion with a m/z of 331.0456, and its molecular formula was accurately determined as C16H12O8. The MS/MS spectra revealed fragment ions at m/z 151 [M-H-180Da]− and m/z 179 [M-H-152Da]−, indicative of retro Diels–Alder (RDA) fission. Based on the current MS/MS data and corroborating literature39, Compound 22 was tentatively identified as laricitrin, and its proposed fragmentation pattern is depicted in Scheme 21 [supporting information].

Compound 25 (rt 11.723 min), 26 (11.865 min), 34 (14.188 min), 40 (15.717 min), 45 (16.416 min), and 48 (16.606 min) were detected as [M-H]− ions at m/z 593.15, all sharing the identical molecular formula C27H30O15, yet eluting at distinct retention times. These compounds were recognized as isomers exhibiting a common characteristic product ion at m/z 285 [M-H-162-146Da]−, indicative of kaempferol aglycone, achieved through the neutral loss of sugar moieties like hexosyl (162) and rhamnosyl (146). Furthermore, in the MS/MS spectra of compounds 25 and 26, product ions at m/z 447 [M-H-146Da]− and 431 [M-H-162Da]− were noted, corresponding to the loss of rhamnosyl and hexosyl, respectively. Based on previously published literature40 and the current MS/MS data, compounds 25 and 26 were tentatively identified as isomers of astragalin 7-rhamnoside (kaempferol-O-rhamnosyl-O-hexoside), while compounds 34, 40, 45, and 48 were discerned as isomers of kaempferol-O-rhamnosyl-hexoside. The fragmentation patterns of these compounds are presented in schemes 26 and 23 [supporting information].

Compound 54 (rt 20.121 min) exhibited an [M-H]− ion at m/z 593.1290, with its molecular formula identified as C30H26O13 through accurate mass measurement analysis. The tandem mass spectra revealed distinctive product ions at m/z 285 [M-H-308Da]−, indicative of kaempferol. The corresponding fragmentation patterns are illustrated in Scheme 39, in the supporting information. Drawing upon the present MS/MS data and corroborating with the literature41, compound 54 was provisionally recognized as tiliroside.

Compounds 30 and 31 were detected as an [M-H]− ion at m/z 595.129 with the same molecular formula C26H28O16. These compounds, identified as isomers, exhibited a common characteristic product ion at m/z 300 [M-H-295Da]−∙, signifying the presence of the quercetin moiety through the loss of arabino glucoside (-C11H19O9). Based on the current MS/MS data and previously documented literature42, these entities were tentatively categorized as isomers of quercetin-3-O-arabinohexoside (Scheme 25, in the supporting information).

Compounds 33 (rt 14.067 min) and 35 (14.354 min) were shown identical precursor ions [M-H]− at m/z 463.087 with the same molecular formula C21H20O12. These two compounds were identified as isomers both displaying the characteristic product ion at m/z 300 [M-H-163Da]−· due to the elimination of glucosyl moiety (–C6H11O5·). Based on the present MS/MS data and findings in the literature43, these two compounds are likely isomers of quercetin-O-hexoside, and their fragmentation pathways are depicted in Scheme 26 in the supporting information.

Compounds 37 (rt 14.723 min) and 38 (rt 15.100 min) were identified by the same [M-H]− ions at m/z 433.077, sharing the identical molecular formula C20H18O11. Analysis of their tandem mass spectral data revealed analogous product ions at m/z 300 [M-H-133Da]−· and 271 [M-H-133-29Da]−, arising from the sequential loss of a sugar moiety, such as arabinosyl (-C5H9O4∙), followed by an HCO moiety, respectively. Accordingly, based on the current MS/MS data and insights from the literature44, these compounds are likely isomers of guaijaverin (Scheme 38, supporting information).

Compounds 39 (rt 15.639 min), 41 (15.938 min), 43 (16.194 min), and 44 (16.255 min) were observed as [M-H]− ions at m/z 447.092, with the common molecular formula C21H20O11. The tandem mass spectra of compounds 39 and 44 exhibited analogous characteristic product ions at m/z 284 [M-H-163Da]−, m/z 255 [M-H-163-29Da]−, and 227 [M-H-163-29-28Da]−, attributed to the loss of the hexosyl moiety (-C6H11O5·), CO+H˙, and CO, respectively. On the contrary, compounds 41 and 43 generated indistinguishable product ions at m/z 300 [M-H-147Da]·−, 271 [M-H-147-29Da]−, and 243 [M-H-147-29-28Da]−, resulting from the loss of rhamnosyl, CO + H˙, and CO, respectively. Through a comprehensive analysis integrating current MS/MS data and corroborating literature sources45,46, compounds 39 and 44 were conclusively identified as isomers of kaempferol-O-hexoside (Ex: Astragalin), while 41 and 43 were unequivocally characterized as isomers of quercetin-O-rhamnoside (Ex: quercitrin). The fragmentation patterns of these compounds are presented in Scheme 44 and 33, in the supporting information.

Compounds 42 (rt 16.120 min) and 47 (16.581 min) were both detected with an identical precursor [M-H]− ion at m/z 579.135, with the same molecular formula C26H28O15. These compounds discerned as isomers, exhibited similar characteristic product ions at 300 [M-H-279Da]− and 301 [M-H-278Da]−, arising from the loss of neutral sugar moieties, specifically rhamnosyl-arabinopyranoside in radical and neutral form, respectively. Based on acquired MS/MS data and referencing pertinent literature47,48,49, both compounds were unequivocally identified as isomers of quercetin-O-rhamnosyl-arabinopyranoside. The corresponding fragmentation pattern is illustrated in scheme 29 (supporting information).

Compounds 46 and 49 exhibited a common precursor [M-H]− ion at m/z 417.0820, sharing the molecular formula C20H18O10, yet eluted at distinct retention times of 16.463 min and 16.728 min, respectively. These compounds were identified as isomers, displaying an identical precursor ion at m/z 284 [M-H-132Da]·− through the loss of the arabinosyl moiety. Upon further fragmentation, they yielded product ions at 255 [M-H-132-29Da]− and 227 [M-H-132-29-28Da]− by the neutral loss of CO + H∙ and CO molecules, respectively. According to the reported literature50,51 and the acquired MS/MS data, these two compounds were identified as isomers of kaempferol-O-arabinoside, and the fragmentation patterns of these compounds are presented in scheme 30 [supporting information].

Compound 50 (rt 17.977 min) was observed as an [M-H]− ion at m/z 563.1399, and its molecular formula was elucidated through precise mass measurements as C26H28O14. The tandem mass spectra unveiled a fragment ion at m/z 431 [M-H-132Da]−, attributed to the detachment of the rhamnosyl moiety (-C5H8O4). Subsequent fragmentation led to ions at 284 [M-H-147Da]− and 255 [M-H-132–147-29Da]−, arising from the loss of the hexosyl moiety (-C6H11O4˙) and CO + H˙, respectively. Based on the current MS/MS data (Scheme 31 [supporting information]) and corroborating with published ligature52,53, compound 50 was tentatively identified as kaempferol-O-rhamnoside-O-xyloside.

Compounds 51 (rt 18.287 min) and 53 (18.644 min) were both identified with an identical [M-H]− ion at m/z 431.098, sharing the molecular formula C21H20O10. These compounds, recognized as isomers, exhibited similar MS/MS spectra, with product ions observed at m/z 284 [M-H-147Da]˙−, 255 [M-H-147-29Da]−, and 227 [M-H-147-29Da]− resulting from the loss of rhamnosyl (-C6H11O4), CO + H˙, and CO, respectively. Utilizing the current MS/MS data (Scheme 32 [supporting information]) and referencing reported literature54, these compounds were conclusively identified as isomers of kaempferol-O-rhamnoside (Afzelin-I).

Compound 52 (rt 18.586 min) displayed a parent ion peak [M-H]− at m/z 301.0350, corresponding to the molecular formula (C15H10O7). In the MS/MS spectra, characteristic fragment ions were observed at m/z 151 [M-H-150Da]− due to the neutral loss of the glycosyl moiety (-C8H6O3) through RDA fission. Subsequently, it further fragmented into m/z 107 [M-H-150–44Da]− by the loss of a CO2 group. The tentative identification of the compound as quercetin was established through a comparative analysis with reported literature55 and the current MS/MS data (Scheme 37) [supporting information].

Compound 55 (22.489 min) was identified with an [M-H]− ion at m/z 723.1711, and its molecular formula was determined to be C39H32O14 through accurate mass measurement analysis. The tandem mass spectra of this compound revealed a distinctive fragment ion at m/z 285 [M-H-439Da]−, corresponding to the kaempferol moiety. Additionally, fragment ions at m/z 559 [M-H-164Da]−, 163 [M-H-560Da]−, and 119 [M-H-560-44Da]− were observed, and the fragmentation patterns are depicted in Scheme 34 [supporting information]. Based on current MS/MS data and an online library match, this compound was tentatively identified as 2″,3″-di-O-p-coumaroylafzelin/platanoside56.

Phenylpropanoid glycosides. Compound 14 (rt: 8.65 min) was tentatively identified as citrusin F by comparing current HRMS data with previously reported literature and online databases (PubChem and HMDB). It was detected as an [M-H]− ion at m/z 519.1712, and the molecular formula was determined to be C22H32O14 using ESI-HRMS analysis. Compound 15 (rt: 9.324 min) was detected as an [M-H]− ion at m/z 441.1758, and the molecular formula C21H30O10 was determined based on accurate mass measurement. This compound was tentatively identified as lusitanicoside by comparing its MS data with previously reported literature57 and online databases [PubChem and HMDB].

Miscellaneous compounds. Compound 7 (rt 4.464 min) was tentatively identified as leonuriside A, based on information from previously reported literature58. It was observed as an [M-H]− ion at m/z 331.1028 with the molecular formula C14H20O9. The MS/MS spectra unveiled a distinctive fragment ion at m/z 168 [M-H-163Da]·-, indicative of the loss of the hexose (-C6H11O5) moiety in the radical form, and further fragmented into m/z 153 [M-H-163–15Da]·- by the loss of methyl radical (-CH3˙), as illustrated in Scheme 7 [supporting information]. Compound 13, with a retention time of 8.571 min, was identified as an [M-H]− ion with a m/z value of 401.1456. The accurate mass measurement led to the determination of its molecular formula as C18H26O10. Further analysis through MS/MS spectra revealed a characteristic product ion at m/z 269 [M-H-132Da]−, indicative of the neutral loss of one of the sugar moieties (-C5H9O4). Based on comprehensive high-resolution MS and MS/MS fragmentation patterns [Scheme 13 (supporting information)], this compound was tentatively identified as benzyl ꞵ-primeveroside59.

Compound 27 (rt 11.88 min) manifested as an [M-H]− ion at m/z 523.2177, with the ascribed molecular formula C26H36O11. The tandem mass spectral analysis revealed a distinctive fragment ion at m/z 361 [M-H-162Da]−, attributable to the neutral loss of a hexosyl moiety (-C6H10O5). Additionally, it underwent further fragmentation into m/z 346 through the loss of a methyl radical moiety [-CH3·]. Based on the current MS/MS data and corroborating literature60, this compound was tentatively identified as mascaroside [Scheme 27 (supporting information)].

Compound 56, eluting with a retention time of 24.87 min, was detected as [M-H]− ion at m/z 297.2433, and its molecular formula was determined to be C18H34O3 using precise mass measurements from HRMS analysis. The tandem mass spectra revealed a distinctive fragment at m/z 183 [M-H-C7H14O]−, indicative of γ-hydrogen transfer through McLafferty rearrangement. Consistent with the available MS/MS data [Scheme 40 (supporting information)] and reported literature61, this compound has been tentatively identified as ricinoleic acid.

Conclusion

This study offers the first comprehensive analysis of the chemical composition of traditionally used Neolitsea pallens leaves. A total of 56 compounds, including four organic acids, five phenolic acids and derivatives, six tannins, two phenylpropanoid glycosides, four lignan glycosides, thirty-one flavonoids, and four miscellaneous compounds, were identified. Utilizing UPLC-QTOF-MS/MS enabled detailed compound characterization by matching their fragmentation patterns with databases and literature. Among these, phenolics, represented by tannins, phenylpropanoids, lignans, and flavonoids, constituted the major class of compounds, which hold promise for various fields such as pharmaceuticals, nutraceuticals, and cosmetics. Moving forward, future studies should focus on exploring the specific biological activities and potential applications of these compounds. By probing deeper into the pharmacological and therapeutic properties of N. pallens leaves constituents, new opportunities for drug development, dietary supplements, and skincare products can be uncovered. Overall, our findings pave the way for interdisciplinary investigations aimed at harnessing the full potential of N. pallens leaves in diverse applications.