Phytochemical exploration of Neolitsea pallens leaves using UPLC-Q-TOF-MS/MS approach

Neolitsea pallens (D. Don) Momiyama & H. Hara (Family: Lauraceae), commonly known as Pale Litsea, is an evergreen small tree, distributed in India at altitudes of 1500–3000 m. Traditionally utilized for various purposes, its leaves and bark are used as spices, and the plant is valued in preparing a hair tonic from freshly pressed juice. Secondary metabolites of the leaves have not comprehensively been analysed so far. The objective of the study was to determine the chemical composition of the leaves by analysing their 25% aqueous methanol extract with the aid of ultra-performance liquid chromatography quadrupole time of flight tandem mass spectrometry. Overall, 56 compounds were identified in the study. Phenolics represented by phenolic acids, phenolic glycosides, proanthocyanidins, and flavonoids were the main components of the extract.


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.
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 C 18 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 www.nature.com/scientificreports/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:// pubch em.ncbi.nlm.nih.gov), ChemSpider (http:// www.chems pider.com/), Food Database (http:// foodb.ca/), Mass Bank (http:// www.massb ank.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, thirtyone flavonoids, and four miscellaneous compounds.4][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.  9) was identified as chlorogenic acid, supported by the comprehensive analysis of MS/MS data and corroborated by findings from previous reports 14,20,21   O 5 ] and it was identified as gallic acid (Scheme 6 [supporting information]).Consequently, based on the acquired MS/MS data and corroborating literature 23,24 , 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 C 30 H 26 O 12 .They were tentatively identified as proanthocyanidin dimers based on previously reported literature [25][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 H 2 O 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 reports [25][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]).

Discussion
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 C 45 H 36 O 18 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 literature 25,[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 C 30 H 24 O 12 based on accurate mass measurements.This compound was identified as a proanthocyanidin dimer, relying on previously reported literature 25 .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 C 26 H 34 O 11 .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 (-C 6 H 10 O 5 ).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 (-OCH 3 ) 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 (-H 2 O) 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 (-CH 3 ) followed by a methoxy group (-CH 3 O) from m/z 359.The fragmentation patterns of these three compounds were presented in Schemes 17, 18, and 29 (supporting information).According to published reports 36 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 (C 28 H 38 O 13 ).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 (−C 6 H 10 O 5 ).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 (−CH 3 ˙) followed by a methoxy group (-CH 3 O˙), respectively (Scheme 20 in the Supporting Information).Considering the current MS/MS data and relevant literature 37 , compound 21 was tentatively identified as lyoniresinol 9-glucoside.
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 C 26 H 28 O 15 .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 literature [47][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 C 20 H 18 O 10 , 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 literature 50,51 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 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 C 22 H 32 O 14 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 C 21 H 30 O 10 was determined based on accurate mass measurement.This compound was tentatively identified as lusitanicoside by comparing its MS data with previously reported literature 57 and online databases [PubChem and HMDB].

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.
H 6 O 5 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 (−H 2 O) followed by the elimination of a carbon dioxide molecule (−CO 2 ) 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 acid 11 .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 C 7 H 8 O 7 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 H 2 O, H 2 O + CO 2 , and H 2 O + 2CO 2 from the parent ion.Notably, the fragment ion at m/z 97 [M-H-H 2 O-2CO 2 ] − 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 (C 4 H 6 O 4 ) through MS spectral comparison with published data 12 .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 H 2 O and CO 2 , 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 C 7 H 12 O 6 .The MS/MS fragment ions at m/z 173, m/z 129, and m/z 111 corresponded to neutral losses of H 2 O, H 2 O + CO 2 , and 2H 2 O + CO 2 , respectively.The current MS/MS data, along with reported literature 893 min) displayed an [M-H] − ion at m/z 457.1344, with the molecular formula C 20 H 26 O 12 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-C 11 H 18 O 9 ] − , representing the loss of deoxyribosyl-rhamnosyl (−294 Da) moiety, further fragmented into m/z 119 by the loss of a CO 2 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] 16-19 .Compound 9 (rt 7.158 min) with the molecular formula C 16 H 18 O 9 , 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-C 9 H 6 O 3 ] − 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 (
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 C 14 H 20 O 9 .The MS/MS spectra unveiled a distinctive fragment ion at m/z 168 [M-H-163Da] •-, indicative of the loss of the hexose (-C 6 H 11 O 5 ) moiety in the radical form, and further fragmented into m/z 153 [M-H-163-15Da] •-by the loss of methyl radical (-CH 3 ˙), 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 C 18 H 26 O 10 .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 (-C 5 H 9 O 4 ).Based on comprehensive high-resolution MS and MS/ MS fragmentation patterns [Scheme 13 (supporting information)], this compound was tentatively identified as benzyl ꞵ-primeveroside 59 .Compound 27 (rt 11.88 min) manifested as an [M-H] − ion at m/z 523.2177, with the ascribed molecular formula C 26 H 36 O 11 .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 (-C 6 H 10 O 5 ).Additionally, it underwent further fragmentation into m/z 346 through the loss of a methyl radical moiety [-CH 3 • ].Based on the current MS/MS data and corroborating literature 60 , 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 C 18 H 34 O 3 using precise mass measurements from HRMS analysis.The tandem mass spectra revealed a distinctive fragment at m/z 183 [M-H-C 7 H 14 O] − , indicative of γ-hydrogen transfer through McLafferty rearrangement.Consistent with the available MS/MS data [Scheme 40 (supporting information)] and reported literature 61 , this compound has been tentatively identified as ricinoleic acid.

Table 1 .
22tention 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.*Basepeakorcharacteristicpeak.formula was determined to be C 17 H 22 O 10 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 CH 3 , CO 2 , CO 2 + CH 3 , and CO 2 + 2CH 3 , 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 C 13 H 16 O 10 .The MS/ MS spectra unveiled distinct product ions at m/z 168 [M-H-164Da] − and m/z 125 [M-H-C 6 H 12 O 5 -44Da] − .The characteristic fragment ion was observed at m/z 168 due to loss of rhamnose [-C 6 H 12 . 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 609.14, sharing the identical molecular formula C 27 H 30 O 16 .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 glucosylrhamnosyl (-C 12 H 21 O 9 ).Moreover, in compound 20, an additional product ion at m/z 462 [M-H-147Da] − was observed, indicative of the loss of rhamnosyl moiety (-C 6 H 11 O 4 ).Through the integration of the present MS/MS data and insights from documented literature 38 , 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 C 16 H 12 O 8 .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 literature 39 , 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 (14.188min), 40 (15.717min), 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 C 27 H 30 min), 32 (13.82 min), and 36 (14.479 min), were identified as [M-H] − ions at m/z 44splaying the characteristic product ion at m/z 300 [M-H-163Da] −• due to the elimination of glucosyl moiety (-C 6 H 11 O 5• ).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 C 20 H 18 O 11 .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 (-C 5 H 9 O 4 • ), 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).