Identification of Daphnane Diterpenoids from Wikstroemia indica Using Liquid Chromatography with Tandem Mass Spectrometry

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for the rapid identification of compounds within natural resources. Daphnane diterpenoids, a class of natural compounds predominantly found in plants belonging to the Thymelaeaceae and Euphorbiaceae families, have attracted much attention due to their remarkable anticancer and anti-HIV activities. In the present study, the presence of daphnane diterpenoids in Wikstroemia indica, a plant belonging to the Thymelaeaceae family, was investigated by LC-MS/MS analysis. As a result, 21 daphnane diterpenoids (1–21) in the stems of W. indica were detected. Among these, six major compounds (12, 15, 17, 18, 20, and 21) were isolated and their structures were unequivocally identified through a comprehensive analysis of the MS and NMR data. For the minor compounds (1–11, 13, 14, 16, and 19), their structures were elucidated by in-depth MS/MS fragmentation analysis. This study represents the first disclosure of structurally diverse daphnane diterpenoids in W. indica, significantly contributing to our understanding of bioactive diterpenoids in plants within the Thymelaeaceae family.


Introduction
Liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HR-MS/MS), usually equipped with an electrospray ionization source, has high adaptability across a broad spectrum of compounds, offering high mass accuracy and sensitivity.Moreover, it provides information-rich fragmentation through product ion spectra, thereby potentially revealing details about the molecular formula and structure of diverse secondary metabolites found in plants [1].The conventional phytochemical research process often necessitates substantial amounts of accessible plant materials and time-consuming purification procedures, whereas applying LC-MS/MS analysis on crude plant extracts at the early stage of phytochemical investigations allows for the rapid identification of the compounds [2,3].
Wikstroemia indica (L.) C. A. Mey. is a semi-evergreen shrub mainly distributed in southeastern China, which has long been used as a traditional Chinese medicine for the treatment of bronchitis, hepatitis, and cancer [21].Recent studies have revealed that the extract of this plant exhibited antiallergic [22], anti-inflammatory [23], and antineoplastic properties [24], therefore heightening interest in its pharmacological exploration.While previous phytochemical investigations of W. indica have yielded coumarins [25], flavonoids [26], lignans [27], and sesquiterpenoids [28], the presence of daphnane diterpenoids has yet to be documented.
During our ongoing research aimed at discovering biological diterpenoids from plants of the Thymelaeaceae family [5,20,29,30], this study comprehensively examined and identified daphnane diterpenoids in the stems of W. indica using LC-MS/MS analysis.

Detection of Daphnane Diterpenoids in W. indica by LC-MS/MS
Due to the limited availability of plant material, the presence of daphnane diterpenoids in W. indica was initially examined by LC-MS/MS analysis.The criteria for validating that the detected peaks represented daphnane diterpenoids were established based on a synthesis of our previous studies and literature review [20,31,32].These criteria included: (1) In the mass spectra, protonated molecular ions ([M + H] + ) and/or ammonium adduct ions ([M + NH 4 ] + ) were observed in positive ion mode, while deprotonated molecular ions ([M-H] − ) and/or formate adduct ions ([M + HCOO] − ) were observed in negative ion mode.(2) In the product ion spectrum obtained from the protonated molecular ion as a precursor ion, a diagnostic ion at m/z 253 (C 17 H 17 O 2 ) or 269 (C 17 H 17 O 3 ) was observed in the positive ion mode [31].(3) The characteristic C 17 product ions derived from C 20 skeletons with the neutral loss of C 3 H 4 O 2 were observed [32].(4) When the ion peaks originated from a macrocyclic daphnane orthoester (MDO), the second and third criteria were not applicable.Instead, product ion peaks derived from continuous losses of H 2 O and CO were observed at the mass range of m/z 250-350 and m/z 400-550, respectively [20].
To enhance the detecting sensitivity, a crude diterpenoid fraction was prepared from the 95% EtOH extract using a sequence of procedures, including EtOAc-H 2 O partition and Diaion HP-20 column chromatography.Subsequent LC-MS/MS analysis of the crude diterpenoid fraction, guided by the aforementioned criteria, resulted in the detection of three major daphnane diterpenoid peaks (15, 20, and 21), strongly suggesting the occurrence of daphnane diterpenoids in W. indica stems.It was noteworthy that detecting daphnane diterpenoids can be challenging due to their chromatographic behavior, which was sometimes similar to common plant constituents, such as fatty acids, acylglycerols, and chlorophyll [33].

Identification of Minor Daphnane Diterpenoids by MS/MS Fragmentation Elucidation
To identify the minor daphnane diterpenoids (1-11, 13, 14, 16, and 19), which could not be isolated, MS/MS fragmentation elucidation was performed.These daphnane diterpenoids exhibited abundant ions in the product ion spectra derived from the protonated molecular ion as a precursor ion.Consequently, a detailed interpretation of the MS/MS fragmentation pathways in positive mode for these peaks was conducted (Figures 3 and 4).The identification of those peaks was confirmed by the LC-MS data which were in full accordance with the corresponding compounds isolated in our previous studies (Table 1) [5,29,30].

Identification of Minor Daphnane Diterpenoids by MS/MS Fragmentation Elucidation
To identify the minor daphnane diterpenoids (1-11, 13, 14, 16, and 19), which could not be isolated, MS/MS fragmentation elucidation was performed.These daphnane diterpenoids exhibited abundant ions in the product ion spectra derived from the protonated molecular ion as a precursor ion.Consequently, a detailed interpretation of the MS/MS fragmentation pathways in positive mode for these peaks was conducted (Figures 3 and 4).The identification of those peaks was confirmed by the LC-MS data which were in full accordance with the corresponding compounds isolated in our previous studies (Table 1) [5,29,30].In the product ion spectra of peaks 3 and 16, the characteristic product ion was observed at m/z 253 (C17H17O2), which was produced by the loss of the 6,7-epoxy moiety, along with the oxymethylene at C-20, as a C3H4O2 unit due to cleavage occurring at the Bring.This observation suggested that both 3 and 16 were daphnane diterpenoids lacking a substituent at C-12 (Figures 3A and S22).Furthermore, the product ions at m/z 207 (C14H23O), 95 (C6H7O), and 81 (C5H5O) for peak 3, and at m/z 179 (C12H19O), 95 (C6H7O), and 81 (C5H5O) for peak 16 indicated that a 2E,4E-tetradecadienoyl moiety was esterlinked to the C-ring in peak 3 and a 2E,4E-dodecadienoyl moiety was ester-linked in peak 16.However, the molecular formula of peak 3 was 18 Da (H2O) larger than that of the orthoester daphnane, huratoxin ( 21) [42] and the fragment ion of [M + H-H2O] + appeared with greater intensity in the mass spectrum of peak 3 (Figure S23).Based on these In the product ion spectra of peaks 3 and 16, the characteristic product ion was observed at m/z 253 (C 17 H 17 O 2 ), which was produced by the loss of the 6,7-epoxy moiety, along with the oxymethylene at C-20, as a C 3 H 4 O 2 unit due to cleavage occurring at the B-ring.This observation suggested that both 3 and 16 were daphnane diterpenoids lacking a substituent at C-12 (Figures 3A and S22).Furthermore, the product ions at m/z 207 (C 14 H 23 O), 95 (C 6 H 7 O), and 81 (C 5 H 5 O) for peak 3, and at m/z 179 (C 12 H 19 O), 95 (C 6 H 7 O), and 81 (C 5 H 5 O) for peak 16 indicated that a 2E,4E-tetradecadienoyl moiety was ester-linked to the C-ring in peak 3 and a 2E,4E-dodecadienoyl moiety was ester-linked in peak 16.However, the molecular formula of peak 3 was 18 Da (H 2 O) larger than that of the orthoester daphnane, huratoxin (21) [42] and the fragment ion of [M + H-H 2 O] + appeared with greater intensity in the mass spectrum of peak 3 (Figure S23).Based on these observations, it was concluded that peak 3 represented a polyhydroxy daphnane type compound, which lacks the orthoester moiety at the C-ring.Thus, peaks 3 and 16 were identified as wikstroelide M (3) [14] and wikstrotoxin B (16) [11], respectively.
In the product ion spectra of peaks 6, 9, 10, 14, and 19, the product ion generated by the loss of the C 3 H 4 O 2 unit was consistently observed at m/z 269 (C 17 H 17 O 3 ), indicating that these peaks corresponded to orthoester daphnanes with a substituent attached to C-12 (Figures 3B and S24).The product ions corresponding to substituents observed in these peaks were assignable as follows: a cinnamoyl moiety at m/z 131 (  4E,6E-decatrienoyl moieties were present in peak 6, the coumaroyl and 2E,4E-decadienoyl moieties in peak 9, the feruloyl and 2E,4E-decadienoyl moieties in peak 10, and the cinnamoyl and 2E,4E,6Edecatrienoyl moieties in peak 14.In peak 19, only the product ions due to the 2E,4Edodecadienoyl moiety were observed, but the molecular formula and the observation of product ions derived from the neutral loss of C 2 H 4 O 2 suggested the presence of the acetyl moiety.Thus, peaks 6, 9, 10, 14, and 19 were identified as acutilobin C (6) [36], daphneodorin D (9) [29], acutilobin D (10) [36], 12-O-(E)-cinnamoyl-9,13,14-ortho-(2E,4E,6E)decatrienylidyne-5β,12β-dihydroxyresiniferonol-6α,7α-oxide ( 14) [40], and wikstroelide A (19) [13]. Peaks and 13 were identified as MDOs by their characteristic MS/MS fragmentation patterns.Although the number of oxygen functional group varied among these compounds, they were all characterized by the abundance of C 30 to C 28 product ions observed in the range of m/z 400-550.The molecular formula of peak 7 indicated the absence of acyl groups.In the product ion spectrum, the neutral loss associated with the macrocyclic ring was assigned to be C 10 H 16 O as in compounds 18 and 20.In addition, a series of C 20 to C 18 product ions were observed with successive losses of H 2 O and CO from m/z 327 (C 20 H 23 O 4 ) (Figures 4A and S25).Peak 7 was suggested to possess the cyclopentanone A-ring structure based on the degree of unsaturation and was identified as pimelea factor S 6 (7) [37].Peak 5 had a molecular formula that was 16 Da (OH) larger than peaks 18 and 20.The product ion spectrum of peak 5 exhibited a series of C 20 to C 18 product ions below m/z 350, as observed in 18 and 20 (Figures 4B and S25).However, the neutral loss associated with the macrocyclic ring differed from 18 and 20, where it was C 10 H 16 O rather than C 10 H 14 O in 5.These observations indicated that 5 possesses an additional hydroxyl group at C-2 of the macrocyclic ring compared to 18 and 20, and was further identified as kraussianin (5) [35].
The pair of peaks 1 and 2, as well as the pair of peaks 8 and 11, had the same molecular formula and exhibited similar product ion spectra, indicating that each pair, like compounds 18 and 20, was in a regioisomeric relationship.The product ion spectra of peaks 1 and 2 revealed three molecules of C 7 H 6 O 2 neutral loss originating from benzoyl acids and two molecules of C 2 H 4 O 2 neutral loss originating from acetic acids, as well as the neutral loss of C 10 H 10 O associated with the macrocyclic ring.In addition, a series of C 30 to C 28 product ions were observed with successive losses of H 2 O and CO from m/z 507 (C 30 H 35 O 7 ) (Figures 4C and S26).These observations suggested that peaks 1 and 2 correspond to daphneodorin B (1) or daphneodorin C (2) with the same molecular formula and combinations of acyl groups.By comparison of retention times, peaks 1 and 2 were identified as daphneodorin C (1) and daphneodorin B (2), respectively [29].The product ion spectra of peaks 8 and 11 revealed the elimination of two molecules of C 7 H 6 O 2 , indicating the presence of two benzoyloxy moieties (Figures 4C and S26).Additionally, the neutral loss associated with the macrocyclic ring was assigned to be C 10 H 14 O as in 5 and the product ion pattern below m/z 350 was the same as 12, which were identified as the regioisomer, stelleralide H (8) [38] and gnidimacrin (11) [39], respectively.
Peak 4 had a molecular weight 14 Da greater than peak 7 but the neutral loss associated with the macrocyclic ring was assigned to be C 10 H 16 O as in peak 7, suggesting that the daphnane skeleton was different from peak 7 (Figures 4D and S27).Based on the molecular formula and degree of unsaturation, peak 4 was identified as pimelotide C [34], which has the bicyclo[2.2.1]heptane A-ring structure.In the product ion spectrum of peak 13, the observation of a loss of C 7 H 6 O 2 suggested the presence of a benzoyloxy moiety (Figures 4E and S27).Furthermore, the product ions observed within the range of m/z 400-550 were 2 Da smaller than those of peak 4. Thus, peak 13 was identified as stelleralide C [5], which shared the bicyclo[2.2.1]heptane A-ring structure of peak 4 and a benzoyloxy moiety attached to C-18.

Plant Material
The stems of W. indica were collected at Guangxi Province, People's Republic of China in February 2018 and identified by Dong Liang (Kunming Plant Classification and Biotechnology Co., Ltd., Kunming, China).A voucher specimen (accession number: 20201021) had been deposited in the herbarium of Shenyang Pharmaceutical University.

LC-MS/MS Conditions
The LC-MS/MS analysis was performed using the same instruments and column as in previous experiments [20].For LC conditions, the mobile phase comprised eluent A (distilled water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid), programmed as follows: 0-15 min, a linear gradient from 50% to 100% B, 15-18 min, 100% B, followed by column re-equilibration at 50% B for 10 min before the subsequent injection.For MS conditions, the in-source CID was set at 0 eV, and the resolution was 70,000 for full MS and 35,000 for full MS/data dependent (dd)-MS/MS modes.The AGC was established at 1E6 for full MS and 1E5 for dd-MS/MS.Data-dependent scanning was performed using HCD with the normalized collision energy at 15 eV.The extracted ion spectra were generated by extracting the following base peaks of each compounds within ±  (21).All data collected in the profile mode were acquired and processed using Thermo Xcalibur 4.1 software.

Conclusions
This study represents the first comprehensive identification of 21 daphnane diterpenoids from the stems of W. indica through a combination of LC-MS guided isolation and MS/MS fragmentation elucidation.The investigation revealed that W. indica contained structurally diverse daphnane diterpenoids, including orthoester daphnane type, polyhydroxy daphnane type, and macrocyclic daphnane orthoester type compounds.The application of MS/MS fragmentation elucidation for structural analysis enabled the rapid and precise identification of these diterpenoids within crude plant extracts.This methodology holds great promise for future research endeavors aimed at discovering bioactive diterpenoids from plants of the Thymelaeaceae family.

Figure 1 .
Figure 1.(A) Total ion chromatogram in the positive ion mode and (B) extracted ion chromatogram from the crude diterpenoid fraction of the stems of W. indica.