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Article

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

by
Mi Zhang
1,
Kouharu Otsuki
1,*,
Reo Takahashi
1,
Takashi Kikuchi
1,
Di Zhou
2,
Ning Li
2 and
Wei Li
1,*
1
Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi 274-8510, Chiba, Japan
2
Key Laboratory of Innovative Traditional Chinese Medicine for Major Chronic Diseases of Liaoning Province, Key Laboratory for TCM Material Basis Study and Innovative Drug Development of Shenyang City, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
*
Authors to whom correspondence should be addressed.
Plants 2023, 12(20), 3620; https://doi.org/10.3390/plants12203620
Submission received: 2 October 2023 / Revised: 16 October 2023 / Accepted: 17 October 2023 / Published: 19 October 2023
(This article belongs to the Special Issue Plant-Based Bioactive Substances Identification, Extraction, and Application)
Browse Figures
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Abstract

:
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.

1. 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].
Daphnane diterpenoids, characterized by their trans-fused 5/7/6-tricyclic skeleton, have garnered attention for their diverse biological activities, including anticancer [4], anti-HIV [5], analgesic [6], anti-inflammatory [7], and neurotrophic activities [8,9]. These diterpenoids are predominantly found in plants of the Thymelaeaceae and Euphorbiaceae families, with the majority of them sourced from the Thymelaeaceae family [10]. Previous phytochemical investigations on plants of the Thymelaeaceae family have reported the isolation of daphnane diterpenoids from 16 genera, such as Daphne, Pimelea, Stellera, and Wikstroemia. Among these, the Wikstroemia genus, comprising over 70 species, holds significant potential as a source of daphnane diterpenoids. Isolation of daphnane diterpenoids has hitherto been reported from a number of species, including W. monticola [11], W. mekongenia [12], W. retusa [13,14], W. polyantha [15], W. chamaedaphne [16,17,18], W. chuii [19], and W. ligustrina [20]. It is evident that the Wikstroemia genus remains relatively underexplored in the research into daphnane diterpenoids.
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.

2. Results and Discussion

2.1. 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 + NH4]+) 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 (C17H17O2) or 269 (C17H17O3) was observed in the positive ion mode [31]. (3) The characteristic C17 product ions derived from C20 skeletons with the neutral loss of C3H4O2 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 H2O 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–H2O 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].
To further enhance the sensitivity of LC-MS/MS detection of daphnane diterpenoids, a portion of the crude diterpenoid fraction underwent additional fractionation through gradient HPLC. As a result, a total of 21 daphnane diterpenoid peaks (1–21) were detected from three out of twelve subfractions (Figure S1). Importantly, all these peaks were subsequently confirmed to be present in the crude diterpenoid fraction through extracted ion chromatogram (XIC) analysis (Figure 1, Table 1).

2.2. LC-MS Guided Isolation and Structural Determination of Major Daphnane Diterpenoids

An LC-MS guided isolation was carried out to obtain daphnane diterpenoids. The crude diterpenoid fraction was subjected to ODS column chromatography and eluted with a stepwise gradient of MeOH–H2O. The fractions, in which daphnane diterpenoids were detected by LC-MS/MS analysis, were subjected to silica gel column chromatography and eluted with a gradient of n-hexane–EtOAc–MeOH–HCOOH. Those fractions containing daphnane diterpenoids were further purified by preparative HPLC and resulted in the isolation of six major daphnane diterpenoids (12, 15, 17, 18, 20, and 21) (Figure 2).
The isolated compounds were identified by detailed NMR and MS spectroscopic analyses. In the 1H- and 13C-NMR spectra, the characteristic resonances for an isopropenyl moiety at δH 4.83–4.99 (Ha-16), 4.94–5.17 (Hb-16), and 1.72–1.84 (H3-17), an epoxy group at δH 3.32–3.57 (H-7), δC 60.0–61.0 (C-6), and 63.5–64.4 (C-7), and an orthoester group at 116.5–119.8 (C-1′), indicated that they were daphnane diterpenoids (Tables S1 and S2) [10].
For compounds 15, 17, and 21, the characteristic resonances of α,β-unsaturated carbonyl moiety at δH 7.56–7.61 (H-1), δC 160.5–161.4 (C-1), 136.6–136.9 (C-2), and 209.5–209.9 (C-3) indicated they belong to orthoester daphnane diterpenoids (Table S1). The presence of a decanoate moiety at C-1′ in 15 was deduced from the proton resonance for the aliphatic methylene multiplets at δH 1.23–1.93 and a terminal methyl triplet at δH 0.86 (H3-10′). The 2E,4E-tetradecadienylidyne moiety of 21 was defined from the proton resonances for a conjugated diene at δH 5.89 (d, J = 15.5 Hz, H-2′), 6.68 (dd, J = 15.5, 10.6 Hz, H-3′), 6.04 (dd, J = 15.2, 10.6 Hz, H-4′), and 5.83 (dt, J = 15.2, 7.2 Hz, H-5′), and a n-nonyl moiety including eight methylenes at δH 1.24–2.07 and a terminal methyl group at δH 0.86 (t, J = 6.9 Hz, H3-14′). The presence of cinnamoyloxy moiety of 17 was deduced from a trans-olefinic moiety at δH 6.35 (d, J = 15.9 Hz, H-2″) and 7.62 (d, J = 15.9 Hz, H-3″), and a phenyl moiety at δH 7.37–7.51 (each multiplet, H-5″ to H-9″), as well as the carbon resonance for an ester carbonyl at δC 165.8 (C-1″), and a 2E,4E-decadienylidyne moiety was confirmed by the proton resonances at δH 5.65 (d, J = 15.4 Hz, H-2′), 6.66 (dd, J = 15.4, 10.6 Hz, H-3′), 6.04 (dd, J = 15.1, 10.6 Hz, H-4′), and 5.85 (dt, J = 15.1, 7.0 Hz, H-5′), and a n-pentyl moiety including four methylenes at δH 2.08 (H-6′), 1.37 (H-7′), and 1.27 (H-8′, 9′), and a terminal methyl group at δH 0.87 (t, J = 6.8 Hz, H3-10′). The location of the cinnamoyl moiety at C-12 was confirmed by the HMBC correlation from H-12 to C-1″. Thus, 15, 17, and 21 were determined as simplexin (15) [41], 12-O-(E)-cinnamoyl-9,13,14-ortho-(2E,4E)-decadienylidyne-5β,12β-dihydroxyresiniferonol-6α,7α-oxide (17) [40], and huratoxin (21) [42].
On the other hand, compounds 12, 18, and 20 belong to MDOs since the resonances of H-1 were observed as the methine proton at δH 2.11–2.97 and the methyl proton resonances at δH 0.79–1.26 of H3-10′ were observed as doublet (Table S2) [10]. The presence of benzoyl moieties of 12, 18, and 20 were determined by the aromatic proton resonances at δH 8.02–8.16 (H-2′,6′), 7.35–7.47 (H-3′,5′), and 7.53–7.59 (H-4′). The locations of benzoyl moieties at C-3, C-18, and C-7′ of 12, C-20 of 18, and C-3 of 20 were confirmed by the HMBC experiment. Thus, 12, 18, and 20 were determined as stelleralide G (12) [38], wikstromacrin (18) [20], and pimelea factor P2 (20) [41].

2.3. 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 (Figure 3 and Figure 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 B-ring. This observation suggested that both 3 and 16 were daphnane diterpenoids lacking a substituent at C-12 (Figure 3A and Figure 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 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 (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 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 C3H4O2 unit was consistently observed at m/z 269 (C17H17O3), indicating that these peaks corresponded to orthoester daphnanes with a substituent attached to C-12 (Figure 3B and Figure S24). The product ions corresponding to substituents observed in these peaks were assignable as follows: a cinnamoyl moiety at m/z 131 (C9H7O), a coumaroyl moiety at m/z 147 (C9H7O2), a feruloyl moiety at m/z 177 (C10H9O3), a 2E,4E-decadienoyl moiety at m/z 151 (C10H15O), 95 (C6H7O), and 81 (C5H5O), a 2E,4E,6E-decatrienoyl moiety at m/z 149 (C10H13O) and 107 (C9H9O), and a 2E,4E-dodecadienoyl moiety at m/z 179 (C12H19O), 95 (C6H7O), and 81 (C5H5O). Namely, the feruloyl and 2E,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,6E-decatrienoyl moieties in peak 14. In peak 19, only the product ions due to the 2E,4E-dodecadienoyl moiety were observed, but the molecular formula and the observation of product ions derived from the neutral loss of C2H4O2 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 1, 2, 4, 5, 7, 8, 11, 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 C30 to C28 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 C10H16O as in compounds 18 and 20. In addition, a series of C20 to C18 product ions were observed with successive losses of H2O and CO from m/z 327 (C20H23O4) (Figure 4A and Figure S25). Peak 7 was suggested to possess the cyclopentanone A-ring structure based on the degree of unsaturation and was identified as pimelea factor S6 (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 C20 to C18 product ions below m/z 350, as observed in 18 and 20 (Figure 4B and Figure S25). However, the neutral loss associated with the macrocyclic ring differed from 18 and 20, where it was C10H16O rather than C10H14O 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 C7H6O2 neutral loss originating from benzoyl acids and two molecules of C2H4O2 neutral loss originating from acetic acids, as well as the neutral loss of C10H10O associated with the macrocyclic ring. In addition, a series of C30 to C28 product ions were observed with successive losses of H2O and CO from m/z 507 (C30H35O7) (Figure 4C and Figure 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 C7H6O2, indicating the presence of two benzoyloxy moieties (Figure 4C and Figure S26). Additionally, the neutral loss associated with the macrocyclic ring was assigned to be C10H14O 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 C10H16O as in peak 7, suggesting that the daphnane skeleton was different from peak 7 (Figure 4D and Figure 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 C7H6O2 suggested the presence of a benzoyloxy moiety (Figure 4E and Figure 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.

3. Materials and Methods

3.1. General Experimental Procedures

The NMR spectra were collected on a JEOL ECA-500 spectrometer (JEOL Ltd., Tokyo, Japan) with the deuterated solvent used as the internal reference. The 1H-NMR spectra were performed at 500 MHz, and the 13C-NMR spectra were generated at 125 MHz. HRESIMS was conducted using a Q-Exactive Hybrid Quadrupole Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA). The following columns were utilized for column chromatography: Diaion HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan), ODS (Chromatorex DM1020T, Fuji Silysia Chemical Ltd., Aichi, Japan) and silica gel (Chromatorex PEI MB 100-40/75, Fuji Silysia Chemical Ltd., Aichi, Japan) columns. For gradient HPLC, two JASCO/PU-2080 Plus Intelligent HPLC pumps (JASCO Corporation, Tokyo, Japan), equipped with an MX-2080-32 dynamic mixer (JASCO Corporation, Tokyo, Japan), a JASCO UV-970 Intelligent UV/vis detector, and an SSC-6800 fraction collector (JASCO Corporation, Tokyo, Japan), were utilized. For preparative HPLC, a Waters 515 HPLC pump (Waters Corporation, Massachusetts, USA), equipped with an ERC RefractoMax520 differential refractometer detector (Thermo Scientific, Waltham, MA, USA) and a Shimadzu SPD-10A UV-vis detector (Shimadzu, Kyoto, Japan), was utilized. For normal-phase HPLC separations, a silica gel column (YMC-Pack SIL, 5 µm, 250 × 20 mm) was utilized with a flow rate of 5.0 mL/min. For reversed-phase HPLC separations, an RP-C18 silica gel column (YMC-Actus Triart C18, 5 µm, 150 × 20 mm) was utilized, with a flow rate of 8.0 mL/min.

3.2. 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.

3.3. Extraction and Isolation

The air-dried whole plants of W. indica (1000 g) were cut into small pieces and extracted with 95% EtOH at room temperature to give an EtOH extract and a residue. The EtOH extract was concentrated (63.0 g), suspended in H2O, and then partitioned with EtOAc. The EtOAc fraction (30.0 g) was subjected to Diaion HP-20 column chromatography, eluted with a stepwise gradient of MeOH/H2O (from 5:5 to 10:0, v/v) to afford three fractions (E1 to E3). The E3 fraction (10.8 g) was subjected to ODS column chromatography, eluted with a stepwise gradient of MeOH–H2O (from 7:3 to 10:0, v/v) to afford four subfractions (E3-1 to E3-4). Subfraction E3-2 (1840.3 mg) was subjected to silica gel column chromatography, eluted with a gradient of n-hexane–EtOAc–MeOH–HCOOH, to afford four subfractions (E3-2-1 to E3-2-5). Subfraction E3-2-3 (193.4 mg) was purified by RP-HPLC (70% CH3CN, 80% CH3CN) to give 12 (0.6 mg). Subfraction E3-2-2 (73.0 mg) was purified by RP-HPLC (85% CH3CN) to give six subfractions (E3-2-2-1 to E3-2-2-6). Subfraction E3-2-2-3 (16.6 mg) was purified by RP-HPLC (80% CH3CN), followed by NP-HPLC (n-hexane/AcOEt, 3:7) to give 15 (7.6 mg), 17 (1.6 mg), and 18 (1.3 mg). Subfraction E3-2-2-4 (4.4 mg) was purified by RP-HPLC (85% CH3CN) to give 20 (1.9 mg) and 21 (1.0 mg).

3.4. 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 ± 5 ppm mass tolerance: m/z 1028.4283 [M + NH4]+ (1), m/z 1028.4264 [M + NH4]+ (2), m/z 585 [M + H–H2O]+ (3), m/z 547.2888 [M + H]+ (4), m/z 655.3469 [M + H]+ (5), m/z 719.3062 [M + H]+ (6), m/z 533.3104 [M + H]+ (7), m/z 775.3690 [M + H]+ (8), m/z 691.3133 [M + H]+ (9), m/z 721.3203 [M + H]+ (10), m/z 775.3687 [M + H]+ (11), m/z 912.4147 [M + NH4]+ (12), m/z 667.3107 [M + H]+ (13), m/z 673.2999 [M + H]+ (14), m/z 533.3107 [M + H]+ (15), m/z 557.3109 [M + H]+ (16), m/z 675.3151 [M + H]+ (17), m/z 639.3527 [M + H]+ (18), m/z 643.3458 [M + H]+ (19), m/z 639.3525 [M + H]+ (20), and m/z 585.3409 [M + H]+ (21). All data collected in the profile mode were acquired and processed using Thermo Xcalibur 4.1 software.

4. 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.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants12203620/s1, Figure S1: Total ion chromatograms and extracted ion chromatograms in the positive ion mode from the (A) E3-2 fraction, (B) E3-3 fraction, and (C) E3-4 fraction; Tables S1 and S2: 1H- (500 MHz) and 13C- (125 MHz) NMR data of compounds 12, 15, 17, 18, 20, and 21 (CDCl3); Figures S2–S21: NMR spectra and HRESIMS data of compounds 12, 15, 17, 18, 20, and 21; Figures S22–S27: Product ion spectra of peaks 1–11 and 13–21 obtained from the protonated molecular ion peak in positive ion mode, and 12 obtained from the ammonium adduct ion peak positive ion mode.

Author Contributions

Conceptualization, W.L.; methodology, M.Z., K.O. and W.L.; formal analysis, M.Z. and K.O.; investigation, M.Z., R.T. and K.O.; resources, D.Z. and N.L.; writing—original draft preparation, M.Z. and K.O.; writing—review and editing, K.O., T.K., N.L. and W.L.; supervision, K.O., T.K. and W.L.; project administration, K.O. and W.L.; funding acquisition, M.Z. and W.L. All authors critically reviewed and revised the manuscript draft and approved the final version for submission. All authors have read and agreed to the published version of the manuscript.

Funding

The investigation was supported by the Japan Society for the Promotion of Science KAKENHI 21K06619 (W.L.). This work was also supported by the Sasakawa Scientific Research Grant from The Japan Science Society (No. 2022-3015) (M.Z.).

Data Availability Statement

All new research data were presented in this contribution.

Acknowledgments

All authors express their appreciation to the editorial board and reviewer for their suggestions and efforts in reviewing this paper. The authors also appreciate the editor for his assistance during the review process.

Conflicts of Interest

The authors declare no competing financial interest.

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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.
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.
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Figure 2. Structures of daphnane diterpenoids 1–21.
Figure 2. Structures of daphnane diterpenoids 1–21.
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Figure 3. Proposed ESI-MS/MS fragmentation pathways for minor daphnane diterpenoids in positive mode. (A) peaks 3 and 16, and (B) peaks 6, 9, 10, 14, and 19.
Figure 3. Proposed ESI-MS/MS fragmentation pathways for minor daphnane diterpenoids in positive mode. (A) peaks 3 and 16, and (B) peaks 6, 9, 10, 14, and 19.
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Figure 4. Proposed ESI-MS/MS fragmentation pathways for minor daphnane diterpenoids in positive mode. (A) peak 7, (B) peak 5, (C) peaks 8, 11, (D) peak 4, and (E) peak 13.
Figure 4. Proposed ESI-MS/MS fragmentation pathways for minor daphnane diterpenoids in positive mode. (A) peak 7, (B) peak 5, (C) peaks 8, 11, (D) peak 4, and (E) peak 13.
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Table 1. Daphnane diterpenoids 1–21 identified from the stems of W. indica.
Table 1. Daphnane diterpenoids 1–21 identified from the stems of W. indica.
No.Rt (min)Molecular
Formula		[M + H]+ (m/z)ESI-MS/MS (m/z) (%) cIdentification
Detected
Mass (m/z)		Error
(ppm)
1 a7.39C55H62O181011.3984−2.97793 (36), 775 (22), 765 (19), 747 (20), 731 (34), 703 (23), 689 (16), 671 (47), 659 (12), 653 (17), 643 (17), 625 (24), 609 (18), 567 (24), 549 (100), 531 (15), 521 (39), 507 (36), 503 (37), 493 (10), 489 (66), 479 (18), 477 (18), 471 (37), 461 (68), 459 (23), 443 (80), 433 (24), 431 (25), 425 (23), 415 (31), 403 (27), 397 (19), 375 (22), 363 (18), 339 (23), 325 (16), 307 (37), 295 (22), 291 (21), 279 (37), 263 (22), 221 (21), 183 (18), 181 (47), 153 (16), 141 (25), 105 (65)daphneodorin C [29]
2 a7.96C55H62O181011.3984−2.43793 (52), 775 (30), 765 (19), 747 (19), 731 (22), 707 (16), 689 (19), 671 (57), 653 (23), 643 (20), 629 (21), 625 (23), 611 (28), 593 (18), 583 (17), 549 (72), 531 (17), 521 (22), 507 (29), 503 (20), 489 (73), 479 (22), 471 (54), 461 (68), 453 (22), 443 (56), 425 (18), 415 (19), 375 (15), 363 (29), 307 (37), 291 (26), 279 (16), 221 (25), 181 (47), 163 (41), 141 (22), 105 (100)daphneodorin B [29]
3 a8.54C34H50O9603.35270.36585 (29), 361 (10), 343 (23), 325 (20), 315 (6), 313 (5), 307 (10), 297 (16), 295 (5), 279 (11), 271 (7), 269 (6), 267 (8), 253 (15), 207 (100), 203 (5), 107 (8), 95 (8), 81 (6)wikstroelide M [14]
4 a8.86C30H42O9547.2888−2.45529 (35), 511 (30), 501 (32), 493 (27), 483 (59), 467 (29), 465 (64), 449 (21), 447 (29), 439 (25), 437 (40), 423 (23), 421 (25), 419 (29), 405 (28), 395 (20), 341 (27), 323 (24), 295 (26), 283 (29), 255 (29), 239 (21), 236 (23), 235 (100), 233 (47), 227 (22), 215 (36), 203 (28), 199 (21), 193 (24), 187 (27), 161 (29), 135 (36), 133 (33)pimelotide C [34]
58.88C37H50O10655.3469−1.24619 (10), 515 (28), 497 (79), 479 (92), 469 (38), 467 (14), 461 (21), 451 (100), 443 (16), 439 (11), 433 (71), 423 (24), 421 (12), 415 (20), 405 (27), 367 (12), 311 (19), 293 (45), 275 (16), 265 (31), 263 (10), 251 (14), 247 (10), 225 (10), 211 (16), 133 (18), 123 (11), 105 (77)kraussianin [35]
6 a9.15C40H46O12719.3062−1.33507 (3), 489 (4), 359 (4), 341 (12), 323 (18), 311 (3), 305 (4), 295 (12), 277 (4), 269 (8), 177 (75), 149 (95), 121 (11), 107 (100), 81(3)acutilobin C [36]
7 a9.15C30H44O8533.3104−1.00515 (36), 497 (66), 479 (100), 469 (28), 467 (16), 461 (66), 451 (53), 449 (25), 443 (20), 433 (59),425 (24), 423 (13), 421 (16), 415(21), 407 (14), 405 (21), 403 (16), 309 (14), 291 (12), 281 (13), 263 (12), 211 (13), 187 (19), 185 (16), 159 (13), 135 (13), 133 (27)pimelea factor S6 [37]
8 a9.32C44H54O12775.36900.20635 (15), 617 (21), 599 (22), 545 (63), 527 (33), 495 (47), 477 (85), 459 (100), 449 (21), 447 (28), 441 (37), 431 (50), 429 (20), 423 (90), 419 (18), 413 (29), 405 (24), 401 (23), 319 (18), 309 (24), 291 (24), 281 (18), 279 (23), 263 (19), 251 (18), 151 (27), 105 (88)stelleralide H [38]
9 a9.77C39H46O11691.3133−2.63509 (4), 505 (2), 491 (5), 359 (7), 341 (20), 323 (21), 313 (4), 311 (3), 305 (3), 297 (2), 295 (13), 277 (4), 269 (9), 267 (3), 261 (3), 151 (100), 147 (75), 133 (3)daphneodorin D [29]
10 a10.00C40H48O12721.3203−1.60509 (4), 491 (7), 359 (6), 341 (22), 323 (23), 313 (3), 311 (3), 305 (5), 295 (16), 277 (4), 269 (11), 267 (4), 261 (3), 177 (95), 151 (100), 95 (6), 81 (6)acutilobin D [36]
11 a10.00C44H54O12775.3687−0.09563 (28), 545 (100), 513 (35), 495 (60), 477 (41), 467 (45), 465 (24), 449 (78), 441 (16), 437 (35), 431 (34), 425 (20), 423 (20), 421 (24), 419 (19), 391 (18), 309 (17), 291 (19), 263 (19), 105 (40)gnidimacrin [39]
12 b10.39C51H58O14912.4147−1.98773 (48), 651 (47), 633 (58), 615 (34), 543 (100), 511 (57), 493 (82), 481 (20), 475 (58), 465 (54), 463 (36), 447 (90), 435 (43), 429 (38), 421 (47), 419 (21), 417 (22), 327 (23), 309 (40), 291 (26), 279 (22), 105 (55)stelleralide G [38]
13 a10.59C37H46O11667.3107−0.93545 (100), 527 (66), 509 (59), 499 (24), 491 (37), 483 (26), 481 (52), 465 (55), 463 (37), 453 (23), 447 (28), 445 (31), 435 (35), 419 (21), 417 (21), 357 (23), 321 (27), 295 (22), 235 (96), 231 (61), 203 (54), 185 (27), 173 (20), 153 (21), 105 (45)stelleralide C [5]
14 a10.86C39H44O10673.2999−1.27359 (1), 341 (3), 323 (4), 295 (3), 277 (1), 269 (2), 149 (100), 131 (6), 107 (21)12-O-(E)-cinnamoyl-9,13,14-ortho-(2E,4E,6E)-decatrienylidyne-5β,12β-dihydroxyresiniferonol-6α,7α-oxide [40]
15 b11.34C30H44O8533.3107−0.23361 (9), 343 (24), 325 (49), 307 (35), 297 (28), 279 (30), 267 (58), 253 (100), 203 (16), 155 (6)simplexin [41]
1611.44C32H44O8557.3109−0.08361 (8), 343 (23), 325 (33), 313 (7), 307 (30), 297 (19), 295 (11), 285 (6), 279 (20), 277 (6), 267 (55), 261 (6), 253 (77), 251 (7), 249 (11), 225 (6), 179 (100)wikstrotoxin B [11]
17 b11.68C39H46O10675.3151−1.85675 (9), 509 (2), 507 (2), 491 (2), 359 (5), 341 (15), 323 (21), 313 (4), 311 (3), 305 (4), 295 (13), 277 (5), 269 (11), 267 (3), 265 (2), 261 (2), 249 (2), 241 (2), 237 (2), 209 (2), 151 (100), 133 (4)12-O-(E)-cinnamoyl-9,13,14-ortho-(2E,4E)-decadienylidyne-5β,12β-dihydroxyresiniferonol-6α,7α-oxide [40]
18 a12.21C37H50O9639.3527−0.10621 (12), 499 (30), 481 (93), 463 (100), 453 (32), 445 (35), 436 (23), 435 (57), 417 (34), 407 (15), 311 (5), 293 (16), 265 (23), 105 (67)wikstromacrin [20]
19 a12.78C36H50O10643.3458−2.97365 (2), 359 (4), 341 (11), 323 (11), 313 (3), 305 (2), 295 (10), 277 (4), 269 (6), 267 (3), 207 (100), 189 (3), 107 (3)wikstroelide A [13]
20 b12.99C37H50O9639.3525−0.28621 (16), 499 (28), 481 (73), 463 (100), 453 (31), 451 (12), 445 (55), 435 (55), 433 (14), 423 (12), 417 (34), 409 (12), 407 (10), 405 (16), 311 (13), 293 (24), 275 (12), 265 (24), 251 (12), 133 (12), 105 (99)pimelea factor P2 [41]
21 b13.27C34H48O8585.3409−2.14361 (14), 343 (20), 325 (41), 313 (10), 307 (25), 297 (23), 295 (13), 279 (25), 267 (59), 253 (99), 249 (16), 207 (100)huratoxin [42]
a Identifications were confirmed with the daphnane diterpenoids isolated in our previous studies. b Isolated daphnane diterpenoids in this study. c ESI-MS/MS of [M + H]+ ion for peaks 1–11 and 13–21 and ESI-MS/MS of [M + NH4]+ ion for peak 12.
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Zhang, M.; Otsuki, K.; Takahashi, R.; Kikuchi, T.; Zhou, D.; Li, N.; Li, W. Identification of Daphnane Diterpenoids from Wikstroemia indica Using Liquid Chromatography with Tandem Mass Spectrometry. Plants 2023, 12, 3620. https://doi.org/10.3390/plants12203620

AMA Style

Zhang M, Otsuki K, Takahashi R, Kikuchi T, Zhou D, Li N, Li W. Identification of Daphnane Diterpenoids from Wikstroemia indica Using Liquid Chromatography with Tandem Mass Spectrometry. Plants. 2023; 12(20):3620. https://doi.org/10.3390/plants12203620

Chicago/Turabian Style

Zhang, Mi, Kouharu Otsuki, Reo Takahashi, Takashi Kikuchi, Di Zhou, Ning Li, and Wei Li. 2023. "Identification of Daphnane Diterpenoids from Wikstroemia indica Using Liquid Chromatography with Tandem Mass Spectrometry" Plants 12, no. 20: 3620. https://doi.org/10.3390/plants12203620

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