Guided Isolation of Isopentenyl Flavonoids from Daphne giraldii Based on the Combination of GNPS and SMART

Abstract

Six flavonoids (1–6), including 3 previously undescribed compounds (1–3), were isolated from the dried roots and stem skins of Daphne giraldii Nitsche. The strategy of LC-tandem mass spectrometry-based Global Natural Products Social Molecular Networking (GNPS) molecular network technology and NMR-based Small Molecule Accurate Recognition Technology (SMART) technology facilitated the precise separation of isopentenyl flavonoids in D. giraldii. The structures were determined through comprehensive spectroscopic analysis. Furthermore, all compounds were evaluated for their cytotoxic activity against Hep3B cells. Specifically, compounds 1 and 3 exhibited significant cytotoxicity with IC₅₀ values of 5.52 ± 0.57 and 2.53 ± 0.49 μM, respectively, compared to the positive control sorafenib (IC₅₀ = 7.08 ± 0.23 μM).

Introduction

Daphne giraldii Nitsche (D. giraldii), a member of the Thymelaeaceae family, is a widely distributed ethnic medicine used in China for managing pain, rheumatoid arthritis, and numbness of the limbs.^1–5) Our research group previously isolated and identified a series of isopentenyl flavones from D. giraldii, among which Daphnegiravone D (DGD) exhibits significant antitumor activity by inducing apoptosis and reactive oxygen species generation through targeting ataxia telangiectasia and Rad3-related protein in hepatocellular carcinoma cells (HCCs).^2,3,6–10)

GNPS (https://gnps.ucsd.edu), as a pivotal tool for the comprehensive analysis of mass spectrometry data, facilitates the rediscovery and characterization of known natural products.^11–13) This cheminformatics tool, created in 2012, has become very popular for visualizing and annotating aligned MS2 spectra to connect related molecules based on similarity scores.^13–15) The Small Molecule Accurate Recognition Technology (SMART) (https://smart.ucsd.edu/classic), first proposed in 2017, is an automated annotation system that demonstrates a distinct advantage in leveraging nonuniform sampling in heteronuclear single quantum coherence (HSQC) alongside deep convolutional neural networks, providing robust mechanisms for accurate recognition of molecular structures.^11,14,15) The integration of GNPS technology and SMART technology facilitates the rapid assessment of the chemical composition of each primary fraction, as well as the identification of the principal chemical constituents in the secondary fraction associated with the target fraction.

To isolate the isopentenyl flavonoid components from the primary fractions of D. giraldii, GNPS technology was employed for chemical component identification in the 4 primary fractions, ultimately identifying the target fraction. SMART technology was utilized to target regions rich in isopentenyl flavonoids for further analysis precisely and to isolate the isopentenyl-rich flavonoid fractions from the 7 secondary fractions isolated from the target primary fraction. Additional separation led to 6 isopentenyl flavonoids (1–6). Their structures were elucidated using a variety of spectroscopic techniques. The cytotoxic activity against the human HCC line Hep3B was evaluated in this investigation.

Experimental

General Experimental Procedures

UV spectra were measured using a Shimadzu UV-2600 spectrometer (Shimadzu, Kyoto, Japan). The optical rotations were recorded using an Anton Paar MCP 200 polarimeter (Anton Paar, Graz, Austria). High resolution electrospray ionization (HR-ESI) data were acquired using a Micro Q-TOF spectrometer (Bruker Daltonics, Billerica, MA, U.S.A.). The NMR spectra were recorded using Bruker AV-600 spectrometers in dimethyl sulfoxide (DMSO)-d₆, with tetramethylsilane (TMS) as the internal standard. Chromatographic silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and reversed-phase C18 silica gel (50 μm, YMC Company, Kyoto, Japan) were employed for column chromatography (CC). HPLC separations were performed on a Shimadzu L6AD series pumping system equipped (Shimadzu) with an SPD-20A UV detector and performed with a YMC Pack ODS-A column (250 × 10 mm, 5 μm, YMC Company). All solvents for extraction and chromatography were commercially purchased. The cells were cultured in a constant temperature CO₂ incubator (Thermo Fisher Scientific, Waltham, MA, U.S.A.). The inhibitory activities were tested on a Varioskan Flash (Bio-Rad Model 680, Thermo Fisher Scientific).

Plant Material

The dried root and stem skins of D. giraldii were collected in October 2019 from Gansu province, China. A voucher specimen of this species was deposited in the herbarium of the Nature Products Laboratory (under the accession number Dg-20191022).

Extraction and Isolation

Dried stem and root skins of D. giraldii (100 kg) were extracted with 70% EtOH (3 × 75 L) under reflux 3 times. The EtOH extrwact (10.7 kg) was partitioned sequentially with ethyl acetate and n-BuOH. The EtOAc extract (1.2 kg) and the n-BuOH (2.7 kg) extract were combined and subjected to a silica gel column and eluted with CH₂Cl₂/MeOH (100 : 1 to 1 : 1, v/v) of increasing polarity to produce 4 fractions (Frs. A–D). After analysis with GNPS, GNPS is directed to Fr. A. Fr. A was purified using CC on HP-20 macroporous resin, followed by separation of 20–90% EtOH fractions through ODS CC with elution using a 20–90% EtOH-H₂O gradient it was fractionated into 7 components, which are Fr. A1 (10.5 g), Fr. A2 (14.5 g), Fr. A3 (12.5 g), Fr. A4 (18.0 g), Fr. A5 (20.4 g), Fr. A6 (22.7 g), and Fr. A7 (15.8 g). The SMART guidance should be utilized to identify the 7 components corresponding to Fr. A5 (20.4 g). Fr. A5 was purified through preparative HPLC (MeOH/H₂O, 75 : 25, v/v) to give A5.1 (137 mg), Fr. A5.2 (134 mg), Fr. A5.3 (210 mg), Fr. A5.4 (290 mg), and Fr. A5.5 (104 mg). Compounds 1–6 were isolated from Fr. A5.1, Fr. A5.2, Fr. A5.3, Fr. A5.4, and Fr. A5.5. Compound 1 (48.6 mg, t_R = 37.9 min) was obtained from Fr. A5.1 by semi-preparative HPLC eluted with CH₃CN-H₂O (38 : 62, v/v). Compound 2 (33.9 mg, t_R = 39.6 min) was isolated from Fr. A5.2 by RP-HPLC (CH₃CN-H₂O, 25:75, v/v). Compound 3 (30.4 mg, t_R = 35.7 min) was obtained from Fr. A5.2 by RP-HPLC (CH₃CN-H₂O, 27 : 78, v/v). Compound 4 (50.5 mg, t_R = 37.9 min) was obtained from Fr. A5.3 by semi-preparative HPLC eluted with CH₃CN-H₂O (27 : 73, v/v).^16,17) Compound 5 (47.5 mg, t_R = 44.3 min) was isolated from Fr. A5.4 by RP-HPLC (CH₃CN-H₂O, 25 : 75, v/v).¹⁸⁾ Compound 6 (15.7 mg, t_R = 34.2 min) was obtained from Fr. A5.5 by RP-HPLC (CH₃CN-H₂O, 27: 78, v/v).¹⁹⁾

Broussoflavonol C (1): Yellow amorphous powder; UV (MeOH) λ_max (log _ε) 208 (2.76), 277 (2.55), 334 (2.46) nm; ¹H- and ¹³C-NMR data see Tables 1 and 2; HR-ESI-MS: m/z 437.1972 [M +H]⁺ (calcd for C₂₆H₂₉O₆: 437.1959).

Table 1. ¹H-NMR Data for Compounds 1–3 in DMSO-d₆

No.	1	2	3	No.	1	2	3
2	—	4.55, d (7.9)	4.89, dd (10.4, 2.3)	3ʹʹ	—	5.73, d (9.8) 6.38, d (9.8)	—
3	—	3.90, m	2.14, 2.06, m	4ʹʹ	1.62, s		—
4	—	2.81, 2.63, m	2.89, 2.73, m	5ʹʹ	1.73, s	1.37, s	1.42, s
5	—	6.84, d (8.2)	6.93, d (8.2)	6ʹʹ	—	1.37, s	1.42, s
6	—	6.29, dd (8.2, 8.4)	6.38, overlap	1ʹʹʹ	3.49, d (6.5)	3.19, d (7.4)	3.28, d (7.4)
7	—	—	—	2ʹʹʹ	5.11, t (6.5)	5.21, t (7.4)	5.29, t (7.4)
8	—	6.17, d (2.4)	6.39, s	3ʹʹʹ	—	—	—
2′	7.91, d (8.9)	6.97, d (2.0)	6.89, d (2.1)	4ʹʹʹ	1.62, s	1.66, s	1.73, s
3′	6.93, d (8.9)	—	—	5ʹʹʹ	1.71, s	1.67, s	1.73, s
4′	—	—	—	6ʹʹʹ	—	—	—
5′	6.93, d (8.9)	—	—	3-OCH3	3.77, s	—	—
6′	7.91, d (8.9)	6.91, d (2.0)	7.01, d (2.1)	5-OH	12.91, br s	—	—
1ʹʹ	3.30, d (6.9)	—	6.31, d (9.8)	OH groups	4.71, s
2ʹʹ	5.14, t (6.9)	—	5.61, d, (9.8)

Table 2. ¹³C-NMR Data for Compounds 1–3 in DMSO-d₆

No.	1	2	3	No.	1	2	3
2	155.1	81.3	78.1	6′	129.9	123.2	127.5
3	137.4	66.2	30.0	1ʹʹ	21.4	—	122.7
4	177.8	33.1	24.8	2ʹʹ	122.6	75.9	130.9
5	155.9	130.0	103.3	3ʹʹ	130.5	130.9	76.3
6	111.4	108.2	107.9	4ʹʹ	25.4	122.2	—
7	160.1	156.6	154.9	5ʹʹ	17.8	27.6	28.2
8	106.2	102.2	103.7	6ʹʹ	—	27.6	28.1
9	151.8	154.6	156.2	1ʹʹʹ	21.7	27.9	28.5
10	103.8	111.2	114.4	2ʹʹʹ	123.0	122.6	122.8
1′	121.0	131.2	133.2	3ʹʹʹ	130.7	131.2	132.1
2′	129.9	128.6	122.1	4ʹʹʹ	25.5	25.5	25.9
3′	115.6	127.7	121.0	5ʹʹʹ	17.9	17.8	18.0
4′	160.1	149.5	150.4	6ʹʹʹ	—	—	—
5′	115.6	120.3	129.3	3-OCH3	59.6	—	—

Daphnegiranol G (2): Yellow oily solid; ${\left[\alpha \right]}_D^{20}$ −110.6 (c 1, MeOH); circular dichroism (CD) (MeOH) nm (Δε) 240 (+1.64), 280 (−7.04); UV (MeOH) λ_max (log ε) 206 (2.66), 228 (0.82) nm; ¹H- and ¹³C-NMR data see Tables 1 and 2; HRESIMS: m/z 415.1880 [M + Na]⁺ (calcd for C₂₅H₂₈NaO₄: 415.1878).

Daphnegiranol E (3): Yellow oily solid; ${\left[\alpha \right]}_D^{20}$−26.4 (c 0.5, MeOH); CD (MeOH) nm (Δε) 280 (−3.35); UV (MeOH) (log _ε) 216 (2.20), 227 (2.12) nm; ¹H- and ¹³C-NMR data see Tables 1 and 2; HR-ESI-MS: m/z 399.1917 [M + Na]⁺ (calcd for C₂₅H₂₈NaO₃: 399.1931).

Electronic Circular Dichroism (ECD) Calculations

The conformational analysis of 2 and 3 was conducted using Spartan software with the Molecular Merck Force Field (MMFF94).^5,6) These conformers were optimized at the B3LYP/6-31G(d) level. The optimized conformers were calculated using the time-dependent density-functional theory method at the B3LYP/6-311++G(2d,p) level. The calculated ECD curves were simulated using SpecDis 1.51 software.

Cell Culture

The Hep3B cell line was obtained from the American Type Culture Collection (U.S.A.). The cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone, U.S.A.) supplemented with 10% fetal bovine serum (Biological Industries, Israel) in a 5% CO₂ incubator at 37°C.

Growth Inhibition Assay

Growth inhibition was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Ameresco, Framingham, MA, U.S.A.) assay with compounds in 96-well plates at a density of 5 × 10³ cells per well in medium and allowed to attach for about 12 h. Cells were treated with a range of concentrations (12.5, 25, 50, and 100 μM). After 48 h, 20 μL of MTT was added to the wells, and the cells were incubated for another 4 h at 37°C. The medium was removed, and DMSO (150 μL/well) was added to each well. Cell viability was measured at 490 nm. All tests needed 3 separate experiments.

Statistical Analysis

All results and data were confirmed in at least 3 separate experiments. SPSS (Statistical Package for the Social Sciences) software was used to determine significant differences via ANOVA followed by Student’s t-test. The data were expressed as means ± standard deviation, and values of p < 0.05 were considered statistically significant.

Results and Discussion

Molecular Networking-Guided of Isopentenyl Flavonoids

To identify isopentenyl flavonoids in D. giraldii, Frs. A–D (as described in “Extraction and Isolation”) were characterized using LC-tandem mass spectrometry (LC-MS/MS) analysis. The obtained LC-MS/MS data were subsequently uploaded to the GNPS platform and subjected to molecular network analysis for precise identification of flavonoid-containing fractions within the 4 primary fractions.⁵⁾ The resulting data (https://cytoscape.gnps2.org/process?task=26cde62de52b49a9a82070ca4e21bb64) were then uploaded to the GNPS web platform and visualized using Cytoscape 3.7.1 (Fig. 1). Our findings revealed that a majority of the flavonoids were present in Fr. A (represented by orange nodes in the network) and Fr. B (represented by green nodes in the network), prompting us to conduct further comprehensive investigations on the Fr. A fraction.

Fig. 1. The Molecular Network of D. giraldii

Fraction A was subsequently subjected to chromatography, resulting in the isolation of 7 fractions (Frs. A1–7). The data from HSQC spectra of Frs. A1–7 were collected and settled as CSV files, which were subsequently uploaded into the SMART system (https://smart.ucsd.edu/classic) to recognize potential structure types within Fr. A5.^7,20) The generated structure hypotheses revealed that 3 out of the top 4 cosine similarity score structures analyzed by SMART belonged to the isopentenyl flavonoids category (Fig. 2). Based on the SMART analysis results, it was inferred that Fr. A5 might contain a significant amount of isopentenyl flavonoids. Subsequently, 6 isopentenyl flavonoids (Fig. 3) were isolated from Fr. A5, and their structures were determined.

Fig. 2. SMART-Based Prioritization of the Isolation and the SMART Result for Fr. A5

Three of the top 4 structures based on cosine similarity score.

Fig. 3. The Structures of Compounds 1–6

Structural Elucidation of New Compounds 1–3

Compound 1, a yellow amorphous powder (methanol), HR-ESI-MS: m/z 437.1972 [M + H]⁺ (calcd. for C₂₆H₂₉O₆: 437.1959), was assigned the molecular formula of C₂₆H₂₉O₆ by ¹H- and ¹³C-NMR, requiring 13 degrees of unsaturation. The ¹H-NMR data of 1 (Table 1) showed the signals for the set of AA′BB′-coupled aromatic proton signals (H-2ʹ, H-6ʹ, H-3ʹ, and H-5ʹ), as well as 2 groups of isopentenyl substituent proton signals (H-1ʹʹʹ, H-1ʹʹ, H-2ʹʹ, H-2ʹʹʹ, H-4ʹʹʹ, H-5ʹʹ, and H-5ʹʹʹ). The ¹³C-NMR data of compound 1 (Table 2) revealed the presence of 26 carbon resonances, which were identified as 15 aromatic carbon signals originating from the flavonoid stem nucleus, along with 2 sets of isopentenyl carbon signals (C-1ʹʹ, C-1ʹʹʹ, C-2ʹʹ, C-2ʹʹʹ, C-3ʹʹ, C-3ʹʹʹ, C -4ʹʹ, C-4ʹʹʹ, C-5ʹʹ, and C-5ʹʹʹ). The elucidation of the compound's gross structure was accomplished by key heteronuclear multiple bond connectivity (HMBC) correlations (Fig. 4) from H-2ʹ and H-6ʹ to C-2 and C-4ʹ, and from H-3ʹ and H-5ʹ to C-1ʹ and C-4ʹ, suggesting that the B ring in the flavonoid possessed an AAʹBBʹ coupling system.²¹⁾ The long-range correlation observed between H-OCH₃ and C-3 suggests that the methoxy group is substituted at the 3-position of the flavonoid nucleus. The correlation from H-1ʹʹ to C-5/C-6/C-7 indicated the isopentenyl substitution on the C-6 position. The correlation from H-1ʹʹʹ to C-7/C-8/C-9 indicated isopentenyl substitution in the C-8 position.²²⁾ The integration of mass spectrometry data indicates that the 5ʹ, 7ʹ, and 4ʹ positions are all modified with hydroxyl groups. In conclusion, based on these findings, compound 1 was successfully identified (Fig. 3) and named broussoflavonol C.

Fig. 4. Key HMBC Correlations of Compounds 1–3

Compound 2, a yellow oily solid (methanol), was assigned the molecular formula of C₂₅H₂₈O₄ by the HR-ESI-MS ion at m/z 415.1880 [M + Na]⁺ (calcd. C₂₅H₂₈O₄Na: 415.1878), requiring 12 degrees of unsaturation. The ¹H-NMR data of 2 (Table 1) displayed the presence of flavone-3-alcohol parent nucleus signals (H-2, H-3, and H-4).²¹⁾ At the same time, the larger coupling constant of H-2 (J_2,3 = 7.9 Hz) indicates that H-2 and H-3 are in a trans-configuration.^22–24) Additionally, the 2ʹʹ, 2ʹʹ-dimethylpyran group signals (H-3ʹʹ, H-4ʹʹ, H-5ʹʹ, and H-6ʹʹ) and an isopentenyl group signal (H-1ʹʹʹ, H-2ʹʹʹ, H-4ʹʹʹ, and H-5ʹʹʹ) were observed. The ¹³C-NMR spectrum (Table 2) showed 25 carbon signals. The elucidation of the compound's gross structure was accomplished by key HMBC correlations (Fig. 4) from H-3 to C-2/C-10, indicating that the hydroxyl substitution occurs at position 3, suggesting that the parent nucleus of the compound is flavan-3-ol. In addition, the 2ʹʹ, 2ʹʹ-dimethylpyran group was located at C-4ʹ and C-5ʹ by the HMBC interactions from H-6ʹ to C-4ʹ/C-4ʹʹ, H-4ʹʹ to C-4ʹ/C-5ʹ/C-6ʹ.^25,26) The HMBC correlation observed from H-1‴ to C-2ʹ, C-3ʹ, and C-4ʹ indicated the isopentenyl group was located at C-3ʹ.^25,27) To determine the absolute configuration of compound 2, the CD was measured. The results showed a positive Cotton effect at 240 nm and a negative Cotton effect at 280 nm, indicating that the absolute configurations are 2R and 3S.²⁴⁾ The calculated ECD curve for 2 matched well with the experimental one (Fig. 5), implying that its absolute configuration is 2R, 3S. Thus, the gross structure of 2 was determined (Fig. 3) and named daphnegiranol G.

Fig. 5. Calculated and Experimental ECD Spectra of 2 and 3 in MeOH

Compound 3, a yellow oily solid (methanol), was assigned the molecular formula of C₂₅H₂₈O₃, as measured by HR-ESI-MS m/z 399.1917 [M + Na]⁺ (calcd. for C₂₅H₂₈O₃Na: 399.1931). The examination of the ¹H- and ¹³C-NMR spectra showed that 3 was similar to those of 2 (Table 1), except for the lack of a hydroxyl in C-3 [δ_H 2.14 2.06 (2H, H-3); δ_C 30.0 (C-3)]. HMBC was used to further elucidate the configuration of compound 3 (Fig. 4). The calculated ECD curve for 3 matched well with the experimental one (Fig. 5), implying that its absolute configuration is 2R. Thus, compound 3 was determined and named daphnegiranol E.

The structure elucidation of the known compounds 4–6 was carried out by a combination of spectroscopic methods (¹H- and ¹³C-NMR) and comparison with published data. Therefore, compounds 4–6 were determined to be cathayanon G (4),²⁸⁾ daphnegiravonol A (5),⁷⁾ and isolicoflavonol (6).²⁵⁾

Cytotoxicity Assay

All isolates were assessed for their inhibitory effects (Table 3) against the human hepatoma cell line Hep3B. The cytotoxic effect of compound 3 against Hep3B cells was found to be the most potent, with an IC₅₀ value of 2.53 μM. A comparison between compounds 2 and 3 reveals a significant attenuation in the cytotoxic activity of flavone-3-ol upon substitution of the 3 hydroxyl groups on the flavan nucleus.^29–31)

Table 3. Cytotoxic [IC₅₀ (μM]] Activities of Isolated Compounds against Hep3B Cell

No.	IC50	No.	IC50
1	5.52 ± 0.57	5	>25
2	>25	6	8.43 ± 0.56
3	2.53 ± 0.49	Sorafenib	7.08 ± 0.23
4	>25

Conclusion

In summary, 6 flavonoids (1–6), including 3 previously undescribed compounds (1–3), were isolated from the dried root and stem skins of D. giraldii. Their structures were determined through a combination of spectroscopic techniques. All compounds underwent evaluation for their cytotoxic activity against Hep3B cells. Among them, compounds 1, 3, and 6 exhibited moderate cytotoxic activities. This experimental method integrates core structural skeleton-related NMR data and the high-sensitivity LC-MS/MS analytical technique, along with structural information from the fractions obtained, to achieve precise and targeted identification of potential compounds. A combination of faster isolation speed and better targeting performance is realized by our integrated strategy, which enables the isolation of novel compounds.

Acknowledgments

This work was supported by the Innovation Team and Talents Cultivation Program of the National Administration of Traditional Chinese Medicine (No.: ZYYCXTD-D-202208) and the Song Shaojiang Expert Workstation of Yunnan Province (No. 202305AF150030).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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