Bioassay and UPLC-Q-Orbitrap-MS/MS guided isolation of polycyclic polyprenylated acylphloroglucinols from St. John's wort and their neuroprotective activity

2.1.1 Chemical

We purchased analytical grade petroleum ether, ethyl acetate, dichloromethane, acetone, and methanol from General-reagent Corporation (Shanghai, China). High-performance liquid chromatography (HPLC) grade acetonitrile, methanol, and water were purchased from Xinlanjing International Corporation (Pennsylvania, USA). Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were obtained from GIBCO Invitrogen Corporation (Carlsbad, USA). DMSO, streptomycin, penicillin, corticosterone (CORT), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were bought from Sigma-Aldrich (USA).

2.1.2 General experimental procedures

Optical rotation was measured on Autopol-VI for compound 2 at 20 °C. Infrared radiation (IR) spectrum of compound 2 was obtained on a Nicolet Is10 FT-IR spectrometer with KBr pellets. A Shimadzu UV-2401PC spectrometer was applied to record ultraviolet (UV) spectrum of compound 2 at room temperature. An Applied Photophysics Chirascan spectrometer was used to acquire Circular Dichroism (CD) spectrum of compound 2. ¹H (600 MHz) and ¹³C (150 MHz) NMR (nuclear magnetic resonance) spectra for compounds 1–10 were recorded on a Bruker Avance III 600 MHz spectrometer with TMS as internal standard. The HMQC (heteronuclear multiple quantum correlation), HMBC (heteronuclear multiple bond correlation), ¹H−¹H COSY (correlation spectroscopy), and ROESY (rotating frame overhauser effect spectroscopy), spectra for compound 2 were also acquired on a Bruker Avance III 600 MHz spectrometer. High resolution electrospray ionization mass spectroscopy (HR-ESI-MS) data of compound 2 was acquired on an Agilent 6500 QSTAR Q-Exactive Orbitrap mass spectrometer. Semi-preparative HPLC was carried out on a Waters-C600 with a Waters C18 column (150 mm × 10 mm, 10 µm). Column chromatography was performed using silica gel (40–80, 200–300, and 300–400 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, People’s Republic of China), MCI gel (75–150 μm, Mitsubishi Chemical Corporation, Japan), and Sephadex LH-20 (25–100 μm, Amersham Biosciences, Sweden), respectively.

2.1.3 Plant material

The aerial parts of H. perforatum were collected in 2017 from Leishan county, Guizhou Province, China. The plant was identified by Dr. Wei Gu, and a voucher specimen was deposited in the Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, Guiyang, China (the voucher specimen: No. 20170321).

2.2.1 UPLC-Q-Orbitrap-MS/MS

The chromatographic analysis was performed on a Thermo Scientific Dionex (Sunnyvale, USA) Ultimate 3000 UHPLC system equipped with an Ultimate 3000 Pump, an Ultimate 3000 Degasser, an Ultimate 3000 RS Autosampler, and an Ultimate 3000 RS column compartment. The samples were separated using a Hypersil GOLDTM C18 column (3.0 μm, 2.1 × 150 mm; Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase consisted of methanol (A) and water (B). The flow rate was 0.2 mL/min, the column temperature was set at 40 °C, and the total run time was 30 min. An isocratic mode of 70% phase A was applied for 0 ∼ 3 min, followed with a linear gradient from 70% to 100% phase A between 3 and 25 min, and 100% phase A was maintained over the last 5 min.

Mass spectrometry with an electrospray ionization source (ESI) was conducted in the positive mode. High-resolution ESI-MS/MS experiments were performed on a Q-Exactive Orbitrap mass spectrometer equipped with an ESI source controlled by the Xcalibur 2.3 software (Thermo Fisher, Waltham, MA, USA). The mass resolution was 17,500 FWHM (full width at half maximum) at 200. Argon was used as the collision gas, and high-purity nitrogen gas was used as the nebulizer and drying gas was set at a flow rate of 4 L/min and a pressure of 0.3 bar, respectively. The heated electrospray ion source was used for the ionization with the interface conditions were as follows: spray voltage, 3.0 kV; and capillary temperature, 320 °C; sheath gas flow rate, 35 units; auxiliary gas flow rate, 10 units; and S lens RF level 50.

2.2.2 Isolation and purification

The air-dried aerial parts (8.8 kg) of SJW were smashed to powders (10–50 mesh), which were further percolated extract with MeOH at room temperature for three times (40 L × 3) and then filtered. The combined MeOH extracts were concentrated under vacuum to give a crude residue (495.0 g), which was suspended in water and then partitioned successively with ethyl acetate (EA) to get the corresponding extracts: EA (150.0 g) and water (WT) (300.0 g) parts. Both EA and WT fractions were screened for the neuroprotective activity (Fig. 1). The active EA fraction was further separated by silica gel column chromatography and eluted with petroleum ether-ethyl acetate (from 1:0 to 0:1) to obtain a high content PPAP (1, 5 g) and eight fractions (Frs. A–H). This known PPAP (1) was an active PPAP after biological test.

Compound 1 was used as a referenced compound and applied to UPLC-Q-Orbitrap-MS. Eight fractions (Frs. A–H) were further analysed using UPLC-Q-Orbitrap-MS. Fraction A of EA extract might have PPAPs according to the results of UPLC-Q-Orbitrap-MS analysis. Therefore, Fr. A (65.1 g) was subjected to column chromatography and eluted with petroleum ether-ethyl acetate (100:1 to 1:1) to obtain seven fractions (Fr. A1–A7). Fr. A3 was further purified by semi-preparative HPLC (MeOH-H₂O, 95:5) to yield compounds 9 (5.2 mg, t_R = 32 min) and 10 (16.3 mg, t_R = 28.4 min). Fr. A4 (32.5 g) was applied to a silica gel column eluted with petroleum ether-acetone (50:1 to 1:1) to yield compound 3 (1.0 g) and five fractions (Frs. A4A–A4E). Fr. A4C (4.8 g) was purified by semi-preparative HPLC (CH₃CN-H₂O, 90:10) to obtain compounds 2 (3.6 mg, t_R = 19.6 min) and 4 (5.2 mg, t_R = 22.8 min). Fr. A5 (10 g) was then separated over an MCI-gel column (MeOH-H₂O from 7:3 to 10:0) to yield seven fractions (Frs. A5A–A5G). Fr. A5G (116 mg) was further purified by semipreparative HPLC (MeOH-H₂O, 85:15) to afford compounds 5 (9.4 mg, t_R = 34.1 min), 6 (11.3 mg, t_R = 27 min), 7 (8.2 mg, t_R = 29.5 min), and 8 (50 mg, t_R = 37.6 min).

2.3.1 ¹³C NMR calculation of 2

Conformational searching of 2 was undertaken with the Spartan software using the Merck molecular force field (MMFF) with standard parameters (Hanwell et al., 2012). All conformers were subjected to density functional theory (DFT) geometry optimization at B3LYP/6-31G (d) level of theory in the gas phase using the Gaussian 16 package. Frequency analyses of all optimized conformers were undertaken at the same level of theory to ensure that a Boltzmann distribution ≥ 5% were chosen, and then they were initially optimized at B3LYP/6–31 + G (d, p) levels in chloroform using the polarizable continuum model (PCM) solvent model in the Gaussian 16 program package (Zhao et al., 2021). The gauge-independent orbital (GIAO) shielding constants of these conformers were converted into unscaled chemical shifts (δ_u) by referencing to tetramethylsilane (TMS) (δ_u = σ_TMS– σ_Cal), where the σ_TMS was the shielding constant of TMS calculated at the same level. The scaled chemical shifts (δ_s) were computed by the formula δ_s = (δ_u − b)/m, where m and b are the slope and intercept, respectively. The linear correlation coefficient was calculated to evaluate deviations between the experimental and calculated results (Chen et al., 2020).

2.3.2 ECD calculation of 2

Conformational searching for 2 was obtained from the same method as ¹³C NMR calculation of 2. The conformers with Boltzmann-population of over 5% were chosen for electronic circular dichroism (ECD) calculations, and then the conformers were initially optimized at B3LYP/6–31 g (d, p) level in MeOH using the CPCM polarizable conductor calculation model. The theoretical calculation of ECD was conducted in MeOH using Time-dependent Density functional theory (TD-DFT) at the B3LYP/6–31 + g (d, p) level for all conformers of 1. Rotatory strengths for a total of 30 excited states were calculated. ECD spectra were generated using the program SpecDis 1.71 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV (Lou et al., 2018).

3.1.1 Bioassay-evaluation of crude extracts and a high content PPAP

The neuroprotective activities for EA extract and WT extract were screened using a CORT-induced PC12 cellular injury model (Fig. 1). The results showed that the EA fraction exhibited neuroprotective activity at the concentrations from 1 to 5 μg/ml, while the WT fraction was inactive. The active EA fraction was further separated by silica gel column chromatography to obtain a high content PPAP (1, 5 g) and eight fractions (Frs. A–H). Compound 1 (10 μM) was evaluated for the neuroprotective effect and the result indicated that this compound showed a significant protective effect by increasing the injured cell viability to 65.1%.

3.1.2 Characterization of PPAPs from the fractions by UPLC-Q-Orbitrap-MS/MS

PPAPs were considered as the characteristic active chemical constituents in SJW. UPLC-Q-Orbitrap-MS/MS was an important method to identify and analyse the typical chemical constituents of plant extracts. So, a high content compound 1 was used as a referenced compound and was applied to UPLC-Q-Orbitrap-MS/MS. In the mass spectrum, major product ion at m/z 349 (1a) and 293 (1b) were detected, which were generated by successive loosing of C₁₅H₂₄ (204 u) unit and C₄H₈ (56 u) unit from compound 1 in Fig. 2A. Alternatively, 1b could be obtained via loosing C₁₉H₃₂ (260 u) unit from 1 directly (Figure 2B), which is commonly observed with [M + Na]⁺ ion. As shown in Figure 2C, a major product ion at m/z 315 (1d) was obtained from the precursor [M + Na]⁺ ion for 1 by loosing of C₁₉H₃₂ (260 u) unit (Fig. 2C). Thus, neutral loss of 260 u together with characteristic product ions (293 u, 349 u, 371 u, and 315 u) could be applied for targeted PPAPs detection.

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Fig. 2

(A) Product ion scan of the selected precursor [M + H]⁺ ion at m/z 553 for 1 (20% NCE); (B) The proposed major fragmentation patterns of [M + H]⁺ for 1; (C) Product ion scan of the selected precursor [M + Na]⁺ ion at m/z 575 for 1 (23% NCE); (D) The proposed major fragmentation patterns of [M + Na]⁺ for 1.

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Different fractions (Frs. A–H) obtained from the silica gel column were analysed by UPLC-Q-Orbitrap-MS/MS, and more than 30 analogues (X1-X35) were identified or tentatively characterized from fraction A of EA extract, on the basis of their elemental constituents, fragmentation patterns, the product ion profiles, and the neutral loss unit (Table 1 and Figure S2).

3.1.3 Structure elucidation of compounds 1–10

Further isolation for fraction A of the EA extract led to the isolation of 10 PPAPs, including a new PPAP (Fig. 3). By comparison of the spectroscopic data with those reported in the literature, nine known compounds were identified to be 8-hydroxyhyperforin-8,1-hemiacetal (1) (Verotta et al., 2000), hyphenrone A (3) (Yang et al., 2014), hyphenrone F (4) (Yang et al., 2014), furohyperforin isomer 2 (5) (Hashida et al., 2010), 27-epifurohyperforin isomer (6) (Hashida et al., 2010), furohyperforin isomer (7) (Hashida et al., 2010), hyperfol F (8) (Lou et al., 2020), (2R,3R,4S,6R)-6-methoxycarbonyl-3-methyl-4,6-di(3-methyl-2-butenyl)-2-(2-methyl-1-oxopropyl)-3-(4-methyl-3-pentenyl)cyclohexanone (9) (Verotta et al., 2000), and (2R,3R,4S,6R)-3-methyl-4,6-di(3-methyl-2-butenyl)-2-(2-methyl-1-oxopropyl)-3-(4-methyl-3- pentenyl)cyclohexanone (10) (Verotta et al., 2000).

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Fig. 3

Chemical structures of 1–10.

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Hyperforen A (2) was obtained as colorless oil. Its molecular formula was fixed as C₃₄H₅₄O₄ based on ¹³C NMR and HR-ESI-MS (high resolution electrospray ionization mass spectroscopy) data (m/z 549.3918, [M + Na]⁺), indicating eight degrees of unsaturation. The infrared radiation (IR) spectrum of 2 exhibited strong absorption bands of the hydroxyl (3439 cm⁻¹) and carbonyl (1710 cm⁻¹) groups. The ¹H NMR spectrum of 2 (Table 2) showed the presence of three olefinic protons [δ_H 4.64 (d, J = 9.0 Hz), 4.69 (t, J = 7.2 Hz), and 4.99 (t, J = 7.2 Hz)] and two isopropyl groups. Combined with its HSQC and HMBC spectra, the ¹³C NMR data of 2 (Table 2) revealed 34 signals, including three nonconjugated carbonyls and three nonconjugated double bonds. All the above evidence indicated that 2 was a PPAP derivative (Yang et al., 2014) containing a bicyclic system according to the remaining two degrees of unsaturation.

The planar structure of 2 was established by extensive analysis of its HSQC, ¹H–¹H COSY, and HMBC spectra (Fig. 4). In the HMBC spectrum, the correlations from Me-34 (δ_H 1.14) to C-8 (δ_C 41.6), C-9 (δ_C 47.2), and C-29 (δ_C 46.6), together with the ¹H–¹H COSY correlations of H-8/H-24/H-30/H₂-29, constructed a cyclopentane moiety (A-ring) with a methyl group at C-9. The correlations from Me-27 (δ_H 1.62, s) and Me-28 (δ_H 1.52, s) to C-26 (δ_C 131.3) and C-25 (δ_C 128.6), along with the ¹H–¹H COSY correlations of H-24/H-25 implied that an isobutenyl group is linked to C-24. Furthermore, a nine-membered carbon ring (B-ring) was assigned based on the HMBC correlations of H-1 (δ_H 3.85, s) to C-8 (δ_C 41.6), C-9 (δ_C 47.2), and C-2 (δ_C 201.3), H₂-14 to C-2 (δ_C 201.3), C-3 (δ_C 88.2), and C-4 (δ_C 210.3), and H₂-6 to C-4, as well as the proton spin system of H-20/H₂-19/H-5/H₂-6/H₂-7/H-8 in the ¹H–¹H COSY spectrum. In addition, a hydroxyl group at C-3 was defined from a proton of 3-OH (δ_H 4.35) to C-2, C-3, and C-4 in the HMBC spectrum. Likewise, the HMBC correlations from Me-17 (δ_H 1.59) and Me-18 (δ_H 1.64) to C-15 (δ_C 115.4) and C-16 (δ_C 137.1), and from Me-22 (δ_H 1.63) and Me-23 (δ_H 1.68) to C-20 (δ_C 121.7) and C-21 (δ_C 133.5) revealed that two prenyl groups were placed at C-3 and C-5, respectively. Finally, the HMBC correlations of H-1 (δ_H 3.85) and Me-12 (δ_H 0.98) to C-10 (δ_C 206.7) implied the linkage of isopropyl to C-1 through C-10. Thus, the planar structure of 2 was identified to be an unprecedented PPAP with a bicyclo[7.3.0]dodecane core. To the best of our knowledge, this PPAP represents the first PPAP with the largest pure carbon ring (a nine-membered carbocyclic system).

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Fig. 4

HMBC, ¹H–¹H COSY, and ROESY correlations of 2.

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The relative configuration of compound 2 was determined by analysis of its ROESY spectrum (Fig. 4). The correlations of 3-OH/H-5, H-5/H-7β, H-7β/Me-34, Me-34/H-30, and H-30/H-24 revealed that those groups were on the same side of molecular and were randomly assigned as β-orientation. In addition, the ROESY correlations of H-14α/H-1 and H-1/H-8 indicated the α-orientation of these protons. Hence, the relative configuration of 2 was determined as depicted in Fig. 3.

Quantum chemical calculations were performed to further confirm the correctness of this compound. The ¹³C NMR shielding constants of compound 2 were calculated with the GIAO method at the B3LYP/6–31 + g (d, p) level in chloroform with PCM. The computational ¹³C NMR data were finally obtained using the linear regression analysis method, which was in good agreement with the experimental data (Table S4), with a correlation coefficient (R) of 0.9971 (Fig. 5A) (Xie et al., 2019). Finally, the absolute configuration of compound 2 was fixed by comparison of the experimental and calculated ECD spectra (Zeng et al., 2018; Ma et al., 2017). The ECD spectra of (1S, 3S, 5S, 8S, 9S, 24S, 30S)-2a and (1R, 3R, 5R, 8R, 9R, 24R, 30R)-2b were calculated using time-dependent density functional theory (TDDFT) at the B3LYP-PCM/6–31 + G(d) levels with PCM in MeOH, and the calculated ECD spectra of 2b matched well with the experimental ECD curve of 2 (Fig. 5B). Therefore, the absolute configuration of 2 was fixed as 1R, 3R, 5R, 8R, 9R, 24R, and 30R (Fig. 3).

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Fig. 5

(A) Regression analysis of the experimental versus alculated ¹³C NMR chemical shifts of 2. (B) Calculated and experimental ECD spectra of 2.

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3.1.4 Plausible biosynthetic pathway for 1–10

The plausible biosynthetic pathway for compounds 1–10 was proposed in Scheme 1 with hyperforin (i) as the precursor, which could be derived from 2,4,6-trihydroxybenzophenone via a cascade of isopentylation and intramolecular cyclization (Yang et al., 2018). The key intermediate (i) underwent an oxidation at C-3 and a hemiketal cyclization between OH-3 and a carbonyl of C-9 to obtain compound 1, which further underwent a retrol aldol rearrangement to yield compound 3 (Verotta et al., 2000; Wang et al., 2019). A five-membered lactone ring of 3 was opened via hydrolysis, and the carboxylic group was lost to obtain compound 4. Then, compound 4 underwent a double bond migration of the prenyl group (Liaw et al., 2019) and oxidation at the allylic methylene to the obtain key intermediate iii. Then, the hydroxyl was lost to get a carbonium ion at C-29, which underwent a Westphalen rearrangement (Blunt et al., 1964) to get the intermediate v with a carbonium ion at C-8. This intermediate further underwent a Wagner-Meerwein rearrangement to get intermediate vi (Li et al., 2005; Arriola et al., 2020; Yang and Grossman, 2020). Then, a new carbonium ion at the allylic methylene formed to yield vii, followed by a Wagner-Meerwein rearrangement and cyclization between the two prenyls to get compound 2. Moreover, compound 5 was obtained via oxidation of the C-15, C-16 double bond of intermediate i and intramolecular cyclization between OH-2 and OH-15. Intermediate ii could be easily obtained from i through keto-enol tautomerism, which was further transformed into 6 and 7 by the same procedures as that of compound 5. Similarly, intermediate ii was converted to 8 via oxidation of the C-20, C-21 double bond and intramolecular cyclization between OH-4 and OH-20. In addition, compound 9 could be obtained by the oxidative breakdown of the C-1-C2 and C-3-C-4 carbon–carbon bonds of intermediate i, which underwent hydrolyzation of the ester and decarboxylation to get compound 10.

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Scheme 1

Plausible biosynthetic pathway to 1–10.

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