Polycyclic Polyprenylated Acylphloroglucinols from Hypericum himalaicum

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Quantum chemical calculations
• Cell culture
• Cell viability
• Reverse transcription–polymerase chain reaction (RT–PCR) analysis
• Western blotting
• Detection of NO production
• TNF-α and IL-6 ELISA
• Detection of ROS production
• Nuclear transport of NF-κB/p65
• Statistical analysis
• Contributorsʼ Statement
• References

Abstract

Six previously undescribed polycyclic polyprenylated acylphloroglucinols (PPAPs) with a vicinal diol moiety (1–6) were isolated from the whole plant of Hypericum himalaicum. Their structures were established through a comprehensive analysis of HRMS and 1D and 2D NMR data, while the absolute configurations were determined using the Mo₂(OAc)₄-induced circular dichroism (ICD), ECD, and NMR calculations. Compound 1 attenuated the secretion of NO, TNF-α, and IL-6, downregulated the protein expression of COX-2 and iNOS, and inhibited the release of ROS in LPS-induced RAW264.7 macrophages. Further investigation revealed that the anti-inflammatory effects may be attributed to the inhibition of the NF-κB and NLRP3 signaling pathways.

Introduction

Hypericum plants from the Hypericaceae family are abundant in chemical constituents, including polycyclic polyprenylated acylphloroglucinols (PPAPs), xanthones, naphthodianthrones, flavonoids, bianthraquinones, chromones, and meroterpenoids [1]. As unique constituents of Hypericaceae and Clusiaceae plants, PPAPs have attracted great attention due to extensive biological activities, such as anti–inflammation, anti-depressant, anti-tumor, and anti–HIV activities [2], [3]. Hypericum himalaicum N. Robson is mainly distributed in the high-altitude regions of the Himalayas and nearby areas, including Tibet (China), Pakistan, Bhutan, Nepal, India, and Sikkim [4], [5]. Previous phytochemical studies on H. himalaicum led to the discovery of a series of PPAPs with potential therapeutic properties for the treatment of type 2 diabetes [6].

Inflammation is a multifaceted process involving injury, anti-injury, and repair mechanisms. During inflammation, multiple pro-inflammatory mediators are released by cells such as macrophages and microglia [7], [8]. COX-2 is responsible for generating prostaglandin E2 [9], while iNOS can catalyze the production of NO by deaminating L-arginine [10]. Their excessive release can lead to various diseases, such as asthma, allergies, inflammatory bowel disease (IBD), and neurodegenerative disorders [11]. NF-κB, a key pro-inflammatory signaling pathway [12], is primarily activated when Toll-like receptor 4 (TLR-4) recognizes various microbial pathogens, leading to the activation of the NF-κB pathway [13], [14]. Upon receiving the activation signal, the IκB protein is phosphorylated by the IκB kinase (IKK) complex, ubiquitinated, and degraded, resulting in the release of the NF-κB dimer into the nucleus to promote inflammation [15], [16]. NF-κB activation upregulates the transcription of inflammasome-related components, such as NLRP3, caspase-3, and ASC, which then assemble into complexes triggering a series of inflammatory reactions [17], [18], [19]. Therefore, targeting the NF-κB pathway and inhibiting NLRP3 inflammasome activation are also considered an effective approach for the prevention and treatment of related diseases.

Given the complexity of inflammatory mechanisms and their association with various diseases, identifying potential anti-inflammatory natural products and elucidating their mechanisms of action are of importance. In this study, we report the isolation of six novel PPAPs (1–6) from H. himalaicum, along with a detailed evaluation of their anti-inflammatory activities. These findings lay a scientific foundation for the potential therapeutic applications of PPAPs and H. himalaicum.

Results and Discussion

Whole plants of H. himalaicum were extracted with 95% EtOH. Fractionation of the extract by a combination of chromatographic methods afforded six compounds (1–6) ([Fig. 1]). Compound 1 was obtained as a colorless oil, and its molecular formula was determined as C₃₈H₅₂O₇ by HRESIMS (m/z 643.3617 [M + Na]⁺, calcd for C₃₈H₅₂O₇Na, 643.3605). The IR spectrum showed the presence of hydroxyl (3421 cm⁻¹) and carbonyl (1732 cm⁻¹) groups. The ¹H NMR data ([Table 1]) revealed two olefinic protons of isopentenyl groups (δ _H 4.88, 1H, t, J = 7.5 Hz; 5.05, 1H, t, J = 7.5 Hz), nine singlet methyls, and a mono-substituted benzene ring (δ _H 7.45, 2H, d, J = 7.3 Hz; 7.21, 2H, t, J = 7.8 Hz; 7.37, 1H, t, J = 7.4 Hz). The ¹³C and DEPT NMR data ([Table 1]) showed twelve non-protonated carbons, including three carbonyls (δ _C 205.7, 194.0, 193.7), ten methines including two oxygen-bearing ones (δ _C 89.9, 79.1), six methylenes, and nine methyls. The above-mentioned spectroscopic data indicated that 1 was a PPAP derivative on the basis of literature comparison [4].

The key ¹H-¹H COSY correlations ([Fig. 2]) of H-27/H-28/H-29, H-24/H-25, H-20/H-19/H-3/H-4, H-14/H-12/H-13/H-15/H-16, and H-34/H-35, combined with the HMBC cross peaks from H-17 and H-18 to C-1, C-2, and C-3, from H-34 to C-6, C-7, C-8, C-35, and C-36, from H-24 to C-4, C-5, C-6, C-9, C-25, and C-26, from H-4 to C-3 and C-5, and from H-3 to C-19 and C-20, revealed the planar structure of 1. The structure of compound 1 was similar to that of attenuatumione C [20], differing only in the oxidation of the isopentenyl chain at the C-3 position to a vicinal diol. The NOESY correlations ([Fig. 3]) of Hα-4 with H-25/Hα-24 revealed the relative configuration of C-1, C-5, and C-25.

According to Hu et al. [21], the orientation of the isoprenyl group at the C-3 position can be determined from the NMR data of C-4, C-17, C-18, and C-19, along with their protons. When the isoprenyl group is axial, the H-4 proton resonates above 2.00 ppm. In contrast, when the C-3 substituent is equatorial, the axial H-4 proton resonates at 1.50 ppm. The axial methyl at C-2 is much more shielded when the C-3 substituent is equatorial due to the γ-gauche interaction [22]. The corresponding relevant ¹H and ¹³C NMR data for compound 1 were similar to those of garcinol, and thus, the substituent at C-3 was assigned as in axial orientation ([Fig. 4]). The absolute configuration of the vicinal 20, 21-diol moiety was determined by Snatzkeʼs method, a Mo₂(OAc)₄-induced circular dichroism (ICD) in DMSO solution. According to this method, the sign of the Cotton effect at 300 nm indicated the absolute configuration of the chiral center in the vicinal diol moiety [23], [24]. Therefore, the 20R configuration was determined by the induced negative signs of band IV (297 nm) (Figure 1S, Supporting Information).

At this point, the configuration at C-26 in compound 1 remained unresolved. Computational chemistry methods were employed to finalize its determination ([Fig. 5]). First, the electronic circular dichroism (ECD) spectra were calculated for all four possible absolute configurations (1a, 1b, 1c, and 1 d). The results showed that, at both the WB97XD/DGDZVP and B3LYP/6 – 31+G** levels, the Cotton effects and curve fitting of configurations 1a and 1c were consistent with the experimental data. Subsequently, the ¹³C NMR data for configurations 1a and 1c were calculated. The calculations revealed that, at the mPW1WP91/6 – 31+G** level, the 26S configuration exhibited smaller mean absolute error (MAE) and root mean square error (RMSE) values, with a DP4+ probability of 100%. Therefore, the absolute configuration of 1 was determined to be 1S, 3R, 5R, 20R, 25R, and 26S, as shown in [Fig. 1].

Compound 2 was isolated as a colorless oil, and its molecular formula was determined as C₃₅H₅₄O₇ (m/z 609.3767 [M + Na]⁺). The 1D NMR spectra of compounds 1 and 2 were similar, except for the resonances of the group attached to C-10. Compound 1 contains a benzene ring (δ _H 7.45, 2H, d, J = 7.3 Hz; 7.21, 2H, t, J = 7.8 Hz; 7.37, 1H, t, J = 7.4 Hz), while compound 2 has an isopropyl group attached to C-10 (δ _H 1.97, 1H, m; 1.09, 3H, d, J = 6.5 Hz; 0.97, 3H, d, J = 6.5 Hz) ([Table 1]). The ¹H-¹H COSY and HMBC ([Fig. 2]) correlation signals of compound 2 were essentially identical to those of compound 1, except for the difference near the C-10 substituent position. Specifically, the HMBC correlations from H-11 to C-1 and Me-12/Me-13 to C-10/C-11 confirmed this distinction. Additionally, the ROESY correlations ([Fig. 3]) of H-22 with Hβ-4/H-24/H-29 and of H-3 with Hα-4/Hβ-4/Me-15 indicated the overall configuration of the skeleton. In the case of the vicinal 17,18-diol moiety, the ICD spectrum of compound 2 exhibited a negative Cotton effect at 297 nm, which corresponds to the 17R configuration (Figure 1S, Supporting Information).

Similarly, the configuration of C-23 could not be directly determined. Therefore, computational chemistry methods were employed to establish the absolute configuration of compound 2 ([Fig. 6]). First, the ECD spectra for all four possible absolute configurations (2a, 2b, 2c, and 2 d) were calculated. The results showed that configurations 2b and 2 d both provided good agreement with the experimental data. Next, the NMR data for 2b and 2 d were calculated and compared with the experimental values. The analysis revealed that the calculated NMR data for the 2b configuration had significantly smaller MAE and RMSE values compared to 2 d, and the DP4+ probability for 2b was 100%. These computational results indicated that compound 2 possesses the opposite core configuration compared to compound 1, which was consistent with the difference in their specific rotations (compound 1: [α]_D ²⁰ + 37.3, compound 2: [α]_D ²⁰ − 32.0). As a result, the structure of compound 2 was determined to be 2b, with the configuration 1R, 3S, 5S, 17R, 22S, and 23R, as shown in [Fig. 1].

Compound 3 was isolated as a colorless oil, and its molecular formula was determined as C₃₅H₅₄O₇ (m/z 609.3762 [M + Na]⁺). The 1D NMR data of 3 were almost identical to those of hypericumoxide B [25]. The ¹H-¹H COSY correlation signals ([Fig. 2]) of H-3/H-4/H-21, H-15/H-16/H-17, H-26/H-27, and H-31/H-32, combined with the HMBC correlations ([Fig. 2]) of H-11 to C-1 and C-10, of H-14 and H-15 to C-1 and C-2, of H-3 to C-21 and C-22, of H-26 to C-4, C-5, and C-6, and of H-31 to C-6, C-7, and C-8, revealed the same planar structure, while ROESY data ([Fig. 3]) indicated that compound 3 possessed the same α-orientation of H-32 as revealed by the correlation of Me-12/Me-34. Compound 3 showed a positive Cotton sign at band IV (307 nm) in the ICD spectrum revealing the 17S configuration (Figure 1S, Supporting information). Finally, the whole absolute configuration of 3 was determined by comparison of the calculated and experimental ECD spectra (Figure 2S, Supporting Information).

Compound 4 was isolated as a colorless oil, and its molecular formula was determined as C₃₅H₅₄O₇ (m/z 609.3765 [M + Na]⁺). The 1D and 2D NMR data of 4 were very similar to those of 3, revealing the same planar structure of these compounds. The ROESY spectrum ([Fig. 3]) indicated that compound 4 possessed a β-orientation of H-32 as supported by the correlation of Me-12/H-32. The ICD spectrum of 4 exhibited a negative band IV (307 nm) indicating the 17R configuration (Figure 1S, Supporting information). The whole absolute configuration of 4 was finally determined by comparison of the calculated and experimental ECD spectra (Figure 2S, Supporting Information).

Compound 5 was obtained as a colorless oil, and its molecular formula was determined as C₃₅H₅₄O₇ on the basis of ¹³C NMR data ([Table 2]) and the positive HRESIMS ion at m/z 609.3765 [M + Na]⁺ that indicated nine degrees of unsaturation. Comprehensive comparison of the 1D NMR data of compound 5 was similar to those of uralione L [26], except for the resonances in the vicinity of C-17 (C-15, Δδ + 1.2 ppm; C-19, Δδ + 2.5 ppm; [Table 2]). The HMBC correlations ([Fig. 2]) from H-22 to C-21 and C-3 and from H-21 to C-3 proved the planar structure of compound 5, which was the same as that of hypericumoxide E [25]. The ROESY cross-peaks ([Fig. 3]) of Me-34/Me-30 indicated that H-32 was α-oriented. The ICD spectrum of 5 showed a positive Cotton effect at band IV (303 nm) indicating the 22S configuration (Figure 1S, supporting information) in contrast to the 22R configuration in hypericumoxide E [25]. Therefore, the structure of 5 was established in [Fig. 1].

Compound 6 was isolated as a colorless oil and possessed a molecular formula of C₃₅H₅₄O₇ based on the ion at m/z 609.3767 [M + Na]⁺ in HRESIMS (calcd for C₃₅H₅₄O₇Na, 609.3767) and ¹³C NMR data ([Table 2]). Comprehensive comparison of the 1D NMR data revealed that compound 6 was similar to attenuatumione G [27]. The ¹H-¹H COSY cross-peaks ([Fig. 2]) of H-3/H-4/H-21/H-22 and H-15/H-16/H-17, combined with the HMBC correlations ([Fig. 2]) from H-3 to C-21 and C-22, and from H-15 to C-1 and C-2, suggested the positions of the groups at C-2 and C-3. The ROESY cross-peaks ([Fig. 3]) of Hβ-4/H-15 and Hβ-4/H-27 indicated that H-27 was β-oriented. The ICD spectrum of 6 showed a positive Cotton effect at band IV (301 nm) indicating the 22S configuration (Figure 1S, Supporting information), while the absolute configuration of the remaining chiral centers was determined by comparison of the calculated and experimental ECD spectra (Figure 2S, Supporting Information). Thus, the structure of 6 was established as shown in [Fig. 1].

To explore the anti-inflammatory activity, cell viability after treatment by compounds 1 – 6 was first assessed. The results showed that only compound 1 was non-toxic to RAW264.7 macrophages within the range of administration (15, 30, and 60 µM) ([Fig. 7 a]). Therefore, compound 1 was selected for the investigation of its effects on the expression of inflammatory factors produced by LPS-induced RAW264.7 macrophages. The release of NO decreased after the administration of compound 1 ([Fig. 7 b]). At the same time, ELISA and Western blotting experiments also showed that the production of inflammatory factors TNF-α, IL-6 was inhibited after treatment with compound 1 ([Fig. 7 c]), which was consistent with the expression of proinflammatory factor mRNA in RT-PCR ([Fig. 7 d]). In summary, these data indicate that compound 1 can inhibit the production of LPS-induced NO, TNF-α, and IL-6. Then, Western blotting experiments showed that compound 1 could also decrease LPS-induced COX-2 and iNOS production ([Fig. 8 a, c]). Consistent with the decreased iNOS protein levels, the mRNA expression of iNOS was also downregulated ([Fig. 8 b]). At the same time, immunofluorescence data showed that, compared with the LPS group, the green fluorescence was weakened after addition of compound 1, indicating that the release of ROS decreased ([Fig. 8 d]).

Compared with the LPS-induced RAW264.7 macrophages group, Western blotting results showed that the protein expression of IL-1β, TLR4, p-IκB, and p-NF-κB was reduced after administration of compound 1 ([Fig. 9 a]), and the immunofluorescence data showed that the red fluorescence entering the nucleus was weakened ([Fig. 9 b]), This suggests that compound 1 may inhibit inflammation by blocking the NF-κB pathway. In order to explore whether the NLRP3 pathway is involved in the regulation, Western blotting experiments were carried out. Compared with the LPS group, the expression of NLRP3 and ASC in RAW264.7 macrophages decreased at the protein level after administration of compound 1 ([Fig. 9 c]). Taken together, the data suggest that compound 1 may inhibit the inflammatory response through the NLRP3 pathway.

Materials and Methods

General experimental procedures

Optical rotations were measured on a Perkin Elmer Model 341 Polarimeter. UV spectra were recorded on a Shamashim UV 2401 spectrometer. IR spectra were recorded on a PerkinElmer one FT–IR spectrometer with KBr disks. CD spectra were determined on an Applied Photophysics Chirascan spectrometer. Also, 1D and 2D NMR spectra were recorded on a Bruker Ascend 400/600 spectrometer with TMS as an internal standard. HRESIMS experiments were performed on a Waters Vion IMS Q–Tof mass spectrometer. Analytical HPLC was performed on a Waters Acquity UPLC H–Class with a PDA detector and a Welch Ultimate XB–C18 (4.6 × 250 mm, 5 µm) column. Semi-preparative HPLC was performed on an LC 3000 liquid chromatography system (Chuang Xing Tong Heng Science and Technology Co., Beijing) with a Welch Ultimate XB–C18 (10 × 250 mm, 5 µm) column. Preparative HPLC was performed on a Waters with UV/Visible Detector 2489, Binary Gradient Module 2545 and a Welch Ultimate XB–C18 (21.2 × 250 mm, 5 µm) column. Silica gel (100 – 200 and 200 – 300 mesh, Qingdao Marine Chemical Co., Ltd) was used for column chromatography. Fractions were monitored by TLC (GF254, Qingdao Marine Chemical Co., Ltd.,), and spots were visualized by heating silica gel plates immersed in 10% sulfuric acid alcohol solution.

Plant material

The whole plants of Hypericum himalaicum were collected from Nyingchi, Tibet Autonomous Region, Peopleʼs Republic of China, in September 2017. The plant was identified by Dr. Drolma Dawa (Tibet Autonomous Region Institute for Food and Drug control, Lhasa, Tibet) and a voucher specimen (No. CIB20170912T2) was deposited at Chengdu Institute of Biology, Chinese Academy of Sciences.

Extraction and isolation

The air-dried plant material (30.0 kg) was powdered and extracted with 95% EtOH (3 × 60 L, 3 days each) at room temperature. After filtration, the solution was evaporated under reduced pressure on a rotary evaporator to give a crude extract (4.0 kg). The extract was applied to silica gel column chromatography (12.0 kg, 100 – 200 mesh) eluting with petroleum ether–ethyl acetate (1 : 0 – 0 : 1, v/v) to afford six fractions (A – G). Fr. E (128.0 g) was loaded on a RP–C₁₈ column (MeOH-H₂O, 40 : 60 – 90 : 10) to yield 22 fractions (Fr. E1 – E22). Fr. 18 (2.6 g) was loaded on a silica gel column petroleum ether–acetone (20 : 1 – 0 : 1) to yield four fractions (Fr. E18a–E18d). Fr. E18b (903 mg) was purified by preparative HPLC (MeCN-H₂O, 75 : 25, 3.0 ml/min) to afford compounds 2 (6.1 mg, t_R = 33.2 min), 6 (26.0 mg, t_R = 48.0 min), and 4 (7.1 mg, t_R = 36.9 min). Fr. 19 (3.1 g) was loaded on a silica gel column eluted with petroleum ether–EtOAc (20 : 1 – 0 : 1) to yield four fractions (Fr. E19a–E19e). Fr. E19c (1.02 g) was purified by preparative HPLC (MeCN-H₂O, 75 : 25, 3.0 ml/min) to afford compound 3 (6.2 mg, t_R = 28.2 min). Fr. 20 (2.3 g) was loaded on a silica gel column eluted with petroleum ether–EtOAc (20 : 1 – 0 : 1) to yield four fractions (Fr. E20a–E20f). Fr. E20c (1.23 g) was purified by preparative HPLC (MeOH-H₂O, 85 : 15, 3.0 ml/min) to afford compounds 5 (25.0 mg, t_R = 40.1 min) and 1 (8.6 mg, t_R = 45.7 min).

Compound 1 : colorless oil; [α]_D ²⁰ + 37.3 (c 0.1, MeOH); UV (MeOH) λ _max (log ɛ) 203 (4.92), 245(4.78), 270 (4.71) nm; CD (DMSO) λ _max (Δɛ) 274 (+ 92.9), 306 (– 8.1), 329 (+ 2.0) nm; IR (KBr): ν _max 3420, 2973, 2931, 1732, 1625, 1448, 1384 cm⁻¹; ¹H and ¹³C NMR data, see [Table 1]; HR–ESI–MS m/z 643.3617 [M + Na]⁺ (calcd for C₃₈H₅₂O₇Na, 643.3611).

Compound 2 : colorless oil; [α]_D ²⁰ – 32.0 (c 0.1, MeOH); UV (MeOH) λ _max (log ɛ) 202 (4.86), 271 (4.72) nm; CD (DMSO) λ _max (Δɛ) 279 (– 53.8), 309 (+ 20.6), 337 (– 4.4) nm; IR (KBr): ν _max 3421, 2972, 2930, 1732, 1626, 1449, 1384 cm⁻¹; ¹H and ¹³C NMR data, see [Table 1]; HRESIMS m/z 609.3758 [M + Na]⁺ (calcd for C₃₅H₅₄O₇Na, 609.3767).

Compound 3 : colorless oil; [α]_D ²⁰ + 36.7 (c 0.1, MeOH); UV (MeOH) λ _max (log ɛ) 203 (4.92), 276 (4.78) nm; CD (DMSO) λ _max (Δɛ) 279 (+ 79.5), 307 (– 29.5) nm; IR (KBr): ν _max 3422, 2973, 2931, 1723, 1616, 1446, 1384 cm⁻¹; ¹H and ¹³C NMR data, see [Table 1]; HRESIMS m/z 609.3762 [M + Na]⁺ (calcd for C₃₅H₅₄O₇Na, 609.3767).

Compound 4 ; colorless oil; [α]_D ²⁰ + 37.1 (c 0.1, MeOH); UV (MeOH) λ _max (log ɛ) 203 (4.95), 276 (4.82) nm; CD (DMSO) λ _max (Δɛ) 279 (+ 54.5), 307 (– 23.5) nm; IR (KBr): ν _max 3434, 2972, 2930, 1727, 1616, 1446, 1384 cm⁻¹; ¹H and ¹³C NMR data, see [Table 2]; HRESIMS m/z 609.3765 [M + Na]⁺ (calcd for C₃₅H₅₄O₇Na, 609.3767).

Compound 5 : colorless oil; [α]_D ²⁰ + 29.7 (c 0.1, MeOH); UV (MeOH) λ _max (log ɛ) 200 (4.67), 269 (4.54) nm; CD (DMSO) λ _max (Δɛ) 278 (+ 78.8), 303 (– 71.7), 334 (+ 6.1) nm; IR (KBr): ν _max 3435, 2974, 2931, 1731, 1607, 1445, 1384 cm⁻¹; ¹H and ¹³C NMR data, see [Table 2]; HRESIMS m/z 609.3765 [M + Na]⁺ (calcd for C₃₅H₅₄O₇Na, 609.3767).

Compound 6 : colorless oil; [α]_D ²⁰ + 88.4 (c 0.2, MeOH); UV (MeOH) λ _max (log ɛ) 202 (4.78), 270 (4.64) nm; CD (DMSO) λ _max (Δɛ) 275 (+ 73.0), 304 (– 19.3), 332 (+ 3.2) nm; IR (KBr): ν _max 3443, 2974, 2929, 1732, 1625, 1447, 1377 cm⁻¹; ¹H and ¹³C NMR data, see [Table 2]; HRESIMS m/z 609.3767 [M + Na]⁺ (calcd for C₃₅H₅₄O₇Na, 609.3767).

Quantum chemical calculations

ECD calculations were based on a previous method [28] using Gaussian 16. A conformational search was conducted within a 5 kcal/mol energy window using xtb-CREST [29]. All conformations within a 2 kJ range from the lowest energy conformation were selected for structure optimization and frequency calculations. The theoretical calculation of ECD was performed using time-dependent density functional theory (TD-DFT) at WB97XD/DGDZVP and B3LYP/6 – 31+G** levels in methanol. The ECD spectra were obtained by weighing the Boltzmann distribution rate of each geometric conformation. SpecDis 1.71 [30] was used to sum up single CD spectra after a Boltzmann statistical weighting for the Gauss curve generation (σ = 0.3 eV) and for the comparison with experimental data.

For NMR calculations, density functional theory (DFT) based theoretical NMR calculation using Gaussian 16 was applied to optimize the structure and calculate the energy. Then, the gauge–including atomic orbital (GIAO) shielding constants were calculated for all conformers using the mPW1WP91/6 – 31+G** level in the chloroform solvent model. The Boltzmann distribution was calculated based on the Gibbs free energy. Then, Boltzmann-weighted averages of the chemical shifts were compared with the experimental values. The DP4+ probability analysis [31] was used to predict the dominant configuration.

Cell culture

RAW264.7 macrophages were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences, and cultured in DMEM medium containing 10% fetal bovine serum and 1% streptomycin and penicillin. The incubator was maintained at 37 °C and 5% CO₂.

Cell viability

Logarithmically growing RAW264.7 macrophages were placed in 96-well cell-culture plates with 10 000 cells per well. After the RAW264.7 macrophages were adherent, different concentrations of compound 1 mixed in the culture medium were incubated for 48 h. Then, 10 µL Cell Counting Kit-8 (CCK8) solution was added to each well and incubated in the dark for 1 h. Finally, the absorbance at 450 nm was measured with a plate reader. Cell viability was calculated using the formula: cell viability(%)= (OD sample – OD blank)/(OD control – OD blank) × 100%.

Reverse transcription–polymerase chain reaction (RT–PCR) analysis

Logarithmically growing RAW264.7 macrophages were placed in 6-well plates with 100,000 cells per well. After the RAW264.7 macrophages were adherent, they were incubated with different concentrations of compound 1 for 2 h and then with LPS (1 µg/mL) for 6 h. Then, 1 mL TRIzol reagent was added to lyse the cells and extract total RNA. RNA purity and concentration were measured with an ultramicrospectrophotometer. After that, genomic DNA was removed with 4 × gDNA wiper mix and reverse transcripted with 5 × HiScriptⅡ qRT SuperMixⅡ. cDNA amplification was carried out with the ChamQ Universal SYBR qPCR Master Mix, in which the 2 × ChamQ Universal SYBR qPCR Master Mix was 5 µL, the DNase-free ddH₂O was 2.1 µL, the Template cDNA was 2.5 µL and the primers IL-6 (forward primer: 5′-GGGACTGATGCTGGTGACAAC-3′, reverse primer: 5′-CAACTCTTTTCTCATTTCCACGA-3′), TNF-α (forward primer: 5′-CCCTCCAGAAAAGACACCATG-3′, reverse primer: 5′-CACCCCGAAGTTCAGTAGACAG-3′), GAPDH (forward primer: 5′-GCAAGTTCAACGGCACAG-3′, reverse primer: 5′-CGCCAGTAGACTCCACGAC-3′) was 0.2 µL.

Western blotting

Logarithmically growing RAW264.7 macrophages were placed in 6-well plates with 100 000 cells per well. After the RAW264.7 macrophages were adherent, they were incubated with different concentrations of compound 1 for 2 h and then with LPS (1 µg/mL) for 24 h. The medium was aspirated, and the cells were washed twice with PBS. Cells were lysed by adding 120 µL of RIPA lysis buffer. The lysed cells were scraped off and centrifuged at 20 000 × g for 15 min at 4 °C. The supernatant was taken, and the total protein concentration was detected using the BCA kit. Protein sample loading buffer (1×) was used at 95 °C for 10 min to prevent denaturation. Then the total proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membrane containing proteins was blocked by a rapid blocking solution for 30 min and the primary antibody was incubated overnight at 4 °C. The primary antibodies were as follows: COX-2 (1 : 1,000, Abcam, ab179800), iNOS (1 : 1,000, NOVCCS, NB300-605SS), NLRP3 (1 : 1,000, CST, 15 101S), ASC/TMS1 (1 : 1000, Proteintech, 69 494-1-Ig), TLR4 (1 : 4000, Proteintech, 66 350 – 1-Ig), p-NF-κB (1 : 1000, CST, 3033S), p-IκB (1 : 1000, CST, 5209S), GAPDH (1 : 50 000, Proteintech, 60 004-1-Ig), and tubulin (1 : 50 000, Proteintech, 66 031-1-Ig). The proteins were then incubated with a secondary antibody at room temperature for 2 h. At last, protein bands were detected by a high-sensitivity ECL chemiluminescence detection kit and analyzed by ImageJ software.

Detection of NO production

Logarithmically growing RAW264.7 macrophages were placed in 6-well plates with 100 000 cells per well. After the RAW264.7 macrophages were adherent, they were incubated with different concentrations of compound 1 for 2 h and then with LPS (1 µg/mL) for 24 h. The supernatant was taken out and the production of NO was detected at 540 nm with the Griess reagent.

TNF-α and IL-6 ELISA

Logarithmically growing RAW264.7 macrophages were placed in 6-well plates with 100 000 cells per well. After the RAW264.7 macrophages were adherent, they were incubated with different concentrations of compound 1 for 2 h and then with LPS (1 µg/mL) for 24 h. The supernatant was taken out and the inflammatory factors TNF-α and IL-6 were assayed by ELISA kits.

Detection of ROS production

Logarithmically growing RAW264.7 macrophages were placed in 12-well plates containing climbing plates with 50 000 cells per well. After the RAW264.7 macrophages were adherent, they were incubated with different concentrations of compound 1 for 2 h and then with LPS (1 µg/mL) for 24 h. Basal medium containing DCFH-DA (10 µM) reagent was added for 30 min at 37 °C. Cells were washed twice with PBS, and fixed with PFM for 15 min. After washing with PBS twice, DAPI reagent was added for nuclear staining for 8 min. Finally, the plates were observed under a fluorescence microscope (Olympus FV1200).

Nuclear transport of NF-κB/p65

Logarithmically growing RAW264.7 macrophages were placed in 12-well plates containing climbing plates with 50 000 cells per well. After the RAW264.7 macrophages were adherent, different concentrations of compound 1 were incubated for 2 h and then with LPS (1 µg/mL) for 24 h. An NF-κB Activation, Nuclear Translocation Assay Kit was used for subsequent experiments. After the original culture medium, cells were washed once with PBS and fixed with PFM for 15 min. The fixative was washed away by adding PBS three times, and the immunostaining blocking solution was used for 1 h. Next, NF-κB p65 antibody was added and incubated at 4 °C overnight. Cells were washed three times at room temperature for 10 min each time. Anti-rabbit Cy3 was added and incubated at room temperature for 1 h. The nucleus was stained for 5 min by adding DAPI and the plates were read under a fluorescence microscope (Olympus, IX73).

Statistical analysis

All data were analyzed by GraphPad Prism 7.0 software and expressed as the mean ± standard deviation (SD) of three replicates from independent experiments. The data were from three independent experiments.

Contributorsʼ Statement

Conception and design of the work: Y. Zhou, D. L. Guo, H. B. Sun; Data collection: G. H. Liu, F. Wu, X. Y. Huo; Analysis and interpretation of the data: F. Wu, G. H. Liu, X. Y. Huo; Statistical analysis: F. Wu, G. H. Liu, X. Y. Huo; Visualization: F. Wu, Z. L. Jin, H. B. Sun; Drafting the manuscript: G. H. Liu, F. Wu, X. Y. Huo; Critical revision of the manuscript: Z. L. Jin, F. Wu, Y. Zhou, D. L. Guo; Supervision: Y. Zhou, D. L. Guo, Y. C. Gu;

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

Thank you Syngenta for the Ph.D. Fellowship awarded to Fan Wu. This paper is dedicated to Professor Youyou Tu, the 2015 Nobel Prize Laureate of Physiology or Medicine on the occasion of her 95th birthday.

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Correspondence

Publication History

Received: 24 June 2024

Accepted after revision: 27 April 2025

Accepted Manuscript online:
28 April 2025

Article published online:
20 May 2025

© 2025. Thieme. All rights reserved.

Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany

• References

• 1 Bridi H, Meirelles GC, von Poser GL. Structural diversity and biological activities of phloroglucinol derivatives from Hypericum species . Phytochemistry 2018; 155: 203-232
  PubMedSearch in Google Scholar
• 2 Zhou ZB, Li ZR, Wang XB, Luo JG, Kong LY. Polycyclic polyprenylated derivatives from Hypericum uralum: Neuroprotective effects and antidepressant-like activity of uralodin A. J Nat Prod 2016; 79: 1231-1240
  PubMedSearch in Google Scholar
• 3 Yang XW, Robert BG, Xu G. Research progress of polycyclic polyprenylated acylphloroglucinols. Chem Rev 2018; 118: 3508-3558
  PubMedSearch in Google Scholar
• 4 Editorial Committee of Flora Reipublicae Popularis Sinicae. Flora Reipublicae Popularis Sinicae, Vol. 50. Beijing: Science Press; 1990: 57
  Search in Google Scholar
• 5 Zhou XT, Yang YC, Cheng HT, Pang KJ, Cheng M, Song P, Yang XZ, Yuan Y. Study on chemical components of Hypericum himalaicum and mechanism of anti-inflammatory based on network pharmacology and molecular docking technology. China J Chin Mater Med 2024; 49: 951-960
  PubMedSearch in Google Scholar
• 6 Cheng HT, Yao YH, Cheng HJ, Zhao P, Kang XY, Zhou XT, Liu WQ, Yang XZ. Discovery of bicyclic polyprenylated acylphloroglucinols from Hypericum himalaicum with glucose transporter 4 translocation activity. Bioorg Chem 2022; 129: 106160
  PubMedSearch in Google Scholar
• 7 Agita A, Alsagaff MT. Inflammation, Immunity, and Hypertension. Acta Med Indones 2017; 49 (02) 158-165
  PubMedSearch in Google Scholar
• 8 Kim SU, de Vellis J. Microglia as immune effectors of the central nervous system: Expression of cytokines and chemokines. Clin Exp Neuroimmunol 2010; 1: 61-69
  PubMedSearch in Google Scholar
• 9 Vane JR, Mitchell JA, Appleton I. Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation. Pharmacology 1994; 91: 2046-2050
  PubMedSearch in Google Scholar
• 10 Ohshima H, Bartsch H. Chronic infections and inflammatory processes as cancer risk factors: Possible role of nitric oxide in carcinogenesis. Mutat Res 1994; 305: 253-264
  PubMedSearch in Google Scholar
• 11 Zhao M, Chu J, Feng S, Guo C, Xue B, He K, Li L. Immunological mechanisms of inflammatory diseases caused by gut microbiota dysbiosis: A review. Biomed Pharmacother 2023; 164: 114985
  PubMedSearch in Google Scholar
• 12 Lawrence T. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol 2009; 1: a001651
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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
• 19 Franchi L, Munoz-Planillo R, Nunez G. Sensing and reacting to microbes through the inflammasomes. Nat Immunol 2012; 13: 325-332
  PubMedSearch in Google Scholar
• 20 Zhou ZB, Zhang YM, Pan K, Luo JG, Kong LY. Cytotoxic polycyclic polyprenylated acylphloroglucinols from Hypericum attenuatum . Fitoterapia 2014; 95: 1-7
  PubMedSearch in Google Scholar
• 21 Hu LK, Sim KY. Sampsoniones A–M, a unique family of caged polyprenylated benzoylphloroglucinol derivatives, from Hypericum sampsonii . Tetrahedron 2000; 56: 1379-1386
  PubMedSearch in Google Scholar
• 22 Gustafson KR, Blunt JW, Munro MHG, Fuller RW, McKee TC, Cardellina IIJH, McMahon JB, Cragg GM, Boyd MR. The guttiferones, HIV-inhibitory benzophenones from Symphonia globulifera, Garcinia livingstonei, Garcinia ovalifolia and Clusia rosea . Tetrahedron 1992; 48: 10093-10102
  CrossrefPubMedSearch in Google Scholar
• 23 Snatzke G, Wagner U, Wolff HP. Circulardichroism–LXXV1: Cottonogenic derivatives of chiral bidentate ligands with the complex [Mo₂ (O₂CCH₃)₄]. Tetrahedron 1981; 37: 349-361
  PubMedSearch in Google Scholar
• 24 Bari LD, Pescitelli G, Pratelli C, Pini D, Salvadori P. Determination of absolute configuration of acyclic 1, 2-diols with Mo₂(OAc)₄. 1. Snatzkeʼs method revisited. J Org Chem 2001; 66: 4819-4825
  PubMedSearch in Google Scholar
• 25 Li X, Li Y, Luo J, Zhou Z, Xue G, Kong LY. New phloroglucinol derivatives from the whole plant of Hypericum uralum . Fitoterapia 2017; 123: 59-64
  PubMedSearch in Google Scholar
• 26 Zhou ZB, Zhang YM, Luo JG, Kong LY. Cytotoxic polycyclic polyprenylated acylphloroglucinol derivatives and xanthones from Hypericum attenuatum . Phytochem Lett 2016; 15: 215-219
  PubMedSearch in Google Scholar
• 27 Liu RD, Su YL, Yang JB, Wang AG. Polyprenylated acylphloroglucinols from Hypericum scabrum . Phytochemistry 2017; 142: 38-50
  PubMedSearch in Google Scholar
• 28 Guo DL, Qiu L, Feng D, He X, Li XH, Cao ZX, Gu YC, Mei L, Deng F, Deng Y. Three new α-pyrone derivatives induced by chemical epigenetic manipulation of Penicillium herquei, an endophytic fungus isolated from Cordyceps sinensis . Nat Prod Res 2020; 34 (07) 958-964
  PubMedSearch in Google Scholar
• 29 Pracht P, Caldeweyher E, Ehlert S, Grimme S. A robust non-parametric density functional tight binding method for structure optimizations, molecular dynamics simulations, and property predictions. J Chem Theory Comput 2020; 16 (03) 2115-2143
  PubMedSearch in Google Scholar
• 30 Pescitelli G, Bruhn T. Good Computational Practice in the Assignment of Absolute Configurations by TDDFT Calculations of ECD Spectra. Chirality 2016; 28 (06) 466-474
  CrossrefPubMedSearch in Google Scholar
• 31 Ermanis K, Parkes KEB, Agbackb T, Goodman JM. Expanding DP4: application to drug compounds and automation. Org Biomol Chem 2016; 14: 3943
  PubMedSearch in Google Scholar

• Supplementary Material