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Prenylated acylphloroglucinols from the fruits of Hypericum patulum

  • Yu-Feng Qiu 1† ,  
  • Yi Zhou 1† ,  
  • Cheng Chen 3 ,  
  • Juan Huang 2 ,  
  • Xing-Wei Yang 1
    •     
Received date: 6 January 2026; Accepted: 12 March 2026; Published online: 23 April 2026
†Yu-Feng Qiu and Yi Zhou have contributed equally to this work.
Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1007/s13659-026-00629-9.
Supplementary material 1. 1H and 13C NMR data for compounds 2 and 3 in CDCl3 (Table S1), Original MS and NMR spectra of compounds 1–4 (Figs. S1–S44).

Abstract

Twenty-eight prenylated acylphloroglucinols, including the new hypulatones C–F (1–4), have been isolated from the fruits of Hypericum patulum and structurally characterized. Compound 1 represents a rare meroterpenoid formed through the addition of a prenylated acylphloroglucinol unit and a sesquiterpenoid moiety. Spirocyclic polycyclic polyprenylated acylphloroglucinol 2 contains six chiral centers, and its relative configuration was established based on 1H–1H coupling constants, conformational analysis, and NOE correlations. The cytotoxic activities of all isolates against two human carcinoma cell lines (Huh-7 and Panc-1) were evaluated using the CCK-8 assay. Bioassay results indicated that compounds 6, 8, and 15 exhibited moderate antiproliferative activity.

Graphical Abstract

Keywords

Prenylated acylphloroglucinols    Hypericum patulum    Structural determination    Cytotoxicity    
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1 Introduction

Polyprenylated acylphloroglucinols (PAPs) are a class of structurally fascinating hybrid natural products that exhibit diverse bioactivities, such as tumor inhibitive, antimicrobial, HIV preventative, antioxidant, and antidepressant [1, 2]. Plants of the genus Hypericum (Hypericaceae) are known to be a rich source of PAPs. Hypericum patulum, commonly known as "Jinsimei", is a Hypericum species primarily distributed in southwestern China, notably in Yunnan, Guizhou, and Sichuan provinces [3]. It is a traditional Chinese medicine commonly used for the treatment of hepatitis, bacterial diseases, and epistaxis [4]. Due to the structural diversity of its chemical constituents and their potential pharmacological effects, this plant has attracted widespread scientific interest [5–17]. As we have a longstanding interest in the investigation of structures and bioactivities of PAPs [1, 18–30], we selected the fruit of H. patulum for further investigation. Four undescribed PAPs, hypulatones C–F (1–4), were isolated together with twenty-four known analogues (5–28, Figs. 1 and 2). Compound 1 represents a rare meroterpenoid formed through the addition of a prenylated acylphloroglucinol unit and a sesquiterpenoid moiety. To date, only eight enantiomeric pairs of this type of meroterpenoids have been reported [14, 31]. Herein are described the isolation, structure determination, and inhibitory activities against two human carcinoma cells (Huh-7 and Panc-1) of all the compounds isolated. This study provides an effective method for configurational assignments of spirocyclic polycyclic polyprenylated acylphloroglucinols (PPAPs) that bear six chiral centers.

Fig. 1

The structures of compounds 1–4

Fig. 2

The structures of compounds 5–28

2 Results and discussion

Hypulatone C (1) was obtained as yellow gum. Its molecular formula C41H60O5 with 12 degrees of unsaturation was established by analysis of 13C NMR (Table 1) and HRESIMS data (m/z 671.4082, [M + K]+, cacld for 671.4072). The FTIR spectrum displayed absorption bands due to carbonyl (1719 and 1699 cm−1) functionalities. The 1H NMR spectrum (Table 1) exhibited the signals of an isopropyl (δH 3.07, sept; 1.16, d; 1.09, d; J = 6.9 Hz), four olefinic protons (δH 4.51–5.01), and nine singlet methyls (δH 0.64–1.67). Analysis of its 13C and DEPT-NMR data revealed a total of 41 carbon resonances, including a shielded sp2 carbon at δC 115.3 (C-1) and three deshielded carbons at δC 178.7 (C-6), 199.3 (C-2), and 203.2 (C-7), which were indicative of the presence of an enol-β-triketo system. The above signals, in combination with a nonconjugated ketone at δC 207.4 (C-4) and two quaternary carbons at δC 57.4 (C-3) and 62.6 (C-5), suggested that compound 1 should possess a dearomatized acylphloroglucinol core. This assumption was further confirmed by the HMBC correlations from a singlet methyl at δH 1.31 (Me-16) to C-2, C-3, and C-4, and from δH 3.29 and 2.28 (H2-17) to C-4, and C-5, and C-6. An isoprenyl group (δC 39.5, C-11; 118.6, C-12; 135.3, C-13; 26.0, C-14; 17.4, C-15) linked to C-3 was deduced by the correlation of δH 4.82 (H-12)/δH 2.67 and 2.31 (H2-11) in the 1H–1H COSY spectrum, together with the correlations of both δH 1.58 (Me-14) and 1.53 (Me-15) with C-11 and C-12, of H2-11 with C-2, C-3, and C-4 in the HMBC spectrum. Moreover, C-17 was proved to be the head of a geranyl group (δC 40.3, C-17; 116.6, C-18; 141.4, C-19; 17.6, C-20; 39.9, C-21; 26.3, C-22; 123.9, C-23; 131.5, C-24; 25.7, C-25; 17.7, C-26) by the HMBC correlations from δH 1.62 (Me-20) to C-18, C-19, and C-21, from both δH 1.67 (Me-25) and 1.59 (Me-26) to C-23 and C-24, coupled with the proton spin systems of H2-17/δH 4.74 (H-18) and δH 1.97 (H2-21)/δH 2.03 (H2-22)/δH 5.02 (H-23) in the 1H–1H COSY spectrum. The HMBC correlations of both doublet methyls at δH 1.16 and 1.09 with C-7 assigned the location of the isopropyl group (δC 39.8, C-8; 18.5, C-9; 18.1, C-10) (Fig. 3).

Table 1

13C (150 MHz) and 1H NMR (600 MHz) data of compound 1 in CDCl3

No δC, type δH mult. (J in Hz) No δC, type δH mult. (J in Hz)
1 115.3, C 23 123.9, CH 5.02, t (6.3)
2 199.3, C 24 131.5, C
3 57.4, C 25 25.7, CH3 1.67, s
4 207.4, C 26 17.7, CH3 1.59, s
5 62.6, C 1′ 92.4, C
6 178.7, C 2′ 59.4, CH 2.63, d (2.0)
7 203.2, C 3′ 64.2, CH 2.85, d (2.0)
8 39.8, CH 3.07, sept (6.9) 4′ 33.8, C
9 18.5, CH3 1.09, d (6.9) 5′ 39.3, CH2 1.57, overlap
10 18.1, CH3 1.16, d (6.9) 1.24, m
11 39.5, CH2 2.67, dd (13.8, 7.8) 6′ 20.2, CH2 1.57, overlap
2.31, dd (13.8, 7.8) 1.41, m
12 118.6, CH 4.82, t (7.8) 7′ 39.5, CH2 2.02, overlap
13 135.3, C 1.76, t (12.6)
14 26.0, CH3 1.58, s 8′ 135.5, C
15 17.4, CH3 1.53, s 9′ 123.6, CH 5.13, dd (10.2, 4.0)
16 21.8, CH3 1.31, s 10′ 23.3, CH2 2.41, m
17 40.3, CH2 3.29, dd (15.0, 9.3) 1.95, overlap
2.28, dd (15.0, 4.0) 11′ 40.3, CH2 1.97, overlap
18 116.6, CH 4.74, dd (9.3, 4.0) 1.71, m
19 141.4, C 12′ 41.0, CH2 2.31, s
20 17.6, CH3 1.62, s 13′ 25.3, CH3 1.00, s
21 39.9, CH2 1.97, m 14′ 22.4, CH3 0.64, s
22 26.3, CH2 2.03, m 15′ 16.3, CH3 1.64, s

Fig. 3

1H–1H COSY and HMBC correlations of 1–4

Besides the aforementioned 26 carbon signals in the 13C and DEPT NMR spectra of 1, the remaining 15 resonances assignable to three nonprotonated carbons (δC 135.5, C-8′; 92.4, C-1′; and 33.8, C-4′), three methines (δC 123.6, C-9′; 64.2, C-3′; and 59.4, C-2′), six methylenes, and three methyls indicated a humulane-type sesquiterpenoid moiety. This deduction was further confirmed by the correlations of 2.63 (H-2′)/δH 2.85 (H-3′), and δH 1.24 (H-5′)/δH 1.41 (H-6′)/δH 1.76 (H-7′), and δH 5.13 (H-9′)/δH 2.41 (H-10′)/δH 1.71 (H-11′) in the 1H–1H COSY spectrum, along with the HMBC correlations from δH 2.31 (H-12′) to δC 92.4 (C-1′), 59.4 (C-2′), and 40.3 (C-11′), from both singlet methyls at δH 0.64 (Me-14′) and 1.00 (Me-13′) to δC 64.2 (C-3′), 33.8 (C-4′), and 39.3 (C-5′), and from singlet methyl at δH 1.64 (H-15′) to δC 39.5 (C-7′), 135.5 (C-8′), and 123.6 (C-9′) (Fig. 3).

The linkage of C-5/C-12′ was deduced by the HMBC correlations from H2-12′ to C-4, C-5, and C-6, which combined the acylphloroglucinol and sesquiterpenoid moieties. The formation of the 2′,3′-epoxide and deduced furan ring were indicated by the indices of hydrogen deficiency along with the special chemical shifts of C-1′ (δC 92.4), C-2′ (δC 59.4), C-3′ (δC 64.2), and C-6 (δC 178.7). So far, the planar structure of 1 was elucidated as shown (Fig. 1).

Comparison of the structure of 1 with that of hyperkouytin C (6) [31] indicated that the benzoyl and prenyl groups in 6 were replaced by an isobutyryl and a methyl in 1, respectively. The 13C chemical shifts of carbons around C-1′, C-2′ C-3′, and C-5 chiral centers are very close to those of 6, which suggests that the relative configurations of C-1′, C-2′ C-3′, and C-5 are identical to those of 6. Furthermore, the chemical shift of H-17a (δH 3.29) was 1.01 ppm downfield of H-17b (δH 2.28), indicating that compound 1 had a close O2′/H-17a contact and the geranyl group was located at the same side of C-2′. This deduction was confirmed by a computationally optimized model (Fig. 4) and the crystallographic data of hypulatone B and hyperkouytins A and B [14, 31]. These evidences further confirmed the relative configurations four chiral carbons mentioned above. Finally, the NOESY correlation between Me-16 and H-18 established the configuration of C-3, thus assigning the relative configuration of 1 as 3S*, 5S*, 1'R*, 2'R*, 3'S* (Fig. 4). Considering that all the meroterpenoids of this type were reported in enantiomeric pairs [14, 31], the optical rotation of 1 ([α]D = + 19) suggested it might be scalemic mixtures. Nevertheless, the lack of sufficient sample quantities precluded the further chiral separation.

Fig. 4

Configuration optimized molecular model of 1. yellow arrows, key NOE correlation; yellow dashed line, close O2′/H-17a contact

Hypulatone D (2) was assigned the molecular formula C26H38O5 by analysis of its 13C NMR (Table 2) and HREIMS data (m/z 429.2643 [M − H]−). Comparing the 1H and 13C NMR data of 2 to those of hyperpatulone E [15] indicates that they are structurally similar. The sec-butyl group in hyperpatulone E is replaced by an isopropyl (δH 1.21, d; 1.13, d; 3.51, sept.; J = 6.8 Hz) in 2, as evidenced by the HMBC correlations from Me-25 (δH 1.21) and Me-26 (δH 1.13) to δC 206.7 (C-23). It is worth noting that one could barely determine the relative configuration of spirocyclic PPAPs characterized by six chiral centers, like 2, unless one used a combination of 1H–1H coupling constants, conformational analysis, and NOE correlations. Firstly, in the 1H spectrum of 2 measured in CDCl3 (Table S1), the 3J coupling constant of H-14b (δH 1.23, t, J = 13.0 Hz) was 13.0 Hz. So, the corresponding six-membered ring adopted chair conformations (Fig. 5), and H-14a (δH 2.00, brd, J = 13.0 Hz) and Me-15 were equatorial while H-14b and H-13 (δH 1.25) were axial. Secondly, the NOE contacts of Me-15 with H-11eq (δH 1.86) and of H-11ax (δH 1.11) with H-8 (δH 1.57) indicated the trans configuration of the octahydro-indene moiety, as well as the relationship between H-12 and H-13. Thirdly, due to the constraints of the spirocyclic framework, the phloroglucinol ring is perpendicular to the octahydro-indene moiety, which itself lies nearly coplanar with the plane formed by the two C-6 substituents. The NOE correlations of H-14a/Me-22 (δH 1.43), H-18 (δH 4.70)/H-7b (δH 1.79), and H-7b/Me-16 (δH 1.28) measured in CD3OD defined the relative configurations of C-6 and C-9. Thus, the structure of 2 was determined as shown and named as hypulatone D (Fig. 1).

Table 2

13C (150 MHz) and 1H NMR (600 MHz) data of compounds 2–4

No 2 (CD3OD) 3 (CD3OD) 4 (CDCl3)
δC, type δH mult. (J in Hz) δC, type δH mult. (J in Hz) δC, type δH mult. (J in Hz)
1 198.5, C 105.6, C 79.7, C
2 112.9, C 191.1, C 193.6, C
3 198.8, C 111.3, C 116.7, C
4 66.4, C 179.3, C 171.8, C
5 210.0, C 54.8, C 58.4, C
6 57.5, C 198.9, C 40.2, CH2 2.90, dd (14.0, 6.0)
1.93, d (14.0)
7 27.1, CH2 1.86, t (13.0) 42.8, CH 3.18, dd (12.4, 3.2) 43.9, CH 1.84, overlap
1.79, m
8 51.3, CH 1.54, m 41.1, CH2 1.83, m 49.1, C
1.55, m
9 79.8, C 73.2, C 204.7, C
10 41.2, CH2 1.79, overlap 28.0, CH2 1.78, t (12.5) 194.0, C
1.71, m 1.43, m
11 27.9, CH2 1.84, m 23.5, CH2 1.67, m 137.0, C
1.09, m 1.61, m
12 49.9, CH 1.23, overlap 50.8, CH 1.48, m 128.1, CH 7.43, d (7.7)
13 36.4, CH 1.22, overlap 73.2, C 127.9, CH 7.22, t (7.8)
14 45.0, CH2 2.06, brd (10.1) 27.2, CH3 1.15, s 132.0, CH 7.38, t (7.7)
1.21, overlap
15 20.3, CH3 0.87, d (5.0) 26.8, CH3 1.14, s 127.9, CH 7.22, t (7.8)
16 26.7, CH3 1.28, s 28.0, CH3 1.16, s 128.1, CH 7.43, d (7.7)
17 40.3, CH2 2.64, dd (13.3, 8.2) 40.1, CH2 2.63, dd (14.0, 7.3) 22.4, CH2 3.11, m
2.45, dd (13.3, 8.2) 2.58, dd (14.0, 6.4) 3.04, dd (14.1, 7.2)
18 118.9, CH 4.70, t (8.2) 120.5, CH 4.84, overlap 120.2, CH 5.09, overlap
19 138.1, C 135.4, C 132.9, C
20 26.1, CH3 1.55, s 26.2, CH3 1.54, s 25.8, CH3 1.62, s
21 17.9, CH3 1.45, s 18.1, CH3 1.58, s 17.7, CH3 1.65, s
22 23.0, CH3 1.43, s 24.8, CH3 1.32, s 36.4, CH2 2.59, m
1.71, overlap
23 206.7, C 208.5, C 102.4, CH 6.05, d (5.6)
OH 3.62, brs
24 35.6, CH 3.51, sept (6.8) 36.8, CH 3.98, sept (6.8) 25.5, CH2 2.28, m
1.99, m
25 20.1, CH3 1.21, d (6.8) 19.5, CH3 1.11, d (6.8) 124.7, CH 5.09, overlap
26 19.3, CH3 1.13, d (6.8) 19.3, CH3 1.10, d (6.8) 131.1, C
27 25.8, CH3 1.63, s
28 18.0 CH3 1.65, s
29 13.7, CH3 1.19, s
30 36.4, CH2 2.21, overlap
1.41, td (14.0, 5.0)
31 27.1, CH2 2.23, overlap
1.84, overlap
32 122.2, CH 5.02, t (7.1)
33 133.4, C
34 25.5, CH3 1.59, s
35 17.8, CH3 1.71, s

Fig. 5

Configuration optimized molecular model of 2. Pink arrows, coupling constants; yellow arrows, NOE correlations

The molecular formula of hypulatone E (3) was determined as C26H40O6 by analysis of its 13C NMR (Table 2) and HRESIMS data (m/z 447.2745 [M–H]−). The 1H and 13C NMR data of 3 resembled those of hyperhenone E (15) [32]. Instead of two olefinic carbons in hyperhenone E, an oxygen-bearing quaternary carbon at δC 73.2 (C-13) and a methyl at δC 27.2 (Me-14) appeared in 3, suggesting that 3 could be derived from hyperhenone E by adding water across the Δ13,14 double bond of the latter. This suggestion was further supported by the correlations of both Me-14 (δH 1.15) and Me-15 (δH 1.14) with C-13 and C-12 (δC 50.8). The NOE contacts of H-7 (3.18) with H-12 (1.48) and Me-16 (1.16) indicated that the relative configurations of C-7, C-9, and C-12 were identical to those of hyperhenone E. Furthermore, the well matched ECD curves of 3 and hyperhenone E (15) suggested that their absolute configuration of C-5 was identical [32–34]. Considering that compounds 3 and 15 were co-isolated and the absolute configuration of 15 was determined by X-ray diffraction data [33], the absolute configuration of 3 could be defined as 5R, 7R, 9R, 12S (Fig. 1).

The molecular formula of hypulatone F (4) was determined to be C35H44O5 from its HRESIMS and 13C NMR data (Table 2). On the basis of analysis of its 1D and 2D NMR data, compound 4 was assigned to possess the same backbone as hypseudohenrin F [35]. The structural novelty of 4 involved the presence of a hemiacetal hydroxyl (δH 3.62, OH-23) rather than a methoxy group, which was confirmed by the 1H–1H COSY correlations of H-22a (δH 2.59) with H-23 (δH 6.05), in combination with the HMBC correlations from H-22a to C-5 (δC 58.4) and C-6 (δC 40.2) and C-9 (δC 204.7), and from H-23 to C-4 (δC 171.8) (Fig. 3). The 2D NMR data showed that the other structural features of 4 were identical to those of hypseudohenrin F.

Twenty-four known compounds were identified as hypulatone A (5) [14], (+)-hyperkouytin C (6) [31], hypulatone B (7) [14], (−)-hyperkouytin D (8) [31], tomoeone A (9) [36], tomoeone B (10) [36], chipericumin D (11) [37], chipericumin E (12) [38], hypercohone G (13) [39], spirohypatone A (14) [13], hyperhenone E (15) [32], bellumone I (16) [40], hyphenrone J (17) [23], hyphenrone K (18) [23], hyphenol J (19) [34], uralione E (20) [41], hookerione K (21) [42], attenuatumione D (22) [43], sampsonione H (23) [44], hypersampsone D (24) [45], sampsonione D (25) [44], sampsonione C (26) [44], hypersampsone I (27) [46], and hypersampsonone G (28) [47], by comparison of their spectroscopic and physical data with those of related literature (Fig. 2).

All the isolates (compounds 1–28) were tested for their cytotoxic activities on Huh-7 and Panc-1 cell lines by CCK-8 assay. Sorafenib and paclitaxel were used as the positive control. As shown in Table 3, compounds 6, 8, and 15 showed moderate inhibitory activity against two human cancer cell lines with IC50 values in the range of 9.7–19.2 µM.

Table 3

Cytotoxicity of compounds 1–28 on two cancer cell lines with IC50 values (μM)

Compound Huh-7 Panc-1 Compound Huh-7 Panc-1
1 35.9 ± 3.3 48.4 ± 6.9 16 42.1 ± 2.8 45.4 ± 4.9
2 40.2 ± 1.1 > 50 17 23.0 ± 2.6 18.6 ± 1.2
3 > 50 41.4 ± 2.1 18 34.6 ± 0.4 23.4 ± 1.3
4 32.3 ± 3.7 35.5 ± 1.6 19 24.9 ± 2.5 > 50
5 17.3 ± 0.7 20.7 ± 1.8 20 17.5 ± 0.5 22.8 ± 2.7
6 19.2 ± 1.0 12.6 ± 0.8 21 36.8 ± 3.6 37.3 ± 2.5
7 21.8 ± 1.0 17.4 ± 0.4 22 10.0 ± 0.4 > 50
8 10.4 ± 0.2 15.5 ± 0.8 23 > 50 > 50
9 36.7 ± 0.7 > 50 24 > 50 > 50
10 43.0 ± 3.0 > 50 25 41.9 ± 2.3 48.5 ± 3.4
11 29.6 ± 0.9 > 50 26 27.3 ± 0.6 21.7 ± 1.6
12 > 50 > 50 27 30.8 ± 0.7 42.0 ± 3.8
13 27.2 ± 0.8 > 50 28 44.0 ± 2.5 16.3 ± 1.0
14 20.3 ± 0.5 20.4 ± 0.6 Sorafenib 7.4 ± 0.4 8.3 ± 1.3
15 9.7 ± 1.0 11.5 ± 0.5 Paclitaxel 8.1 ± 0.7 3.2 ± 1.3

In summary, four previously undescribed PAPs, hypulatones C–F (1–4), together with twenty-four known analogues, were isolated from the fruit of Hypericum patulum and structurally characterized. Compounds 6, 8, and 15 showed moderate inhibitory activity against two human cancer cell lines with IC50 values in the range of 9.7–19.2 µM. Our findings enriched the structural diversity of natural PAPs, and also provided a useful method for configurational assignments of spirocyclic PPAPs that bear six chiral centers.

3 Experimental procedures

3.1 General experimental procedures

Optical rotations were measured on a Jasco P-1020 polarimeter. UV spectra were recorded on a Shimadzu UV-2401PC spectrometer. IR spectra were recorded on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. 1D and 2D NMR spectra were recorded on a Bruker DRX-600 spectrometer using TMS as an internal standard. Unless otherwise specified, chemical shifts (δ) are expressed in ppm with reference to the solvent signals. ESIMS and HREIMS data were acquired on Waters Xevo TQS and Waters AutoSpec Premier P776 mass spectrometers, respectively. Semi-preparative HPLC was performed on an Agilent 1100 HPLC with a Zorbarx SB-C18 (9.4 × 250 mm) column. Silica gel (200–300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, People's Republic of China) were used for column chromatography. Fractions were monitored by TLC (GF 254, Qingdao Marine Chemical Co., Ltd.), and spots were visualized by heating silica gel plates immersed in H2SO4 in EtOH.

3.2 Plant materials

The fruits of Hypericum patulum were collected in Xishan of Kunming County, Yunan Province, People's Republic of China, in July 2020. The plant was identified by Dr. L. Zhang, and a voucher specimen (KIB 20200701) has been deposited at the Kunming Institute of Botany.

3.3 Extraction and isolation

The dried fruits of Hypericum patulum (3.12 kg) were powdered and percolated with MeOH (3 × 10 L) at room temperature for 24 h to yield an extract (680 g) after evaporation in vacuo. The residue was suspended in H2O and partitioned with EtOAc and H2O to yield the EtOAc fraction (350 g). This fraction was subjected to column chromatography over silica gel eluted with a petroleum ether–EtOAc in gradient (19:1, 9:1, 8:2, 7:3, 1:1, and 0:1) to obtain 10 fractions (Fr. A–J).

Fr. A (18.6 g) was fractionated by MCI gel column chromatography (MeOH–H2O, 70:30–100:0) to provide five subfractions (Fr. A1–A5). Fr. A3 (3.1 g) was fractionated by silica column chromatography using petroleum ether–EtOAc (100:1–0:1) as eluents to provide four subfractions (Fr. A3.1–A3.4). Fr. A3.2 (715 mg) was then purified by semipreparative HPLC (MeOH–H2O, 92:8) to produce compounds 23 (9.1 mg) and 27 (30.8 mg). Fr. A4 (1.9 g) was then fractionated by silica gel column chromatography using petroleum ether–EtOAc (50:1–0:1) as eluents to provide four subfractions (Fr. A4.1–A4.4). Fr. A4.2 (540 mg) was then purified by semipreparative HPLC eluting with MeOH–H2O (95:5), in combination with preparative TLC, to afford compounds 1 (2.6 mg), 7 (4 mg), 21 (7 mg), 24 (9.9 mg) and 25 (6 mg). Fr. A4.3 (175 mg) was then fractionated by semipreparative HPLC (MeOH–H2O, 94:6) to produce compounds 5 (8 mg), 6 (1.3 mg), and 8 (15.7 mg). A portion of Fr. B (6.8 g) was fractionated by MCI gel column chromatography (MeOH–H2O, 60:40–100:0) to provide five subfractions (Fr. B1–B5). Fr. B5 (3.5 g) was chromatographed on a silica gel column, eluting with petroleum ether–EtOAc (20:1–0:1), to gather Fr. B5.1–B5.3. Compounds 16 (3.9 mg), 17 (6 mg), and 18 (4.1 mg) were obtained from Fr. B5.2 by semipreparative HPLC (MeOH–H2O, 95:5). Using semipreparative HPLC (MeCN–H2O, 85:15 and 70:30, respectively), Fr. B4 (639 mg) afforded compounds 10 (4.8 mg) and 15 (89.4 mg). Fr. C (38.5 g) was fractionated by using MCI gel column chromatography (MeOH–H2O, 40:60–100:0) to gather Fr. C1–C5. Fr. C5 was chromatographed on a silica gel column (petroleum ether–EtOAc, 20:1–0:1) to provide five subfractions (Fr. C5.1–C5.5). Using semipreparative HPLC (MeCN–H2O, 85:15) and preparative TLC (petroleum ether–EtOAc), compounds 11 (33 mg), 20 (5 mg), 28 (28.1 mg) from Fr. C5.4 (889.5 mg), and compounds 4 (12.3 mg), 9 (3.2 mg), 22 (18.3 mg), and 26 (28.3 mg) from Fr. C5.5 (48.9 mg) were isolated. Similarly, compounds 2 (3.1 mg), 3 (4.5 mg), 12 (13.2 mg), 13 (3.1 mg), 14 (6.3 mg), and 19 (3.9 mg) were obtained from Fr. E (60 g) by using silica gel column, semipreparative HPLC (MeCN − H2O, 70:30), and preparative TLC.

Hypulatone C (1): Yellow gum; [α]D25 + 19 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (4.28), 230 (4.31), 275 (4.28) nm; IR (KBr) νmax 2964, 2930, 2872, 2857, 1719, 1699, 1625, 1456, 1383, 1250, 1184, 1095, 1030, 908, 803 cm−1; 1H and 13C NMR data, see Table 1. ESIMS m/z 671 [M + K]+; HRESIMS m/z 671.4082 (calcd for C41H60O5K, 671.4072).

Hypulatone D (2): Yellow gum; [α]D25 − 12 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 240 (2.89), 283 (3.02) nm; CD (c 3 × 10−4, MeOH) λmax nm (Δε) 201 (− 5.23), 221 (+ 10.95), 244 (+ 9.96), 272 (+ 9.50), 295 (− 1.82), 305 (+ 1.13), 334 (− 2.83); IR (KBr) νmax 3317, 2945, 2831, 1668, 1533, 1448, 1279, 1022 cm−1; 1H and 13C NMR data, see Table 2. ESIMS m/z 429 [M − H]−; HRESIMS m/z 429.2643 (calcd for C26H37O5, 429.2641).

Hypulatone E (3): Light yellow gum; [α]D25 + 112 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (3.07), 238 (3.02), 279 (2.90), 327 (2.97) nm; CD (c 3 × 10−4, MeOH) λmax nm (Δε) 199 (− 18.6), 228 (+ 4.52), 248 (+ 0.29), 270 (+ 7.74), 317 (+ 10.39), 350 (+ 5.35); IR (KBr) νmax 3334, 3084, 1637, 1502, 1022 cm−1; 1H and 13C NMR data, see Table 2. ESIMS m/z 447 [M − H]−; HRESIMS m/z 447.2745 (calcd for C26H39O6, 447.2747).

Hypulatone F (4): Light yellow gum; [α]D25 − 23 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (4.25), 248 (4.30), 275 (4.18) nm; IR (KBr) νmax 3437, 2967, 2925, 2859, 1728, 1695, 1627, 1448, 1340, 1317, 1227, 1190,1142, 1057,869, 799, 768, 689 cm−1; 1H and 13C NMR data, see Table 2. HRESIMS m/z 583.2826 (calcd for C35H44O5K, 583.2820).

3.4 Cytotoxicity assay

Cells (Huh-7 and Panc-1) were seeded in 96-well plates at a density of 1 × 104 cells per well, incubated for 24 h, and treated with the different concentrations of all isolated compounds (3.125, 6.25, 12.5, 25, and 50 μM) at 37 ℃ in 5% CO2 for another 24 h. A 10 μL amount of the Cell Counting Kit-8 (Biosharp, Shanghai, China) was added to the medium and incubated for 2 − 4 h; then the absorbance was read at a wavelength of 450 nm using a microplate reader (Shenzhen Sanli Technology Co. Ltd, Shenzhen, China). Sorafenib (G-CLONE, Beijing, China) and paclitaxel (G-CLONE, Beijing, China) were used as positive control [27]. The half-maximal inhibitory concentration (IC50) value was measured and calculated by GraphPad Prism 8 software.

Notes

Acknowledgements

The authors would like to thank the Natural Sciences Foundation of Guangdong Province (No. 2025A1515010551) and the Shenzhen Medical Research Funds (A2303041). The project was also funded by Shanghai Pujiang Program (No. 2021PJD091) and Shenzhen High-level Hospital Construction Fund (GSP-ZDSYS-009, GSP-QNPY-A2025004).

Author contributions

Xing-Wei Yang and Juan Huang conceived and designed the study. Yu-Feng Qiu and Yi-Zhou performed the isolation and purification of the compounds. Yu-Feng Qiu and Cheng Chen performed the pharmacological experiments and analysis of the data. The manuscript has been drafted by Yu-Feng Qiu and revised by Xing-Wei Yang. All authors have read and agreed to the published version.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Competing interests

The authors declare no competing financial interest.

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Authors and Affiliations

  1. 1. School of Pharmaceutical Sciences (Shenzhen), Sun Yat-Sen University, Shenzhen 518107, People's Republic of China
  2. 2. State Key Laboratory of Cardiovascular Disease, Fuwai Shenzhen Hospital, Chinese Academy of Medical Sciences, Shenzhen 518057, China
  3. 3. Department of Pharmacology, Shanghai Medicilon Inc., Shanghai, China