1 Introduction

The genus Hypericum (Hypericaceae Juss.) comprises nearly 500 species and is subdivided into 36 taxonomic sections [1]. The plants of this genus have been proved to be rich sources of prenylated phenolic metabolites with diverse phloroglucinols, xanthones, and flavonoids structures as well as valuable bioactivities such as antidepressant, antibacterial, and antiproliferative activities [2,3,4]. Our group has been engaged in the systematic studies of isoprenylated polyphenols from Hypericum species for more than ten years, and has discovered a series of fascinating chemical structures with intriguing biological activities [5,6,7,8,9,10,11,12,13,14,15]. Hypericum faberi R. Keller is distributed in the south and west of China [16]. Detailed literature research shows that it has never been phytochemical studied before. To further explore the new and bioactive natural polyphenols from Hypericum species, H. faberi was selected to carry out a phytochemical study and six new polyphenols, hyperfaberols A–F (1–6), together with additional seven known related derivatives 5,7-dihydroxy-2-isopropylchromon (7) [17], madeleinol A (8) [18], empetrifranzinans A/B (9/10) [18], otogirin (11) [19], dulxanthone D (12) [20], and toxyloxanthone B (13) [21] were isolated. It’s noteworthy that these isolates can be divided to three group based on their structural characters: isoprenylated xanthones (1–2 and 12–13), isoprenylated acylphloroglucinols (3–5 and 8–11), and chromones (6–7). In the bioactive assay, compounds 1 and 11 showed cytotoxities against the human esophageal cancer cell line (ECA-109) and the pancreatic tumor cell line (PANC-1) in vitro, respectively. Herein, we report the results of the isolates from the whole plant of H. faberi and their biological evaluation.

2 Results and Discussion

Hyperfaberol A (1) was obtained as a yellow amorphous powder. The HRESIMS data showed a deprotonated molecular ion at m/z 461.1979 [M–H]−, indicating a molecular formula of C28H30O6 (calcd for C28H29O6, 461.1970). The IR spectrum showed absorption bands of hydroxy (3423 cm−1) and conjugated carbonyl (1634 cm−1) functionalities. The 1H NMR spectrum showed a hydroxy group (δH 13.24), two aromatic protons (δH 6.14 and 6.73), two protons of a terminal double bond (δH 4.37, 4.48, 1H each, s), and five methyls (δH 1.34–1.64, s) (Table 1). The 13C and DEPT NMR data showed 28 carbon signals which were sorted by HSQC techniques as five methyls, three methylenes (including one terminal double bond, δC 111.0 and 147.2), six methines, and 14 quaternary carbons. The typical thirteen signals for a carbonyl (δC 181.6) and two penta-substitued phenyl units (δC 162.5, 160.3, 154.0, 153.5, 152.4, 138.2, 119.5, 106.0, 104.5, 102.4, 102.0, 97.1) can be easiliy distunguished in the 13C NMR, which indicated a xanthone skeleton of 1 as hinted by the UV absorption bands at 203, 267 and 333 [22, 23]. In the HMBC spectrum, the correlations from 1-HO to C-1, C-2, C-9, and C-9a; from H-2 to C-1, C-3, C-4, C-9, and C-9a; from H-5 to C-6, C-7, C-8a, C-9, and C-10a, and from H2-11 to C-3, C-4, and C-4a confirmed the xanthone framework (Fig. 2).

Table 1 13C (150 MHz) and 1H (600 MHz) NMR Data of 1 (in DMSO)
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Carefully analysis of the 1D NMR spectra indicated the remianing 15 carbon signals (from C-11 to C-25) should assigned to three isoprenyl derived units. Two of which were deduced to form a lavandulyl moiety by the 1H-1H COSY cross-peaks of H2-11/H-12/H2-13/H-14 together with the HMBC correlations from H2-20 to C-12, C-18 and C-19, and from H3-16/H3-17 to C-14 and C-15. The location of this lavandulyl moiety at C-4 was evidenced by the HMBC correlations of H-11 with C-3, C-4, and C-4a [24]. The third isoprenyl group was elucidated to form a characteristic pyran ring between C-7 and C-8 as revealed by the HMBC correlations from H-21 to C-7, C-8, C-8a, and C-23, and from H3-24/H3-25 to C-7, C-22, and C-23. Consequently, the structure of 1, hypefaberol A, was established as shown (Fig. 1).

Fig. 1
figure 1

Structures of 1–13

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Hyperfaberol B (2), a yellow amorphous powder, had the molecular formula C19H16O6 as deduced from its HRESIMS m/z 339.0881 [M–H]− (calcd for 339.0874) and 13C NMR data. The IR spectrum showed absorption bands of hydroxy (3370 cm−1) and conjugated carbonyl (1664 cm−1). The 1H NMR revealed the presence of one hydroxy (δH 13.15, s), three aromatic protons [δH 6.43 (1H, s), 6.97 (1H, d, J = 8.8 Hz), and 7.90 (1H, d, J = 8.8 Hz)], two protons of a terminal double bond (δH 4.94, 5.09, 1H each, s), one methyl (δH 1.76, 3H, s), and one methoxy group (δH 4.09, 3H, s) (Table 2). The 13C NMR spectrum exhibited 19 signals, including 15 sp2 carbons [one carbonyl, 12 aromatic carbons, and a terminal double bond signals (δC 113.0 and 143.3)] and four sp3 carbons (one methyl, one methoxy, one methylene, and one oxygenated methine). Side-by-side analysis of the 1H and 13C NMR spectra indicated that 2 were closely similar to the known xanthone, 2,3-dihydro-4-hydroxy-9-methoxy-2-(1-methylethenyl)-6H-furo[3,2-b]xanthen-5-one [25]. The only difference was that the aromatic proton (H-3) in the latter was replaced by a hydroxy group in 2, which can be confirmed by the downfiled chemical shift of 154.3 (C-3), the obvious HMBC correlations from the two ortho-coupled aromatic protons H-1/H-2 to C-3, from H-1 to C-9, C-9a, and C-4a, from H-2 to C-3, C-4, C-9, and C-9a, from H-5 to C-6, C-7, C-8a, C-9, and C-10a, from HO-8 to C-7, C-8, C-8a, and C-9, from H2-12 to C-6, C-7, and C-8, from H2-16 to C-13, C-14, and C-15, and the 1H-1H COSY between H2-12 and H-13. Thus, the structure of 2 was elucidated and named hyperfaberol B (Fig. 1).

Table 2 13C (150 MHz) and 1H (600 MHz) NMR Data of 2 (in CDCl3)
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Hyperfaberol C (3) was isolated as a yellow gum. The molecular formula was established as C21H32O6 from its 13C NMR (Table 3) and HRESIMS data (m/z 379.2136, [M–H]−, calcd for 379.2126). The IR absorptions implied hydroxy (3417 cm−1) and carbonyl (1622 cm−1) functionalities. Its 1H NMR spectrum showed three hydroxy groups (δH 10.26, 10.51 and 14.14), an isopropyl group (δH 1.05, 6H, d, J = 6.7 Hz; 3.89, sept., J = 6.7 Hz), three singlet methyls (δH 0.94, 1.01, and 1.66), a methoxy group (δH 3.03), one olefinic proton (δH 5.11), and one aromatic proton (δH 5.97). The 13C and DEPT NMR spectrum showed 21 carbon signals including 10 signals assignable to an isobutyryl (δC 209.5, C-7; 38.0, C-8; and 19.3, C-9/10) and a phloroglucinol core (δC 102.7, C-1; 163.8, C-2; 106.0, C-3; 162.2, C-4; 94.2, C-5; and 159.6, C-6), which indicated that 3 could be an acylphloroglucinol derivative. The location of the acyl group at C-1 was evidenced by the HMBC correlation of HO-2 with C-7 and further indicated the formation of intramolecular hydrogen bonding between the hydroxyl and the carbonyl group. In addition, the HMBC correlations from HO-2 to C-1, C-2, and C-3, from HO-4 to C-3, C-4, and C-5, and from HO-6 to C-1, C-5, and C-6 were all presented, which confirmed the phloroglucinol core furthermore. The remaining eleven signals was assigned to three methyls, one methoxy, three methylenes, two methines (including one sp2 methine), and two quaternary carbons (including one sp2 carbon). The 1H-1H COSY correlations of H2-11/H-12 and H2-15/H2-16/H-17, conjugated with the HMBC correlations from H3-14 to C-12, C-13, and C-15, from H3-19/H3-20 to C-17 and C-18, and from H3-21 to C-18, established the 6-hydroxy-7-methoxy-3,7-dimethyl-2-octenyl partial structure for the fifteen signals moiety (Fig. 2) [26]. Moreover, the HMBC correlations from H2-11 to C-2, C-3 and C-4 established the attachment of the moiety at C-3. The NOE correlations of H2-11/Me-14 and H2-12/H2-15 in the ROESY spectrum elucidated the 12E-configured double bond of the moiety. Thus, the structure of hyperfaberol C (3) was elucidated (Fig. 1).

Table 3 13C (150 MHz) and 1H (600 MHz) NMR Data of 3 and 4 (in DMSO)
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Fig. 2
figure 2

Key HMBC, 1H–1H COSY, and ROESY correlations of 1, 3, and 6

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Hyperfaberol D (4), was obtained as a yellow gum, the molecular formula, C20H28O5, was established by HRESIMS data (m/z 347.1871 [M–H]−, calcd for 347.1864). Comparison of the 1D NMR data indicated that the structures of 4 and 3 were very similar to each other (Table 3). The difference lied in that compound 3 has one more methoxy group than 4, meanwhile, the oxygenated quaternary carbon (δC 76.8, C-18) and Me-20 (δC 19.6) in 3 were also replaced by a terminal double bond (δC 148.2, C-18; and 109.8, C-20) in 4, which could be confirmed by the 1H-1H COSY cross-peaks of H2-11/H-12 and H2-15/H2-16/H-17, and the HMBC correlations from H3-14 to C-12, C-13, and C-15, from H2-20 to C-17, C-18, and C-19, and from H2-11 to C-2, C-3, and C-4. In the ROESY spectrum, an NOE cross-peak between Me-14 and H2-11 was observed, indicating the E configuration of C-12/13 double bond of the moiety (Fig. 2). Thus, the structure of 4 was therefore established to be that show in Fig. 1.

Hyperfaberol E (5) gave the molecular formula of C20H28O5 by the HRESIMS m/z 347.1876 [M–H]− (calcd for 347.1864). Its structure was elucidated to possess similar acylphloroglucinol scaffold with that of empetrikathiforin by detailed analysis of its MS and 1D NMR data [27]. The only difference lies in that the sec-butyl group in empetrikathiforin was replaced by an isopropyl in 5 [δH 3.86 (1H, sept., J = 6.7 Hz, H-8), 1.02 (3H each, d, J = 6.7 Hz, H-9/H-10); δC 38.1, C-8; 19.3, C-9/10] (Table 4), which was confirmed by the 1H-1H COSY cross-peaks of H-8/H3-9/H3-10 and the HMBC correlations of Me-9/Me-10 to C-7 and C-8 (Fig. 2).

Table 4 13C (150 MHz) and 1H (600 MHz) NMR Data of 5 (in DMSO)
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Hyperfaberol F (6) was isolated as an amorphous powder. Its molecular formula of C13H14O4 was assigned on the basis of its 13C NMR data and the observed [M–H]− ion at m/z 233.0823 (calcd for C13H13O4, 233.0819). The IR absorptions implied the presence of hydroxy (3417 cm−1) and conjugated carbonyl (1622 cm−1) groups. The 1H NMR revealed the presence of two aromatic protons at δH 6.14 (1H, d, J = 1.6 Hz) and 5.97 (1H, d, J = 1.6 Hz), and a singlet olefinic proton at δH 5.94 (1H, s), as well as a sec-butyl group at δH 2.49 (1H, m), 1.03 (3H, d, J = 6.9 Hz), 1.38 and 1.50 (2H, each m), and 0.68 (3H, s). The 13C NMR spectrum exhibited 13 signals, including nine sp2 carbons (one carbonyl, six aromatic carbons, and two olefinic carbons) and four sp3 carbons (two methyls, one methylene, and one methine) (Table 5). The nine downfield sp2 signals indicated that 6 possessed a chromone skeleton, while the four signals for sp3 carbons relatively upfield confirmed the presence of the sec-butyl group observed in the 1H NMR spectrum. The 1D NMR data of 6 was similar to those of 5, 7-dihydroxy-2-isopropylchromon (7) [17], the difference lies in that the isopropyl in 7 was substituted by a sec-butyl group (δC 39.4, C-9; 17.5, C-10; 26.9, C-11; and 11.4, C-12) in 6, which can be proved by the correlations from H-9 to C-2 in the HMBC spectrum, as well as the long linkage of H-9/H3-10/H2-11/H3-12 in the 1H-1H COSY spectrum (Fig. 2). Thus, the structrue of 6 was assigned as shown.

Table 5 13C (150 MHz) and 1H (600 MHz) NMR Data of 6 (in DMSO)
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Empetrifranzinan A/B (9/10) have been previously described from Hypericum empetrifolium [18]. However, the relative configurations of the two compounds were not given in previous literature, and which are demonstrated herein. In the ROESY spectrum of 9, the NOE correlations of H-11/H-12a, H-12a/H-15b/H-17, and H-12a/H3-14 suggested the β-orientation of H-11, Me-14, and H-17. The same relative configuration of 10 was also established as shown in Fig. 3.

Fig. 3
figure 3

Key NOESY correlations of 9 and 10

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The inhibitory activities of the isolates were examined against the five human tumor cell lines (ECA-109, PANC-1, BIU-87, and BEL-7402) with taxol as the positive control (IC50 values of 1.2, 3.5, 2.1 and 1.9 μM, respectively) [28]. Compound 1 showed cytotoxicity against the human esophageal cancer cell line (ECA-109) with an IC50 value of 12.8 μM, while compound 11 exhibited activity against the pancreatic tumor cell line (PANC-1) with an IC50 value of 12.0 μM. Additionally, other isolates showed no activities against the five human tumor cell lines with IC50 values > 30 μM.

3 Experimental

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. The chemical shifts (δ) were expressed in ppm with reference to the solvent signals. ESIMS and HR-ESIMS analysis were carried out on Waters Xevo TQS and Agilent G6230 TOF mass spectrometers, respectively. Preparative and semi-preparative HPLC were performed on Waters 1525 HPLC (column: SunFire OBD-C18, 19 × 250 mm) and Agilent 1100 HPLC (column: Zorbax SB-C18, 9.4 × 250 mm), respectively. Silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical Co., Ltd., People’s Republic of China), and MCI gel (75–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan) 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 ethanol.

3.2 Plant Material

The whole plants of H. faberi were collected in Yiliang County, Zhaotong City, Yunnan Province, China. It was identified by Dr. Yong-Zeng Zhang in Kunming Institute of Botany. A voucher specimen (no. 201707H01) has been deposited at the Kunming Institute of Botany.

3.3 Extraction and Isolation

The dried whole plants of H. faberi (15.0 kg) were powdered and percolated with MeOH at room temperature and filtered. The filtrate was evaporated in vacuo to be concentrated. The crude extract (3.2 kg) was subjected to a silica gel column chromatography (CC) using elution of successive CHCl3 and EtOAc to give a non-polar fraction (163.0 g) and a polar fraction (192 g). The CHCl3 fraction was separated by an MCI-gel CC (MeOH–H2O, 70–100%) to yield six fractions (Fr. 1–6, polarity from small to large). Fr. 3 (10.2 g) was separated by a Rp-18 column (49 × 460 mm, MeOH–H2O, 80–100%) to obtain eleven subfractions (Fr. 3-1 to Fr. 3-11) on the basis of TLC analysis. Fr. 3-7 (226.1 mg) was fractioned by preparative HPLC (MeOH–H2O, 88%) to afford Fr. 3-7-1, which was further purified by semipreparative HPLC (MeOH–H2O, 85%) to give the 11 (5.6 mg). Fr. 3-8 (271.7 mg) was fractioned by preparative HPLC (MeOH–H2O, 86%) to afford Fr. 3-8-1, which was further separated by semipreparative HPLC (MeOH–H2O, 80%) to obtain compound 1 (4.7 mg). Fr. 3-10 (184.2 mg) was purified by preparative HPLC (MeOH–H2O, 80%) to yield compound 9 (8.4 mg) and 10 (5.0 mg). Fr. 4 (11.5 g) was separated by a Rp-18 column (MeOH–H2O, 70–100%) to obtain thirteen subfractions (Fr. 4–1 to Fr. 4–13). Fr. 4–7 (394.6 mg) was fractioned by preparative HPLC (MeOH–H2O, 93%) to afford Fr. 4–7–1, which was then purified by semipreparative HPLC (MeOH–H2O, 90%) to afford the 2 (1.3 mg).

The EtOAc fraction (192 g) was separated by an MCI-gel CC (MeOH–H2O, 45–90%) to obtain five fractions (Fr. E1–E5, polarity from small to large). Fr. E1 (25.1 g) was separated by silica gel CC (petroleum ether/EtOAc, from 400:1 to 0:1) to give eight subfractions, Fr. E1–1 to Fr. E1–8. Fr. E1–4 (603.1 mg) was purified by repeated preparative HPLC (MeOH/H2O: 80%) to yield the 3 (2.6 mg). Fr. E2 (11.7 g) was separated by silica gel CC (petroleum ether/EtOAc, from 50:1 to 0:1) to give eleven subfractions, Fr. E2–1 to Fr. E2–11. Fr. E2–3 (142.3 mg) was further purified by preparative HPLC (MeOH/H2O: 75%) to yield the 5 (1.8 mg). Fr. E2–4 (1.4352 g) was further isolated sequentially using Sephadex LH-20 (Acetone), preparative HPLC (MeOH/H2O: 70%) to yield the 12 (1.7 mg). Fr. E3 (10.5 g) was separated by silica gel CC (petroleum ether/EtOAc, from 25:1 to 0:1) to obtain ten subfractions, Fr. E3–1 to Fr. E3–10. Fr. E3–3 (533.5 mg), Fr. E3–4 (392.1 mg), and Fr. E3–6 (1.2 g) was separated by Sephadex LH-20 (Acetone), and purified by prep-HPLC (MeOH/H2O: 70%) to afford compounds 6 (1.8 mg), 7 (2.3 mg), 8 (2.1 mg), 13 (1.5 mg). Fr. E3–8 (562.7 mg) was separated by silica gel CC (petroleum ether/EtOAc, from 10:1 to 0:1) to give eight subfractions, Fr. E3–8–1 to Fr. E3–8–8. Fr. E3–8–2 (405.2 mg) was further subjected to a silica gel CC (CDCl3/MeOH, from 400:1 to 0:1) to give Fr. E3–8–2–1 to Fr. E3–8–2–8. Fr. E3–8–2–4 (62.4 mg) was further purified by semipreparative HPLC (MeOH/H2O: 70%) to yield the 4 (3.3 mg).

3.3.1 Hyperfaberol A (1)

Yellow amorphous powder; [α] 22D +19 (c 0.12, MeOH); UV (MeOH) λmax (log ε): 385 (3.74), 333 (4.25), 267 (4.36), 245 (4.33), 203 (4.52) nm; IR (KBr) νmax: 3423, 2968, 2925,1634, 1574, 1454 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 461 [M–H]−; HR-ESIMS m/z 461.1979 [M–H]− (calcd for C28H29O6, 461.1970).

3.3.2 Hyperfaberol B (2)

Yellow amorphous powder; [α] 20D +6 (c 0.13, MeOH); UV (MeOH) λmax (log ε): 323 (4.4), 283 (4.02), 248 (4.66), 214 (4.40) nm; IR (KBr) νmax: 3370, 3083, 2925, 2854, 2731, 1664, 1613, 1518, 1403, 1374, 1228, 1161, 1032 cm−1; 1H and 13C NMR data, see Table 2; ESIMS m/z 339 [M–H]−; HR-ESIMS m/z 339.0881 [M–H]− (calcd for C19H15O6, 339.0874).

3.3.3 Hyperfaberol C (3)

Yellow gum; [α] 20D −34 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 291 (4.16), 197 (4.31) nm; IR (KBr) νmax 3417, 2975, 2932, 2874, 2125, 1622, 1519, 1431, 1383, 1354, 1301 cm−1; 1H and 13C NMR data, see Table 3; ESIMS m/z 379 [M–H]−; HR-ESIMS m/z 379.2136 [M–H]− (calcd for C21H31O6, 379.2126).

3.3.4 Hyperfaberol D (4)

Yellow gum; [α] 20D −23 (c 0.16, MeOH); UV (MeOH) λmax (log ε) 291 (3.86), 196 (4.06) nm; IR (KBr) νmax 3427, 2969, 2929, 2876, 2730, 1602, 1519, 1384, 1296, 1140, 1034 cm−1; 1H and 13C NMR data, see Table 3; ESIMS m/z 347 [M–H]−; HR-ESIMS m/z 347.1871 (calcd for C20H27O5, 347.1864).

3.3.5 Hyperfaberol E (5)

Yellow gum; [α] 20D −19 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 290 (3.23), 228 (3.15), 197 (3.39) nm; IR (KBr) νmax 3428, 2966, 2932, 2873, 1623, 1518, 1433, 1354, 1238, 1171, 1023 cm−1; 1H and 13C NMR data, see Table 4; ESIMS m/z 347 [M–H]−; HR-ESIMS m/z 347.1876 [M–H]− (calcd for C20H27O5, 347.1864).

3.3.6 Hyperfaberol F (6)

Yellow powder; [α] 20D −12 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 294 (3.82), 250 (4.19), 227 (4.14), 202 (4.31) nm; IR (KBr) νmax 3417, 3093, 3071, 2967, 2934, 2878, 2726, 2633, 1622, 1107, 1011 cm−1; 1H and 13C NMR data, see Table 5; ESIMS m/z 233 [M–H]−; HR-ESIMS m/z 233.0823 [M–H]− (calcd for C13H13O4, 233.0819).

3.3.7 Cytotoxcity Assay

The cytotoxic assay with taxol as a positive control was conducted by MTT method in 96-well plates reported previously (Biological Assay, Supporting Information) [7].