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Erecricins A–E, prenylated acylphloroglucinols from the roots of Hypericum erectum
Fitoterapia 114 (2016) 188–193
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Erecricins A–E, prenylated acylphloroglucinols from the roots of Hypericum erectum Shuangxin Lu a, Naonobu Tanaka a,b,⁎, Yutaka Tatano c, Yoshiki Kashiwada a,⁎⁎ a b c
Graduate School of Pharmaceutical Sciences, Tokushima University, Tokushima 770-8505, Japan Graduate School of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8513, Japan Division of Immunobiology, Department of Pharmaceutical Sciences, International University of Health and Welfare, Tochigi 324-8501, Japan
a r t i c l e
i n f o
Article history: Received 30 June 2016 Received in revised form 21 August 2016 Accepted 25 August 2016 Available online 26 August 2016 Keywords: Acylphloroglucinol Hypericum erectum Hypericaceae Erecricins A–E Adotogirin
a b s t r a c t Six new prenylated acylphloroglucinols, erecricins A–E (1–5) and adotogirin (6), were isolated from the roots of Hypericum erectum (Hypericaceae). Their structures were assigned on the basis of spectroscopic evidences. Erecricins A–E (1–5) are bicyclic prenylated acylphloroglucinols possessing a chromane or a chromene skeleton. Adotogirin (6) is a simple achylphloroglucinol with an O-geranyl moiety. Antimicrobial activities of these acylphloroglucinols were also evaluated. © 2016 Elsevier B.V. All rights reserved.
1. Introduction
2. Experimental
The genus Hypericum (Hypericaceae) comprises almost 500 species germinating as herbs, shrubs, and trees, which are growing in temperate sections and mountains in the tropical areas [1]. Hypericum plants have been used as herbal remedies for the treatment of cuts, burns, melancholia, anxiety, abdominal, and urogenital pains [2–5]. A variety of prenylated acylphloroglucinols with interesting biological activities such as antibacterial, antiviral, antiproliferative, antiinflammatory, antioxidative, and antidepressant activities have been isolated from Hypericum plants to date [4–7]. Hypericum erectum is distributed in Japan, China, and Korea. The aerial parts of H. erectum are of medicinal values to heal hurts, sooth bruises, and malarial fever [2]. Some antibacterial, antiplasmoidal, and antihemorrhagic prenylated phloroglucinols have been isolated from this species [8–10]. In our continuing research on the constituents of Hypericum plants [6,11–13], the roots of H. erectum were investigated, which resulted in the isolation of six new prenylated acylphloroglucinols, erecricins A–E (1–5) and adotogirin (6). Herein, we describe the isolation, structure elucidation, and antimicrobial activities of 1–6.
2.1. General experimental procedures
⁎ Correspondence to: N. Tanaka, Graduate School of Pharmaceutical Sciences, Tokushima University, Tokushima 770-8505, Japan. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (N. Tanaka), [email protected] (Y. Kashiwada).
http://dx.doi.org/10.1016/j.fitote.2016.08.014 0367-326X/© 2016 Elsevier B.V. All rights reserved.
Optical rotations were measured on a JASCO P-2200 digital polarimeter. MS were obtained on a Waters LCT PREMIER 2695. NMR spectra were measured with a Bruker AVANCE-500 instrument using tetramethylsilane as an internal standard. Column chromatography was performed with silica gel 60 N (63–210 μm, Kanto Chemical), MCI gel CHP-20P (75–150 μm, Mitsubishi Chemical), YMC gel ODS-A (S-50 μm, YMC Co., Ltd.), and Sephadex LH-20 (25–100 μm, GE Health Care). HPLC was performed on YMC-Triart C18 (5 μm, ϕ20 × 250 mm; YMC Co., Ltd.), Mightysil RP-18 GP (5 μm, ϕ20 × 250 mm; Kanto Chemical), Mightysil RP-18 GP-II (5 μm, ϕ20 × 250 mm), COSMOSIL 5C18-AR-II (5 μm, ϕ20 × 250 mm, Nacalai Tesque), and COSMOSIL 5C18-MS-II (5 μm, ϕ20 × 250 mm). 2.2. Plant material Hypericum erectum was cultivated at the medicinal herb garden of Tokushima University, and was collected in July 2014. A voucher specimen (HYE201407) was deposited in the herbarium of Graduate School of Pharmaceutical Sciences, Tokushima University. 2.3. Extraction and isolation The dried roots (2.0 kg) of H. erectum were extracted with MeOH at room temperature to give the extract (170.7 g), which was partitioned
S. Lu et al. / Fitoterapia 114 (2016) 188–193
with EtOAc and water. The EtOAc-soluble material was partitioned with n-hexane and 90% MeOH aq. The n-hexane-soluble material was subjected to silica gel column chromatography (n-hexane/EtOAc, 9:1 to 0:10) to give nine fractions (frs. 1–9). Fr. 2 was fractionated by MCI CHP-20P column chromatography (MeOH/H2O, 0:1 to 1:0) to afford three fractions (frs. 2.1–3). Fr. 2.3 was applied to an ODS column (MeOH/H2O, 0:1 to 1:0), and purified by HPLC on YMC-Triart C18 (MeOH/H2O, 95:5) to yield frs. 2.3.3.1–8, including erecricin B (2, 23.8 mg, fr. 2.3.3.7). Erecricins A (1, 3.1 mg) and C (3, 1.5 mg) were isolated from fr. 2.3.3.5 and fr. 2.3.3.6, respectively, using HPLC on COSMOSIL 5C18-MS-II (MeOH/H2O, 95:5). Fr. 2.2 was separated by ODS column chromatography (MeOH/H2O, 0:1 to 1:0) and purified by HPLC on COSMOSIL 5C18-MS-II (MeOH/H2O, 95:5) to give erecricins D (4, 5.3 mg) and E (5, 22.0 mg). Fr. 3 was separated by chromatography on an ODS column (MeOH/H2O, 0:1 to 1:0), an MCI CHP-20P column (MeOH/H2O, 0:1 to 1:0), and a silica gel column (n-hexane/acetone, 95:5 to 0:10), and purified by HPLC on COSMOSIL 5C18-MS-II (MeOH/ H2O, 80:20) to yield adotogirin (6, 22.5 mg) and otogirin (7, 3.3 mg). 2.4. Erecricin A (1) Colorless oil; [α]D + 58.9 (c 0.34, CHCl3); HRESIMS m/z 479.3151 [M − H]− (calcd for C31H43O4, 479.3161); 1H and 13C NMR data (Table 1). 2.5. Erecricin B (2) Colorless oil; [α]D + 69.4 (c 2.70, CHCl3); HRESIMS m/z 517.3317 [M + Na]+ (calcd for C32H46O4Na, 517.3294); 1H and 13C NMR data (Table 1).
189
2.6. Erecricin C (3) Colorless oil; [α]D − 60.0 (c 0.18, CHCl3); HRESIMS m/z 517.3269 [M + Na]+ (calcd for C32H46O4Na, 517.3294); 1H and 13C NMR data (Table 1).
2.7. Erecricin D (4) Colorless oil; [α]D − 25.2 (c 0.44, CHCl3); HRESIMS m/z 479.3151 [M − H] − (calcd for C 31H43 O4 , 479.3161); 1 H and 13C NMR for major tautomer 4a (Table 2); 1H NMR for minor tautomer 4b: δ H 18.66 (1H, s, 1-OH), 6.53 (1H, d, J = 10.1 Hz, H-23), 5.86 (1H, dd, J = 15.5, 10.9 Hz, H-12), 5.50 (1H, d, J = 10.9 Hz, H-11), 5.33 (1H, d, J = 10.1 Hz, H-24), 5.30 (1H, m, H-13), 4.99 (1H, brt, J = 6.9 Hz, H-18), 4.09 (1H, sept, J = 6.8 Hz, H-29), 2.57 (1H, m, H-8), 2.32 (1H, m, H-7a), 2.18 (1H, m, H-14), 1.97 (1H, m, H-17a), 1.90 (1H, m, H-7b), 1.87 (1H, m, H-17b), 1.65 (3H, s, H 3 -21), 1.56 (3H, s, H3-20), 1.53 (3H, s, H3-27), 1.52 (3H, s, H3-10), 1.43 (3H, s, H3 -26), 1.41 (3H, s, H3 -22), 1.15 (3H, d, J = 6.8 Hz, H3-30), 1.11 (3H, d, J = 6.8 Hz, H3-31), 0.94 (3H, d, J = 6.7 Hz, H 3-15), and 0.92 (3H, d, J = 6.7 Hz, H 3 -16); 13C NMR for minor tautomer 4b: δC 209.1 (C-28), 197.5 (C-1), 179.7 (C-5), 165.0 (C-3), 139.9 (C-13), 137.1 (C-9), 132.4 (C-19), 129.0 (C-11), 123.7 (C-24), 122.7 (C-12), 122.5 (C-18), 116.4 (C-23), 109.9 (C-4), 109.6 (C-6), 79.1 (C-25), 47.0 (C-2), 40.1 (C-7), 38.4 (C-8), 35.4 (C-29), 33.3 (C17), 31.3 (C-14), 29.3 (C-27), 28.3 (C-26), 26.9 (C-22), 25.7 (C-21), 22.1 (C-15), 22.4 (C-16), 18.9 × 2 (C-30 and C-31), 18.4 (C-10), and 17.9 (C-20).
Table 1 1 H and 13C NMR data for erecricins A–C (1–3) in CDCl3. 1
2
3
Position
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1 2 3 4 5 5-OH 6 7
197.7 48.0 171.6 115.6 189.3 – 105.4 31.0
197.8 48.0 171.5 115.7 189.3 – 106.3 30.8
40.3 86.0 19.1 133.2 126.7 124.3 137.0 18.4 26.0 29.5
18 19 20 21 22 23 24 25 26 27 28 29 30 31
121.5 133.7 17.9 25.8 28.5 21.2 121.9 131.7 17.9 25.7 208.0 35.4 18.8 18.9
– – – – – 19.10 (1H, s) – 2.18 (1H, dd, 14.4, 3.9) 1.44 (1H, d, 14.4) 2.03 (1H, m) – 1.17 (3H, s) 5.73 (1H, d, 15.3) 6.54 (1H, dd, 15.3, 10.9) 5.86 (1H, d, 10.9) – 1.80 (3H, s) 1.78 (3H, s) 2.07 (1H, m) 1.69 (1H, m) 5.05 (1H, m) – 1.57 (3H, s) 1.68 (3H, s) 1.41 (3H, s) 3.05 (2H, m) 5.04 (1H, m) – 1.68 (3H, s) 1.64 (3H, s) – 3.73 (1H, qt, 6.8, 6.8) 1.16 (1H, d, 6.8) 1.71 (1H, m) 1.38 (1H, m) 0.85 (3H, t, 7.4)
197.8 48.5 172.6 114.4 189.4 – 106.3 30.7
8 9 10 11 12 13 14 15 16 17
– – – – – 19.04 (1H, s) – 2.20 (1H, dd, 14.3, 3.8) 1.46 (1H, d, 14.3) 2.04 (1H, m) – 1.19 (3H, s) 5.74 (1H, d, 15.3) 6.65 (1H, dd, 15.3, 10.9) 5.88 (1H, d, 10.9) – 1.80 (3H, s) 1.82 (3H, s) 2.10 (1H, m) 1.72 (1H, m) 5.07 (1H, m) – 1.58 (3H, s) 1.70 (3H, s) 1.42 (3H, s) 3.07 (2H, m) 5.05 (1H, m) – 1.70 (3H, s) 1.66 (3H, s) – 3.90 (1H, sept, 6.8) 1.11 (3H, d, 6.8) 1.18 (3H, d, 6.8)
– – – – – 19.06 (1H, s) – 2.05 (1H, m) 1.41 (1H, m) 2.04 (1H, m) – 1.62 (3H, s) 5.44 (1H, d, 15.3) 6.32 (1H, dd, 15.3, 10.8) 5.73 (1H, d, 10.8) – 1.71 (3H, s) 1.75 (3H, s) 2.13 (1H, m) 1.74 (1H, m) 5.11 (1H, m) – 1.60 (3H, s) 1.71 (3H, s) 1.43 (3H, s) 3.09 (2H, m) 5.08 (1H, m) – 1.68 (3H, s) 1.62 (3H, s) – 3.75 (1H, qt, 6.8, 6.8) 1.17 (1H, d, 6.8) 1.72 (1H, m) 1.39 (1H, m) 0.86 (3H, t, 7.4)
32
40.3 85.9 18.9 133.1 126.7 124.3 137.0 18.3 26.1 29.5 121.5 133.7 17.9 25.8 28.4 21.2 121.9 131.7 17.9 25.7 207.2 41.7 17.0 26.6 11.9
41.7 86.5 25.8 128.9 127.8 124.4 136.9 18.3 26.0 29.9 121.6 133.7 17.9 25.9 28.9 21.3 121.6 131.8 17.9 25.6 206.9 41.6 17.1 26.7 11.8
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S. Lu et al. / Fitoterapia 114 (2016) 188–193
2.8. Erecricin E (5)
Table 3 1 H and 13C NMR data for adotogirin (6) in CDCl3.
Colorless oil; [α]D − 3.8 (c 2.17, CHCl3); HRESIMS m/z 533.3223 [M + Na]+ (calcd for C32H46O5Na, 533.3243); 1H and 13C NMR for major tautomer 5a (Table 2); 1H NMR for minor tautomer 5b: δH 18.82 (1H, s, 1-OH), 6.52 (1H, d, J = 10.0 Hz, H-23), 5.86 (1H, dd, J = 14.9, 10.9 Hz, H-12), 5.49 (1H, d, J = 10.9 Hz, H-11), 5.33 (1H, d, J = 10.0 Hz, H-24), 5.27 (1H, dd, J = 14.9, 7.1 Hz, H-13), 4.99 (1H, brs, H18), 4.00 (1H, qt, J = 6.6, 6.6 Hz, H-29), 2.57 (1H, m, H-8), 2.31 (1H, m, H-7a), 2.18 (1H, m, H-14), 1.99 (1H, m, H-17a), 1.90 (1H, m, H-7b), 1.86 (1H, m, H-17b), 1.71 (1H, m H-31a), 1.65 (3H, s, H3-21), 1.55 (3H, s, H3-20), 1.55 (3H, s, H3-10), 1.52 (3H, s, H3-27), 1.38 (3H, s, H326), 1.40 (3H, s, H3-22), 1.36 (1H, m H-31b), 1.12 (3H, d, J = 6.6 Hz, H3-30), 0.94 (3H, d, J = 6.6 Hz, H3-15), 0.92 (3H, d, J = 6.6 Hz, H3-16), and 0.89 (3H, dd, J = 12.7, 6.9 Hz, H3-32); 13C NMR for minor tautomer 5b: δC 208.6 (C-28), 198.2 (C-1), 180.4 (C-5), 164.9 (C-3), 139.8 (C-13), 137.0 (C-9), 132.1 (C-19), 128.9 (C-11), 123.4 (C-24), 122.6 (C-12), 122.4 (C-18), 116.3 (C-23), 109.6 (C-6), 109.3 (C-4), 79.1 (C-25), 47.0 (C-2), 42.0 (C-29), 39.9 (C-7), 38.3 (C-8), 33.1 (C-17), 31.1 (C-14), 29.2 (C-27), 28.3 (C-26), 27.3 (C-22), 26.3 (C-31), 25.6 (C-21), 22.4 (C-16), 22.3 (C-15), 18.4 (C-10), 17.8 (C-20), 16.4 (C-30), and 11.7 (C32).
Position
δC
δH (J in Hz)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
160.2a) 103.9b) 162.5 92.5 160.8a) 104.3b) 7.2 65.3 118.9 141.4 16.7 39.4 26.2 123.6 131.9 17.7 25.6 210.4 45.9 16.7 27.0
22
11.9
– – – 5.96 (1H, s) – – 2.02 (3H, s) 4.52 (2H, d, 6.3) 5.44 (1H, t, 6.3) – 1.71 (3H, s) 2.08 (2H, m) 2.12 (2H, m) 5.08 (1H, t, 6.6) – 1.60 (3H, s) 1.67 (3H, s) – 3.77 (1H, qt, 6.8, 6.8) 1.16 (3H, d, 6.8) 1.84 (1H, m) 1.41 (1H, m) 0.91 (3H, t, 7.4)
a,b)
Signals may be interchangeable.
2.9. Adotogirin (6) 2.10. Antimicrobial assay Pale yellow amorphous solid; [α]D + 7.7 (c 0.21, MeOH); HRESIMS m/z 383.2199 [M + Na]+ (calcd for C22H32O4Na, 383.2198); 1H and 13 C NMR data (Table 3). Table 2 1 H and 13C NMR data for major tautomers (4a and 5a) of erecricins D (4) and E (5) in CDCl3. 4a
5a
Position
δC
δH (J in Hz)
δC
δH (J in Hz)
1 2 3 4 5 5-OH 6 7
193.9 51.5 172.6 105.1 185.9 – 105.8 40.1
193.9 51.4 172.2 104.8 185.9 – 106.4 39.9
8 9 10 11 12 13 14 15 16 17
38.4 137.1 18.5 129.0 122.7 139.9 31.3 22.1 22.4 33.3
– – – – – 19.08 (1H, s) – 2.32 (each 1H, m) 1.90 (1H, m) 2.50 (1H, m) – 1.54 (3H, s) 5.50 (1H, d, 10.9) 5.86 (1H, dd, 15.5, 10.9) 5.28 (1H, dd, 15.5, 6.6) 2.18 (1H, m) 0.95 (3H, d, 6.8) 0.92 (3H, d, 6.8) 1.97 (1H, m) 1.87 (1H, m) 4.99 (1H, brt, 6.9) – 1.56 (3H, s) 1.65 (3H, s) 1.29 (3H, s) 6.45 (1H, d, 10.1) 5.38 (1H, d, 10.1) – 1.43 (3H, s) 1.56 (3H, s) – 3.86 (1H, sept, 6.9) 1.13 (3H, d, 6.9) 1.12 (3H, d, 6.9)
– – – – – 19.01 (1H, s) – 2.31 (each 1H, m) 1.90 (1H, m) 2.50 (1H, m) – 1.55 (3H, s) 5.49 (1H, d, 10.9) 5.86 (1H, dd, 14.9, 10.9) 5.28 (1H, dd, 14.9, 6.8) 2.18 (1H, m) 0.94 (3H, d, 6.7) 0.92 (3H, d, 6.7) 1.99 (1H, m) 1.86 (1H, m) 4.99 (1H, brs) – 1.55 (3H, s) 1.65 (3H, s) 1.28 (3H, s) 6.45 (1H, d, 10.1) 5.37 (1H, d, 10.1) – 1.43 (3H, s) 1.56 (3H, s) – 3.75 (1H, qt, 6.9, 6.9) 1.10 (1H, d, 6.9) 1.71 (1H, m) 1.36 (1H, m) 0.89 (3H, brt, 7.8)
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
122.5 132.4 17.9 25.7 27.5 115.0 123.3 80.7 28.3 29.3 207.7 35.4 18.9 18.9
38.3 137.0 18.4 128.9 122.6 139.8 31.1 22.0 22.4 33.1 122.4 132.1 17.8 25.6 27.3 115.0 123.2 80.7 28.3 29.2 207.1 41.7 11.9 26.3 11.7
2.10.1. Test microorganisms Seven clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) strains, three clinical isolates of methicillin-sensitive S. aureus (MSSA) strains, S. aureus 209P and Smith, one Bacillus subtilis strain, and four Escherichia coli strains were used in this study. All the strains of microorganisms used in this work were kindly supplied by Dr. C. Sano, the School of Medicine, Shimane University (Shimane, Japan).
2.10.2. Susceptibility testing The MICs were determined by broth microdilution method in 96well microtiter plates with cation-supplemented Mueller-Hinton broth (CAMHB; Ca2+, 25 μg/mL; Mg2+, 12.5 μg/mL; Becton Dickinson, Sparks, MD) according to the current guidelines of the Clinical and Laboratory Standards Institute (CLSI). All the strains of microorganisms were inoculated at a final bacterial density of about 5 × 105 CFU/mL. Then, S. aureus strains were incubated at 35 °C for 20 h, and B. subtilis and E. coli strains were incubated at 37 °C for 20 h before the MICs
21 31
30
O
29 28 6
O
1
22
18 8
20
17
15
2
5
12 9 11
4
HO
19
7
O
14 13
16
10 23 24 25 27
1H-1H
26
1
COSY HMBC NOESY
Fig. 1. Selected 2D NMR correlations of erecricin A (1).
S. Lu et al. / Fitoterapia 114 (2016) 188–193
2
7
8
17
22 10 9
3
NOESY
1
Fig. 2. Selected NOESY correlations and the relative stereochemistry for the chromane core of erecricin A (1) (protons of methyl groups were omitted).
were determined. The MICs of the test compounds were reported as an MIC, MIC range, and MIC50. 3. Results and discussion The MeOH extract of H. erectum roots was partitioned with EtOAc and water. The EtOAc-soluble material was further partitioned with 90% MeOH aq. and n-hexane. Repeated chromatographic separations of the n-hexane-soluble material gave six new prenylated acylphloroglucinols, erecricins A–E (1–5) and adotogirin (6), together with one known prenylated acylphloroglucinol (7) which was identified as otogirin by comparison of the spectroscopic data with the literature data [8]. Erecricin A (1) was obtained as an optical active colorless oil {[α]D + 58.9 (c 0.34, CHCl3)}, and its molecular formula was established as C31H44O4 by the HRESIMS (m/z 479.3151 [M − H]−, Δ −1.0 mmu). The 1H NMR spectrum showed the signals of five olefinic protons, three sp3 methylenes, two sp3 methines, and ten methyls as well as the characteristic down-field shifted signal (δH 19.04, s) due to a hydrogen-bonded hydroxy group (Table 1), indicating the existence of a β-diketone moiety with an enol form [10]. The 13C NMR and DEPT spectra suggested the presence of 31 carbons including two enols, four
191
olefins, two ketone carbonyl carbons, one quaternary carbon, and one oxygenated tertiary carbon (Table 1). From these observations, erecricin A (1) was deduced to be a prenylated acylphloroglucinol with four isoprene units. Comparison of the 1D NMR spectra of 1 with the literature data implied that 1 had a structure similar to hypelodin A [11], a prenylated acylphloroglucinol possessing the chromane core, but had different substituents at C-2 and C-6. The substituent at C-2 in 1 was assigned as a methyl group by HMBC interpretations of H3-22 with C1, C-2, C-3, and C-7 (Fig. 1), while a 2-methylpropanoyl group at C-6 was revealed by 1H-1H COSY cross-peaks of H-29 with H3-30 and H331 and HMBC correlations for H3-30 with C-28, OH-5 with C-4, C-5, C6 and C-28. Therefore, the gross structure of 1 was elucidated as shown in Fig. 1. NOESY correlations for H-8/H3-22, H-7β/H3-10, and H-17b/H3-10 suggested the pseudochair conformation of the tetrahydropyran ring (C-2, C-3, and C-7–C-9) and the pseudoaxial orientations for H-7β, H8, 10-Me, and 22-Me (Fig. 2). This was underpinned by a large 3J value of the vicinal coupling for H-7β/H-8 (J = 14.3 Hz) (Table 1). Thus, the relative stereochemistry for 1 was assigned as shown in Chart 1. The HRESIMS revealed the molecular formula of erecricin B (2) to be C32H46O4 {m/z 517.3317 [M + Na]+, Δ +2.3 mmu}, larger by 14 mass units than that of 1. Though the 1D NMR spectra of 2 were similar to those of 1, the resonances of a 2-methylbutanoyl group were observed for 2 in place of the signals of the 2-methylpropanyl moiety for 1. The presence of the 2-methylbutanoyl group at C-6 in 2 was confirmed by HMBC analysis. The relative configuration of C-29 remains to be assigned, since any NOESY correlations were not observed. Thus, erecricin B was elucidated to be 2 (Chart 1). The molecular formula of erecricin C (3) was assigned as C32H46O4 by the HRESIMS {m/z 517.3269 [M + Na]+, Δ −2.5 mmu}, which was identical to that of 2. Analysis of the 1H and 13C NMR spectra (Table 2) indicated 3 to be a diastereomer of 2 at the tetrahydropyran moiety. NOESY correlations of H3-22/H-8 and H-7β/H-11 revealed the pseudoaxial orientations of H-7β, 22-Me, and the substituent at C-9 (Fig. 3). Therefore, the structure of 3 was assigned as shown in Chart 1. Erecricins D (4) and E (5) were individually isolated as optical active colorless oil {[α]D − 25.2 (c 0.44, CHCl3) for 4; [α]D − 3.8 (c 2.17, CHCl3) for 5}. The molecular formula of 4 was assigned to be C31H44O4 in light of the HRESIMS (m/z 479.3151 [M − H]−, Δ −1.0 mmu). In the 1H NMR
21
R 30 29 28
O
19
O
18
22
O
20
7
1 6
8
15
17
4
O
21
14
9 3
HO
O
O
2 5
11
12
13
HO
16
19
R
O
10
23
OH
31
6
7
11
16
2 8
20
22
24
1 18
HO
O
4
13 9
10
15 12
14
17
25 27
26
R 1 2
R
H Me
3
7 6
H Me
R O O HO
15
10
O
22 7
1
8
6
2
5
3
11
12
13
16
R
6 5
17
O
4
25
23 24
O
18 26
OH 1
14 9
O
19
20
21
27
4a 5a
R H Me
R 4b 5b
H Me
Chart 1. Structures of erecricins A–E (1–5), adotogirin (6), and otogirin (7) (4a/5a and 4b/5b are major and minor tautomers of 4 and 5, respectively).
S. Lu et al. / Fitoterapia 114 (2016) 188–193
OH
192
O 8
21
7
22
11
2
3
HO
10
6
15
10 22 12
O 5
4
HO
2 3
7
8 17
O
14
9 11
13
16
18 27
25
23
19 24
21
26
1H-1H
4a
O
8
9
15
10 12
14
17
1H-1H
spectrum of 4, a pair of down-field shifted hydroxy proton signals (δH 19.08 and 18.66) were observed in a ratio of ca. 3:1, indicating the presence of two tautomers (4a and 4b). The feature of the 1D NMR spectra of 4 were similar to those of hyperguinone B [14], a prenylated acylphlorglucinol with a chromene core, whereas the signals of the different substituent consisting of 15 carbons were observed for 4 in place of the resonances due to the prenyl group at C-2 in hyperguinone B. The substituent was elucidated by 2D NMR analysis (Fig. 4). The 1H-1H COSY spectrum indicated the connectivities of C-7–C-8, C-11–C-14, C-14–C15, and C-14–C-16, while the connectivities among C-8, C-10, and C11 via C-9 were suggested by HMBC correlations for H3-10 with C-8, C-9, and C-11. HMBC correlations for H3-21 with C-18, C-19, and C-20 and 1H-1H cross-peaks of H-8/H2-17 and H2-17/H-18 revealed the presence of a prenyl group at C-8. Similarly, erecricin E (5) was elucidated to be a prenylated acylphloroglucinol possessing the same chromene core as seen in 4 with a 2-methylbutanoyl group at C-6. Thus, the planar structures of erecricins D (4) and E (5) were assigned as shown in Chart 1, while the stereochemistries for 4 and 5 were not elucidated. The 1H and 13C NMR spectra of adotogirin (6) (Table 3), C22H32O4, resembled to those of a known acylphloroglucinol otogirin (7), except for the presence of the signals due to a sec-butyl group in place of those due to the isopropyl group in 7. These observations implied that 6 is an prenylated acylphloroglucinol with a 2-methylbutanoyl group, a methyl group, and an O-geranyl group. The structure of adotogirin (6) was confirmed by analysis of the 2D NMR spectra (Fig. 5). Erecricins A–E (1–5), adotogirin (6), and otogirin (7) were evaluated for their antimicrobial activities on strains of Staphylococcus aureus (MRSA and MSSA), Bacillus subtilis, and Escherichia coli. Among the tested compounds, adotogirin (6) and otogirin (7) exhibited an antimicrobial activity against MRSA {6: MIC range 0.5–4.0 μg/mL (MIC50 1.0 μg/mL); 7: MIC range 0.5–8.0 μg/mL (MIC50 0.5 μg/mL)}, MSSA {6: MICs 1.0 μg/mL for all strains; 7: MIC range 0.5–1.0 μg/mL (MIC50
1
4
NOESY
Fig. 3. Selected NOESY correlations and the relative stereochemistry for the chromane core of erecricin C (3) (protons of methyl groups were omitted).
6
16
11 13
3
O
7
2
6
19 20
9
22
29 28
1
18
COSY HMBC
Fig. 4. Selected 2D NMR correlations for major tautomer 4a of erecricin D (4).
COSY HMBC NOESY
Fig. 5. Selected 2D NMR correlations for adotogirin (6).
0.5 μg/mL)}, and Bacillus subtilis (MIC each 2.0 μg/mL), while antiplasmodial activity, cytotoxicity, and antagonistic activity against thromboxane A2 and leukotriene D4 of 7 have been reported [8]. In contrast, erecricins A–E (1–5) did not show any antimicrobial activity. Investigation of the MeOH extract from the roots of Hypericum erectum afforded five new bicyclic prenylated achylphloroglucinols, erecricins A–E (1–5), and one prenylated achylphloroglucinols with an O-geranyl moiety, adotogirin (6), together with a known prenylated acylphlotoglucinol, otogirin (7), whose structures were elucidated by spectroscopic analysis. O-Prenylated acylphloroglucniols are relatively rare than C-prenylated acylphlorogluciniols. Antimicrobial activities of various phloroglucinols possessing O-prenylated, dimeric or more complex structures have been reported to date [6,15]. Adotogirin (6) and otogirin (7) appeared to be potent antibacterial agents against Staphylococcus aureus, and Bacillus subtilis. Conflict of interest The authors declare no conflict of interest. Acknowledgment This work was partly supported by a Grant-in-Aid for Scientific Research (No. 15K18885) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References [1] N.M. Nürk, S. Madriñán, M.A. Carine, M.W. Chase, F.R. Blattner, Molecular phylogenetics and morphological evolution of St. John's wort (Hypericum; Hypericaceae), Mol. Phylogenet. Evol. 66 (2013) 1–16. [2] C. Wiart, Anti-inflammatory plants, Ethnopharmacology of Medicinal Plants: Asia and the Pacific, Humana Press Inc., Totowa, New Jersey 2006, pp. 1–50. [3] J. Sarris, Herbal medicines in the treatment of psychiatric disorders: a systematic review, Phytother. Res. 21 (2007) 703–716. [4] G. Laakmann, C. Schüle, T. Baghai, M. Kieser, St. John's wort in mild to moderate depression: the relevance of hyperforin for the clinical efficacy, Pharmacopsychiatry 31 (1998) 54–59. [5] P. Avoto, A survey on the Hypericum genus: secondary metabolites and bioactivity, in: A. Atta-ur-Rahman (Ed.), Studies in Natural Products Chemistry, Elsevier Science BV, Amsterdam 2005, pp. 603–634. [6] N. Tanaka, J. Kobayashi, Prenylated acylphloroglucinols and meroterpenoids from Hypericum plants, Heterocycles 90 (2015) 23–40. [7] Y.-B. Chen, A.E. Fazary, Y.-C. Lin, I.-W. Lo, S.-C. Ong, S.-Y. Chen, C.-T. Chien, Y.-J. Lin, W.-W. Lin, Y.-C. Shen, Hyperinakin, a new anti-inflammatory phloroglucinol derivative from Hypericum nakamurai, Nat. Prod. Res. 27 (2013) 727–734. [8] M. Tada, K. Chiba, H. Yamada, H. Maruyama, Phloroglucinol derivatives as competitive inhibitors against thromboxane A2 and leukotriene D4 from Hypericum erectum, Phytochemistry 30 (1991) 2559–2562. [9] T. Kosuge, H. Ishida, T. Satoh, Studies on antihemorrhagic substances in herbs classified as hemostatics in Chinese medicine, IV. On antihemorrhagic principles in Hypericum erectum Thunb, Chem. Pharm. Bull. 33 (1985) 202–205. [10] H.I. Moon, Antiplasmodial and cytotoxic activity of phloroglucinol derivatives from Hypericum erectum Thunb, Phytother. Res. 24 (2010) 941–944. [11] C. Hashida, N. Tanaka, K. Kawazoe, K. Murakami, H.D. Sun, Y. Takaishi, Y. Kashiwada, Hypelodins A and B, polyprenylated benzophenones from Hypericum elodeoides, J. Nat. Med. 68 (2014) 737–742.
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[14] K. Winkelmann, J. Heilmann, O. Zerbe, T. Rali, O. Sticher, New prenylated bi- and tricyclic phloroglucinol derivatives from Hypericum papuanum, J. Nat. Prod. 64 (2001) 701–706. [15] I.P. Singh, S.B. Bharate, Phloroglucinol compounds of natural origin, Nat. Prod. Rep. 23 (2006) 558–591.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Erecricins A–E, prenylated acylphloroglucinols from the roots of Hypericum erectum Shuangxin Lu a, Naonobu Tanaka a,b,⁎, Yutaka Tatano c, Yoshiki Kashiwada a,⁎⁎ a b c
Graduate School of Pharmaceutical Sciences, Tokushima University, Tokushima 770-8505, Japan Graduate School of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8513, Japan Division of Immunobiology, Department of Pharmaceutical Sciences, International University of Health and Welfare, Tochigi 324-8501, Japan
a r t i c l e
i n f o
Article history: Received 30 June 2016 Received in revised form 21 August 2016 Accepted 25 August 2016 Available online 26 August 2016 Keywords: Acylphloroglucinol Hypericum erectum Hypericaceae Erecricins A–E Adotogirin
a b s t r a c t Six new prenylated acylphloroglucinols, erecricins A–E (1–5) and adotogirin (6), were isolated from the roots of Hypericum erectum (Hypericaceae). Their structures were assigned on the basis of spectroscopic evidences. Erecricins A–E (1–5) are bicyclic prenylated acylphloroglucinols possessing a chromane or a chromene skeleton. Adotogirin (6) is a simple achylphloroglucinol with an O-geranyl moiety. Antimicrobial activities of these acylphloroglucinols were also evaluated. © 2016 Elsevier B.V. All rights reserved.
1. Introduction
2. Experimental
The genus Hypericum (Hypericaceae) comprises almost 500 species germinating as herbs, shrubs, and trees, which are growing in temperate sections and mountains in the tropical areas [1]. Hypericum plants have been used as herbal remedies for the treatment of cuts, burns, melancholia, anxiety, abdominal, and urogenital pains [2–5]. A variety of prenylated acylphloroglucinols with interesting biological activities such as antibacterial, antiviral, antiproliferative, antiinflammatory, antioxidative, and antidepressant activities have been isolated from Hypericum plants to date [4–7]. Hypericum erectum is distributed in Japan, China, and Korea. The aerial parts of H. erectum are of medicinal values to heal hurts, sooth bruises, and malarial fever [2]. Some antibacterial, antiplasmoidal, and antihemorrhagic prenylated phloroglucinols have been isolated from this species [8–10]. In our continuing research on the constituents of Hypericum plants [6,11–13], the roots of H. erectum were investigated, which resulted in the isolation of six new prenylated acylphloroglucinols, erecricins A–E (1–5) and adotogirin (6). Herein, we describe the isolation, structure elucidation, and antimicrobial activities of 1–6.
2.1. General experimental procedures
⁎ Correspondence to: N. Tanaka, Graduate School of Pharmaceutical Sciences, Tokushima University, Tokushima 770-8505, Japan. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (N. Tanaka), [email protected] (Y. Kashiwada).
http://dx.doi.org/10.1016/j.fitote.2016.08.014 0367-326X/© 2016 Elsevier B.V. All rights reserved.
Optical rotations were measured on a JASCO P-2200 digital polarimeter. MS were obtained on a Waters LCT PREMIER 2695. NMR spectra were measured with a Bruker AVANCE-500 instrument using tetramethylsilane as an internal standard. Column chromatography was performed with silica gel 60 N (63–210 μm, Kanto Chemical), MCI gel CHP-20P (75–150 μm, Mitsubishi Chemical), YMC gel ODS-A (S-50 μm, YMC Co., Ltd.), and Sephadex LH-20 (25–100 μm, GE Health Care). HPLC was performed on YMC-Triart C18 (5 μm, ϕ20 × 250 mm; YMC Co., Ltd.), Mightysil RP-18 GP (5 μm, ϕ20 × 250 mm; Kanto Chemical), Mightysil RP-18 GP-II (5 μm, ϕ20 × 250 mm), COSMOSIL 5C18-AR-II (5 μm, ϕ20 × 250 mm, Nacalai Tesque), and COSMOSIL 5C18-MS-II (5 μm, ϕ20 × 250 mm). 2.2. Plant material Hypericum erectum was cultivated at the medicinal herb garden of Tokushima University, and was collected in July 2014. A voucher specimen (HYE201407) was deposited in the herbarium of Graduate School of Pharmaceutical Sciences, Tokushima University. 2.3. Extraction and isolation The dried roots (2.0 kg) of H. erectum were extracted with MeOH at room temperature to give the extract (170.7 g), which was partitioned
S. Lu et al. / Fitoterapia 114 (2016) 188–193
with EtOAc and water. The EtOAc-soluble material was partitioned with n-hexane and 90% MeOH aq. The n-hexane-soluble material was subjected to silica gel column chromatography (n-hexane/EtOAc, 9:1 to 0:10) to give nine fractions (frs. 1–9). Fr. 2 was fractionated by MCI CHP-20P column chromatography (MeOH/H2O, 0:1 to 1:0) to afford three fractions (frs. 2.1–3). Fr. 2.3 was applied to an ODS column (MeOH/H2O, 0:1 to 1:0), and purified by HPLC on YMC-Triart C18 (MeOH/H2O, 95:5) to yield frs. 2.3.3.1–8, including erecricin B (2, 23.8 mg, fr. 2.3.3.7). Erecricins A (1, 3.1 mg) and C (3, 1.5 mg) were isolated from fr. 2.3.3.5 and fr. 2.3.3.6, respectively, using HPLC on COSMOSIL 5C18-MS-II (MeOH/H2O, 95:5). Fr. 2.2 was separated by ODS column chromatography (MeOH/H2O, 0:1 to 1:0) and purified by HPLC on COSMOSIL 5C18-MS-II (MeOH/H2O, 95:5) to give erecricins D (4, 5.3 mg) and E (5, 22.0 mg). Fr. 3 was separated by chromatography on an ODS column (MeOH/H2O, 0:1 to 1:0), an MCI CHP-20P column (MeOH/H2O, 0:1 to 1:0), and a silica gel column (n-hexane/acetone, 95:5 to 0:10), and purified by HPLC on COSMOSIL 5C18-MS-II (MeOH/ H2O, 80:20) to yield adotogirin (6, 22.5 mg) and otogirin (7, 3.3 mg). 2.4. Erecricin A (1) Colorless oil; [α]D + 58.9 (c 0.34, CHCl3); HRESIMS m/z 479.3151 [M − H]− (calcd for C31H43O4, 479.3161); 1H and 13C NMR data (Table 1). 2.5. Erecricin B (2) Colorless oil; [α]D + 69.4 (c 2.70, CHCl3); HRESIMS m/z 517.3317 [M + Na]+ (calcd for C32H46O4Na, 517.3294); 1H and 13C NMR data (Table 1).
189
2.6. Erecricin C (3) Colorless oil; [α]D − 60.0 (c 0.18, CHCl3); HRESIMS m/z 517.3269 [M + Na]+ (calcd for C32H46O4Na, 517.3294); 1H and 13C NMR data (Table 1).
2.7. Erecricin D (4) Colorless oil; [α]D − 25.2 (c 0.44, CHCl3); HRESIMS m/z 479.3151 [M − H] − (calcd for C 31H43 O4 , 479.3161); 1 H and 13C NMR for major tautomer 4a (Table 2); 1H NMR for minor tautomer 4b: δ H 18.66 (1H, s, 1-OH), 6.53 (1H, d, J = 10.1 Hz, H-23), 5.86 (1H, dd, J = 15.5, 10.9 Hz, H-12), 5.50 (1H, d, J = 10.9 Hz, H-11), 5.33 (1H, d, J = 10.1 Hz, H-24), 5.30 (1H, m, H-13), 4.99 (1H, brt, J = 6.9 Hz, H-18), 4.09 (1H, sept, J = 6.8 Hz, H-29), 2.57 (1H, m, H-8), 2.32 (1H, m, H-7a), 2.18 (1H, m, H-14), 1.97 (1H, m, H-17a), 1.90 (1H, m, H-7b), 1.87 (1H, m, H-17b), 1.65 (3H, s, H 3 -21), 1.56 (3H, s, H3-20), 1.53 (3H, s, H3-27), 1.52 (3H, s, H3-10), 1.43 (3H, s, H3 -26), 1.41 (3H, s, H3 -22), 1.15 (3H, d, J = 6.8 Hz, H3-30), 1.11 (3H, d, J = 6.8 Hz, H3-31), 0.94 (3H, d, J = 6.7 Hz, H 3-15), and 0.92 (3H, d, J = 6.7 Hz, H 3 -16); 13C NMR for minor tautomer 4b: δC 209.1 (C-28), 197.5 (C-1), 179.7 (C-5), 165.0 (C-3), 139.9 (C-13), 137.1 (C-9), 132.4 (C-19), 129.0 (C-11), 123.7 (C-24), 122.7 (C-12), 122.5 (C-18), 116.4 (C-23), 109.9 (C-4), 109.6 (C-6), 79.1 (C-25), 47.0 (C-2), 40.1 (C-7), 38.4 (C-8), 35.4 (C-29), 33.3 (C17), 31.3 (C-14), 29.3 (C-27), 28.3 (C-26), 26.9 (C-22), 25.7 (C-21), 22.1 (C-15), 22.4 (C-16), 18.9 × 2 (C-30 and C-31), 18.4 (C-10), and 17.9 (C-20).
Table 1 1 H and 13C NMR data for erecricins A–C (1–3) in CDCl3. 1
2
3
Position
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1 2 3 4 5 5-OH 6 7
197.7 48.0 171.6 115.6 189.3 – 105.4 31.0
197.8 48.0 171.5 115.7 189.3 – 106.3 30.8
40.3 86.0 19.1 133.2 126.7 124.3 137.0 18.4 26.0 29.5
18 19 20 21 22 23 24 25 26 27 28 29 30 31
121.5 133.7 17.9 25.8 28.5 21.2 121.9 131.7 17.9 25.7 208.0 35.4 18.8 18.9
– – – – – 19.10 (1H, s) – 2.18 (1H, dd, 14.4, 3.9) 1.44 (1H, d, 14.4) 2.03 (1H, m) – 1.17 (3H, s) 5.73 (1H, d, 15.3) 6.54 (1H, dd, 15.3, 10.9) 5.86 (1H, d, 10.9) – 1.80 (3H, s) 1.78 (3H, s) 2.07 (1H, m) 1.69 (1H, m) 5.05 (1H, m) – 1.57 (3H, s) 1.68 (3H, s) 1.41 (3H, s) 3.05 (2H, m) 5.04 (1H, m) – 1.68 (3H, s) 1.64 (3H, s) – 3.73 (1H, qt, 6.8, 6.8) 1.16 (1H, d, 6.8) 1.71 (1H, m) 1.38 (1H, m) 0.85 (3H, t, 7.4)
197.8 48.5 172.6 114.4 189.4 – 106.3 30.7
8 9 10 11 12 13 14 15 16 17
– – – – – 19.04 (1H, s) – 2.20 (1H, dd, 14.3, 3.8) 1.46 (1H, d, 14.3) 2.04 (1H, m) – 1.19 (3H, s) 5.74 (1H, d, 15.3) 6.65 (1H, dd, 15.3, 10.9) 5.88 (1H, d, 10.9) – 1.80 (3H, s) 1.82 (3H, s) 2.10 (1H, m) 1.72 (1H, m) 5.07 (1H, m) – 1.58 (3H, s) 1.70 (3H, s) 1.42 (3H, s) 3.07 (2H, m) 5.05 (1H, m) – 1.70 (3H, s) 1.66 (3H, s) – 3.90 (1H, sept, 6.8) 1.11 (3H, d, 6.8) 1.18 (3H, d, 6.8)
– – – – – 19.06 (1H, s) – 2.05 (1H, m) 1.41 (1H, m) 2.04 (1H, m) – 1.62 (3H, s) 5.44 (1H, d, 15.3) 6.32 (1H, dd, 15.3, 10.8) 5.73 (1H, d, 10.8) – 1.71 (3H, s) 1.75 (3H, s) 2.13 (1H, m) 1.74 (1H, m) 5.11 (1H, m) – 1.60 (3H, s) 1.71 (3H, s) 1.43 (3H, s) 3.09 (2H, m) 5.08 (1H, m) – 1.68 (3H, s) 1.62 (3H, s) – 3.75 (1H, qt, 6.8, 6.8) 1.17 (1H, d, 6.8) 1.72 (1H, m) 1.39 (1H, m) 0.86 (3H, t, 7.4)
32
40.3 85.9 18.9 133.1 126.7 124.3 137.0 18.3 26.1 29.5 121.5 133.7 17.9 25.8 28.4 21.2 121.9 131.7 17.9 25.7 207.2 41.7 17.0 26.6 11.9
41.7 86.5 25.8 128.9 127.8 124.4 136.9 18.3 26.0 29.9 121.6 133.7 17.9 25.9 28.9 21.3 121.6 131.8 17.9 25.6 206.9 41.6 17.1 26.7 11.8
190
S. Lu et al. / Fitoterapia 114 (2016) 188–193
2.8. Erecricin E (5)
Table 3 1 H and 13C NMR data for adotogirin (6) in CDCl3.
Colorless oil; [α]D − 3.8 (c 2.17, CHCl3); HRESIMS m/z 533.3223 [M + Na]+ (calcd for C32H46O5Na, 533.3243); 1H and 13C NMR for major tautomer 5a (Table 2); 1H NMR for minor tautomer 5b: δH 18.82 (1H, s, 1-OH), 6.52 (1H, d, J = 10.0 Hz, H-23), 5.86 (1H, dd, J = 14.9, 10.9 Hz, H-12), 5.49 (1H, d, J = 10.9 Hz, H-11), 5.33 (1H, d, J = 10.0 Hz, H-24), 5.27 (1H, dd, J = 14.9, 7.1 Hz, H-13), 4.99 (1H, brs, H18), 4.00 (1H, qt, J = 6.6, 6.6 Hz, H-29), 2.57 (1H, m, H-8), 2.31 (1H, m, H-7a), 2.18 (1H, m, H-14), 1.99 (1H, m, H-17a), 1.90 (1H, m, H-7b), 1.86 (1H, m, H-17b), 1.71 (1H, m H-31a), 1.65 (3H, s, H3-21), 1.55 (3H, s, H3-20), 1.55 (3H, s, H3-10), 1.52 (3H, s, H3-27), 1.38 (3H, s, H326), 1.40 (3H, s, H3-22), 1.36 (1H, m H-31b), 1.12 (3H, d, J = 6.6 Hz, H3-30), 0.94 (3H, d, J = 6.6 Hz, H3-15), 0.92 (3H, d, J = 6.6 Hz, H3-16), and 0.89 (3H, dd, J = 12.7, 6.9 Hz, H3-32); 13C NMR for minor tautomer 5b: δC 208.6 (C-28), 198.2 (C-1), 180.4 (C-5), 164.9 (C-3), 139.8 (C-13), 137.0 (C-9), 132.1 (C-19), 128.9 (C-11), 123.4 (C-24), 122.6 (C-12), 122.4 (C-18), 116.3 (C-23), 109.6 (C-6), 109.3 (C-4), 79.1 (C-25), 47.0 (C-2), 42.0 (C-29), 39.9 (C-7), 38.3 (C-8), 33.1 (C-17), 31.1 (C-14), 29.2 (C-27), 28.3 (C-26), 27.3 (C-22), 26.3 (C-31), 25.6 (C-21), 22.4 (C-16), 22.3 (C-15), 18.4 (C-10), 17.8 (C-20), 16.4 (C-30), and 11.7 (C32).
Position
δC
δH (J in Hz)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
160.2a) 103.9b) 162.5 92.5 160.8a) 104.3b) 7.2 65.3 118.9 141.4 16.7 39.4 26.2 123.6 131.9 17.7 25.6 210.4 45.9 16.7 27.0
22
11.9
– – – 5.96 (1H, s) – – 2.02 (3H, s) 4.52 (2H, d, 6.3) 5.44 (1H, t, 6.3) – 1.71 (3H, s) 2.08 (2H, m) 2.12 (2H, m) 5.08 (1H, t, 6.6) – 1.60 (3H, s) 1.67 (3H, s) – 3.77 (1H, qt, 6.8, 6.8) 1.16 (3H, d, 6.8) 1.84 (1H, m) 1.41 (1H, m) 0.91 (3H, t, 7.4)
a,b)
Signals may be interchangeable.
2.9. Adotogirin (6) 2.10. Antimicrobial assay Pale yellow amorphous solid; [α]D + 7.7 (c 0.21, MeOH); HRESIMS m/z 383.2199 [M + Na]+ (calcd for C22H32O4Na, 383.2198); 1H and 13 C NMR data (Table 3). Table 2 1 H and 13C NMR data for major tautomers (4a and 5a) of erecricins D (4) and E (5) in CDCl3. 4a
5a
Position
δC
δH (J in Hz)
δC
δH (J in Hz)
1 2 3 4 5 5-OH 6 7
193.9 51.5 172.6 105.1 185.9 – 105.8 40.1
193.9 51.4 172.2 104.8 185.9 – 106.4 39.9
8 9 10 11 12 13 14 15 16 17
38.4 137.1 18.5 129.0 122.7 139.9 31.3 22.1 22.4 33.3
– – – – – 19.08 (1H, s) – 2.32 (each 1H, m) 1.90 (1H, m) 2.50 (1H, m) – 1.54 (3H, s) 5.50 (1H, d, 10.9) 5.86 (1H, dd, 15.5, 10.9) 5.28 (1H, dd, 15.5, 6.6) 2.18 (1H, m) 0.95 (3H, d, 6.8) 0.92 (3H, d, 6.8) 1.97 (1H, m) 1.87 (1H, m) 4.99 (1H, brt, 6.9) – 1.56 (3H, s) 1.65 (3H, s) 1.29 (3H, s) 6.45 (1H, d, 10.1) 5.38 (1H, d, 10.1) – 1.43 (3H, s) 1.56 (3H, s) – 3.86 (1H, sept, 6.9) 1.13 (3H, d, 6.9) 1.12 (3H, d, 6.9)
– – – – – 19.01 (1H, s) – 2.31 (each 1H, m) 1.90 (1H, m) 2.50 (1H, m) – 1.55 (3H, s) 5.49 (1H, d, 10.9) 5.86 (1H, dd, 14.9, 10.9) 5.28 (1H, dd, 14.9, 6.8) 2.18 (1H, m) 0.94 (3H, d, 6.7) 0.92 (3H, d, 6.7) 1.99 (1H, m) 1.86 (1H, m) 4.99 (1H, brs) – 1.55 (3H, s) 1.65 (3H, s) 1.28 (3H, s) 6.45 (1H, d, 10.1) 5.37 (1H, d, 10.1) – 1.43 (3H, s) 1.56 (3H, s) – 3.75 (1H, qt, 6.9, 6.9) 1.10 (1H, d, 6.9) 1.71 (1H, m) 1.36 (1H, m) 0.89 (3H, brt, 7.8)
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
122.5 132.4 17.9 25.7 27.5 115.0 123.3 80.7 28.3 29.3 207.7 35.4 18.9 18.9
38.3 137.0 18.4 128.9 122.6 139.8 31.1 22.0 22.4 33.1 122.4 132.1 17.8 25.6 27.3 115.0 123.2 80.7 28.3 29.2 207.1 41.7 11.9 26.3 11.7
2.10.1. Test microorganisms Seven clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) strains, three clinical isolates of methicillin-sensitive S. aureus (MSSA) strains, S. aureus 209P and Smith, one Bacillus subtilis strain, and four Escherichia coli strains were used in this study. All the strains of microorganisms used in this work were kindly supplied by Dr. C. Sano, the School of Medicine, Shimane University (Shimane, Japan).
2.10.2. Susceptibility testing The MICs were determined by broth microdilution method in 96well microtiter plates with cation-supplemented Mueller-Hinton broth (CAMHB; Ca2+, 25 μg/mL; Mg2+, 12.5 μg/mL; Becton Dickinson, Sparks, MD) according to the current guidelines of the Clinical and Laboratory Standards Institute (CLSI). All the strains of microorganisms were inoculated at a final bacterial density of about 5 × 105 CFU/mL. Then, S. aureus strains were incubated at 35 °C for 20 h, and B. subtilis and E. coli strains were incubated at 37 °C for 20 h before the MICs
21 31
30
O
29 28 6
O
1
22
18 8
20
17
15
2
5
12 9 11
4
HO
19
7
O
14 13
16
10 23 24 25 27
1H-1H
26
1
COSY HMBC NOESY
Fig. 1. Selected 2D NMR correlations of erecricin A (1).
S. Lu et al. / Fitoterapia 114 (2016) 188–193
2
7
8
17
22 10 9
3
NOESY
1
Fig. 2. Selected NOESY correlations and the relative stereochemistry for the chromane core of erecricin A (1) (protons of methyl groups were omitted).
were determined. The MICs of the test compounds were reported as an MIC, MIC range, and MIC50. 3. Results and discussion The MeOH extract of H. erectum roots was partitioned with EtOAc and water. The EtOAc-soluble material was further partitioned with 90% MeOH aq. and n-hexane. Repeated chromatographic separations of the n-hexane-soluble material gave six new prenylated acylphloroglucinols, erecricins A–E (1–5) and adotogirin (6), together with one known prenylated acylphloroglucinol (7) which was identified as otogirin by comparison of the spectroscopic data with the literature data [8]. Erecricin A (1) was obtained as an optical active colorless oil {[α]D + 58.9 (c 0.34, CHCl3)}, and its molecular formula was established as C31H44O4 by the HRESIMS (m/z 479.3151 [M − H]−, Δ −1.0 mmu). The 1H NMR spectrum showed the signals of five olefinic protons, three sp3 methylenes, two sp3 methines, and ten methyls as well as the characteristic down-field shifted signal (δH 19.04, s) due to a hydrogen-bonded hydroxy group (Table 1), indicating the existence of a β-diketone moiety with an enol form [10]. The 13C NMR and DEPT spectra suggested the presence of 31 carbons including two enols, four
191
olefins, two ketone carbonyl carbons, one quaternary carbon, and one oxygenated tertiary carbon (Table 1). From these observations, erecricin A (1) was deduced to be a prenylated acylphloroglucinol with four isoprene units. Comparison of the 1D NMR spectra of 1 with the literature data implied that 1 had a structure similar to hypelodin A [11], a prenylated acylphloroglucinol possessing the chromane core, but had different substituents at C-2 and C-6. The substituent at C-2 in 1 was assigned as a methyl group by HMBC interpretations of H3-22 with C1, C-2, C-3, and C-7 (Fig. 1), while a 2-methylpropanoyl group at C-6 was revealed by 1H-1H COSY cross-peaks of H-29 with H3-30 and H331 and HMBC correlations for H3-30 with C-28, OH-5 with C-4, C-5, C6 and C-28. Therefore, the gross structure of 1 was elucidated as shown in Fig. 1. NOESY correlations for H-8/H3-22, H-7β/H3-10, and H-17b/H3-10 suggested the pseudochair conformation of the tetrahydropyran ring (C-2, C-3, and C-7–C-9) and the pseudoaxial orientations for H-7β, H8, 10-Me, and 22-Me (Fig. 2). This was underpinned by a large 3J value of the vicinal coupling for H-7β/H-8 (J = 14.3 Hz) (Table 1). Thus, the relative stereochemistry for 1 was assigned as shown in Chart 1. The HRESIMS revealed the molecular formula of erecricin B (2) to be C32H46O4 {m/z 517.3317 [M + Na]+, Δ +2.3 mmu}, larger by 14 mass units than that of 1. Though the 1D NMR spectra of 2 were similar to those of 1, the resonances of a 2-methylbutanoyl group were observed for 2 in place of the signals of the 2-methylpropanyl moiety for 1. The presence of the 2-methylbutanoyl group at C-6 in 2 was confirmed by HMBC analysis. The relative configuration of C-29 remains to be assigned, since any NOESY correlations were not observed. Thus, erecricin B was elucidated to be 2 (Chart 1). The molecular formula of erecricin C (3) was assigned as C32H46O4 by the HRESIMS {m/z 517.3269 [M + Na]+, Δ −2.5 mmu}, which was identical to that of 2. Analysis of the 1H and 13C NMR spectra (Table 2) indicated 3 to be a diastereomer of 2 at the tetrahydropyran moiety. NOESY correlations of H3-22/H-8 and H-7β/H-11 revealed the pseudoaxial orientations of H-7β, 22-Me, and the substituent at C-9 (Fig. 3). Therefore, the structure of 3 was assigned as shown in Chart 1. Erecricins D (4) and E (5) were individually isolated as optical active colorless oil {[α]D − 25.2 (c 0.44, CHCl3) for 4; [α]D − 3.8 (c 2.17, CHCl3) for 5}. The molecular formula of 4 was assigned to be C31H44O4 in light of the HRESIMS (m/z 479.3151 [M − H]−, Δ −1.0 mmu). In the 1H NMR
21
R 30 29 28
O
19
O
18
22
O
20
7
1 6
8
15
17
4
O
21
14
9 3
HO
O
O
2 5
11
12
13
HO
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19
R
O
10
23
OH
31
6
7
11
16
2 8
20
22
24
1 18
HO
O
4
13 9
10
15 12
14
17
25 27
26
R 1 2
R
H Me
3
7 6
H Me
R O O HO
15
10
O
22 7
1
8
6
2
5
3
11
12
13
16
R
6 5
17
O
4
25
23 24
O
18 26
OH 1
14 9
O
19
20
21
27
4a 5a
R H Me
R 4b 5b
H Me
Chart 1. Structures of erecricins A–E (1–5), adotogirin (6), and otogirin (7) (4a/5a and 4b/5b are major and minor tautomers of 4 and 5, respectively).
S. Lu et al. / Fitoterapia 114 (2016) 188–193
OH
192
O 8
21
7
22
11
2
3
HO
10
6
15
10 22 12
O 5
4
HO
2 3
7
8 17
O
14
9 11
13
16
18 27
25
23
19 24
21
26
1H-1H
4a
O
8
9
15
10 12
14
17
1H-1H
spectrum of 4, a pair of down-field shifted hydroxy proton signals (δH 19.08 and 18.66) were observed in a ratio of ca. 3:1, indicating the presence of two tautomers (4a and 4b). The feature of the 1D NMR spectra of 4 were similar to those of hyperguinone B [14], a prenylated acylphlorglucinol with a chromene core, whereas the signals of the different substituent consisting of 15 carbons were observed for 4 in place of the resonances due to the prenyl group at C-2 in hyperguinone B. The substituent was elucidated by 2D NMR analysis (Fig. 4). The 1H-1H COSY spectrum indicated the connectivities of C-7–C-8, C-11–C-14, C-14–C15, and C-14–C-16, while the connectivities among C-8, C-10, and C11 via C-9 were suggested by HMBC correlations for H3-10 with C-8, C-9, and C-11. HMBC correlations for H3-21 with C-18, C-19, and C-20 and 1H-1H cross-peaks of H-8/H2-17 and H2-17/H-18 revealed the presence of a prenyl group at C-8. Similarly, erecricin E (5) was elucidated to be a prenylated acylphloroglucinol possessing the same chromene core as seen in 4 with a 2-methylbutanoyl group at C-6. Thus, the planar structures of erecricins D (4) and E (5) were assigned as shown in Chart 1, while the stereochemistries for 4 and 5 were not elucidated. The 1H and 13C NMR spectra of adotogirin (6) (Table 3), C22H32O4, resembled to those of a known acylphloroglucinol otogirin (7), except for the presence of the signals due to a sec-butyl group in place of those due to the isopropyl group in 7. These observations implied that 6 is an prenylated acylphloroglucinol with a 2-methylbutanoyl group, a methyl group, and an O-geranyl group. The structure of adotogirin (6) was confirmed by analysis of the 2D NMR spectra (Fig. 5). Erecricins A–E (1–5), adotogirin (6), and otogirin (7) were evaluated for their antimicrobial activities on strains of Staphylococcus aureus (MRSA and MSSA), Bacillus subtilis, and Escherichia coli. Among the tested compounds, adotogirin (6) and otogirin (7) exhibited an antimicrobial activity against MRSA {6: MIC range 0.5–4.0 μg/mL (MIC50 1.0 μg/mL); 7: MIC range 0.5–8.0 μg/mL (MIC50 0.5 μg/mL)}, MSSA {6: MICs 1.0 μg/mL for all strains; 7: MIC range 0.5–1.0 μg/mL (MIC50
1
4
NOESY
Fig. 3. Selected NOESY correlations and the relative stereochemistry for the chromane core of erecricin C (3) (protons of methyl groups were omitted).
6
16
11 13
3
O
7
2
6
19 20
9
22
29 28
1
18
COSY HMBC
Fig. 4. Selected 2D NMR correlations for major tautomer 4a of erecricin D (4).
COSY HMBC NOESY
Fig. 5. Selected 2D NMR correlations for adotogirin (6).
0.5 μg/mL)}, and Bacillus subtilis (MIC each 2.0 μg/mL), while antiplasmodial activity, cytotoxicity, and antagonistic activity against thromboxane A2 and leukotriene D4 of 7 have been reported [8]. In contrast, erecricins A–E (1–5) did not show any antimicrobial activity. Investigation of the MeOH extract from the roots of Hypericum erectum afforded five new bicyclic prenylated achylphloroglucinols, erecricins A–E (1–5), and one prenylated achylphloroglucinols with an O-geranyl moiety, adotogirin (6), together with a known prenylated acylphlotoglucinol, otogirin (7), whose structures were elucidated by spectroscopic analysis. O-Prenylated acylphloroglucniols are relatively rare than C-prenylated acylphlorogluciniols. Antimicrobial activities of various phloroglucinols possessing O-prenylated, dimeric or more complex structures have been reported to date [6,15]. Adotogirin (6) and otogirin (7) appeared to be potent antibacterial agents against Staphylococcus aureus, and Bacillus subtilis. Conflict of interest The authors declare no conflict of interest. Acknowledgment This work was partly supported by a Grant-in-Aid for Scientific Research (No. 15K18885) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References [1] N.M. Nürk, S. Madriñán, M.A. Carine, M.W. Chase, F.R. Blattner, Molecular phylogenetics and morphological evolution of St. John's wort (Hypericum; Hypericaceae), Mol. Phylogenet. Evol. 66 (2013) 1–16. [2] C. Wiart, Anti-inflammatory plants, Ethnopharmacology of Medicinal Plants: Asia and the Pacific, Humana Press Inc., Totowa, New Jersey 2006, pp. 1–50. [3] J. Sarris, Herbal medicines in the treatment of psychiatric disorders: a systematic review, Phytother. Res. 21 (2007) 703–716. [4] G. Laakmann, C. Schüle, T. Baghai, M. Kieser, St. John's wort in mild to moderate depression: the relevance of hyperforin for the clinical efficacy, Pharmacopsychiatry 31 (1998) 54–59. [5] P. Avoto, A survey on the Hypericum genus: secondary metabolites and bioactivity, in: A. Atta-ur-Rahman (Ed.), Studies in Natural Products Chemistry, Elsevier Science BV, Amsterdam 2005, pp. 603–634. [6] N. Tanaka, J. Kobayashi, Prenylated acylphloroglucinols and meroterpenoids from Hypericum plants, Heterocycles 90 (2015) 23–40. [7] Y.-B. Chen, A.E. Fazary, Y.-C. Lin, I.-W. Lo, S.-C. Ong, S.-Y. Chen, C.-T. Chien, Y.-J. Lin, W.-W. Lin, Y.-C. Shen, Hyperinakin, a new anti-inflammatory phloroglucinol derivative from Hypericum nakamurai, Nat. Prod. Res. 27 (2013) 727–734. [8] M. Tada, K. Chiba, H. Yamada, H. Maruyama, Phloroglucinol derivatives as competitive inhibitors against thromboxane A2 and leukotriene D4 from Hypericum erectum, Phytochemistry 30 (1991) 2559–2562. [9] T. Kosuge, H. Ishida, T. Satoh, Studies on antihemorrhagic substances in herbs classified as hemostatics in Chinese medicine, IV. On antihemorrhagic principles in Hypericum erectum Thunb, Chem. Pharm. Bull. 33 (1985) 202–205. [10] H.I. Moon, Antiplasmodial and cytotoxic activity of phloroglucinol derivatives from Hypericum erectum Thunb, Phytother. Res. 24 (2010) 941–944. [11] C. Hashida, N. Tanaka, K. Kawazoe, K. Murakami, H.D. Sun, Y. Takaishi, Y. Kashiwada, Hypelodins A and B, polyprenylated benzophenones from Hypericum elodeoides, J. Nat. Med. 68 (2014) 737–742.
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[14] K. Winkelmann, J. Heilmann, O. Zerbe, T. Rali, O. Sticher, New prenylated bi- and tricyclic phloroglucinol derivatives from Hypericum papuanum, J. Nat. Prod. 64 (2001) 701–706. [15] I.P. Singh, S.B. Bharate, Phloroglucinol compounds of natural origin, Nat. Prod. Rep. 23 (2006) 558–591.