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Sesquiterpenes from the fruits of Illicium jiadifengpi and their anti-hepatitis B virus activities
Fitoterapia 104 (2015) 41–44
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Sesquiterpenes from the fruits of Illicium jiadifengpi and their anti-hepatitis B virus activities Ji-Feng Liu a,⁎, Lei Wang c, Ya-Feng Wang a,b, Xu Song a, Li-Jia Yang a, Yan-Bing Zhang a,⁎ a b c
School of Pharmaceutical Science, Zhengzhou University, Ke Xue Da Dao 100, Zhengzhou 450001, People's Republic of China Henan Key laboratory for Pharmacology of Liver Disease, Academy of Medical and Pharmaceutical Science, Zhengzhou University, 40 Daxue Road, Zhengzhou 450052, People's Republic of China College of Chemistry and Chemical Engineering, Xuchang University, Henan, Xuchang 461000, People's Republic of China
a r t i c l e
i n f o
Article history: Received 26 February 2015 Accepted in revised form 2 May 2015 Accepted 6 May 2015 Available online 9 May 2015 Keywords: Illiciaceae Illicium jiadifengpi Sesquiterpenes Anti-HBV activity
a b s t r a c t Two new compounds, 2S-hydroxyl-jiadifenolide (1) and jiadifenlactone acid (2), and eight known compounds were isolated from the fruits of Illicium jiadifengpi. Their structures were analysed using several spectroscopic techniques, including 1D-, 2D-NMR and HR-ESI-MS experiments. The anti-hepatitis B virus (HBV) activities of the isolates were evaluated via HBV transfection of the Hep G2.2.15 cell line. The inhibitory rates of the most active compounds, compounds 4 and 5, on the HBeAg and HBsAg expression were 28.85 ± 3.15% and 17.53 ± 1.81% and 37.93 ± 2.74% and 23.47 ± 9.52% at concentrations of 64.94 μM and 61.35 μM, respectively. © 2015 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Illicium species are rich in biosynthetically unique seco-prezizaanesesquiterpenes and prenylated C6–C3 compounds [1,2]. The former are considered to be characteristic chemical markers of Illicium species [3,4]. Several seco-prezizaane-sesquiterpenes have been reported to exhibit diverse biological activities, including anti-HBV activity. In a previous paper, we reported the structure and anti-HBV activities of henrylactones A–G, which were isolated from Illicium henryi [5,6]. As part of our ongoing studies on biologically active substances of the genus Illicium, we have investigated the chemical components of the ethanol extracts of the fruits of Illicium jiadifengpi, which is a mediumsized tree that is primarily found in South China. Its bark has been used as a traditional Chinese medicine for the treatment of rheumatism [7]. Previous chemical studies on I. jiadifengpi have indicated the presence of essential oils [7,8], sesquiterpenes [9–14], diterpenoids [15–17], and flavones [18]. In this paper we described two new seco-prezizaane-sesquiterpenes, 2S-hydroxyl-jiadifenolide (1) and jiadifenlactone acid (2), together with eight known compounds (3–10) and investigated their anti-HBV activities
2.1. General experimental procedures
⁎ Corresponding authors at: School of Pharmaceutical Science, Zhengzhou University, Ke Xue Da Dao 100, Zhengzhou 450001, People's Republic of China. E-mail addresses: [email protected] (J.-F. Liu), [email protected] (Y.-B. Zhang).
http://dx.doi.org/10.1016/j.fitote.2015.05.004 0367-326X/© 2015 Elsevier B.V. All rights reserved.
Optical rotations were determined on a Horiba SEPA-300 polarimeter (Horiba, Tokyo, Japan). UV spectra were measured on a Shimadzu UV-210A spectrophotometer (Shimadzu, Kyoto, Japan). IR (KBr) spectra were recorded on a Bio-Rad FTS-135 spectrometer (Bio-Rad, California, USA). 1D and 2D NMR spectra were recorded on Bruker AM-400 NMR spectrometers with TMS as internal standard (Bruker, Bremerhaven, Germany). MS spectra were run on a VG Auto Spec-3000 spectrometer (VG, Manchester, England) and API Qstar Pulsar (Applied Biosystems, Foster City, USA). Silica gel (200–300 mesh) for column chromatography was obtained from Qingdao Meigao Chemical Company, Qingdao, China. Sephadex LH-20 (20–150 μm) was purchased from Pharmacia Fine Chemical Co. Ltd., Sweden.
2.2. Plant material The fruits of I. jiadifengpi B. N. Chang were collected from Guangdong Province, P.R. China, in September 2011 and identified by Dr. Mengqi Liu, School of Pharmaceutical Science, Henan University of Traditional Chinese Medicine. A voucher specimen of the fruits (2011-09-02) was deposited in the School of Pharmaceutical Science at Zhengzhou University.
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J.-F. Liu et al. / Fitoterapia 104 (2015) 41–44
Table 1 1 H-(400 MHz) and 13C-(100 MHz) NMR data for 1 and 2 in C5D5N. Position
1 δH (mult, J in Hz)
2 δC
1 2
3.92 (m) 4.29 (m)
46.9 74.7
3
2.61 (1H, dd, 13.5, 9.4) 2.40 (1H, dd, 13.4, 5.4) – – – 5.03 (1H, d, 5.9) 2.76 (1H, dd, 12.8, 6.0) 2.49 (1H, d, 12.8) – – – – 1.47 (3H, s) 4.82 (1H, d, 9.3) 3.90 (1H, d, 9.3) 1.67 (3H, d, 7.2)
42.8
4 5 6 7 8 9 10 11 12 13 14 15
92.6 47.4 78.4 77.1 32.1 56.6 102.0 172.7 176.9 20.2 73.4 12.5
δH (mult, J in Hz)
δC
3.31 (m) 2.17 (1H, m) 1.80 (1H, m) 1.97 (1H, m) 2.00 (1H, m) – – – 5.10 (1H, d, 6.1) 3.09 (1H, d, 11.6) 2.98 (1H, dd, 11.7, 6.2) – 4.83 (1H, s) – – 1.65 (3H, s) 4.60 (1H, d, 8.7) 4.03 (1H, d, 8.7) 1.36 (3H, d, 7.5)
33.4 29.6 29.8 82.7 48.2 78.9 81.9 27.0 61.9 80.0 175.4 180.1 21.1 75.1 13.0
2.3. Extraction and isolation Powdered fruits of I. jiadifengpi (5.0 kg) were extracted three times with 95% EtOH (20 L) under reflux for 2 h each time. The extract was concentrated under a vacuum to obtain a residue. The residue was dissolved in H2O and then extracted successively with CHCl3 (three times, each 2.5 L) and n-BuOH (three times, each 2.5 L). The CHCl3 fraction (90 g) was subjected to silica gel column chromatography (CC) (9 × 40 cm, 1000 g, 200–300 mesh) and eluted with a CHCl3/ MeOH (100:0, 90:10, 80:20, v/v, each 10 L) gradient to produce nine fractions (Frs. 1–9). Fr. 2 (10 g) was further subjected to silica gel CC (5 × 35 cm, 280 g; petroleum ether/EtOAc, 100:0–60:30 gradient) to produce six sub-fractions (Frs. 2a–2f). Compounds 1 (20 mg), 3 (15 mg) and 4 (10 mg) were obtained from the Fr. 2b (1.0 g) by silica gel CC (2.5 × 40 cm, 80 g; CHCl3/Me2CO, 95:5–70:30 gradient). Fr. 2c (2.3 g) was chromatographed through a silica gel column (2.6 × 50 cm, 100 g) and eluted with petroleum ether/acetone (98:2, 90:10, 80:20, 70:30, v/v, each 1.0 L) to yield compounds 2 (8 mg), 5 (35 mg) and 10 (50 mg). Fr. 3 (7 g) was subjected to silica gel CC (5 × 32 cm, 200 g; petroleum ether/acetone, 90:10–70:30 gradient) followed by Sephadex LH-20 CC (MeOH) to obtain compounds 6 (9 mg) and 8 (5 mg). Fr. 4 (10 g) was also subjected to a silica gel column (5 × 32 cm, 200 g) and eluted with a CHCl3/Me2CO (100:0, 90:10,
80:20, v/v, each 3 L) gradient to obtain six fractions (Frs. 4a–4f). Fr. 4b (1.5 g) was further separated through column chromatography on silica gel (2.4 × 30 cm, 60 g) with petroleum ether/acetone (60:40) to furnish compound 9 (8 mg). Fr. 5 (2 g) was chromatographed through a silica gel column (2.4 × 30 cm, 60 g, CHCl3:MeOH = 90:10) and further purified by Sephadex LH-20 CC (MeOH) to provide compound 7 (38 mg). Each of the purified compounds had a degree of purity N 92%. 2S-hydroxyl-jiadifenolide (1) Needle crystals (MeOH); [α]24.9 D–103.30 (c 0.087, MeOH); UV (CHCl3): λmax (log ε) = 206 (2.76), 215 (2.59) nm; IR (KBr): νmax = 3406, 1784, 1630, 1250, 1036 cm− 1; 13 1 H and C NMR data see Table 1; HRESI-MS: m/z = 349.0897 [M + Na]+ (calcd. for C15H18O8Na: 349.0899). Jiadifenlactone acid (2) White amorphous solid; [α]16.7 D − 43.80 (c 0.089, MeOH); UV (CHCl3): λmax (log ε) = 208 (2.99), 215 (2.88) nm; IR (KBr): νmax = 3430, 2925, 1763, 1625, 1383, 1023 cm− 1; 1H and 13C NMR data see Table 1; HR-ESI-MS: m/z = 335.1107 [M + Na]+ (calcd. for C15H20O7Na: 335.1107).
2.3.1. X-ray diffraction analysis of compound 1 The crystal structure data for 1: λ = 1.54184 Å, T = 291 (2) K, space group P21, a = 7.3608 (3) Å, b = 13.4259 (6) Å, c = 13.9975 (6) Å, V = 1383.30 (3) Å, Z = 4, Dc = 1.567 g·cm− 3, crystal size 0.20 × 0.16 × 0.16 mm. These data had been deposited in the Cambridge Crystallographic Date Center (deposition number: 1047242) and can be obtained free via http://www.ccdc.cam.ac.uk/deposit. An anti-HBV assay was performed according to a previous report [19]. Hep G2.2.15 cells were grown in 96-well plates and were allowed to form monolayers. Aliquots of 200 mL of media containing varying concentrations of test media at doses below the CC50 were added to each well. Cells that were not exposed to test compounds served as cell controls. Lamivudine (3TC) was used as a reference compound. The cells were grown in the presence of the studied compounds for 9 days; their media were renewed on the 6th day. Suspensions were immediately collected for antigen assays. The concentrations of HBsAg and HBeAg in the cell culture supernatants were detected using a commercially available ELISA kit (Kehua Bio-engineering Corporation, Shanghai,
Fig. 1. Structures of compounds 1–10.
J.-F. Liu et al. / Fitoterapia 104 (2015) 41–44
43
Fig. 2. Key HMBC correlations of compounds 1 and 2.
China) according to the manufacturer's protocol. Inhibition rates (%) were calculated using the following formula: [(A450 of the control wells without drug − A450 of the experimental wells) / (A450 of the control wells without drug − A450 of blank control wells without cells)] × 100% (see details in Supporting information). Fig. 4. X-ray crystal structure of compound 1.
2.4. Supporting information The NMR data of compounds 3–10 and original spectra for compounds 1–2, as well as the procedure for anti-HBV assay of the tested compounds are available as Supporting information. 3. Results and discussion Compound 1 was obtained as needle crystals (MeOH). Its molecular formula was defined as C15H18O8, as determined by HR-ESI-MS (m/z 349.0897 [M + Na]+, calcd. 349.0899). The IR spectrum showed absorption bands at 3406 and 1784 cm−1, which were respectively indicative of hydroxyl and carbonyl groups. The 1H and 13C NMR analysis of compound 1 indicated the presence of a tertiary methyl group (δH 1.47), a secondary methyl group [δH 1.67 (d, J = 7.2 Hz)], an oxymethylene [δH 3.90 and 4.82 (each d, J = 9.3 Hz); δC 73.4 (C-14)], and two oxymethines [δH 4.29 (m); δC 74.7 (C-2); δH 5.03 (d, J = 5.9 Hz), δC 77.1 (C-7)]. A comparison of the NMR data that was generated for compound 1 with NMR data of jiadifenolide (3) [13] suggested that these two molecules shared a similar skeleton. The main difference between compound 1 and jiadifenolide (3) was the existence of a hydroxyl at C-2 in compound 1, which was indicated in the 1H NMR spectrum by the presence of an oxymethine proton signal [δH 4.29 (m); δC 74.7 (C-2)] and the absence of methylene proton signals. This was further supported by the correlations of H-15/H-1, H-1/H-2 and H-2/H-3 in its 1H–1H COSY spectrum and the correlations between H-15 with C-1, C-2, C-9 and H-2 with C-15 in its HMBC spectrum (Fig. 2). The configuration of the 2-OH group was determined as α based on an interaction between H-15 and H-2 in the NOESY spectrum (Fig. 3). To further corroborate the structure, a single crystal of compound 1 was obtained from MeOH, and X-ray crystallographic analysis with copper radiation was successfully performed (CCDC 1047242), which clearly indicated that the configuration of the 2-hydroxyl group was 2S. Accordingly, the structure of 1 was determined as 2S-hydroxyljiadifenolide (Fig. 4).
Compound 2, a white amorphous powder with [α]16.7 D–43.8 (c 0.089, MeOH), was determined by positive HR-ESI-MS (m/z 335.1107 [M + Na]+, calcd. 335.1107) to possess the molecular formula C15H20O7. Its IR spectrum showed absorption bands at 3430 cm−1 and 1763 cm−1, which were ascribed to hydroxyl and carbonyl functional groups, respectively. A 13C NMR spectrum (Table 1) displayed 15 carbon signals, including two methyl, four methylene, three methine and six quaternary carbons. A comparison of its NMR data with that of neomajucin (6) [20] suggested that the structure of compound 2 was almost identical to neomajucin except for the absence of a δ-lactone ring unit. This was also supported by the absence of a correlation of H-7 to C-11 in the HMBC. The downfield shift of C-10 (δC 80.0) and the HMBC correlation of H-7 to C-10 allowed us to connect C-7 and
Table 2 Anti-HBeAg/HBsAg effects of selected compounds in HepG2.2.15.a Compounds
Concentration (μM)
HBeAgb inhibition rate (%)
HBsAgc inhibition rate (%)
1
61.35 12.27 2.45 64.10 12.82 2.56 64.52 12.90 2.58 64.94 12.99 2.59 61.35 12.27 2.45 64.10 12.82 2.56 64.10 12.82 2.56 61.35 12.27 2.45 100 10
17.60 ± 3.64 9.18 ± 4.01 5.49 ± 4.17 7.58 ± 1.21 6.06 ± 3.67 4.85 ± 2.41 18.82 ± 3.60 17.41 ± 3.66 17.96 ± 3.64 28.85 ± 3.15 15.70 ± 3.74 5.80 ± 4.17 37.93 ± 2.74 16.42 ± 3.69 11.84 ± 3.89 22.13 ± 3.44 18.41 ± 3.60 11.25 ± 3.92 12.58 ± 3.86 4.55 ± 4.22 4.70 ± 4.21 10.15 ± 5.56 7.70 ± 1.99 3.17 ± 2.72 36.86 ± 1.67 30.71 ± 12.00
12.43 ± 1.92 7.37 ± 2.25 5.21 ± 1.66 16.66 ± 4.24 4.73 ± 4.79 1.89 ± 2.41 21.54 ± 2.03 21.49 ± 2.84 9.94 ± 0.89 17.53 ± 1.81 16.39 ± 1.84 9.11 ± 3.86 23.47 ± 9.52 18.71 ± 6.34 9.22 ± 3.34 15.61 ± 0.53 14.20 ± 9.45 5.73 ± 4.51 24.66 ± 9.30 15.45 ± 2.05 14.67 ± 1.33 23.97 ± 6.09 15.14 ± 1.29 8.12 ± 2.87 26.47 ± 5.16 15.20 ± 4.02
2
3
4
5
6
7
10
3TCd
Fig. 3. Selected NOESY correlations of compounds 1 and 2.
a Results are expressed as the mean ± standard deviation (SD) from three independent experiments. b HBsAg: HBV surface antigen. c HBeAg: HBV e antigen. d 3TC: lamivudine, an antiviral agent used as a positive control.
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J.-F. Liu et al. / Fitoterapia 104 (2015) 41–44
C-10 through an ether bond, resulting in the formation of an oxolane ring. The relative configuration of compound 2 was defined by a NOESY experiment (Fig. 3) in which correlations between H-14 and H-10, H-8 and H-1, and H-1 and H-2 were exhibited. Thus, compound 2 possessed the same relative configuration as neomajucin (6), and the configuration of its H-10 was determined as β. Consequently, the structure of compound 2 was characterised as shown in Fig. 1 and named as jiadifenlactone acid. The eight known compounds were identified as being jiadifenolide (3) [13], 2-oxo-3,4-dehydroxyneomajucin (4) [20], 2-oxoneomajucin (5) [20], neomajucin (6) [20], majucin (7) [20], (2S)-hydroxyneomajucin (8) [20], (2R)-2-hydroxyneomajucin (9) [21] and (1R,2S)-1,2epoxyneomajucin (10) [21], respectively (see details in Supporting information). Compounds 1–7 and 10 were isolated and then tested for their potential anti-HBV activities according to their abilities to inhibit the secretion of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in HBV-infected HepG2.2.15 cells under non-cytotoxicity concentrations and the results are summarised in Table 2 (the IC50 values of the isolates were not measured) [19]. Lamivudine (3TC) was purchased from Glaxo SmithKline (Suzhou, China, purity N 98.5%) and used as a positive control. The results revealed that the tested seco-prezizaane-sesquiterpenes possessed weak anti-HBV activities. The most active compounds, compounds 4 and 5, against the expression of HBeAg and HBsAg produced inhibitions of 28.85 ± 3.15% and 17.53 ± 1.81% and 37.93 ± 2.74% and 23.47 ± 9.52% at concentrations of 64.94 μM and 61.35 μM, respectively, which suggested that the carbonyl group at the 2-position enhanced anti-HBV activity. Additionally, the presence of a δ-lactone ring moiety was found to be a structural requirement for anti-HBV activity. Conflict of interest The authors indicated no potential conflicts of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (NSFC no. 81172961). The authors are grateful to Dr. Bing Zhao (School of Pharmaceutical Science, Zhengzhou University) for all of the spectral measurements. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2015.05.004.
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Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Sesquiterpenes from the fruits of Illicium jiadifengpi and their anti-hepatitis B virus activities Ji-Feng Liu a,⁎, Lei Wang c, Ya-Feng Wang a,b, Xu Song a, Li-Jia Yang a, Yan-Bing Zhang a,⁎ a b c
School of Pharmaceutical Science, Zhengzhou University, Ke Xue Da Dao 100, Zhengzhou 450001, People's Republic of China Henan Key laboratory for Pharmacology of Liver Disease, Academy of Medical and Pharmaceutical Science, Zhengzhou University, 40 Daxue Road, Zhengzhou 450052, People's Republic of China College of Chemistry and Chemical Engineering, Xuchang University, Henan, Xuchang 461000, People's Republic of China
a r t i c l e
i n f o
Article history: Received 26 February 2015 Accepted in revised form 2 May 2015 Accepted 6 May 2015 Available online 9 May 2015 Keywords: Illiciaceae Illicium jiadifengpi Sesquiterpenes Anti-HBV activity
a b s t r a c t Two new compounds, 2S-hydroxyl-jiadifenolide (1) and jiadifenlactone acid (2), and eight known compounds were isolated from the fruits of Illicium jiadifengpi. Their structures were analysed using several spectroscopic techniques, including 1D-, 2D-NMR and HR-ESI-MS experiments. The anti-hepatitis B virus (HBV) activities of the isolates were evaluated via HBV transfection of the Hep G2.2.15 cell line. The inhibitory rates of the most active compounds, compounds 4 and 5, on the HBeAg and HBsAg expression were 28.85 ± 3.15% and 17.53 ± 1.81% and 37.93 ± 2.74% and 23.47 ± 9.52% at concentrations of 64.94 μM and 61.35 μM, respectively. © 2015 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Illicium species are rich in biosynthetically unique seco-prezizaanesesquiterpenes and prenylated C6–C3 compounds [1,2]. The former are considered to be characteristic chemical markers of Illicium species [3,4]. Several seco-prezizaane-sesquiterpenes have been reported to exhibit diverse biological activities, including anti-HBV activity. In a previous paper, we reported the structure and anti-HBV activities of henrylactones A–G, which were isolated from Illicium henryi [5,6]. As part of our ongoing studies on biologically active substances of the genus Illicium, we have investigated the chemical components of the ethanol extracts of the fruits of Illicium jiadifengpi, which is a mediumsized tree that is primarily found in South China. Its bark has been used as a traditional Chinese medicine for the treatment of rheumatism [7]. Previous chemical studies on I. jiadifengpi have indicated the presence of essential oils [7,8], sesquiterpenes [9–14], diterpenoids [15–17], and flavones [18]. In this paper we described two new seco-prezizaane-sesquiterpenes, 2S-hydroxyl-jiadifenolide (1) and jiadifenlactone acid (2), together with eight known compounds (3–10) and investigated their anti-HBV activities
2.1. General experimental procedures
⁎ Corresponding authors at: School of Pharmaceutical Science, Zhengzhou University, Ke Xue Da Dao 100, Zhengzhou 450001, People's Republic of China. E-mail addresses: [email protected] (J.-F. Liu), [email protected] (Y.-B. Zhang).
http://dx.doi.org/10.1016/j.fitote.2015.05.004 0367-326X/© 2015 Elsevier B.V. All rights reserved.
Optical rotations were determined on a Horiba SEPA-300 polarimeter (Horiba, Tokyo, Japan). UV spectra were measured on a Shimadzu UV-210A spectrophotometer (Shimadzu, Kyoto, Japan). IR (KBr) spectra were recorded on a Bio-Rad FTS-135 spectrometer (Bio-Rad, California, USA). 1D and 2D NMR spectra were recorded on Bruker AM-400 NMR spectrometers with TMS as internal standard (Bruker, Bremerhaven, Germany). MS spectra were run on a VG Auto Spec-3000 spectrometer (VG, Manchester, England) and API Qstar Pulsar (Applied Biosystems, Foster City, USA). Silica gel (200–300 mesh) for column chromatography was obtained from Qingdao Meigao Chemical Company, Qingdao, China. Sephadex LH-20 (20–150 μm) was purchased from Pharmacia Fine Chemical Co. Ltd., Sweden.
2.2. Plant material The fruits of I. jiadifengpi B. N. Chang were collected from Guangdong Province, P.R. China, in September 2011 and identified by Dr. Mengqi Liu, School of Pharmaceutical Science, Henan University of Traditional Chinese Medicine. A voucher specimen of the fruits (2011-09-02) was deposited in the School of Pharmaceutical Science at Zhengzhou University.
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Table 1 1 H-(400 MHz) and 13C-(100 MHz) NMR data for 1 and 2 in C5D5N. Position
1 δH (mult, J in Hz)
2 δC
1 2
3.92 (m) 4.29 (m)
46.9 74.7
3
2.61 (1H, dd, 13.5, 9.4) 2.40 (1H, dd, 13.4, 5.4) – – – 5.03 (1H, d, 5.9) 2.76 (1H, dd, 12.8, 6.0) 2.49 (1H, d, 12.8) – – – – 1.47 (3H, s) 4.82 (1H, d, 9.3) 3.90 (1H, d, 9.3) 1.67 (3H, d, 7.2)
42.8
4 5 6 7 8 9 10 11 12 13 14 15
92.6 47.4 78.4 77.1 32.1 56.6 102.0 172.7 176.9 20.2 73.4 12.5
δH (mult, J in Hz)
δC
3.31 (m) 2.17 (1H, m) 1.80 (1H, m) 1.97 (1H, m) 2.00 (1H, m) – – – 5.10 (1H, d, 6.1) 3.09 (1H, d, 11.6) 2.98 (1H, dd, 11.7, 6.2) – 4.83 (1H, s) – – 1.65 (3H, s) 4.60 (1H, d, 8.7) 4.03 (1H, d, 8.7) 1.36 (3H, d, 7.5)
33.4 29.6 29.8 82.7 48.2 78.9 81.9 27.0 61.9 80.0 175.4 180.1 21.1 75.1 13.0
2.3. Extraction and isolation Powdered fruits of I. jiadifengpi (5.0 kg) were extracted three times with 95% EtOH (20 L) under reflux for 2 h each time. The extract was concentrated under a vacuum to obtain a residue. The residue was dissolved in H2O and then extracted successively with CHCl3 (three times, each 2.5 L) and n-BuOH (three times, each 2.5 L). The CHCl3 fraction (90 g) was subjected to silica gel column chromatography (CC) (9 × 40 cm, 1000 g, 200–300 mesh) and eluted with a CHCl3/ MeOH (100:0, 90:10, 80:20, v/v, each 10 L) gradient to produce nine fractions (Frs. 1–9). Fr. 2 (10 g) was further subjected to silica gel CC (5 × 35 cm, 280 g; petroleum ether/EtOAc, 100:0–60:30 gradient) to produce six sub-fractions (Frs. 2a–2f). Compounds 1 (20 mg), 3 (15 mg) and 4 (10 mg) were obtained from the Fr. 2b (1.0 g) by silica gel CC (2.5 × 40 cm, 80 g; CHCl3/Me2CO, 95:5–70:30 gradient). Fr. 2c (2.3 g) was chromatographed through a silica gel column (2.6 × 50 cm, 100 g) and eluted with petroleum ether/acetone (98:2, 90:10, 80:20, 70:30, v/v, each 1.0 L) to yield compounds 2 (8 mg), 5 (35 mg) and 10 (50 mg). Fr. 3 (7 g) was subjected to silica gel CC (5 × 32 cm, 200 g; petroleum ether/acetone, 90:10–70:30 gradient) followed by Sephadex LH-20 CC (MeOH) to obtain compounds 6 (9 mg) and 8 (5 mg). Fr. 4 (10 g) was also subjected to a silica gel column (5 × 32 cm, 200 g) and eluted with a CHCl3/Me2CO (100:0, 90:10,
80:20, v/v, each 3 L) gradient to obtain six fractions (Frs. 4a–4f). Fr. 4b (1.5 g) was further separated through column chromatography on silica gel (2.4 × 30 cm, 60 g) with petroleum ether/acetone (60:40) to furnish compound 9 (8 mg). Fr. 5 (2 g) was chromatographed through a silica gel column (2.4 × 30 cm, 60 g, CHCl3:MeOH = 90:10) and further purified by Sephadex LH-20 CC (MeOH) to provide compound 7 (38 mg). Each of the purified compounds had a degree of purity N 92%. 2S-hydroxyl-jiadifenolide (1) Needle crystals (MeOH); [α]24.9 D–103.30 (c 0.087, MeOH); UV (CHCl3): λmax (log ε) = 206 (2.76), 215 (2.59) nm; IR (KBr): νmax = 3406, 1784, 1630, 1250, 1036 cm− 1; 13 1 H and C NMR data see Table 1; HRESI-MS: m/z = 349.0897 [M + Na]+ (calcd. for C15H18O8Na: 349.0899). Jiadifenlactone acid (2) White amorphous solid; [α]16.7 D − 43.80 (c 0.089, MeOH); UV (CHCl3): λmax (log ε) = 208 (2.99), 215 (2.88) nm; IR (KBr): νmax = 3430, 2925, 1763, 1625, 1383, 1023 cm− 1; 1H and 13C NMR data see Table 1; HR-ESI-MS: m/z = 335.1107 [M + Na]+ (calcd. for C15H20O7Na: 335.1107).
2.3.1. X-ray diffraction analysis of compound 1 The crystal structure data for 1: λ = 1.54184 Å, T = 291 (2) K, space group P21, a = 7.3608 (3) Å, b = 13.4259 (6) Å, c = 13.9975 (6) Å, V = 1383.30 (3) Å, Z = 4, Dc = 1.567 g·cm− 3, crystal size 0.20 × 0.16 × 0.16 mm. These data had been deposited in the Cambridge Crystallographic Date Center (deposition number: 1047242) and can be obtained free via http://www.ccdc.cam.ac.uk/deposit. An anti-HBV assay was performed according to a previous report [19]. Hep G2.2.15 cells were grown in 96-well plates and were allowed to form monolayers. Aliquots of 200 mL of media containing varying concentrations of test media at doses below the CC50 were added to each well. Cells that were not exposed to test compounds served as cell controls. Lamivudine (3TC) was used as a reference compound. The cells were grown in the presence of the studied compounds for 9 days; their media were renewed on the 6th day. Suspensions were immediately collected for antigen assays. The concentrations of HBsAg and HBeAg in the cell culture supernatants were detected using a commercially available ELISA kit (Kehua Bio-engineering Corporation, Shanghai,
Fig. 1. Structures of compounds 1–10.
J.-F. Liu et al. / Fitoterapia 104 (2015) 41–44
43
Fig. 2. Key HMBC correlations of compounds 1 and 2.
China) according to the manufacturer's protocol. Inhibition rates (%) were calculated using the following formula: [(A450 of the control wells without drug − A450 of the experimental wells) / (A450 of the control wells without drug − A450 of blank control wells without cells)] × 100% (see details in Supporting information). Fig. 4. X-ray crystal structure of compound 1.
2.4. Supporting information The NMR data of compounds 3–10 and original spectra for compounds 1–2, as well as the procedure for anti-HBV assay of the tested compounds are available as Supporting information. 3. Results and discussion Compound 1 was obtained as needle crystals (MeOH). Its molecular formula was defined as C15H18O8, as determined by HR-ESI-MS (m/z 349.0897 [M + Na]+, calcd. 349.0899). The IR spectrum showed absorption bands at 3406 and 1784 cm−1, which were respectively indicative of hydroxyl and carbonyl groups. The 1H and 13C NMR analysis of compound 1 indicated the presence of a tertiary methyl group (δH 1.47), a secondary methyl group [δH 1.67 (d, J = 7.2 Hz)], an oxymethylene [δH 3.90 and 4.82 (each d, J = 9.3 Hz); δC 73.4 (C-14)], and two oxymethines [δH 4.29 (m); δC 74.7 (C-2); δH 5.03 (d, J = 5.9 Hz), δC 77.1 (C-7)]. A comparison of the NMR data that was generated for compound 1 with NMR data of jiadifenolide (3) [13] suggested that these two molecules shared a similar skeleton. The main difference between compound 1 and jiadifenolide (3) was the existence of a hydroxyl at C-2 in compound 1, which was indicated in the 1H NMR spectrum by the presence of an oxymethine proton signal [δH 4.29 (m); δC 74.7 (C-2)] and the absence of methylene proton signals. This was further supported by the correlations of H-15/H-1, H-1/H-2 and H-2/H-3 in its 1H–1H COSY spectrum and the correlations between H-15 with C-1, C-2, C-9 and H-2 with C-15 in its HMBC spectrum (Fig. 2). The configuration of the 2-OH group was determined as α based on an interaction between H-15 and H-2 in the NOESY spectrum (Fig. 3). To further corroborate the structure, a single crystal of compound 1 was obtained from MeOH, and X-ray crystallographic analysis with copper radiation was successfully performed (CCDC 1047242), which clearly indicated that the configuration of the 2-hydroxyl group was 2S. Accordingly, the structure of 1 was determined as 2S-hydroxyljiadifenolide (Fig. 4).
Compound 2, a white amorphous powder with [α]16.7 D–43.8 (c 0.089, MeOH), was determined by positive HR-ESI-MS (m/z 335.1107 [M + Na]+, calcd. 335.1107) to possess the molecular formula C15H20O7. Its IR spectrum showed absorption bands at 3430 cm−1 and 1763 cm−1, which were ascribed to hydroxyl and carbonyl functional groups, respectively. A 13C NMR spectrum (Table 1) displayed 15 carbon signals, including two methyl, four methylene, three methine and six quaternary carbons. A comparison of its NMR data with that of neomajucin (6) [20] suggested that the structure of compound 2 was almost identical to neomajucin except for the absence of a δ-lactone ring unit. This was also supported by the absence of a correlation of H-7 to C-11 in the HMBC. The downfield shift of C-10 (δC 80.0) and the HMBC correlation of H-7 to C-10 allowed us to connect C-7 and
Table 2 Anti-HBeAg/HBsAg effects of selected compounds in HepG2.2.15.a Compounds
Concentration (μM)
HBeAgb inhibition rate (%)
HBsAgc inhibition rate (%)
1
61.35 12.27 2.45 64.10 12.82 2.56 64.52 12.90 2.58 64.94 12.99 2.59 61.35 12.27 2.45 64.10 12.82 2.56 64.10 12.82 2.56 61.35 12.27 2.45 100 10
17.60 ± 3.64 9.18 ± 4.01 5.49 ± 4.17 7.58 ± 1.21 6.06 ± 3.67 4.85 ± 2.41 18.82 ± 3.60 17.41 ± 3.66 17.96 ± 3.64 28.85 ± 3.15 15.70 ± 3.74 5.80 ± 4.17 37.93 ± 2.74 16.42 ± 3.69 11.84 ± 3.89 22.13 ± 3.44 18.41 ± 3.60 11.25 ± 3.92 12.58 ± 3.86 4.55 ± 4.22 4.70 ± 4.21 10.15 ± 5.56 7.70 ± 1.99 3.17 ± 2.72 36.86 ± 1.67 30.71 ± 12.00
12.43 ± 1.92 7.37 ± 2.25 5.21 ± 1.66 16.66 ± 4.24 4.73 ± 4.79 1.89 ± 2.41 21.54 ± 2.03 21.49 ± 2.84 9.94 ± 0.89 17.53 ± 1.81 16.39 ± 1.84 9.11 ± 3.86 23.47 ± 9.52 18.71 ± 6.34 9.22 ± 3.34 15.61 ± 0.53 14.20 ± 9.45 5.73 ± 4.51 24.66 ± 9.30 15.45 ± 2.05 14.67 ± 1.33 23.97 ± 6.09 15.14 ± 1.29 8.12 ± 2.87 26.47 ± 5.16 15.20 ± 4.02
2
3
4
5
6
7
10
3TCd
Fig. 3. Selected NOESY correlations of compounds 1 and 2.
a Results are expressed as the mean ± standard deviation (SD) from three independent experiments. b HBsAg: HBV surface antigen. c HBeAg: HBV e antigen. d 3TC: lamivudine, an antiviral agent used as a positive control.
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J.-F. Liu et al. / Fitoterapia 104 (2015) 41–44
C-10 through an ether bond, resulting in the formation of an oxolane ring. The relative configuration of compound 2 was defined by a NOESY experiment (Fig. 3) in which correlations between H-14 and H-10, H-8 and H-1, and H-1 and H-2 were exhibited. Thus, compound 2 possessed the same relative configuration as neomajucin (6), and the configuration of its H-10 was determined as β. Consequently, the structure of compound 2 was characterised as shown in Fig. 1 and named as jiadifenlactone acid. The eight known compounds were identified as being jiadifenolide (3) [13], 2-oxo-3,4-dehydroxyneomajucin (4) [20], 2-oxoneomajucin (5) [20], neomajucin (6) [20], majucin (7) [20], (2S)-hydroxyneomajucin (8) [20], (2R)-2-hydroxyneomajucin (9) [21] and (1R,2S)-1,2epoxyneomajucin (10) [21], respectively (see details in Supporting information). Compounds 1–7 and 10 were isolated and then tested for their potential anti-HBV activities according to their abilities to inhibit the secretion of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in HBV-infected HepG2.2.15 cells under non-cytotoxicity concentrations and the results are summarised in Table 2 (the IC50 values of the isolates were not measured) [19]. Lamivudine (3TC) was purchased from Glaxo SmithKline (Suzhou, China, purity N 98.5%) and used as a positive control. The results revealed that the tested seco-prezizaane-sesquiterpenes possessed weak anti-HBV activities. The most active compounds, compounds 4 and 5, against the expression of HBeAg and HBsAg produced inhibitions of 28.85 ± 3.15% and 17.53 ± 1.81% and 37.93 ± 2.74% and 23.47 ± 9.52% at concentrations of 64.94 μM and 61.35 μM, respectively, which suggested that the carbonyl group at the 2-position enhanced anti-HBV activity. Additionally, the presence of a δ-lactone ring moiety was found to be a structural requirement for anti-HBV activity. Conflict of interest The authors indicated no potential conflicts of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (NSFC no. 81172961). The authors are grateful to Dr. Bing Zhao (School of Pharmaceutical Science, Zhengzhou University) for all of the spectral measurements. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2015.05.004.
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