Four new sesquiterpenes from the stems of Pogostemon cablin

Fitoterapia 86 (2013) 183–187

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Four new sesquiterpenes from the stems of Pogostemon cablin Fei Li a, Chuang-Jun Li a, Jie Ma a, Jing-Zhi Yang a, Hui Chen a, Xi-Ming Liu b, Yan Li a, Dong-Ming Zhang a,⁎ a State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China b Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China

a r t i c l e

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Article history: Received 12 January 2013 Accepted in revised form 6 March 2013 Available online 19 March 2013 Keywords: Pogostemon cablin Sesquiterpene Patchoulene Guaiane Hepatoprotective activity

a b s t r a c t Four new sesquiterpenes (1–4), together with four known compounds (5–8), were isolated from the stems of Pogostemon cablin (Blanco.) Benth. Their chemical structures were elucidated by means of spectroscopic methods, including 1D and 2D NMR, HRESIMS, IR, and UV. Hepatoprotective activities of these compounds were investigated, by studying the protective effect on HL-7702 cellular injury induced by DL-galactosamine. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Pogostemon cablin (Blanco.) Benth., an annual herb, is a member of genus Pogostemon, Lamiaceae family. It is mostly distributed in tropical and subtropical areas and also fostered in Guangdong, Guangxi, Taiwan and Fujian provinces in China. P. cablin is a traditional Chinese medicine recorded in Chinese Pharmacopoeia [1] for its therapeutic function for heat and dampness elimination, nerve smoothness and fatigue alleviation. It was also used as a component of some proprietary Chinese medicines against indigestion, headache, and fever [1]. In addition, the patchouli alcohol, which is the most abundant in the plant, is an important ingredient in many fine fragrance products like perfumes, as well as in soaps and cosmetic products [2]. Chemical and pharmacological researches on P. cablin have been carried out in recent years. A

⁎ Corresponding author at: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Xicheng District, Beijing 100050, China. Tel./fax: +86 10 63165227. E-mail addresses: [email protected] (F. Li), [email protected] (D.-M. Zhang). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.03.010

number of mono- and sesquiterpenoids [3–7], triterpenoids and steroids [8–10], flavonoids [11–14], alkaloids [15] and phenylpropanoid glycosides [16] were reported from the title plant. The constituents exhibited marked activities such as antibacterial [17–19], anti-influenza virus [20], antiinflammatory [21], cytotoxic [13], antimutagenic [12], inhibitory activity against PAF-induced platelet aggregation [22], and insecticidal activity [23]. As a part of our research on bioactive constituents of Traditional Chinese Medicine (TCM), a detailed phytochemical investigation on the 70% ethanol extract of P. cablin was carried out. Here, we reported the isolation and characterization of three new patchoulenetype and a new guaiane-type sesquiterpenes (Fig. 1). Their hepatoprotective activities were also displayed in this paper. 2. Experimental procedure 2.1. General Optical rotations were recorded on a Jasco P-2000 automatic digital polarimeter. UV spectra were measured in CH3OH on a Jasco V650 spectrophotometer (JASCO, Inc., Easton, USA).

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IR spectra were measured by means of FT-IR Microscope Transmission, on an FT-IR spectroscope (Nicolet 5700, Thermo Nicolet Corporation, Madison, USA). The 1H, 13C NMR, 1H– 1H COSY, HSQC, HMBC and NOESY spectra were obtained on an INOVA spectrometer (Varian Medical Systems, Inc., Palo Alto, California, USA) at 500 and 125 MHz for 1H and 13C, respectively. ESIMS experiments were performed on an Agilent 1100 Series LC/MSD Trap-SL mass spectrometer. HRESIMS data were measured by an Agilent Technologies 6520 Accurate Mass Q-Tof LC/MS spectrometer (Agilent Technologies, Santa Clara, USA). HPLC was performed on a Shimadzu LC-6 AD instrument with a SPD-20A detector (Shimadzu Corporation, Kyoto, Japan). A reverse-phase C18 column (YMC-Pack ODS-A Φ 20 × 250 mm, 5 μm, YMC Co., Ltd, Kyoto, Japan) was employed. Column chromatography was performed with PRP-512 resin (Beijing Jufu Resin Company, Beijing, China) and silica gel (100– 200 mesh, Qingdao Marine Chemical Inc., Qingdao, China). TLC was carried out on precoated silica gel GF254 plates. Spots were visualized under UV light (254 or 365 nm) or by spraying with 10% H2SO4 in 90% EtOH followed by heating. 2.2. Plant material The stems of P. cablin (Blanco.) Benth. were collected in April 2010, from Guangdong province, China, and identified by Prof. Lin Ma. Its voucher specimen (ID-S-2319) is deposited at the Institute of Materia Medica, Chinese Academy of Medical Sciences. 2.3. Extraction and isolation The air-dried powdered stems of P. cablin (20 kg) were refluxed with 70% ethanol for 3 times (2 h each time). The EtOH extract was evaporated under reduced pressure to yield a dark green residue. The residue was suspended in water, and then partitioned with PE, EtOAc, and BuOH, successively. After the removal of the solvent, the EtOAc portion (130 g) was chromatographed on silica gel (100– 200 mesh, 13.5 × 100 cm) column and eluted with CHCl3– CH3OH system (100:0, 95:5, 9:1, 8:2, 7:3, and 0:100) to give F1–F8. F2 (31 g) was refined by PRP-512 resin, and eluted with 50%, 70%, 90% and 100% CH3OH–H2O. The 70% portion (F2–70, 4.5 g) was chromatographed over reversed-phase silica gel, eluting with a gradient of increasing CH3OH (50%– 100%) in H2O, to yield 15 subfractions (F2-70-1–F2-70-15). F2-70-12 (131 mg) was separated by HPLC (30% CH3CN–H2O,

8 mL/min, 210 nm) to afford compounds 2 (15 mg), 3 (13 mg), and a mixture of 4 and 5. Phenyl-bonded phase preparative column was employed to separate 4 and 5. With a retained time of 235 min, the mixture peak finally split into two peaks in the preparation diagram (13% CH3CN–H2O, 8 mL/min, 210 nm). F2-70-14 (78 mg) was purified by HPLC (60% CH3OH–H2O) to yield 1 (8 mg), 6 (10 mg), 7 (4 mg), and 8 (5 mg). Compound 1 colorless oil; [α]D20 − 180.4 (MeOH, c 0.10); UV (MeOH) λmax (log ε) 252 (1.43) nm; IR νmax 3492, 2970, 2953, 1692, 1641, 1372, 1104, 914 cm −1; HRESIMS: m/z 235.1690 [M + H] + (calcd for C15H23O2, 235.1693). 1H NMR (500 MHz, DMSO) and 13C NMR (125 MHz, DMSO) spectral data see Tables 1 and 2. Compound 2 colorless oil; [α]D20 + 19.7 (MeOH, c 0.10); UV (MeOH) λmax (log ε) 240 (1.47) nm; IR νmax 3448, 2972, 2933, 1688, 1618, 1391, 1195, 946 cm −1; HRESIMS: m/z 235.1687 [M + H] + (calcd for C15H23O2, 235.1693). 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectral data see Tables 1 and 2. Compound 3 colorless oil; [α]D20 − 28.3 (MeOH, c 0.10); UV (MeOH) λmax (log ε) 240 (1.50) nm; IR νmax 3416, 2973, 2922, 1673, 1621, 1367, 1189, 989 cm −1; HRESIMS: m/z 235.1689 [M + H] + (calcd for C15H23O2, 235.1693). 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectral data see Tables 1 and 2. Compound 4 colorless oil; [α]D20 − 49.5 (MeOH, c 0.10); UV (MeOH) λmax (log ε) 238 (1.31) nm; IR νmax 3482, 2969, 2922, 1687, 1644, 1374, 1233, 896 cm −1; HRESIMS: m/z 235.1691 [M + H] + (calcd for C15H23O2, 235.1693). 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectral data see Tables 1 and 2.

2.4. Protective effect on cytotoxicity induced by DL-galactosamine in HL-7702 Cells The hepatoprotective effects of compounds 1–8 were determined by a MTT colorimetric assay [24] in HL-7702 cells. Each cell suspension of 2 × 104 cells in 200 μL of RPMI 1640 containing fetal calf serum (10%), penicillin (100 U/mL), and

Fig. 1. Structures of compounds 1–4.

F. Li et al. / Fitoterapia 86 (2013) 183–187

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Table 1 1 H NMR spectra data of compounds 1–4 (δ in ppm, J in Hz, 500 MHz). Proton

1a

H-2

α.2.13, 1H, m β.1.51, 1H, m 1.78, 2H, m

H-3

H-5

2.27, 1H, dd (J = 10.4, 10.4 Hz) α.1.45, 1H, dt (J = 13.2, 10.4 Hz) β.2.04, 1H, dt (J = 13.2, 6.4 Hz) 2.32, 1H, br d (J = 6.4 Hz)

H-6

H-7

2b

3b

4b

α.2.57, 1H, d (J = 17.5 Hz) β.2.49, 1H, d (J = 17.5 Hz)

2.52, 2H, br s

2.56, 2H, br s

α.2.11, 1H, dd (J = 19.5, 2.0 Hz) β.2.70, 1H, dd (J = 19.5, 3.0 Hz) 1.95, 1H, m

α.2.27, 1H, dd (J = 19.5, 2.0 Hz) β.2.56, 1H, br d overlapped 1.93, 1H, m α.1.28, 1H, m β.2.00, 1H, m α.1.73, 1H, m β.1.81, 1H, m

α.2.42, 1H, dd (J = 16.0, 12.0 Hz) β.2.67, 1H, dd (J = 16.0, 2.0 Hz) 2.01, 1H, dt (J = 12.0, 2.0 Hz) 1.83, 2H, m

H-9

5.69, 1H, s

α.1.18, 1H, m β.2.00, 1H, m 1.94, 2H, m

H-10 CH3-12

0.88, 3H, s

0.88, 3H, s

0.81, 3H, s

CH3-13 CH3-14 CH3-15

0.87, 3H, s 1.23, 3H, s 1.97, 3H, s

0.92, 3H, s 1.44, 3H, s 1.29, 3H, s

0.92, 3H, s 1.42, 3H, s 1.29, 3H, s

−OH

4.36, 1H, br s

H-8

a b

α.1.57, 1H, m β1.77, 1H, m 3.01, 1H, m 4.73, 1H, br s 4.76, 1H, br s 1.79, 3H, s 1.43, 3H, s 1.01, 3H, d (J = 7.5 Hz)

Compound 1 was measure in DMSO, and TMS was used as internal standard. Compounds 2, 3, 4 were measured in CDCl3, and TMS was used as internal standard.

streptomycin (100 μg/mL) was placed in a 96-well microplate and precultured for 24 h at 37 °C under a 5% CO2 atmosphere. Fresh medium (100 μL) containing bicyclol and test samples was added, and the cells were cultured for 1 h. Then, the cultured cells were exposed to 25 mM DL-galactosamine for 24 h. Then, 100 μL of 0.5 mg/mL MTT was added to each well after the withdrawal of the culture medium and incubated for an additional 4 h. The resulting formazan was dissolved in 150 μL of DMSO after aspiration of the culture medium. The

Table 2 13 C NMR data (δ) of compounds 1–4 (125 MHz). Carbon

1a

2b

3b

4b

C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15

64.8 23.9 44.4 78.6 67.6 27.2 63.3 201.1 124.8 173.5 50.2 25.8 20.8 25.3 21.4

145.8 203.6 53.2 74.3 172.8 30.8 43.2 29.2 41.1 45.4 43.8 19.3 22.9 26.5 13.2

145.6 204.1 53.0 74.0 173.4 30.8 43.2 29.0 41.2 43.9 43.2 19.1 22.8 26.1 12.9

146.0 204.6 51.0 76.6 173.4 32.3 46.7 30.9 32.6 26.9 151.0 109.4 20.7 26.6 17.2

a Compound 1 was measured in DMSO, and TMS was used as internal standard. b Compounds 2, 3, 4 were measured in CDCl3, and TMS was used as internal standard.

optical density (OD) of the formazan solution was measured on a microplate reader at 492 nm. All values were expressed as ±SD. The Student's t-test for unpaired observations between normal or control and tested samples was carried out to identify statistical differences; p values less than 0.05 were considered as significantly different. 3. Results and discussion Compound 1 was isolated as a colorless oil. Its molecular formula, C15H22O2, was deduced from the HRESIMS for the [M + H]+ ion peak at m/z 235.1690 (calcd for C15H23O2, 235.1693), implying five degrees of unsaturation. The UV spectrum showed absorbance at λmax 252 nm, and the IR spectrum exhibited the presence of a hydroxyl (3492 cm−1) and an α, β-unsaturated carbonyl moiety (1692 and 1641 cm−1). An olefinic proton δH 5.69 (1H, s), a hydroxy group δH 4.36 (1H, s), and four single methyls δH 0.87 (3H, s), δH 0.88 (3H, s), δH 1.23 (3H, s), and δH 1.97 (3H, s), were observed in the 1H NMR spectrum. The 13C NMR and HSQC spectra disclosed signals corresponding to 15 carbons, including one carbonyl (δC 201.1), one olefinic group (δC 173.5, 124.8), three quaternary carbons (δC 78.6, 64.8, 50.2) including one (δC 78.6) connected to an oxygen atom, two methines (δC 67.6, 63.3), three methylenes (δC 44.4, 27.2, 23.9), and four methyl groups (δC 25.8, 25.3, 21.4, 20.8). Based on the NMR data, the structure of compound 1 was established as a patchoulene-type sesquiterpene [25]. In the 1H– 1H COSY spectrum, two spin systems were detected, with the first fragment running from H-2 to H-3, and the second connecting H-5 to H-7 (Fig. 2). In the HMBC, long-range correlations: H-14 to C-3, C-4 and C-5, H-12 and H-13 to C-1, C-7 and C-11, and H-15 to C-1, C-9 and C-10, confirmed the

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Fig. 2. Important correlations observed in HMBC and 1H–1H COSY of compounds 1–4.

locations of the four methyls. The methyl groups H-12 and H-13 were determined to be germinal at C-11 based on mutual HMBC correlations to each other. The hydroxyl δH 4.36 (1H, s) was determined to be connected to C-4 since it was correlated to C-3, C-4, C-5 and C-14 in HMBC. Furthermore, the correlations of H-6, 7/C-8, H-7/C-9, and H-2, 5/C-10 established the position of α, β-unsaturated carbonyl. The relative configuration was determined by NOESY experiment (Fig. 3). The spatial correlation from H-14 (3H, s) to H-6β suggested that the H-14 was β-oriented. Thus, 4-OH was α-oriented. Since 4-OH/H-5, H-12/ H-14, and H-12/H-6β were correlated with each other, H-14 and the bridge between C-1 and C-7 via C-11 were on the same side of the ring system. Therefore, the structure of 1 was determined to be 8-keto-9(10)-α-patchoulene-4α-ol. Compound 2 was obtained as a colorless oil. The HRESIMS at m/z 235.1687 [M + H]+ displayed the molecular formula to be C15H22O2 (calcd for C15H23O2, 235.1693). The UV absorbance at λmax 240 nm suggested an enone moiety. The IR spectral data indicated the presence of OH (3448 cm−1) and C_O (1688 cm−1). The 1H NMR, 13C NMR, and HSQC spectra were all similar to those of 1 except that the chemical shift of CH3-15 was shifted from δH 1.97 to 1.29, and the olefinic proton δH 5.69 (1H, s) disappeared. Due to 1H– 1H COSY spectra, a fragment of \CH2(6)\CH(7)\CH2(8)\CH2(9)\ was deduced (Fig. 2), which indicated that the bridge carbon of the ring system should be changed. Combined analyzes of HMBC spectrum, correlations of H-6/C-11, H-7/C-10 and C-11, and H-9/C-11 revealed the presence of an internal bridge from C-7 to C-10 via C-11. The connectives observed in HMBC between H-3, 6, 9, 15/ C-1, H-3/C-2, and H-3, 6, 7/C-5 determined the location of the enone moiety at C-1, 2, and 5. Thus, the planar structure of 2 was established (Fig. 2). The relative configuration of 2 was confirmed through the NOESY method (Fig. 3). NOEs were observed between H-14 and H-6α, and H-12 and H-6β. These correlations suggested that H-14 and the germinal methyl

groups were on the opposite side of the ring system. Thus, the structure of 2 was finally determined as 2-keto-1(5)-βpatchoulene-4β-ol. Compound 3 gave the same molecular formula C15H22O2 as 2 for the [M + H] + ion at m/z 235.1689 in HRESIMS (calcd for C15H23O2, 235.1693). The UV, IR and NMR spectroscopic data were all similar to those of 2, which indicated that 3 may be an isomer of 2. Detailed 1H NMR spectrum showed that H-3 changed into a broad singlet comparing to the double doublets of 2, which suggested the chemical atmosphere around H-3 may changed. The HSQC, 1H– 1H COSY, and HMBC spectra confirmed that the planar structure of 3 was also 2-keto-1(5)-β-patchoulene-4-ol. With NOESY correlations from H-14 to H-6β and H-12 to H-6β, the 4-OH was determined to be α-oriented. Therefore, compound 3 was established as 2-keto-1(5)-β-patchoulene-4α-ol (Fig. 1). Compound 4 was also obtained as a colorless oil, with C15H22O2 as its molecule formula, indicated by HRESIMS at m/z 235.1691 [M + H] + (calcd for C15H23O2, 235.1693). The NMR spectra of 4 were closely similar with 4-hydroxy-10epirotundone [26], except that the signals of H-6 and H-7 moved to lower fields than the known compound. The enhancement of integration values of H-6β, H-3, and H-7 was observed when 4-OH (solvent DMSO-d6) was irradiated, and irradiation of CH3-15 resulted in the enhancement of H-6α in the NOE difference spectral experiments (Fig. 3). Thus, 4β-OH, 7β-H, and 15α-CH3 were verified. Therefore, the structure of 4 was established as 2-keto-4β-hydroxyguai-1, 11-diene. The known compounds 5–8 were identified as 4hydroxy-10-epirotundone [26], tschimganical A [27], 5, 7dihydroxy-3′, 4′-dimethoxyflavanone [28], and 3-hydroxy-4methoxycinnamaladehyde [29] by comparison of their physicochemical data (UV, IR, NMR, MS, and [α]D20) with the reported values.

Fig. 3. Key NOEs in compounds 1–4.

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Compounds 1–8 were bioassayed for their hepatoprotective activities against DL-galactosamine-induced toxicity in HL-7702 cells, using the hepatoprotective activity drug bicyclol as the positive control. At 10 μM, compounds 6, 7, and 8 reduced DL-galactosamine (GalN)-induced HL-7702 cells damage by 65.4% ± 0.047, 73.6% ± 0.050, and 70.7% ± 0.016, respectively, while the positive control bicyclol gave an 87.8% ± 0.049 survival rate. However, sesquiterpenes 1–5 showed moderate activities with survival rate at 33.0% ± 0.026, 40.5% ± 0.043, 32.4% ± 0.036, 32.3% ± 0.016, and 35.4% ± 0.059, respectively. Acknowledgments We thank our colleagues of our institute: Professor X. J. Jin for NMR measurements, and Professor J. B. Li for MS measurements. This research program was supported by the National Natural Science Foundation of China (no. 21132009), the National Science and Technology Project of China (2011ZX09307-002-01), the National Basic Research Program of China (2009CB523004), the Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (IRT1007). References [1] China Pharmacopoeia Committee, Chinese Pharmacopoeia, China Medical Science Press: Beijing; 2010, vol. 1, p. 42. [2] Bauer K, Garbe D, Surburg H. Preparation, properties and used. Common fragrance and flavour materials. 3rd ed. Weinheim: Wiley-VCH; 1997. p. 205. [3] Terhune SJ, Hogg JW, Laurence BM. Tetrahedron Lett 1973;47:4705.

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