New sesquiterpenes from the rhizomes of homalomena occulta

    New sesquiterpenes from the rhizomes of homalomena occulta Feng Zhao, Chao Sun, Li Ma, Ya-Nan Wang, Yuan-Fang Wang, JuFeng Sun, Gui-Ge Hou, Wei Cong, Hong-Juan Li, Xiao-Hua Zhang, Yan Ren, Chun-Hua Wang PII: DOI: Reference:

S0367-326X(15)30144-1 doi: 10.1016/j.fitote.2015.12.015 FITOTE 3324

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

10 November 2015 15 December 2015 17 December 2015

Please cite this article as: Feng Zhao, Chao Sun, Li Ma, Ya-Nan Wang, Yuan-Fang Wang, Ju-Feng Sun, Gui-Ge Hou, Wei Cong, Hong-Juan Li, Xiao-Hua Zhang, Yan Ren, ChunHua Wang, New sesquiterpenes from the rhizomes of homalomena occulta, Fitoterapia (2015), doi: 10.1016/j.fitote.2015.12.015

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ACCEPTED MANUSCRIPT New sesquiterpenes from the rhizomes of Homalomena occulta Feng Zhao a, Chao Sun b, Li Ma b, Ya-Nan Wang c, Yuan-Fang Wang a, Ju-Feng Sun a, Gui-Ge

The Key Laboratory of Traditional Chinese Medicine Prescription Effect and Clinical Evaluation of State

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a

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Hou a, Wei Cong a, Hong-Juan Li a, Xiao-Hua Zhang a, Yan Ren a, Chun-Hua Wang a*

Administration of Traditional Chinese Medicine, School of Pharmacy, Binzhou Medical University, Guanai Road 346, Yantai 264003, China Yantai Stomatological Hospital, Yantai 264001, China

c

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica,

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b

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Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

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[Abstract] Six new sesquiterpenes (1-6), along with eight known ones (7-14) were isolated from

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the rhizomes of Homalomena occulta. Structure elucidation of the new compounds was achieved through 1D NMR, 2D NMR spectroscopic techniques and HRESIMS, while the absolute

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configurations of compounds 1, 2 and 5 were confirmed by X-ray crystallographic analysis. All of the isolates were evaluated for their activity against LPS-induced production of nitrogen oxide (NO) in macrophage cells, and compounds 1 and 5 showed inhibitory effect on NO production with the IC50 values of 21.2 and 15.4 M, respectively.

Key words: Homalomena occulta; sesquiterpene; crystallographic analysis; NO production inhibition



Corresponding authors. Tel.: +86 535 6913406; fax: +86 535 6913718. E-mail addresses: [email protected] (F. Zhao); [email protected] (C. Wang).

ACCEPTED MANUSCRIPT 1. Introduction The genus Homalomena (Araceae) is widely distributed in America and the tropical areas of

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Asia and comprises over 140 species, but only four species belonging to this genus were found in

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China: Homalomena aromatic, Homalomena occulta, Homalomena kelungensis and Homalomena hainanensis [1]. H. occulta is widely distributed in Guangdong, Guangxi and Yunnan Provinces, the rhizomes of which have long been used as Chinese Traditional Medicine (TCM) for treatment

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of stomach aches, gastroenteritis, and rheumatic arthritis, and also for strengthening the tendons

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and bones [1,2]. Previous investigation revealed that H. occulta was a rich source of essential oil [3-5], and a few sesquiterpenes were also obtained from this herb, some of which exhibited

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antibacterial and stimulative effect on cellular processes of osteoblasts [6-9]. As part of our search

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for bioactive compounds from Chinese medicinal plants, the rhizomes of H. occulta have been

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investigated. Herein, we report the isolation and structure elucidation of six new sesquiterpenes (1-6) and eight known compounds (7-14) (Fig. 1), among them compound 1 was a rare example of

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a naturally occurring axane-type sesquiterpene, and compound 6 was a trihydroxyeudesmane semi-fumarate. These compounds were evaluated for their inhibitory effects on the production of NO through a macrophage-mediated bioassay.

2. Experimental details 2.1. General experimental procedures Optical rotations were recorded on a Rudolph Research Autopol III automatic polarimeter. IR spectra were recorded on a JASCO FT-IR-6300 spectrophotometer or a PerkinElmer Frontier

ACCEPTED MANUSCRIPT Mid-IR FTIR spectrophotometer with KBr disks. NMR spectra were obtained at 500 or 600 MHz for 1H, and 125 or 150 MHz for 13C, respectively, on Bruker Avance III 500 or Bruker Avance III

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HD 600 NMR spectrometer in acetone-d6 or MeOH-d4 using TMS as the internal standard.

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HRESIMS data were measured using a Micromass Autospec-Ultima ETOF spectrometer. Column chromatography was performed with silica gel (100-200 or 200-300 mesh, Qingdao Marine Chemical Inc. China) and Sephadex LH-20 (Pharmacia). HPLC separation was performed on a

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Waters 600 controller, a Waters 600 pump, and a Waters 2489 dual λ absorbance detector with an

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Ultimate XB-C18 (5 m, 250 × 10 mm) column. TLC was carried out with glass precoated silica gel GF254 plates (Yantai Chemical Industry Research Institute, China). Spots were visualized by

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2.2. Plant Material

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spraying with 10% H2SO4 in EtOH followed by heating.

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The rhizomes of Homalomena occulta were purchased at Anguo medicinal herb market of Hebei province, and identified by Professor Ying-Lin Wang from School of Combined Traditional

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Chinese and Western Medicine, Binzhou Medical University. A voucher specimen (S-QNJ-2010) is deposited at Herbarium of the School of Pharmacy, Binzhou Medical University. 2.3. Extraction and Isolation The dried rhizomes of Homalomena occulta (9 kg) were powdered and extracted with 95% EtOH at room temperature and evaporated under reduced pressure to yield a residue (400 g), which was suspended in H2O and then partitioned with ethyl acetate (EtOAc). The EtOAc extract (198 g) was subjected to CC over silica gel (100-200 mesh, petroleum ether-acetone, 10:0, 10:0.5, 10:1, 2:1, 1:1, 0:1) to give six fractions (Fr. 1Fr. 6). Fr. 3 (45.5 g) was chromatographed over silica gel with a gradient of increasing ethyl acetate in petroleum ether (10:1, 10:2, 2:1, 1:1) to

ACCEPTED MANUSCRIPT yield four subfractions, Fr. 3-1Fr. 3-4. Fr. 3-2 was recrystallized in CHCl3 to afford 12 (6.6 mg). Fr. 3-3 was recrystallized in acetone to afford 7 (2.1 g). Fr. 4 (23 g) was subjected to further silica

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gel CC with gradient petroleum ether-acetone (100:0-0:100) to give twenty subfractions, Fr.

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4-1Fr. 4-20. Fr. 4-3 (0.15 g) was loaded on CC over Sephadex LH-20 using petroleum ether-CHCl3-CH3OH (5:5:1) to give ten subfractions. Fr. 4-3-7 (50 mg) was purified by preparative TLC (CHCl3-acetone, 3:1) to afford 10 (20 mg). Fr. 4-4 (0.2 g) was chromatographed

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over Sephadex LH-20 using petroleum ether-CHCl3-CH3OH (5:5:1) to give ten subfractions. Fr.

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4-4-5 was recrystallized in acetone to afford 9 (9.1 mg). Fr. 4-4-6 was separated by preparative TLC (CHCl3-acetone, 3:1) and semi-preparative HPLC with MeOH-H2O (75:25, 2 ml/min) to

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afford 1 (2.2 mg). Fr. 4-4-7 was purified by semi-preparative HPLC with MeOH-H2O (80:20, 2

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ml/min) to afford 5 (9.7 mg). Fr. 4-6 (1.2 g) was chromatographed over Sephadex LH-20 using

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petroleum ether-CHCl3-CH3OH (5:5:1) to give five subfractions. Fr. 4-6-2 (15 mg) was purified by HPLC with MeOH-H2O (70:30, 2 ml/min) to afford 11 (9.7 mg). Fr. 4-6-5 (44 mg) was purified

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by preparative TLC (CHCl3-acetone, 3:1) to afford 2 (9 mg) and 3 (12 mg). Fr. 4-9 (2 g) was chromatographed over Sephadex LH-20 using petroleum ether-CHCl3-CH3OH (5:5:1) to give nine subfractions. Fr. 4-9-4 was purified by HPLC with MeOH-H2O (70:30, 2 ml/min) to afford 4 (4.7 mg). Fr. 4-9-5 was purified by HPLC with MeOH-H2O (70:30, 2 ml/min) to afford 13 (8 mg). Fr. 4-9-6 was purified by HPLC with MeOH-H2O (65:35, 2 ml/min) to afford 14 (8 mg). Fr. 4-9-7 was purified by HPLC with MeOH-H2O (70:30, 2 ml/min) to afford 6 (7 mg). Fr. 4-9-8 was recrystallized in acetone to afford 8 (1.1 g). 2.3.1. (1S,4S,5R,6R,10S)-ax-1,4,11-triol (1) Colorless gum; [] D = +34.2 (c = 0.2, MeOH); IR (KBr) max 3325, 2969, 2947, 2860, 1464, 20

ACCEPTED MANUSCRIPT 1365, 1049 cm-1; 1H NMR (MeOH-d4, 600 MHz) data, see Table 1;

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C NMR (MeOH-d4, 150

MHz) data, see Table 2; (+)-HRESIMS m/z 279.1926 [M+Na]+ (calcd for C15H28NaO3, 279.1931).

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2.3.2. (1S,4R,5S,6R,7R,10S)-opposit-1,4,7-triol (2)

Colorless needles; [] D = 24.1 (c = 0.1, MeOH); IR (KBr) max 3397, 2952, 2872, 1464,

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1383, 1075, 1031, 916 cm-1; 1H NMR (acetone-d6, 500 MHz) data, see Table 1;

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C NMR

C15H28NaO3, 279.1931).

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2.3.3. 7R-5βH,10α-opposit-1α,4α,7-triol (3)

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(acetone-d6, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 279.1926 [M+Na]+ (calcd for

Colorless gum; [] D = +35.2 (c = 0.2, MeOH); IR (KBr) max 3375, 2958, 2872, 1464, 1371, 20

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1073, 1027, 948 cm-1; 1H NMR (acetone-d6, 500 MHz) data, see Table 1; 13C NMR (acetone-d6,

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125 MHz) data, see Table 2; (+)-HRESIMS m/z 279.1926 [M+Na]+ (calcd for C15H28NaO3,

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279.1931).

2.3.4. 2α-hydroxyhomalomenol A (4) White amorphous powder; [] D = +31.6 (c = 0.15, MeOH); IR (KBr) max 3439, 2957, 2868,

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1561, 1386, 1040, 1020, 836 cm-1; 1H NMR (MeOH-d4, 500 MHz) data, see Table 1;

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(MeOH-d4, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 277.1774 [M+Na]+ (calcd for C15H26NaO3, 277.1774). 2.3.5. (1S,4R,5R,6R,7R,10S)-isodauc-6,7,10-triol (5) Colorless needles; [] D = +6.4 (c = 0.38, MeOH); IR (KBr) max 3412, 2935, 2872, 1453, 20

1372, 1010, 907 cm-1; 1H NMR (MeOH-d4, 500 MHz) data, see Table 1; 13C NMR (MeOH-d4, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 279.1927 [M+Na]+ (calcd for C15H28NaO3, 279.1931). 2.3.6. Eudesma-4β,7α-diol-1β-fumarate (6)

ACCEPTED MANUSCRIPT Colorless gum; [] D = 18.8 (c = 0.4, MeOH); IR (KBr) max 3406, 2935, 2865, 1699, 1642, 20

1262, 1174, 977, 905 cm-1; 1H NMR (MeOH-d4, 600 MHz) data, see Table 1; 13C NMR (MeOH-d4,

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150 MHz) data, see Table 2; (+)-HRESIMS m/z 377.1944 [M+Na]+ (calcd for C19H30NaO6,

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377.1935). 2.4. Single crystal X-ray diffraction analysis

Crystal analysis were performed on an Agilent Gemini S Ultra CCD diffractometer using Cu

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Kα radiation ( = 1.54178 Å). The structures were solved by direct methods and refined by

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full-matrix least squares on F2 using the SHELXL-97 program. Crystallographic data for the structures in this paper have been deposited with the Cambridge Crystallographic Data Centre

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2.4.1. Crystal data for 1

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(CCDC 1051600 for 1; CCDC 1421857 for 2; CCDC 1420363 for 3).

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C15H28O3 (fw = 256.37); monoclinic, space group P21; a = 6.3040(3) Å, b = 14.7161(8) Å, c = 8.1547(5) Å, α = γ = 90, β = 90.0410(10); V = 756.51(7) Å3, T = 293(2) K, Z = 2, Dc = 1.125

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mg/cm3, F (000) = 284. A total of 4355 reflections were collected within the range 5.42    66.19, in which 2431 reflections were observed [I > 2 (I)]. The final R = 0.0491, RW = 0.1195, and S = 1.023. Flack parameter = 0.1(3). 2.4.2. Crystal data for 2 C15H28O3 (fw = 256.37); orthorhombic, space group P21212; a = 12.2340(10) Å, b = 15.8951(18) Å, c = 7.8362(5) Å, α = β = γ = 90; V = 1523.8(2) Å3, T = 293(2) K, Z = 4, Dc = 1.117 mg/cm3, F (000) = 568. A total of 3105 reflections were collected within the range 4.56    66.04, in which 2167 reflections were observed [I > 2 (I)]. The final R = 0.0665, RW = 0.1139, and S = 1.028. Flack parameter = 0.0(6).

ACCEPTED MANUSCRIPT 2.4.3. Crystal data for 5 C15H29O3.50 (fw = 265.38); monoclinic, space group P21; a = 7.1531(5) Å, b = 19.0058(17) Å,

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c = 11.4810(9) Å, α = γ = 90, β = 96.341(2); V = 1551.3(2) Å3, T = 293(2) K, Z = 4, Dc = 1.136

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mg/cm3, F (000) = 588. A total of 7900 reflections were collected within the range 3.87    66.19, in which 4771 reflections were observed [I > 2 (I)]. The final R = 0.0959, RW = 0.1630, and S = 1.073. Flack parameter = 0.0(6).

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2.5. NO production inhibitory assay

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The NO production inhibitory assay was performed according to a method described in the literature [10], and dexamethasone was employed as the positive control.

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3. Results and discussion

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Compound 1 was obtained as a colorless gum. Its molecular formula was determined to be

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C15H28O3 based on the pseudomolecular peak observed by HRESIMS at m/z 279.1926 [M+Na]+ (calcd for C15H28NaO3, 279.1931) and

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C NMR spectra. Its IR spectrum showed characteristic

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absorption band for hydroxy group at 3324 cm-1. The 1H NMR spectrum of 1 exhibited resonances for an oxymethine (H 3.61, dd, J = 11.2, 4.4 Hz, H-1), and four tertiary methyls. The 13C NMR and HSQC spectrum of 1 displayed 15 carbon signals, including four methyls, five methylenes, three methines (one oxygenated), and three quaternary carbons (two oxygenated) (Tables 1 and 2). The above data indicated that 1 was a bicyclic sesquiterpene and had a hydrindane skeleton [6,9,11]. Comparison of the NMR data of 1 with those of known compound bullatantriol (7) [9,12] implied that they shared the same planar structure, and this deduction was further confirmed by 1

H-1H COSY and HMBC spectra. The 1H-1H COSY spectrum of 1 displayed correlations for the

spin systems of H-1/H2-2/H2-3, H-5/H-6/H2-8/H2-9 and H-6/H2-7 (Fig. 2). The HMBC

ACCEPTED MANUSCRIPT correlations from H-1 (H 3.61) to C-9 (C 39.5) and C-14 (C 22.0), from H3-15 (H 1.32) to C-3 (C 36.6), C-4 (C 73.5) and C-5 (C 65.8), and from both H3-12 (H 1.17) and H3-13 (H 1.18) to

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C-7 (C 54.6) and C-11 (C 72.3) (Fig. 2), in combination with the shifts of these protons and

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carbons, confirmed connections of the structural units and quaternary carbons. Thus, the planar structure of 1 was established as shown in Fig. 1. In the NOESY spectrum of 1, the correlations between H-5 and H3-14 suggested that 1 had a cis-hydrindane (axane) base skeleton, versus

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bullatantriol (7) possessing a trans-hydrindane (oppositane) base skeleton [13]. In addition,

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correlations of H-1/H-6 indicated that the protons were cofacial and -oriented, with reference to the literatures describing the axane-type sesquiterpenens isolated from plants [14,15]. While

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correlations of H-5/H-7, H-5/H3-14 and H3-14/H3-15 revealed that these protons were placed at

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the opposite side of the molecule and α-oriented (Fig. 3). In order to define the absolute

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configuration of 1, a good-quality crystal of 1 was obtained by slow evaporation from MeOH and was submitted for X-ray diffraction analysis using Cu Kα radiation while the Flack absolute

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structure parameter was refined to a value of 0.1(3) (Fig. 4). Consequently, the structure of 1 was determined as (1S,4S,5R,6R,10S)-ax-1,4,11-triol. Compound 2 was obtained as colorless needles and had a [M+Na]+ ion peak at m/z 279.1926 in its HRESIMS, corresponding to the same molecular formula of C15H28O3 as 1. The NMR spectroscopic data of 2 were similar to those of 1, except for the presence of an additional oxymethine (H 3.62, dd, J = 9.6, 2.2 Hz, H-7; C 75.6, C-7), and two methyls (H 0.89, 0.99, d, J = 6.8 Hz, each 3H) assignable to an isopropyl group (Tables 1 and 2). These data suggested that the hydroxy group at C-11 in 1 was placed at C-7 in 2, which was confirmed by the 1H-1H COSY correlations of H-6 (H 2.30) with H-7, and H-7 with H-11 (H 1.58), as well as HMBC

ACCEPTED MANUSCRIPT correlations from H-7 to C-11 (C 32.0), C-12 (C 18.8) and C-13 (C 20.3), and from both H3-12 and H3-13 to C-7 and C-11 (Fig. 2). The large coupling constant of H-5 (J5,6 = 9.6 Hz) indicated

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that H-5 and H-6 were trans-oriented. Furthermore, NOESY correlations of H-1/H-5 and

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H-1/H-3b revealed that these protons had the same orientation on the ring system, opposite H-6, CH3-14 and CH3-15 deduced from the correlations of H-6 with H3-14, H-6 with H3-15, and H3-15 with H-3a, indicating the trans-fused ring junction of 2 (Fig. 3). In addition, X-ray diffraction

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analysis using Cu Kα radiation unambiguously established the absolute configuration of 2 with the

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Flack absolute structure parameter refined to a value of 0.0(6) (Fig. 4). Therefore, the structure of 2 was elucidated to be (1S,4S,5S,6R,7R,10S)-opposit-1,4,7-triol.

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Compound 3 was obtained along with 2 by preparative TLC as a colorless gum. Its molecular

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C NMR, HSQC and HMBC spectroscopic data of 3 were quite similar to

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The IR, 1H NMR,

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formula of C15H28O3 was established from HRESIMS ([M+Na]+ m/z 279.1926) to be the same as 2.

those of 2 (Tables 1 and 2) except for the higher frequency shifts of H3-15 (H 1.34) and C-15 (C

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31.5), indicating the -orientation of the CH3-15 at C-4 in 3, which was confirmed by the NOESY correlations of H-1/H-5, H-5/H-8 and H-5/H3-15. The closely identical 1D and 2D NMR spectra suggested that 3 and 2 had the same relative configurations for C-1, C-5, C-6 and C-10. Slight difference between the chemical shifts of C-7 (C 1.0 ppm) indicated that 2 and 3 had the same R configuration at C-7, which was deduced from the conclusion that larger differences of approximately 6 ppm were observed between 7 R and 7S by a comparison of the 13C NMR values of dracunculifoliols with those of their 7-epimers [13]. Thus, 3 was established as 7R-5βH,10α-opposit-1α,4α,7-triol. Compound 4 was obtained as a white amorphous powder, and had a molecular formula of

ACCEPTED MANUSCRIPT C15H26O3 as determined by HRESIMS ([M+Na]+ m/z 277.1774, calcd for C15H26NaO3, 277.1774) and NMR experiments. The 1H and 13C NMR spectroscopic data of 4 (Tables 1 and 2) were very

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similar to those of homalomenol A previously isolated from H. aromatica [11]. Detailed

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comparison of the NMR data between 4 and homalomenol A demonstrated that the resonance for H-1 was changed from the double doublet of homalomenol A into a doublet of 4 (H 3.10, d, J = 9.3 Hz), and the resonance for C-1 was significantly deshielded (C 84.0). This infromation

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combined with the signals for an additional oxymethine (H 3.90, ddd, J = 5.2, 9.3, 11.5 Hz; C

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69.2) revealed the presence of a hydroxy group at C-2 in 4. The observed HMBC correlations from H3-15 to C-1, from H-1 to C-2, and from H-2 to C-1 and C-3 confirmed this conclusion. The

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relative configuration of 4 was established to be same as homalomenol A based on the large

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coupling constant of H-1 (J1,2 = 9.3 Hz), as well as the NOESY correlations of H-1/H-9α, H-1/H-5,

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H-5/H-3α and H-3α/H3-15, and correlations of H-6/H3-14, H3-14/H-2, H-2/H-3β, H-3β/H3-15 and H3-14/H-9β. Thus, 4 was determined as 2α-hydroxyhomalomenol A.

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Compound 5 was obtained as colorless needles with a molecular formula of C15H28O3 based on HRESIMS ([M+Na]+ m/z 279.1927) and NMR experiments. Its IR spectrum showed characteristic absorption band for hydroxy group at 3421 cm-1. The 1H NMR spectrum of 5 revealed resonances for two oxymethines (H 3.36, brs, H-6; H 3.98, brd, J = 9.8 Hz, H-10), two tertiary methyls, and two methyls (H 0.91, 0.98, d, J = 6.7 Hz, each 3H) assignable to an isopropyl group. The 13C NMR and HSQC spectrum of 5 displayed 15 carbon signals, including four methyls, four methylenes, five methines (two oxygenated), and two quaternary carbons (one oxygenated) (Tables 1 and 2). The above data indicated that 5 was an isodaucane-type sesquiterpene [7,8,16]. The NMR spectroscopic features of 5 were similar to those of

ACCEPTED MANUSCRIPT Homalomenol C isolated from H. aromatica [17], except for the absence of the epoxy bridge between C-6 and C-10, as evidenced by the upfield shifted C-10 (C 79.2) and that the molecular

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formula of 5 had two hydrogen atoms more than that of Homalomenol C, indicating that two

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hydroxy groups were placed at C-6 and C-10 in 5, respectively. This conclusion was further verified by the observed 1H-1H COSY correlations of H2-8/H2-9/H-10, as well as the HMBC correlations from H-6 to C-1, C-4, C-7, C-8 and C-15, and from H-10 to C-1, C-2, C-8, C-9 and

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C-14 (Fig. 2). Thus, the gross structure of 5 was established as shown in Fig. 1. In the NOESY

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spectrum, correlations between H-5 and H3-14 revealed the cis-fused ring junction of 5. In addition, correlations of H-5/H-6, H3-14/H-3β and H3-14/H-9β indicated that these protons had the

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same orientation on the ring system, opposite H-4 and H-10 based on the correlations of H-4/H-3α

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and H-4/H-9α (Fig. 2). The absolute configuration of 5 was finally confirmed by X-ray diffraction

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analysis using Cu Kα radiation with the Flack absolute structure parameter refined to a value of 0.0(6) (Fig. 4). According to the above evidences, the structure of 5 was established as

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(1S,4R,5R,6R,7R,10S)-isodauc-6,7,10-triol. Compound 6 was obtained as a colorless gum, and had a molecular formula of C19H30O6 deduced from HRESIMS ([M+Na]+ m/z 377.1944, calcd for C19H30NaO6, 377.1935) and NMR experiments. Its IR spectrum exhibited absorption bands for OH (3406 cm-1), C=O (1699 cm-1) and C=C (1642 cm-1) groups. The 1H and 13C NMR spectra (Tables 1 and 2) of the basic skeleton in 6 were quite identical to those of the known compound 1β,4β,7α-trihydroxyeudesmane (8) [11,12], except for the extra proton signals at H 6.74 (1H, d, J = 15.7 Hz) and H 6.77 (1H, d, J = 15.7 Hz), and carbon resonances at C 168.4, 166.5, 135.9 and 134.4, which were attributed to a fumaryl moiety [18]. The signals displayed in 1H and 13C NMR spectrum of 6 were calculated for

ACCEPTED MANUSCRIPT C19H29O5, the remaining OH unit combined with the deshielded H-1 (H 4.66, dd, J = 11.5, 3.4 Hz) and C-1 (C 84.1) resonances indicated that 6 was a 1-semifumarate of 8, which was confirmed by 13

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C NMR data of 6 were assigned

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the HMBC correlation from H-1 to C-1. All of the 1H and

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unambiguously based on 1H-1H COSY, HSQC and HMBC spectra, and the relative configuration of 6 was assigned as being identical with that of 8 by analysis of its NOESY spectra. Accordingly, compound 6 was determined as eudesma-4β,7α-diol-1β-fumarate.

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In addition to the six above-mentioned new compounds, eight known sesquiterpenes were

NMR

data,

their

structures

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isolated from H. occulta. Through ESIMS analysis and comparisons with previously reported were

confirmed

as

bullatantriol

(7)

[9,12],

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1β,4β,7α-trihydroxyeudesmane (8) [11,12], 1β,4β,6α-trihydroxyeudesmane (9) [7], pterodontriol

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(10) [19], 4α-hydroxy-homalomenol C (11) [20], 6α,7α,10α-trihydroxyisodaucane (12) [8],

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cadinane-4β,5α,10β-triol (13) [9], and 1β,4β-dihydroxy-11,12,13-trinor-8,9-eudesmen-7-one (14) [20]. All of the isolates were evaluated for their biological activities against LPS-induced

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production of NO in macrophage cells. Among the tested compounds, 1 and 5 showed moderate inhibitory effects with IC50 values of 21.2 μM and 15.4 μM, respectively, whereas the positive control (dexamethasone) gave an IC50 value of 0.9 μM. Germacrene D was recognized as an important biogenetic precursor of many sesquiterpene structures, which was discussed in the literature [21]. On the basis of skeletal rearrangements, ()-germacrene D was attempted to derive the axane (1), oppositane (4, 7), isodaucane (5, 11, 12), eudesmane (6, 8, 9, 10 and 14), and cadinane (13) groups of sesquiterpenes [14,21]. The proposed biogenetic pathways for compounds 1-14 were shown in Fig. 5. In particular, the H-5 and Me-14 in compounds 2 and 3 were β- and α-oriented, respectively, demonstrating 2 and 3 to be rare

ACCEPTED MANUSCRIPT examples of 5βH,10α-oppositanes, versus 4 and 7 possessing 5α-H and 10β-Me. Based on analysis of the single-crystal structure of 2, we assumed that 5βH,10α-oppositanes were biosynthesized

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from (+)-germacrene D by the similar pathway described for 4 and 7 (Fig. 5). Compound 6

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represented a rare occurrence of semi-fumarate of isoprenoids, which could be a dehydration product of malate, in view of the presence of 1β,4β,7α-trihydroxyeudesmane-1β-methyl malate in H. occulta [9]. Compound 1 is an axane-type sesquiterpene, which has rarely been reported from

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natural sources. To the best of our knowledge, this is the first report of confirming the absolute

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configuration for the axane-type sesquiterpenes obtained from plants by X-ray crystallographic analysis, demonstrating a relative configuration in accord with that of the derivatives formed in the

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D

biosynthetic pathway (Fig. 5) [21].

Conflict of interest

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The authors declared no conflict of interest.

Acknowledgments This work was financially supported by Shandong Provincial Natural Science Foundation, China (ZR2011HL067, ZR2012CQ041 and 2014CGZH1316), and a project of Shandong Province Higher Educational Science and Technology Program (J13LL80).

ACCEPTED MANUSCRIPT References [1] Editorial Committee of the Chinese Academy of Science, ‘Flora of China’, Science Press &

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Missouri Botanical Garden Press, Beijing, China, 2010, Vol. 23, p. 17-8.

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[2] Chinese Pharmacopoeia Commission. Chinese pharmacopoeia, Beijing, Chemical Industry Press, 2005, Vol. 1, p. 24.

[3] Zhou CM, Yao C, Sun HL, Qiu SX, Cui GY. Volatile constituents of the rhizome of

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Homalomena occulta. Planta Med 1991; 57: 391-2.

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[4] Chen YZ, Xue DY, Li ZL, Han H. Chemical constituents of the essential oil of Homalomena occulta (Lours) Schott. Sepu; 1986; 4: 324-7.

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[5] Zeng LB, Zhang ZR, Luo ZH, Zhu JX. Antioxidant activity and chemical constituents of

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essential oil and extracts of Rhizoma Homalomenae. Food Chem 2011; 125: 456-63.

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[6] Wang YF, Wang XY, Lai GF, Lu CH, Luo SD. Three new sesquiterpenoids from the aerial parts of Homalomena occulta. Chem Biodivers 2007; 4: 925-31.

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[7] Hu YM, Liu C, Cheng KW, Sung HH, Williams LD, Yang ZL, Ye WC. Sesquiterpenoids from Homalomena occulta affect osteoblast proliferation, differentiation and mineralization in vitro. Phytochemistry 2008; 69: 2367-73. [8] Hu YM, Yang ZL, Wang H, Ye WC. A new sesquiterpenoid from rhizomes of Homalomena occulta. Nat Prod Res 2009; 23: 1279-83. [9] Xie XY, Wang R, Shi YP. Sesquiterpenoids from the rhizomes of Homalomena occulta. Planta Med 2012; 78: 1010-4. [10] Zhao F, Wang SJ, Lin S, Zhu CG, Yuan SP, Ding XY, et al. Anthraquinones from the roots of Knoxia valenrianoides. J Asian Nat Prod Res 2011; 13: 1023-9.

ACCEPTED MANUSCRIPT [11] Sung TV, Steffan B, Steglich W, Klebe G, Adam G. Sesquiterpenoids from the roots of Homalomena aromatica. Phytochemistry 1992; 31: 3515-20.

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[12] Hu YM, Yang ZL, Ye WC, Chong QH. Studies on the constituents in rhizome of

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Homalomena occuta. Chin J Chin Mater Med 2003; 28: 342-4.

[13] Weyerstahl P, Marschall H, Schneider K. Terpenes and terpene derivatives, XXXIII. Synthesis of rac-cis- and -trans- dracunculifoliol and their 10-epimers. Liebigs Ann 1995; 2:

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231-40.

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[14] García A, Delgado G. Cytotoxic cis-fused bicyclic sesquiterpenoids from Jatropha neopauciflora. J Nat Prod 2006; 69: 1618-21.

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[15] Al-Rehaily AJ, Ahmad MS, Mossa JS, Muhammad I. New axane and oppositane

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sesquiterpenes from Teclea nobilis. J Nat Prod 2002; 65: 1374-6.

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[16] Tchuendem MH, Mbah JA, Tsopmo A, Ayafor JF, Sterner O, Okunjic CC, et al. Anti-plasmodial sesquiterpenoids from the African Reneilmia cincinnata. Phytochemistry

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1999; 52: 1095-9.

[17] Sung TV, Kutschabsky L, Porzel A, Steglich W, Adam G. Sesquiterpenes from the roots of Homalomena aromatica. Phytochemistry 1992;31:1659-61. [18] Zhu X, Zheng D, Zhang X, Peng H. Synthesis and characterization of mono-n-alkyl fumarate. Chem Res and Appl 2012; 24: 1413-7. [19] Zhao Y, Yue J, Lin Z, Ding J, Sun H. Eudesmane sesquiterpenes from Laggera pterodonta. Phytochemistry 1997: 44: 459-64. [20] Henchiri H, Bodo B, Deville A, Dubost L, Zourgui L, Raies A, et al. Sesquiterpenoids from Teucrium ramosissimum. Phytochemistry 2009; 70: 1435-41.

ACCEPTED MANUSCRIPT [21] Bülow N, & König WA. The role of germacrene D as a precursor in sesquiterpene biosynthesis: investigations of acid catalyzed, photochemically and thermally induced

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rearrangements. Phytochemistry 2000; 55: 141-68.

ACCEPTED MANUSCRIPT OH

10

HO 10

8

6

HO 15

13

H

11 7

1

H

OH R 1 R2 HO H 12 R1 R 2 2 OH Me 3 Me OH

OH

HO

H 12 11

OH

8

9

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7

HO

HO

H

O H

OH

H

HO

OH

H

11

12

OH

OH

H

HO

H 14

13

TE OH

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OH

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OH

1

Fig. 2.

OH

H HO

HO

HO

HO 2

5

1

H-1H COSY (bold) and key HMBC (arrow) correlations of 1 and 5.

H OH

HO H

H

H OH

H

1

H

H OH

HO

H

H

HO H 2

Fig. 3. Selected NOESY correlations of 1, 2 and 5.

HO H H H

H H

H

OH H OH

5

O

OH 13 11 12

6

OH

Structures of compounds 1-14.

OH

H

10

D

Fig. 1.

H

HO

OH

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HO

7

HO 15

HO

OH

8

5

OH

H

HO

10 3

OH 15

13

COOH

1

OH

5

OH

H

HO

6

4

OH H

8

5

H

OH

HO

1

3

O 14

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5

3'

1'

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3

O

HO

14

T

OH

OH 14 1

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Fig. 4. X-ray ORTEP drawings of 1, 2 and 5.

ACCEPTED MANUSCRIPT 10

9

[O]

14

H Eudesmanes H

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or

Me H

Me

Me H

H

Me H

H

H

H

Me

H

H

Me

+ H2

[O]

TE

H

[O]

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Me H

2, 3 10 ,5  H-oppositanes

[O]

4, 7 10  ,5  H-oppositanes

5, 11 Isodaucanes

13 Cadinanes

D

Me

[O]

[O]

H H

W agner -Meerw ein rearrangement H

H

H

H

H

H

H

H

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cyclization

H

H

H

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H

H

H

(-)-Germacrene D

(+)-Germacrene D

6

H

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H

H

COOH H2 O

HO

H

OH

OH

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OPP

O

C 3 H7 + malic acid 8 H2 O

[O]

or H

O

[O]

[O]

H

12 Isodaucane [O]

1 Axane

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Fig. 5. Proposed biogenetic pathways for compounds 1-14.

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ACCEPTED MANUSCRIPT

5

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1.59 m

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Table 1. 1H NMR data for compounds 1-6 (δ in ppm and J in Hz). position 1 2 3 4 1 3.61 dd 3.37 dd 3.31 dd 3.10 d (9.3) (11.2, 4.4) (10.5, 4.4) (11.0, 3.9) 2 1.69 m; 1.57 m 1.75 m; 3.90 ddd 1.56 m 1.49 m (5.2, 9.3, 11.5) 3 1.75 m; 1.73 dd 1.72 m; 1.94 dd 1.56 m (9.1, 2.9); 1.48 m (13.6, 5.2) 1.52 m 1.43 dd (13.5, 11.5) 4 5 1.41 d (8.7) 1.53 d 1.10 d 1.21 d (12.4) (11.5) (11.3) 6 2.31 m 2.30 m 2.37 m 3.06 m

1.72 m; 1.58 m

1.71 m; 1.38 m

11 12 13 14 15 2 3

5.13 (9.5)

1.75 m; 1.52 m

2.12 m; 1.27 m

1.53 m; 1.20 m

1.56 dd (10.7, 8.3); 1.34 m

D

1.58 m

1.17 s 1.18 s 1.01 s 1.32 s

3.59 dd (7.5, 2.5)

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1.57 m; 1.25 m

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10

1.85 m; 1.35 m

1.60 m

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9

3.62 dd (9.6, 2.2)

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8

1.98 dd (14.0, 1.7); 1.49 dd (14.0, 8.6) 2.04 m; 1.37 m

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7

0.89 d (6.8) 0.99 d (6.8) 0.88 s 1.21 s

1.86 m 1.91 dd (7.9, 1.6) 3.36 brs

1.55 overlapped 1.53 m; 1.39 m

brd

1.95 dt (3.1, 13.9); 1.44 m

brd

1.59 m 1.68 d (1.1) 1.69 d (1.0) 1.09 s 1.15 s

1.64 m; 1.55 m 1.35 m

2.13 m; 1.33 m 3.98 (9.8)

1.86 dp (2.3, 6.9) 0.90 d (7.0) 0.94 d (7.0) 1.06 s 1.34 s

6 4.66 dd (11.5, 3.4) 2.00 m; 1.60 m

0.91 d (6.7) 0.98 d (6.7) 0.93 s 1.22 s

1.58 m 0.95 d (6.4) 0.95 d (6.4) 1.13 s 1.14 s 6.74 d (15.7) 6.77 d (15.7)

Data were measured in MeOH-d4 at 600 MHz for 1 and 6, in acetone-d6 at 500 MHz for 2 and 3, and in MeOH-d4 at 500 MHz for 4 and 5.

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6 84.1 24.5 40.2 71.8 44.8 29.2 74.7 29.9 35.8 39.3 40.5 17.4 17.5 13.2 29.7 166.5 135.9 134.4 168.4

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5 51.0 42.0 28.3 52.6 50.5 83.2 74.8 36.9 30.1 79.2 34.0 20.0 23.2 21.3 31.1

Data were measured in MeOH-d4 at 600 MHz for 1 and 6, in acetone-d6 at 500 MHz for 2 and 3, and in MeOH-d4

at 500 MHz for 4 and 5.

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C-10 of 4 was overlapped by signals of solvent, and data was obtained from the correlation in HMBC spectrum.

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b

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a

D

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Table 2. 13C NMR data for compounds 1-6 (δ in ppm) a. position 1 2 3 4b 1 71.7 79.1 78.8 84.0 2 29.2 29.9 28.2 69.2 3 36.6 42.5 40.7 48.8 4 73.5 71.7 70.4 71.3 5 65.8 54.2 57.2 59.5 6 37.3 40.3 42.8 34.3 7 54.6 75.6 76.6 132.6 8 32.7 19.2 23.6 30.0 9 39.5 39.1 37.4 38.6 10 48.1 46.4 47.1 47.4 11 72.3 32.0 30.7 127.9 12 29.3 18.8 14.8 16.8 13 30.8 20.3 20.9 24.6 14 22.0 14.4 14.3 14.5 15 30.4 22.2 31.5 29.6 1 2 3 4

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ACCEPTED MANUSCRIPT

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Graphical abstract

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