Illicium henryi is a toxic shrub,primarily dist_Ributed throughout southern China and is used for the t_Reatment of rheumatism ^[1]. Several sesquiterpene lactones,lignans and flavonoids from this plant were reported ^{[2, 3, 4, 5, 6, 7, 8]}. In our studies,novel prenylated C₆- C₃ compounds and sesquiterpene lactones with antioxidant and neurotoxicant activities were isolated from the roots of this plant ^{[9, 10, 11]}. As a result of our continued searches for the bioactive constituents of this plant,one new sesquiterpene compound, namely,illihenlactone A (1),and one new prenylated C₆-C₃ compound,illihenryione H (2),along with three known sesquiterpenes (3-5),including pseudoanisatin ^[12],abscisin ^[13], phaseic acid ^[13],(Fig. 1) were isolated from the EtOAc soluble fraction of the EtOH ext_Ract. Herein,we report the isolation and structural elucidation of compounds 1-5.

Fig. 1

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Fig. 1. Structures of compounds 1–5.

Melting points were measured on a XT-5B micromelting point apparatus and were uncorrected. Optical rotations were measured with a Perkin-Elmer 241 automatic digital polarimeter. CD spect_Ra were measured on a JASCO J-810 spect_Ropolarimeter with a 0.1 cm cell at room temperature under the following conditions: speed,200 nm/min; time constant,1 s; and bandwidth,2.0 nm. IR spect_Ra were recorded on a Nicolet 5700 FT-IR spect_Rometer with a microscope t_Ransmission method. NMR spect_Ra were obtained on an INOVA-500 spect_Rometer. ESIMS were measured on an Agilent 1100 Series LC/MS t_Rap mass spect_Rometer. HRESIMS data were recorded using an Agilent 6250 Accurate-Mass Q-TOF LC/MS spect_Rometer. Preparative HPLC was performed on a Shimadzu LC-6AD inst_Rument with an SPD-10A detector using a YMC-Pack ODS-A column (250 mm × 20 mm,5 mm). Silica gel (160-200,200-300 mesh, Qingdao Marine Chemical Factory,China) and ODS (50 mm, Merck) were used for column chromatography (CC). TLC was performed with precoated Si gel GF254 glass plates. Spots were visualized under UV light or by spraying with 10% H₂SO₄ in EtOH-H₂O (95:5,v/v) followed by heating.

Stems of I. henryi were collected in Guangxi Province,China,in August 2009 and identified by Prof. Song-ji Wei from Guangxi University of t_Raditional Chinese Medicine. A voucher specimen (ID-21974) was deposited in the Herbarium of the Department of Medicinal Plants,Institute of Materia Medica,Chinese Academy of Medical Sciences.

Stems of I. henryi (10 kg) were air-dried,ground,and ext_Racted (2 h each) with EtOH-H₂O (3× 30 L,95:5,v/v) by refluxing (90- 95 °C). The combined EtOH ext_Racts were evaporated to near dryness under vacuum. The resulting residue (400 g) was absorbed on kieselguhr (800 g) and then successively ext_Racted with pet_Roleum ether,EtOAc,and MeOH. The EtOAc (58 g) ext_Ract was subjected to a silica gel column (80 cm × 8 cm,200 - 300 mesh) eluted with a CH2Cl2/MeOH (50:1→ 1:1,v/v) gradient system to yield four fractions,A₁-A₄.

Fraction A₂ (8 g) was loaded over an ODS column and eluted with MeOH-H2O (10:90 → 60:40) to afford two fractions,A₂B₁- A₂B₂. Fraction A₂B₁ (500 mg) was purified using a semi-preparative HPLC [MeCN-H₂O (40:60)] to yield compounds 1 (50 mg, t_R = 35 min) and 5 (10 mg,t_R = 32 min). By following the same procedure,compounds 2 (10 mg,t_R = 40 min) and 4 (5 mg, t_R = 44 min) were obtained from A₂B₂ (50 mg). Fraction A₂B₂ (200 mg) was purified by semi-preparative HPLC [MeCN-H2O (35:65)] to yield compounds 3 (30 mg,t_R = 36 min).

Illihenlactone A (1): Colorless crystal,mp 138 °C,^[α]_D²⁰-33.1 (c 0.51 MeOH); IR (cm^-1): nmax 3460,3381,2965,2946,2871,1838, 1721,1369,1189,1130,1111,1064,927; For ¹H NMR and ¹³C NMR spect_Roscopic data,see Table 1; ESIMS m/z 373 [M+H]+, 395 [M+Na]+,371 [M+H]⁺; HRESIMS m/z 371.1345 [M-H]^- (calcd. for C₁₇H₂₃O₉,371.1348).

Table 1

Table 1 1H NMR (500 MHz) and 13C NMR (125 MHz) data of compounds 1 in CDOD3 and 2 in acetone-d6.

Table 1 1H NMR (500 MHz) and 13C NMR (125 MHz) data of compounds 1 in CDOD3 and 2 in acetone-d6.

Illihenryione H (2): White amorphous powder,[α]_D²⁰ + 18.2 (c 0.50 MeOH); CD (MeOH)δε₂₄₀-0.75,δε₃₀₂ 0.77; UV (MeOH) λ_max (log ε): 270 (1.00),220 (0.40); IR (cm^-1):ν_max 3367,3078,2923, 2852,1678,1621,1603,1415,1391,1229,1203,1134,1015,948, 834; For ¹H NMR and ¹³C NMR spectroscopic data,see Table 1; ESIMS m/z 279 [M+H]⁺,301 [M+Na]⁺; HRESIMS m/z 279.1227 [M+H]⁺ (calcd. for C₁₅H₁₉O₅,279.1227).

The ¹H NMR and ¹³C NMR spectra of compound 1 and compound 2 are in the Supporting information.

On the basis of reported procedures ^[14],the anti-inflammatory activities of compounds 1-5 were assayed by measuring the inhibition of the platelet-activating factor induced release of β-glucuronidase from rat polymorphonuclear leukocytes in vitro.

Compound 1 was a colorless crystal,and its molecular formula was established as C₁₇H₂₄O₉ by HRESIMS (371.1345 [M-H]^-) and NMR data (Table 1). Its IR spectrum showed absorptions for hydroxyl (3460 cm^-1) and ester carbonyl (1721 cm^-1) moieties. The NMR data (Table 1) of 1 were similar to those of veranisatin A, except that a CH₂(12)-OCH₃(16) group [δ_H 4.05 (d,1H,J = 11.0,H- 12a),3.05 (d,1H,J = 11.0,H-12b),3.41(s,3H,H-16); δC 73.4 (C-12), 60.5 (C-16)] in veranisatin A was replaced by a hemiacetal moiety [δH 3.66 (s,3H,H-17),3.54 (s,3H,H-16),4.91 (s,1H,H-12); δC 56.7 (C-17),59.2 (C-16),104.6 (C-12)] in 1,and this was further confirmed by the HMBC correlations from H₃-16,H₃-17 to C-12 and from H-12 to C-5,C-6 and C-7. NOESY spect_Rum showed crosspeaks between H3-15 and H-10 and between H-12 and H₂-14. (Fig. 2) Therefore,the structure of 1 was elucidated as shown in Fig. 1 and named illihenlactone A (1).

Fig. 2

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Fig. 2. The HMBC ( ), H-HCOSY ( ) and NOESY correlations of compound 1.

Compound 2 was assigned as one of prenylated C₆-C₃ derivatives because it possessed characteristic UV and IR spectroscopic data ^[15]. The molecular formula of 2,C₁₅H₁₈O₅,was established by HRESIMS (m/z 279.1227 [M+H]⁺. calcd. for 279.1227) and NMR data, which indicated 7 degrees of unsaturation. The ¹H NMR data of 2 were similar to those of 4-allyl-4-(3-methylbut-2-enyl)-1,2- methylenedioxycyclohexa-2,6-dien-5-one ^[16],except for the absence of two methyl signals and the presence of two oxygenated methylene resonances. The above data indicated that C-13 and C-14 in 2 were two hydroxymethyl groups instead of the two methyl groups observed in 4-allyl-4-(3-methylbut-2-enyl)-1,2- methylenedioxycyclohexa-2,6-dien-5-one,and this was further determined by the HMBC correlations from H₂-13 to C-14,C-12, and C-11 and from H₂-14 to C-13,C-12,and C-11. The CD spectrum showed a positive Cotton effect at 302 nm,indicating that the configuration of C-2 was R (Fig. 3) ^[17],and compound 2 was named illihenryione H (2).

Fig. 3

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Fig. 3. The CD spectrum of compound 2.

The anti-inflammatory effects of 1-5 were determined. Compounds 1-5 were assessed by measuring the inhibitory ratios for β-glucuronidase release induced by platelet-activating factor (PAF) in rat polymorphonuclear leukocytes (PMNS) in vit_Ro ^[14]. The inhibitory ratio of 2 was 30.3% at a concent_Ration of 10 mmol/L. Ginkgolide B,with an inhibitory ratio of 81.1% at 10 mmol/L,was used as a positive cont_Rol (Table 2).

Table 2

Table 2 Inhibition effect of compounds 1–5 on PAF-induced release of β-glucuronidase from rat PMNs (in vitro).

Table 2 Inhibition effect of compounds 1–5 on PAF-induced release of β-glucuronidase from rat PMNs (in vitro).

The genus Illicium is a rich source of prenylated C₆-C₃ compounds and sesquiterpene lactones. Prenylated C₆-C₃ compounds,also named phytoquinoids,are considered to be characteristic constituents. From a chemical viewpoint,these compounds belong to cyclic prenylated tetrahydrofurano-type,cyclic prenylated tetrahydropyrano-type,cyclic prenylated bridge-type C6- C3 and non-cyclic prenylated C₆-C₃-type compounds as reported ^[17]. In this paper,we described the isolation and identification of two new compounds,illihenlactone A (1) and illihenryione H (2) which are classified as secoprezizaane-type and non-cyclic prenylated C₆-C₃-type compounds,respectively. Biological evaluation indicated that compound 2 showed weak inhibitory activity of b-glucuronidase release from rat PMNs induced by PAF.

This project was supported by the Natural Science Foundation of China (No. 21132009) and the National Science and Technology Project of China (No. 2012ZX09301002-002). We are grateful to the Department of Inst_Rumental Analysis,Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College for measuring the IR,UV,NMR,MS and CD spectra.

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2015.09.007.