Halophilols A and B, Two New Stilbenes from Iris halophila

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

A new monomeric stilbene, halophilol A (1), and a new tetrastilbene, halophilol B (2), along with three known oligostilbenes were isolated from the seeds of Iris halophila. Their structures were established on the basis of the spectral data. The oligostilbene skeleton is encountered for the first time in the Iridaceae family. Bioactivity tests showed that 1 had moderate cytotoxicity against KB and HMEC cell lines (IC₅₀ = 17.28 μM, 22.47 μM respectively), while 2 was inactive.[*]

The stilbenoids have aroused considerable interest for their bioactivities, such as chemopreventive, antifungal, anti-inflammatory, lipid-lowering activities, cytotoxicity against human tumor cell lines, and protein tyrosine kinase (PTK56^lck) inhibition [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. Stilbenoids are, therefore, regarded as one of the important candidates for drug development. In the course of phytochemical investigation on the seeds of Iris halophila Pall., we obtained two new stilbenoids, halophilols A (1) and B (2), along with three known compounds, resveratrol (3), ε-viniferin (4) and γ-2-viniferin (5). This is the first report of oligostilbenes from the family Iridaceae. Herein, we describe the isolation and characterization of the new compounds.

Halophilol A (1) was obtained as an amorphous powder. In the HR-EIMS, the molecular ion peak was observed at m/z = 302.1166 suggesting the molecular formula C₁₇H₁₈O₅ (C₁₇H₁₈O₅ requires 302.1154). The ¹H-NMR spectrum exhibited signals due to a couple of meta-coupled aromatic protons [δ = 6.64 and 6.86 (each 1H, d, J = 1.8 Hz)], one 1,2,3-trisubstituted benzene ring protons [δ = 7.12 (1H, dd, J = 7.9, 1.8 Hz), δ = 7.02 (1H, t, J = 7.9 Hz), δ = 6.89 (1H, dd, J = 7.9, 1.8 Hz)], and a pair of trans double bond protons [δ = 7.03 and 7.19 (each 1H, d, J = 16.4 Hz)]. Moreover, three signals at δ = 3.93, 3.92 and 3.83 (each 3H, s) were attributed to aromatic methoxy groups. The above findings suggested that 1 was a pentasubstituted trans-resveratrol stilbene. The ¹³C-NMR spectrum displayed 14 sp² carbon signals in agreement with the stilbene skeleton. Those positions of three methoxy and two hydroxy groups were elucidated by the HMQC and HMBC spectra. The HMBC spectrum demonstrated the long-range correlations at H-7/C-6 and C-2; H-6/C-4, C-7 and C-2; H-8/C-9 and C-10; H-14/C-10 and C-12; H-13/C-9 and C-11; 10-OMe/C-10; 4-OMe or 5-OMe/C-5 and C-4. Thus, compound 1 was identified as 4,5,10-trimethoxy-3, 11-dihydroxy-E-stilbene.

Halophilol B (2), [α]_D ²⁵: + 152.4° (c 1.28, MeOH), brown amorphous powder, was found to have the molecular formula C₅₆H₄₂O₁₂ deduced from the quasimolecular ion peak at m/z = 907.2753 ([M + H]⁺) in its positive HR-ESI-MS, which was consistent with the ¹³C- and DEPT NMR spectra (Table [1]). The ¹H-NMR spectrum (Table [1]) indicated the presence of three 1,4-disubstituted benzene rings, two 1,3,5-trisubstituted benzene rings, one 1,3,4-trisubstituted benzene ring, and two 1,2,3,5-tetrasubstituted benzene rings. Two olefinic protons at δ = 6.03 and 6.18 (each 1H, d, J = 12.2 Hz) were ascribed to a cis double bond functionality. Three sets of AB spin system signals at δ = 3.63 and 5.19 (each 1H, d, J = 4.9 Hz), δ = 3.99 and 5.30 (each 1H, d, J = 5.0 Hz), and δ = 4.33 and 5.24 (each 1H, d, J = 10.0 Hz), respectively, suggested the presence of three dihydrobenzofuran moieties bearing 4-oxyphenyl and 3,5-dioxyphenyl groups of oligostilbenes biosynthesized from resveratrol molecules. The planar structure of halophilol B was thus deduced as 2, which was further confirmed by the HMQC and HMBC spectra (Fig. [1]). The relative configuration was assigned by the detailed analysis of the ¹H-NMR data of the dihydrobenzofuran rings. The coupling constants J _7,8 = 4.9, J _{7′ , 8′} = 10.0, and J _{7′ ′′ , 8′ ′′} = 5.0 Hz were similar to those of (+)-vitisin C [4], disclosing the E configuration of H-7 and H-8, H-7′′′ and H-8′′′, and Z configuration of H-7′ and H-8′, as shown in structure 2.

Both 1 and 2 were evaluated for cytotoxic effects using procedures as previously reported [11]. Moderate activity was observed for 1 against two human cancer cell lines KB and HMEC (human microvascular endothelial cell), with IC₅₀ values of 17.28 μM and 22.47 μM, respectively [etoposide (1.88 μM/KB cells), and hydroxycamptothecin (0.31 μM/HMEC cells) as control], but no consistent activity was found for 2.

Materials and Methods

Optical rotations were measured using a Perkin-Elmer 241 MC polarimeter. IR spectra were recorded on a Nicolet Magna 750 FTIR (KBr) spectrophotometer. All MS data were obtained with a MAT-95 mass spectrometer. NMR spectra were recorded on a Bruker AM400 instrument with TMS as internal standard, the chemical shift values are reported in ppm (δ) and coupling constants (J) are given in Hz. Silica gel (200 - 300, 400 mesh) and precoated plates of silica gel (HSGF₂₅₄) (Qingdao Haiyang Chemical Group Co., Qingdao, People’s Republic of China) were used for column chromatography (CC) and for TLC, respectively. The dried seeds of Iris halophila Pall. were collected in Tashikurgan, Xinjiang Province, China, in August, 2000, and identified by Prof. Guang-Xing Yang of Kashi Institute of Drug Research of Xinjiang. A voucher (No. 0058) specimen was deposited in our laboratory.

The dried seeds of I. halophila Pall. (5 kg) were extracted with 95 % EtOH (5 L × 3) at room temperature. The concentrated extract was suspended in MeOH/H₂O (20/80, v/v, 1 L) and partitioned successively with petroleum ether, chloroform, EtOAc and n-BuOH (each 1 L × 3). The CHCl₃-soluble fraction (50 g) was subjected to CC over silica gel eluted with petroleum ether-EtOAc gradients (10 : 1, 5 : 1, 2 : 1, finally 1 : 1, each 2 L) to obtain frs. 1 - 4. The petroleum ether-EtOAc (5 : 1) fraction (fr. 2, 5g) was chromatographed over silica gel with CHCl₃-acetone (10 : 1, 3 L) to yield frs. 2.1 - 2.10. The fr. 2.1 and fr. 2.3 were further purified on Sephadex LH-20 (CHCl₃-MeOH, 2 : 1, each 300 mL) to afford 1 (40 mg, R_f: 0.72 in CHCl₃-MeOH, 10 : 1) and 3 (15 mg), respectively. The separation of the EtOAc extract (30 g) over silica gel column using a CHCl₃-MeOH gradient (50 : 1→1 : 1, each 1L) led to five subfractions. The CHCl₃-MeOH (15 : 1) (4 g) subfraction was applied successively on silica gel and Sephadex LH-20 columns to obtain 5 (30 mg). The CHCl₃-MeOH (10 : 1) subfraction (1.3 g) was separated on an RP C-18 column (3.5 × 4.5 cm) eluted with MeOH-H₂O (1 : 1, 1 L), collecting 50 mL aliquots, and monitored by TLC (solution system, CHCl₃-MeOH, 5 : 1, iodine vapor for detection), to yield 2 (R_f: 0.35, 20 mg), and 4 (50 mg).

Halophilol A (1): White amorphous powder. UV (CHCl₃): λ_max = 200.0, 307.0 nm. IR (film): ν_max = 3417, 2923, 1720, 1585, 1467, 1385, 1103, 995, 744 cm^-1. EIMS: m/z (rel. int.) = 302 (M⁺, 100), 287 (8), 255 (35), 237 (12), 227 (22), 85 (10), 83 (16). HR-EIMS: found m/z 302.1166 (M⁺); calcd. for C₁₇H₁₈O_{5 :} 302.1154. ¹H-NMR (CDCl₃, 400 MHz): δ = 3.83 (3H, s, 10-OCH₃), 3.92 and 3.93 (each 3H, s, 4-OCH₃ and 5-OCH₃ or reversed), 6.64 (1H, d, J = 1.8 Hz, H-6), 6.86 (1H, d, J = 1.8 Hz, H-2), 6.89 (1H, dd, J = 7.9, 1.8 Hz, H-12), 7.02 (1H, t, J = 7.9 Hz, H-13), 7.03 (1H, d, J = 16.4 Hz, H-7), 7.12 (1H, dd, J = 7.9, 1.8 Hz, H-14), 7.19 (1H, d, J = 16.4 Hz, H-8). ¹³C-NMR (CDCl₃, 100 MHz): δ = 55.9 (5-OCH₃), 61.0 (4-OCH₃), 61.7 (10-OCH₃), 103.1 (C-6), 106.0 (C-2), 114.6 (C-12), 118.0 (C-14), 122.2 (C-8), 124.9 (C-13), 130.2 (C-7), 130.5 (C-9), 133.6 (C-1), 135.5 (C-4), 144.8 (C-10), 149.1 (C-11), 149.4 (C-3), 152.4 (C-5).

Halophilol B (2): Brown amorphous powder, m. p. > 300 °C, [α]_D ²⁰: + 152.4° (c 1.28, MeOH). UV (MeOH): λ_max = 202.0, 284.0 nm. IR (film): ν_max = 3405, 2920, 1614, 1514, 1444, 1172, 1130, 1007, 833 cm^-1. HR-ESI-MS: m/z 907.2753 (calcd. for C₅₆H₄₃O_{12 :} 907.2755). ¹H-NMR (acetone-d ₆, 400 MHz) and ¹³C-NMR (acetone-d ₆, 100 MHz) NMR data see Table [1].

Acknowledgements

We thank Professor Guang-Xing Yang of Kashi Institute of Drug Research of Xinjiang, for the plant collection and identification, and Dr. Ze-Hong Miao and Miss Mei-Hong Li of our institute for conducting the cytotoxicity assays.

References

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Prof. Da-Yuan Zhu

Shanghai Institute of Materia Medica

Chinese Academy of Sciences

294 Taiyuan Road

Shanghai 200031

P. R. China

Fax: +86-21-64370269

Email: dyzhu@mail.shcnc.ac.cn

• 1 Jiang M, Cai L, Udeani G, Slowing K V, Thomas C F, Beecher C WW, Fong H HS, Farnsworth N R, Kinghorn A D, Mehta R G, Moon R C, Pezzuto J M. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes.  Science. 1997;  275 218-20
  CrossrefPubMedSearch in Google Scholar
• 2 Kitanaka S, Ikezawa T, Yasukanwa K, Yamanouchi S, Takido M, Sung H K, Kim I H. (+)-α-Viniferin, an anti-inflammatory compound from Caragana chamlagu root.  Chemical & Pharmaceutical Bulletin. 1990;  38 432-5
  CrossrefPubMedSearch in Google Scholar
• 3 Jayatilake G, Jayasuriya H, Lee E -S, Koonchanok N M, Geahlen R L, Ashendei C L, Mclaughlin J L, Chang C J. Kinase inhibitors from Polygonum cuspidatum .  Journal of Natural Products. 1993;  56 1805-10
  CrossrefPubMedSearch in Google Scholar
• 4 Ito J, Niwa M. Absolute structure of new hydroxystilbenoids, vitisin C and viniferal, from Vitis vinifera .  Tetrahedron. 1996;  52 9991-8
  CrossrefPubMedSearch in Google Scholar
• 5 Korhammer S, Reniero F, Mattivi F. An oligostilbene from Vitis roots.  Phytochemistry. 1995;  38 1501-4
  CrossrefPubMedSearch in Google Scholar
• 6 Breuil A G, Adrian M, Pirio N, Meunier P, Bessis R, Jeandet P. Metabolism of stilbene phytoalexins by Botrytis cinerea .  Tetrahedron Letters. 1998;  39 537-40
  CrossrefPubMedSearch in Google Scholar
• 7 Robert H C, Samir A.K; Mark T H. Dimerization of resveratrol by the grapevine pathogen Botrytis cinerea .  Journal of Natural Products. 2000;  63 29-33
  CrossrefPubMedSearch in Google Scholar
• 8 Lee S -H, Shin N H, Kang S H, Park J S, Chung S R. α-Viniferin, a prostaglandin H₂ synthase inhibitor from root of Carex humilis .  Planta Medica. 1998;  64 204-7
  PubMedSearch in Google Scholar
• 9 Yan K -X, Terashima K, Takaya Y, Niwa M. A novel oligostilbene named (+)-viniferol from the stem of Vitis vinifera .  Tetrahedron. 2001;  57 2711-5
  CrossrefPubMedSearch in Google Scholar
• 10 Ito T, Tanaka T, Nakaya K, Iinuma M, Takahashi Y, Naganawa H, Ohyama M, Nakanishi Y, Bastow K F, Lee K -H. A new resveratrol octamer, vateriaphenol A, in Vateria indica .  Tetrahedron Letters. 2001;  42 5909-12
  CrossrefPubMedSearch in Google Scholar
• 11 Xiao D, Tan W F, Li M H, Ding J. Antiangiogenic potential of 10-hydroxycamptothecin.  Life Science. 2001;  69 619-28
  PubMedSearch in Google Scholar

Prof. Da-Yuan Zhu

Shanghai Institute of Materia Medica

Chinese Academy of Sciences

294 Taiyuan Road

Shanghai 200031

P. R. China

Fax: +86-21-64370269

Email: dyzhu@mail.shcnc.ac.cn