HETEROCYCLES

Short Paper | Regular issue | Vol. 83, No. 4, 2011, pp. 849-854
Received, 29th December, 2010, Accepted, 9th February, 2011, Published online, 14th February, 2011.
DOI: 10.3987/COM-10-12131

■ The First Hydroperoxydihydrochalcone in the Etlingera Genus: Etlinglittoralin from the Rhizomes of Etlingera littoralis

Chotika Jeerapong, Sarot Cheenpracha, Wisanu Maneerat, Uma Prawat, Thongchai Kruahong, and Surat Laphookhieo*

Natural Products Research Laboratory, School of Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai 57100, Thailand

Abstract

Etlinglittarolin (6), a monoterpene-substituted hydroperoxy dihydrochalcone, together with five known dihydrochacones including 2′, 6′-dihydroxy-4′-methoxydihydrochalcone (1), 2′,4′,6′-trihydroxydihydrochalcone (2), 2′,6′,4-trihydroxy-4′-methoxydihydrochalcone (3), methyllinderatin (4), and adunctin E (5), was isolated from the rhizomes of Etlingera littoralis. Their structures were elucidated by spectroscopic analysis.

Plants of Zingiberaceae are widely distributed throughout the tropical forests. Many of them are used for food, spices, medicines, dyes, perfume and aesthetics.1 Some metabolites from Zingiberaceae plants have found to be interesting biological activities, for example anti-malaria,2 anti-tumor3 and anti-HIV-1 protease inhibitory.4 Etlingera littoralis is one of the Zingiberaceae plants which are found in several parts of Thailand. Its rhizome decoction has been used for the treatment of stomachache, carminative, and heart tonic.5 As part of our study of chemical constituents and biological activity from medicinal plants, we now report the structure elucidation of a new monoterpene-substituted dihydrochalcone, etlinglittoralin (6) along with five known dihydrochalcones (Figure 1) isolated from the rhizomes of E. littoralis.

Compound 6, [α]D25 -22 (c 0.006, CHCl3), was obtained as a white amorphous powder (mp 102.0-103.6 °C). The molecular formula of C26H32O6 was deduced from the ESITOFMS data, exhibiting the [M+H]+ ion peak at m/z 441.2266 (calcd. for 441.2277). Its IR spectrum revealed absorption bands for hydroxy (3398 cm-1) and carbonyl (1631 cm-1) groups. The UV absorption bands at λmax 236, 282 and 342 nm supported the presence of a conjugated carbonyl in the structure. The 13C NMR and DEPT spectrum of 6 indicated the signals of the dihydrochalcone (12 aromatic carbons, 2 aliphatic carbons and one carbonyl carbon)6 and contained ten additional resonances with three methyls [δC 22.2 (C-7′′), 21.9 (C-10′′), 15.6 (C-9′′)], two methylenes [δC 32.0 (C-2′′), 17.3 (C-3′′)], four methines [δC 87.7 (C-6′′), 46.5 (C-4′′), 39.9 (C-5′′), 27.3 (C-8′′)] and a quaternary carbon [δC 81.1 (C-1′′)]. The latter signals suggested that the presence of a hydroperoxy group at C-1′′ was supported by the molecular formula C26H32O6 and the downfield chemical shift of the oxygenated carbon C-1′′ at δC 81.1.7-9 From above data, the dihydrochalcone was substituted by a saturated cyclic monoterpene moiety (Table 1). The 1H NMR spectroscopic data (Table 1) showed signals characteristic of isopropyl unit at δH 1.83 (1H, m, H-8′′), 0.85 (3H, d, J = 6.8 Hz, H3-9′′), and 0.83 (3H, d, J = 6.8 Hz, H3-10′′) and a tertiary methyl group at δH 1.43 (3H, s, H3-7′′). This data was quite similar to a p-menthane unit.10 In the 1H NMR spectroscopic data, the signals characteristic of a 2′,6′-dihydroxy-4′-methoxydihydrochalcone derivative were clearly observed at δH 13.23 (1H, s, OH), 7.17-7.30 (5H, m), 6.04 (1H, s, H-3′), 3.81 (3H, s, 4′-OCH3), 3.34 (2H, m, H-α), and 3.02 (2H, t, J = 7.6 Hz, H-β).9
The attachment of the p-menthane unit on the dihydrochalcone moiety was established with the combined results of COSY, HMQC and HMBC experiments. In the HMBC spectrum, the p-methine proton H-5′′ correlated with C-4′, C-5′, and C-6′ suggesting that the p-menthane was C-linked to the dihydrochalcone core between C-5′′ and C-5′. The deshielded oxymethine carbon C-6′′ (δC 87.7) and the aromatic carbon C-6′ (δC 162.0) implied to O-linkage between C-6′′ and C-6′. Thus, the p-menthane unit and the dihydrochalcone core formed a five-membered ring as in agreement with the spectral data of adunctin E, previously isolated from Piper aduncum.8

The relative location of the 4′-OMe was confirmed by an HMBC experiment in which a correlation was observed for OMe protons with C-4′ (δC 161.9). In addition, OMe showed the cross-peaks with H-3′ (δH 6.04) and H3-10′′ (δH 0.83) in a 2D NOESY experiment (Figure 2). The cross-peaks between H3-7′′/H-3′′, H-3′′/H-5′′, H-5′′/H3-10′′, H-5′′/H-6′′ and H-6′′/H3-7′′ indicated the relative configurations at C-1′′, C-4′′, C-5′′ and C-6′′ as 1′′S*, 4′′R*, 5′′S* and 6′′R*, respectively. Accordingly, the structure of 6 was determined to be (1′′S*, 4′′R*, 5′′S*, 6′′R*)-etlinglittoralin.

The known compounds were identified as 2′, 6′-dihydroxy-4′-methoxydihydrochalcone (1),8,9 2′,4′,6′-trihydroxydihydrochalcone (2),4 2′,6′,4-trihydroxy-4′-methoxydihydrochalcone (3),11 methyllinderatin (4),12 adunctin E (5).6All of them were identified by exhaustive spectral analysis (1D and 2D NMR spectra) and also comparison with their spectroscopic data with those reported in the literature. All compounds were tested for their antimalarial activity but, unfortunately, they were inactive.

EXPERIMENTAL
GENERAL
The optical rotation [α]D values were determined with a Bellingham & Stanley ADP440 polarimeter. UV spectra were recorded with a Perkin-Elmer UV-Vis spectrophotometer. The IR spectra were recorded with a Perkin-Elmer FTS FT-IR spectrophotometer. The NMR spectra were recorded using 400 MHz Bruker spectrometer. Chemical shifts were recorded in parts per million (δ) in CDCl3 with tetramethylsilane (TMS) as an internal reference. The ESITOFMS was obtained from a MicroTOF, Bruker Daltonics mass spectrometer. Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60 H (Merck, 5-40 μm) and silica gel 100 (Merck, 63-200 μm), respectively. Precoated plates of silica gel 60 F254 were used for analytical purposes.

PLANT MATERIAL
The rhizomes of E. littoralis were collected in June 2009 from Surat Thani Province, southern part of Thailand. Botanical identification was made by Assistant Professor Dr. Chatchai Ngamriabsakul and a specimen (number MFU-NPR 0015) was deposited at Natural Products Research Laboratory, School of Science, Mae Fah Luang University.
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EXTRACTION AND ISOLATION
Chopped-fresh rhizomes (3.89 kg) of E. littoralis were extracted with CH2Cl2−MeOH (1:1, v/v), over the period of 3 days at room temperature. The mixture was filtered and then evaporated to dryness under reduced pressure and partition with CH2Cl2 to afford the CH2Cl2 extracted (22.80 g). A portion of CH2Cl2 extract (11.80 g) was subjected to quick column chromatography (QCC) over silica gel and eluted with a gradient of n-hexane−EtOAc (100% n-hexane−100% EtOAc) to afford thirteen fractions (F1-F13). Fraction F3 (755.10 mg) was resubmitted to column chromatography (CC) eluting with EtOAc−n-hexane (1:9, v/v) to afford four subfractions (F3A-F3D). Subsraction F3A (48.8 mg) was further purified by CC with CH2Cl2−n-hexane (1:4, v/v) to give compound 5 (3.0 mg) and three subfractions (F3A1-F3A3). Compound 4 (7.1 mg) derived from subfraction F3A1 (12.8 mg) whereas 6 (3.0 mg) obtained from subfraction F3A3 (8.7 mg) by prep. TLC developed with EtOAc−n-hexane (1:4, v/v) and prep. TLC with acetone−n-hexane (1:9, v/v), respectively. Fraction F8 (322.4 mg) was washed with CH2Cl2−n-hexane (1:4, v/v) yielding a pale-yellow solid which was further separated by CC with CH2Cl2−n-hexane (7:3, v/v) to give compound 1 (50.1 mg). Fraction F12 (1.83 g) was resubmitted to QCC over silica gel eluting with a gradient of n-hexane−EtOAc (1:5-3:5, v/v) to afford seven subfractions (F12A-F12G). Subfraction F12E (220.0 mg) was purified by CC eluting with EtOAc−CH2Cl2 (1:100, v/v) to give compound 2 (108.6 mg). Subfraction F12G (98.0 mg) was purified by CC on silica gel eluting with EtOAc−CH2Cl2 (1:100, v/v) to give compound 3 (47.5 mg).

Etlinglittoralin (6): White amorphous powder. Mp 102.0-103.6 °C. [α]D25 -22 (c 0.006, CHCl3). UV (CHCl3) (log ε): 236 (4.23), 282 (4.40), 342 (3.36) and 337 (3.32) nm. IR (neat) νmax: 3398, 2954, 2927, 1631, 1602 cm-1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) see Table 1; ESITOFMS (m/z): [M+H]+ m/z 441.2266 (calc. for C26H33O6, 441.2277).

ANTI-MALARIAL ASSAY
Anti-malarial activity was evaluated against the parasite Plasmodium falciparum (K1 strain, multidrug resistant), using the method of Trager and Jensen (1976).13 Quantitative assessment of in vitro malarial activity was determined by means of the microculture radioisotope technique based on the method described by Desjardins et al. (1979).14 The inhibitory concentration (IC50) represented the concentration that caused 50% reduction in parasite growth which was indicated by the in vitro uptake of [3H]-hypoxanthine by P. falciparum. The standard compounds were dihydroartemisinin (IC50 1.58 nM) and mefloquine (IC50 0.0282 μM).

ACKNOWLEDGEMENTS
CJ and TK thank the Center of Excellence for Innovation in Chemistry (PERCH-CIC) and Faculty of Science and Technology, Suratthani Rajabhat University, for financial support. The Natural Products Research Laboratory, School of Science, Mae Fah Luang University, is also gratefully acknowledged for partial financial support and laboratory facilities. We are indebted to Mr. Nitirat Chimnoi, Chulabhorn Research Institute, Bangkok, for recoding the mass spectra, and Ms. Nareerat Thongtip, Department of Chemistry, Phuket Rajabhat University, for recording the NMR spectra. We also thank Bioassay Research Facility of BIOTEC (Thailand) for anti-malarial test.

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