Norisoprenoid, phenolic and steroidal constituents from mallotus luchenensis - Nguyen Huu Toan Phan

558 Journal of Chemistry, Vol. 47 (5), P. 558 - 562, 2009 NORISOPRENOID, PHENOLIC AND STEROIDAL CONSTITUENTS FROM MALLOTUS LUCHENENSIS Received 8 July 2008 NGUYEN HUU TOAN PHAN1, CHAU VAN MINH2, NGUYEN HAI DANG2, DINH THI THU THUY2, NGUYEN PHUONG THAO2, PHAN VAN KIEM2, VU KIM THU3, YVAN VANDER HEYDEN4 AND JOËLLE QUETIN-LECLERCQ5 1Tay Nguyen Institute of Biology, Da Lat 2Institute of Natural Products Chemistry, VAST, Vietnam 3Hanoi University of Mining and Geology, Vietnam 4Vrije Universiteit Brussel, Laarbeeklaan, 103 B-1090 Brussels, Belgium 5Analytical Chemistry, Drug Analysis and Pharmacognosy Unit, Université Catholique de Louvain, Avenue E. Mounier, 72 B-1200 Brussels, Belgium ABSTRACT A C13-norisoprenoid malloluchenoside (1), two phenolics gallic acid (2) and corillagin (3), and two steroids daucosterol (4) and stigmast-5-enee-3-O-(6-O-octadecanoyl-β-D- glucopyranoside) (5) were isolated from the MeOH extract of Mallotus luchenensis. Their structures were elucidated on the basis of spectral and physicochemical data. Among which, 1 was isolated for the first time from the nature. Key words: Mallotus luchenensis, norisoprenoid, phenolic, steroid, malloluchenoside. I - INTRODUCTION Mallotus luchenensis is widely distributed in the Northern part of Vietnam. In particular, it has been found in Lang Son, Hoa Binh, Ha Nam, Ninh Binh provinces [1, 2]. Studies on the chemical constituents of the Mallotus genus revealed that flavonoids, phenolics and benzopyrans are the most common components [3 - 6]. In the course of our continuing studies for the chemical components of Mallotus species, we have isolated and identified five compounds including two steroids, two phenolics, and a new C13-norisoprenoid from M. luchenensis species. C13-norisoprenoids are well known as important aroma constituents of grape juices and wines. They were also found in the tea leaves as aroma precursors [7] but rarely found in the Mallotus genus. This paper deals with the structural elucidation of a new C13-norisoprenoid along with the other isolated compounds from M. luchenensis. II - MATERIALS AND METHODS 1. General experiment procedures The 1H-NMR (500 MHz) and 13C-NMR (125 MHz) spectra were recorded on a Bruker AM500 FT-NMR spectrometer. Chemical shifts are referenced to δ using tetramethylsilan (TMS) as an internal standard. The Electronspray Ionization (ESI) mass spectrum was obtained using an AGILENT 1200 LC- MSD Trap spectrometer. Column 559 chromatography (CC) was performed on silica gel 230 - 400 mesh (0.040 - 0.063 mm, Merck) or YMC RP-18 resins (30 - 50 μm, Fujisilisa Chemical Ltd.). Thin layer chromatography (TLC) was performed on DC-Alufolien 60 F254 (Merck 1.05715) or RP18 F254 (Merck) plates. 2. Plant material The aerial parts of M. luchenensis Metc., 1914 were collected in Son La, Vietnam in May 2006 and identified by Dr. Tran Huy Thai, Institute of Ecology and Biological Resources, VAST, Vietnam. An authentic sample was deposited at the Institute of Natural Products Chemistry, VAST, Vietnam. 3. Extraction and isolation The dried aerial parts of M. luchenensis were extracted three times with MeOH by a sonicator. The extract was dried under reduced pressure to give MeOH extract (20 g). The MeOH extract was then dissolved in water and partitioned in turn with hexane and then with EtOAc obtaining fractions hexane (3.5 g) and EtOAc (5.6 g). The water layer was passed through a dianion column eluted with MeOH- H2O (gradient: 0/100, 25/75, 50/50, 75/25, and 100/0, v/v) to give five fractions W1 (1.8 g), W2 (2.5 g), W3 (1.9 g), W4 (2.6 g), and W5 (1.0 g), respectively. The n-hexane fraction (3.5 g) was chromatographed on a silica gel column using n-hexane-acetone gradient system (from 100:1 to 1:1, v/v) to obtain fractions H1 (1.2 g), H2 (0.8 g), and H3 (1.5 g). The H2 fraction was then subjected to a silica gel column chromatography using n-hexane-acetone (5:1, v/v) system to afford 4 (10.5 mg). The H3 fraction was subjected to a YMC column chromatography with acetone-H2O (1:4, v/v) as eluant to yield 5 (16 mg). Compound 1 (10 mg) was purified by using a YMC column chromatography with MeOH- H2O (1:3, v/v) system on the W2 fraction. Similarly, by repeated column chromatography of the W1 fraction, 2 (17 mg) was purified. Column chromatography of W4 fraction on silica gel normal and then reversed phases led to the isolation of 3 (13 mg) as white powder. Malloluchenoside (1): White powder; ESI- MS m/z: 529 [M+Na]+; 1H-NMR (500 MHz, CD3OD) and 13C-NMR (125 MHz, CD3OD): See Table 1. Gallic acid (2): Colorless needles; mp: 235- 240oC; ESI MS m/z: 171 [M+H]+, 169 [M-H]-; 1H-NMR (500MHz, CD3OD) δ: 7.08 (brs, H-2, H-6); 13C-NMR (125 MHz, CD3OD) δ: 122.0 (C-1), 110.4 (C-2, C-6), 139. 6 (C-3, C-5),146.3 (C-6), and 170.3 (C-7). Corilagin (3): White needles; mp: 208oC, [α]D20 -250o (c, 0.3 in MeOH), UV: [base] λmax 240, 326; ESI-MS m/z: 657 [M+Na]+, 633 [M- H]-; 1H-NMR (500 MHz, CD3OD) δ: 6.71 (s, H- 3′), 6.68 (s, H-3′′) 7.07 (s, H-2′), 6.38 (d, J = 2.0 Hz, H-1), 4.01 (s, H-2), 4.58 (m, H-3), 4.48 (d, J = 3.0 Hz, H-4), 4.54 (m, H-5), and 4.98/4.17 (m, H-6); 13C-NMR (125 MHz, CD3OD) δ: 95.0(C-1), 69.4 (C-2), 71.6 (C-3), 62.5 (C-4), 76.2 (C-5), 65.0 (C-6), 117.2 (C-1′), 125.5 (C- 2′), 110.2 (C-3′), 145.3 (C-4′), 137.7 (C-5′), 146.4 (C-6′), 170.1 (C-7′), 116.7 (C-1′′), 125.5 (C-2′′), 108.3 (C-3′′), 145.2 (C-4′′), 138.2 (C- 5′′), 145.6 (C-6′′), 168.5 (C-7′′), 120.6 (C-1′′′), 111.0 (C-2′′′), 146.4 (C-3′′′), 140.4 (C-4′′′), 146.4 (C-5′′′), 111.0 (C-6′′′), and 166.7 (C-7′′′). Daucosterol (4): White powder; mp: 284- 286oC; [α]D26 -41,5o (c, 0.4 in Py); IR(KBr) νmax cm-1: 3420, 1460, 1090; 1H-NMR (500 MHz, CD3OD) δ: 3.52 (1H, dd, J = 11.7, 5.1 Hz, H-3), 5.35 (1H, br d, J = 5.0 Hz, H-6), 0.68 (3H, s, H- 18), 1.00 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.84 (3H, t, J = 7.6 Hz, H-26), 0.81 (3H, d, J = 6.8 Hz, H-28), 0.83 (3H, d, J = 7.3 Hz, H-29), and 4.30 (1H, d, J = 7.8 Hz, H-1′); 13C- NMR (125MHz, CD3OD) δ: 36.8 (C-1), 31.3 (C-2), 76.9 (C-3), 39.3 (C-4), 140.4 (C-5), 121.1 (C-6), 31.4 (C-7), 31.3 (C-8), 49.9 (C-9), 36.1 (C-10), 20.5 (C-11), 38.2 (C-12), 41.8 (C-13), 55.2 (C-14), 25.4 (C-15), 29.2 (C-16), 56.0 (C- 17), 11.6 (C-18), 19.0 (C-19), 35.4 (C-20), 18.5 (C-21), 33.3 (C-22), 27.7 (C-23), 45.0 (C-24), 28.9 (C-25), 19.6 (C-26), 18.9 (C-27), 22.5 (C- 28), 11.7 (C-29), 100.7 (C-1′), 73.4 (C-2′), 76.7 (C-3′), 70.0 (C-4′), 76.6 (C-5′), and 61.0 (C-6′). 560 Stigmast-5-ene-3-O-(6-O-octadecanoyl-β- D-glucopyranoside) (5): White powder; ESI MS m/z: 893 [M+Na]+; 1H-NMR (500 MHz, CDCl3) δ: 3.47 (m, H-3), 5.37 (t, J = 3.0 Hz, H- 6), 0.68 (s, H-18), 1.00 (s, H-19), 0.91 (d, J = 6.5 Hz, H-21), 0.79 (d, J = 6.0 Hz, H-26, H-27), 0.80 (d, J = 6.5 Hz, H-29), 4.37 (d, J = 7.5 Hz, H-1′), 3.29 - 3.58 (m, H-3′, H-5′), 4.27 (br d, J = 12.0 Hz, H-6′a), 4.44 (dd, J = 5.0, 12.0 Hz, H-6′b), and 0.88 (d, J = 6.5 Hz, H-20′′); 13C- NMR (125MHz, CDCl3) δ: 37.3 (C-1), 29. 6 (C- 2), 79.6 (C-3), 38.9 (C-4), 140.3 (C-5), 122.2 (C-6), 32.0 (C-7), 32.0 (C-8), 56.1 (C-9), 36.8 (C-10), 21.1 (C-11), 39.8 (C-12), 42.4 (C-13), 56.8 (C-14), 24.3 (C-15), 28.3 (C-16), 56.1 (C- 17), 12.0 (C-18), 19.4 (C-19), 36.2 (C-20), 18.8 (C-21), 34.3 (C-22), 26.2 (C-23), 45.9 (C-24), 29.2 (C-25), 19.8 (C-26), 19.1 (C-27), 23.1 (C- 28), 12.0 (C-29), 101.2 (C-1′), 73.9 (C-2′), 76.8 (C-3′), 73.6 (C-4′), 76.0 (C-5′), 63.2 (C-6′), 174.7 (C-1′′), 34.3 (C-2′′), 24.3 (C-3′′), 28.3 - 29.7 (C-4′′-15′′), 25.0 (C-16′′), 29.4 (C-17′′), 31.9 (C-18′′), 22.7 (C-19′′), and 14.1 (C-20′′). O OH O O HO HO OH OOH HO H3C OH H3C CH3 H3C 1'2' 3' 4' 5' 6' 1" 2" 4" Ara Glc 1 3 5 69 10 13 1211 2 4 5'' 1 O OH O OH O O OH OH OH O O HO OH OHHO OH HO O 1'' 2'' 3'' 4'' 6'' 5'' 1' 2'3' 4' 5' 6' 7" 8"9'' 1"' 2"' 3"' 4"' 5"'6"' 1 3 6 3 OHHO OH OHO 7 4 2 1 35 6 2 O O HO HO OH OR 17 18 19 20 22 5 24 26 27 25 29 3 4: R = H 5: R = CO-(CH2)18-CH3 Figure 1: Structures of compounds 1 - 5 III - RESULTS AND DISCCUSION The molecular formula of 1 was assigned as C24H42O11 by examination of ESI MS and NMR spectra. The positive ESI MS of 1 displayed the pseudomolecular ion peak at m/z 529 [M+Na]+. The 1H-NMR spectrum of 1 presented signals of four methyl groups at δH 1.05 (s), 1.08 (s), 1.22 (d, J = 6.5 Hz), and 1.66 (s). The signals of methine, methylene, oxygenated methine and methylene groups were also observed in this spectrum. The 13C-NMR spectrum of 1 has the resonances due to the presences of 11 carbons belonging to the two sugar units. The signals of the aglycon were ascribable to an 3-hydroxy- 7,8-dihydro-β-ionol [7]. Comparing the NMR data of 1 with those of (3R,9R)-3-hydroxy-7,8- dihydro-ionyl-9-O-β-D-apiofuranosyl-β-D- glucopyranoside [7] revealed that structure of 1 was very closed to the reported compound except for chemical shift data of the apiose unit. These observations indicated that the second 561 sugar unit of 1 was not apiofuranoside. The NMR data of this sugar unit (δ 109.0, 83.0, 78.9, 86.0, and 63.1) were very similar to those of arabinofuranose in 1-O-trans-cinamoyl-α-L- arabinofuranosyl-(1→6)-β-D-glucopyranose (δ 110.8, 83.7, 79.3, 86.6, and 63.8) [12], confirming that the sugar partial structure of 1 was α-L-arabinofuranosyl-(1→6)-β-D- glucopyranose. Furthermore, the H-C correlations between H-1′ and C-9, H-1′′ and C- 6′ were observed in the HMBC spectrum confirming the linkage position of the aglycon and sugar moieties (Table 1). All the NMR data of the aglycon of 1 were completed agreement with those of (3R,9R)-3-hydroxy-7,8-dihydro- ionyl-9-O-β-D-apiofuranosyl-β-D- glucopyranoside (table 1) [7] suggesting that the absolute configurations at C-3 and C-9 were both determined as R. Therefore, 1 was deduced as (3R,9R)-3-hydroxy-7,8-dihydro-ionyl-9-O-α- L-arabinofuranosyl-β-D-glucopyranoside, which was named as malloluchenoside. To our best knowledge, this is the first report of this compound from the nature. Table 1: 1H- and 13C-NMR spectral data and HMBC correlations of 1 C δC* δC a, b δH a, c (J in Hz) HMBC (H → C) Aglycon 1 40.0 38.8 2 49.8 49.8 1.37 (t, 12.0)/1.70 (m) 3 65.8 65.7 3.86 (m) 4 40.5 42.9 1.95 (dd, 11.0, 16.5)/ 2.20 (dd, 5.0, 16.5) C-5, 6, 2 5 125.4 125.3 6 138.6 138.5 7 25.4 25.3 1.98/ 2.30 (m) 8 38.9 38.8 1.50/ 1.70 (m) 9 76.4 76.4 3.84 (m) 10 19.9 19.9 1.22 (s) C-8, 9 11 30.4 30.4 1.08 (s) C-1, 6, 12 12 29.0 28.9 1.05 (s) C-1, 2, 11, 6 13 20.2 20.1 1.66 (s) C-4, 5, 6 Glc Glc 1′ 102.4 102.3 4.34 (d, 8.0) C-9 2′ 75.2 75.2 3.16 (t, 9.0) 3′ 78.0 78.1 3.83 (m) 4′ 71.8 72.2 3.38(dd, 3.0, 9.0) 5′ 76.8 76.8 3.44 (m) 6′ 68.5 68.2 3.62/ 4.03 (m) Api Ara 1′′ 110.9 109.9 4.99 (dd, 1.5, 4.5) C-6′ 2′′ 78.2 83.0 4.01 (m) 3′′ 80.6 78.9 3.85 (m) 4′′ 75.3 86.0 3.98 (m) 5′′ 65.8 63.1 3.60/3.74 (m) aMeasured in CD3OD, b125 MHz, c500 MHz, *δC of (3R,9R)-3-hydroxy-7,8-dihydro-ionyl-9-O-β-D-apiofuranosyl-β-D- glucopyranoside [7], Chemical shifts in ppm. 562 The 1H-NMR spectrum of 3 showed two singlet signals at δH 6.68 and 6.70 which were assigned to two aromatic protons. A broad singlet at δH 7.07 was assigned to a proton of a gallyol residue. The presence of an anomeric proton signal was upfield shifted to δH 6.38 (d, J = 2.0 Hz). The 13C-NMR and DEPT spectra of 3 showed the signals of three aromatic rings at the range of δC 108.3 – 146.4, three carbonyl groups at δH 168.5, 166.7 and 170.1, and a sugar unit at the range of δC 62.5 – 95.1. Crosspeaks between H-1 (δH 6.38)/H-2′′′ (δH 7.07) and the cacbonyl group of one galloyl unit (δC 166.7), and between H-3 (δH 4.58) and the cacbonyl group of a phenoyl unit (δC 168.5), H-6 (δH 4.98) and the carbonyl group of another phenoyl unit (δC 170.1) were observed in the HMBC spectrum. The ESI MS of 3 displayed the fragment ion peaks at m/z 657 [M+Na]+, 487 [M+Na-galloyl]+ (in the positive mode) and at m/z 633 [M-H]-, m/z 463 [M-H-galloyl]- (in the negative mode). From the above evidence and comparison with published data, 3 was determined to be 1-O-galloyl-3,6-(R)-hexahy- droxydiphenoyl-β-D-glucose or corilagin, a substance isolated from Punica gratanum [9]. By carrying out the same structural elucidation methods, and comparison with the reported data [8], the structure of 2 and 4 were identified as gallic acid [8] and daucosterol [10], respectively. Compound 5 was purified from the n- hexane fraction of M. luchenensis. The 1H- NMR spectrum of 5 showed a signal of one olefinic double bond at δH 5.37 (t, J = 3.0 Hz), overlapped signals of an aliphatic chain at δH 1.3 - 2.5, an anomeric signal at δH 4.37 (d, J = 7.5 Hz), and hydroxylated methylene signals at δH 4.27 (br d, J = 12.0 Hz)/4.43 (dd, J = 5.0, 12.0 Hz). Signals of the methyl groups were also observed in this spectrum at δC 0.68, 0.79, 0.80, 0.88, 0.91, and 1.00. The 13C-NMR and DEPT spectra of 5 displayed signals of 55 carbons, in which six carbons were assigned to a sugar unit, twenty nine carbons were assigned to an aglycon part, one carbonyl carbon (δC 174.7), and nineteen carbons were assigned to an aliphatic chain. The NMR data of the aglycon and sugar parts of 5 were compared with those of daucosterol and found to match well. Therefore, structure of 5 was suggested to be an acylated daucosterol. The correlations of protons and carbons of 5 were determined by HSQC and HMBC spectra. The positive ESI MS of 5 showed the pseudomolecular ion peak at m/z 893 [M+Na]+ which was assigned to the molecular formula C55H98O7 (M = 870). From the above evidence and comparison with the reported data, 5 was determined as stigmast-5- ene-3-O-(6-O-octadecanoyl-β-D- glucopyranoside), an isolated compound from Stelmatocrypton khasianum [11], however, this is the first report of 5 from the genus Mallotus. REFERENCES 1. L. D. Moi, T. M. Hoi, D. D. Huyen, T. H. 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