- freely available
Int. J. Mol. Sci. 2012, 13(12), 16430-16443; doi:10.3390/ijms131216430
Abstract: Four new endiandric acid analogues, tsangibeilin C (1), tsangibeilin D (2), tricyclotsangibeilin (3) and endiandric acid M (4), one new lignan, beilschminol B (5) and two new sesquiterpenes, (+)-5-hydroxybarbatenal (6) and (4R,5R)-4,5-dihydroxycaryophyll-8(13)-ene (7), together with four known compounds (8–11), were isolated from the roots of Beilschmiedia tsangii (Lauraceae). The structures of 1–7 were determined by spectroscopic techniques. Among the isolates, endiandric acid M (4) exhibited moderate iNOS inhibitory activity, with an IC50 value of 31.70 μM.
Over 40 species of Formosan lauraceous plants have been screened for anti-inflammatory activity using an inducible nitric oxide synthase (iNOS) assay. A methanolic extract of the roots of B. tsangii Merr. (Lauraceae) has shown potent inhibition of NO production, with no cytotoxicity against RAW 264.7 cells. B. tsangii is a medium-sized evergreen tree, distributed throughout Tonkin, Vietnam, southern and western China and southern Taiwan. About 200 Beilschmiedia species are found in these tropical regions, including two species in Taiwan . Our previous study reported two new tetrahydrofuran-type lignans, beilschmin A and beilschmin B, two new 1-phenylbutyl benzoates, tsangin A and tsangin B, together with thirteen known compounds isolated from the stem of this plant . One year later, three new epoxyfuranoid lignans, 4α,5α-epoxybeilschmin A, 4α,5α-epoxybeilschmin B and beilschmin D, together with nine known compounds, were obtained from the leaves . More recently, six new endiandric acid analogues, tsangibeilin A, tsangibeilin B, endiandramide A, endiandric acid K, endiandric acid L and endiandramide B, two new lignans, beilschminol A and tsangin C, and six known compounds have been obtained from the roots of this species . In this continuation of our research, four new endiandric acid analogues, tsangibeilin C (1), tsangibeilin D (2), tricyclotsangibeilin (3) and endiandric acid M (4), one new lignan, beilschminol B (5) and two new sesquiterpenes: (+)-5-hydroxybarbatenal (6) and (4R,5R)-4,5-dihydroxycaryophyll-8(13)-ene (7), together with four known compounds (8–11), have been isolated from the roots of this plant. The structures of 1–7 were determined by spectroscopic techniques. This paper elucidates the structures of 1–7 (Figure 1). Endiandric acid M (4) exhibited moderate iNOS inhibitory activity, with an IC50 value of 31.70 μM.
2. Results and Discussion
2.1. Structure Elucidation
Compound 1 was isolated as a light yellowish oil. Its molecular formula was established as C20H26O3 by ESIMS and HRESIMS, with eight unsaturated degrees. The IR spectrum showed absorptions at 1696 cm−1 for carbonyl groups (δ 201.6, C-4 and δ 168.3, C-14) and at 3432 cm−1 for a hydroxy group of carboxylic acid. These findings were supported by 13C NMR spectrum. The 1H, 13C NMR (Table 1), COSY (Figure 2), HSQC and HMBC (Figure 2) spectra of 1 were similar to those of beilschmiedic acid D  and also contained 13 skeletal signals of an endiandric acid moiety. The characteristic two cis olefinic protons at δ 5.56 (ddd, J = 10.2, 3.0, 1.8 Hz, H-8) and 5.85 (ddd, J = 10.2, 4.2, 3.0 Hz, H-9) in 1 were similar to those of beilschmiedic acid D, but the signal for another olefinic proton in 1 was shifted upfield to δ 6.70 (d, J = 1.2 Hz, H-5), because a carbonyl group (δC 201.6, C-4) in 1 replaced a methylene group [δ 2.06 (m, Ha-4) and 2.54 (dt, J = 8.8, 3.1 Hz, Hb-4)] in beilschmiedic acid D. The length of the alkyl side chain at C-11 of 1 was two methylenes less than beilschmiedic acid D, as supported by the molecular formula of 1 (C20H26O3). The rigid tetracyclic skeleton was indicated by HMBC correlations, including: H-5 to C-3, C-6, C-7 and C-14, H-3 to C-4, and C-7, H-13 to C-8 and C-10, H-8 to C-6 and C-10, H-9 to C-7, H-2 to C-3, C-4, C-11 and C-13, H-1 to C-3 and C-13, H-12 to C-3, C-9 and C-11 and H-11 to C-9. The relative configuration of 1, rel-(1S,3R,7R,10R,11S,12S,13S), was supported on the basis of NOESY (Figure 3) and structural similarity with beilschmiedic acid D . There are seven chiral centers in 1. However, in view of the observed optical rotation ([α]23D ± 0), 1 should be racemic, as are many endiandric acid analogues [6–9]. According to the above data, the structure of 1 was elucidated and named tsangibeilin C.
Analysis of the ESIMS and HREIMS of 2 revealed a molecular formula of C19H26O3, representing seven degrees of unsaturation, one carbon and one degree of unsaturation less than compound 1 (C20H26O3). The IR spectrum indicated the presence of a hydroxy group (3397 cm−1) and a carbonyl group (1692 cm−1). The rigid tetracyclic skeleton of 2 was the same as that of 1, according to its 1H and 13C NMR spectra (Table 1), inclusive of COSY (Figure 2), NOESY (Figure 3), HSQC and HMBC (Figure 2) experiments. The major differences between 1 and 2 were two hydroxy groups at C-6 and C-7 in 2 were substituted for the carboxyl group (δ 168.3, C-14) and H-7 [δH 3.51 (1H, br s)] at C-6 and C-7 in 1, as supported by HRESIMS, IR and DEPT spectra. The NOESY spectrum (Figure 3) showed correlations between Ha-2, H-3 and H-11, but these three protons showed no correlations with H-1, Hb-2, H-10, H-12 and H-13. This suggested that Ha-2, H-3 and H-11 are on the same side of the molecule, and that H-1, Hb-2, H-10, H-12 and H-13 are on the opposite side of the molecule. The β-orientation of the hydroxy group at C-7 was attributed according to the structural similarity with endiandric acid analogues and biogenetic consideration, where the rings A/B, B/C, C/D and B/D were cis-fusion and ring A/C was trans-fusion [4,8,9]. The relative configuration of 2, rel-(1S,3R,7R,10R,11S,12S,13S) was supported on the basis of NOESY and structural similarity with beilschmiedic acid D . On the basis of the observations above, compound 2 was elucidated and named tsangibeilin D.
Compound 3 was obtained as an optically active light yellowish oil with [α]24D + 36.3 (c 0.024, CHCl3). IR absorption bands at 3422 cm−1 (OH) and 1729 cm−1 (ester carbonyl) were observed. The ESIMS analysis of 3 showed the [M+Na]+ ion at m/z 341, in agreement with the molecular formula of C20H30O3, with six degrees of unsaturation as confirmed by HRESIMS. The 13C NMR (Table 2) and DEPT spectra indicated that 3 contains one methyl, eight methylenes, ten methines and one quaternary carbon. The HSQC and COSY (Figure 2) spectra revealed three fragments, C1-C2-C3-C4-C5-C6, C1-C13-C11-C12-C1 and C-13-C14-C15-C9-C10, and the HMBC (Figure 2) correlations, H-10 to C-9, C-12, C-13 and C-15 and H-9 to C-11, connected the fragments C1-C13-C11-C12-C1 and C-13-C14-C15-C9-C10 to form a cyclohexane ring fused with a cyclobutane ring. The carboxyl group (δ 173.5, C-7) connected the fragment C1-C2-C3-C4-C5-C6 with the cyclohexane ring, as supported by HMBC correlations, H-6 to C-4, C-5 and C-7, H-5 to C-3 and C-7 and H-9 to C-7. According to the above evidence, the carboxyl group, the fragment C1-C2-C3-C4-C5-C6, the cyclohexane ring and the cyclobutane ring become a cyclododecane ring, which is also supported by six degrees of unsaturation. The HMBC spectrum showed correlations between H-1′ and C-1, C-11, C-2′ and C-3′, thus establishing the presence of an alkyl chain (C-1′~C-6′) to be connected at C-12. Finally, the HRESIMS, IR, and DEPT spectra indicated the presence of a hydroxy group located at C-10. The NOESY spectrum (Figure 3) showed correlations between H-9, H-10 and H-12, suggesting these three protons were on the same side of the molecule, but H-1, H-11 and H-13 showed no correlations with H-9, H-10 and H-12, which should therefore be on the opposite side of the molecule. The relative configuration of 3, rel-(1S,9R,10S,11R,12S,13S), was supported on the basis of NOESY and its structural similarity with know endiandric acid analogues. Based on further spectrographic evidence, the structure of 3 was elucidated and designated as tricyclotsangibeilin.
ESIMS and HRESIMS data of compound 4 indicated the molecular formula C22H22O4, four methylenes less than endiandric acid L  with 12 degrees of unsaturation, which was the same as endiandric acid L. The UV, IR, 1H NMR (Table 2) and 13C NMR (Table 2) spectra were similar to those of endiandric acid L, except that a methylene group [δ 2.78 (1H, dd, J = 15.6, 7.8 Hz, Ha-1′), 2.81 (1H, br d, J = 7.8 Hz, Hb-1′)] in 4 replaced the pentamethylene group in endiandric acid L. The relative configuration of 4 was supported by NOESY spectrum and its structural similarity with endiandric acid L . Thus, compound 4 was named endiandric acid M. Compounds 1, 2 and 4 were racemic, and all structures were determined by 13C NMR, NOESY, COSY, HSQC and HMBC experiments (see Figures 2 and 3).
Compound 5 was obtained as an optically active light yellowish oil, [α]25D –18.6 (c 0.18, MeOH), and its molecular formula was established as C23H30O7 by ESIMS and HRESIMS. UV absorptions at 209, 240 sh, and 270 nm, in conjunction with a bathochromic shift after the addition of alkali, indicated a phenolic benzenoid moiety. An IR absorption band at 3415 cm−1 (OH) was also observed. The 1H NMR signals for a 7,7′-epoxylignan moiety [δ 1.07 (3H, d, J = 6.0 Hz, H-9′), 1.08 (3H, d, J = 6.0 Hz, H-9), 1.78 (1H, m, H-8), 1.78 (1H, m, H-8′), 4.61 (1H, d, J = 8.8 Hz, H-7) and 4.63 (1H, d, J = 9.2 Hz, H-7′)] were similar to those of beilschminol A , except that OCH3-3′ [δ 3.88 (3H, s)] and OCH3-4′ [δ 3.83 (3H, s)] in 5 replaced a methylenedioxy group [δ 5.96 (2H, each d, J = 1.4 Hz)] in beilschminol A. Thus, the planar structure of 5 was proposed to be 3-hydroxy-4,5,3′,4′,5′-pentamethoxy-7,7′-epoxylignan. The relative configuration of 5 was based on the NOESY spectrum and its structural similarity with (7R,8R,7′R,8′R)-3-hydroxy-3′,4′-methylenedioxy-4,5,5′-trimethoxy-7,7′-epoxylign . Thus, compound 5 was elucidated as rel-(7R,8R,7′R,8′R)-3-hydroxy-4,5,3′,4′,5′-pentamethoxy-7,7′-epoxylignan and named beilschminol B.
Compound 6 was isolated as an optically active light yellowish oil, [α]25D + 123.8 (c 0.06, CHCl3). The HRESIMS analysis of 6 gave a quasi-molecular ion peak at m/z 257.1516 [M+Na]+ (calcd. for C15H22O2Na, 257.1517), consistent with a molecular formula of C15H22O2 with five degrees of unsaturation. The IR, 1H, and 13C NMR (Table 3) spectra resembled those of (+)-barbatenal , except that a hydroxy group and an oxymethine proton [δ 4.09 (1H, d, J = 3.6, H-5)] in C-5 (δc 71.2) of 6 replaced a methylene [δ 1.65 (1H, dd, J = 21.1, 3.3 Hz, Ha-5), 2.04 (1H, dd, J = 20.1, 2.8 Hz, Hb-5)] in C-5 (δc 41.9) of (+)-barbatenal, resulting in the downshifting of the olefinic proton (δH 6.60, H-4) in 6 from δH 5.87 (H-4) in (+)-barbatenal. The relative configuration of 6 was determined by NOESY spectrum, where Ha-1 showed correlations with H-2, H-12, H-13 and H-14, but no correlation with H-5. It has been suggested that Ha-1, H-2, H-12, H-13 and H-14 are on the same side of the molecule and that Hb-1 and H-5 are on the opposite side of the molecule. Furthermore, H-5 showed a correlation with H-14 due to a spatial distance between these two protons of <4 Å, as supported by simulating the ChemDraw 3D Ultra (version 10.0). Based on the NOESY spectrum, the optical rotation and comparison to values in the literature, the relative configuration of 6 was determined to be rel-(2β, 5α, 12β, 13β, 14β). Hence, the structure of 6 was elucidated and designated as (+)-5-hydroxybarbatenal, which was further confirmed by DEPT, HSQC, NOESY (Figure 3) and HMBC (Figure 2) techniques.
Compound 7 was isolated as an optically active light yellowish oil with [α]25D –23.9 (c 0.032, CHCl3). IR absorption bands at 3396 cm−1 (OH) and 1637 cm−1 (C=C) were observed. From the HRESIMS data, the molecular formula was determined to be C15H26O2 (m/z 261.1828 [M+Na]+; calcd. 261.1830). The 13C NMR (Table 3) and DEPT spectra indicated that 7 contains three methyls, six methylenes, three methines and three quaternary carbons, suggesting a sesquiterpene skeleton. The HSQC and COSY (Figure 2) spectra revealed the correlations from C1-C9, C2-C3 and C-5-C6-C7 and the HMBC (Figure 2) correlations, H-1 to C-3, C-8 and C-11, H-10 to C-8, C-9 and C-11, H-9 to C-1 and C-8, H-7 to C-5, C-6, C-8 and C-9, H-6 to C-4, C-5 and C-8, H-3 to C-4 and C-5 and H-2 to C-1, C-3, C-4 and C-9, connected the fragments C1-C9, C2-C3 and C-5-C6-C7 to form a cyclononane ring and a cyclobutane ring. HMBC correlations, H-15 to C-1, C-10 and C-11, H-14 to C-10, C-11 and C-15, H-12 to C-3, C-4 and C-5, H-13 to C-7, C-8 and C-9, confirmed that three methyl groups were connected at C-4 and C-11 and that a terminal double bond was present at C-8. Finally, the HRESIMS, IR, DEPT spectra indicated two hydroxy groups located at C-4 and C-5, respectively. The NOESY spectrum (Figure 3) showed correlations between Hb-7, H-9, H-12 and H-14, suggesting these three protons were on the same side of the molecule, but H-1, H-5, Ha-7 and H-15 showed no correlations with Hb-7, H-9, H-12 and H-14 and, therefore, should be on the opposite side of the molecule. The structure of 7 was elucidated as (4R, 5R)-4,5-dihydroxycaryophyll-8(13)-ene and identified with its synthetic compound . Though compound 7 has previously been reported as a reduction intermediate , our study is the first time to have isolated this compound from a natural source.
The known compounds, 3,4,5-trimethoxybenzaldehyde (8) , octahydro-4-hydroxy-3α-methyl-7-methylene-α-(1-methylethyl)-1H-indene-1-methanol (9) [13,14], eudesm-4(15)-ene-1β,6α-diol (10) [15,16] and ursolic acid (11)  were identified by comparison of their physical and spectroscopic data with values reported in the literature.
2.2. Anti-inflammatory Activities
Emax (%) and IC50 (μM) values of iNOS inhibitory activity of five compounds, including compounds 4, 5 and 11, and the two compounds, 6β-hydroxystigmast-4-en-3-one and rel-(7S,8S,7′R,8′R)-3,3′,4,4′,5,5′-hexmethoxylignan, reported by Huang et al., 2011 , were obtained at the concentration range of 0.1 to 100 μM. Results are shown in Table 4. The technique for the anti-iNOS activity assay was the same as in our previous study . Among the isolates, endiandric acid M (4) exhibited moderate iNOS inhibitory activity, with IC50 value of 31.70 μM. Endiandramide B , endiandric acids K , L  and M, with the same skeleton but with different substituents at C-8, exhibited ascending degrees of iNOS inhibitory activity in this order: endiandramide B (16.40 μM) > endiandric acid M (4) (31.70 μM) > endiandric acid L (39.56 μM) > endiandric acid K (58.21 μM). This suggests that the potency of the substituent at C-8 was an N-isobutylamido group > an α,β-unsaturated carboxylic acid group > a carboxylic acid group. Endiandric acid M (4), with four fewer methylenes than endiandric acid L, showed stronger potency than endiandric acid L. This suggests that fewer methylenes in the alkyl side of endiandric acid analogues result in greater potency of iNOS inhibitory activity.
3. Experimental Section
3.1. General Experimental Procedures
All melting points were measured on a Yanaco micro-melting apparatus and were uncorrected. Optical rotations were measured on a Jasco P-1020 digital polarimeter. The UV spectra were obtained with a Jasco V-530 UV/VIS spectrophotometer, and IR spectra (KBr or neat) were taken on a Perkin-Elmer System 2000 FT-IR spectrometer. 1D (1H, 13C, DEPT) and 2D (COSY, NOESY, HSQC, HMBC) NMR spectra using CDCl3 or acetone-d6 or CD3OD as solvent were recorded on Varian Germini 2000-200 (200 MHz for 1H NMR, 50 MHz for 13C NMR), Varian Unity Plus 400 (400 MHz for 1H NMR, 100 MHz for 13C NMR) and Varian VNMRS-600 (600 MHz for 1H NMR, 150 MHz for 13C NMR) spectrometers. Chemical shifts were internally referenced to the solvent signals in CDCl3 (1H, δ 7.26; 13C, δ 77.0) or acetone-d6 (1H, δ 2.05; 13C, δ 205.1) or CD3OD (1H, δ 3.31; 13C, δ 49.0), with TMS as the internal standard. ESIMS were obtained on an API 3000 mass spectrometer (Applied Biosystems) and HRESIMS on a Bruker Daltonics APEX II 30e mass spectrometer. Silica gels (70–230, 230–400 mesh) (Merck) were used for column chromatography (CC), and silica gel 60 F-254 (Merck) was used for analytical and preparative TLC. A spherical C18 100A column (20–40 μM) (Silicycle) was used for medium-pressure liquid chromatography.
3.2. Plant Material
Roots of B. tsangii were collected at Mudan, Pingtung County, Taiwan, in April 2009 and identified by one of the authors (I.-S.C.). A voucher specimen (Chen 6120) was deposited in the Herbarium of the School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China.
3.3. Extraction and Isolation
The dried roots (7.9 kg) of B. tsangii were sliced and extracted three times with cold MeOH (10 L) at room temperature. The concentrated MeOH extract (280 g) inhibited nitrite production below 25%, with no observed cytotoxicity at 100 μg/mL. The MeOH extract was partitioned using EtOAc-H2O (1:1) to obtain EtOAc-soluble (120 g) and H2O-soluble (80 g) fractions. The EtOAc solubles (100 g) were applied to a silica gel CC and eluted with n-hexane-EtOAc gradient solvent system (15:1 to 100% EtOAc) to obtain 13 fractions (A-1–A-13). Fractions A-4–A-13 showed inhibition of nitrite production using the anti-iNOS assay, and the H2O-soluble fraction showed no inhibitory activity. CC of fraction A-8 (9.6 g) on silica gel, eluting with a gradient of CH2Cl2–EtOAc, yielded fractions A-8-1 to A-8-10. Fraction A-8-5 (416 mg) was subjected to MPLC (RP-18), eluting with acetone:H2O (3:2), to obtain fractions A-8-5-1 to A-8-5-14. Fraction 8-5-6 (11.7 mg) was applied to preparative TLC (n-hexane:acetone, 3:1) to obtain 6 (2.8 mg). Fraction A-8-5-14 (210 mg) was applied to MPLC (RP-18) eluting with acetone:H2O (2:1) to give 20 fractions (A-8-5-14-1–A-8-5-14-20). Fraction A-8-5-14-10 (20.8 mg) was separated over MPLC eluting with n-hexane:acetone (8:1) to afford 3 (1.2 mg). Fraction A-8-7 (1.97 g) was submitted to MPLC (RP-18), eluting with MeOH:H2O (10:1), to obtain fractions A-8-7-1 to A-8-7-14. Fraction A-8-7-6 (223 mg) was applied to MPLC and eluted with n-hexane: acetone (4:1) to obtain nine fractions (A-8-7-6-1–A-8-7-6-9). Fraction A-8-7-6-7 (5.5 mg) was purified by preparative TLC (RP-18) developed with MeOH:H2O (15:1) to give 4 (4.4 mg). Fraction A-8-7-11 was subjected to MPLC and eluted with n-hexane: acetone (5:1) to yield 11 (5.7 mg). Fraction A-9 (17.8 g), on silica gel CC, eluting with a gradient of n-hexane–acetone, provided fractions A-9-1 to A-9-16. Fraction A-9-4 (104 mg) was applied to MPLC (RP-18) and eluted with acetone:H2O (1:1) to provide 7 (1.6 mg) and 15 fractions (A-9-4-1–A-9-4-15). Fraction A-9-4-12 (3.6 mg) was purified by preparative TLC (RP-18) and developed with n-hexane:EtOAc (2:1) to give 10 (3.2 mg). Fraction A-9-4-4 was treated with the same step as fraction A-9-4-12 to yield 9 (1.1 mg). Fraction A-9-9 (587 mg) was separated over MPLC (RP-18), eluting with a system of acetone:H2O (1:1), to afford fractions A-9-9-1 to A-9-9-21. Fraction A-9-9-2 (5 mg) and fraction A-9-9-10 (13.3 mg) were applied by preparative TLC developed with CH2Cl2:MeOH (70:1) and CH2Cl2:EtOAc (8:1) to give 8 (2.5 mg) and 5 (8.9 mg), respectively. Fraction A-9-9-20 (462 mg) was submitted to MPLC (RP-18) and eluted with acetone:H2O (3:1) to afford fractions A-9-9-20-1 to A-9-9-20-15. Fraction A-9-9-20-6 (63.8 mg) was subjected to MPLC (RP-18) and eluted with acetone:H2O (2:1) to afford fractions A-9-9-20-6-1 to A-9-9-20-6-12. Fraction A-9-9-20-6-3 (3.8 mg) and fraction A-9-9-20-6-7 (13.3 mg) were purified by preparative TLC (RP-18), developed with MeOH:H2O (5:1) and acetone: H2O (2:1), to give 1 (3.7 mg) and 2 (3.5 mg), respectively.
Tsangibeilin C (1): Light yellowish oil; [α]23D ±0 (c 0.07, CHCl3); IR (neat) νmax cm−1: 3432 (hydroxy group of carboxylic acid), 1696 (C=O); for 1H and 13C NMR spectroscopic data, see Table 1; ESIMS: m/z 337 [M + Na]+; HRESIMS: m/z 337.1782 [M + Na]+ (calcd. for C20H26O3Na, 337.1780).
Tsangibeilin D (2): Light yellowish oil; [α]24D ±0 (c 0.07, CHCl3); IR (neat) νmax cm−1: 3397 (OH), 1692 (C=O); for 1H and 13C NMR spectroscopic data, see Table 1; ESIMS: m/z 325 [M + Na]+; HREIMS: m/z 302.1879 [M]+ (calcd. for C19H26O3, 302.1882).
Tricyclotsangibeilin (3): Light yellowish oil; [α]24D + 36.3 (c 0.024, CHCl3); IR (neat) νmax cm−1: 3422 (OH), 1729 (C=O); for 1H and 13C NMR spectroscopic data, see Table 2; ESIMS: m/z 341 [M + Na]+; HRESIMS: m/z 341.2096 [M + Na]+ (calcd. for C20H30O3Na, 341.2093).
Endiandric acid M (4): Light yellowish oil; [α]25D ±0 (c 0.09, CHCl3); IR (neat) νmax cm−1: 3461 (hydroxy group of carboxylic acid), 1694 (C=O), 1040, 937 (OCH2O); for 1H and 13C NMR spectroscopic data, see Table 2; ESIMS: m/z 373 [M + Na]+; HRESIMS: m/z 373.1418 [M + Na]+ (calcd. for C22H22O4Na, 373.1416).
Beilschminol B (5): Light yellowish oil; [α]25D –18.6 (c 0.18, MeOH); UV (MeOH) λmax (log ɛ): 209 (4.63), 240 sh (3.92), 270 (3.12) nm; UV (MeOH+KOH) λmax (log ɛ): 210 (4.71), 242 sh (3.97), 280 (3.25) nm; IR (neat) νmax cm−1: 3415 (OH), 1593, 1508, 1461 (benzene ring); 1H NMR (400 MHz, CDCl3) δ 1.07 (3H, d, J = 6.0 Hz, H-9′), 1.08 (3H, d, J = 6.0 Hz, H-9), 1.78 (2H, m, H-8, H-8′), 3.83 (3H, s, OCH3-4′), 3.88 (12H, s, OCH3-3′, OCH3-4, OCH3-5, OCH3-5′), 4.61 (1H, d, J = 8.8 Hz, H-7), 4.63 (1H, d, J = 9.2 Hz, H-7′), 5.78 (1H, br s, D2O exchangeable, OH-3), 6.55 (1H, d, J = 1.8 Hz, H-6), 6.61 (2H, s, H-2′, H-6′), 6.64 (1H, d, J = 1.8 Hz, H-2); 13C NMR (100 MHz, CDCl3) δ 13.9 (C-9′), 14.0 (C-9), 50.9 (C-8′), 51.0 (C-8), 55.9 (OCH3-5), 56.1 (OCH3-3′, OCH3-5′), 60.8 (OCH3-4′), 60.9 (OCH3-4), 88.2 (C-7), 88.5 (C-7′), 101.6 (C-6), 102.9 (C-2′, C-6′), 105.8 (C-2), 134.7 (C-4), 137.2 (C-4′), 138.0 (C-1′), 138.7 (C-1), 149.1 (C-3), 152.4 (C-5), 153.2 (C-3′, C-5′); ESIMS: m/z 441 [M + Na]+; HRESIMS: m/z 441.1887 [M + Na]+ (calcd. for C23H30O7Na, 441.1889).
(+)-5-Hydroxybarbatenal (6): Light yellowish oil; [α]25D +123.8 (c 0.06, CHCl3); IR (neat) νmax cm−1: 3408 (OH), 1681 (C=O); for 1H and 13C NMR spectroscopic data, see Table 3; ESIMS: m/z 257 [M + Na]+; HRESIMS: m/z 257.1516 [M + Na]+ (calcd. for C15H22O2Na, 257.1517).
(4R,5R)-4,5-Dihydroxycaryophyll-8(13)-ene (7): Light yellowish oil; [α]25D –23.9 (c 0.032, CHCl3); IR (neat) νmax cm−1: 3396 (OH), 1637 (C=C); for 1H and 13C NMR spectroscopic data, see Table 3; ESIMS: m/z 261 [M + Na]+; HRESIMS: m/z 261.1828 [M + Na]+ (calcd. for C15H26O2Na, 261.1830).
There are about 200 Beilschmiedia species in the tropical regions mentioned earlier, including two species in Taiwan . Except for the two species in Taiwan, previous studies of nine Beilschmiedia species in the world have revealed endiandric acids [5,18,19], flavonoids [20,21], alkaloids [22–24], benzopyrans , arylpropanoids  and sesquiterpenoids . Several isolates have shown antibacterial [19,21] and antimalarial  activities. Phytochemical studies of the two Formosan Beilschmiedia species have been carried out by our group, analyzing the stems , the leaves  and the roots  of B. tsangii, together with the roots [8,9] of B. erythrophloia. Both species possess endiandric acids, 7,7′-epoxylignans, 7′,8′-seco-7,7′-epoxylignans, benzopyrans, arylpropanoids, amides, sesquiterpenoids and steroids. In this study, endiandric acids, 7,7′-epoxylignans, benzenoid, sesquiterpenoids and triterpenoid were also reported. The constituents of the roots of the two Formosan Beilschmiedia species showed 19 new endiandric acid analogues as key secondary metabolites. The roots of other Beilschmiedia species found in other regions have not yet been studied. However, several isolates from two Formosan Beilschmiedia species have shown cytotoxic , anti-tubercular [3,9] or anti-inflammatory activities  in previous studies. These three bioactivities have not yet been examined in other members of the Beilschmiedia genus.
This research was kindly supported by the National Science Council of the Republic of China (NSC 99-2320-B-037-009) and the Kaohsiung Medical University Research Foundation (KMUER013).
- Conflict of InterestThe authors declare no conflict of interest.
- Liao, J.C. Lauraceae. In Flora of Taiwan, 2nd ed; Editorial Committee of the Flora of Taiwan: Taipei, Taiwan1996; Volume 2, pp. 433–499.
- Chen, J.J.; Chou, E.T.; Duh, C.Y.; Yang, S.Z.; Chen, I.S. New cytotoxic tetrahydrofuran- and dihydrofuran-type lignans from the stem of Beilschmiedia tsangii. Planta Med 2006, 72, 351–357. [Google Scholar]
- Chen, J.J.; Chou, E.T.; Peng, C.F.; Chen, I.S.; Yang, S.Z.; Huang, H.Y. Novel epoxyfuranoid lignans and antitubercular constituents from the leaves of Beilschmiedia tsangii. Planta Med 2007, 73, 567–571. [Google Scholar]
- Huang, Y.T.; Chang, H.S.; Wang, G.J.; Chen, C.H.; Lin, C.H.; Yang, Y.Z.; Chen, I.S. Anti-inflammatory endiandric acid analogues from the roots of Beilschmiedia tsangii. J. Nat. Prod 2011, 74, 1875–1880. [Google Scholar]
- Chouna, J.R.; Nkeng-Efouet, P.A.; Lenta, B.N.; Wansi, J.D.; Kimbu, S.; Sewald, N. Endiandric acid derivatives from the stem bark of Beilschmiedia anacardioides. Phytochem. Lett 2010, 3, 13–16. [Google Scholar]
- Bandaranayake, W.M.; Banfield, J.E.; Black, D.S.C.; Fallon, G.D.; Gatehouse, B.M. Constituents of Endiandra species. I. endiandric acid, a novel carboxylic acid from Endiandra introrsa (Lauraceae), and a derived lactone. Aust. J. Chem 1981, 34, 1655–1667. [Google Scholar]
- Bandaranayake, W.M.; Banfield, J.E.; Black, D.S.C.; Fallon, G.D.; Gatehouse, B.M. Constituents of Endiandra species. III. 4-[(E,E)-5′-phenylpenta-2′,4′-dien-1′-yl]tetracyclo[5,4,0,02,5,03,9]undec-10-ene-8-carboxylic acid from Endiandra introrsa (Lauraceae). Aust. J. Chem 1982, 35, 567–579. [Google Scholar]
- Yang, P.S.; Cheng, M.J.; Chen, J.J.; Chen, I.S. Two new endiandric acid analogs, a new benzopyran, and a new benzenoid from the root of Beilschmiedia erythrophloia. Helv. Chim. Acta 2008, 91, 2130–2138. [Google Scholar]
- Yang, P.S.; Cheng, M.J.; Peng, C.F.; Chen, J.J.; Chen, I.S. Endiandric acid analogues from the roots of Beilschmiedia erythrophloia. J. Nat. Prod 2009, 72, 53–58. [Google Scholar]
- Adio, A.M.; Konig, W.A. Sesquiterpenoids and norsesquiterpenoids from three liverworts. Tetrahedron 2007, 18, 1693–1700. [Google Scholar]
- Collado, I.G.; Hanson, J.R.; Hitchcock, P.B.; Macias-Sanchez, A.J. Stereochemistry of epoxidation of some caryophyllenols. J. Org. Chem 1997, 62, 1965–1969. [Google Scholar]
- Pouchert, C.J.; Behnke, J. The Aldrich Library of13C and1H FT NMR Spectra, 1st ed; Aldrich Chemical Co., Inc.: Wisconsin, WI, USA, 1993; Volume 2, p. 964. [Google Scholar]
- Iijima, T.; Yaoita, Y.; Kikuchi, M. Five new sesquiterpenoids and a new diterpenoid from Erigeron annuus (L.) Pers., Erigeron philadelphicus L. and Erigeron sumatrensis Retz. Chem. Pharm. Bull 2003, 51, 545–549. [Google Scholar]
- Zhang, H.J.; Tan, G.T.; Santarsiero, B.D.; Mesecar, A.D.; Van Hung, N.; Cuong, N.M.; Soejarto, D.D.; Pezzuto, J.M.; Fong, H.H.S. New sesquiterpenes from Litsea verticillata. J. Nat. Prod 2003, 66, 609–615. [Google Scholar]
- Kamel, A. 7-epi-Eudesmanes from Teucrium polium. J. Nat. Prod 1995, 58, 428–431. [Google Scholar]
- Bilia, A.R.; Mendez, J.; Morelli, I. Phytochemical investigations of Licania genus. Flavonoids and triterpenoids from Licania carii. Pharm. Acta Helv 1996, 71, 191–197. [Google Scholar]
- Min, B.S.; Kim, Y.H.; Lee, S.M.; Jung, H.J.; Lee, J.S.; Na, M.K.; Lee, C.O.; Lee, J.P.; Bae, K.H. Cytotoxic triterpenes from Crataegus pinnatifida. Arch. Pharm. Res 2000, 23, 155–158. [Google Scholar]
- Banfield, J.E.; Black, D.S.C.; Collins, D.J.; Hyland, B.P.M.; Lee, J.J.; Pranowo, S.R. Constituents of some species of Beilschmiedia and Endiandra (Lauraceae): New endiandric acid and benzopyran derivatives isolated from B.oligandra. Aust. J. Chem 1994, 47, 587–607. [Google Scholar]
- Chouna, J.R.; Nkeng-Efouet, P.A.; Lenta, B.N.; Devkota, K.P.; Neumann, B.; Stammler, H.G.; Kimbu, S.; Sewald, N. Antibacterial endiandric acid derivatives from Beilschmiedia anacardioides. Phytochemistry 2009, 70, 684–688. [Google Scholar]
- Harborne, J.B.; Mendez, J. Flavonoids of Beilschmiedia miersii. Phytochemistry 1969, 8, 763–764. [Google Scholar]
- Lenta, B.N.; Tantangmo, F.; Devkota, K.P.; Wansi, J.D.; Chouna, J.R.; Soh, R.C.F.; Neumann, B.; Stammler, H.G.; Tsamo, E.; Sewald, N. Bioactive constituents of the stem bark of Beilschmiedia zenkeri. J. Nat. Prod 2009, 72, 2130–2134. [Google Scholar]
- Kitagawa, I.; Minagawa, K.; Zhang, R.S.; Hori, K.; Doi, M.; Inoue, M.; Ishida, T.; Kimura, M.; Uji, T.; Shibuya, H. Dehatrine, an antimalarial bisbenzylisoquinoline alkaloid from the Indonesian medicinal plant Beilschmiedia madang, isolated as a mixture of two rotational isomers. Chem. Pharm. Bull 1993, 41, 997–999. [Google Scholar]
- Tillequin, F.; Koch, M.; Pusset, J.; Chauviere, G. Two new morphinane alkaloids from Beilschmiedia oreophila Schlechter (Lauraceae). Heterocycles 1985, 23, 1357–1361. [Google Scholar]
- Johns, S.R.; Lamberton, J.A.; Sioumis, A.A.; Tweeddale, H.J. New aporphine alkaloids from Beilschmiedia podagrica. Aust. J. Chem 1969, 22, 1277–1281. [Google Scholar]
- Kumamoto, J.; Scora, R.W. Structure of sarisan, an isomer of myristicin, isolated from the leaf oil of Beilschmiedia miersii. J. Agric. Food Chem 1970, 18, 544–545. [Google Scholar]
|δC||δH (J in Hz)||δC||δH (J in Hz)|
|1||41.6 (CH)||2.36, m||42.0 (CH)||2.41, br dd (12.0, 6.0)|
|2||31.5 (CH2)||1.56, td (12.0, 6.0)|
1.65, br dd (12.0, 5.4)
|31.0 (CH2)||1.57, td (12.0, 6.0)|
1.62, br dd (12.0, 5.4)
|3||50.5 (CH)||3.05, ddd (13.5, 12.0, 5.4)||54.3 (CH)||2.94, ddd (13.2, 12.0, 5.2)|
|4||201.6 (C)||-||203.7 (C)||-|
|5||135.8 (CH)||6.70, d (1.2)||132.3 (CH)||6.54, s|
|6||151.5 (C)||-||158.5 (C)||-|
|7||36.2 (CH)||3.51, br s||71.2 (C)||-|
|8||123.5 (CH)||5.56, ddd (10.2, 3.0, 1.8)||125.9 (CH)||6.01, br d (10.2)|
|9||131.5 (CH)||5.85, ddd (10.2, 4.2, 3.0)||132.6 (CH)||5.87, dd (10.2, 4.2)|
|10||35.8 (CH)||2.40, m||35.9 (CH)||2.47, m|
|11||48.0 (CH)||1.47, m||47.9 (CH)||1.45, m|
|12||35.2 (CH)||2.87, m||32.9 (CH)||2.97, br dd (8.4, 6.0)|
|13||44.8 (CH)||2.35, m||51.9 (CH)||2.24, ddd (13.2, 8.4, 1.2)|
|1′||38.4 (CH2)||1.54, m||38.2 (CH2)||1.54, m|
|2′||28.3 (CH2)||1.28, m||28.0 (CH2)||1.25, m|
|3′||31.0 (CH2)||1.28, m||30.4 (CH2)||1.28, m|
|4′||33.2 (CH2)||1.28, m||33.0 (CH2)||1.28, m|
|5′||23.9 (CH2)||1.28, m||23.7 (CH2)||1.30, m|
|6′||15.0 (CH3)||0.88, t (7.2)||14.4 (CH3)||0.90, t (6.9)|
|δC||δH (J in Hz)||δC||δH (J in Hz)|
|1||41.9 (CH)||2.37, m||43.7 (CH)||2.72, br dd (12.0, 5.4)|
|2||31.8 (CH2)||1.28, m|
1.99, ddd (14.4, 9.6, 4.8)
|41.3 (CH)||2.45, dt (9.0, 5.4)|
|3||132.3 (CH)||5.50, ddd (15.6, 9.6, 6.0)||41.1 (CH)||1.72, m|
|4||130.1 (CH)||5.24, ddd (15.6, 9.6, 5.4)||41.2 (CH)||2.25, br t (7.8)|
|5||31.2 (CH2)||2.38, m||41.0 (CH)||2.36, m|
|6||34.3 (CH2)||2.29, ddd (11.4, 5.4, 2.4)|
|39.9 (CH2)||1.63, br d (12.3)|
1.87, br dd (12.3, 5.4)
|7||173.5 (C)||-||43.4 (CH)||1.89, br dd (9.0, 5.4)|
|8||-||-||48.2 (CH)||2.91, br dd (8.2, 3.6)|
|9||70.1 (CH)||4.87, dt (5.6, 2.4)||38.8 (CH)||2.53, m|
|10||67.2 (CH)||3.96, br d (7.2)||134.2 (CH)||6.19, br t (7.2)|
|11||40.8 (CH)||2.21, m||131.9 (CH)||6.25, br t (7.2)|
|12||33.3 (CH)||3.18, br dt (15.6, 7.5)||-||-|
|13||30.9 (CH)||3.02, m||-||-|
|14||136.0 (CH)||6.16, dd (10.4, 6.0)||-||-|
|15||123.9 (CH)||6.25, dd (10.4, 5.6)||-||-|
|1′||36.3 (CH2)||1.47, q (7.5)||43.1 (CH2)||2.78, dd (15.6, 7.8)|
2.81, br d (7.8)
|2′||27.7 (CH2)||1.27–1.33, m||136.5 (C)||-|
|3′||29.5 (CH2)||1.27–1.33, m||110.5 (CH)||6.75, d (1.8)|
|4′||31.9 (CH2)||2.24, m||149.2 (C)||-|
|5′||22.7 (CH2)||1.27–1.33, m||147.3 (C)||-|
|6′||14.1 (CH3)||0.90, t (6.9)||109.3 (CH)||6.74, d (7.8)|
|7′||-||-||123.1 (CH)||6.68, dd (7.8, 1.8)|
|1″||-||-||155.3 (CH)||6.72, dd (15.6, 8.2)|
|2″||-||-||121.3 (CH)||5.71, dd (15.6, 0.9)|
|OCH2O||-||-||102.3 (CH2)||5.94, s|
|δC||δH (J in Hz)||δC||δH (J in Hz)|
|1||36.9 (CH2)||1.37, br d (12.0)|
1.69, dd (12.0, 4.2)
|56.9 (CH)||1.65, ddd (10.2, 3.6, 1.8)|
|2||42.8 (CH)||2.64, d (4.2)||23.2 (CH2)||1.33, m|
|3||147.7 (C)||-||40.8 (CH2)||1.56, ddd (15.0, 8.4, 1.8)|
1.93, ddd (15.0, 11.1, 2.1)
|4||147.5 (CH)||6.60, dd (3.6, 0.6)||75.1 (C)||-|
|5||71.2 (CH)||4.09, d (3.6)||73.3 (CH)||3.60, br dd (6.0, 2.4)|
|6||48.7 (C)||-||32.6 (CH2)||1.57, m|
|7||56.9 (C)||-||34.7 (CH2)||2.05, m|
2.43, ddd (13.2, 9.0, 4.2)
|8||36.2 (CH2)||1.10, m|
|9||38.6 (CH2)||1.25, m||42.3 (CH)||2.37, q (10.2)|
|10||27.1 (CH2)||1.32, br dd (6.9, 0.9)|
|36.0 (CH2)||1.58, m|
1.74, t (10.2)
|11||54.9 (C)||-||34.1 (C)||-|
|12||26.9 (CH3)||1.089, s||21.5 (CH3)||1.14, s|
|13||24.2 (CH3)||0.96, s||110.5 (CH2)||4.92, d (1.2)|
4.94, d (1.2)
|14||18.7 (CH3)||1.087, s||22.1 (CH3)||0.98, s|
|15||193.3 (CH)||9.54, s||30.1 (CH3)||1.00, s|
|OH||-||-||-||2.07, br s|
|OH||-||-||-||2.29, br d (3.0)|
|Compounds||Emax (%) a||IC50 (μM) b|
|endiandric acid M (4)||97.03 ± 1.30||31.70 ± 0.25|
|beilschminol B (5)||55.62 ± 1.96||95.37 ± 0.52|
|ursolic acid (11)||36.55 ± 4.66||>100|
|6β-hydroxystigmast-4-en-3-one c||11.69 ± 2.91||>100|
|rel-(7S,8S,7′R,8′R)-3,4,5,3′,4′,5′-hexmethoxylignanc||52.67 ± 5.05||98.26 ± 0.13|
|aminoguanidine d (a selective iNOS inhibitor)||80.35 ± 0.26||26.55 ± 0.48|
|Nω-nitro-L-arginine d (a nonselective iNOS inhibitor)||43.72 ± 0.76||152.46 ± 10.53|
aEmax indicates mean maximum inhibitory effect, at a concentration of 100 μM, expressed as a percentage inhibition of nitrite production induced by LPS (200 ng/mL) in the presence of vehicle;bIC50 means the concentration producing 50% Emax. (n = 4–6 in each group);cThis compound was reported in Huang et al., 2011 ;dpositive control.
© 2012 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).