Lignans from the Roots of Taxus wallichiana and Their Î±-Glucosidase

Article pubs.acs.org/jnp

Lignans from the Roots of Taxus wallichiana and Their α‑Glucosidase Inhibitory Activities Phu H. Dang,† Hai X. Nguyen,† Hanh H. T. Nguyen,† Thai D. Vo,† Tho H. Le,† Trong H. N. Phan,† Mai T. T. Nguyen,†,‡ and Nhan T. Nguyen*,†,‡ †

Faculty of Chemistry, VNUHCM−University of Science, 227 Nguyen Van Cu Street, District 5, Ho Chi Minh City, Vietnam Cancer Research Laboratory, Vietnam National University, Ho Chi Minh City, 227 Nguyen Van Cu Street, District 5, Ho Chi Minh City, Vietnam

‡

S Supporting Information *

ABSTRACT: From an EtOAc-soluble extract of the roots of Taxus wallichiana, six new (1−6) and 11 known lignans were isolated. The structures of the new compounds were elucidated based on interpretation of spectroscopic data. (+)-7′-epiTsugacetal (1) is a rare aryltetralin-type lignan having a cis-orientation of H-7′ and H-8′. Compounds 3−6 were identified as the first naturally occurring tetrahydrofuranoid lignans having a cis-orientation of H-7 and H-8. All tested compounds were found to possess α-glucosidase inhibitory activity, with formosanol (9) showing the most potent effect with an IC50 value of 35.3 μM.

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RESULTS AND DISCUSSION The powdered roots of T. wallichiana were refluxed in MeOH. The MeOH-soluble extract was suspended in H2O and successively partitioned with EtOAc to yield an EtOAc fraction. Further separation and purification of the EtOAc-soluble fraction led to the isolation of 17 compounds including six new lignans (1−6). The known lignans were identified as (−)-7′-O-methyltanegool (7),13 α-conidendrin (8),4 formosanol (9),14 (+)-tsugacetal (10),14 α-intermedianol (11),14 oxabicyclooctalignan (12),15 lanceolatanin C (13),16 lanceolatanin D (14),16 matairesinol (15),14 7-methoxymatairesinol (16),17 and oxomatairesinol (17).17 Compound 1 was isolated as a white, amorphous solid and showed a sodiated molecular HRESIMS ion at m/z 395.1475 [M + Na]+ (calcd for C21H24O6Na, 395.1471). Its IR spectrum showed the presence of hydroxy (3400 cm−1) and aromatic (1450 cm−1) groups. The 1H NMR spectrum displayed signals for a 1,3,4-trisubstituted [δH 6.70 (d, J = 8.1 Hz), 6.58 (d, J = 1.9 Hz), and 6.32 (dd, J = 8.1, 1.9 Hz)] and a 1,2,4,5tetrasubstituted [δH 8.21 and 7.93 (each 1H, s)] aromatic moiety, three methoxy singlets (δH 3.83, 3.75, and 3.28), an

Taxus wallichiana Zucc. grows in many countries in Asia including Afghanistan, Pakistan, India, Nepal, Bhutan, the People’s Republic of China, Indonesia, Malaysia, Myanmar, Vietnam, and the Philippines.1 Its wood powder is used for the traditional remedy of diabetes in Vietnam.2 A previous study on the chemical constituents of T. wallichiana, collected at Lam Dong Province in Vietnam, led to the isolation mainly of taxoids and the determination of their α-glucosidase inhibitory activity.3 Lignans were isolated from T. wallichiana for the first time in 1982.4 These lignans were identified as α-conidendrin, β-conidendrin, hydroxymatairesinol, isoliovil, texiresinol, isotexiresinol, and (−)-secoisolariciresinol.4−6 Moreover, a lignan supplement has been shown to enhance glycemic control in animals.7 In a continued study on the screening of medicinal plants for α-glucosidase inhibitory activity,8−12 it was found that an EtOAc-soluble extract of the roots of T. wallichiana showed potent α-glucosidase inhibitory activity, with an IC50 value of 85.0 μg mL−1. Thus, a further fractionation study was carried out on its chemical constituents, leading to the isolation of 17 lignans including the six new analogues 1−6. In this work, the isolation and structural elucidation of these compounds by spectroscopic methods are described as well as their αglucosidase inhibitory activity evaluation. © 2017 American Chemical Society and American Society of Pharmacognosy

Received: February 27, 2017 Published: June 5, 2017 1876

DOI: 10.1021/acs.jnatprod.7b00171 J. Nat. Prod. 2017, 80, 1876−1882

Journal of Natural Products

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Chart 1

Table 1. NMR Spectroscopic Data for Compounds 1 and 2 1a position

a

δC, type

1 2 3 4 5 6 7 8 9

128.5, 132.3, 117.7, 146.0, 147.8, 112.6, 29.0, 40.1, 104.9,

C C CH C C CH CH2 CH CH

1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′

134.8, 115.0, 147.3, 145.7, 115.3, 123.7, 45.8, 42.9, 69.9,

C CH C C CH CH CH CH CH2

OMe-3 OMe-5 OMe-9 OMe-3′ OH-4 OH-4′

2b

56.4, CH3 54.7, CH3 56.2, CH3

δH (J in Hz)

δC, type

6.41, s

6.77, 2.85, 2.16, 4.77,

s m dddd (13.1, 11.1, 6.2, 4.7) d (4.7)

6.58, d (1.9)

6.70, 6.32, 4.26, 2.57, 4.00, 3.16,

d (8.1) dd (8.1, 1.9) d (6.0) dddd (13.1, 10.4, 8.0, 6.0) dd (8.0, 8.0) dd (10.4, 8.0)

3.75, 3.28, 3.83, 7.18, 7.36,

s s s s s

142.9, 107.2, 151.4, 145.0, 113.5, 126.2, 46.7, 43.6, 69.8,

C CH C C CH C CH CH CH2

125.4, 114.8, 145.7, 146.0, 111.2, 131.5, 77.3, 52.1, 194.0,

C CH C C CH C CH CH C

δH (J in Hz) 6.77, s

7.35, s 3.82, 3.35, 4.18, 3.87,

dd (3.6, 1.1) dddd (5.9, 5.5, 3.6, 1.0) dd (9.0, 5.9) dd (9.0, 1.0)

6.62, s

6.59, s 5.02, d (5.5) 3.28, ddd (5.5, 5.5, 1.1)

56.4, CH3

4.00, s

56.1, CH3

3.79, s 5.58, s 5.56, s

δ values in acetone-d6. bδ values in CDCl3.

oxymethylene [δH 4.00 (dd, J = 8.0, 8.0 Hz) and 3.16 (dd, J = 10.4, 8.0 Hz)], an acetal proton [δH 4.77 (d, J = 4.7 Hz)], a bisbenzylic methine proton doublet [δH 4.26 (d, J = 6.0 Hz)], a benzylic methylene group [δH 2.85 (m)], and two methine

protons [δH 2.57 (dddd, J = 13.1, 10.4, 8.0, 6.0 Hz) and 2.16 (dddd, J = 13.1, 11.1, 6.2, 4.7 Hz)] (Table 1). The 13C NMR spectrum showed the presence of resonances for 12 aromatic carbons (δC 112.6−147.8), two aromatics (δC 56.4, 56.2) and 1877



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Figure 1. Significant HMBC correlations (solid arrows) observed for 1−6.

Figure 2. Key NOESY correlations (blue solid arrows) observed for 1−6.

benzene units (δC 107.2−151.4), an oxymethine (δC 77.3), an oxymethylene (δC 69.8), three methines (δC 52.1, 46.7, 43.6), and two methoxy groups (δC 56.4, 56.1). The 1H and 13C NMR data of 2 closely resembled those of lanceolatanin C (13),16 except for the presence of a C-7′ benzylic oxymethine in 2 instead of a benzylic methylene group in 13. Moreover, the HMBC correlations between H-9/C-7′ and H-7′/C-9 suggested the presence of a C-9(7′)-oxido bridge in 2 (Figure 1). The positions of the methoxy groups were assigned as C-3 and C-3′ based on the observed HMBC correlations. The presence of a C-9′ carbonyl group was based on the HMBC correlations between H-5/C-9′, H-2/H-9′, and H-8′/C-9′. The relative configuration of 2 was suggested based on those of lirionol derivatives.19 The NOESY correlations between H-7/ H-2, H-7/H-5′, and H-7/H-8 together with the small Wcoupling between H-7 and H-8′ suggested their β-orientations (Figure 2). Moreover, H-7′ showed correlations with H-2′ and H-8′ in the NOESY spectrum, suggesting its equatorial βorientation. Compound 2 showed a positive specific rotation ([α]25 D +35.6); thus, the structure of 2 was assigned as (+)-9,7′oxidolanceolatanin C. A plausible biosynthesis pathway of 2 has been proposed from the concomitantly isolated oxabicyclooctalignan (12).16 Compound 3 was deduced to have the molecular formula C21H26O7 based on its HRESIMS ion at m/z 413.1574 [M + Na]+ (calcd for C21H26O7Na, 413.1576). Its IR spectrum showed the presence of hydroxy (3415 cm−1) and aromatic (1510 cm−1) groups. Its 1H NMR spectrum (Table 2) showed signals for two 1,3,4-trisubstituted aromatic rings [δH 6.71− 6.98], two benzylic oxymethine protons [δH 4.63 (d, J = 7.1 Hz) and 3.99 (d, J = 9.1 Hz)], two oxymethylene groups [δH 4.14 (dd, J = 8.9, 4.3 Hz), 3.88 (dd, J = 8.9, 7.2 Hz), and 3.28 (m), 3.16 (m)], two methine protons [δH 2.51 (dddd, J = 9.1, 7.2, 5.9, 4.3 Hz) and 1.81 (dddd, J = 7.1, 5.9, 5.9, 4.3 Hz)], and three methoxy singlets [δH 3.84, 3.82, and 3.12 (each 3H, s)]. The 13C NMR spectrum showed signals for 12 aromatic carbons (δC 110.9−148.6), two oxymethines (δC 86.7, 84.0), two oxymethylenes (δC 71.5, 62.1), two methines (δC 53.5, 49.8), and three methoxy groups (δC 56.4, 56.4, 56.3). These were characteristic of those reported for tetrahydrofuranoid-

an aliphatic (δC 54.7) methoxy group, an acetal carbon (δC 104.9), an oxymethylene (δC 69.9), three methines (δC 45.8, 42.9, 40.1), and a methylene (δC 29.0) carbon. Comparison of the 13C NMR data of 1 with those of (+)-tsugacetal (10)14 suggested that the differences between them involved the signals for the C-8 and C-8′ methine carbons (δC 40.1 and 42.9 in 1 and δC 46.0 and 50.9 in 10) (Figure 40S, Supporting Information). The observed HMBC correlations (Figure 1) permitted assignment of the 2,7′-cyclolignan structural feature. The locations of the three methoxy groups were suggested to be at C-3′, C-5, and C-9 from the HMBC spectrum. The HMBC correlations between H-9/C-9′ and H-9′/C-9 suggested the presence of a C-9(9′) tetrahydrofuran unit. The relative configuration of 1 was determined based on NOESY correlations (Figure 2). The NOESY correlations between H-7′/H-8′, H-8′/H-9′β, and H-8′/OMe-9 indicated their β-orientations with respect to the central six-membered ring. Moreover, the cis-orientation of H-7′ and H-8′ was supported by the large variations in ΔδC‑8 and ΔδC‑8′ values for compounds 1 and 10 (−5.9 and −8.0, respectively), which were in agreement with reported data.18 The C-9 acetal proton and the C-8 methine proton were α-oriented based on their NOESY correlation. Compound 1 showed a positive specific rotation ([α]25 D +48.5), which was similar to that of 10. Therefore, the structure of 1 was assigned as (+)-7′-epitsugacetal. Compound 1 represents a rare aryltetralin-type lignan having the cis-orientation of H-7′ and H-8′. Compound 2 was isolated as a white, amorphous solid. Its molecular formula was determined to be C20H18O6 by the HRESIMS ion at m/z 377.0992 [M + Na]+ (calcd for C20H18O6Na, 377.1001). The 1H NMR spectrum showed signals for two 1,2,4,5-tetrasubstituted benzene moieties [δH 7.35, 6.77, 6.62, 6.59 (each 1H, s)], a bisbenzylic methine proton [δH 3.82 (dd, J = 3.6, 1.1 Hz)], a benzylic oxymethine proton [δH 5.02 (d, J = 5.5 Hz)], an oxymethylene group [δH 4.18 (dd, J = 9.0, 5.9 Hz) and 3.87 (dd, J = 9.0, 1.0 Hz)], two methine protons [δH 3.35 (dddd, J = 5.9, 5.5, 3.6, 1.0 Hz) and 3.28 (ddd, J = 5.5, 5.5, 1.1 Hz)], and two methoxy groups [δH 4.00 and 3.79 (each 3H, s)] (Table 1). Its 13C NMR spectrum showed resonances for a carbonyl carbon (δC 194.0), two 1878


a

56.3, CH3

56.4, CH3 56.4, CH3

OMe-3 OAc-9

OMe-3′ OMe-7′ OH-4 OH-4′

1879

d (8.1) dd (8.1, 1.9) d (7.1) dddd (7.1, 5.9, 5.9, 4.3) m m

3.82, 3.12, 7.33, 7.45,

6.77, 6.71, 3.99, 2.51, 4.14, 3.88, 3.84,

s s s s

d (8.0) dd (8.0, 1.9) d (9.1) dddd (9.1, 7.2, 5.9, 4.3) dd (8.9, 7.2) dd (8.9, 4.3) s

6.85, d (1.9)

6.77, 6.84, 4.63, 1.81, 3.28, 3.16,

6.98, d (1.9)

δH (J in Hz)

δ values in acetone-d6. bδ values in CDCl3.


132.6, 111.7, 148.6, 147.3, 115.5, 121.7, 86.7, 49.8, 71.5,

1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′


δC, type

135.5, 110.9, 148.3, 146.7, 115.5, 119.8, 84.0, 53.5, 62.1,

1 2 3 4 5 6 7 8 9

position

3a



56.1, CH3 56.7, CH3

56.2, CH3

131.2, 109.6, 147.0, 145.7, 114.3, 121.3, 85.5, 49.9, 70.9,

133.7, 108.9, 146.8, 145.4, 114.3, 119.5, 84.0, 51.9, 63.0,

δC, type

Table 2. NMR Spectroscopic Data for Compounds 3−6

3.86, 3.20, 5.67, 5.62,

6.87, 6.74, 4.03, 2.54, 4.15, 4.02, 3.86,

δH (J in Hz)

d (8.0) m d (7.9) dddd (7.9, 6.2, 5.9, 5.8) dd (10.8, 5.9) dd (10.8, 5.8)

s s s s

d (8.1) dd (8.1, 1.9) d (8.2) dddd (8.2, 7.8, 6.2, 5.0) dd (9.2, 5.0) dd (9.2, 7.8) s

6.78, m

6.85, 6.78, 4.49, 1.95, 3.33, 3.29,

6.78, m

4b


56.1, CH3 56.1, CH3

56.1, CH3

131.5, 109.0, 147.2, 146.1, 114.5, 121.1, 86.6, 52.7, 70.2,


δC, type 132.7, 109.0, 146.9, 145.6, 114.4, 119.6, 84.7, 56.0, 63.7,

3.89, 3.18, 5.83, 5.83,

6.88, 6.80, 3.94, 2.59, 3.64, 3.57, 3.90,

δH (J in Hz)

m m d (9.2) dddd (9.2, 9.1, 7.4, 3.7) m m

s s brs brs

m dd (8.0, 1.9) d (10.0) dddd (10.0, 8.7, 8.4, 7.4) m dd (8.7, 8.7) s

6.83, m

6.88, 6.83, 4.37, 2.27, 3.73, 3.63,

6.88, m

5b

56.1, 20.8, 170.8, 56.1, 56.7,

131.7, 109.2, 147.1, 145.8, 114.3, 121.1, 85.7, 49.7, 70.9,

CH3 CH3 CO CH3 CH3



δC, type 133.3, 109.0, 146.7, 145.4, 114.4, 119.5, 84.2, 49.1, 64.0,

δH (J in Hz)

d (8.2) dd (8.2, 1.7) d (7.8) dddd (7.8, 6.2, 6.2, 4.9) dd (11.3, 6.2) dd (11.3, 4.9)

3.87, 3.18, 5.61, 5.59,

6.85, 6.70, 3.95, 2.41, 4.27, 3.97, 3.91, 1.87,

s s s s

d (8.0) dd (8.0, 1.9) d (9.0) dddd (9.0, 7.2, 6.2, 4.1) dd (9.3, 7.2) dd (9.3, 4.1) s s

6.75, d (1.9)

6.88, 6.82, 4.48, 2.02, 3.75, 3.70,

6.88, d (1.7)

6b

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Figure 3. Selected NOESY correlations (blue solid arrows) on the most stable conformers of 3−6 using PM3 calculations.

The structure of 5 was assigned as 7′S*-methoxy-7-epilariciresinol based on the NOESY correlations of H-7′/H-9. Both compounds 4 and 5 showed negative specific rotations ([α]25 D −67.3 and −21.9, respectively), which was similar to that of 3. These compounds represent the first naturally occurring tetrahydrofuranoid-type lignan having a cis-orientation of H-7 and H-8. Compound 6 showed a sodiated molecular ion at m/z 455.1690 [M + Na]+ (calcd for C23H28O8Na, 455.1682) in the HRESIMS. The 1H and 13C NMR spectra of 6 (Table 2) closely resembled those of 4, except for the presence of an Oacetyl group [δH 1.87 (s), δC 170.8, 20.8]. The HMBC correlations between the oxymethylene protons [δH 3.75 (dd, J = 11.3, 6.2 Hz) and 3.70 (dd, J = 11.3, 4.9 Hz)] and the acetoxy carbonyl carbons at δC 170.8 suggested that the location of acetylation is at C-9 (Figure 1). The NOESY spectrum of 6 suggested the same C-7, C-8, and C-8′ relative configurations as those of 4 (Figure 2). Compound 6 showed a negative specific rotation ([α]25 D −51.7); thus, the structure of 6 was assigned as (−)-(7′R*)-9-O-acetyl-7′-methoxy-7-epi-lariciresinol based on the insignificant variations in ΔδC‑7′, ΔδC‑8′, and ΔδC‑9′ values for compounds 6 and 4 (+0.2, −0.2, and 0.0, respectively) and the absence of the correlations between H-7′/H-9 in the NOESY spectrum of 6 (Figure 3). The 7-epi-lariciresinol-type lignans (4−6) are biosynthesized from epi-pinoresinol according to the reported pathway.21 Thus, epi-pinoresinol, which was isolated from a plant of the Taxus genus,22 was proposed to be a precursor in the biosynthesis of the tetrahydrofuranoid-type lignans for the first time. The isolated compounds were tested for their α-glucosidase inhibitory activity. Acarbose, which is used to control the blood glucose level of patients, was used as the positive control in this study. Compounds 1−3, 6, 7, 12, and 16 were not tested due to the insufficient amounts isolated. All tested compounds were found to possess α-glucosidase inhibitory activity (Table 3). Formosanol (9) and tsugacetal (10) showed moderate activity, with IC50 values of 35.3 and 38.8 μM, respectively.

type lignans; moreover, the data of 3 closely resembled those of (−)-7′-O-methyltanegool (7),13 except for signals at δC‑8 53.5, δC‑8′ 49.8, and δC‑9 62.1 in 3 and δC‑8 56.1, δC‑8′ 51.1, and δC‑9 63.4 in 7 (Figure 38S, Supporting Information). Compound 3 showed a negative specific rotation ([α]25 D −13.8) similar to that of 7. The locations of the methoxy groups were assigned at C-3, C-3′, and C-7′ based on the HMBC spectrum (Figure 1). The C-7 and C-8 protons were cis-oriented based on the variations in ΔδC‑8, ΔδC‑8′, and ΔδC‑9 values for compounds 3 and 7 (−2.6, − 1.3, and −1.3, respectively)20 and their NOESY correlations (Figure 2). The trans-orientation of H-8/H-8′ was suggested via the cross-peak of H-8′/H-9 in the NOESY spectrum. The most stable conformers of the 7′R and 7′S diastereomers of 3 were established by using PM3 calculations in MOPAC2016 (Figure 3). The NOESY spectrum of 3 showed a correlation between H-7′/H-8, which indicated the structure of 3 as (−)-7′R*-O-methyl-7-epi-tanegool. Compounds 4 and 5 have the same molecular formula, C21H26O7, as that of 3. These were found to be different based on comparison of their 1H and 13C NMR data (Table 2). In their 13C NMR data, the conspicuous differences involved the signals of the C-8 and C-8′ methine carbons (δC 51.9 and 49.9 in 4, δC 56.0 and 52.7 in 5, and δC 53.5 and 49.8 in 3). The HMBC spectra of 4 and 5 were also similar to those of 3 (Figure 1). Owing to differences in their NOESY spectra, it was deduced that 4 and 5 were stereoisomers of 3. In contrast with the trans-orientation of H-7 and H-8′ in 3, their cis-orientation was suggested in both 4 and 5 based on their NOESY correlation and the variations in ΔδC‑8 values for compounds 4 and 3, and 5 and 3 (−1.6 and +2.5, respectively).13 The NOESY correlation between H-7/H-8 indicated their cisorientation (Figure 2) in both 4 and 5, which was supported by the large variations in their ΔδC‑9 values and that of 7′methoxylariciresinol13 (+2.6 and +3.3, respectively). Thus, the C-7′ configuration was different in the structures of 4 and 5; these were suggested based on their most stable conformers using PM3 calculations (Figure 3). The absence of the correlations between H-7′/H-9 in the NOESY spectrum of 4 indicated the structure of 4 as 7′R*-methoxy-7-epi-lariciresinol. 1880



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with EtOAc−n-hexane (20:80 → 100:0) mixtures, to afford compound 2 (3.0 mg). Fraction Fr.9 (17.2 g) was passed over a silica gel column (5 × 120 cm), eluted with CHCl3−n-hexane (30:70 → 100:0) and EtOAc−n-hexane (20:80 → 100:0) mixtures, to yield nine subfractions (Fr.9.1−9.9). Subfraction Fr.9.1 was separated by column chromatography with EtOAc−n-hexane (20:80 → 100:0) mixtures, to give compounds 10 (4.0 mg) and 11 (3.5 mg). Subfractions Fr.9.5 and Fr.9.6 were chromatographed over a silica gel column with EtOAc−nhexane (20:80 → 100:0) and MeOH−CHCl3 (5:95 → 20:80) mixtures and were then purified by preparative TLC using MeOH− CHCl3 (2:98), to afford compounds 4 (4.0 mg), 5 (5.0 mg), 13 (3.5 mg), and 14 (3.5 mg). Subfraction Fr.9.9 was purified by preparative TLC using MeOH−CHCl3 (5:95), to afford compound 12 (2.0 mg). (+)-7′-epi-Tsugacetal (1): white, amorphous solid; [α]25 D +48.5 (c 0.01, MeOH); IR νmax (KBr) 3400, 3005, 1600, 1450 cm−1; 1H and 13 C NMR (acetone-d6, 500 MHz, see Table 1); HRESIMS m/z 395.1475 [M + Na]+ (calcd for C21H24O6Na, 395.1471). (+)-9,7′-Oxidolanceolatanin C (2): white, amorphous solid; [α]25 D +35.6 (c 0.01, MeOH); IR νmax (KBr) 3405, 3000, 1735, 1605, 1455 cm−1; 1H and 13C NMR (CDCl3, 500 MHz, see Table 1); HRESIMS m/z 377.0992 [M + Na]+ (calcd for C20H18O6Na, 377.1001). (−)-7′R*-O-Methyl-7-epi-tanegool (3): white, amorphous solid; [α]25 D −13.8 (c 0.01, MeOH); IR νmax (KBr) 3415, 2935, 1510, 1265, 1160 cm−1; 1H and 13C NMR (acetone-d6, 500 MHz, see Table 2); HRESIMS m/z 413.1574 [M + Na]+ (calcd for C21H26O7Na, 413.1576). 7′R*-Methoxy-7-epi-lariciresinol (4): white, amorphous solid; [α]25 D −67.3 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 210 (2.53), 230 (1.52), 280 (0.61) nm; IR νmax (KBr) 3410, 2950, 1480, 1300, 1150 cm−1; 1H and 13C NMR (CDCl3, 500 MHz, see Table 2); HRESIMS m/z 413.1579 [M + Na]+ (calcd for C21H26O7Na, 413.1576). 7′S*-Methoxy-7-epi-lariciresinol (5): white, amorphous solid; [α]25 D −21.9 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 210 (2.47), 230 (1.50), 280 (0.65) nm; IR (KBr) νmax 3400, 2900, 1550, 1205, 1105 cm−1; 1H and 13C NMR (CDCl3, 500 MHz, see Table 2); HRESIMS m/z 413.1566 [M + Na]+ (calcd for C21H26O7Na, 413.1576). (−)-(7′R*)-9-O-Acetyl-7′-methoxy-7-epi-lariciresinol (6): white, amorphous solid; [α]25 D −51.7 (c 0.01, MeOH); IR (KBr) νmax 3445, 2980, 1730, 1555, 1120 cm−1; 1H and 13C NMR (CDCl3, 500 MHz, see Table 2); HRESIMS m/z 455.1690 [M + Na]+ (calcd for C23H28O8Na, 455.1682). α-Glucosidase Inhibitory Assay. The inhibitory activity of αglucosidase was determined according to a published method.11 Thus, 3 mM p-nitrophenyl-α-D-glucopyranoside (25 μL) and 0.2 U/mL αglucosidase (25 μL) in 0.01 M phosphate buffer (pH = 7.0) were added to the sample solution (625 μL) to start the reaction. Each reaction was carried out at 37 °C for 30 min and stopped by adding 0.1 M Na2CO3 (375 μL). Enzymatic activity was quantified by measuring absorbance at 401 nm. The IC50 value was defined as the concentration of an α-glucosidase inhibitor that inhibited 50% of αglucosidase activity. Acarbose, a known α-glucosidase inhibitor, was used as a positive control.

Table 3. α-Glucosidase Inhibitory Activity of the Isolated Compoundsa compound

IC50 (μM)

compound

IC50 (μM)

4 5 8 9 10 11

42.9 170 194 35.3 38.8 133

13 14 15 17 acarboseb

52.2 135 175 79.1 215

a Compounds 1−3, 6, 7, 12, and 16 were not tested due to the insufficient amounts isolated. bPositive control.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on an A. Krüss Optronic P3000 polarimeter (A. Krüss Optronic GmbH, Hamburg, Germany). The IR spectra were measured with a Shimadzu IR-408 infrared spectrometer (Shimadzu Pte., Ltd., Singapore). The NMR spectra were recorded on a Bruker Avance III 500 spectrometer (Bruker BioSpin AG, Bangkok, Thailand) with tetramethylsilane as internal standard, and the chemical shifts are expressed as δ values. HRESIMS was performed on a Bruker micrOTOF QII mass spectrometer (Bruker Singapore Pte., Ltd., Singapore). Column chromatography was carried out using silica gel 60, 0.06−0.2 mm (Scharlau, Barcelona, Spain), and LiChroprep RP18, 40−63 μm (Merck KGaA, Darmstadt, Germany). Analytical and preparative TLC were carried out on precoated Kieselgel 60F254 or RP18 plates (Merck KGaA, Darmstadt, Germany). Other chemicals were of the highest grade available. Molecular Modeling. Molecular geometric properties were calculated by the PM3 method, with an EF optimizer and a minimum RMS of 0.01. Plant Material. The roots of T. wallichiana were collected in the Don Duong District, Lam Dong Province, Vietnam, in March 2008 and were identified by Dr. Ngot Van Pham, Department of Botany, Faculty of Biology, Ho Chi Minh City University of Pedagogy, Vietnam. A voucher specimen (MCE0048) has been deposited at the Division of Medicinal Chemistry, Faculty of Chemistry, VNUHCM− University of Science. Extraction and Isolation. The powdered roots of T. wallichiana (2.0 kg) were refluxed with MeOH (5 L, 3 h × 3), and the MeOHsoluble extract (503.7 g) was suspended in H2O (3 L) and successively partitioned with EtOAc (2 L) to give an EtOAc-soluble fraction (400 g). A portion (100 g) of this fraction was subjected to silica gel column chromatography (10 × 150 cm) and eluted with EtOAc−n-hexane (0:100 → 100:0) and MeOH−CHCl3 (5:95 → 20:80) mixtures, to obtain 14 fractions (Fr.1−Fr.14). Fraction Fr.4 (0.56 g) was purified using preparative TLC with CHCl3−n-hexane (10:90), to give compound 17 (3.5 mg). Fraction Fr.5 (2.5 g) was chromatographed over a silica gel column (3 × 60 cm) with CHCl3−n-hexane (10:90 → 100:0) and EtOAc−n-hexane (20:80 → 100:0) mixtures, and the resulting fractions were purified by preparative TLC using EtOAc−nhexane (20:80) and MeOH−CHCl3 (1:99), to afford compounds 3 (2.0 mg) and 7 (3.0 mg). Fraction Fr.6 (0.51 g) was passed over a silica gel column (2 × 60 cm), eluted with CHCl3−n-hexane (20:80 → 100:0) and EtOAc−n-hexane (20:80 → 100:0) mixtures, to yield eight subfractions (Fr.6.1−6.8). Subfractions Fr.6.1, Fr.6.2, and Fr.6.5 were purified using preparative TLC with CHCl3−n-hexane (20:80), to afford compounds 1 (2.5 mg), 6 (2.0 mg), 8 (50 mg), and 9 (3.5 mg). Fraction Fr.7 (5.62 g) was chromatographed over a silica gel column (4 × 60 cm) with EtOAc−n-hexane (20:80 → 100:0) and MeOH− CHCl3 (5:95 → 20:80) mixtures, to afford five subfractions (Fr.7.1− 7.5). Subfractions Fr.7.1 and Fr.7.3 were subjected to silica gel column chromatography with EtOAc−n-hexane (20:80), to give compounds 15 (4.0 mg) and 16 (2.0 mg). Fraction Fr.8 (7.1 g) was passed over a silica gel column (5 × 80 cm) with EtOAc−n-hexane (20:80 → 100:0) and MeOH−CHCl3 (5:95 → 20:80) mixtures, to yield five subfractions (Fr.8.1−8.5). Subfraction Fr.8.3 was chromatographed

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00171. Copies of spectroscopic data for 1−6 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail (N. T. Nguyen): [email protected]. Tel: +84-907426-331. Fax: +84-838-353-659. ORCID

Mai T. T. Nguyen: 0000-0001-8006-4028 Nhan T. Nguyen: 0000-0001-5142-4573 1881



Article

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by a grant from the Vietnam National University Ho Chi Minh City (No. A2015-18-02), to M.T.T.N.

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REFERENCES

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Lignans from the Roots of Taxus wallichiana and Their Î±-Glucosidase

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