Malaysianol B, an oligostilbenoid derivative from Dryobalanops lanceolata

Fitoterapia 83 (2012) 1569–1575

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Malaysianol B, an oligostilbenoid derivative from Dryobalanops lanceolata A. Wibowo a, N. Ahmat a,⁎, A.S. Hamzah a, A.L.M. Low a, S.A.S. Mohamad a, H.Y. Khong b, A.S. Sufian c, N. Manshoor c, H. Takayama d a b c d

Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor Darul Ehsan, Malaysia Faculty of Applied Sciences, Universiti Teknologi MARA, Jalan Meranek, 94300, Kota Samarahan, Sarawak, Malaysia Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Kuala Selangor, Selangor, Malaysia Graduate School of Pharmaceutical Sciences, Chiba University, 1‐33 Yayoi-cho, Inage-ku, Chiba 263‐8522, Japan

a r t i c l e

i n f o

Article history: Received 24 May 2012 Accepted in revised form 3 September 2012 Available online 13 September 2012 Keywords: Dipterocarpaceae Dryobalanops lanceolata Oligostilbenoid Tetramer Malaysianol B Antibacterial

a b s t r a c t A new oligostilbenoid tetramer, malaysianol B (1), was isolated from the acetone extract of the stem bark of Dryobalanops lanceolata along with seven oligostilbenoids tetramers; hopeaphenol (2), stenophyllol A (3), nepalensinol B (4), vaticanol B (5) and C (6), upunaphenol D (7), and flexuosol A (8). The structures of the isolated compounds were established on the basis of their spectroscopic data evidence. The antibacterial activity of the isolated compounds was evaluated using resazurin microtitre-plate assay. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Dryobalanops lanceolata (Dipterocarpaceae) is a major species in the emergent canopy in Lambir Forest and Sarawak lowland Dipterocarp forest [1]. As other genus in Dipterocarpaceae family, Dryobalanops has been shown to be a rich source of oligostilbenoids [2–4]. Stilbenoid oligomers are receiving considerable chemical and biological attention owing to their structural complexity as well as their array of bioactivities exhibited such as antifungal, anti-HIV [5], cytotoxic [6,7], anti-inflammatory [8] and antibacterial effects. Our previous study reported the isolation of stilbenoid oligomer derivatives from Dryobalanops aromatica as well as their cytotoxic activity [3]. In continuation of our interest in the phytochemical study of stilbenoid oligomers in Dryobalanops plant, we report here the structure of a new stilbenoid tetramer from the stem bark

⁎ Corresponding author. Tel.: +60 3 55444643; fax: +60 3 55444562. E-mail address: [email protected] (N. Ahmat). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2012.09.004

of Dryobalanops lanceolata, antibacterial activity, and its biogenetic pathway.

2. Experimental 2.1. General experimental procedure The following instruments were used: CD spectrum was recorded on a spectropolarimeter (JASCO J-720WI). UV and IR spectrum were measured with a Varian Conc. 100 instruments and a Perkin Elmer Spectrum One FTIR spectrometer, respectively. HRESI-MS was obtained with a JEOL AccuTOF-T100LP mass spectrometer. The 1H and 13C NMR spectrum were recorded with a Bruker Advance Model [300 MHz (1H) and 75 MHz (13C)], and the melting points were measured on a Melting-Point Apparatus with microscope JM628. The following adsorbents were used for purification: vacuum liquid chromatography (Si-gel 60, Merck catalog number: 1.07747) and radial chromatography (Si-gel 60 GF254, Merck catalog number: 1.07749), and TLC analysis (Merck, Kieselgel 60 F254

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0.25 mm). Solvents used in this research are of analytical grade and technical grade that were distilled before used.

(8.8 g) using the same methodology gave 2 (155.6 mg), 3 (15.3 mg), 5 (30.0 mg) and 6 (20.0 mg).

2.2. Plant material

2.3.1. Malaysianol B (1) Dark brown solid; IR (KBr) νmax cm−1: 3338, 2920, 1606, 1605, 1513, 1451, 1342, 1235, 1167, 1127, 1006, and 835; 1 HNMR (d6-acetone) see Table 1; 13C NMR (d6-acetone) see Table 1; HRESI-MS m/z: [M +Na]+ 945.24924(Calc. for C56H42O13-Na, 945.25231). CD (c =0.05, MeOH) nm: 226.4 [ Δε = −78.4], 284.6 [−5.7], 238.6 [−27.6], and 295.6 [−18.1].

The stem bark of D. lanceolata was collected from Sarawak, Malaysia, and a voucher specimen (UiTMKS3001) was deposited at the herbarium of Universiti Teknologi MARA, Malaysia (Sarawak campus). 2.3. Extraction and isolation

2.4. Antibacterial (resazurin microtitre-plate assay) The dried powder of the stem bark of D. lanceolata (4 kg) was macerated with acetone (4x10 L), and evaporated under reduced pressure to give a dark brown residue (268 g). The dried acetone extract was dissolved in a small volume of MeOH (± 200 mL), and added with diethyl ether to a volume ± 2 L to give MeOH-diethyl ether soluble fraction (88.6 g) after decantation and evaporation, and an insoluble fraction (179.4 g). A part of the soluble fraction (60 g) was subjected to vacuum liquid chromatography (VLC), (silica gel, 400 g; eluted with mixtures of n-hexane/EtOAc 40% to 100%, and EtOAc/MeOH 10%, 20% and 100%) to give seven major fractions (DL1–DL7). Fraction DL4 (9.9 g) was refractionated further with VLC using eluent, n-Hexane:EtOAc and EtOAc: MeOH as gradient to give eight major fractions (DL4.1–DL4.8). Fraction DL4.3 (2.9 g) was refractionated using radial chromatography (eluent, n-Hexane:EtOAc:MeOH = 35:60:5) to give six fractions (DL4.3.1–DL4.3.6), and purification of fraction DL4.3.1 (0.95 g) with radial chromatography (eluent, CHCl3:MeOH = 15%) yielded 8 (453.2 mg), fraction DL4.3.2 (0.50 g) yielded 7 (45.3 mg), while fraction DL4.3.3 (0.74 g) yielded 4 (172.2 mg) and 1 (50.4 mg). Further purification of fifth fraction DL5

Table 1 1 H and 13C NMR data of 1 in acetone-d6 (300 MHz for 1H and 75 MHz for δH (mul., J in Hz) δC

HMBC ( H⇔ C)

No

δH (mul., J in Hz)

δC

HMBC (1H⇔13C)

1a 2a/6a 3a/5a 4a 7a 8a 9a 10a 11a 12a 13a 14a 1b 2b/6b 3b/5b 4b 7b

– 7.12 6.78 – 5.48 4.13 – – – 6.34 – 6.38 – 6.86 6.58 – 5.10

1c 2c/6c 3c/5c 4c 7c 8c 9c 10c 11c 12c 13c 14c 1d 2d/6d 3d/5d 4d 7d

– 6.99 6.51 – – 4.42 – – – 6.17 – 6.38 – 6.82 6.79 – 5.06

131.3 132.7 116.6 162.7 199.3 55.5 137.7 123.5 163.3 97.5 160.1 109.6 134.3 128.1 117.1 158.8 95.6

– C-1c, C-3c/5c, C-4c, C-7c C-1c, C-2c/6c, C-4c, C-7c – – C-7c, C-9c, C-10c, C-14c, C-7b, C-8b – – – C-9c, C-10c, C-11c, C-13c, C-14c – C-8c, C-9c, C-10c, C-11c, C-12c, C-13c – C-1d, C-3d/5d, C-4d, C-7d C-1d, C-2d/6d, C-4d – C-1d, C-2d/6d, C-8d, C-9d, C-10c, C-11c

8b

4.54 (dd, 4.2; 10.2) – – – 5.71 (d, 1.8) – 5.58 (d, 2.1)

– C-1a, C-3a/5a, C-4a, C-7a C-1a, C-2a/6a, C-4a – C-1a, C-2a/6a, C-8a, C-9a, C-10b, C-11b 1a, 7a, 9a, 10a, 14a, C-9b, C-10b – – – C-10a, C-11a, C-13a, C-14a – C-8a, C-10a, C-11a, C-12a, C-13a – C-1b, C-3b/5b, C-4b, C-7b C-1b, C-2b/6b, C-4b – C-1b, C-2b/6b, C-8b, C-9b, C-9a, C-10a, C-8c C-7b, C-9b, C-10b, C-14b, C-10a, C-7c, C-8c – – – C-9b, C-10b, C-11b, C-13b, C-14b – C-8b, C-10b, C-12b, C-13b

8d

3.37 (d, 4.2)

57.6

9d 10d 11d 12d 13d 14d

– 5.70 (m) – 6.24 (t, 2.1) – 5.68 (m)

149.1 109.1 160.9 103.3 160.9 107.4

C-1d, C-8d, C-9d, C-10d, C-14d, C-9c, C-10c, C-11c – C-8d, C-12d – C-10d, C-11d, C-13d, C-14d – C-8d, C-12d

9b 10b 11b 12b 13b 14b

(d, 12.8) (d, 12.6)

(d, 2.1) (d, 2.1) (d, 8.7) (d, 8.7) (d, 3.9)

131.5 131.3 116.9 159.6 89.0 50.8 142.9 121.9 157.2 103.0 159.4 107.6 135.0 129.8 116.3 156.8 41.2 47.6 139.0 119.8 160.0 97.7 158.3 112.5

13

C).

No

(d, 8.7) (d, 8.4)

1

13

In the antimicrobial screening, a modified resazurin microtitre-plate assay was used as reported by Sarker [9]. Overnight shaken test organisms were adjusted to an absorbance of 0.7–0.8 at 600 nm and 630 nm prior to use. Assay was conducted in 96-well plate and labeled accordingly. 10 μL of test organisms was placed in each well. 40 μL (1 mM) of isolated compounds (dissolved in 10% DMSO) was pipetted into wells. After 18 h of incubation (37 °C) of the plate, 20 μL of 0.5 mM resazurin was added and re-incubated 8 h for Staphylococcus epidermidis (ATCC 12228), Staphylococcus aureus (ATCC 25923) and Staphylococcus xylosus (ATCC 29971), and overnight for Shigella flexneri (ATCC 12022), Salmonella typhimurium (ATTCC 14028), Escherichia coli (ATCC 6633). Chloramphenicol (1 mM, dissolved in 10% DMSO) was used as positive control whereas 10% DMSO as negative control. The color change was then assessed visually. The growth was indicated by color changes from purple to pink or colorless (does not have antibacterial activity). Furthermore, the minimum inhibition concentration (MIC) of active compounds (at 1 mM) is analyzed using the same methodology in various

(d, 8.7) (d, 9.0)

(d, 10.2)

(d, 2.1) (d, 2.1) (d, 8.7) (d, 8.7) (d, 4.2)

HO

H O

HO

OH

H O

OH

HO

HO

HO

H H

O

H

HO

H H

H

HO

H

OH

HO O H

H H

H

OH

OH

HO

OH

H

HO OH

OH

OH OH

H

H

OH

H

OH

H

H

H O

HO

O H

HO

O H

HO

OH

OH

HO

1

OH

2

O

3

HO OH

OH

HO

OH

H

OH O HO

H H

H H

H

H

H

OH

H

HO

O HO

HO

OH H

H

H OH

H

HO

H

H H

4

H H

H H

HO

HO

HO

6

HO

HO H

HO

H

H

OH

HO

OH

O

OH OH

H O

H OH

HO

OH

OH

H

H

O

O

H O O

OH

OH

O

5 HO

HO

H

H

OH

HO

OH

OH

H O

HO

A. Wibowo et al. / Fitoterapia 83 (2012) 1569–1575

OH OH

HO

H

H OH

H

OH

OH H O

OH OH

O H

HO

OH

7

8 Fig. 1. Oligostilbenes of Dryobalanops lanceolata.

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A. Wibowo et al. / Fitoterapia 83 (2012) 1569–1575

HO

12b

OH

O

HO

7a

HO

14b 7c

8a

O

10d

OH

14a

8c

8b

14d

7b

HO

8d 14c

7d

12a

OH

OH

O

HO

12c

OH

HMBC COSY 5c

7a

HO

O

H

2a

12b

HO

C1

OH

A1

OH

HO

B2 8a 6a

14a

H

O

D2

2c

10d

14d

A2

HO

2b

H H

5b

7b

B1 6b

8b

8c

7d

H 14c

H C2

O

OH

HO

HO

H 8d

6d

D1

NOESY OH Fig. 2. Key COSY, HMBC and NOESY correlations in 1.

concentrations (0.80–0.025 mM). The lowest concentration at which color change occurred was taken as the MIC value. 3. Results and discussion The acetone extract of the stem bark of D. lanceolata was separated by combination of vacuum liquid and radial chromatographies to give altogether eight compounds, including a new oligostilbenoid (1) and seven known

oligostilbenoids (hopeaphenol (2) [10], stenophyllol A (3) [11], nepalensinol B (4) [12], vaticanol B (5) and C (6) [13], upunaphenol D (7) [14], and flexuosol A (8) [15]). The known compounds (2–8) exhibited physical and spectral data identical to values in the literature. Malaysianol B (1), obtained as a brown amorphous powder, with the molecular formula of C56H42O13 which was established by an [M+ Na]+ ion peak at m/z 945.24924 in the high resolution of electron spray ionization (HR-ESI-MS) together

A. Wibowo et al. / Fitoterapia 83 (2012) 1569–1575

with the NMR spectral data suggesting a resveratrol tetramer type of compound. The 13C NMR spectrum of 1 (Table 1) showed 46 signals representing 56 carbon atoms that are distributed into 11 oxyaryl, 10 quaternary aromatic, 25 methine aromatic, seven methine alkyl, and a carbonyl ketone. The 13C NMR spectrum indicated 1 as a tetramer with modification of one methine aliphatic carbon to ketone group. The 1H NMR spectral data of 1 (Table 1) showed the presence of eight sets of ortho-coupled aromatic signals in AA'XX’ spin system assignable to four p-hydroxybenzene rings at δH 7.12/6.78, 6.86/6.58, 6.99/6.51 and 6.82/6.79 (rings A1, B1, C1, and D1, respectively), and three sets of meta-coupled aromatic protons for three units of disubstituted-3,5-dihydoxybenzene rings at δH 6.34/6.38, 5.71/5.58 and 6.17/6.38 (rings A2, B2, and C2, respectively). A triplet and two broad singlet signals observed at δH 6.24, 5.70 and 5.68 belong to a monosubstituted-3,5-dihydoxybenzene ring (ring D2). The NMR spectrum also disclosed the presence of two pairs of doublet characteristic for two 1,2dihydrobenzofuran moieties at δH 5.48/4.13 (H7a/H8a), and 5.06/3.37(H7d/H8d), as well as three signal of methine protons at δH 5.10 (d, J=3.9 Hz, H7b), 4.54 (dd, J=4.2; 10.2 Hz, H8b) and 4.42 (d, J=10.2 Hz, H8c) for neighboring CH–CH–CH unit. The above evidence suggested 1 as a tetramer resveratrol with two units of 1,2-dihydrobenzofuran rings, and one cycloheptane ring (Fig. 1). The HMBC spectrum of 1 showed long-range correlations between H7a/C2a/6a, and H7d/C2d/6d, establishing the attachment of rings A1 and D1 to oxymethine carbons C7a and C7d, which is part of the benzofuran ring, while position of rings A2 and D2 were determined by the correlations between H8a/ C14a, and H8d/C10d/14d, respectively. The correlations between H7b/C9b/10a and H8b/C9a indicated that H7b and H8b are part of a cycloheptane ring, which is formed between C7b/C8a and rings A2/B2 (ampelopsin A type of skeleton [16]), while correlations of H7b/C1b/2b/6b and H8b/C9b/10b/14b established the position of rings B1 and B2. Furthermore, the HMBC spectrum also revealed correlations between carbonyl carbon at δC 199.3 (C7c) with H2c/6c, and C8c with H14c indicating the attachment of rings C1 and C2 to carbons C7c and C8c respectively, while correlation of C10c/11c and H7d/8d indicated that the unit of resveratrol D is attached to C10c/11c of ring C2 (ε-viniferin type of skeleton [17]). Finally, the important correlations between H8c and C7b/8b established the link between C8b in the cycloheptane ring (ampelopsin A type) and C8c in the other dimer unit (ε-viniferin type) to form a

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tetramer (Fig. 2). Thus, the structure of 1 was determined as a new compound assigned as malaysianol B. The NOE correlations between H7a/H10a/14a and H8a/H2a/6a, and H7d/H10d/14d and H8d/H2d/6d confirmed the trans relationships of H7a/H8a and H7d/H8d, respectively, while correlations between H8a/H2b/6b and H2a/6a/H2b/6b established the trans- and cis-orientations of H7b/H8a and H7a/H7b, respectively (Fig. 2). The spatial relations between H7b/H8b, H8b/H14c, H8c/H2c/6c, and H3c/5c/H12c further confirmed the cis-orientation of H7b/H8b, and trans-orientation of H8b/H8c, while correlation between H2c/6c/H10d/14d established the cis-orientation of protons H8c/ H8d. The orientation of proton H8b, H8c and H8d resulted in the syn-orientation of three aromatic rings (rings B2, C1 and D2) which cause the protons H12b/H14b (ring B2) and H10d/H14d (ring D2) to be shielded due to the anisotropic effect. Based on these NOE observations, the relative configuration of seven asymmetric carbons in 1, C7a/C8a, C7b/C8b/C8c, and C7d/C8d, were determined as rel-R/R, R/S/R, and R/R respectively (Fig. 2). All of the isolated compounds except 3 were screened for their antibacterial activity against Gram-positive and Gramnegative bacterial strains by using resazurin microtitre-plate assay (Table 2). Compounds 7 and 8 exhibited promising antibacterial property as they inhibited the cell growth of three Gram-positive strains, Staphylococcus epidermidis, S. aureus and S. xylosus wit MIC 25/75, 50/100 and 25/75 μM, respectively. The presence of free resveratrol in the skeletons of 7 and 8 showed significance effect to their activity, however the activity is still far from chloramphenicol as positive control. In addition, the position of isolated free resveratrol in 8 gave less activity than terminal in 7 due to a disruption of the double bond resonance in the free resveratrol. Biogenetically, the structures of the eight resveratrol tetramers (1–8) of Dryobalanops lanceolata showed close relationships in which all compounds are formed from the condensation of two ε-viniferin molecules as a precursor which differ just at the type of condensation (Fig. 3). Compounds 1, 2, 3 and 4 possess identical condensation type, where they are initiated from oxidative coupling reaction of two radical species at aliphatic carbon C8 (C8–C8 type) of two ε-viniferin molecules, while 5, 6 and 8 from the oxidative coupling reaction of radicals of aliphatic carbon C8 and aromatic carbon C14 (C8–C14 type) of two ε-viniferin molecules (Fig. 3). The condensation types of all compounds above are commonly found in Dipterocarpaceae plants, but the condensation type of 7 is different as it is formed from the

Table 2 Antibacterial screening of isolated compounds by microtitre-plate resazurin assay. Compounds

Malaysianol B (1) Hopeaphenol (2) Nepalensinol B (4) Vaticanol B (5) Vaticanol C (6) Upunaphenol D (7) Flexuosol A (8) Chloramphenicol

Bacterial strains (MIC, μM) SE

SA

SX

SF

ST

EC

− − − − − +(25) +(75) +(0.025)

− − − − − +(50) +(100) +(0.025)

− − − − − +(25) +(75) +(0.062)

+(625) − − +(625) +(625) +(500) +(375) +(0.031)

− − − − − − − +

− − − − − − − +

Gram-positive: SE, Staphylococcus epidermidis (ATCC 12228); SA, Staphylococcus aureus (ATCC 25923); SX, Staphylococcus xylosus (ATCC 29971). Gram-negative, SF, Shigella flexneri (ATCC 12022); ST, Salmonella typhimurium (ATTCC 14028); EC, Escherichia coli (ATCC 6633). (+) active, (−) not active at 1000 μM.

OH

HO

H H HO

H

H

H

H

OH HO

H

O HO

7b

d

7c

d

H

c/d

a

OH

10d

OH

H

Cyclization C14-C7

b

H H

O

OH

HO

OH

HO

OH

O

H+

H

O

HO

1

H

H H

4

H+

OH

C8-C8 type

+ e-viniferin H O

HO

OH

O

HO

OH

H H

H

HO

g

HO

H

C8-C3 type & benzofuran formation

OH

7

OH

OH OH

O H

13

OH

HO OH

OH

8

e -viniferin C8-C14 type

OH

OH

OH

H

HO H

HO

O

8c 14b

h

7b

HO

5

OH O

O OH

h/j

H

8b

O H

H H

H H

HO

OH

10d

j

h/i Cyclization C7-C8 & C7-C10

OH H

7c

H

H

H

i

HO

H H

O

O

H O

H

H

H

OH HO

OH

H O

HO

H

O H

HO

O

O H

OH

OH

H

H

C8-C14 type & benzofuran formation

H O

O

e

HO

O

g 4

HO

7

OH

e/f

OH f

OH

3

H

H

H

14

8

OH

O

HO

HO

e

HO

OH OH

H O

HO

H

H

HO

OH

H O

OH

Cyclization C7-C8 & C7-C14

HO

HO O

OH

H OH O

OH

Fig. 3. Proposed biogenetic pathway of the resveratrol tetramer of D. lanceolata.

HO

H OH

6

A. Wibowo et al. / Fitoterapia 83 (2012) 1569–1575

OH

10a

Cyclization C10-C7 Oxidation at C7

OH

HO

H 8c

O H

HO

HO OH

OH

8b

HO OH

OH

3

c a/c

OH

H H

H

O

OH

O H 14c

14b

H

O

H

HO

HO

HO

HO

OH HO

Cyclization C10-C7 Oxidation at C4

Cyclization C10-C7

H O OH

OH

H

H

H

a/b

HO

2

O H

OH a/b

HO

HO

H HO

HO OH

OH

H

OH

OH

OH

H O

H O

HO

O H

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H O

HO

A. Wibowo et al. / Fitoterapia 83 (2012) 1569–1575

radical species of aliphatic carbon C8 and aromatic carbon C3 (C8–C3 type) of two ε-viniferin molecules (Fig. 3). Interestingly, this type of condensation is not common in Dipterocarpaceae family and only found in other family that produce resveratrol oligomers type of compounds, such as Vitaceae, Cyperaceae, Fabaceae and Gnetaceae [18]. This suggests the chemotaxonomic correlation of genus Dryobalanops with those families. Acknowledgments The authors would like to thank The Ministry of Higher Education, Malaysia for the financial support under the Fundamental Research Grant Scheme [600-RMI/ST/FRGS/5/ 3/Fst (12/2010)] and Faculty of Applied Sciences, Universiti Teknologi MARA, UiTM for providing research facility. References [1] Terauchi R. Jpn J Genet 1994;69:567-76. [2] Syah YM, Aminah NS, Hakim EH, Aimi N, Kitajima M, Takayama H, et al. Phytochemistry 2003;63:913-7.

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