Phytochemistry 89 (2013) 125–130

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Phytochemistry

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Ent-pimarane and ent-trachylobane diterpenoids from Mitrephora albaand their cytotoxicity against three human cancer cell lines

Kanok-on Rayanil ⇑, Suphaluck Limpanawisut, Pittaya TuntiwachwuttikulDepartment of Chemistry, Faculty of Science, Silpakorn University, Nakorn Pathom 73000, Thailand

a r t i c l e i n f o

Article history:Received 21 September 2012Received in revised form 15 January 2013Accepted 29 January 2013

Keywords:Mitrephora albaAnnonaceaeEnt-pimarane diterpeneEnt-trachylobane diterpeneCytotoxicity

0031-9422/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.phytochem.2013.01.012

⇑ Corresponding author. Tel.: +66 34255797; fax: +E-mail addresses: krdzb1@yahoo.com, kanok-on@s

a b s t r a c t

Bioassay-guided fractionation of the hexane extract of the branches of Mitrephora alba led to the isolationof five diterpenoids: ent-8b-hydroxypimar-15-en-18-oic acid, ent-15,16-dihydroxypimar-8(14)-en-18-oic acid, ent-3b-hydroxytrachyloban-18-oic acid, ent-3b-hydroxytrachyloban-18-al and methyl ent-3b-hydroxytrachyloban-18-oate, together with five related known diterpenoids. The structures wereelucidated by spectroscopic analysis and comparison with literature data. All isolated compounds wereevaluated for their cytotoxic activities against three human cancer cell lines. The results showed thatthree ent-trachylobane diterpenes had moderate cytotoxicity against NCI-H187 cancer cells.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The genus Mitrephora belongs to the Annonaceae family andcomprises approximately 48 species distributed throughout Asiaand Australia. Twelve species are found in Thailand and someplants in this genus have been used as Thai folk medicine for tonicpurposes (Chalermglin, 2001; Smitinand, 2001). Previous phyto-chemical investigations established that Mitrephora species con-tain diterpenoids (Deepralard et al., 2007; Li et al., 2005, 2009;Meng et al., 2007; Supudompol et al., 2004; Zgoda-Pols et al.,2002), polyacetylene carboxylic acids/esters (Li et al., 2009; Zgodaet al., 2001), fatty acids (Brophy et al., 2004), lignans (Deepralardet al., 2007; Ge et al., 2008; Moharam et al., 2010), sesquiterpenes(Deepralard et al., 2007; Ge et al., 2008) and alkaloids (Deepralardet al., 2007; Lee et al., 1990; Moharam et al., 2010; Mueller et al.,2009; Yu et al., 2005). Among these compounds, the diterpenoidsand alkaloids showed significant antimicrobial, antimalarial, anti-platelet aggregation and cytotoxic activities.

Mitrephora alba Ridl. (Annonaceae), a 6–8 meter tall tree withwhite flowers, is widely distributed in southern Thailand andMalaysia. However, its pharmaceutical use remains unknown. Tothe best of our knowledge, no phytochemical study of M. albahas hitherto been reported. A preliminary study on several ThaiMitrephora species in our laboratory indicated that the hexane ex-tract of the branches of M. alba inhibits growth of NCI-H187 and KBhuman cancer cell lines with the percentage inhibition of 93% and

ll rights reserved.

66 34271356.u.ac.th (K.-o. Rayanil).

88%, respectively (observed at 50 lg/mL), and so the chemicalinvestigation of this extract was an attractive undertaking, aimingto discover new potent anticancer constituents. Described hereinare the isolation, characterization and cytotoxicity study of fivenew diterpenoids (1–2, 4–6 Fig. 1), together with a cytotoxicitystudy of known ones.

2. Results and discussion

2.1. Structure elucidation and identification

The air-dried branches of M. alba were extracted with EtOH atroom temperature. The EtOH extract was successively partitionedbetween hexane, EtOAc, n-BuOH and water. Fractionation of thebioactive hexane extract using silica gel and C18 reversed-phasechromatography led to isolation of five new diterpenoids includingtwo pimarane diterpenoids: ent-8b-hydroxypimar-15-en-18-oicacid (1), ent-15,16-dihydroxypimar-8(14)-en-18-oic acid (2), andthree trachylobane diterpenoids: ent-3b-hydroxytrachyloban-18-oic acid (4), ent-3b-hydroxytrachyloban-18-al (5) and methyl ent-3b-hydroxytrachyloban-18-oate (6), together with five knownditerpenoids. By comparison of spectroscopic and physical datawith those in the literature, the known compounds were identifiedas ent-pimara-8(14),15-dien-18-oic acid (3) (Xie et al., 1989;Wenkert and Buckwalter, 1972), ent-trachyloban-18-oic acid (7)(Leong and Harrison, 1997), ent-trachyloban-3b,19-diol (8)(Kapingu et al., 2000, reported as 3a,19-dihydroxytrachylobane),ent-trachyloban-3b,18-diol (9) (Scher et al., 2010) and ent-trachy-loban-3b-ol (10) (Scher et al., 2010), respectively.

H

HHO2C

H

HR1 R2

R3

12

3 45

6

7

89

10

1112

13

14

15 16

1720

19

12

34

5

6

7

8

9

10

11

12

131415

16 17

18

19

20

18

H

HHO2C

H

HHO2C

HO OH

31 2

R1 R2 R345678910

CO2HCHOCO2CH3CO2HCH3CH2OHCH3

CH3CH3CH3CH3CH2OHCH3CH3

H

OHOHOH

OHOHOH

OH

Fig. 1. Chemical structures of diterpenes 1–10.

126 K.-o. Rayanil et al. / Phytochemistry 89 (2013) 125–130

Compound 1 was obtained as a white amorphous solid. Themolecular formula was established as C20H32O3 from the [M+Na]+

ion at m/z 343.2285 (calc. for C20H32O3Na, 343.2249), suggestingfive degrees of unsaturation in the molecule. The 1H NMR spectrumshowed resonances for three methyl singlets at d 0.90, 0.96 and1.18 and a vinyl group at d 5.09 (dd, J = 10.8, 1.2 Hz, 1H), 5.14(dd, J = 18.0, 1.2 Hz, 1H) and 5.97 (dd, J = 18.0, 10.8 Hz, 1H) (Ta-ble 1). The 13C NMR and DEPT spectra indicated 20 carbons, includ-ing three methyls, eight methylenes, two methines, four

Table 11H (300 MHz) and 13C (75 MHz) NMR spectroscopic data for 1–2 in CDCl3 (J in Hz inparentheses).

Position 1 2

dH dC dH dC

1 0.95 m 38.6 1.12 m 38.31.70 m 1.72 m

2 1.43 m 17.2 1.54 m 18.21.53 m

3 1.60 m 37.0 1.64 m 37.11.75 m 1.77 m

4 47.5 47.25 1.72 m 50.5 1.93 dd (12.3,2.4) 49.16 1.10 m 20.9 1.28 m 25.0

1.72 m 1.45 m7 1.30 m 41.4 2.14 m 35.9

1.72 m 2.25 dd (14.1,2.7)8 72.9 138.79 0.98 m 56.3 1.81 m 50.3

10 36.6 38.011 1.48 m 17.6 1.56 m 18.6

1.65 m12 1.23 m 35.9 1.19 m 31.8

2.01 dd (13.5,2.1) 1.59 m13 36.5 37.114 1.25 m 53.3 5.30 s 128.0

1.65 m15 5.97 dd (18.0,10.8) 147.5 3.58 dd (9.3,1.8) 78.716 5.09 dd (10.8,1.2) 112.1 3.51 dd (10.2,9.3) 63.2

5.14 dd (18.0,1.2) 3.70 dd (10.2,1.8)17 0.90 s 32.4 0.90 s 23.518 185.2 182.219 1.18 s 16.3 1.19 s 17.020 0.96 s 15.8 0.82 s 15.3

quaternary carbons (one oxygenated), one carboxyl (d 185.2) anda vinyl double bond (Table 1). The above information was inaccordance with a pimarane diterpenoid skeleton (Wenkert andBuckwalter, 1972). However, compound 1 has a negative opticalrotation (½a�25

D �16.4 (c 0.65, CHCl3), which was the same sign asthat of ent-pimarane derivatives (Matsuo et al., 1976). Therefore,compound 1 was determined to be an ent-pimarane rather thana pimarane. Comparison of the NMR spectroscopic data of 1 withthose of the known ent-pimara-8(14),15-dien-18-oic acid (3), alsoisolated from this plant, established that they were closely related.The main differences were the absence of a C-8/C-14 double bondsignal and the presence of an oxygenated quaternary carbon reso-nance at dC 72.9 in compound 1. The aforementioned data impliedthat the C-8/C-14 double bond in 3 was replaced by a C-8 hydroxylgroup in compound 1. This assumption was further confirmed byHMBC correlations between C-8 (d 72.9) and H-9 (d 0.98, m) andH-14 (d 1.25, m; 1.65, m). Unambiguous assignment of the C-8 hy-droxyl orientation was established by comparing the 13C NMRspectrum of 1 with that of (�)-thermarol, ent-pimar-15-en-8b,19-diol, (Matsuo et al., 1976). The C-8 resonance in the 13CNMR spectrum of (�)-thermarol occurred at d 72.5, which was al-most identical to that in 1 (d 72.9). This lent strong support that theC-8 hydroxyl group in 1 is in the b-orientation. Further support ofthe C-8 hydroxyl orientation was obtained by comparing the 13CNMR spectrum of 1 with that of 8b-hydroxypimar-15-en-19-oicacid (Ramos et al., 1984). The resonances in the 13C NMR spectrawere almost identical. The main differences occurred at C-3-C-6and C-18-C-20, an indication of different influences exerted byfunctional groups at C-4. Moreover, the same ABX pattern of C-15 and C-16 protons in the 1H NMR spectrum of 8b-hydroxypi-mar-15-en-19-oic acid (dA = 5.10, dB = 5.13, dX = 5.97; JAB = 1.4 Hz,JAX = 14.2 Hz, JBX = 18.0 Hz) and compound 1 (see above) suggesteda cis-relationship between the C-8 hydroxyl and the C-13 vinylgroups (see references for the trans-relationship; Chen et al., 2009;Rao and Rao, 1978). Therefore, it was concluded that the C-8 hydro-xyl group in 1 is in the b-orientation and compound 1, a new diter-pene, was found to be ent-8b-hydroxypimar-15-en-18-oic acid.

Compound 2 was obtained as a white amorphous solid and itsmolecular formula was determined to be C20H32O4 by HRESI-TOFMS, consistent with the molecular ion peak at m/z 359.2217[M+Na]+ (calc. for C20H32O4Na, 359.2198). Its spectroscopic data

K.-o. Rayanil et al. / Phytochemistry 89 (2013) 125–130 127

(IR, 1H NMR and 13C NMR) were closely related to those of 3. Inaddition, similar to 1 and 3, compound 2 also has a negative opticalrotation, ½a�25

D �37.0 (c 0.65, MeOH), suggesting that it was also anent-pimarane derivative. Compound 2 showed an olefinic protonsignal at d 5.30 (s) in the 1H NMR spectrum and olefinic carbon res-onances at d 138.7 (C-8) and 128.0 (C-14) in the 13C NMR spectrum(Table 1). These signals were also observed in 3, indicating that 2was an ent-pimar-8(14)-ene (Cambie et al., 1975). This conclusionwas confirmed by the HMBC correlations of H-14 (d 5.30) to C-7 (d35.9), C-9 (d 50.3), C-12 (d 31.8), C-13 (d 37.1), C-15 (d 78.7) and C-17 (d 23.5). In contrast to the observation of a vinyl substitution incompound 3, the 1H NMR spectrum of 2 indicated presence of car-binolic proton resonances at d 3.70 (dd, J = 10.2, 1.8 Hz), 3.58 (dd,J = 9.3, 1.8 Hz) and 3.51 (dd, J = 10.2, 9.3 Hz). This indicated that 2was an ent-pimar-8(14)-ene bearing a 1,2-dihydroxyethyl moietyat C-13 (Lee et al., 2008; Luo et al., 2001; Sam et al., 1991). Thisassumption was supported by the HMBC correlations between H-15 (d 3.58) and C-12 (d 31.8), C-13 (d 37.1), C-14 (d 128.0), C-16(d 63.2), and C-17 (d 23.5). In addition, comparison of the 13CNMR spectrum of compound 2 with that of ent-15,16-dihydroxypi-mar-8(14)-en-19-oic acid, previously isolated from Aralia cordata(Lee et al., 2008), established only a difference in the chemical shiftof a methyl group located at C-4. The chemical shift of the C-4methyl group of compound 2 occurred at dC 17.0 versus dC 29.7in ent-15,16-dihydroxypimar-8(14)-en-19-oic acid. This suggestedthat compound 2 is a C-4 epimer of ent-15,16-dihydroxypimar-8(14)-en-19-oic acid. This conclusion was confirmed by theinteraction between H-19 (d 1.19) and H-20 (d 0.82) in the NOESYspectrum, which indicated that the C-4 methyl group was in anaxial position, and hence, the carboxylic group was in an equatorialposition, as observed in compounds 1 and 3. Thus, 2 was elucidatedas ent-15,16-dihydroxypimar-8(14)-en-18-oic acid, hithertounreported in the literature.

Compound 4 was obtained as a white amorphous solid, ½a�25D

�63.3 (c 0.28, CHCl3). Its molecular formula was established asC20H30O3 by HRESITOFMS (observed m/z 341.2097 [M+Na]+),

Table 21H (300 MHz) and 13C (75 MHz) NMR spectroscopic data for 4–6 in CDCl3 (J in Hz in pare

Position 4 5

dH dC dH

1 0.96 m 37.1 0.93 m1.58 m 1.58 m

2 1.57 m 26.2 1.61 m3 3.95 dd (11.4,5.1) 75.6 3.37 dd (10.5,4.4 53.45 1.50 m 50.6 1.25 m6 1.14 m 22.6 0.93 m

1.48 m 1.38 m7 1.38 m 38.3 1.40 m

1.48 m8 40.79 1.15 m 53.0 1.15 m

10 37.411 1.65 m 19.7 1.65 m

1.89 m 1.93 ddd (14.4,112 0.58 br d 20.4 0.59 br d13 0.83 dd (7.2,2.7) 24.2 0.83 dd (7.8,3.014 1.15 m overlapped 33.3 1.15 m overlapp

2.02 d (12.0) 2.02 d (12.0)15 1.25 d overlapped 50.1 1.25 d (11.4)

1.38 d (11.4) 1.39 d overlappe16 22.517 1.13 s 20.5 1.13 s18 182.1 9.34 s19 1.12 s 10.6 1.03 s20 0.96 s 15.1 0.98 s

CO2CH3

requiring 6 degrees of unsaturation. The IR spectrum showedstrong bands at 3447 and 1705 cm�1, characteristic of hydroxyland carboxyl groups, respectively. The 13C NMR spectrum dis-played only a carboxyl carbonyl group (d 182.1) and a secondaryalcohol (d 75.6), without any other functionality in the sp2 region(Table 2). This suggested that 4 could be a pentacyclic diterpene.Analysis of the 1H NMR spectrum established the presence of threemethyl groups at d 0.96 (s), 1.12 (s) and 1.13 (s). Furthermore, thetwo highfield resonances at dH 0.58 (br d) and 0.83 (dd, J = 7.2,2.7 Hz) indicated the presence of a cyclopropane ring, characteris-tic of the trachylobane structures (Fraga, 1994). Comparison of theNMR data of 4 with those of ent-trachyloban-18-oic acid (7) (Leongand Harrison, 1997) suggested that their structures were mostlyidentical, except that the former has one additional secondaryalcohol in the molecule. This secondary alcohol in compound 4was placed at C-3 by HMBC correlations between the methinehydrogen (d 3.95, dd, J = 11.4, 5.1 Hz, H-3) and C-1(d 37.1), C-4 (d53.4), C-18 (d 182.1) and C-19 (d 10.6). The strong cross-peak be-tween H-3 and H-5, and the correlation between Me-19 and Me-20, observed in the NOESY spectrum, permitted us to conclude thatthe C-3 hydroxyl and the C-19 methyl groups are both in the b-ori-entation. Thus, the structure of compound 4, now reported for thefirst time, was established as ent-3b-hydroxytrachyloban-18-oicacid.

Compound 5 was obtained as a white amorphous solid, ½a�25D

�31.0 (c 0.19, CHCl3) and its molecular formula was determinedto be C20H30O2 from its molecular ion peak at m/z 325.2036[M+Na]+ (calc. for C20H30O2Na, 325.2143) in the HRESITOFMS.The NMR spectra of 5 closely resembled those of 4, except forthe presence of a formyl group (dH 9.34, dC 207.1) in the former in-stead of a carboxyl functionality (Table 2). Our presumption wasfurther confirmed by the HMBC correlations between the formylhydrogen (dH 9.34) and C-3 (d 72.0), C-4 (d 55.2), C-5 (d 48.0),and C-19 (d 8.8). Key NOESY correlation cross-peaks for 5 wereobserved between H-3 and H-5, as well as between Me-19 andMe-20. From the above information and literature data (Scher

ntheses).

6

dC dH dC

37.1 0.97 m 37.21.58 m

25.8 1.55 m 26.18) 72.0 3.95 dd (11.1,5.1) 75.5

55.2 53.748.0 1.47 m 51.022.2 0.98 m 22.6

1.45 m38.1 1.42 m 38.3

40.7 40.753.0 1.15 m 53.036.9 37.519.6 1.66 m 19.7

1.1,3.0) 1.89 ddd (14.7,11.4,2.7)20.4 0.58 br d 20.5

) 24.2 0.82 dd (7.5,2.7) 24.2ed 33.4 1.15 m overlapped 33.3

2.02 d (12.0)50.3 1.22 d (11.1) 50.3

d 1.36 d overlapped22.5 22.420.5 1.12 s 20.5

207.1 178.18.8 1.11 s 10.7

14.9 0.95 s 15.1

3.69 s 52.1

Table 3Cytotoxic activities of the isolated compounds against NCI-H187, KB and MCF7 cancer cell lines.

Compound Cytotoxicity (IC50, lM)

NCI-H187 KB MCF7

Ent-8b-hydroxypimar-15-en-18-oic acid (1) >150 >150 >150Ent-15,16-dihydroxypimar-8(14)-en-18-oic acid (2) >150 >150 >150Ent-pimara-8(14),15-dien-18-oic acid (3) >150 >150 >150Ent-3b-hydroxytrachyloban-18-oic acid (4) >150 >150 >150Ent-3b-hydroxytrachyloban-18-al (5) 55.9 69.4 92.0Methyl ent-3b-hydroxytrachyloban-18-oate (6) 47.2 >150 >150Ent-trachyloban-18-oic acid (7) >150 >150 >150Ent-trachyloban-3b,19-diol (8) >150 92.3 >150Ent-trachyloban-3b,18-diol (9) 49.8 62.1 106.4Ent-trachyloban-3b-ol (10) 94.2 >150 >150Ellipticinea 4.3 1.8 –Doxorubicina 0.2 1.1 17.9Tamoxifena – – 21.3

a Positive control.

128 K.-o. Rayanil et al. / Phytochemistry 89 (2013) 125–130

et al., 2010), compound 5 was elucidated as ent-3b-hydroxytrach-yloban-18-al.

Compound 6 was isolated as a white amorphous solid, ½a�25D

�32.7 (c 0.70, CHCl3) and its molecular formula was establishedas C21H32O3 from the molecular ion peak at m/z 355.2150[M+Na]+ in the HRESITOFMS. The only difference between 6 and4 spectroscopic data (Table 2) was the presence of a methyl esterin the former instead of a carboxylic acid at C-18 position. Accord-ingly, the NMR spectrum of 6 contained a singlet signal at dH 3.69,which showed one bond 1H/13C connectivity with the carbon at dC

52.1 and HMBC correlation with a carbonyl resonance at dC 178.1,confirming the presence of a methoxycarbonyl group. The HMBCcorrelations between C-18 (d 178.1) and H-3 (d 3.95, dd, J = 11.1,5.1 Hz) and H-19 (d 1.11, s) supported our conclusion. The struc-ture of 6 was thus established as methyl ent-3b-hydroxytrachylo-ban-18-oate.

2.2. Biological evaluation

All isolated compounds were evaluated for their cytotoxicityagainst NCI-H187 (human small cell lung cancer), KB (human car-cinoma of the nasopharynx) and MCF7 (human breast cancer) celllines. The results of their cytotoxic activities are shown in Table 3.Overall, ent-trachylobane derivatives isolated from M. alba exhib-ited moderate to weak cytotoxic activities against the three humancell lines while all ent-pimarane diterpenoids were inactive.Methyl ent-3b-hydroxytrachyloban-18-oate (6) showed the high-est activity against NCI-H187 cell lines (IC50: 47.2 lM) followedby 9 (IC50: 49.8 lM) and 5 (IC50: 55.9 lM). Compound 9 alsoshowed moderate activity against KB cell lines with an IC50 valueof 62.1 lM, while the rest of the trachylobanes were weakly activeor inactive.

3. Conclusions

This first phytochemical investigation of M. alba establishedthat diterpenoids were the major chemical constituents. Fivenew diterpenoids (1–2, 4–6) and five known ones (3, 7–10) wereisolated for the first time from this genus. These findings also com-plemented previous reports of the occurrence of diterpenes in theMitrephora genus; however, the ent-pimarane derivatives havebeen isolated from this genus for the first time. Interestingly, onthe basis of human cancer cell cytotoxicity data, the ent-trachylo-bane structure seems to enhance a greater degree of anticanceractivity than the ent-pimarane skeleton.

4. Experimental

4.1. General experimental procedure

Melting points were measured on a Kofler hot stage apparatusand are uncorrected. Optical rotations were measured on a JascoP1010 digital polarimeter. UV spectra were recorded on a HewlettPackard 8453 UV–VIS spectrometer. IR spectra were obtained on aPerkin Elmer GX FT-IR spectrophotometer. 1D and 2D NMR exper-iments were recorded on a Brüker AVANCE 300 MHz spectrometeroperating at 300 MHz for proton and 75 MHz for carbon. Massspectra were recorded on a Micromass LCT mass spectrometer,and the lock mass calibration was applied for the determinationof accurate masses. Column chromatography (CC) was carriedout on either silica gel (Merck, 70–230 or 230–400 mesh) orLiChroprep RP-18 (Merck, 40–63 mesh). TLC was performed onMerck precoated silica gel 60 F254 plates and spots were visualizedunder UV light (254 and 365 nm) or by spraying with 1% CeSO4 in10% aqueous H2SO4 followed by heating.

4.2. Plant material

Branches of M. alba were collected in January 2009 from TonPariwat Wildlife Conservation Area, Phang-nga Province, Thailandand were identified by Dr. Piya Chalermglin of the Thailand Insti-tute of Scientific and Technological Research. A voucher specimen(SS614/272) was deposited at the Herbarium of the Faculty ofScience and Technology, Phuket Rajabhat University, Phuket,Thailand.

4.3. Extraction and isolation of compounds

Air-dried and crushed branches of M. alba (2.5 kg) were ex-tracted with EtOH–H2O (3� 5 L, 95:5, v/v) at room temperatureand dried by rotary evaporation to give the ethanolic extract(230 g). The latter was diluted with water and partitioned into hex-ane, EtOAc and n-BuOH. Evaporation of the respective solventsgave the hexane (50 g), EtOAc (6 g) and n-BuOH (19 g) extracts.The hexane extract (50 g) was subjected to silica gel flash CC sep-aration, using a gradient system of hexane–EtOAc (100:0 to 0:100)as eluent to afford 22 fractions (fraction size 1 L). Fraction 1(10.15 g) was purified by silica gel CC using a gradient of hexane/EtOAc (100:0 to 30:70) to give ent-pimara-8(14),15-dien-18-oicacid (3, 432 mg) and ent-trachyloban-18-oic acid (7, 620 mg). Frac-tion 7 (2.00 g) was separated by silica gel CC using hexane/EtOAc(7:1) as mobile phase to afford 13 subfractions (Fr. 7.1–7.13).

K.-o. Rayanil et al. / Phytochemistry 89 (2013) 125–130 129

Subfraction Fr. 7.8 was purified further by additional reversedphase C18 CC, eluted with MeOH/CH3CN (7:1), to afford ent-trach-yloban-3b-ol (10, 10.0 mg). Fraction 8 (1.71 g) was applied to a sil-ica gel column using hexane/EtOAc (15:1, 12:1, 10:1, 8:1 and 5:1)as eluent to yield compound 10 (63.4 mg). Fraction 9 (3.04 g) wassubjected to a silica gel CC using hexane/EtOAc/benzene (5:1:1) aseluent to give five subfractions (Fr. 9.1–9.5). Further purification ofFr. 9.3 (230 mg) by silica gel CC using hexane/EtOAc (5:1) as eluentafforded methyl ent-3b-hydroxytrachyloban-18-oate (6, 37.2 mg).Fraction 11 (0.73 g) was separated by a silica gel CC employinghexane/EtOAc as gradient mixtures to afford eight subfractions(Fr. 11.1–11.8). Fraction 11.2 (259 mg) was further purified by sil-ica gel CC using a gradient of hexane/EtOAc (4:1 to 2:1) to give ent-8b-hydroxypimar-15-en-18-oic acid (1, 41.6 mg) and ent-trachylo-ban-3b,19-diol (8, 39.9 mg). Fraction 12 (1.23 g) was purified usingsilica gel CC, eluted with hexane/EtOAc (5:1) to give 11 subfrac-tions (Fr. 12.1–12.11). Further separation of Fr. 12.8 (230 mg) bysilica gel CC using hexane/EtOAc/benzene (5:1:1) as mobile phasegave ent-3b-hydroxytrachyloban-18-al (5, 64.3 mg). Fraction 13(1.80 g) was separated by silica gel CC using hexane/EtOAc (2:1)as mobile phase to afford ent-trachyloban-3b-18-diol (9,20.5 mg). Fraction 15 (2.02 g) was subjected to silica gel CC usingCH2Cl2/MeOH/H2O (100:3:1) as eluent, yielding seven subfractions(Fr. 15.1–15.7). Further purification of Fr.15.5 by silica gel CC usingCH2Cl2/hexane/EtOAc (2:1:1) as mobile phase afforded ent-15,16-dihydroxypimar-8(14)-en-18-oic acid (2, 10.2 mg) and ent-3b-hydroxytrachyloban-18-oic acid (4, 8.4 mg).

4.3.1. Ent-8b-hydroxypimar-15-en-18-oic acid (1)White amorphous solid; ½a�25

D �16.4 (c 0.65, CHCl3); IR (KBr):mmax 3445, 2919, 1692, 1635, 1275, 1184 cm�1; HRESITOFMS m/z343.2285 [M+Na]+ (calc. for C20H32O3Na, 343.2249); for 1H and13C NMR (300 and 75 MHz, CDCl3) spectroscopic analysis, seeTable 1.

4.3.2. Ent-15,16-dihydroxypimar-8(14)-en-18-oic acid (2)White amorphous solid; ½a�25

D �37.0 (c 0.65, MeOH); IR (KBr):mmax 3453, 2940, 2871, 1695, 1639, 1249, 1020, 876 cm�1; HRESI-TOFMS m/z 359.2217 [M+Na]+ (calc. for C20H32O4Na, 359.2198);for 1H and 13C NMR (300 and 75 MHz, CDCl3) spectroscopic analy-sis, see Table 1.

4.3.3. Ent-3b-hydroxytrachyloban-18-oic acid (4)White amorphous solid; ½a�25

D �63.3 (c 0.28, CHCl3); IR (KBr):mmax 3447, 2916, 2849, 1705, 1243, 1156 cm�1; HRESITOFMS m/z341.2097 [M+Na]+ (calc. for C20H30O3Na, 341.2093); for 1H and13C NMR (300 and 75 MHz, CDCl3) spectroscopic analysis, seeTable 2.

4.3.4. Ent-3b-hydroxytrachyloban-18-al (5)White amorphous solid; ½a�25

D �31.0 (c 0.19, CHCl3); IR (KBr):mmax 3397, 2917, 2849, 1723, 1267, 1047 cm�1; HRESITOFMS m/z325.2036 [M+Na]+ (calc. for C20H30O2Na, 325.2143); for 1H and13C NMR (300 and 75 MHz, CDCl3) spectroscopic analysis, seeTable 2.

4.3.5. Methyl ent-3b-hydroxytrachyloban-18-oate (6)White amorphous solid; ½a�25

D �32.7 (c 0.70, CHCl3); IR (KBr):mmax 3217, 2922, 2859, 1745, 1642, 1440, 1241, 1152, 1083 cm�1;HRESITOFMS m/z 355.2150 [M+Na]+ (calc. for C21H32O3Na,355.2249); for 1H and 13C NMR (300 and 75 MHz, CDCl3) spectro-scopic analysis, see Table 2.

4.4. Cytotoxicity bioassays

NCI-H187 (Human small cell lung carcinoma, ATCC CRL-5804)was determined by a MTT assay (Plumb et al., 1989). Briefly, cellswere diluted to 105 cells/mL. The test compounds were diluted indistilled H2O and added to microtiter plates in a total volume of100 lL. Plates were incubated at 37 �C, 5% CO2 for 5 days. Fiftymicroliters of a 2 lg/lL MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue) was addedto each well of the plate. The plates were wrapped with aluminumfoil and incubated for 4 h. After incubation, the microplates werespun down at 200g for 5 min. MTT was then removed from thewells and the formazan crystals were dissolved in DMSO(200 lL) and Sorensen’s glycine buffer (25 lL). The OD was readin a microtiter plate reader at the wavelength of 570 nm. MCF7(Human breast adenocarcinoma, ATCC HTB-22) and KB (Humancarcinoma of the nasopharynx, ATCC CCL-17) were determinedby a colorimetric cytotoxicity assay that measured the level of cellgrowth from the cellular protein content according to literature(Skehan et al., 1990). Ellipticin, doxorubicin and tamoxifen wereused as positive controls. DMSO was used as negative control.Briefly, cells at a logarithmic growth phase were harvested and di-luted to 105 cells/mL with fresh medium and mixed gently. Thetest compounds were diluted in distilled H2O and placed intomicrotiter plates in a total volume of 200 lL. The plates were incu-bated at 37 �C, 5% CO2 for 72 h. After the incubation period, thecells were fixed by CCl3CO2H:H2O (1:1). The plates were incubatedat 4 �C for 30 min, washed with tap H2O and air-dried at room tem-perature. Plates were stained with 0.05% sulforhodamine B dis-solved in 1% AcOH for 30 min. After the staining period, SRB wasremoved with 1% AcOH. The plates were air-dried before the bounddye was dissolved with 10 mM Tris base for 5 min on a shaker. TheOD was read on a microtiter plate reader at the wavelength of510 nm.

Acknowledgements

The work was financially supported by Grant MRG5480032from the Thailand Research Fund (TRF), the Commission on HigherEducation (CHE) and the Faculty of Science, Silpakorn University,Nakorn Pathom, Thailand. The authors are grateful to Dr. Piya Chal-ermglin, Thailand Institute of Scientific and Technological Researchand Assoc. Prof. Dr. Uma Prawat, Phuket Rajabhat University, fortheir support in collecting and identifying the plant material.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.phytochem.2013.01.012.

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