Food Chemistry 131 (2012) 972–976

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Food Chemistry

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Norditerpenoids from Agathis macrophylla

Ya Li a, Tong-Tong Wang a, Jie Zhao a, Xiang Shi b, Shu-Chen Hu a, Kun Gao a,⇑a State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR Chinab School of Life Sciences, Lanzhou University, Lanzhou 730000, PR China

a r t i c l e i n f o

Article history:Received 17 June 2011Received in revised form 12 July 2011Accepted 21 September 2011Available online 28 September 2011

Keywords:Agathis macrophyllaNorditerpenoidsPTP1BCytotoxicity

0308-8146/$ - see front matter Crown Copyright � 2doi:10.1016/j.foodchem.2011.09.089

⇑ Corresponding author. Tel.: +86 9318912592; faxE-mail address: npchem@lzu.edu.cn (K. Gao).

a b s t r a c t

Three new norditerpenoids, (4S,5R,9S,10R)-methyl 19-hydroxy-15,16-dinorlabda-8(17),11E-dien-13-oxo-18-oate (1), (4R,5R,9R,10R,13S/R)-13-hydroxypodocarp-8(14)-en-19-oic acid (2/3) were isolatedfrom Agathis macrophylla, together with 11 known diterpenoids. The structures of these compounds,1–14, were established, mainly by spectroscopic analysis. The inactivation of protein tyrosine phospha-tase 1B (PTP1B) by compounds 5–9 and 11–13 was tested and the cytotoxicity of compounds 1–14against HL60 cell and SMMC-7721 cell were evaluated. Biological screening studies indicated that com-pounds 5, 7/8 and 9 were mild inactivators of PTP1B, whilst compounds 1, 4, 6, 7/8, and 10–12 exhibitedmoderate activity against both of the tested cell lines.

Crown Copyright � 2011 Published by Elsevier Ltd. All rights reserved.

1. Introduction

The genus Agathis (Araucariaceae) is composed of 20 speciesgrowing over the tropical zone such as; the Philippines,Viet Nam, Malay Peninsula and Australia (Chen, Li, & Chen,1997). Many of these species are used widely in traditional medi-cine in China, and the resulting phytochemical investigations have,in turn, uncovered a variety of diterpenoids and biflavones fromthese species (Cambie et al., 1989; Khan et al., 1972; Smith, Marty,& Peters, 1981). Diterpenoids exhibit diverse biological activitiessuch as antibacterial, antifungal, antiprotozoal, anti-inflammatoryactivities and cytotoxic activity (Abe et al., 2006; Cheung, Miyase,Lenguyen, & Smal, 1993; Dimas et al., 2006; Linhua, Heras, & Hoult,1996). Moreover, there have been few reports on the inhibitoryactivity of diterpenoids against protein tyrosine phosphatase 1B(PTP1B) (Han et al., 2005; Kate, Aubry, Tremblay, & Kerr, 2008;Kim et al., 2006; Na et al., 2006), and diterpenoids have been ofinterest to natural product scientists as leads for new therapeuticagents. PTP1B acts as a negative regulator of insulin signalling,and there is substantial evidence supporting PTP1B as the criticalPTP controlling insulin signalling pathway. Selective inhibition ofPTP1B has served as a potential drug target for the treatment oftype 2 diabetes. Although there have been a number of reportson the design and development of synthetic PTP1B inhibitors, onlya few studies have been reported on PTP1B inhibitors derived fromplants (Han et al., 2005; Kate et al., 2008; Kim et al., 2006; Na et al.,2006).

011 Published by Elsevier Ltd. All r

: +86 9318912582.

Agathis macrophylla is a tall, evergreen arbor and commonlygrows to a height of over 20 m. No chemical constituent of thisspecies has been reported. We conducted a systematic phytochem-ical study on the branches and leaves of A. macrophylla, and haveisolated and identified eight labdane and six podocarpane diterpe-noids. The inactivation of PTP1B by compounds 5–9 and 11–13 wastested and the cytotoxicity of compounds 1–14 (Fig. 1) against theHL60 cell and the SMMC-7721 cell was evaluated. In the presentpaper, work was undertaken to deal with isolation and character-ization of three new norditerpenoids, one labdane (1), and twopodocarpane derivatives (2 and 3) from A. macrophylla. Bioactiveevaluations of compounds 1–14 are also described herein.

2. Materials and methods

2.1. General methods

Optical rotations were taken on a Perkin–Elmer 341 polarime-ter. UV detections were measured on a Shimadzu UV-260 spectro-photometer. CD spectra were obtained on an Olis DSM 1000spectrometer. IR spectra were recorded on a Nicolet NEXUS 670FT-IR spectrometer. NMR spectra were recorded on VarianMercury-300BB NMR (300 MHz) and Varian Mercury plus-400(400 MHz) spectrometers with TMS as internal standard. EIMS datawere collected on a HP5988A GCMS spectrometer. HRESIMS datawere measured on a Bruker Daltonics APEX II 47e spectrometer.Silica gel (200–300 mesh) used for column chromatography (CC)and silica gel GF254 (10–40 lm) used for thin layer chromatogra-phy (TLC) were supplied by Qingdao Marine Chemical Factory,Qingdao, PR China. Sephadex LH-20 (Mitsubishi Co., Tokyo, Japan)

ights reserved.

Fig. 1. The structures of compounds 1–14.

Y. Li et al. / Food Chemistry 131 (2012) 972–976 973

and MCI-gel Diaion HP-20 (Mitsubishi Co., Tokyo, Japan) were usedfor sample pretreatment. Spots were detected on TLC under UVlight, or by heating after spraying with 5% H2SO4 in C2H5OH (v/v).

2.2. Plant material

The air-dried, and partially chopped, aerial parts of A. macrophy-lla were collected in Jianfengling National Forest Park, HainanProvince, PR China, in August 2007. The plant was identified byProf. Guo-Liang Zhang, School of Life Sciences, Lanzhou University.A specimen (No. 20070904) was deposited in the College of Chem-istry and Chemical Engineering, Lanzhou University.

2.3. Extraction and isolation

Leaves and branches of A. macrophylla (7.6 kg) were powderedand extracted with 95% C2H5OH (8 l � 3, each extraction lasted7 days), at room temperature. The combined extracts were concen-trated in vacuum to obtain a residue of 1200 g, which was ex-tracted successively with petroleum ether, EtOAc, and n-BuOH.The EtOAc fraction (450 g) was applied to a Silica gel column andeluted with a gradient of petroleum ether:acetone (1:0, 20:1,10:1, 5:1, 2:1, 1:1, 0:1) to give seven fractions (A–G). Fraction B(30 g) was subjected to column chromatography, using petroleumether:acetone (30:1, 10:1, 5:1, 2:1) to give four crude fractions(B1–B4). Fraction B1 (3.5 g) was further fractionated on a silica gelcolumn (70 g) using petroleum ether:EtOAc (20:1, 10:1, 5:1) toobtain compound 10 (15 mg), 12 (5 mg) and a mixture of 4 and5, which was purified by preparative TLC using petroleumether:acetone (15:1, �3) to give pure 4 (3 mg) and 5 (30 mg). Frac-tion B3 (4 g) was fractionated on a silica gel column (80 g) usingpetroleum ether:acetone (20:1, 15:1, 8:1) to obtain compounds 9(15 mg) and 11 (10 mg). Fraction B4 (2 g) was subjected to column

chromatography, on silica gel (40 g), and eluted with petroleumether:acetone (20:1, 15:1, 8:1) to give compound 6 (15 mg). Frac-tion C (15 g) was chromatographed, using petroleum ether:acetone(15:1, 10:1, 5:1), to give three crude fractions (C1–C3), which weresubjected to passage over a Sephadex LH-20 column (2 � 150 cm),eluted with CHCl3:CH3OH (1:1). Fraction C1 (2.8 g) was furtherfractionated on a silica gel column (56 g) using petroleumether:acetone (13:1) to obtain compound 13 (21 mg) and 14(25 mg). Fraction C3 (4 g) was purified by preparative TLC usingCHCl3:EtOAc (100:8, �6) to give a mixture of 7 and 8 (15 mg). Frac-tion D was firstly subjected to passage over a Sephadex LH-20column (2 � 150 cm), eluted with CHCl3:CH3OH (1:1) to removepigments, and then followed by a gradient of CHCl3:EtOAc (15:1,10:1, 8:1), to give two crude fractions (D1, D2). Fraction D1 (5.0 g)was further fractionated on a silica gel column (50 g), using petro-leum CHCl3:EtOAc (10:1), to obtain compound 1 (10 mg) and amixture of 2 and 3 which was chromatographed with CHCl3:actone(100:8), to give pure compound 2 (10 mg) and 3 (3 mg).

2.4. Characteristic data of compounds

(4S,5R,9S,10R)-Methyl 19-hydroxy-15,16-dinorlabda-8(17),11E-dien-13-oxo-18-oate (1): a colourless oil; ½a�20

D +11 (c 1.0,MeOH); UV/Vis (CHCl3) kmax (loge) 242 (2.38) nm; IR (film) mmax

3429, 2933, 2876, 2832, 1722, 1671 cm�1; 1H (CDCl3, 400 MHz)and 13C (CDCl3, 100 MHz) NMR see Table 1; HRESIMS m/z321.2054 (calcd for C19H29O4, 321.2060).

(4R,5R,9R,10R,13S)-13-Hydroxypodocarp-8(14)-en-19-oic acid(2): a colourless powder; ½a�20

D �11 (c 2.0, MeOH); UV/Vis (CHCl3)kmax (loge) 243 (0.25) nm; IR (film) mmax 3391, 2933, 2866,1695 cm�1; 1H (CDCl3, 400 MHz) and 13C (CDCl3, 100 MHz) NMRsee Table 1; HRESIMS m/z 261.1849 (calcd for C17H25O2, 261.1849).

X-ray crystal data of 2 (CCDC 821746): C17H26O3, Mr = 278.38,orthorhombic, space group P2(1)2(1)2(1), a = 8.057(9) Å, b =8.346(9) Å, c = 23.08(2) Å, V = 1552(3) Å3, Z = 4, Dcalcd = 1.192mg/m3, crystal dimensions 0.25 � 0.22 � 0.21 mm were used formeasurements on a Bruker APEX II area detector diffractometerwith a graphite monochromator, Mo Ka radiation (k = 0.71073 Å).The total number of reflections measured was 7418, of which2879 were unique. Final indices: R1 = 0.0533, wR2 = 0.1214 forobserved reflections, and R1 = 0.0896, wR2 = 0.1417 for allreflections. The crystal structure 2 was solved by direct methods,using SHELX-97, (Sheldrick, G. M. University of Gottingen,Gottingen, Germany, 1990) and expanded using difference Fouriertechniques, refined by SHELX-97.

(4R,5R,9R,10R,13R)-13-Hydroxypodocarp-8(14)-en-19-oic acid(3): a colourless powder; ½a�20

D �9 (c 0.8, MeOH); UV/Vis (CHCl3)kmax (loge) 241 (1.51) nm; IR (film) mmax 3390, 2926, 2855,1694 cm�1; 1H (CDCl3, 400 MHz) and 13C (CDCl3, 100 MHz) NMRsee Table 1; HRESIMS m/z 261.1849 (calcd for C17H25O2, 261.1849).

2.5. Cytotoxicity assay

Cell proliferation was assessed by a sulforhodamine B (SRB)cytotoxicity assay (sulforhodamine B) SRB method. The cytotoxic-ity of HEM and HCPT toward cells HO-8910 was determined in96-well microtiter plates, by the sulforhodamine B method,described by Skehan, Storeng, and Scudiero (1990). Briefly, expo-nentially growing HO-8910 cells were harvested and seeded in96-well plates, with the final volume 100 ll containing 4 � 103

cells per well. After 24 h incubation, cells were treated with variousconcentrations of HEM, or HCPT, for 48 h. The cultures were fixedat 4 �C for 1 h, by addition of ice-cold 50% trichloroacetic acid(TCA), to give a final concentration of 10%. Fixed cells were rinsed5 times with deionized water and stained for 10 min with 0.4% sul-forhodamine B dissolved in 0.1% acetic acid. The wells were

Table 11H and 13C NMR spectroscopic data for compounds 1, 2 and 3 in CDCl3 solution.

No. 1 2 3

dH (J in Hz) dC dH (J in Hz) dC dH (J in Hz) dC

1 1.16 m 32.2 CH2 1.11 m 37.9 CH2 1.14 dd (4.8, 6.4) 38.2 CH2

2.30 br d (8.0) 1.69 m 1.77 m2 1.59 m 19.0 CH2 1.59 m 18.0 CH2 1.59 m 18.1 CH2

1.89 m 1.76 m 1.85 m3 1.08 m 40.4 CH2 1.61 m 37.0 CH2 1.61 dd (3.2 6.4) 37.0 CH2

1.46 m 1.79 m 1.74 m4 50.2 C 47.1 C 47.2 C5 1.52 (dd 2.8, 10.4) 49.6 CH 1.90 m 50.0 CH 1.97 dd (2.4, 12.4) 51.1 CH6 1.71 m 24.9 CH2 1.33 m 24.7 CH2 1.32 m 24.6 CH2

1.92 m 1.48 br dd (8.4, 13.2) 1.62 dd (2.4 12.4)7 2.01 m 36.7 CH2 2.11 m 34.9 CH2 2.12 br d (12.4) 35.1 CH2

2.45 brd (12.0) 2.29 br dd (2.0, 4.4) 2.28 ddd (3.2, 4.4, 12.8)8 147.6 C 140.1 C 143.3 C9 2.50 d (6.4) 60.0 CH 1.94 dd (2.8, 12.4) 48.7 CH 1.85 m 48.6 CH10 39.4 C 37.8 C 37.6 C11 6.84 dd (6.4 16.0) 145.7 CH 1.29 m 19.6 CH2 1.59 m 16.3 CH2

1.77 dd (5.2, 12.4) 1.79 m12 6.08 d (16.0) 133.8 CH 1.23 m 32.6 CH2 1.52 dd (3.6 8.8) 30.4 CH2

2.06 m 1.76 m13 198.0 C 4.13 br s 67.4 CH 4.11 d (2.8) 64.3 CH14 2.33 s 27.3 CH3 5.46 br s 126.7 CH 5.65 t (2.8) 123.6 CH17 4.81 d (1.6) 108.9 CH2

4.30 d (1.6)18 70.8 CH2 1.19 s 16.8 CH3 1.14 s 16.8 CH3

19 3.94 d (6.4) 175.6 C 184.3 C 184.1 C3.49 d (6.4)

20 0.76 s 13.8 CH3 0.77 s 14.7 CH3 0.78 s 15.1 CH3

OCH3 3.70 s 51.6 CH3

974 Y. Li et al. / Food Chemistry 131 (2012) 972–976

washed 5 times with 0.1% acetic acid and left to dry overnight. Theabsorbed sulforhodamine B was dissolved in 150 ll unbuffered 1%Tris base [tris(hydroxymethyl) aminomethane] solution in water(pH 10.5). The absorbance extracted sulforhodamine B at 515 nmwas measured on a microplate reader (Bio-Rad). The experimentswere carried out in triplicate. Each run entailed 5–6 concentrationsof the compounds being tested. The percentage survival rates ofcells exposed to the compounds were calculated by assuming thesurvival rate of untreated cells to be 100%.

2.6. Time-dependent inactivation of PTP1B

Recombinant PTP1B was prepared in Dr. Kent S. Gates’ labora-tory (Departments of Chemistry and Biochemistry, University ofMissouri, Columbia, Missouri) (LaButti, Chowdhury, Reilly, & Gates,2007). Inactivation assays were performed using existing literatureprotocols (Montalibet, Skorey, & Kennedy, 2005). Free thiols wereremoved from a stock solution of purified PTP1B using Zeba minicentrifugal buffer exchange columns (Pierce, catalogue no.89882), according to the manufacturer’s protocol. The exchangebuffer contained 50 mM Bis–Tris, 50 mM Tris, 100 mM NaOAc,10 mM DTPA and 0.5% Tween 80, pH 7.0. In the inactivation reac-tions, PTP1B (�400 nM final concentration) was added as a stocksolution, in exchange buffer, to chemicals at various concentra-tions, in exchange buffer with 10% DMSO at 25 �C. Aliquots(10 ll) were removed at various time points and placed in 490 llof assay buffer (pH 6.0), consisting of 50 mM Bis–Tris, 100 mMNaCl, 10 mM DTPA and p-nitrophenyl phosphate (p-NPP)(20 mM) at 30 �C for 10 min. The enzymatic reaction was quenchedby addition of NaOH (500 ll of a 2 N solution, in water), and theamount of p-nitrophenol released during the assay was deter-mined at 25 �C by measuring the absorbance at 410 nm using aUV/Vis spectrometer (Hewlett–Packard diode array spectropho-tometer 8452A equipped with a temperature-controlled Peltier cellholder). The apparent first order rate constant (kobs) for the inacti-vation of PTP1B by chemicals, at each concentration, was calcu-lated by the method of Voet and co-workers (Kraut et al., 2000).

The apparent first order rate constants (kobs) for the inactivationof PTP1B by chemicals were plotted versus concentration and thesecond-order rate constant (ki), for the inactivation of PTP1B bychemicals, were extracted from the slope. The ki values are themeans ± SD from three independent experiments.

3. Results and discussion

3.1. Phytochemical investigation

Compound 1 was isolated as colourless oil. Its molecular for-mula was established as C19H28O4 by high-resolution electrosprayionization mass spectrometry (HRESIMS) at m/z 321.2054 [M+H]+

(calcd 321.2060), indicating six degrees of unsaturation. The IRabsorption bands at 3429, 1722, 1671 cm�1 implied the presenceof hydroxyl, carbonyl and a,b-unsaturated keto groups. The 1HNMR spectrum (Table 1) showed signals for three methyl groupsat d 0.76 (3H, s), 2.33 (3H, s), 3.70 (3H, s) (one of which was themethoxyl group), an oxygenated CH2 at d 3.49 (1H, d, J = 6.4 Hz)and 3.94 (1H, d, J = 6.4 Hz), an exomethylene group at d 4.30 (1H,d, J = 1.6 Hz) and 4.81 (1H, d, J = 1.6 Hz) (which were characteristicof a labdane-type diterpene), as well as olefinic protons at d 6.08(1H, d, J = 16.0 Hz) and 6.84 (1H, dd, J = 6.4, 16.0 Hz). The 13CNMR and DEPT spectra (Table 1) of 1 supported the above analysis,showing three methyl groups at d 13.8, 27.3 and 51.6, seven meth-ylenes (including an oxygenated CH2 at d 70.8 and an olefinic CH2

at d 108.9), four methines (including two olefinic CH at d 133.8 and145.7), five quaternary carbons (including two carbonyl carbons atd 198.0, 175.6 and an olefinic carbon at d 147.6). The above infor-mation suggested that 1 was a dinorlabdane diterpenoid. In theHMBC spectrum (Fig. 2), the correlations from H-12 (d 6.08) toC-14 (d 27.3), C-9 (d 60.0) and C-13 (d198.0), and from H-11(d 6.84) to C-9 (d 60.0), C-10 (d 39.4) and the carbonyl signal at d198.0 (C-13) allowed the construction of the side-chain (C-11 toC-14), and the correlations from H-OCH3 (d 3.70) to C-18(d 175.6) indicated that OCH3 was attached at C-18. The geometryof D11 was determined to be E on the basis of the large coupling

O

H

HOH

NOE 1D

HHHCOOCH3OH

O

HMBC H C

HOOC

Fig. 2. Selected HMBC and NOE correlations of 1.

Y. Li et al. / Food Chemistry 131 (2012) 972–976 975

constant (J = 16.0 Hz) of H-11 and H-12. In the NOE experiments(Fig. 2), irradiation of H-9 enhanced the signals of H-7a (5.07%),H-12 (1.90%) and H-5 (2.44%), and irradiation of H-19 enhancedthe signals of H-5 (2.62%) and H-2a (3.39%). These informationsindicated that H-5, H-9 and H-19 were on the same side. Com-pound 1 was recognized as members of normal-labdane seriesthrough its positive [a]D value (David, David, Yang, & Cordell,1999). Which was confirmed by applying the octant rule to the po-sitive Cotton effect, observed at 218 nm (De = +1.88), due to theexocyclic double bond in 1 (Fig. 3) (Scott & Wrixon, 1970). Thus,the structure of compound 1 was determined as (4S,5R,9S,10R)-methyl 19-hydroxy-15,16-dinorlabda-8(17),11E-dien-13-oxo-18-oate. To our best knowledge, only seven structurally relatedcompounds have been reported before, all of them being 15,16-dinorditerpenoid with different oxygenation patterns, suggest-ing that they may be oxidized derivatives of a common C20 labdaneprecursor (Yang, Ye, Feng, & Zhao, 2007).

Compound 2 was obtained as a colourless powder. The molecularformula was determined to be C17H26O3 by HRESIMS spectroscopicdata (m/z 261.1849 [M-H2O+H]+, calcd 261.1849). Its IR spectrumshowed absorption bands at mmax 3391 and 1695 cm�1, representinghydroxyl and carboxylic acid groups, respectively. In the 1H and 13CNMR spectra (Table 1), characteristic signals appeared for twomethyl groups [dH 0.77 (3H, s)/dC 14.7, dH 1.21(3H, s)/dC 16.8] anda carboxylic acid group (dC 184.3), no isopropyl group was observedin its NMR spectra. The above data, along with the appearance of 17signals in the 13C NMR spectrum, indicated that 2 is an analogue ofthe podocarpane-type norditerpenoid (Kuo & Chang, 2000; Zhang

Fig. 3. Octant projections and CD spectra of 1 and 2.

et al., 1999). Furthermore, the 1H and 13C NMR spectra of 2 weresimilar to that of 5 (Yang et al., 2008), with the absence of signalsfor a keto group and the presence of signals for a hydroxyl methineat dC 67.4/dH 4.13 (CH-13). On the basis of the above spectroscopicdata, the planar structure of 2 was deduced as 13-hydroxypodo-carp-8(14)-en-19-oic acid. In the NOESY spectrum of 2, the correla-tion of signals at d 0.77 (H-20) with d 1.19 (H-18) and d 4.13 (H-13)showed that compound 2 had OH-13a and COOH-18a substituentsand the relative configuration of 2 were confirmed by X-ray crystal-lography (Fig. 4). The absolute stereochemistry of 2 was substanti-ated by applying the octant rule to the positive Cotton effect,observed at 203 nm (De = +4.95), due to the endo-double bondmoiety in 2 (Fig. 3) (Scott & Wrixon, 1970). Thus, compound 2 wasdeduced to be (4R,5R,9R,10R,13S)-13-hydroxypodocarp-8(14)-en-19-oic acid.

Compound 3 was obtained as a colourless powder. The molecularformula C17H26O3 attributed to 2 was deduced from analysis of HRE-SIMS and 1H and 13C NMR spectroscopic data. The IR spectrumshowed absorption bands for a hydroxyl group at 3390 cm�1 anda carboxylic acid group at 1694 cm�1. The 1H and 13C NMR spectraldata of 3 (Table 1) were similar to those of 2. The major differenceswere that the carbon signals for C-11, C-12, C-13 and C-14 of 3 wereshifted to high field (d 16.3, 30.4, 64.3, 123.6), compared to those of 2(d 19.6, 32.6, 67.4, 126.7). Those values indicated that 2 and 3 wereepimeric at C-13. Thus, compound 3 was assigned to be(4R,5R,9R,10R,13R)-13-hydroxypodocarp-8(14)-en-19-oic acid.

The 11 known compounds were identified as 15-nor-14-oxolab-da-8(17),12E-dien-19-oic acid (4) (Hsieh, Fang, & Cheng, 1998),13-oxo-podocarp-8(14)-en-19-oic acid (5) (Yang et al., 2008), 13-oxo-podocarp-8(14)-en-19-oate (6) (Abad, Arnó, Peiró, & Zaragozá,1991), 16-hydroxy-8(17),13-labdadien-15,16-olid-19-oic acid (7)(Asili et al., 2004), 15n-hydroxypinusolidic acid (8) (Asili et al.,2004), lambertianic acid (9) (Asili et al., 2004), methyl lambertia-nate (10) (Bell & Fetizon, 1976), pinusolidic acid (11) (Asili et al.,2004), pinusolide (12) (Asili et al., 2004), angustanoic acid F (13)(Sy & Brown, 1998), 8,11,13-abietatrien-15-ol (14) (Hsieh et al.,1998), by comparing of their NMR and MS data with those reportedin the literature. Compounds 7 and 8 were isolated as a mixture.

3.2. Inhibitory activity against PTP1B and cytotoxic activity

The inhibitory activities of compounds 5–9 and 11–13 againstPTP1B were evaluated, and the results indicate that compounds5, 7/8 and 9 are moderate time-dependent inactivators of PTP1Bwith ki values of 0.11, 0.07 and 0.058 M�1 s�1.

The cytotoxic activities of compounds 1–14 were evaluatedagainst the HL-60 cell and SMMC-7721 cell, in which mitomycinwas used as the positive control. Unfortunately, none of the tested

Fig. 4. ORTEP diagram of the crystal structure of 2.

Table 2IC50 values (lg/ml) for cytotoxicity of compounds 1–14.

Compounds IC50 (lg /ml)

HL60 cell SMMC-7721 cell

Mitomycin 0.56 ± 0.17 1.85 ± 0.531 89.06 ± 1.39 74.21 ± 5.532 >200 127.55 ± 3.783 >200 74.03 ± 2.984 82.95 ± 1.41 89.20 ± 7.645 >200 90.38 ± 7.206 42.86 ± 0.95 86.94 ± 0.927/8 74.51 ± 3.10 85.67 ± 7.879 69.58 ± 7.32 198.44 ± 5.7210 49.25 ± 1.58 35.36 ± 3.5111 91.03 ± 0.51 124.48 ± 0.8712 31.29 ± 5.28 51.29 ± 0.8913 >200 109.88 ± 6.5614 >200 >200

976 Y. Li et al. / Food Chemistry 131 (2012) 972–976

compounds had significant cytotoxic activity against these celllines (Table 2).

4. Conclusion

Diterpenoids exhibit diverse biological activities, and the genusAgathis is a rich source of diterpenoids. Since 1975, a series of lab-dane-type diterpenoids have been identified and some of themshowed significant cytotoxicities (Zhao et al., 2008). Unfortunatelynone of the labdane and podocarpane diterpenoids isolated from A.macrophylla showed significant cytotoxic activity against thetested cell lines, but some of these diterpenoids are mild time-dependent inactivators of PTP1B. PTPs catalyse hydrolytic removalof tyrosine phosphoryl groups via initial attack of key catalytic cys-teine residue. PTPs work in tandem with PTKs to regulate a varietyof crucial cell signalling pathways in mammals. Inhibition of PTPscan have profound biological effects resulting from modulation ofsignal transduction pathways (Seiner, Labutti, & Gates, 2007). Sev-eral classes of plant-derived secondary metabolites have been de-scribed as PTP1B inhibitors (Kim et al., 2005). To our knowledge,PTP1B inhibitory activity of labdane and podocarpane diterpenoidsis being reported here for the first time in this study, and the pres-ent study reveals the possibility that labdane and podocarpanediterpenoids could be considered as lead compounds for treatingtype 2 diabetes.

The isolation and identification of bioactive compounds fromnatural sources has given insights into the discovery of new drugs,against several diseases. This study may enter into the database ofthe structure–activity relationships of labdane and podocarpanediterpenoids for further development of new therapeutic agents.Natural products continue to play a highly significant role in thedrug discovery and development process. Further investigation ofbiological activities of nature products should be of interest to us.

Acknowledgements

This work was supported by the National Basic ResearchProgramme (973 Programme) of China (No. 2007CB108903), theNational Natural Science Foundation of China (No. 20972062)and the 111 Project of China.

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