Phenolic compounds and their anti-oxidative properties and protein kinase inhibition from the Chinese mangrove plant Laguncularia racemosa

Phytochemistry 71 (2010) 435–442

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Phenolic compounds and their anti-oxidative properties and protein kinase inhibition from the Chinese mangrove plant Laguncularia racemosa Cui Shi a, Min-Juan Xu a, Mirko Bayer b, Zhi-Wei Deng c, Michael H.G. Kubbutat d, Wim Waejen e, Peter Proksch b, Wen-Han Lin a,* a

State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, PR China Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine University, Duesseldorf D-40225, Germany Analytical and Testing Center, Beijing Normal University, Beijing 100875, PR China d ProQinase GmbH, Breisacher Strasse 117, 79106 Freiburg, Germany e Institute of Toxicology, Heinrich-Heine University, Duesseldorf D-40225, Germany b c

a r t i c l e

i n f o

Article history: Received 5 June 2009 Received in revised form 1 October 2009 Available online 18 December 2009 Keywords: Laguncularia racemosa Combretaceae White mangrove Phenolic compounds Structure elucidation Anti-oxidative activity Protein kinase inhibitors Insecticidal activity

a b s t r a c t Phenolic compounds, named integracin D (1), (70 R, 80 S, 8S)-8-hydroxyisoguaiacin (3), (2R, 3R) pinobanksin-3-caffeoylate (5) and threo-8S-7-methoxysyringylglycerol (6), respectively, were isolated from the Chinese mangrove plant Laguncularia racemosa (L) Gaertn. f. (Combretaceae), together with 23 known phenolic metabolites. Their structures were elucidated on the basis of extensive spectroscopic analyses including that of IR, UV, MS, CD, 1D and 2D NMR spectra as well as by comparison with literature data. Compound 5 showed significant anti-oxidative activity in the DPPH and TEAC free-radical-scavenging assays, while several of the phenolic compounds were tested for protein kinase inhibitory activity in an assay involving 24 different human tumor related protein kinases. Compounds 5, 7, and 23 showed potential inhibition with IC50 values between 2.2 and 3.6 lg/mL toward individual kinases. The ellagic acid derivatives were tested for insecticidal activity. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction The white mangrove plant Laguncularia racemosa (L) Gaertn. f. (Combretaceae) is an evergreen tree, and is a common species in mangrove forests along the Pacific and Atlantic coasts and tropical southern Asia (Laura et al., 2004). A bark infusion is historically used as an astringent and tonic, and as a folk remedy for dysentery, aphthae, fever and scurvy (Bandaranayake, 1998; List and Horhammer, 1969–1970). Morton reported that the antitumor activity of this plant was attributed to its tannin content (Morton, 1965), and its leaves possess antibiotic properties against Escherichia coli (Collier et al., 1952). L. racemosa is known to contain high contents of tannins (De-Godoy et al., 1997), significant quantities of b-sitosterol and lupeol (Koch et al., 2003), and polysaccharides (Gladys et al., 1993). However, natural products of this species growing in the mangrove forest of South China have rarely been studied, except for a sulfated nor-sesquiterpene and a sulfated lipid reported recently (Xue et al., 2008). In the course of our systematic investigation of the chemical diversity and the bio-function of Chinese mangrove plants, L. racemosa was collected from the mangrove forest of Hainan Island, PR China. The EtOH extract of its twigs and * Corresponding author. Tel.: +86 10 82806188; fax: +86 10 82802724. E-mail address: [email protected] (W.-H. Lin). 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.11.008

bark showed moderate cytotoxic activities against several tumor cell lines. A comprehensive chromatographic separation of the bioactive extract resulted in the isolation of 27 different phenolic constituents (Fig. 1), including four new compounds (1, 3, 5, and 6). Compound 1 is a new dimeric alkyl aromatic compound of polyketide origin for which we proposed the trivial name integracin D, as its structure is closely related to the potential anti-HIV-1 integrase inhibitors integracins A–C (Singh et al., 2002). Compounds 3, 5, and 6 are the derivatives of cyclolignan, dihydroflavone, and phenylglycerol, respectively. Their structures were determined based on 1D and 2D NMR, IR, and MS spectroscopic data along with comparison with literature data. The isolated compounds were evaluated in various bioassay models, including anti-oxidative potential, protein kinase inhibition, and insecticidal activity. In this paper, we report the isolation and structure elucidation of the new compounds and their bioactivities.

2. Results and discussion 2.1. Structural determination Compound 1, a colorless oil, has the molecular formula of C37H56O11S as determined by analysis of the HRESIMS at m/z

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C. Shi et al. / Phytochemistry 71 (2010) 435–442

R

1

3

17

14

7

O 5

OH

HO

7'

2'

O 8'

1'

15'

6'

1. R = OSO3H 2. R = OH HO

O

7

4 5

OH

O

2

OH

7'

2

3

R2 9 R1

8

7

R3

8'

1'

R5

3' OMe OH 3. R1 = OH, R2 = R3 = CH3, R4 = R5 = H 4. R1 = R3 =CH2OH, R2 = H, R4 = R5 = OCH3 4'

MeO

1"

O 6"

OH OH

O

HO O

R2

R5

MeO

8

HO

O

HO

OH OH

6

O

HO HO HO

R3 R4

13. R1 = R5 = H, R2 = R4 = OMe, R3 = OH 14. R1 = R4 = R5 = H, R3 = OH, R2 = OMe 15. R5 = R2 = R4 = H, R3 = OH, R1 = OMe 16. R1 = CH2OH, R2 = R3 = R4 = R5 = H 17. R1 = R5 = OMe, R3 = OH, R2 = R4 = H 18. R1 = OMe, R2 = R4 = R5 = H, R3 = CH2OH

O

7

R1

O

HO HO

OH

9

O

R

HO OMe R1 R2 O

O

O

R2

O

11. R = H 12. R = OMe

R1

HO

7

OH

8. R1 = H, R2 = OH 9. R1 = R2 = H 10. R1 = R2 = OH

O

OMe

1

4

HO

R1

HO

HO

HO

R2

HO HO

4

R4

O

O

3

7"

O

HO

1

1'

5

OH

5

5'

O

4'

4"

HO

18'

MeO

R4

O R3 O 24. R1 = R2 = R3 = R4 = OH 25. R1 = R2 = R3 = R4 = OMe 26. R1 = R3 = R4 = OMe, R2 = OH 27. R1 = R3 = OMe, R2 = R4 = OH

O

O

R3 HO 19. R1 = R2 = R3 = OMe 20. R1 = R2 = R3 = H O MeO H Glu

HO OMe 23

OH OH

HO OMe HO O

21 OH O OMe 22

Fig. 1. Structures of the phenolic compounds.

707.3448 [MH] and the NMR spectroscopic data. The IR spectrum showed absorption bands at 3400, 1736, 1713, and 1664 cm1, characteristic of hydroxyl, carbonyl, and aromatic groups, whereas the UV absorption maxima at 263 and 299 nm were typical of a benzoate functionality. The 13C NMR and DEPT spectra exhibited resonances (Table 1) including 12 aromatic carbons for two substituted aromatic rings, two oxymethines, two ester carbonyl carbons, and three methyl groups. The remaining signals were attributed to partially overlapped aliphatic methylene groups which accounted for 18 carbons. The 1H NMR spectrum displayed two methyl triplets (d 0.84 and 0.87) typical of terminal methyl protons, two oxymethine protons at d 5.04 and 4.76, and an acetyl methyl singlet at d 1.97. Five aromatic protons for two spin systems were recognized, in which the weak COSY coupling signals between d 6.14 (1H, br s, H-30 ) and 6.13 (1H, br s, H-50 ) were attributed to meta-coupled aromatic protons, while the weak COSY coupling resonances among d 6.38 (1H, br s, H-2), 6.50 (1H, br s, H4), and 6.27 (1H, br s, H-6) were assigned to a 1,3,5-tri-substituted phenyl ring. The long-range COSY correlations between H-2 (or H6) and the methylene protons at d 2.40 (2H, t, J = 7.5 Hz, H-7), and between H-50 and the methylene protons at d 2.55 (2H, m, H-80 ), along with the TOCSY data, indicated that each aromatic ring was connected to an alkyl side-chain. The HMBC correlated the oxymethine at d 5.04 (1H, m, H-14) and Me-17 to C-15 and C-16, and the oxymethine at d 4.76 (1H, m, H-150 ) and Me-180 to C-160 and C-170 (Fig. 2), confirming the positions of the oxygenated car-

bons as C-14 and C-150 , respectively. Additional HMBC correlations established that one of the oxymethines (C-150 ) was connected to an acetyl group, while the other (C-14) formed an ester with a benzoic acid. Comparison of the 1H and 13C NMR spectroscopic data of 1 (Table 1) with those reported for integracin A (2) (Singh et al., 2002) indicated that both compounds were structurally similar. The difference was due to the presence of a sulfate group in 1, as evident from the molecular composition. The downfield shifts of H-2 and H-4, along with the upfield shifted C-3 (d 154.6), compared to corresponding shifts of 2 indicated that the sulfate is at C-3 of the aromatic ring. The ion peak at m/z 359, as derived by cleavage of the benzonate ester bond provided the alkyl length of C11, as was the case for 2. To confirm the alkyl length assignment, compound 1 was hydrolyzed by 1M HCl–MeOH for 15 h at 75 °C to afford the subunits of la and lb (Fig. 3). The ESIMS spectra showed m/z 281 [M+H]+ for 1a and m/z 339 [M+H]+ for 1b, indicating that both subunits contain an undecyl side-chain. Comparison of optical 20 rotation values between 1a/1b (½a20 D +8.7 for 1a, ½aD +6.4 for 1b) 25 and the literature value (½aD +9.7) for (S)-1-phenylundecan-3-ol (Manabu et al., 2000), in contrast to the R form of the latter, allowed the assignment of 14S and 150 S of 1. Thus, the structure of 1 was determined to be the C-3 sulfated integracin A, namely integracin D. The HRESIMS spectrum of 3 provided a pseudo-molecular ion peak at m/z 367.1512 [M+Na]+, which corresponds to the molecular formula C20H24O5, implying nine degrees of unsaturation. The IR

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C. Shi et al. / Phytochemistry 71 (2010) 435–442 Table 1 1 H and 13C NMR spectroscopic data and COSY and HMBC correlations of 1.a dC 1 2 3 4 5 6 7 8 9–11 12 13 14 15 16 17 10 20 30 40 50 60 70 80 90 100 –120 130 140 150 160 170 180 Ac0 a

144.0 111.6 154.6 105.5 157.8 110.5 35.7 31.2 29.2–29.6 25.3 34.1 74.8 36.3 18.5 14.3 109.0 157.7 100.8 157.7 109.2 144.9 169.8 35.0 31.9 29.2–29.6 25.2 34.0 73.5 36.2 18.6 14.2 170.7 21.3

dH

COSY

HMBC

6.38 (br s)

H-4, H-6, H-7

C-3, C-4, C-6, C-7

6.50 (br s)

H-2, H-6

C-2, C-3, C-5, C-6

6.27 (br s) 2.40 (t, 7.5) 1.49 (m) 1.25–1.28 (m) 1.25 (m) 1.57 (m) 5.04 (m) 1.57 (m) 1.35 (m) 0.84 (t, 7.0)

H-2, H-4, H-7 H-2, H-6, H2-8

C-2, C-4, C-5, C-7 C-1, C-2, C-6, C-8, C-9

H2-13, H2-15

C-70 , C-12, C-13, C-15, C-16

H2-16

C-15, C-16

6.14 (br s)

H-50

C-10 , C-20 , C-40 , C-50

6.13 (br s)

H-30 , H2-80

C-10 , C-30 , C-40 , C-80

2.55 (m) 1.45 (m) 1.25–1.28 (m) 1.25 (m) 1.45 (m) 4.76 (m) 1.45 (m) 1.35 (m) 0.87 (t, 7.0)

H-50 , H2-90

C-10 , C-50 , C-60 , C-90 , C-100

H2-140 , H2-16

C@ O(Ac), C-130 , C-140 , C-160 , C-170

H2-170

C-160 , C-170

1.97 (s)

Measured in DMSO-d6.

absorption at 3400 cm1 suggested the presence of a hydroxyl group. The 1H NMR spectrum displayed an ABX aromatic spin system at d 6.58 (1H, d, J = 1.5 Hz), 6.66 (1H, dd, J = 1.5, 8.0 Hz), and 6.85 (1H, d, J = 8.0 Hz), two aromatic singlet protons at d 6.31 (1H, s) and 6.55 (1H, s) typical of a tetra-substituted phenyl ring,

H

HO3SO H

1

3

5

H 7

14

H

OH

HO

O

17 7'

O

2'

H

8'

1'

H HMBC COSY, TOCSY

4'

18'

15'

6'

O

H

OH

O

Fig. 2. Main HMBC, TOCSY and COSY correlations of 1.

HO3SO 5

OH

O

two methoxy groups at d 3.89 (3H, s) and 3.84 (3H, s), two methyl groups at d 0.92 (3H, d, J = 6.5 Hz) and 1.19 (3H, s), and two isolated germinal protons at d 2.83 (1H, d, J = 15 Hz) and 3.06 (1H, d, J = 15 Hz), as well as two vicinal coupled methines at d 2.03 (1H, qd, J = 6.5, 11 Hz) and 3.39 (1H, d, J = 11 Hz). The HMQC and COSY spectra allow the assignment of all protonated carbons in the molecule. Analysis of 1H and 13C NMR spectroscopic data indicated that the structure of 3 was closely related to the known cyclolignan isoguaiacin (King and Wilson, 1964). However, an oxygenated quaternary carbon at C-8 (d 72.4, qC) and the methyl singlet at d 1.19 (3H, s, H-9) of 3 suggested that C-8 was hydroxylated. This was further confirmed by the HMBC correlations from H3-9 to C-7 (d 46.7, t), C-8, and C-80 (d 47.0, d). The positions of the two methoxy groups at C-5 and C-30 of the aromatic rings were determined by the NOE relationship between MeO (d 3.89)/H-6 (d 6.31), MeO (d 3.84)/H-20 (d 6.58), and H-6/H2-7 (d 2.83, 3.06) (Fig. 4). The stereochemistry of 3 was determined from analysis of the J values, NOESY, together with the CD spectrum. The coupling con-

HCl/MeOH

O

HO OH

HO

OH 5

OH

O

O O

1a m/z 281 [M + H]+

O

HO OH OH

Fig. 3. Methanolysis of 1.

1b m/z 339 [M + H]+

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C. Shi et al. / Phytochemistry 71 (2010) 435–442

Fig. 4. NOESY correlations of 3.

stant of JH-70 /H-80 = 11.0 Hz was attributed to the axial-orientation of H-70 and H-80 . Irradiation of H-70 caused NOE enhancements of H390 and H3-9 in 1D NOE (GOESY) spectra and indicated that both methyl groups were positioned on the same side as H-70 . The absolute configuration of C-70 was assessed by CD spectral analysis. Ohashi et al. (1994) reported the Cotton effect (CE) of the first couplets corresponding to the B-absorption band reflect the aryl substituent at C-70 with a negative value for 70 S and a positive value for 70 R. Therefore, the CD data of B-absorption band at 291 (+0.20), together with 274 (1.76) and 239 (2.02) nm of 3 allowed assignment of 70 R. Accordingly, C-8 and C-80 were assigned as S configurations. Thus, the structure of 3 was determined as (70 R, 80 S, 8S)-8-hydroxyisoguaiacin. Compound 5 was isolated as a pale yellow amorphous solid. Its molecular formula was C24H18O8 by HRESIMS data analysis. The UV bands at 299 and 332 nm and the IR asborptions at 3348 and 1711 cm1 suggested the presence of hydroxyl, carbonyl, and conjugated phenyl groups. The 1H NMR spectrum showed three aromatic spin systems, including meta-coupled protons at d 6.40 (1H, br s, H-6) and 6.51 (1H, br s, H-8); the protons of a monosubstituted aromatic ring at d 7.76 (2H, d, J = 7.5 Hz), 7.38 (2H, t,

J = 7.5 Hz), and 7.33 (1H, t, J = 7.5 Hz), and an ABX spin system at d 7.17 (1H, d, J = 8.0 Hz), 7.08 (1H, dd, J = 1.5, 8.0 Hz), and 7.51 (1H, d, J = 1.5 Hz). In addition, the olefinic protons at d 6.58 (1H, d, J = 16.0 Hz) and 7.94 (1H, d, J = 16.0 Hz) indicated these possessed an E-configuration. The vicinal coupled protons at d 5.77 (1H, d, J = 11.5 Hz) and 6.47 (1H, d, J = 11.5 Hz) corresponded to the carbons at d 81.8 (CH) and 72.8 (CH) in HMQC, indicating the presence of two oxymethine groups. The 13C NMR spectrum exhibited, in total, 24 carbon resonances involving 18 aromatic carbons, a ketone, a carbonyl carbon, two oxymethines, and two olefinic carbons. The NMR spectroscopic data in association with HMBC and COSY correlations led to establishment of a flavanone nucleus related to pinobanksin (Ondrias et al., 1997) and a caffeoyl residue. The linkage of the caffeoyl group at C-3 of the parent nucleus was deduced from the HMBC correlation between H-3 (d 6.47) and the carbonyl carbon at d 166.1 (s) (Fig. 5). Thus, 5 was determined as pinobanksin-3-caffeoylate. The coupling constant of JH-2/H-3 = 11.5 Hz was indicative of a trans axial-orientation of H-2 and H-3. Based on Slade’s assignment of the absolute configuration of dihydroflavonols (Slade et al., 2005), the positive CE320 (+16.95) nm for n ? p* transition and the negative CE287 (23.53) nm for p ? p* transition in the CD spectrum of 5 were consistent with 2R, thus, C-3 also possessed the R configuration. The molecular formula of 6 was established as C12H18O6 based on HRESIMS and NMR data. The 1H and 13C NMR spectroscopic data of 6 were closely related to syringylglycerol (Ma and Zhao, 2008), except for the presence of an additional methoxy group. The HMBC correlation between the methoxy protons d 3.13 (3H, s) and d 84.6 (d, C-7) confirmed the methoxy group was linked to C-7. Thus, 6 is 7-methoxysyringylglycerol. The large coupling constant between H-7 and H-8 (J = 6.5 Hz) suggested a threo conformation of C-7/C-8 (Spassov, 1969). The absolute configuration at C-8 was established based on the revised Snatzke’s method (Bari et al., 2001). The induced CD (ICD) spectrum of 6 caused by adding Mo2(AcO)4 in DMSO showed a positive band IV (298 nm, +0.16), a negative band V (265 nm, 1.17), and a negative band III (352 nm, 0.04), which were consistent with 8S. Accordingly, 6 was determined as threo-8S-7-methoxysyringylglycerol. Basis on the 1D and 2D NMR, IR, and MS spectroscopic data and comparison of the spectroscopic data with literature data, 23 further known phenolic compounds were identified. These included a dimeric alkyl aromatic compound integracin A (2) (Singh et al., 2002), a cyclolignan ()-lyoniresinol (4) (Kato, 1960), four favonoids naringenin (7) (Haznagy and Glusin, 1974), apigenin (8)

0.9

compound 5

H H H HO

O O

H H

H

O H OH

O

OH

H H

TROLOX

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

OH

HMBC H Fig. 5. Selected HMBC correlations of 5.

absorbance 734 nm (ABTS)

0.8

0

5

10

15

20

25

concentration (µM) Fig. 6. Antioxidative capacity of 5 (measured at 734 nm in comparison to the synthetic antioxidant TROLOX data are means ± SD (n = 3); *P < 0.05 vs. control).

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C. Shi et al. / Phytochemistry 71 (2010) 435–442

(Leopoldini et al., 2004), crysin (9) (Popova et al., 1967), luteoline (10) (Exarchou et al., 2002), 10 phenolic glycosides (1-O-(3-meth(11) oxy-4-hydroxyphenyl)-6-O-galloyl-b-D-glucopyranoside (Kanji et al., 1987) and its 5-MeO substituted analouge (12) (Lampiro et al., 1998), koaburaside (13) (Burns et al., 2007), tachioside (14) (Muneharu et al., 1973), isotachioside (15) (Muneharu et al., 1973), salicin (16) (Susana and Pinto, 2008), leonuriside A (17) (Otsuka et al., 1989), vanilloloside (18) (Kanchanapoom et al., 2006), nikoenoside (19) (Morikawa et al., 2003), 1-O-phenylmethoxy-bD-glucopyranoside (20) (Kanho et al., 2005); two propanol derivatives C-veratroylglycol (21) (Hoon et al., 2005) and 3-(O-b-D-glucopyranosyl)-2-[4-(3-hydroxypropyl)-3-methoxyphenoxy]propanol (22) (Balasundram et al., 2006), 4-hydroxy-3,5-dimethoxybenzaldehyde (23) (Baltenweck-Guyot et al., 2000), and four ellagic acid analogues ellagic acid (24), ellagic acid tetramethyl ether (25), ellagic acid 3,30 ,4-trimethyl ether (26), and ellagic acid 3,4-dimethyl ether (27) (Ye et al., 2007). 2.2. Anti-oxidative activity All phenolic compounds, apart from 1, 9, and 19, were tested for their anti-oxidative activity using the TLC-DPPH method (Catherine et al., 1996; Re et al., 1999). Most of the investigated compounds showed positive anti-oxidative activity (discoloration of violet color to yellow). Compound 5 was the most active natural product analyzed. Examination of 5 in a photometric DPPH assay resulted in a dose-dependent radical-scavenging activity and an EC50 value of 27.5 lM, which was similar to the positive control quercetin (EC50 = 20.86 lM). A comparison of antioxidant activity between 5 and caffeic acid (EC50 = 28.0 lM) suggested that the active functionality of 5 is attributed to the caffeoyl group. Similar anti-oxidative activity for 5 was also detected in the TEAC assay, another cell-free assay for radical-scavenging (Benavente-García et al., 2000) activity. Compound 5 was comparable to the synthetic vitamin E derivative TROLOX (Fig. 6). However, 5 showed no cytotoxic activity against H4IIE as well as two other cell lines (Hct116, C6) up to a concentration of 25 lM. This result may be due to a weak cellular uptake of this substance in H4IIE cells. 2.3. Inhibition of human tumor related protein kinases Human tumor related protein kinases are of particular interest as targets for the development of novel anticancer drugs (Brauers et al., 2000). We selected compounds 3, 5, 7, 11, and 23 to represent different types of phenolic compounds isolated in this study as candidates to test their inhibitory activity against 24 different human protein kinases. Compounds 5, 7, and 23 inhibited most

of the analyzed protein kinases (Table 2), while compound 11 was more specific inhibiting mainly kinases Aurora-B, FLT3, INSR, and SAK. Compound 3 showed weak inhibitory activity against all 24 protein kinases. Compounds that specifically inhibited protein kinases with IC50 values below 1  106 g/mL included 5 (FLT3, SAK), 7 (SRC, SAK), and 23 (IGF1-R, SRL, FLT3, SAK). Kinase FLT3 plays an important role in the regulation of proliferation of primitive hematopoietic progenitors, while FLT3 is expressed by myeloblasts in 70–100% of cases of acute myelogenous leukemia (AML) (Small, 2006). The significant inhibitory activity of 5 and 7 toward FLT3 implied that these natural products could possibly be developed as lead compounds for treatment of AML. Human SAK (Snk/Plk-akin kinase), a p53-repressed gene, is highly expressed in several kinds of cancers (Li et al., 2005). The potential inhibitory activity of 5, 7, and 23 toward SAK indicated that they may induce the inhibition of tumor cell proliferation. This depiction was partly proved by the recent report that 7 showed cytotoxicity in cell lines derived from various cancers (Kanno et al., 2005). Thus, the antitumor activity of the bark extract is not only attributed to the tannin content (Morton, 1965), but is also due to other phenolic components with a more specific mode of action. 2.4. Insecticidal activity The ellagic acid analogues (24, 26–27) and 3-O-methylellagic acid were analyzed for insecticidal activity by incorporating each compound into an artificial diet at an arbitrarily chosen concentration (215 ppm) and offering the spiked diet to neonate larvae of the vigorous pest insect Spodoptera littoralis in a chronic feeding experiment (Tsevegsuren et al., 2007; Handayani et al., 1997). After 6 days of exposure, larval survival and larval weight were monitored and compared to controls. All compounds tested showed growth inhibition of larvae S. littoralis (90% for 24, 82% for 3-Omethyl ellagic acid, 46% for 27, and 20% for 26, under 215 ppm), and the most active ellagic acid (24) pointed to the free ortho-dihydroxy group as the active site of the molecule. 2.5. Concluding remarks Phenolic compounds in the plant kingdom play an important role in growth and reproduction, providing protection against pathogens and predators, contributing towards the color and sensory characteristics of fruits and vegetables (Nicholson and Hammerschmidt, 1992). The rich content of phenolic compounds in the leaves of mangrove species is well recognized for the purpose of protecting the leaves from the damaging effect of solar UV radiation (Lovelock et al., 1992). However, the abundant phenolic

Table 2 IC50 values of compounds 3, 5, 7, 23 and 11 against 24 different protein kinases. Compound

AKT1

ARK5

Aurora-A

Aurora-B

B-RAF-VE

CDK2/ CycA

CDK4/ CycD1

CK2-alpha1

EGF-R

EPHB4

ERBB2

FAK

3 5 7 23 11

n.a. n.a. n.a. n.a. n.a.

n.a. 3.2  106 2.6  106 2.4  106 n.a.

n.a. 2.3  106 3.4  106 8.4  106 n.a.

n.a. 2.3  106 2.8  106 3.2  106 8.3  106

n.a. 2.4  106 3.4  106 8.2  106 n.a.

n.a. 9.7  106 8.2  106 n.a. n.a.

n.a. 2.8  106 3.1  106 4.7  106 n.a.

n.a. n.a. n.a. n.a. n.a.

n.a. 1.7  106 1.3  106 2.3  106 n.a.

n.a. 4.6  106 5.5  106 3.2  106 n.a.

n.a. 2.1  106 2.2  106 3.0  106 n.a.

n.a. 8.5  106 5.4  106 7.4  106 n.a.

IGF1-R

SRC

VEGF-R2

VEGF-R3

FLT3

INS-R

MET

PDGFR-beta

PLK1

SAK

TIE2

COT

n.a. 1.4  106 1.2  106 4.9  107 6.9  106

n.a. 1.0  106 7.4  107 6.9  107 n.a.

n.a. 1.7  106 1.8  106 1.2  106 n.a.

n.a. 5.0  106 3.2  106 2.9  106 n.a.

n.a. 9.8  107 1.8  106 5.6  107 2.0  106

n.a. 1.8  106 2.6  106 2.2  106 6.3  106

n.a. 5.1  106 4.6  106 4.8  106 n.a.

n.a. 5.1  106 4.6  106 4.8  106 n.a.

n.a. 6.6  106 7.8  106 8.4  106 n.a.

n.a. 8.9  107 9.2  107 4.3  107 2.2  106

n.a. 2.0  106 2.3  106 5.1  106 n.a

n.a. 3.9  106 2.3  106 5.7  106 n.a

3 5 7 23 11

It was determined in biochemical protein kinase activity assays that the inhibitory potential of compounds at various concentrations. IC50 values in g/mL is showed as listed. n.a.: IC50 > 1  105 g/mL.

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derivatives with diverse structural patterns found in the twigs and bark of L. racemosa implied that they may generate diverse and variable effects on ecological interactions. In fact, the antioxidation and insecticide of the diverse phenolic compounds from L. racemosa led to the prediction that the additional ecological functions may be related to herbivores, detritivores, and/or pathogens through oxidative activation.

3. Experimental 3.1. General experimental procedures Optical rotations were recorded on a Pudolph Research Analytical Automatic Polarimeter, whereas IR spectra were obtained on a Thermo Nicolet Nexus 470 FT-IR spectrometer. UV spectra were measured on a SHIMADZU LC-20AD spectrometer coupled with SPD-M20A, whereas 1H and 13C NMR spectra were recorded on a Brucker Avance DRX 500 spectrometer. HRESIMS spectra were measured on a Bruker APEX FT_MS (7.0 T) spectrometer; GF254 silica gel for TLC was purchased from Qingdao Marine Chemistry Co. Ltd. HPLC was performed on Alltech 426 with a UV detector, and the Chromasil C18 column (semi-preparative) was purchased from Pharm Co. Ltd. Perkin–Elmer Lambda 25 UV/VIS Spectrometer was used in DPPH and TEAC free-radical-scavenging assay.

3.2. Plant material Twigs and bark of L. racemosa was collected in the mangrove forest at Dongzhaigong, Hainan Island, PR China in August 2003, and was identified by Prof. P. Lin of Xiamen University. A voucher sample of this plant (HMP-065) was deposited at the State Key Laboratory of Natural and Biomimetic Drugs, Peking University.

3.3. Extraction and isolation Air dried twigs and bark (3.0 kg) of L. racemosa were ground and percolated with aqueous EtOH (5:95, V/V, 10 L) for 96 h at rt. After concentration in vacuo, the EtOH extract (60 g) was partitioned between H2O and EtOAc (V/V = 1:1, three times) and n-BuOH (V/ V = 1:1, three times), successively. The EtOAc fraction (12.0 g) was subjected to an vacuum liquid chromatography (Si gel, 200– 300 mesh, 200 g, 6  50 cm) eluted with a gradient of CHCl3– MeOH (50:1, 2 L; 20:1, 2 L; 6:1, 1.5 L; 1:1, 1.5 L) to afford eight fractions (F1–F8). F2 (234 mg) was subjected to Si gel-H column chromatography CC (40 g, 3  25 cm) eluting with petroleum ether (PE)–EtOAc (2:1, 750 mL) to obtain 3 (3 mg), 4 (4.3 mg), and 25 (6.3 mg). F3 (380 mg) was subjected to Sephadex LH-20 (200 g, 3.5  120 cm) and eluted with MeOH (1.2 L) to yield 8 (7.2 mg) and 7 (2.0 mg), while the remaining portion (45.0 mg) was separated on ODS-18 column (20 g, 1.5  30 cm) with MeOH–H2O (7:3, V/V) as an eluant to obtain 2 (2.0 mg), 1 (5.1 mg), 9 (5.5 mg), and 5 (9.1 mg). F4 (350 mg) was separated on Si gel-H column (20 g, 1.5  30 cm) eluting with a gradient petroleum ether– acetone (5:1, 500 mL; 2:1, 500 mL; 1:1, 500 mL) to afford 21 (5.2 mg), 22 (3.5 mg), and 23 (2.0 mg). F5 (1.2 g) was applied to a Si gel column (200–300 mesh, 25 g, 1.5  30 cm) eluted with CHCl3–MeOH (4:1, 1.5 L) to yield 24 (5.4 mg), 13 (3.0 mg), 14 (24 mg), and 7 (3.0 mg). F6 (195 mg) was separated on a semi-preparative HPLC (ODS) using MeOH–H2O (1:5) as a mobile phase to yield 10 (13 mg), 12 (9.0 mg), 27 (4.6 mg), 20 (5.2 mg), and 6 (4.7 mg), while F7 (380 mg) was treated by the same method as F6 with a mobile phase of MeOH–H2O (1:4) to yield 17 (8.0 mg), 19 (20 mg), 18 (8.3 mg), 16 (6.5 mg), 26 (3.8 mg), and 11 (3.5 mg).

3.4. Spectroscopic data of compounds 3.4.1. Integracin D (1) Colorless oil; ½a20 D +12 (c 1.0, MeOH); UV (MeOH) kmax 233, 263, 299 nm; IR (KBr) mmax 3400, 2929, 2856, 1736, 1713, 1644 and 1617 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 707.3448 [MH]+ (calcd. 707.3450). 3.4.2. (70 R, 80 S, 8S)-8-Hydroxyisoguauacin (3) Colorless oil; ½a20 D 92 (c 0.5, MeOH); UV (MeOH) kmax 282 nm; IR (KBr) mmax 3400, 2926, 2854, 1735 and 1644 cm1; 1H NMR data (500 MHz, CDCl3): d 6.31 (1H, s, H-3), 6.55 (1H, s, H-6), 3.06 (1H, d, J = 15.0 Hz, H-7a), 2.83 (1H, d, J = 15.0 Hz, H-7b), 1.19 (3H, s, H-9), 6.58 (1H, d, J = 1.5 Hz, H-20 ), 6.85 (1H, d, J = 8.0 Hz, H-50 ), 6.66 (1H, dd, J = 1.5, 8.0 Hz, H-60 ), 3.39 (1H, d, J = 11.0 Hz, H-70 ), 2.03 (1H, qd, J = 6.5, 11.0 Hz, H-80 ), 0.92 (3H, d, J = 6.5 Hz, H-90 ), 3.89 (3H, s, OMe), 3.84 (3H, s, OMe); 13C NMR data (125 MHz, CDCl3): d 126.8 (s, C-1), 132.2 (s, C-2), 115.3 (d, C-3), 144.9 (s, C-4), 144.3 (s, C-5), 110.4 (d, C-6), 46.7 (t, C-7), 72.4 (s, C-8), 20.3 (q, C-9), 137.4 (s, C-10 ), 111.2 (d, C-20 ), 146.4 (s, C-30 ), 144.6 (s, C-40 ), 114.0 (d, C-50 ), 122.4 (d, C-60 ), 53.1 (d, C-70 ), 47.0 (d, C-80 ), 12.5 (q, C-90 ), 55.8 (q, OMe), 55.9 (q, OMe); HRESIMS m/z 367.1512 [M+Na]+ (calcd. 367.1516). 3.4.3. (2R, 3R) Pinobanksin-3-caffeoylate (5) Pale yellow amorphous solid; ½a20 D +78 (c 0.13, MeOH); UV (MeOH) kmax 299 and 332 nm; IR (KBr) mmax 3348, 1711 and 1639 cm1; 1H NMR data (500 MHz, C5D5N): d 5.77 (1H, d, J = 11.5 Hz, H-2), 6.47 (1H, d, J = 11.5 Hz, H-3), 6.40 (1H, br s, H6), 6.51 (1H, br s, H-8), 7.76 (2H, d, J = 7.5 Hz, H-20 , H-60 ), 7.38 (2H, t, J = 7.5 Hz, H-30 , H-50 ), 7.33 (1H, t, J = 7.5 Hz, H-40 ), 6.58 (1H, d, J = 16.0 Hz, H-200 ), 7.94 (1H, d, J = 16.0 Hz, H-300 ), 7.51 (1H, br s, H-500 ), 7.17 (1H, d, J = 8.0 Hz, H-800 ), 7.08 (1H, d, J = 8.0 Hz, H900 ); 13C NMR data (125 MHz, C5D5N): d 81.8 (d, C-2), 72.8 (d, C3), 192.4 (s, C-4), 163.3 (s, C-5), 96.6(d, C-6), 169.3 (s, C-7), 98.0 (d, C-8), 165.1 (s, C-9), 101.8 (s, C-10), 136.7 (s, C-10 ), 128.3 (d, C20 , C-60 ), 129.1 (d, C-30 , C-50 ), 129.7 (d, C-40 ), 166.1 (s, C-1”), 113.2 (d, C-2”), 147.7 (d, C-3”), 126.5 (s, C-400 ), 116.0 (d, C-500 ), 147.8 (s, C-600 ), 150.9 (s, C-700 ), 116.7 (d, C-800 ), 122.4 (d, C-900 ); HRESIMS m/ z 433.0923 [MH]+ (calcd. 433.0929). 3.4.4. Threo-8S-7-methoxysyringylglycerol (6) +48.4 (c 0.1, MeOH); UV (MeOH) kmax Colorless oil; ½a20 D 270 nm; IR (KBr) mmax 3367, 2963, 2919, 2850, 1735 and 1613 cm1; 1H NMR data (500 MHz, DMSO-d6): d 6.52 (2H, s, H2, H-6), 4.00 (1H, d, J = 6.5 Hz, H-7), 3.48 (1H, ddd, J = 3.5, 5.5, 6.5 Hz, H-8), 3.09 (1H, dd, J = 5.5, 10.5 Hz, H-9a), 3.23 (1H, dd, J = 3.5, 10.5 Hz, H-9b), 3.28 (6H, s, 2  OMe), 3.13 (3H, s, OMe); 13 C NMR data (125 MHz, DMSO-d6): d 130.0 (s, C-1), 105.2 (d, C2, C-6), 148.1 (s, C-3, C-5), 135.1 (s, C-4), 84.6 (d, C-7), 75.4 (d, C8), 62.8 (t, C-9), 56.4 (q, 2  OMe), 56.8 (q, OMe); HRESIMS m/z 281.0998 [M+Na]+ (calcd. for C12H18O6Na, 281.0996). 3.5. Methanolysis of 1 Compound 1 (2.6 mg) was dissolved in 1 M HCl–MeOH (6 mL), with the whole heated until reflux began, then being maintained for 15 h at 75 °C. The MeOH solution was concentrated and separated on semi-preparative HPLC (ODS) using MeOH–H2O (1:7) as a mobile phase to afford 1a (0.6 mg) and 1b (0.78 mg). 1 Compound 1a, ½a20 D +8.7 (c 0.13, CHCl3); H NMR data (500 MHz, DMSO-d6): d 6.24 (2H, d, J = 1.5 Hz), 6.10 (1H, t, J = 1.5 Hz), 3.40 (1H, m), 2.52 (2H, t, J = 6.5 Hz), 1.52 (2H, m), 1.42–1.44 (4H, m), 1.30 (2H, m), 1.25–1.28 (8H, m), 0.94 (3H, t, J = 7.0 Hz); ESIMS m/ z 281 [M+H]+.

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Compound 1b, ½a20 +6.4 (c 0.18, CHCl3); 1H NMR data D (500 MHz, DMSO-d6): d 6.37 (1H, d, J = 1.5 Hz), 6.28 (1H, d, J = 1.5 Hz), 3.86 (3H, s), 3.42 (1H, m), 2.71 (2H, t, J = 7.0 Hz), 1.53 (2H, m), 1.41–1.44 (4H, m), 1.32 (2H, m), 1.22–1.29 (8H, m), 0.96 (3H, t, J = 7.0 Hz); ESIMS m/z 339 [M+H]+. 3.6. DPPH radical-scavenging activity assay The TLC-DPPH assay was performed on Si gel TLC (Sigma) plates spotting the same concentration (1 mM) of each sample and spraying with 1% 2,20 -diphenyl-1-picrylhydrazyl (DPPH) reagent. The yellow bands of the samples against a violet background indicated a positive reaction. The IC50 values of the samples were measured by the same method as indicated previously (Cotelle et al., 1996). Samples were dissolved in 10 lL MeOH and reacted with 490 lL DPPH solution (100 lM) (Sigma–Aldrich) for 5 min. The difference in absorption between the DPPH blank solution (DPPH solution, 100 lM) and the positive control (propylgallate, 100 lM) was calculated as 100% anti-oxidative activity.

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as a marker for cell viability was measured photometrically (560 nm). 3.10. Biochemical protein kinase activity assay The protein kinase activity was tested in 96-well FlashPlates from Perkin–Elmer/NEN (Boston, MA) in a 50 lL reaction volume (Aly et al., 2008), which contained 20 lL of assay buffer, 5 lL of test compound (in 10% DMSO), 5 lL of ATP solution (in H2O), 10 lL of substrate, and 10 lL of purified recombinant protein kinase in the reaction cocktail. It also contained 60 mM HEPES–NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 lM Na-orthovanadate, 1.2 mM DTT, 50 lg/mL PEG20000, and 1 lM [c-33P]-ATP (ca. 5  105 cpm/ well) in the assay for all enzymes. The substrates used in this study was listed below: GSK3 (14–27), AKT1; tetra (LRRWSLG), Aurora A, B; MEK1 KM, B-Raf; histone H1, CDK2/CycA; Rb-CTF, CDK4/CycD1; Poly(Glu, Tyr)4:1, EGF-R, EPHB4, ERBB2, FAK, IGF1-R, SRC, VEGF-R2, VEGF-R3, Tie2; Poly(Ala, Glu, Lys, Tyr), FLT3, INS-R, Met, PDGF-Rb; casein, PLK1, CK2a1. Autophosphorylation was measured for ARK5, COT, and SAK.

3.7. TEAC-assay 3.11. Insecticidal bioassay TROLOX equivalent anti-oxidative capacity (TEAC) was measured by spectro-photometrically analyzing the decolorisation of a stable radical cation ABTS (2,20 -azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid)) at 734 nm by comparison with the synthetic antioxidant TROLOX. Absorption was measured after 4 min of mixing the isolated compound with the ABTS solution. The significance of changes in the test responses was assessed using a one-way ANOVA followed by LSD post hoc test, differences were considered to be significant at P < 0.05.

Larvae of the polyphagous pest insect S. littoralis (Noctuidae, Lepidoptera), which were from a laboratory colony reared on artificial diet under controlled conditions at 26 °C were used for this insect feeding assay. An artificial diet was treated with different concentrations of samples, and the feeding test was conducted with neonate larvae (n = 20). The survival and weight of surviving larvae were protocolled and compared to controls after a 6-day exposure.

3.8. Intracellular DCF formation

Acknowledgments

As a marker for intracellular oxidative stress, the fluorescent probe 2,7-dichlorodihydrofluorescein (H2DCF) was used. Cells were incubated with 2,7-dichlorodihydrofluorescein diacetate (H2DCF/DA) which is cleaved by cytosolic esterases to H2DCF thus preventing the back-diffusion of the dye into the extracellular space. The oxidation of intracellular nonfluorescent H2DCF to highly fluorescent DCF was measured in a Wallac Victor2 multilabel counter (excitation: 485 nm, emission: 525 nm). 50,000 cells/ well were seeded on a 96-well microplate and allowed to attach for 24 h. For fluorescence measurement, the medium was changed (DMEM medium without phenol red) and cells were incubated with compound 5 or DMSO (vehicle) for 60 min. After this preincubation, the cells were incubated 30 min with 50 AM H2DCF/DA. Then medium was changed and various concentrations of H2O2 were added to all cells. After 60 min, the H2O2-mediated increase in DCF fluorescence was determined.

We are grateful to Mrs. Jie Ouyang (Beijing Normal University) for measuring the NMR spectra. This work was supported by Grants from NSFC (No. 30672607), the National Hi-Tech Projects (2006AA09Z446, 2006DFA31100, 2007AA09Z448, 2006AA09Z405, 2007AA09Z435), China Uni-PhD Base Project (20060001149), and International Cooperation Projects of BMBF-MOST. C. Shi is grateful to the China Scholarship Council for a fellowship.

3.9. Determination of cytotoxicity Metabolically active H4IIE rat hepatoma cells (DMEM, 10% FCS), rat C6 glioma cells (DMEM, 5% FCS) and Hct116 human colon carcinoma cells (RPMI, 10% FCS) were maintained in a humidified atmosphere at 37 °C with 5% CO2. The cell culture medium contained 100 units/mL penicillin and 100 lg/mL streptomycin and was changed twice per week. For determination of cytotoxicity, the MTT-assay was used. The cells were plated on 96-multiwell plates with 10,000 cells/well and allowed to attach for 24 h. Then the cells were treated with different concentrations of 5 for 24 h. After this treatment the medium was changed and the cells were incubated for 2 h under cell culture conditions with 20 lg/mL MTT. After this incubation time the cells were lysed with 50% ethanol/49% water/1% acetic acid. The concentration of reduced MTT

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