Isolation and Characterization of Cancer Chemopreventive Compounds from Barringtonia maunwongyathiae

Abstract

A new plant species, Barringtonia maunwongyathiae. W. Chuakul (Lecythidaceae), was recently discovered in Khuan Thon Forest, Ao Luek District, Krabi Province, Thailand. Chemical investigation of the leaves of this plant led to the isolation of 10 triterpenes, 3 steroids, and a vitamin E derivative. The structures of these compounds were identified as taraxerol (1), 3-(E.)-coumaroyltaraxerol (2), 3-(Z.)-coumaroyltaraxerol (3), 3-(E.)-coumaroyl β.-amyrin (4), 3-(Z.)-coumaroyl β.-amyrin (5), 3-(E.)-coumaroyl α.-amyrin (6), 3-(Z.)-coumaroyl α.-amyrin (7), 3-(E.)-coumaroyllupeol (8), 3-(Z.)-coumaroyllupeol (9), 3,19,24-trihydroxyurs-12-ene-28-oic acid (10), stigma-4,22-dien-3-one (11), β.-stigmasterol (12), 3-O.-β.-D-glucopyranosyl-stigmasta-5,22-diene (13), and α.-tocopherylquinone (14). All compounds were evaluated for their cancer chemopreventive potential based on inhibition of TPA-induced ornithine decarboxylase expression, COX-1 and COX-2 activities, and phorbol ester–induced NF-κB luciferase expression, as well as activation of antioxidant response element–mediated luciferase expression. Compounds 1, 2, and 14 demonstrated greatest promise, while 12 and 13 showed moderate activity.

Keywords:

• Antioxidant responsive element (ARE)
• ARE luciferase reporter system
• Barringtonia maunwongyathiae
• cancer chemoprevention
• coumaroyl triterpenes
• COX-1
• COX-2
• Lecythidaceae
• NF-κ. B luciferase reporter system, ornithine decarboxylase (ODC)g48
• tocopherylquinone

Introduction

A number of cancer chemopreventive agents have been isolated from plants, and these agents function through a variety of mechanisms (Zhang et al., 1992; Aggarwal, 2004; Chun et al., 2004; Ramirez-Mares et al., 2004; Pezzuto et al., 2005). For example, ornithine decarboxylase (ODC) is essential for polyamine synthesis and growth in mammalian cells; it provides putrescine, which is usually converted into the higher polyamines, spermidine and spermine. ODC, which catalyzes the rate-limiting step in the biosynthesis of polyamines, is closely linked with cellular proliferation and carcinogenesis. Accordingly, inhibitors of ODC, like α. -difluoromethylornithine (DFMO), have been used for cancer prevention and therapy (Bachrach & Wang, 2002).

As another example, evidence has accumulated indicating that the beneficial action of some plant food is due, at least in part, to induction of phase II detoxification enzymes. These enzymes detoxify harmful substances by converting them to hydrophilic metabolites that can be excreted (Talalay et al., 2003). There is a strong inverse relationship between tissue levels of NAD(P)H:quinone oxidoreductase and γ-glutamylcysteine synthetase and susceptibility to chemical carcinogenesis. The induction of phase II enzymes is mediated through cis.-regulatory DNA sequences located in the promoter or enhancer region, which are known as antioxidant responsive elements (AREs). A wide range of structurally diverse compounds can activate the AREs. Several studies have shown that dietary antioxidants, such as 1,2-dithiole-3-thiones, phenolic flavonoids, curcuminoids, isothiocyanates, and carotenoids, may function as anticancer agents by activating this transcription system (Ben-Dor et al., 2005).

NF-κ.B is another inducible transcription factor for genes involved in cell survival, cell adhesion, inflammation, differentiation, and growth. In resting cells, NF-κ.B is localized in the cytoplasm by binding to inhibitory Iκ.B proteins that block nuclear localization sequences of NF-κ.B. NF-κ.B is activated by a variety of stimuli such as carcinogens, inflammatory agents, and tumor promoters including cigarette smoke, phorbol esters, okadaic acid, and TNF. Constitutive activation of NF-κ.B may upregulate the expression of inflammatory cytokines, chemokines, cell adhesion molecules, COX-2, and matrix metalloproteinase (MMP)-9 (Li et al., 2005). Many of the target genes that are activated are critical to the establishment of early or late stages of aggressive cancers (Dorai & Aggarval, 2004). Thus, agents that can suppress NF-κ.B activation have the potential to suppress carcinogenesis & thereby have therapeutic potential (Nakanishi & Toi, 2005). Although appropriate levels of NF-κ.B activity are crucial for normal cellular proliferation, constitutive NF-κ.B activation is involved in the enhanced growth of several cancers (Bharti & Aggarwal, 2002).

As a final example, inhibitors of cyclooxygenase activity have been associated with neoplastic transformation (Wang & Dubois, 2006), and inhibition of this activity provides a strategy for the prevention of cancer (Cuendet & Pezzuto, 2000).

The genus Barringtonia. (Lecythidaceae) consists of 56 species distributed in the wild in the tropics from eastern Africa to northern Australia and southern Asia (Payens, 1967; Chantaranothai, 1995). Some species are used in traditional medicine as an anti-inflammatiory, anti-asthmatic (Panthong et al., 1986), anaesthetic, and anti-infective (McClatchey, 1996). Barringtonia acutangula. Gaerth, B. racemosa. Roxb., and B. asiatica. Kurz were chemically investigated and were reported to contain di- and triterpenes (Barua et al., 1961; Sastry et al., 1967; Anjaneyulu et al., 1978; Rao et al., 1986; Pal et al., 1994; Hasan et al., 2000; Herlt et al., 2002; Burton et al., 2003). Various biological activities have been reported for some species of Barringtonia. B. racemosa. exhibited antibacterial (Khan et al., 2001), antitumor (Thomas et al., 2002), and antinociceptive activities (Deraniyagala et al., 2003), whereas B. asiatica. was reported to exhibit antifungal and antibacterial activities (Khan et al., 2002).

Barringtonia maunwongyathiae. W. Chuakul is a new species discovered recently in Khuan Thon Forest, Ao Luek District, Krabi Province, Thailand. It is a tree up to 8 m high, which has been used topically to relieve pain and inflammation caused by Gluta usitata. (Wall.) Ding Hou resin (Chuakul, 2001). No previous phytochemical or biological studies have been performed with this species. Our initial cancer chemopreventive testing showed that extracts from the leaves of B. maunwongyathiae. mediated a strong inhibitory response with COX-2 (IC₅₀ value of 0.89 µg/mL with a petroleum ether extract), as well as potent inhibition of TPA-induced NF-κ.B–dependent reporter gene expression (IC₅₀ value of 0.16 µg/mL for an EtOAc extract). The current paper reports the isolation of triterpenes, steroids, and tocopherylquinone from the leaves of B. maunwongyathiae. as well as their cancer chemopreventive activity.

Materials and Methods

General experimental procedures

Optical rotations were measured with a Perkin-Elmer 241 polarimeter. UV spectra were measured on a Beckman DU-7 spectrometer. IR spectra were run on a Jasco FT/IR-410 spectrometer, equipped with a Specac Silver Gate ATR system by applying a film on a germanium plate. NMR spectra were recorded on a Bruker DPX-300 or a Bruker DPX-400 NMR spectrometer. Chemical shifts (δ) were expressed in ppm with reference to TMS or the solvent signals. All NMR data were obtained by using standard pulse sequences supplied by the vendor. LREIMS and LRESIMS were recorded on a JEOL GC Mate II and a Thermo Finnigan LCQ mass spectrometer, respectively. Preparative reverse-phase HPLC was carried out on a Waters 600E Delivery System pump, equipped with a Waters 996 photodiode detector, and a Phenomenex LUNA C18 (2) column (120 Å, 10 µm, 50 × 250 mm) at 20 mL/min. Silica gel 60 (0.063 µm to 0.2 nm, E Merck) and silica gel RP.-18 (40–63 µm, EM Science) were used for column chromatography. Thin-layer chromatography was performed on Whatman glass-backed plates coated with 0.25-mm layers of silica gel 60. Fractions were monitored by TLC, and spots were visualized by heating silical gel plates sprayed with 10% H₂SO₄ in MeOH.

Plant material

The leaves of B. maunwongyathiae. W. Chuakul were collected from Khuan Thon Forest, Ao Luek District, Krabi Province, Thailand, and were identified by one of the authors (W.C.). A voucher specimen (Chuakul 2953) has been deposited at the herbarium of the Department of Pharmaceutical Botany, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand.

Extraction and isolation

The dry powdered leaves of B. maunwongyathiae. (1.40 kg) were successively extracted in a Soxhlet apparatus with petroleum ether (60–80°C), CHCl₃, EtOAc, and MeOH. Each extract was filtered and evaporated under reduced pressure to yield 44.8, 30.8, 14.0, and 89.6 g of crude extracts, respectively, and subjected to bioassay. A portion of the COX-2 active petroleum ether extract (30.0 g) was subjected to fractionation by vaccum column chromatography over silica gel (0.063 µm to 0.2 nm, 600 g), eluting with petroleum ether, CHCl₃, and EtOAc in an order of increasing polarity to afford 20 fractions [petroleum ether–CHCl₃/100:0 (eluates PF1–PF3, each 0.5 L), 95:5 (eluates PF4–PF6, each 1.0 L), 9:1 (eluate PF7, 1.0 L), 8:2 (eluates PF8–PF9, each 1.0 L), 7:3 (eluate PF10, 1.0 L), 6:4 (eluate PF11, 0.8 L), 1:1 (eluates PF12–PF13, each 0.8 L), and 3:7 (eluate PF14, 0.5 L); CHCl₃-EtOAc/100:0 (eluates PF15–PF16, each 1.0 L), and 9:1 (eluates PF17–PF20, each 1.0 L)]. Re-chromatography of PF15 (1.2 g) over a silica gel (0.063 µm to 0.2 nm, 40.0 g) column, eluting with CHCl₃ (5 × 300 mL), yielded five subfractions (PF21–PF25). Taraxerol (1, Fig. 1; 265.9 mg), a major component, was obtained from the subfraction PF25 by direct crystallization. Fraction PF16 (0.5 g) was re-chromatographed over a silica gel (0.063 µm to 0.2 nm, 14.00 g) column, eluting with CHCl₃ (5 × 120 mL), to yield subfractions PF26–PF30. β.-Stigmasterol (12, 30.4 mg) was crystallized from the subfraction PF28. Fraction PF17 (1.0 g) was subjected to separation over a silica gel (0.063 µm to 0.2 nm, 25.0 g) column by gradient elution of n.-hexane, CHCl₃ and EtOAc to afford subfractions PF31–PF43. Subfraction PF39 (1129.3 mg) was further fractionated over a silica gel (0.063 µm to 0.2 nm, 90.0 g) column, eluting with CHCl₃-EtOAc/95:5 (3 × 200 mL) to afford 3-(Z.)-coumaroyltaraxerol (3, 30.4 mg). Fraction PF18 (7.2 g) was subjected to a silica gel RP-18 (26.0 g) column separation, using gradient solvent system of MeOH, MeCN, and H₂O to give eight subfractions [MeCN-H₂O/9:1 (eluates PF44-PF48, each 1.0 L); MeCN (eluates PF49–PF50, each 2.0 L); MeOH (eluate PF51, 1 L)]. Chromatography of the combined fractions PF46–PF47 over a silica gel (230–400 mesh, 95.0 g) column, eluting with CHCl₃ and CHCl₃-Me₂CO/99:1, afforded six additional subfractions PF48-PF53. Subfractions PF49–PF50 were combined and subjected to reverse-phase preparative HPLC (MeOH 100%) separation to give α.-tocopherylquinone (14, 22.6 mg), stigma-4, 22-dien-3-one (11, 5.6 mg) and a mixture (11.5 mg) of 3-(E.)-coumaroyllupeol (8) and 3-(Z.)-coumaroyllupeol (9). Subfractions PF51 and PF52 were combined and subjected to reverse-phase preparative HPLC (MeOH 100%) separation to give a mixture (3.5 mg) of 3-(E.)-coumaroyl β.-amyrin (4) and 3-(Z.)-coumaroyl β.-amyrin (5) and a mixture (3.8 mg) of 3-(E.)-coumaroyl α.-amyrin (6) and 3-(Z.)-coumaroyl α.-amyrin (7).

Figure. 1 Chemical structures of compounds 1–14.

The NF-κ.B active ethyl acetate extract (14.0 g) was fractionated by silica gel (0.063 µm to 0.2 nm, 300.0 g) column chromatography, eluting with CHCl₃, Me₂CO, and MeOH in an order of increasing polarity to give 50 fractions [CHCl₃ (eluates EF1–EF23, each 0.5 L); CHCl₃-Me₂CO/95:5 (eluates EF24–EF33, each 0.5 L), 9:1 (eluates EF34–EF35, each 1.0 L), and 8:2 (eluates EF36–EF37, each 1.0 L); CHCl₃-MeOH/97:3 (eluates EF38–EF42, each 1.5 L), 95:5 (eluates EF43, 1.5 L), 9:1 (eluates EF44–EF47, each 1.5 L), 8:2 (eluates EF48–EF50, each 1.0 L). Fraction EF40 was dissolved in MeOH and filtered to afford 3-O.-β.-D-glucopyranosyl-stigmasta-5,22-diene (13) as a precipitate (57.5 mg). Further separation of fraction EF33 (0.3 g) on a silica gel (0.063 µm to 0.2 nm, 300.0 g) column, eluting with a gradient system of the mixture between CHCl₃ with Me₂CO or between CHCl₃ and MeOH, gave 19 subfractions (EF51–EF69). The combined subfractions EF59–EF61 were dissolved in MeOH and filtered to afford 3,19,24-trihydroxyurs-12-ene-28-oic acid (10) as a precipitate (106.4 mg).

A portion of initially inactive CHCl₃ extract (8.0 g) was also chromatographed on a silica gel (0.063 µm to 0.2 nm, 200.0 g) column, eluting with n.-hexane, CHCl₃ and EtOAc in an order of increasing polarity to afford 18 fractions [n.-hexane (eluates CF1–CF2, each 0.3 L); n.-hexane-CHCl₃/1:1 (eluates CF3–CF5, each 0.2 L); CHCl₃ (eluates CF6–CF9, each 0.5 L); CHCl₃-EtOAc/9:1 (eluates CF10–CF11, each 0.5 L), and 1:1 (eluates CF12–CF13, each 0.4 L); EtOAc (eluates CF14–CF15, each 0.4 L), CF16 (0.8 L), and CF17–CF18 (each 0.5 L). Fraction CF9 (326.0 mg) was further fractionated over a silica gel (0.063 µm to 0.2 nm, 250.0 g) column by a gradient elution of CHCl₃, Me₂CO, and MeOH to yield 20 subfractions (CF19–CF38). Subfraction CF28 (145.3 mg) was further fractionated over a silica gel (0.063 µm to 0.2 nm, 95.0 g) column, eluting with CHCl₃ (7 × 250 mL) to afford 3-(E.)-coumaroyltaraxerol (2, 22.4 mg).

Identification of isolated compounds

Structures of all isolated compounds were elucidated on the basis of their spectral data (IR, MS, ¹H NMR, ¹³C NMR, DEPT, COSY, HSQC, and HMBC), and confirmed by comparison with literature values. The ¹H NMR and ¹³C NMR spectra are shown in Tables 1 and 2, respectively.

Table 1. ¹H NMR spectral data of compounds 1–9 (400 MHz, J. in Hz, CDCl₃).

Position	1	2	3	4	5 (δ.H)	6	7	8	9
1	1.61 m.	1.05 m.	1.01 m. 1.63m.	1.06 m. 1.65 m.	1.06 m. 1.65 m.	1.12 m. 1.71 m.	1.12 m. 1.71 m.	1.00 m. 1.65 m.	1.00 m. 1.65 m.
2	1.62 m.	1.64 m. 1.69 m.	1.61 m. 1.68 m.	1.67 m. 1.88 m.	1.67 m. 1.88 m.	1.70 m.	1.70 m.	1.64 m.	1.64 m.
3	3.17 dd. (4.7, 11.0)	4.57 dd. (5.5, 10.4)	4.49 dd. (5.2, 11.6)	4.60 dd. (8.2, 7.8)	4.54 dd. (4.9, 11.2)	4.64 dd. (8.1, 7.7)	4.56 dd. (4.7, 10.6)	4.60 dd. (5.5, 10.3)	4.52 dd. (4.5, 11.5)
5	0.76 m.	0.91 m.	0.86 m.	0.87 m.	0.83 m.	1.32 m.	1.32 m.	0.79 m.	0.79 m.
6	1.48 m. 1.58 m.	1.49 m. 1.62 m.	1.45 m. 1.59 m.	1.42 m. 1.53 m.	1.42 m. 1.53 m.	1.41 m. 1.52 m.	1.41 m. 1.52 m.	1.40 m. 1.48 m.	1.40 m. 1.48 m.
7	1.33 m. 2.01 dt. (12.6, 3.2)	1.38 m. 2.01 dt. (12.6, 3.2)	1.35 m. 2.00 dt. (12.6, 3.2)	1.33 m. 1.52 m.	1.33 m. 1.52 m.	1.36 m. 1.57 m.	1.36 m. 1.57 m.	1.36 m.	1.36 m.
9	1.40 m.	1.45 m.	1.44 m.	1.56 m.	1.56 m.	1.57 m.	1.57 m.	1.29 m.	1.29 m.
11	1.42 m. 1.60 m.	1.49 m. 1.62 m.	1.45 m. 1.59 m.	1.68 m. 1.87 m.	1.68 m. 1.87 m.	1.93 m.	1.93 m.	1.41 m.	1.41 m.
12	1.49 m. 1.59 m.	1.52 m. 1.61 m.	1.55 m. 1.60 m.	5.16 m.	5.16 m.	5.13 m.	5.13 m.	1.67 m.	1.67 m.
13	–	–	–	–	–	–	–	1.68 m.	1.68 m.
15	5.51 dd. (3.1, 8.1)	5.51 dd. (3.1, 8.0)	5.51 dd. (3.1, 8.1)	0.95 m. 1.75 m.	0.95 m. 1.75 m.	1.83 brt. (13.4)	1.83 brt. (13.4)	1.00 m. 1.68 m.	1.00 m. 1.68 m.
16	1.62 m. 1.89 dd. (3.1, 14.7)	1.61 m. 1.89 dd. (3.1, 14.7)	1.63 m. 1.90 dd. (3.1, 14.8)	0.78 m. 1.98 m.	0.78 m. 1.98 m.	2.00 brt. (13.4)	2.00 brt. (13.4)	1.47 m.	1.47 m.
18	0.94 m.	0.94 m.	0.93 dd. (4.8, 13.5)	1.02 m. 1.95 m.	1.02 m. 1.95 m.	1.33 m.	1.33 m.	1.33 m.	1.33 m.
19	0.92 m. 1.29 m.	0.96 m. 1.29 m.	0.95 m.1.30 m.	1.70 m.	1.70 m.	0.93 m.	0.93 m.	2.38 dt. (5.8, 10.8)	2.37 dt. (5.8, 10.8)
20	–	–	–	–	–	1.32 m.	1.32 m.	–	–
21	1.23 m. 1.33 m.	1.23 m. 1.33 m.	1.22 m. 1.33 m.	1.08 m. 1.35 m.	1.08 m. 1.30 m.	1.29 m. 1.41 m.	1.29 m. 1.41 m.	1.31 m. 1.90 m.	1.31 m. 1.91 m.
22	0.99 m. 1.35 m.	1.00 m. 1.36 m.	0.99 m. 1.33 m.	1.20 m. 1.44 m.	1.20 m. 1.44 m.	1.30 m. 1.45 m.	1.30 m. 1.45 m.	1.20 m. 1.39 m.	1.20 m. 1.39 m.
23	0.95 s.	0.88 s.	0.84 s.	0.85 s.	0.85 s.	0.88 s.	0.91 s.	0.88 s.	0.85 s.
24	0.78 s.	0.93 s.	0.81 s.	0.97 s.	0.94 s.	0.94 s.	0.82 s.	1.04 s.	1.02 s.
25	0.90 s.	0.95 s.	0.93 s.	0.80 s.	0.94 s.	1.01 s.	0.97 s.	0.88 s.	0.85 s.
26	1.06 s.	1.08 s.	1.06 s.	0.92 s.	0.96 s.	1.02 s.	1.00 s.	0.91 s.	0.80 s.
27	0.88 s.	0.89 s.	0.88 s.	1.12 s.	1.11 s.	1.08 s.	1.06 s.	0.95 s.	0.94 s.
28	0.79 s.	0.80 s.	0.79 s.	0.81 s.	0.81 s.	0.79 s.	0.79 s.	0.79 s.	0.78 s.
29	0.92 s.	0.93 s.	0.93 s.	0.85 s.	0.85 s.	0.80 d. (6.0)	0.80 d. (6.0)	4.57 d. (1.8) 4.68 d. (1.8)	4.57 d. (1.8) 4.68 d. (1.8)
30	0.88 s.	0.89 s.	0.88 s.	0.85 s.	0.85 s.	0.92 d. (5.0)	0.92 d. (5.0)	1.68 s.	1.68 s.
2′		6.27 d. (15.9)	5.80 d. (12.7)	6.28 d. (15.9)	5.81 d. (12.8)	6.30 d. (16.0)	5.84 d. (12.6)	6.29 d. (15.8)	5.83 d. (12.6)
3′		7.57 d. (15.9)	6.80 d. (12.7)	7.58 d. (15.9)	6.80 d. (12.8)	7.61 d. (16.0)	6.82 d. (12.6)	7.60 d. (15.8)	6.83 d. (12.6)
5′		7.40 d. (8.6)	7.60 d. (8.6)	7.41 d. (8.6)	7.63 d. (8.6)	7.44 d. (8.5)	7.64 d. (8.4)	7.42 d. (8.5)	7.62 d. (8.6)
6′		6.80 d. (8.6)	6.76 d. (8.6)	6.81 d. (8.6)	6.77 d. (8.6)	6.83 d. (8.5)	6.79 d. (8.4)	6.83 d. (8.5)	6.78 d. (8.6)
8′		6.80 d. (8.6)	6.76 d. (8.6)	6.81 d. (8.6)	6.77 d. (8.6)	6.83 d. (8.5)	6.79 d. (8.4)	6.83 d. (8.5)	6.78 d. (8.6)
9′		7.40 d. (8.6)	7.60 d. (8.6)	7.41 d. (8.6)	7.63 d. (8.6)	7.44 d. (8.5)	7.64 d. (8.4)	7.42 d. (8.5)	7.62 d. (8.6)

Table 2. ¹³C-NMR spectral data of compounds 1–9 and 14 (100 MHz, CDCl₃).

Position	1	2	3	4	5 (δ.C)	6	7	8	9	14
1	37.7 t.	37.4 t.	37.4 t.	38.2 t.	38.2 t.	38.5 t.	38.5 t.	38.4 t.	38.4 t.	21.3 t.
2	27.2 t.	23.5 t.	23.4 t.	23.5 t.	23.5 t.	23.5 t.	23.5 t.	23.8 t.	23.7 t.	40.2 t.
3	79.1 d.	81.0 d.	81.0 d.	80.8 d.	80.9 d.	80.8 d.	80.9 d.	80.9 d.	81.1d.	72.6 s.
4	38.8 s.	37.9 s.	37.7 s.	37.9 s.	37.7 s.	37.9 s.	37.8 s.	37.9 s.	37.9 s.	42.2 t.
5	55.5 d.	55.6 d.	55.6 d.	55.3 d.	55.3 d.	55.3 d.	55.3 d.	55.4 d.	55.4 d.	21.4 t.
6	18.8 t.	18.7 t.	18.7 t.	18.3 t.	18.3 t.	18.3 t.	18.3 t.	18.2 t.	18.2 t.	37.5 t.
7	41.3 t.	41.2 t.	41.2 t.	32.5 t.	32.5 t.	32.9 t.	32.9 t.	34.2 t.	34.2 t.	32.7 d.
8	39.0 s.	38.9 s.	38.9 s.	41.7 s.	41.7 s.	40.1 s.	40.1 s.	40.8 s.	40.8 s.	37.4 t.
9	49.3 d.	49.1 d.	49.1 d.	47.5 d.	47.5 d.	47.6 d.	47.6 d.	50.3 d.	50.3 d.	24.5 t.
10	38.0 s.	37.9 s.	37.8 s.	36.8 s.	36.8 s.	36.8 s.	36.8 s.	37.1 s.	37.1 s.	37.4 t.
11	17.5 t.	17.5 t.	17.5 t.	23.5 t.	23.5 t.	23.7 t.	23.6 t.	20.9 t.	20.9 t.	32.7 d.
12	33.7 t.	33.6 t.	33.6 t.	121.6 d.	121.6 d.	124.3 d.	124.3 d.	25.1 t.	25.1 t.	37.3 t.
13	37.6 s.	37.5 s.	37.5 s.	145.2 s.	145.2 s.	139.6 s.	139.6 s.	38.0 d.	38.0 d.	24.8 t.
14	158.1 s.	157.9 s.	157.9 s.	39.8 s.	39.8 s.	42.1 s.	42.1 s.	42.8 s.	42.8 s.	39.3 t.
15	116.9 d.	116.9 d.	116.9 d.	26.1 t.	26.1 t.	26.6 t.	26.6 t.	27.4 t.	27.4 t.	28.0 d.
16	37.7 t.	37.7 t.	37.7 t.	26.9 t.	26.9 t.	28.1 t.	28.1 t.	35.6 t.	35.6 t.	22.6 q.
17	35.8 s.	35.7 s.	35.7 s.	32.5 s.	32.5 s.	33.8 s.	33.8 s.	43.0 s.	43.0 s.	26.6 q.
18	48.8 d.	48.7 d.	48.7 d.	47.2 d.	47.2 d.	59.1 d.	59.1 d.	48.3 d.	48.3 d.	19.7 q.
19	36.7 t.	36.6 t.	36.6 t.	46.8 t.	46.8 t.	39.6 d.	39.6 d.	48.0 d.	48.0 d.	19.7 q.
20	28.8 s.	28.8 s.	28.8 s.	31.1 s.	31.1 s.	39.7 d.	39.7 d.	151.0 s.	151.0 s.	22.7 q.
21	33.1 t.	33.1 t.	33.1 t.	34.7 t.	34.7 t.	31.3 t.	31.3 t.	29.7 t.	29.8 t.
22	35.1 t.	35.1 t.	35.1 t.	37.1 t.	37.1 t.	41.5 t.	41.5 t.	40.0 t.	40.0 t.
23	28.0 q.	28.0 q.	28.0 q.	28.1 q.	28.1 q.	28.1 q.	28.1 q.	28.0 q.	28.0 q.
24	15.5 q.	16.7 q.	16.6 q.	15.6 q.	15.6 q.	16.9 q.	16.8 q.	16.0 q.	16.0 q.
25	15.4 q.	15.5 q.	15.5 q.	16.8 q.	16.7 q.	15.8 q.	15.8 q.	16.2 q.	16.2 q.
26	25.9 q.	25.9 q.	25.9 q.	16.8 q.	16.8 q.	16.9 q.	16.9 q.	16.7 q.	16.5 q.
27	21.3 q.	21.3 q.	21.3 q.	25.9 q.	25.9 q.	23.3 q.	23.3 q.	14.5 q.	14.5 q.
28	29.8 q.	29.8 q.	29.8 q.	28.4 q.	28.4 q.	28.8 q.	28.8 q.	18.0 q.	18.0 q.
29	33.4 q.	33.3 q.	33.3 q.	33.3 q.	33.3 q.	17.5 q.	17.5 q.	109.4 t.	109.4 t.
30	29.9 q.	29.9 q.	29.9 q.	23.7 q.	23.7 q.	21.4 q.	21.4 q.	19.3 q.	19.3 q.
1′		167.3 s.	166.4 s.	167.1 s.	166.3 s.	167.2 s.	166.4 s.	167.3 s.	166.6 s.	187.7 s.
2′		116.2 d.	117.8 d.	116.5 d.	117.9 d.	116.5 d.	117.9 d.	116.3 d.	117.8 d.	140.4 s.
3′		144.0 d.	143.1 d.	143.8 d.	142.8 d.	143.8 d.	142.9 d.	143.9 d.	143.2 d.	140.5 s.
4′		127.3 s.	127.5 s.	127.6 s.	127.6 s.	127.6 s.	127.8 s.	127.5 s.	127.5 s.	187.2 s.
5′		129.9 d.	132.3 d.	129.9 d.	132.4 d.	129.9 d.	132.4 d.	129.9 d.	132.3 d.	144.4 s.
6′		115.8 d.	114.9 d.	115.8 d.	114.9 d.	115.8 d.	114.9 d.	115.8 d.	115.0 d.	140.2 s.
7′		157.6 s.	156.6 s.	157.3 s.	156.3 s.	157.3 s.	156.4 s.	157.5 s.	156.7 s.	12.3 q.
8′		115.8 d.	114.9 d.	115.8 d.	114.9 d.	115.8 d.	114.9 d.	115.8 d.	115.0 d.	12.4 q.
9′		129.9 d.	132.3 d.	129.9 d.	132.4 d.	129.9 d.	132.4 d.	129.9 d.	132.3 d.	11.9 q.

Taraxerol(1)

Obtained as colorless plates; : + 8.1° (c. 0.04, CHCl₃); UV (MeOH) λ_max (log ε): 207 (3.8) nm; IR ν_max (dried film): 3482, 2933, 2865, 1473, 1382, 1056, 1035 cm⁻¹; LREIMS (m./z.): 426 [M]⁺ (C₃₀H₅₀O) (17.6), 408 (6.1), 302 (40.1), 204 (100), 189 (28.9).

3-(E)-Coumaroyltaraxerol (2)

Obtained as white powder; : + 18.5° (c. 0.5, CHCl₃); UV (MeOH) λ_max (log ε): 210 (4.0), 312 (4.0) nm; IR ν_max (dried film): 3330, 2935, 2855, 1704, 1604, 1513, 1454, 1375, 1167 cm^−1; LRESIMS (m./z.): 571 [M-H]⁻ (C₃₉H₅₆O₃).

3-(Z)-Coumaroyltaraxerol(3)

Obtained as colorless needles; : + 28.7° (c. 0.1, CHCl₃); UV (MeOH) λ_max (log ε): 207 (4.3), 312 (4.0) nm; IR ν_max (dried film): 3360, 2939, 2863, 1681, 1604, 1513, 1452, 1373, 1168 cm⁻¹; LRESIMS (m./z.): 571 [M-H]⁻ (C₃₉H₅₆O₃).

3-(E)-Coumaroylβ.-amyrin.(4)and 3-(Z)-coumaroyl.β.-amyrin.(5)

Obtained as colorless gum; UV (MeOH) λ_max (log ε): 211 (4.1), 224 (4.0), 311 (4.2) nm; IR ν_max (dried film): 3360, 2945, 2863, 1680, 1604, 1513, 1456, 1363, 1168 cm^−1; LRESIMS (m./z.): 571 [M-H]⁻ (C₃₉H₅₆O₃).

3-(E)-Coumaroylα.-amyrin.(6)and 3-(Z)-coumaroyl.α.-amyrin.(7)

Obtained as colorless gum; UV (MeOH) λ_max (log ε): 207 (3.9), 221 (3.8), 312 (3.9) nm; IR ν_max (dried film): 3364, 2949, 2852, 1679, 1604, 1514, 1453, 1377, 1168 cm⁻¹; LRESIMS (m./z.): 571 [M-H]⁻ (C₃₉H₅₆O₃).

3-(E)-Coumaroyllupeol(8)and 3-(Z)-coumaroyllupeol.(9)

Obtained as colorless gum; UV (MeOH) λ_max (log ε): 207 (4.0), 224 (3.8), 311 (3.9) nm; IR ν_max (dried film): 3362, 2943, 2863, 1683, 1604, 1513, 1454, 1378, 1168 cm⁻¹; LRESIMS (m./z.): 571 [M-H]⁻ (C₃₉H₅₆O₃).

3, 19, 24-Trihydroxyurs-12-ene-28-oic acid(10)

Obtained as white powder; : + 21.0° (c. 0.1, CHCl₃); UV (MeOH) λ_max (log ε): 209 (3.8) nm; IR ν_max (dried film): 3377, 2933, 2869, 1687, 1457, 1377, 1235, 1158, 1046 cm⁻¹; LRESIMS (m./z.): 487 [M-H]⁻ (C₃₀H₄₈O₅).

Stigma-4, 22-dien-3-one(11)

Obtained as white wax; + 32.9° (c. 0.07, CHCl₃); UV (MeOH) λ_max (log ε): 202 (3.6), 239 (3.8) nm; IR ν_max (dried film): 2933, 2866, 1678, 1457, 1378, 1231 cm^−1; LRESIMS (m./z.): 411 [M + H]⁺ (C₂₉H₄₆O).

β.-Stigmasterol (12)

Obtained as colorless needles; : − 20.0° (c. 0.02, CHCl₃); UV (MeOH) λ_max (log ε): 206 (3.8) nm; IR ν_max (dried film): 3314, 2938, 2867, 1678, 1605, 1513, 1459, 1375, 1286, 1169, 1046 cm⁻¹; LRESIMS (m./z.): 412 [M]⁺ (C₂₉H₄₈O).

3-O-β.D.-Glucopyranosyl-stigmasta-5, 22-diene (13.)

Obtained as white powder; : + 83.3° (c. 0.06, CHCl₃); UV (MeOH) λ_max (log ε): 204 (3.6) nm; IR ν_max (dried film): 3403, 2933, 2867, 1456, 1372, 1171, 1073, 1024 cm⁻¹; LRESIMS (m./z.): 597 [M + Na]⁺ (C₃₅H₅₈O₆Na).

α.-Tocopherylquinone. (14)

Obtained as yellow oil; : + 0.0° (c. 0.1, CHCl₃); UV (MeOH) λ_max (log ε): 204 (3.7), 220 (3.5), 261 (3.9), 268 (3.8) nm; IR ν_max (dried film): 3480, 2925, 2865, 1642, 1460, 1375, 1282, 1164 cm⁻¹; LRAPCI (m./z.): 446 [M]⁻ (C₂₉H₅₀O₃).

COX assays

The effect of test compounds on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) was determined by measuring PGE₂ production (Cuendet et al., 2000; Waffo-Teguo et al., 2001; Jang et al., 2002) as previously described (Homhual et al., 2005a). Indomethacin was used as a positive control, yielding IC₅₀ values between 0.05 and 0.1 (1 and 5 µM) with COX-1 and COX-2, respectively.

Luciferase assays

Luciferase assays were conducted as previously described (Homhual et al., 2005a). Data for ARE induction (EC₅₀ values) were expressed as the concentration of compound that provoked activation half-way between baseline (DMSO control) and maximum response. Data for NF-κ.B constructs were expressed as IC₅₀ values (i.e., concentration required to inhibit TPA-activated NF-κ.B activity by 50%). For NF-κ.B, tumor necrosis factor (TNF)-α. was used as a standard activator for NF-κ.B expresion (IC₅₀ 0.06 nM), and for ARE induction sulforaphane (EC₅₀ 4–6 µM), 4′-bromoflavone (EC₅₀ 30 µM), or β.-naphthoflavone (EC₅₀ 8 µM) were used. With the experimental conditions employed, no signs of overt cellular toxicity were observed with the test compounds or extracts.

TPA-induced ornithine decarboxylase activity

Ornithine decarboxylase is the first rate-limiting enzyme for the biosynthesis of polyamines in mammalian cells (McCann et al., 1992). Test materials were assessed for potential to inhibit the induction of ODC by TPA using T24 (ATCC) cells as described previously (Hsieh et al., 1988; Gerhäuser et al., 1995). Standard inhibitors of TPA-induced ODC activity included deguelin (IC₅₀ 0.1 µM), apigenin (IC₅₀ 6 µM), and menadione (IC₅₀ 8.3 µM).

Results and Discussion

Ten triterpenoids (1–10) belonging to friedooleanane, oleanane, ursane, and lupane skeletons, three steroids (11–13), along with α.-tocopherylquinone (14) were isolated from the leaves of B. maunwongyathiae. by silica gel column chromatography as well as reverse-phase preparative HPLC (Fig. 1). Analysis of the spectral data including IR, MS, 1D and 2D NMR spectra determined the structures of these compounds as taraxerol (1), 3-(E.)-coumaroyltaraxerol (2), 3-(Z.)-coumaroyltaraxerol (3) (McLean et al., 1994; Laphookhieo et al., 2004), 3-(E.)-coumaroyl β.-amyrin (4), 3-(Z.)-coumaroyl β.-amyrin (5) (Takahashi et al., 1999), 3-(E.)-coumaroyl α.-amyrin (6), 3-(Z.)-coumaroyl α.-amyrin (7) (Öksüz et al., 1999), 3-(E.)-coumaroyllupeol (8), 3-(Z.)-coumaroyllupeol (9) (Chang et al., 1998), 3,19,24-trihydroxyurs-12-ene-28-oic acid (10) (Nakatani et al., 1989), stigma-4,22-dien-3-one (11) (Almeida et al., 1996), β.-stigmasterol (12), 3-O.-β.-D-glucopyranosyl-stigmasta-5,22-diene (13) (Alam et al., 1996), and α.-tocopherylquinone (14) (Olson et al., 1980), which were confirmed by the comparison with the literature spectral data. For the purpose of comparison, the ¹H and ¹³C NMR data of compounds 1–9 are listed in Tables 1 and 2, respectively. In addition, because no ¹³C NMR data of compound 14 have ever been published in the literature, this information is also included in Table 2. All isolates, except for stigmasterol, are reported for the first time in B. maunwongyathiae..

Of these compounds, coumaroyl β.-amyrin (4 and 5), coumaroyl α.-amyrin (6 and 7), and coumaroyllupeol (8 and 9) were isolated as three pairs of mixtures with each containing cis. and trans. isomers in a ratio of 0.64:1.0, 0.62:1.0, and 1:0.28, respectively. The inseparation of the three pairs of isomers were caused by the interconversion between the cis. and trans. isomers. The phenomena were observed when we tried to separate the individual peak of each cis. and trans. isomers by preparative HPLC. Although the cis. and trans. isomers were separable in the HPLC chromatography, the compound isolated from any single peak turned out to be a mixture of the cis. and trans. isomers. Cis.- and trans.-coumaroyl moieties were easily distinguished in the ¹H NMR spectra by the different coupling constants between the two coumaroyl olefinic protons (for the cis.-coumaroyls, J. ≈ 12.7 Hz; for the trans.-coumaroyls, J. ≈ 15.9 Hz).

In this study, the inhibitory effects on TPA-induced ODC activity, COX-2 activity, and phorbol ester–induced NF-κ.B luciferase expression, as well as the activation effects to ARE-mediated luciferase expression were used for evaluation of cancer chemopreventive potential.

The polyamines are critical for cell proliferation, differentiation, and are involved in DNA, RNA, and protein synthesis, as well as in stabilizing membrane and cytoskeletal structure (Feith et al., 2005). ODC is found in limited amounts in quiescent cells; activity can be rapidly and markedly increased in response to many trophic stimuli. Many different studies from animal models have shown that polyamines biosynthesis inhibition or polyamine analogues have a remarkable potential to block tumor growth.

Cyclooxygenases catalyze the conversion of arachidonic acid to prostaglandins, which are known to play a role in human cancer (Earashi et al., 1995). Therefore, inhibition of COX and the subsequent reduction of PG synthesis provide a viable strategy for the prevention of tumor development.

Nuclear factor-κ.B is composed of a family of inducible factors that, on activation, can induce cell proliferation, block differentiation, and prevent apoptosis (Baldwin, 2001). Accordingly, inhibition of induction could be of benefit.

Finally, activation of the ARE, located at the 5′-flanking region of phase II genes, leads to transcription of genes coding for phase II detoxifying enzymes (Kong et al., 1980). The selective induction of phase II detoxification enzymes is associated with cancer chemopreventive activity.

All isolates were subjected to chemoprevention investigation, and the results are shown in Table 3. Taraxerol (1) demonstrated significant inhibition against COX-2 with an IC₅₀ value of 0.9 µM. Interestingly, although 3-trans-.coumaroyltaraxerol (2) showed potent activity on COX-2 and ARE assays, its cis. isomer (3) showed no activity in any of the assays. While inhibition effects of taraxerol on COX-2 and NF-κ.B are more potent than those of 2, its enhancement effect on ARE expression is much less than that of 2. Other isolated triterpenes (4–10) showed no activity in all the assays. On the other hand, the three steroids (11–13) showed either weak or no activity with the assays used in this study. α.-Tocopherylquinone (14), the major physiologic oxidation product of a potent antioxidant α.-tocopherol, was reported to be an essential enzyme cofactor in the channeled mitochondrial fatty acid desaturation pathway (Infante, 1999) and can protect low-density lipoprotein (LDL) particles from oxidation (Stocker et al., 2003). In the current study, it was found that this compound inhibited TPA-induced ornithine decarboxylase activity with an IC₅₀ value of 5.9 µM and enhanced ARE expression with an EC₅₀ value of 5.2 µM. This is the first report on cancer chemopreventive activity of taraxerol (1), 3-trans.-coumaroyltaraxerol (2), β.-stigmasterol (12), 3-O.-β.-D-glucopyranosyl-stigmasta-5,22-diene (13), and α..-tocopherylquinone (14).

Table 3. Biological activity of compounds 1–14.

			Luciferase activity
Compound	ODC^a. IC50 (µM)	COX-2 IC50 (µM)	ARE^b. EC50 (µM)	NF-κ.B^c. IC50 (µM)
Taraxerol (1)	I^d.	0.92	31.2	10.2
3-(E.)-Coumaroyltaraxerol (2)	I	2.31	2.40	18.2
3-(Z.)-Coumaroyltaraxerol (3)	I	I	I	I
Coumaroyl β.-amyrin (4,5)	I	I	I
Coumaroyl α.-amyrin (6,7)	I	I	I	I
Coumaroyllupeol (8,9)	I	I	I	I
3,19,24-Trihydroxyurs-12-ene-28-oic acid (10)	I	I	I	I
Stigma-4,22-dien-3-one (11)	I	I	I	I
β.-Stigmasterol (12)	I	I	47.3	I
3-O.-β.-D-Glucopyranosyl-stigmasta-5,22-diene (13)	I	26.9	I	35.0
Tocopherylquinone (14)	5.92	I	5.16	I

^a.TPA-induced ornithine decarboxylase activity.

^b.Concentration required to enhance activity by twofold.

^c.Concentration required to inhibit activity by 50%.

^d.I, inactive (ODC, > 20 µg/mL; COX-2, > 10 µg/mL; ARE, > 20µg/mL; NF-κ.B, > 20 µg/mL).

Our experimental results indicate that inhibition of NF-κ.B by taraxerol (1), 3-trans.-coumaroyltaraxerol (2), and 3-O.-β.-D-glucopyranosyl-stigmasta-5,22-diene (13) correlates with the inhibition of NF-κ.B–responsive genes such as COX-2. Inhibition of NF-κ.B activation has been shown for some natural polyphenols (Surh et al., 2001) and other plant-derived agents (Shukla & Gupta, 2004). In summary, taraxerol (1) and 3-trans.-coumaroyltaraxerol (2) are capable of mediating specific biological responses, which, along with our previous chemopreventive study of other triterpenes (Homhual et al., 2005b), may warrant further investigation.

Acknowledgments

This study was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. program (grant no. PHD/5.A.MU/44/D.1) to A.J. and N.B. and by program project P01 CA48112 funded by the National Cancer Institute, NIH, Bethesda, Maryland. The authors are grateful to the NMR Lab of the Department of Medicinal Chemistry and Pharmacognosy, and the Research Resources Center, University of Illinois at Chicago, for access to the Bruker DPX 300 and 400 MHz instruments, as well as the acquisition of MS data.