Cancer Chemopreventive Activity of Rotenoids from Derris trifoliata

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant materials
• Extraction and isolation
• In vitro EBV-EA activation experiment
• In vivo two-stage mouse skin carcinogenesis test
• Results and Discussion
• References

Abstract

A study of the chemical constituents of the stems of Derris trifoliata Lour. (Leguminosae) led to the isolation and identification of one new rotenoid, 6aα,12aα-12a-hydroxyelliptone (3), together with five other known rotenoids. In a search for novel cancer chemopreventive agents (anti-tumor promoters), we carried out a primary screening of five of the rotenoids isolated from the plant for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in Raji cells. The inhibitory activity of 3 was found to be equivalent to that of β-carotene without any cytotoxicity. Deguelin (4) and α-toxicarol (5) exhibited a marked inhibitory effect on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. This investigation indicated that rotenoids might be valuable anti-tumor promoters.

Introduction

Rotenone and related compounds, isolated from the roots of plants such as Derris (Leguminosae), were originally used to paralyze fish before being used as an insecticide [1]. Rotenoids are known not only as toxicants, but also as candidate anticancer agents. Rotenoids have been shown to exhibit extremely potent cytotoxicity against six human cancer cell lines [2]. Deguelin (4), one of the rotenoids, has also been shown to mediate strong inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity in cell culture and to reduce the activity in the two-stage 7,12-dimethylbenz[a]anthracene (DMBA)/TPA skin carcinogenesis model with mice and rats [3], [4], [5], [6]. Furthermore, we have previously reported that some rotenoids including tephrosin (6; Fig. [1]) isolated from plants of the Leguminosae family showed strong inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter TPA, and exhibited significant anti-tumor promotion effects on mouse skin tumors in an in vivo two-stage carcinogenesis test [7]. In this paper, we describe the isolation and identification of one new rotenoid, 6aα,12aα-12a-hydroxyelliptone (3), from the stems of D. trifoliata Lour. We also report the inhibitory effects of rotenoids isolated from the plant on EBV-EA activation and in vivo two-stage carcinogenesis of mouse skin.

Materials and Methods

General experimental procedures

¹H- and ¹³C-NMR, COSY, HMQC, HMBC (J = 8 Hz), and NOE were measured on JNM A-400, A-600 and/or ECP-500 (JEOL) spectrometers. Chemical shifts are shown in δ (ppm) with tetramethylsilane (TMS) as an internal reference. All mass spectra were taken using electron impact (EI) ionization, unless otherwise stated, using an HX-110 (JEOL), and/or a JMS-700 (JEOL) spectrometer with a direct inlet system. UV spectra were recorded on a UVIDEC-610C double-beam spectrophotometer (JASCO) in MeOH, IR spectra on an IR-230 (JASCO) in CHCl₃, optical rotations on a DIP-370 (JASCO) in CHCl₃ at 25 °C, and CD spectra on a J-600 (JASCO) in MeOH. Preparative TLC was performed on Kieselgel 60F₂₅₄ (Merck).

Plant materials

The plant materials used in this study, Derris trifoliata Lour. (Leguminosae), were collected in August 1995. Dr H.T.W. Tan identified the material using keys or by comparison with herbarium sheet specimens at the Herbarium, Department of Biological Sciences, The National University of Singapore (SINU) or at the Herbarium, Botanic Gardens, Singapore (SING). A voucher specimen has been deposited at SINU under number H.T.W. Tan 6 - 1995. The plant is used as a stimulant, antispasmodic and counter-irritant in Singapore.

Extraction and isolation

The dried stems (135 g) of D. trifoliata were extracted with acetone (2 L × 3) at room temperature and the solvent was evaporated under reduced pressure to give the acetone extract (6.5 g), which was subjected to silica gel (l50 g) column chromatography eluted with hexane-acetone (17 : 3, 4 : 1, 7 : 3, 3 : 2, 1 : 1, 3 : 7), acetone, CH₂Cl₂-MeOH (3 : 1) and MeOH gradients (each 0.5 L), successively to obtain fractions 1 - 9. Fraction 2 (hexane-acetone, 4 : 1, 298 mg) was chromatographed on silica gel (10 g) and eluted with hexane-acetone (85 : 15 → 60 : 40, 150 mL for each eluent) yielding 4 (13.8 mg) and 5 (11.0 mg), which were purified by preparative TLC with CH₂Cl₂, respectively. Fraction 3 (hexane-acetone, 7 : 3, 622 mg) was chromatographed on silica gel (12g) and eluted with hexane-acetone (85 : 15 → 50 : 50, 200 mL for each eluent) yielding 4 (27.9 mg), 5 (7.5 mg) and 6 (l2.5 mg). Fraction 4 (hexane-acetone, 3 : 2, 812 mg) was subjected to column chromatography on silica gel (12 g), and elution with hexane-acetone (90 : 10 → 50 : 50, 200 mL for each eluent) to afford 2 (22.2 mg), which was purified by preparative TLC with benzene-MeOH (96 : 4). Fraction 5 (hexane-acetone, 1 : 1, 1.0 g) was chromatographed on silica gel (15g) and elution with hexane-acetone (80 : 20 → 40 : 60) afforded 1 (21.5 mg) and 3 (12.6 mg), which were purified by preparative TLC with hexane-EtOAc (80 : 20) and benzene-acetone (94 : 6), respectively.

6aα,12aα-12a-Hydroxyelliptone (3): Colorless oil; [α]_D ²⁵: -4.4° (c 0.068, CHCl₃); IR (CHCl₃): ν_max = 3504, 1678, 1614 cm^-1; UV (MeOH): λ_max (log ε) = 203 (4.57), 239 (4.46), 277 (3.85), 292 sh (3.70), 319 (3.47) nm; CD (MeOH): [Θ]₂₃₅ = -11 758, [Θ]₂₃₉ = -7617, [Θ]₂₄₄ = -10 621, [Θ]₂₅₂ = -3796, [Θ]₂₆₂ = -5962, [Θ]₂₇₄ = 0, [Θ]₂₉₅ = 9725, [Θ]₃₂₅ = 2943, [Θ]₃₄₃ = 0, [Θ]₃₅₅ = -1766, [Θ]₃₆₉ = 0; ¹H-NMR (CDCl₃, 400 MHz): δ = 7.87 (1H, d, J = 8.8 Hz, H-11), 7.56 (1H, d, J = 2.2 Hz, H-2′), 7.17 (1H, dd, J = 1.1, 8.8 Hz, H-10), 6.91 (1H, dd, J = 1.1, 2.2 Hz, H-3′), 6.55 (1H, s, H-1), 6.49 (1H, s, H-4), 4.74 (1H, br s, H-6a), 4.72 (1H, dd, J = 14.3, 2.6 Hz, H-6), 4.56 (1H, d, J = 14.3 Hz, H-6), 4.49 (1H, br s, 12a-OH), 3.80 (3H, s, 3-OCH₃), 3.71 (3H, s, 2-OCH₃); ¹³C-NMR (CDCl₃,100 MHz): δ = 192.2 (s, C-12), 160.6 (s, C-9), 155.8 (s, C-7a), 151.2 (s, C-3), 148.4 (s, C-4a), 145.1 (d, C-2′), 144.0 (s, C-2), 123.9 (d, C-11), 117.2 (s, C-8), 112.0 (s, C-11a), 109.3 (d, C-1), 108.4 (s, C-12b), 107.1 (d, C-10), 104.8 (d, C-3′), 101.1 (d, C-4), 77.2 (d, C-6a), 67.7 (s, C-12a), 63.8 (t, C-6), 56.4 (q, 2-OCH₃), 55.8 (q, 3-OCH₃); Differential NOE: Irradiation of 3-OCH₃ (δ = 3.80) -19 % enhancement of H-4 (δ = 6.49), irradiation of 2-OCH₃ (δ = 3.71) -11 % enhancement of H-1 (δ = 6.55); El-MS: m/z = 368 [M⁺] (31), 208 [M⁺ - C₉H₄O₃] (base peak), 193 (9), 165 (10), 160 [M⁺ - C₁₁H₁₂O₄] (9); HR-MS: m/z = 368.0913 (calcd. for C₂₀H₁₆O₇ : 368.0895).

In vitro EBV-EA activation experiment

Raji cells in the exponential growth phase were maintained in RPMI-1640 medium (Sigma Chemical Co., MI, USA) supplemented with 10 % fetal bovine serum, harvested by centrifugation and resuspended. To test the effect of the candidate compounds, each concentration of the candidate compounds was added to the Raji cells in medium supplemented with 32 pmol TPA and 4 mM n-butyric acid, and incubated for 48 h at 37 °C. After incubation, each sample was centrifuged at 1,000 g for 10 min and the precipitates were resuspended in PBS and placed on a 76 × 26 mm micro slide glass (Matsunami Glass Ind., Ltd, Tokyo). The smear samples were stained with NPC serum and human IgG serum to examine their specific activation. The activated early antigens were detected by immunohistochemical studies using fluorescence microscopy. The treated cells were observed with trypan blue staining for cell cytotoxicity. For the determination of cytotoxicity, the cell viability was required to be more than 60 % 2 days after treatment with the compounds for an accurate result.

In vivo two-stage mouse skin carcinogenesis test

Female ICR mice were obtained at 5 - 6 weeks of age from SLC Co. Ltd (Shizuoka, Japan). Groups of 15 mice were housed in subgroups of five in polycarbonate cages. Mice were permitted free access to MP solid diet (Oriental yeast Co., Ltd., Chiba, Japan) and drinking water at all times during the study. The back of each mouse was shaved with surgical clippers before the first day of tumor initiation. Tumors on the back of the mice were initiated with DMBA (390 nmol) in acetone (0.1 mL). One week after initiation, the tumors were promoted by twice weekly application of TPA (1.7 nmol) in acetone (0.1 mL). Rotenoid-treated mice were treated with the test compounds (85 nmol) in acetone (0.1 mL) 1 h before each TPA treatment. The incidence of papillomas was observed weekly for 20 weeks. Positive control mice were given topical application of acetone to the same portion of the back as promoter treatment 1 h before TPA application, and rotenoids were also given by the same treatment instead of acetone.

Results and Discussion

The acetone extract of stems of D. trifoliata was fractionated by silica gel column chromatography and preparative TLC to obtain one new rotenoid, 6aα,12aα-12a-hydroxyelliptone (3) as a colorless oil, [α]_D ²⁵: -4.4° (c 0.068, CHCl₃) and its molecular formula was determined as C₂₀H₁₆O₇ by HR-MS. The UV spectrum of 3 was similar to that of elliptone (2). The IR spectrum exhibited bands at ν_max = 3504 and 1678 cm^-1 due to hydroxy and conjugated carbonyl groups. The MS fragmentation peaks occurred at m/z = 208 (base peak, M⁺ - ^·C₉H₄O₃) and 160 (9 %, M⁺ - ^·C₁₁H₁₂O₄) via a retro Diels-Alder process in ring C. The ¹H-NMR spectrum of 3 exhibited two singlet methoxy groups (δ = 3.80 and 3.71) and four aromatic protons [two ortho-coupled protons (δ = 7.87, 7.17) and two singlet protons (δ = 6.55, 6.49)]. It also exhibited the following signals due to protons on carbon atoms bearing oxygen atoms: δ = 4.74 (H-6a), 4.72 (H-6), and 4.56 (H-6). In addition, the characteristic signals of two benzofuranoic protons (δ = 7.56 and 6.91) were observed along with a D₂O exchangeable proton (δ = 4.49, OH). Location of the methoxy groups at C-2, C-3 was indicated by observation of NOEs between a methoxy at δ = 3.80 and an aromatic singlet proton at δ = 6.49 (H-4) and between another methoxy at δ = 3.71 and an aromatic singlet proton at δ = 6.55 (H-l). The presence of a hydroxy group at C-12a was indicated by HMBC, which showed a significant C-H three-bond correlation between a carbonyl carbon at δ_C = 192.2 (C-12) and a hydroxy proton at δ = 4.49 (Fig. [2]) coupled with the MS fragmentations. The H-1 chemical shift value (δ = 6.55) indicated that the B/C ring junction of 3 was cis [8]. The determination of the absolute configuration of 12a-hydroxyrotenoids by CD spectroscopy was studied by Tahara et al. [9]. 12a-Hydroxyrotenone and villosinol having [6aR,12aR] stereochemistry were reported to show positive Cotton effects at 359 nm and 348 nm and negative Cotton effects at 335 nm and 303 nm, respectively. And 12a-hydroxyerythynone having [6aS,12aS] has been known to show a negative Cotton effect at 370 nm and a positive Cotton effect at 332 nm [9]. From analysis of the CD spectrum, 3 was found to give a negative Cotton effect at 355 nm and a positive Cotton effect at 325 nm, therefore the B/C ring absolute stereochemistry was assigned to be [6aS,12aS] [9], [10]. On the basis of these data, the structure of 6aα,12aα-12a-hydroxyelliptone (3) was determined to be as shown in Fig. [1].

Recently, the isolation of the (+)-isomer of 3 from D. malaccensis was reported [11]. Five known rotenoids, rotenone (1), elliptone (2), deguelin (4), α-toxicarol (5), and (-)-tephrosin (6), were also isolated and were identified by comparison of their spectral data with those published in the literature [12]. The absolute configuration of (-)-tephrosin (6), isolated from the seeds of Millettia dura (Dunn), remained unchanged [10]. As to the consideration of the CD spectrum of 6, a positive Cotton effect ([Θ] + 894) at 357 nm and a negative Cotton effect ([Θ] -13 157) at 327 nm were observed; therefore the B/C ring absolute stereochemistry was determined as [6aR,12aR]. Further examination of the constituents of this plant is now in progress.

Five natural rotenoids (1 - 5) were tested for their anti-tumor-promoting activity by using a short-term in vitro assay of TPA-induced EBV-EA activation in Raji cells. The inhibitory effects of these rotenoids on the activation of the virus-genome, and the viabilities of Raji cells, are shown in Table [1]. All tested rotenoids were found to show inhibitory activity on the EBV activation even at a 1 × 10 mol ratio, and showed a significant inhibitory effect at high concentrations (1 × 10³ mol ratio). However, all rotenoids showed only weak cytotoxicity in assays of Raji cells even at the 1 × 10³ mol ratio. The inhibitory effects of rotenoids 1 - 5 were more potent than that of β-carotene, a vitamin A precursor commonly used as a reference in cancer prevention studies [13], at 1 × 10² and 1 × 10 mol ratios (23.2 - 33.6 % and 3.2 - 5.3 % inhibition of activation, respectively). At 1 × 10³ and 5 × 10² mol ratios, rotenoids 1 - 5 showed equivalent or weaker inhibitory effects (86.1 - 100.0 % and 53.8 - 62.6 %, respectively) than that of β-carotene.

Deguelin (4) and α-toxicarol (5) are the major components in the stems of D. trifoliate while the tumor inhibitory activity of deguelin (4) in the mouse skin carcinogenesis model has already been reported by Udeani et al. [4]. In the present study, we demonstrated the inhibitory effect of α-toxicarol (5) to compare it with the effect of deguelin (4) on an in vivo skin carcinogenesis model. At 10 weeks after DMBA-TPA tumor promotion, the incidence of papillomas was 100 % in the control animals. In comparison, the incidence of papillomas in the group pre-treated with α-toxicarol (5) was markedly reduced by 73 and 33 % after 10 and 15 weeks of promotion, respectively (Fig. [3] A). As shown in Fig. [3] B, the number of papillomas per mouse was reduced by about 63 and 34 % after 10 and 20 weeks in α-toxicarol (5) pre-treated mice, respectively, compared to untreated mice. These results suggest that α-toxicarol (5) showed about the same inhibitory activity as deguelin (4) (Figs. 3 A and 3 B), and these rotenoids might be valuable as potential cancer chemopreventive agents (anti-tumor promoters). Intensive investigations on the structure-activity relationship of the rotenoids, and also studies investigating the mechanism of their inhibitory activities, are now in progress.

References

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• 10 Ollis W D, Rhodes C A, Sutherland I O. The extractives of Millettia dura (Dunn). The constitutions of durlettone, durmillone, milldurone, millettone and millettosin.  Tetrahedron. 1967;  23 4741-60
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• 12 Carlson D G, Weisleder D, Tallent W H. NMR investigations of rotenoids.  Tetrahedron. 1973;  29 2731-41
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• 13 Murakami A, Ohigashi H, Koshimizu K. Anti-tumor promotion with food phytochemicals: A strategy for cancer chemoprevention.  Bioscience Biotechnology and Biochemistry. 1996;  60 1-8
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Prof. Masataka Itoigawa

Tokai Gakuen University

Ukigai

Miyoshi-cho

Nishikamo-gun

Aichi 470-0207

Japan

Phone: +81-5613-6-5555

Fax: +81-5613-6-6757

Email: itoigawa@tokaigakuen-u.ac.jp

References

• 1 Kan W S. Manual of Medicinal Plants in Taiwan. Vol. 2 National Research Institute of Chinese Medicine Taiwan; 1972: pp 276-80
  Search in Google Scholar
• 2 Li L, Wang H K, Chang J J, McPhail A T, McPhail D R, Terada H. et al . Antitumor agents, 138. Rotenoids and isoflavones as cytotoxic constituents from Amorpha fruticosa .  Journal of Natural Products. 1993;  56 690-8
  PubMedSearch in Google Scholar
• 3 Udeani G O, Zhao G M, Shin Y G, Kosmeder JW 2 nd, Beecher C WW, Kinghorn A D. et al . Pharmacokinetics of deguelin, a cancer chemopreventive agent in rats.  Cancer Chemotherapy and Pharmacology. 2001;  47 263-8
  CrossrefPubMedSearch in Google Scholar
• 4 Udeani G O, Gerhäuser C, Thomas C F, Moon R C, Kosmeder J W, Kinghorn A D. et al . Cancer chemopreventive activity mediated by deguelin, a naturally occurring rotenoid.  Cancer Research. 1997;  57 3424-8
  PubMedSearch in Google Scholar
• 5 Gerhäuser C, Lee S K, Kosmeder J W, Moriarty R M, Hamel E, Mehta R G. et al . Regulation of ornithine decarboxylase induction by deguelin, a natural product cancer chemopreventive agent.  Cancer Research. 1997;  57 3429-35
  PubMedSearch in Google Scholar
• 6 Gerhäuser C, Mar W, Lee S K, Suh N, Luo Y, Kosmeder J. et al . Rotenoids mediate potent cancer chemopreventive activity through transcriptional regulation of ornithine decarboxylase.  Nature Medicine. 1995;  1 260-6
  CrossrefPubMedSearch in Google Scholar
• 7 Konoshima T, Terada H, Kokumai M, Kozuka M, Tokuda H, Estes J R. et al . Studies on inhibitors of skin tumor promotion, XII. Rotenoids from Amorpha fruticosa .  Journal of Natural Products. 1993;  56 843-8
  CrossrefPubMedSearch in Google Scholar
• 8 Lin L -J, Ruangrungsi N, Cordell G A, Shieh H- L, You M, Pezzuto J M. 6-Deoxyclitoriacetal from Clitoria macrophylla .  Phytochemistry. 1992;  31 4329-31
  CrossrefPubMedSearch in Google Scholar
• 9 Tahara S, Narita E, Ingham J L, Mizutani J. New rotenoids from the root bark of Jamaican dogwood (Piscidia erythrina L.)  Zeitschrift fur Naturforschung. 1990;  45c 154-60
  PubMedSearch in Google Scholar
• 10 Ollis W D, Rhodes C A, Sutherland I O. The extractives of Millettia dura (Dunn). The constitutions of durlettone, durmillone, milldurone, millettone and millettosin.  Tetrahedron. 1967;  23 4741-60
  CrossrefPubMedSearch in Google Scholar
• 11 Thasana N, Chuankamnerdkarn M, Ruchirawat S. A new 12a-hydroxyelliptone from the stems of Derris malaccensis .  Heterocycles. 2001;  55 1121-5
  PubMedSearch in Google Scholar
• 12 Carlson D G, Weisleder D, Tallent W H. NMR investigations of rotenoids.  Tetrahedron. 1973;  29 2731-41
  CrossrefPubMedSearch in Google Scholar
• 13 Murakami A, Ohigashi H, Koshimizu K. Anti-tumor promotion with food phytochemicals: A strategy for cancer chemoprevention.  Bioscience Biotechnology and Biochemistry. 1996;  60 1-8
  PubMedSearch in Google Scholar

Prof. Masataka Itoigawa

Tokai Gakuen University

Ukigai

Miyoshi-cho

Nishikamo-gun

Aichi 470-0207

Japan

Phone: +81-5613-6-5555

Fax: +81-5613-6-6757

Email: itoigawa@tokaigakuen-u.ac.jp