Polyprenylated isoflavanone and isoflavonoids from Ormosia henryi and their cytotoxicity and anti-oxidation activity

Fitoterapia 83 (2012) 161–165

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Polyprenylated isoflavanone and isoflavonoids from Ormosia henryi and their cytotoxicity and anti-oxidation activity Shixiu Feng a, b, Jing Hao a, Zhifang Xu a, Tao Chen b, Samuel X. (Shengxiang) Qiu a,⁎ a Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, 723 Xingke Road, Tianhe District, Guangzhou 510650, People's Republic of China b Laboratory of Southern Subtropical Plant Diversity, Shenzhen Flary Lake Botanical Garden, Chinese Academy of Sciences, 160 Xianhu Road, Luohu District, Shenzhen 518004, People's Republic of China

a r t i c l e

i n f o

Article history: Received 25 July 2011 Accepted in revised form 12 October 2011 Available online 24 October 2011 Keywords: Ormosia Ormosia henryi Prain Isoflavonoids Cytotoxicity Anti-oxidation activity

a b s t r a c t A rare naturally-occurring polyprenylated isoflavanone, designated ormosinol (1), and a new isoflavonoid glycoside, named ormosinoside (2), along with 21 known compounds were isolated from the root bark of Ormosia henryi Prain. The structures of compounds 1 and 2 were determined as 5,7,2′,4′-tetrahydroxyl-6,8,5′-tri-(γ,γ-dimethylallyl)isoflavanone and isoprunetin-7-O-β-D-xylopyranosyl-(1 → 6)-β-D-glucopyranoside on the basis of a combination of 1D-, 2D-NMR and mass spectroscopic measurements. Compound 1 showed significant anti-oxidation activity against DPPH radicals (IC50 28.5 μM) and cancer cell line (A549, LAC, and HepG2) growth inhibitory activity with IC50 ranging from 4.25 to 7.09 μM, while compound 2 found to be inactive to both testing systems. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The genus Ormosia (Leguminosae), comprising about 120 species, is mainly distributed in the topical areas of the word with 35 species being native in China. Many plants of this genus are used for features or ornamental, some species have applications in folk medicine [1]. Previous phytochemical studies of this genus reported the isolation of a variety of secondary metabolites such as flavonoids, alkaloids, and triterpenoids [2–6]. Ormosia henryi Prain, a tall tree, mainly grows in southern provinces of China, with its root, leaves and stem bark used as Chinese folk medicine for the treatment of swallow, pain and inflammation [7]. With an aim to search for new anti-inflammatory natural products, we initiated an intensive phytochemical study on the roots of O. henryi, resulting in the isolation of two new compounds, ormosinol (1) and ormosinoside (2), as well as 21 known compounds (3–23). Herein, we describe the isolation, structure

⁎ Corresponding author. Tel./fax: + 86 20 37081190. E-mail address: [email protected] (S.X.(S.) Qiu). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.10.007

elucidation and biological evaluation of the two new compounds 1 and 2. 2. Experimental 2.1. General Melting points were determined on a Yanagimoto Seisakusho MD-S2 and are uncorrected. Optical rotations were obtained on a Perkin-Elmer 341 polarimeter with MeOH and DMSO as solvent. The UV spectra were recorded in MeOH on a Perkin Elmer Lambda 25 UV–VIS Spectrophotometer. The IR spectra were measured in KBr on a WQF-410 FT-IR spectrophotometer. The 1H (400 MHz), 13C (100 MHz) and 2D NMR spectra were recorded on a Bruker DRX-400 instrument using TMS as an internal standard. The chemical shifts are given in δ (ppm) and coupling constants in Hz. ESI-MS were collected on MDS SCIEX API 2000 LC/GC/MS instrument. HRESI-MS data were obtained on an API QSTAR mass spectrometer. For column chromatography, silica gel 60 (100−200 mesh) and polyamide (80–100 mesh), were produced by Qingdao Marine Chemical Ltd., (Qingdao, People's Republic of

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China), Sephadex LH-20 and Diaion HP-20 porous resin were produced by Mitsubishi Chemical Holdings of Japan. 2.2. Plant material The root bark of Ormosia henryi Prain was collected from Nankang County of Jiangxi Province, People's Republic of China, in October 2007. The plant was identified by Professor Fuwu Xin of SCBG and a voucher was deposited at Laboratory of Phytochemistry, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China. 2.3. Extraction and isolation The air-dried material (34 kg) was smashed into small pieces and extracted 3 times by 95% EtOH at room temperature. The combined extracts were filtered and concentrated under vacuum to obtain a crude residue (2800 g). The residue was suspended in H2O and partitioned in sequence using petroleum ether (60–90 °C), EtOAc, and n-BuOH, respectively, to afford a petroleum ether extract (220 g), EtOAc extract (1150 g), and an n-BuOH extract (280 g). The petroleum ether-soluble extract was dissolved in acetone and a white precipitate was obtained, which was filtered and washed by MeOH and Acetone successively to obtain compound 3 (100 g). The supernatant was subjected to a silica gel column chromatography (CC) and eluted with a gradient petroleum ether–acetone (from 10:0 to 6:4) to yield eight fractions (1–8). Fraction 2 (10.0 g) had white residue, which was filtered and washed by MeOH to get compound 6 (500 mg). Fraction 4 (5.0 g) was further chromatographed on silica gel CC and eluted with CHCl3–MeOH (98:2) to obtain compound 21 (100 mg). The EtOAc extraction was dissolved by 80% EtOH and had white deposition, which was filtered and recrystallized with 80% EtOH to get white crystals of compound 12 (250 g). Then the supernatant was separated by a silica gel CC using CH3Cl–MeOH (from 100:0 to 60:40) to give eight fractions (9–16). Fraction 9 was further subjected to silica gel CC and eluted with petroleum ether–acetone (3:1) to give six subfractions (9a–9f). Subfraction 9a (1.0 g) was purified by CHCl3–MeOH (98:2) on a silica gel CC to yield compounds 3 (130 mg) and 4 (150 mg). Subfraction 9b (21 g) and 9f (100 g) both had white deposition, which was filtered and recrystallized with 80% EtOH to obtain compounds 5 (5 g) and 9 (31 g), respectively. Subfraction 9c (3 g) was subjected to recrystallization with MeOH to yield compound 7 (30 mg); while the supernatant was further purified by silica gel CC eluting with CH3Cl–MeOH (92:8) to get compound 1 (160 mg). Fraction 10 (5.5 g) was applied to silica gel CC using CHCl3– acetone (3:1) and purified by Sephadex LH-20 CC using MeOH to obtain compounds 16 (56 mg), 18 (39 mg) and 20 (89 mg). Fraction 11 (20.6 g) was purified with the same method to get compounds 8 (1.0 g), 11 (40 mg), 10 (300 mg), 19 (200 mg) and 17 (393 mg). Fraction 12 (21.8 g) had white needles, which was recrystallized with 80% EtOH to yield compound 22 (13.5 g); the supernatant was subjected to silica gel CC eluting with CHCl3–MeOH–H2O (10:3:1) and purified by Sephadex LH-20 (MeOH) to obtain compound 13 (80 mg). Fr.13 (5.5 g) was subjected to silica gel CC

eluting with CHCl3–MeOH–H2O (7:3:1) and purified by Sephadex LH-20 (MeOH) to yield compound 14 (80 mg). The n-BuOH-soluble extract (280 g) was chromatographed on a Diaion HP-20 resin column and eluted successively with H2O, 50% EtOH and finally EtOH. The 50% EtOH eluent was collected and concentrated under vacuum to afford a residue (150 g) which was then subjected to a silica gel CC and eluted with CHCl3–MeOH–H2O (7:3:1) to give four fractions (17–20). Fraction 19 was separated by polyamide CC eluting with 20% MeOH, and further purified by Sephadex LH-20 CC (MeOH) to yield compounds 23 (120 mg), 15 (45 mg) and 2 (50 mg). Ormosinol (1): white power, mp 99–104 °C; [α] 20D + 1° (c 2.0, MeOH). UV/Vis λmax (MeOH) nm (log ε): 203 (2.90), 295 (2.40). IR vmax (KBr) (cm − 1): 3380, 2969, 2913, 1700, 1631, 1509, 1442 and 1378. 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) data see Table 1. HR-ESIMS m/z 493.2609 [M+ H]+ (calcd for C30H37O6, 493.2584). Ormosinosides A (2): white power, mp 225–228 °C, [α] 20D − 57° (c 1.0, DMSO). UV/Vis λmax (MeOH) nm (log ε): 202 (3.69), 250 (3.75). IR vmax (KBr) (cm − 1): 3390, 2919, 1637, 1515, 1461, 1428 and 1375. 1H NMR (DMSO-d6, 400 MHz) δ: 9.51 (1H, s, OH-4′), 8.11 (1H, s, H-2), 7.30 (2H, d, J = 8 Hz, H-2′ and 6′), 6.78 (2H, d, J = 8 Hz, H-3′ and 5′), 6.73 (1H, d, J = 2 Hz, H-8), 6.55 (1H, d, J = 2 Hz, H-6), 5.04 (1H, d, J = 6.8 Hz, H-1″), 4.16 (1H, d, J = 7.2 Hz, H-1′″), 3.67 (3H, s, 5-OCH3), 2.0–5.0 (10H, m, sugar protons); 13C NMR (DMSO-d6, 100 MHz) data see Table 2. HR-ESI-MS m/z 577.1556 [M − H] − (calcd for C27H29O14, 577.1562). 2.4. Cytotoxicity assay The MTT assay was performed according to literature with slight modification [8]. Human cancer cells were plated at 1 × 10 4 cells per well in 96 well microtiter plates and incubated for 24 h at 37 °C, 5% CO2. Some wells of the plate were added with only 100 μl of culture medium as a background well, different concentrations (50, 25, 12.5, 6.5 and 3.125 μmol) of the compound were added. After 3 days of incubation at 37 °C, 5% CO2, 20 μl MTT reagent (5 mg/ml) was added. After incubating at 37 °C for 4 h, MTT reagent was removed and DMSO (150 μl) was added to each well and shaken for another 10 min. The absorbance was then determined by a CENios microplate reader (TECAN, Genios) at a wavelength of 570 nm. Control wells received only the media without the test samples. The conventional anticancer drug, doxorubicin was used as positive control in this study. The inhibition of cell growth by the samples tested was calculated as percentage anticancer activity and was calculated using the following formula: percentage anticancer activity (Ac − As/Ac) × 100%. Ac and As refer to the absorbance of control and the sample, respectively. 2.5. Antioxidant activities assay The DPPH-scavenging assay was carried out according to literature [79] with minor modification, namely, 200 µl of reaction mixtures containing 20 µl test samples dissolved in DMSO and 180 µl of DPPH (0.1 mM) were plated in 96-cell plates incubated in the dark for 60 min. After the reaction, absorbance was measured at 515 nm, and percent inhibition was calculated. The antioxidant activity of each sample was

S. Feng et al. / Fitoterapia 83 (2012) 161–165 Table 1 1 H-(400 MHz) and Positiona 2

C-(100 MHz) NMR data of compound 1 DMSO-d6. δC 69.8

3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 1′′′ 2′′′ 3′′′ 4′′′ 5′′′ 1′′′′ 2′′′ 3′′′ 4′′′ 5′′′ 5-OH 7-OH 2′-OH 4′-OH a

13

46.2 198.3 158.9 107.9 157.7 107.0 161.2 102.3 117.8 154.8 102.5 153.6 112.1 130.3 21.4 122.8 130.3 17.7 21.0 17.5 123.3 130.7 25.5 27.2 17.7 123.0 130.3 25.4 27.1

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Table 2 13 C NMR (100 MHz) data of compounds 2 and 13.

δH (mult, J = Hz)

Position

13

2

4.40 (dd, 4.8, 10.4) 4.40 (dd, 5.6, 10.4) 4.08 (dd, 5.6, 4.4)

2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 1′′′ 2′′′ 3′′′ 4′′′ 5′′′ 5-OCH3

150.9 124.9 173.9 160.8 97.2 161.4 95.6 157.1 109.6 122.6 130.3 114.9 158.8 114.9 130.3 99.9 73.1 76.6 69.8 77.3 60.7

150.8 124.9 173.9 160.8 97.0 161.3 95.7 157.1 109.6 122.6 130.2 114.9 158.9 114.9 130.2 99.7 76.4 76.6 69.8 75.7 65.7 104.2 73.1 73.4 69.5 68.8 56.2

6.34 (s)

6.62 (s) 3.19 (d, 7.6) 5.09 (m) 1.67 1.69 3.21 5.09

(s) (s) (d, 7.6) (m)

1.61 1.59 3.02 5.15

(s) (s) (d, 6.8) (t, 3.6)

1.59 (s) 1.54 (s) 12.61 (s) 9.56 (br) 9.14 (s) 9.24 (s)

Signals were assigned from the 13C–1H COSY, HMBC and NOESY spectra.

expressed in terms of IC50 (μM required to inhibit DPPH radical formation by 50%) and calculated from the log-dose inhibition curve [9]. 3. Results and discussion The EtOH extract of the roots of O. henryi was fractioned with petroleum ether, EtOAc, and n-BuOH. Separation by a combination of silica gel, Sephadex LH-20 and ODS column chromatography afforded a new polyprenylated isoflavanone (1), and a new isoflavonoid glycosides (2), together with 21 known compounds, lupeol (3) [10], 7-O-methylbiochanin A (6) [13], octadecyl caffeate (21) [22], from the petroleum ether-soluble fraction, lupenone (4) [11], biochanin A (5) [12], 4′,7-di-O-methyldaidzein (7), genistein (8), daidzein (9), sissotrin (10), daidzin (11) [13], isoprunetin (12), isoprunetin-7-O-β-D-glucoside (13) [14], isoformononetin (14) [15], naringenin (16) [17], aromadendrin (17) [18], apigenin (18) [19], kaempferol (19) [20], (−)-syringaresinol (20) [21], kakkanin (22) [23], from the EtOAc-soluble fraction, and sophoricoside (15) [16], 6″-β-D-xylose-genistin (23) [24], from the n-BuOH-soluble fraction. The structure of the known compounds was determined by interpretation for their spectroscopic data as well as by a comparison with reported data.

56.1

Compound 1, obtained as a red solid, has a molecular formula of C30H36O6 as determined by HR-ESI-MS where in a protonated molecule [M+ H]+ was detected at m/z 493.2609 (calcd for C30H37O6 493.2584). The IR spectrum shows the absorption of a hydroxyl group (3380 cm − 1), a conjugated carbonyl group (1631 cm − 1), and olefinic functionality (1509 cm − 1). An isoflavanone skeleton is suggested from the proton signals at δH 4.08 (1H, dd, H-3) and 4.40 (2H, dd, H-2α and H-2β) in the 1H NMR spectrum, coupled with 13C signals arising from a carbonyl group (δC 198.3), a methylene group (δC 46.2) and a methine group (δC 69.8) from the 13C NMR spectrum [25]. The 1H NMR displayed indicative group signals at δH 1.54 (3H, s, H3-5″″), 1.59 (6H, s, H3-5′′′ and H3-4″″), 1.61 (3H, s, H3-4′′′), 1.67 (3H, s, H3-4″), 1.69 (3H, s, H3-5″), 3.02 (2H, d, H2-1″″), 3.19 (2H, d, H2-1″), 3.21 (2H, d, H2-1′′′), 5.09 (2H, m, H-2′′′ and 2″″) and 5.15 (1H, t, H-2″), characteristic of three dimethylallyl (pentenyl) groups, and the chemical shift of the methylene protons (3.02–3.21 ppm) indicated that the prenyl group was bonded to an aromatic ring; four phenolic hydroxyl groups at δH 12.61 (1H, s, 5-OH), 9.56 (1H, brs, 7-OH), 9.24 (1H, s, 4′-OH) and 9.14 (1H, s, 2′-OH); two aromatic singles at δH 6.34 (1H, s, H-3′) and 6.62 (1H, s, H-6′), suggesting 1 is a flavanone (dihydroflavone) derivative with four hydroxyl and three pentenyl appendages, although their precise positions needed to be established. The attachment of the two hydroxyl groups are readily assigned to C-5 and C-7 based on the long-range 1H– 13C correlations (HMBC) observed between δH 12.61 (5-OH)/δC 158.9 and 107.9 (C-5, 6); δH 9.56 (7-OH)/δC 157.7 and 107.0 (C-7, 8), as well as correlations between δH 3.21 (4H, m, H-1″,H-1′′′) to δC 157.7/158.9 and 157.7/161.2 (C-7/5 and C-7/9), respectively. By analogy, the long-range 1H–13C

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S. Feng et al. / Fitoterapia 83 (2012) 161–165 1

HO

O

OH H

OH

O

H

OH

Fig. 1. Key HMBC correlations of 1.

correlations between H-2″/C-6 and H-2′′′/C-8 suggested that the two pentenyl appendages being attached at C-6 and C-8, respectively. The long-range 1H– 13C correlation of δH 6.62 (H-6′) to δC 46.2, 154.8, 153.6 (C-3, 2′, 4′) indicated that there is an aromatic proton at C-6′. The presence of two hydroxyl group at C-2′ and 4′ respectively is evident based on the longrange 1H– 13C correlations between δH 9.14 (2′-OH)/δC 154.8, 117.8, 102.5 (C-2′, 1′, 3′) and δH 9.24 (4′-OH)/δC 153.6, 102.5, 112.1 (C-4′, 3′, 5′). The attachment of the third pentenyl group to C-5′ could be deduced from the long-range 1H– 13C correlations between δH 3.02 (H-1′′′′) and δC 153.6/130.2 (C-4′/6′). Other salient HMBC correlations are depicted in Fig. 1, which are in full agreement with the proposed structure. Furthermore, mutual NOE cross peaks between H-3′ and 2′-OH, H-3′ and 4′-OH observed in the NOESY spectrum confirmed the positions of two hydroxyl groups. Compound 1 has a nearly zero [α] 20D (+1°, c 2.0, MeOH) indicating it being a racemic mixture and the absolute configuration of C-3 was assigned to 3R and 3S as it is the only chiral center in the molecule. Thus, compound 1 was identified as 5,7,2′,4′-tetrahydroxyl-6,8,5′-tri-(γ,γ-dimethylallyl) isoflavanone (or 6,8,5′-tri-(γ,γγ-dimethylallyl)dalbergioidin), named ormosinol. Compound 2, which was obtained as a white powder, exhibited a molecular formula of C27H30O14 as determined by HR-ESI-MS wherein a deprotonated molecule [M − H] − of m/z 577.1556 (calcd C27H29O14 577.1562). The IR absorptions revealed the presence of hydroxyl groups (3399 cm− 1) and an unsaturated carbonyl group (1614 cm − 1). From the examination of the molecular formula, compound 2 apparently comprises a flavone or isoflavone nucleus with two sugar moieties. Presumably, the molecular frame of the molecule is isoflavone since the isoflavone nature was confirmed by

H NMR spectroscopy, which showed the typical C-2 proton signal at δH 8.11 ppm. The isoflavone nucleus could be readily assigned to isoprunetin by a comparison of the 13C NMR data of 2 with that of isoprunetin [14], which were virtually identical except for the slight difference of the signals arising from C-6, C-7 and C-8, assuming due to the introduction of the sugar chain at C-7, the so-called ‘glycosylation effects’ [26]. In the 1H NMR spectrum, two doublets at δH 5.04 and 4.16 were assigned to the anomeric protons of the two sugar units, respectively. The large 3JH1H2 coupling constants of these protons (6.8 and 7.2 Hz respectively) indicated a β-anomeric configuration of the sugar moieties. To get insight on the identities of the aglycone and sugar moiety, a partial mild acidic hydrolysis experiment was performed on TLC. Upon the exposure to hydrochloride vapor, compound 2 afforded two hydrolysate products, namely isoprunetin (12) and isoprunetin-7-O-β-D-glucoside (13), as monitored by co-TLC with authentic standards which were also isolated and identified from the title plant. A comparison of the 13C NMR data of 2 with those of 13 revealed that the remaining hexose unit was linked to the inner glucose via a 1 → 6 glycosidic linkage based on the diagnostic chemical shift of the 13C signal between 2 and 13 at C-6″ of glucose, which is about 5 ppm downfield from those of 13 due to the well known effects of O-glycosylation, indicating that 2 contained a terminal pentose and a C-6″ substituted β-D-glucopyranosyl group. The terminal pentose was identified as xylose since its 13C NMR data were found to be close to the literature values for methyl β-D-xylopyranoside [27], suggesting that 2 contained the β-D-xylopyranosyl-(1 → 6)-β-D-glucopyranosyl moiety, whose identity was further confirmed by its identical 13C NMR data with those from literature [28]. In the NOESY spectrum, some informative NOE correlations were observed between glucose anomeric proton (δH 5.04, H-1″) with H-6 (δH 6.55) and H-8 (δH 6.73) of isoflavone nucleus as well as between xylose anomeric proton (δH 4.16, H-1′′′) with H-6″ (Fig. 2). Thus, compound 2, named ormosinoside, was identified as isoprunetin-7-O-β-D-xylopyranosyl-(1 → 6)β-D-glucopyranoside. All the compounds isolated were evaluated in vitro for their radical scavenging activities against DPPH and growth inhibitory activity against a panel of human cancer cell lines (A549, LAC, HepG2) using the MTT method. As a result, compound 1 displayed noticeable scavenging activity with IC50 values of 28.5 μM, while compound 2 was inactive (IC50 > 100 μM) (L-ascorbic acid was positive control, IC50 25.6 ± 0.3 μM); compound 1 also showed significant inhibitory activity with the IC50 values of 4.25, 5.22, 7.09 μM, respectively; whereas compound 2 was inactive to any of the cell lines at the concentration of 200 μM (doxorubicin

Fig. 2. Key NOESY correlations of 2.

S. Feng et al. / Fitoterapia 83 (2012) 161–165

as positive control, IC50 15.15, 20.48, 79.5 μM, respectively). Of all the compounds isolated and tested in the current study, only 1 exhibited potent anti-oxidation and cancer cell growth inhibitory activity, which may therefore account not only for the potency of the crude root extract but can also serve to validate, as well as experiments go, the folklore use of this material for certain diseases and symptoms. Acknowledgements This work was jointly financially supported by Chinese National Science Foundation (No. 30973635) and CAS ‘100 Talents Program’ Foundation. Also funded by grants from the Knowledge Innovation Program of the Chinese Academy of Sciences (No.KSCX2-YW-R-217) and the Natural Science Foundation of China (No.30973635), and Guangdong Province Natural Science Foundation (No. 10151065005000026). References [1] Merrill ED, Chen L. The Chinese and Indochinese Species of Ormosia, Sargentia, Flora of China 1943; 3: 77. [2] Iinuma M, Okawa Y, Tanaka T, Ho FC, Kobayashi Y, Miyauchi KI. Phytochemistry 1994;37:889. [3] Davies AP, Hassall CH. Tetrahedron Lett 1966;50:6291. [4] Olivier M, Renault JH, Richard B, Moretti C, Zèches-Hanrot M. Fitoterapia 2001;72:583. [5] Moran NC, Quinn GP, Butler Jr WM. J Pharmacol Exp Ther 1959;125:73.

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