A new coumestan with immunosuppressive activities from Flemingia philippinensis

Fitoterapia 82 (2011) 615–619

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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e

A new coumestan with immunosuppressive activities from Flemingia philippinensis Li. Li a,b, Xueyang Deng a,b, Lixia Zhang c, Pan Shu a,b, Minjian Qin a,b,⁎ a b c

Department of Resources Science of Traditional Chinese Medicines, China Pharmaceutical University, Nanjing 210009, China Key Laboratory of Modern Chinese Medicines (Ministry of Education), China Pharmaceutical University, Nanjing 21009, China The Yunnan Branch, Institute of Medicinal Plant, Chinese Academy of Medical Sciences, Jinghong 666100, China

a r t i c l e

i n f o

Article history: Received 22 November 2010 Accepted in revised form 20 January 2011 Available online 28 January 2011 Keywords: Flemingia philippinensis Leguminosae Coumestan Piscigenin Immunosuppressive activity

a b s t r a c t A new coumestan, 1,3,9-trihydroxy-8-methoxycoumestan, named flemicoumestan A 1 together with nine isoflavone-related compounds were isolated from the ethyl acetate extract of the roots of Flemingia philippinensis. Their structures were elucidated and confirmed by spectroscopic methods and literature data. Compounds 3 and 4 showed strong lymphocyte proliferation inhibitory activity with an IC50 value of 1.04–2.76 μM, and the low cytotoxicity with the CC50 value of 71.01 and 56.36 μM, respectively. © 2011 Elsevier B.V. All rights reserved.

1. Introduction About forty species of Flemingia (Leguminosae) were found through the tropical and subtropical regions of Asia, Africa and Australia. Sixteen species and one variety mainly grow in southwestern China [1]. Among them, ten species have been used as folk medicines for the treatment of various diseases, such as rheumatism, arthropathy, chronic nephritis and menopausal syndrome [2]. Flemingia philippinensis Merr & Rolfe (Leguminosae), as the most widely used specie in Flemingia, is mainly distributed at the southwestern region of China such as Guangxi, Yunnan Province. Previous phytochemistrical investigations have reported that F. philippinensis contained various isoflavones, flavones, isoflavanones and coumaronochromones [3–5], which exhibit estrogenic and anti-estrogenic [6], anti-oxidative and anti-inflammatory activities [7]. In our chemical investigation, one new coumestan flemicou-

⁎ Corresponding author at: Department of Resources Science of Traditional Chinese Medicines, College of Traditional Chinese Medicines, China Pharmaceutical University, No. 24 Tong Jia Xiang, Nanjing 210009, JiangSu Province, China. Tel.: +86 025 86185130; fax: +86 025 85301528. E-mail address: [email protected] (M.J. Qin). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.01.019

mestan A 1, two known coumestan 4,2′-Epoxy-4′,5-dihydroxy7,5′-dimethoxy-3-phenylcoumarin 2 [8], aureol 3 [9] and seven known isoflavonoids: auriculasin 4 [10], biochaninA 5 [11], formononetin 6 [12], piscigenin 7, genistein 8 [13], daidzein 9 [14] and genistin 10 [15] (Fig. 1), were isolated from the ethyl acetate extract of the dried roots. In addition, the compound piscigenin had been found in the Piscidia erythrine [16] and Godoya antiopquiensis [17], but there was only limited spectral data and little detail explanations. In this paper, we replenished 1D and 2D NMR data to elucidate the structure. Furthermore, the isolation, structural elucidation and immunosuppressive activities of the ten compounds were investigated. 2. Experimental 2.1. Generals Melting points were measured on an X-4 micro-melting point apparatus, uncorrected. UV spectra were recorded on a Schimadzu UV-2450 spectrophotometer. IR spectra were determined by a Nicolet Impact-410 spectrophotometer with KBr disks. NMR spectra were carried out on a Bruker ACF-500 spectrometer (500 MHz for 1H and 125 MHz for 13C)

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Fig. 1. Chemical structures of isolated compounds.

with TMS as an internal standard. The mass spectra of the compounds were obtained on an ABI-Mariner (ESI-TOF-MS). For column chromatography, silica gel (100–200 and 200– 300 mesh) was obtained from Qingdao Marine Chemical Co., polyamide (80–100 mesh) was bought from Sinapharm Chemical Reagent Co., and Sephadex LH-20 was chosen from Pharmacia Co. 2.2. Plant material The roots of F. philippinensis were collected in Nanning, Guangxi Province, China, in August, 2007. The plant was identified by Dr. Min-jian Qin, and a voucher specimen (No. Li 2007-001) was deposited in the Herbarium of Medicinal Plants of China Pharmaceutical University, Nanjing, China. 2.3. Extraction and isolation The dried roots of F. philippinensis (9.3 kg) were powdered and extracted with 80% EtOH (3× 50 L) under a refluxing temperature for 2 h. After evaporation of the solvent, the concentrated extract (1345 g) was suspended in warm water (8.0 L) and successively partitioned with ethyl acetate (5× 4.0 L) and n-BuOH (5× 4.0 L). The ethyl acetate fraction (168 g) was separated on a silica gel column (100–200 mesh, 8.0 × 85 cm), using a gradient of CHCl3–MeOH (100:0, 100:1, 50:1, 30:1, 15:1, 10:1, 5:1, 2:1, and 1:1 v/v, each 750 ml), to give nine fractions (A1–A9). The fraction A2 was further subjected to polyamide column (80–100 mesh, 3 × 60 cm) chromatography, and then eluted with CHCl3–MeOH (50:1 v/v, 300 ml) to afford 2 (108 mg) and 4 (56 mg). Fraction A3 was applied to repeated column chromatography (CC) over silica gel (200–300 mesh, 2.0 × 60 cm, petroleum ether–acetone 2:1 v/v, each 150 ml) and then further purified on a Sephadex LH-20 (1.5 × 90 cm,

CHCl3:MeOH, 1:1 v/v, 200 ml) to obtain compound 5 (15 mg) and 6 (48 mg). Fraction 4 was separated by silica CC (200–300 mesh, 3.0 × 80 cm) using CHCl3–acetone (10:1–1:1 v/v, each 120 ml) to give five subfractions (B1 to B5) according to TLC analysis. Subfraction B2 was repeated with silica gel CC eluted with petroleum ether–acetone (2:1 v/v, each 80 ml) to yield 7 (72 mg), and the subfraction B3 was subjected to recrystallize with MeOH to give 8 (2.3 g). Compound 9 (22 mg) and 3 (31 mg) were separated by preparative TLC with CHCl3– acetone (6:1 v/v, 180 ml) from the A5 fraction. Fraction A6 was packed to the CC (200–300 mesh, 2.0 × 60 cm) over silica gel eluting with CHCl3–acetone (10:1 v/v, 300 ml) to yield 1 (219 mg). Fraction 7 was subjected to silica gel CC (200–300 mesh, 3.0 × 80 cm), eluting with CHCl3–MeOH (15:1–1:1) mixture, obtained 10 (1.5 g, each 700 ml). 2.3.1. Flemicoumestan A Pale yellow needles; m.p N 300 °C; UVλmax (MeOH) nm (log ): 335 (3.72), 263 (4.01), 207 (4.53); IRvmax (KBr) cm−1: 3424, 3329, 1698, 1624, 1481, 1415, 1337, 1278, 1153, 1127,1091, 1068, 1030, 1009, 992, 949, 901,841, 808, 768, and 740; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data were shown in Table 1; ESI-MS m/z 313 [M-H]− ; HR-ESI-MS m/z 337.0298 [M+Na]+ (calcd for [C16H10O7Na]+, 337.0319). 2.3.2. Piscigenin Pale yellow needles; m.p: 242–244 °C; UVλmax (MeOH) nm (log ): 264 (4.36), and 206 (4.48) nm; IRvmax (KBr) cm−1: 3426, 2934, 1650, 1617, 1566, 1520, 1453, 1431, 1392, 1366, 1347, 1311, 1290, 1200, 1171, 1149, 1117, 1092, 1052, 914, 839, and 798; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data were shown in Table 2; ESI-MS

L. Li et al. / Fitoterapia 82 (2011) 615–619 Table 1 The spectroscopic data of compound 1 (1H NMR 500 MHz and NMR,125 MHz, DMSO-d6). Position 1 2 3 4 4a 6 6a 6b 7 8 9 10 10a 11a 11b OCH3 1-OH 3-OH 9-OH

δH (m, J in Hz) 6.37 (d, 2.0) 6.40 (d, 2.0)

7.30 (s)

7.20 (s)

3.90 (s) 10.87 10.44 9.51

δC

HMBC

151.0 94.8 161.3 99.1 155.0 157.7 100.9 113.6 101.6 146.2 149.3 101.6 146.7 159.5 95.3 56.1

C-1, 3, 4, 11b

13

2.5. Lymphocyte proliferation and cytotoxicity assay C

7–8-week-old female C57BL/6 mice were killed by cervical dislocation and splenocytes were prepared aseptically to single cell suspensions. Splenic lymphocytes were stimulated with ConcanavalinA (ConA) 2 μg/mL or lipopolysaccharide (LPS) 10 μg/mL, and then incubated for 48 h at 37 °C in a humidified incubator containing 5% CO2 plus the required concentrations of drugs (1, 10 and 100 μM), respectively. After 40 h of preincubation, 0.5 μCi/well [3H]-thymidine was added and pulsed. Finally, cells were harvested onto a glass fiber filter using a HARVESTER96® 96-well cell harvester and incorporated radioactivity was measured by a Beta Scintillation Counter [18,19]. Cytotoxicity was assessed by using the MTT method [18]. Briefly, 20 μL of MTT solution (5 mg/mL) was pulsed 4 h prior to the end of the culture (in a total volume of 160 μL), and then the crystals were dissolved with 80 μL of solvent [10% sodium dodecylsulfate (SDS), and 50% N,N-dimethyl formamide, pH 7.2]. The solution was incubated for another 7 h and the optical densities (OD) were read on a microplate reader at a wavelength of 570 nm.

ROESY

C-2, 3, 4a, 11b

C-6a, 8, 9, 10a

OCH3

C-6a, 8, 9, 10a

C-8

617

H-7

m/z 331 [M+ H]+; HR-ESI-MS m/z 353.0634 [M+ Na]+ (calcd for [C17H14O7Na]+, 353.0632).

3. Results and discussion 3.1. Chemistry

2.4. Animals and housing conditions Female C57BL/6 mice (6–8-week-old) were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences. The mice were housed in specific pathogen-free conditions, which were allowed to acclimatize in our facility for one week before any experiments were started. All experiments were carried out according to the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Bioethics Committee of the Shanghai Institute of Materia Medica.

Table 2 The spectroscopic data of compound 7 (1H NMR 500 MHz and NMR,125 MHz, DMSO-d6). Position

δH (m, J in Hz)

δC

HMBC

ROESY

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

8.38

154.34 120.60 180.07 161.96 98.97 164.27 93.61 157.44 104.38 122.33 106.83 147.70 135.87 147.70 106.83 56.11 56.11

C-3, 4, 9, 1′

H-2′

6.23 (d, 2.0) 6.39 (d, 2.0)

6.85 (s)

6.85 (s) 3.79 (s) 3.79 (s) 12.97 10.80 8.50

13

C-5, 7, 8, 10

C-3, 1′,3′, 4′,6′

H-2, OCH3

C-3, 1′, 2′, 4′, 5′

OCH3 H-2′ H-6′

C-2, 6′

C

Compound 1, obtained as pale yellow needles and displayed an obvious blue fluorescence on the chromatograms under UV(λ254, 365) light. The molecular formula was deduced as C16H10O7, from its HR-ESI-MS spectrum, which showed a pseudo-molecular [M + Na]+ ion at m/z 337.0298 (calcd for [C16H10O7Na]+, 337.0319). The UV spectrum of 1 in MeOH exhibited absorption maxima at 335,263 and 207 nm; its IR spectrum showed characteristic absorption bands for hydroxyls at 3424,3329 cm−1 and conjugated carbonyl at 1698 cm−1. The 1H NMR spectrum further indicated the presence of a meta-coupled doublets at δ 6.37 (1H, d, J = 2.0 Hz) and δ 6.41 (1H, d, J = 2.0 Hz) of the A-ring, two one-proton doublets at δ 7.l0 (1H, s) and 7.20 (1H, s) were attributed to the B-ring, three hydroxyl groups δ 10.80 (1H, s, 1-OH), 10.44 (1H, s, 3-OH), and 9.51 (1H, s, 9-OH) and a methoxyl group at δ 3.90 (3H, s). The 13C NMR spectrum showed 16 signals, including one methoxy carbon at δ 56.1. The result from the chemical reaction, together with MS, UV, IR, 1H NMR and 13C NMR data revealed that the character of 1 was a similar coumestan skeleton [8,9]. Further confirmation was obtained from the HMBC correlations (Fig. 2): the aromatic proton H-7 (δ 7.30) correlated with C-6a (δ 100.9), and C-9 (δ 149.3), the methoxyl proton at δ 3.90 correlated with C-8 (δ 148.6), indicating the placement of the methoxyl group at C-8. This was also proved by ROESY spectrum, which showed NOE interaction between the H-7 (δ 7.30) and the proton at δ 3.90. Thus, we concluded that the structure of 1 was 1,3,9-trihydroxy-8-methoxycoumestan, named flemicoumestan A (Fig. 1). Detailed assignments of protons and carbons of 1 were shown in Table 1. Compound 7, obtained as yellow needles and the positive result of the spray reagent of AlCl3 suggested a flavone-like compound. The molecular formula was determined as C17H14O7 from the analysis of positive HR-ESI-MS spectrum,

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Fig. 2. The key HMBC and ROESY correlations of compounds 1 and 7.

which showed a quasi-molecular [M + Na]+ ion at m/z 353.0634 (calcd for [C17H14O7Na]+, 353.0632). The UV spectrum exhibited absorption maxima at 264,206 nm in MeOH and one aromatic singlet proton at δ 8.38 in the 1H NMR spectrum (Table 2) of 7 indicated that it might be an isoflavone. Furthermore, its IR spectrum exhibited a chelated hydroxyl group at 3426 cm−1 and a chelated carbonyl group at 1651 cm−1. The 1H NMR spectrum showed the presence of one hydroxyl group at δ 12.90 (1H, s), which suggested that it substituted a group of hydroxyl at C-5. The aromatic protons of meta-coupled doublets at δ 6.23 and δ 6.56 (each 1H, d, J = 2.1 Hz) corresponded to the H-6 and H-8 positions of ring A. Therefore, we can deduce that the character of compound 7 was a 5,7-dioxygenated isoflavonoid. In addition, two chemically equivalent aromatic protons at δ 6.85 (2H, s) revealed that 4′-hydroxyl was substituted at the B ring. The 1 H NMR spectrum also showed two hydroxyl proton signals at δ 10.80 (1H, s, 7-OH) and δ 8.40 (1H, s, 4′-OH), two methoxyl groups at δ 3.79 (6H, s). The 13C NMR spectrum exhibited 17 carbon signals, including two methoxyl carbons at δ 56.71 and one carbonyl carbon at δ 180.07 (C-4). In the HMBC spectrum, the methoxy protons at δ 3.79 (6H, s) showed correlation with two carbon signals at δ 148.8 (C-3′, C-5′), respectively. The protons at δ 6.85 (2H, s) were observed to couple with two carbons at δ 120.3 (C-3) and 122.0 (C-1′). Hence the methoxy group was placed at C-3′ and C-5′. This was futher confirmed by the correlation to the proton signals at δ 6.85 (H-2′, H-6′) observed in the ROESY spectrum. Following detailed analysis of the HMBC spectra, compound 7 was elucidated by 5,7, 4′-trihydroxy-3′, 5′dimethox isoflavones, named piscigenin.

Table 3 Inhibition splenocyte proliferation and cytotoxicity of the isolated compounds a. Compounds

1 2 3 4 5 6 7 8 9 10 a

IC50 values (μm). T cells

B cells

2.75 7.73 2.76 1.88 5.58 1.67 13.54 3.54 47.63 5.15

0.23 2.41 1.20 1.04 2.75 62.84 0.72 0.14 1.18 2.03

3.2. Biological activities There are various kinds of immunosuppressants which have been utilized in the organ transplantation and autoimmune system diseases. However, clinical application has shown that they might increase the risks of tumors arising, and induce cardiovascular disease and microorganism infections [20,21]. Therefore, we put our effort on the Traditional Chinese Medicine (TCM), which has notable curative effects with less side effects. The present research has proved that T and B cells contribute a lot during the pathologic change of transplant rejection and autoimmune diseases, which indicates that inhibiting the activation and proliferation of these cells may prove to be an effective settlement for those disorder [22,23]. In order to seek for some new immunosuppressive agents with lower cytotoxicity and higher efficacy, we assessed the compounds in vitro for their inhibitory activities on ConA-induced T cell and LPS-induced B cell proliferation. Cytotoxicity on lymphocyte was evaluated with MTT assay. All the results were presented at Table 3. Among the active compounds, compounds 1, 3, 4 and 8 showed strong activity with an IC50 value of 0.14 to 3.54 μM, while 2, 5, 7 and 10 appeared moderate activation with an IC50 value of 3.54 to 13.54 μM. Compound 9 exhibited only weak inhibitory activity against T cell proliferation (IC50, 47.63 μM) while 6 showed low inhibitory activity against B cell proliferation (IC50, 47.63 μM). From the cytotoxicity results, we found that the CC50 of compounds 3 and 4 were 26 to 59 times higher than their IC50, which revealed they had a favorable safety range as potential safe immunosuppressants. Our study suggested the possibility of developing compound 3 and 4 as potential immunosuppressants, and the further studies are necessary to elucidate the details of the mechanism. Acknowledgment

CC50 values (μm).

50.10 57.45 71.01 56.36 55.08 42.15 30.47 29.01 Inactive b 84.49

The 50% cytotoxic concentration (CC50) and the 50% inhibitory concentration (IC50). b Inactive b50% inhibition. Compounds were tested at a maximum concentration of 100 um.

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