Fitoterapia 94 (2014) 114–119
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Phenolic constituents from the leaves of Cratoxylum formosum ssp. pruniflorum Juan Xiong a, Xin-Hua Liu b, Van-Binh Bui c,d,e, Zhi-Lai Hong a, Li-Jun Wang a, Yun Zhao c,d, Hui Fan c,d, Guo-Xun Yang a, Jin-Feng Hu a,⁎ a b c d e
Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, PR China Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, PR China Department of Chemistry, East China Normal University, Shanghai 200062, PR China Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, East China Normal University, Shanghai 200062, PR China Department of Chemistry, Hoa Lu University, Ninh Binh 40000, Viet Nam
a r t i c l e
i n f o
Article history: Received 3 January 2014 Accepted in revised form 6 February 2014 Available online 15 February 2014 Keywords: Cratoxylum formosum ssp. pruniflorum Clusiaceae Phenolic compounds Anti-neuroinflammation Neuroprotection
a b s t r a c t One (formosumone A, 1) new and fifteen (2–16) known phenolic compounds were isolated from the leaves of Cratoxylum formosum ssp. pruniflorumm, a substitute for the popular bitter nail tea (“Kuding Tea”) generally used in Southeast Asia. Their structures were determined by extensive spectroscopic analysis and by comparison with literature data. Compound 1 possesses a rare scaffold of a flavanone coupled with a phloroglucinol moiety, representing the first example of such a scaffold from the Clusiaceae family. Among the isolates, toxyloxanthone B (11) and vismione D (12) were found to show remarkable anti-neuroinflammatory effects by inhibiting nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated murine BV-2 microglial cells. Additionally, toxyloxanthone B (11) exhibited significant neuroprotective effect against β-amyloid25–35 (Aβ25–35)-induced cell viability decrease in SH-SY5Y neuroblastoma cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction People believe that natural tea drink is beneficial for postponing human aging and expanding their lifespan. “Kuding (bitter nail tea)” as a famous functional beverage has been ordinarily used in southern and southwestern China for a long time. “Kuding” (“ku” means bitter, while “ding” describes its nail shape in Chinese) is appreciated more for its valuable health-care function than its special bitter-taste. It has been traditionally applied for heat-clearing, quenching thirst, detoxifying, preventing obesity, reducing blood fat, and lowering blood pressure [1,2]. There are 17 species which belong to 5 families generally used as the original materials of “Kuding Tea”, mainly including Ilex kudingcha, Ligustrum robustum, Ligustrum pedunculare, Listea coreana, and Ehretia thyrsiflora [1]. Cratoxylum formosum ssp. pruniflorum (Kurz) Gogelin
⁎ Corresponding author. Tel./fax: +86 21 51980172. E-mail address: [email protected] (J.-F. Hu).
http://dx.doi.org/10.1016/j.fitote.2014.02.002 0367-326X/© 2014 Elsevier B.V. All rights reserved.
(family Clusiaceae) is widely distributed in the tropical region of Southeast Asia. Its leaves are commonly used as a substitute for “Kuding Tea” in Yunnan Province of China and its neighborhood in Vietnam. However, the chemistry and pharmacology of the leaves have been so far not studied. As a continuation of our interest in discovering new agents for the treatment of aging-associated diseases from natural products [3,4], the leaves from the title plant were phytochemically investigated. Reported herein are the isolation and structure determination of a new (1) and fifteen known (2–16) phenolic compounds (Fig. 1) and their anti-neuroinflammatory and neuroprotective activities. 2. Experimental 2.1. General experimental procedures Optical rotations were determined on a Perkin-Elmer 341 polarimeter. UV and IR spectra were recorded on a Shimadzu
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115
Fig. 1. Chemical structures of compounds 1–16.
UV-2550 and an Avatar 360 ESP FTIR spectrometer, respectively. CD spectra were measured on a Jasco 810 spectrometer. NMR spectra were recorded on a Bruker AM-400 or DRX-500 instruments; Chemical shifts are expressed in δ (ppm), and referenced to the residual solvent signals. ESI-MS were measured on an Agilent 1100 series mass spectrometer; and HR-ESI-MS were measured on a Bruker Daltonics micrOTOF-QII mass spectrometer. Column chromatography (CC) was performed using silica gel (200–300 mesh, Kang-Bi-Nuo Silysia Chemical Ltd., China), macroporous adsorption resin D101 (0.3–1.25 mm, Cang Zhou Bon Adsorber Technology Ltd., China), MCI gel CHP20P (75–150 μm, Mitsubishi Chemical Industries, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden). Silica gel-precoated plates (GF254, 0.25 mm) were used for TLC detection. Spots were visualized using UV light (254 and/or 366 nm) and by spraying with 15% (v/v) H2SO4–EtOH followed by heating to 120 °C. 2.2. Plant material The leaves of C. formosum ssp. pruniflorum were collected from Ninh Binh, Vietnam, by one of the authors (V.-B. Bui) in July 2011. The plant was identified by Prof. Bao-Kang Huang
(Department of Pharmacognosy, The second Military Medical University, Shanghai, China). A voucher specimen (No. 20110601) was deposited at the Herbarium of the Department of Natural Products Chemistry, School of Pharmacy at Fudan University. 2.3. Extraction and isolation The dried and powdered leaves (7.5 kg) were extracted with 95% EtOH (18 L × 3) at room temperature to give a black crude extract (750 g, semi-dry). The entire EtOH extract was subjected to a D101 macroporus resin column eluted successively by MeOH/H2O (80:20, v/v), EtOH/H2O (95:5, v/v), and acetone (neat). The 95% EtOH fraction (ca. 380 g) was fractionated by CC over silica gel [petroleum ether (PE)–EtOAc, 4:1 → 0:1, v/v] to give five fractions (Frs. 1–5). Fr. 1 (20.9 g) was applied to silica gel CC with PE-acetone (8:1 → 4:1, v/v) followed by gel permeation chromatography (GPC) on Sephadex LH-20 in MeOH to furnish compound 13 (4.8 mg). Fr. 2 (130.5 g) was subjected to silica gel (CH2Cl2/MeOH, 70:1 → 35:1, v/v), yielding Frs. 2.1– 2.5. Purification of Fr. 2.1 by CC over silica gel (CH2Cl2/MeOH, 70:1, v/v) gave compounds 9 (5.6 mg) and 10 (27.8 mg). Accordingly, compound 8 (7.5 mg) was isolated from Fr. 2.2. CC
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of Fr. 2.3 on silica gel (CH2Cl2/MeOH, 50:1, v/v) followed by GPC on Sephadex LH-20 (MeOH) afforded compounds 1 (8.0 mg), 7 (39.8 mg), and 11 (8.5 mg). Fr. 2.4 was subjected to silica gel (CH2Cl2/MeOH, 50:1 → 30:1, v/v), furnishing compounds 2 (40.2 mg), 5 (37.8 mg), and 6 (14.5 mg). Fr. 3 (35.0 g) was chromatographed on silica gel (CH2Cl2/MeOH, 30:1 → 10:1, v/v) to afford compounds 3 (10.3 mg) and 4 (14.1 mg). The acetone fraction (ca. 150 g) was decolorized by a MCI gel column with MeOH/H2O (8:2 → 0:1, v/v), giving three fractions (Frs. 6–8). Compounds 14 (7.0 mg) and 15 (2.6 mg) were obtained from Fr. 6 (32.5 g) by repeated CC over silica gel (PE/acetone 10:1 → 5:1, v/v). Fr. 7 (21.9 g) was applied to silica gel CC (PE/acetone 6:1 → 3:1, v/v) to afford compounds 12 (2.6 mg) and 16 (2.8 mg). Methyl 3-((2R,3S)-5,7-dihydroxy-2-(4-hydroxyphenyl)4-oxochroman-3-yl)-2, 4,6-trihydroxybenzoate (Formosumone A, 1): yellow amorphous powder; [α]20 D −59.0 (c = 0.061, MeOH); UV (MeOH) λmax (log ε) 290 (3.68), 310 (3.51) nm; IR (KBr): 3386 (br), 2964, 2767, 1643, 1463, 1397, and 1128 cm−1; CD (c 3.4 × 10− 4 M, MeOH) λmax (Δε): 284 (+ 23.1), 310 (− 6.7) nm; for 1H and 13C NMR data, see Table 1; ESIMS: m/z 493 [M + K]+, 477 [M + Na]+, 455 [M + H], 453 [M − H]−; HR-ESIMS: m/z 493.0520 [M + K]+ (calcd. for C23H18O10K, 493.0532). 2.4. Measurement of NO production and cell viability in LPS-activated BV-2 cells The mouse microglia BV-2 cell line was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA), and maintained in Dulbecco's modified Eagle's medium containing 1800 mg/L NaHCO3, supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. The anti-neuroinflammatory activity in BV-2 cells was evaluated according to the reported protocol with modification [4,5]. The NO production was quantified by nitrite accumulation in the culture medium using the Griess reaction kit (Beyotime Biotechnology, China) according to the manufacturer's instructions. Briefly, the BV-2 cells were pretreated with different concentrations (3.125, 6.25, 12.5, 25, 50, and 100 μM) of indicated compounds for 4 h, and then stimulated with or without lipopolysaccharide (LPS) (1 μg/mL, Sigma-Aldrich) for 24 h. The isolated supernatants were mixed with an equal volume of Griess reagent. NaNO2 was used to generate a standard curve, and NO production was determined by measuring the optical density at 540 nm by a microplate reader (M200, TECAN, Austria GmbH, Austria). The cell viability was measured using a 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [4,6]. NG-monomethyl-L-arginine (L-NMMA, Beyotime, purity ≥ 99%), a well-known NO synthase inhibitor, was used as the positive control. The IC50 values were determined by GraphPad Prism 5. 2.5. Neuroprotective activity assay in SH-SY5Y cells The cell viability of SH-SY5Y cells was evaluated according to the reported protocol with modification [3]. The cells were high passages from the ATCC maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cells were seeded into 96-well
Table 1 1 H (500 MHz) and
13
C (125 MHz) NMR dataa of compound 1.
No.
δHb (J values in Hz)
δHc (J values in Hz)
δCb
δCc
2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ OCH3 5-OH 7-OH 4′-OH 2″-OH 4″-OH 6″-OH
5.77 (1H, d, 12.0) 4.67 (1H, d, 12.0)
5.89 (1H, d, 12.0) 4.80 (1H, d, 12.0)
5.88 (1H, d, 2.1)
5.96 (1H, d, 2.0)
5.91 (1H, d, 2.1)
5.98 (1H, d, 2.0)
7.14 (1H, d, 8.5) 6.64 (1H, d, 8.5)
7.25 (1H, d, 8.5) 6.72 (1H, d, 8.5)
7.14 (1H, d, 8.5) 6.64 (1H, d, 8.5)
6.72 (1H, d, 8.5) 7.25 (1H, d, 8.5)
5.84 (1H, s)
5.91 (1H, s)
3.85 (3H, s) 12.20 (1H, s) 10.56 (1H, s) 9.27 (1H, s) 10.91 (1H, s) 10.30 (1H, s) 9.80 (1H, s)
4.03 (3H, s) 12.32 (1H, s) ND ND 10.51 (1H, s) ND ND
81.4 47.5 197.3 100.9 163.6 96.0 166.3 95.0 163.0 128.3 128.7 114.7 157.5 114.7 128.7 93.3 162.4 100.9 162.2 95.1 160.3 170.5 52.2
82.7 48.9 198.4 102.9 165.5 96.9 166.9 95.8 164.5 130.2 129.8 115.5 158.5 115.5 129.8 94.0 161.3 102.3 ND 95.9 162.3 171.2 53.1
c
a Assignments were made by a combination of 1D- and 2D-(COSY, HSQC, HMBC) NMR experiments. b Measured in DMSO-d6 (328 K). c Measured in acetone-d6 (298 K); ND: Not detectable.
plates at a density of 2.5 × 105 cells/mL in MEM/F12 medium supplemented with 10% (v/v) FBS. After 24 h, the serum-free MEM/F12 medium was used to substitute the original medium. The test compounds and the positive control (epigallocatechin3-gallate, ECGG) were made to 10−2 M stock solutions with DMSO and then diluted to corresponding concentrations with the cell culture medium. Cells were incubated with test compounds (1 or 10 μM) or ECGG (10 μM, Sigma, purity N 98%) for 2 h prior to treatment with 10 μM Aβ25–35 for another 24 h without changing the culture medium. 10 μL of MTT (5 mg/mL, Sigma, purity: 98%) was then added to each well and incubated at 37 °C for 3 h. The cells were finally lysed with 100 μL of DMSO, and the amount of MTT formazan was measured at 490 nm using a microplate reader (M200, TECAN, Austria GmbH, Austria). Data were evaluated for statistical significance with a one-way ANOVA followed by the Least Significant Difference (LSD) test using a computerized statistical package. 3. Results and discussion The dried leaves of the title plant were extracted with 95% EtOH at room temperature to give a crude extract, which was found to show preliminary anti-neuroinflammatory effect by inhibiting nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine microglia BV-2 cells. From this material, seven flavonoids (1–7), four xanthones (8–11), and five anthranoids (12–16) (Fig. 1) were purified. Comparing their spectroscopic data and physicochemical properties with those
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reported in the literature, the known compounds were identified as naringenin-(3 → 8)5,7-dihydroxychromone (2) [7], GB-1 (3) [8], GB-2 (4) [8], GB-1a (5) [8], GB-2a (6) [8], 2,3-trans-dihydro-kaemferol (7) [9], 2′-hydroxymethyl-3′,3′dimethyl-dihydrofuran-1,5-dihydroxy xanthone (pruniflorone M, 8) [10,11], formoxanthone C (9) [12], 2′-hydroxy-4′,4′dimethyl-pyran-1,5-dihydroxyxanthone (pruniflorone N, 10) [10], toxyloxanthone B (11) [13], vismione D (12) [14,15], vismione B (13) [14,16], parietin (14) [17], vismiaquinone (15) [17], and 11-hydroxy-5-methoxy-2,2,9-trimethyl-2H-anthra [1,2-b]-pyran-7,12-dione (16) [18]. Compound 1 was obtained as a yellow amorphous powder. The molecular formula of 1 was determined to be C23H18O10 based on a pseudo-molecular ion peak at m/z 493.0520 [M + K]+ in its positive mode HR-ESIMS. The IR spectrum exhibited absorption bands (νmax) at 3386 and 1643 cm−1 attributed to hydroxy and conjugated carbonyl groups. The 1H NMR spectrum (Table 1) of 1 displayed signals of a typical 1,4-disubstituted benzene group with an AA′BB′ system [δ 7.14 (2H, d, J = 8.5 Hz, H-2′, 6′); 6.64 (2H, d, J = 8.5 Hz, H-3′, 5′)], a tetrasubstituted aromatic ring with the protons located at the meta-positions [δ 5.88 (1H, d, J = 2.1 Hz, H-6), 5.91 (1H, d, J = 2.1 Hz, H-8)], and two methines at δ 5.77 (1H, d, J = 12.0 Hz, H-2) and 4.67 (1H, d, J = 12.0 Hz, H-3). The above data were characteristic for the presence of a 4′,5,7-trihydroxy flavanone framework, indicating that compound 1 was a naringenin derivative similar to biflavonoids 2–6 [7,8]. The remaining signals including an olefinic proton at δ 5.84 (1H, s, H-5″) and a methoxy group at 3.85 (3H, s) in the 1H NMR spectrum of 1 were thus assigned to the C-3 substituent (Table 1). In addition to the fifteen carbon signals involved in the core structure (naringenin-type nucleus), the 13C NMR spectrum (in DMSO-d6, 328 K) of 1 exhibited eight additional carbon signals including six olefinic carbons [δ 93.3 (qC, C-1″), 162.4 (qC, C-2″), 100.9 (qC, C-3″), 162.2 (qC, C-4″), 95.1 (CH, C-5″), 160.3 (qC, C-6″)], an ester carbonyl at δ 170.5, and a methoxy group at δ 52.2 (Table 1). From the above evidences, a methyl 2,4,6-trihydroxybenzoate unit was proposed as the substitutional moiety bound to C-3 in compound 1 as depicted in Fig. 1. The linkage position between the flavanone moiety and the phloroglucinol unit in 1 was confirmed by the observation of a clear 3J correlation of H-3 (δ 4.67)/C-3″ (δ 100.9) in its HMBC spectrum (Fig. 2). Additionally, HMBC correlations from 2″-OH (δ 10.91) to C-1″ (δ 93.3)/C-3″ (δ 100.9) and from OMe (δ 3.85) to C-7″ (δ 170.5) were also observed. Due
Fig. 2. Observed key HMBC correlations of 1.
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to the lack of correlations from H-5″ to its neighboring protons (in the NOESY spectrum) and carbons (in the HMBC spectrum), the structure of the C-3 substituent was further secured by comparing its 13C NMR data (Table 1) with those of methyl 2,4,6-trihydroxybenzoate [δ 92.3 (C-1), 164.4 (C-2), 95.3 × 2 (C-3, C-5), 162.1 (C-4), 161.9 (C-6), 169.3 (\CO), 51.8 (\OMe)] [19]. The trans configuration between H-2 and H-3 in 1 can be easily determined by the large coupling constant (J2,3 = 12.0 Hz). The experiment circular dichroism (CD) spectrum of 1 showed a positive cotton effect at 284 nm and a negative one at 310 nm, indicative of a (2R)-configuration [20,21]. Thus, the entire structure of 1 was unambiguously elucidated to be methyl 3-((2R,3S)-5,7-dihydroxy-2-(4-hydroxy-phenyl)-4oxochroman-3-yl)-2,4,6-trihydroxybenzoate (formosumone A). Naturally occurring flavonoids conjugated with a phloroglucinol moiety are quite rare. To our knowledge, only two examples have been previously reported from the skin of Allium cepa [22], and both of which are 2,3-epoxyflavanone derivatives possessing either a phloroglucinoyl or a 2,4,6-trihydroxyphenylglyoxylate unit at C-3. Unlike compound 1, biflavonoids 2–6 showed two sets of signals in their 1H and 13C NMR spectra recorded at room temperature due to their rotameric behavior (atropisomerism) [7,23,24], which resulted from the hindered rotation between the flavanone and the flavanonol moieties around the C-3/C-8″ axis (Fig. 1). The absolute configurations of biflavonoids 2–6 were also determined by comparison of their CD spectra (See Supporting information) with those reported in the literature [21,24]. Undoubtedly, compounds 1–6 have the same (2R,3S)-naringenin scaffold. Among the isolates, pruniflorone M (8), formoxanthone C (9), pruniflorone N (10), vismione D (12), vismiaquinone (15), and 11-hydroxy-5-methoxy-2,2,9-trimethyl-2H-anthra [1,2-b]-pyran-7,12-dione (16) have been previously obtained either from the fruits [10] or from the barks [25] of C. formosum ssp. pruniflorum. Vismione B (13) was firstly isolated from the fruits of C. cochinchinense [26]. The remaining known compounds (i.e., 2–7 and 14) were reported herein for the first time from plants of the Cratoxylum genus. Neuroinflammation has proven to be implicated in the pathogenesis of neurological disorders, such as Alzheimer's
Table 2 Inhibitory effects on NO production in LPS-stimulated BV-2 cells. Compound
IC50a (μM)
Cell viabilityb (%)
1 2 3 4 8 11 12 c L-NMMA
57.3 59.7 71.5 48.8 17.2 12.4 8.3 14.5
100.3 96.6 94.1 98.0 93.0 99.9 91.9 98.4
± ± ± ± ± ± ± ±
0.5 2.4 1.7 1.3 0.6 0.1 0.7 0.3
± ± ± ± ± ± ± ±
5.3 5.0 5.7 5.0 0.5 0.8 5.0 2.2
a IC50 value of each compound was defined as the concentration (μM) of indicated compound that caused 50% inhibition of NO production in LPSstimulated RAW264.7 cells. b Cell viability after treatment with 50 μM of each compound was expressed as a percentage (%) of untreated control cells. The results are averages of three independent experiments. c G L-NMMA (N -monomethyl-L-arginine): Positive control.
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disease (AD) and Parkinson's disease (PD) [27,28]. A potential therapeutic approach for these neurodegenerative (inflammation- or aging-associated) diseases is inhibition of the synthesis or release of inflammatory mediators [e. g. NO] in overactivated microglia [29]. In this study, the 95% EtOH extract from the leaves of the title plant was found to show antineuroinflammatory activity by significantly (P b 0.01) reducing NO production in LPS-stimulated BV-2 cells when compared with the control group treated by LPS only. Therefore, all the purified compounds from this extract were evaluated for their anti-neuroinflammatory effects on NO production in LPS-activated BV-2 cells. The inhibitory activities of the tested compounds on NO production were expressed as 50% inhibition concentration (IC50). As shown in Table 2, four flavonoids (1–4), two xanthones (8, 11) and a prenylated anthranoids (12) could significantly decrease NO production in BV-2 cells without influence on cell viability at concentrations up to 50 μM, while others were inactive (IC50 N 100 μM). Among them, toxyloxanthone B (11) and vismione D (12) demonstrated the most potent NO inhibitory activity (IC50 = 12.4, 8.3 μM, resp.), better than that of the positive control, NG-monomethyl-L-arginine (L-NMMA, IC50 = 14.5 μM). Interestingly, xanthones 8–10 have been previously reported to possess potent inhibitory activity against LPS-induced NO release in macrophages RAW 264.7 cells [10]. All the isolates were also evaluated for their neuroprotective effects against β-amyloid25–35 (Aβ25–35)-induced cell viability decrease in SH-SY5Y neuroblastoma cells [3]. Only toxyloxanthone B (11) was found to show significant neuroprotective effect at 10 μM (22.31% of increase in cell viability) (Fig. 3).
Supporting information The NMR spectra and HR-ESI-MS for compound 1 and the ECD spectra for compounds 1–6 are available as Supporting Information.
Fig. 3. The neuroprotective effect of compound 11 against Aβ25–35-induced neurotoxicity in SH-SY5Y cells. ##P b 0.01 vs. control; **P b 0.01 vs. Aβ25–35 group. ECGG (epigallocatechin-3-gallate) was used as a positive control.
Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments The authors gratefully acknowledge Prof. Bao-Kang Huang (Department of Pharmacognosy at School of Pharmacy, The second Military Medical University, Shanghai, China) for the plant identification. This work was supported by NSFC grants (Nos. 81273401, 81202420), a STCSM grant (No. 11DZ1921203), grants from the Ph.D. Programs Foundation of Ministry of Education (MOE) of China (Nos. 20120071110049, 20120071120049), a MOST grant (No. 2011ZX09307-002-01), and the National Basic Research Program of China (973 Program, Grant No. 2013CB530700). References [1] Na Z. Chemical constituents of volatile oil from leaf of Cratoxylum formosum subsp. pruniflorum in Xishuangbanna of Yunna Province. J Plant Resour Environ 2007;16:75–7. [2] Vo VV. A dictionary of medicinal plants in Vietnam. Ho Chi Minh City: Y Hoc Publisher; 1997 435. [3] Tang Y, Fu Y, Xiong J, Li M, Ma G-L, Yang G-X, et al. Casuarinines A-J, lycodine-type alkaloids from Lycopodiastrum casuarinoides. J Nat Prod 2013;76:1475–84. [4] Wang L-J, Xiong J, Liu S-T, Liu X-H, Hu J-F. Sesquiterpenoids from Chloranthus henryi and their anti-neuroinflammatory activities. Chem Biodivers 2014. http://dx.doi.org/10.1002/cbdv.201300283 (in press). [5] Kim CS, Kim SY, Moon E, Lee MK, Lee KR. Steroidal constituents from the leaves of Hosta longipes and their inhibitory effects on nitric oxide production. Bioorg Med Chem Lett 2013;23:1771–5. [6] Wu SB, Wen Y, Pang F, Zhang H-F, Zhao Z, Hu J-F. Antiproliferative and apoptotic activities of linear furocoumarins from Notopterygium incisum on cancer cells. Planta Med 2010;76:82–5. [7] Kumar V, Brecht V, Frahm AW. Conformational analysis of the biflavonoid GB2 and a polyhydroxylated flavanone–chromone of Cratoxylum neriifolium. Planta Med 2004;70:646–51. [8] Gunatilaka AAL, Sriyani HTB, Sotheeswaran S, Waight ES. 2,5Dihydroxy-1,6-dimethoxyxanthone and biflavonoids of Garcinia thwaitesii. Phytochemistry 1983;22:233–5. [9] Prescott AG, Stamford NPJ, Wheeler G, Firmin JL. In vitro properties of a recombinant flavonol synthase from Arabidopsis thaliana. Phytochemistry 2002;60:589–93. [10] Boonnak N, Khamthip A, Karalai C, Chantrapromma S, Ponglimanont C, Kanjana-Opas A, et al. Nitric oxide inhibitory activity of xanthones from the green fruits of Cratoxylum formosum subsp. pruniflorum. Aust J Chem 2010;63:1550–6. [11] Fun H-K, Chantrapromma S, Boonnak N, Karalai C, Chantrapromma K. Redetermination and absolute configuration of pruniflorone M monohydrate. Acta Crystallogr 2011;E67:o1916–7. [12] Surat L, Wisanu M, Sorwaporn K. Antimalarial and cytotoxic phenolic compounds from Cratoxylum maingayi and Cratoxylum cochinchinense. Molecules 2009;14:1389–95. [13] Ishiguro K, Fukumoto H, Nakajima M, Isoi K. Xanthones in cell suspension cultures of Hypericum paturum. Phytochemistry 1993;33:839–40. [14] Botta B, Delle Monache F, Delle Monache G, Marini Bettolo GB, Oguakwa JU. 3-Geranyloxy-6-methyl-1,8-dihydroxyanthraquinone and vismiones C, D and E from Psorospermum febrifugum. Phytochemistry 1983;22:539–42. [15] Sibanda S, Nyanyira C, Nicoletti M, Galeffi C. Vismiones L and M from Ochna pulchra. Phytochemistry 1993;34:1650–2. [16] Nicoletti M, Marini-Bettolo GB. Keto-enolic tautomerism and spectral data of prenylated anthranoids from Vismia genus. Tetrahedron 1982;38:3679–86. [17] MdeLS Goncalves, Mors WB. Vismiaquinone, a Δ1-isopentenyl substituted anthraquinone from Vismia reichardtiana. Phytochemistry 1981;20:1947–50. [18] Delle Monache F, Ferrari F, Marini-Bettolo GB, Maxfield P, Cerrini S, Fedeli W, et al. Vismiones from Vismia baccifera var. dealbata (H.B.K.): chemistry and X-ray structure determination. Gazz Chim Ital 1979; 109:301–10. [19] Lee YR, Wang X. First concise synthesis of biologically interesting nigrolineabenzopyran A, (±)-blandachromene II, and (±)-daurichromene D. Bull Kor Chem Soc 2007;208:2061–4.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Phenolic constituents from the leaves of Cratoxylum formosum ssp. pruniflorum Juan Xiong a, Xin-Hua Liu b, Van-Binh Bui c,d,e, Zhi-Lai Hong a, Li-Jun Wang a, Yun Zhao c,d, Hui Fan c,d, Guo-Xun Yang a, Jin-Feng Hu a,⁎ a b c d e
Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, PR China Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, PR China Department of Chemistry, East China Normal University, Shanghai 200062, PR China Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, East China Normal University, Shanghai 200062, PR China Department of Chemistry, Hoa Lu University, Ninh Binh 40000, Viet Nam
a r t i c l e
i n f o
Article history: Received 3 January 2014 Accepted in revised form 6 February 2014 Available online 15 February 2014 Keywords: Cratoxylum formosum ssp. pruniflorum Clusiaceae Phenolic compounds Anti-neuroinflammation Neuroprotection
a b s t r a c t One (formosumone A, 1) new and fifteen (2–16) known phenolic compounds were isolated from the leaves of Cratoxylum formosum ssp. pruniflorumm, a substitute for the popular bitter nail tea (“Kuding Tea”) generally used in Southeast Asia. Their structures were determined by extensive spectroscopic analysis and by comparison with literature data. Compound 1 possesses a rare scaffold of a flavanone coupled with a phloroglucinol moiety, representing the first example of such a scaffold from the Clusiaceae family. Among the isolates, toxyloxanthone B (11) and vismione D (12) were found to show remarkable anti-neuroinflammatory effects by inhibiting nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated murine BV-2 microglial cells. Additionally, toxyloxanthone B (11) exhibited significant neuroprotective effect against β-amyloid25–35 (Aβ25–35)-induced cell viability decrease in SH-SY5Y neuroblastoma cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction People believe that natural tea drink is beneficial for postponing human aging and expanding their lifespan. “Kuding (bitter nail tea)” as a famous functional beverage has been ordinarily used in southern and southwestern China for a long time. “Kuding” (“ku” means bitter, while “ding” describes its nail shape in Chinese) is appreciated more for its valuable health-care function than its special bitter-taste. It has been traditionally applied for heat-clearing, quenching thirst, detoxifying, preventing obesity, reducing blood fat, and lowering blood pressure [1,2]. There are 17 species which belong to 5 families generally used as the original materials of “Kuding Tea”, mainly including Ilex kudingcha, Ligustrum robustum, Ligustrum pedunculare, Listea coreana, and Ehretia thyrsiflora [1]. Cratoxylum formosum ssp. pruniflorum (Kurz) Gogelin
⁎ Corresponding author. Tel./fax: +86 21 51980172. E-mail address: [email protected] (J.-F. Hu).
http://dx.doi.org/10.1016/j.fitote.2014.02.002 0367-326X/© 2014 Elsevier B.V. All rights reserved.
(family Clusiaceae) is widely distributed in the tropical region of Southeast Asia. Its leaves are commonly used as a substitute for “Kuding Tea” in Yunnan Province of China and its neighborhood in Vietnam. However, the chemistry and pharmacology of the leaves have been so far not studied. As a continuation of our interest in discovering new agents for the treatment of aging-associated diseases from natural products [3,4], the leaves from the title plant were phytochemically investigated. Reported herein are the isolation and structure determination of a new (1) and fifteen known (2–16) phenolic compounds (Fig. 1) and their anti-neuroinflammatory and neuroprotective activities. 2. Experimental 2.1. General experimental procedures Optical rotations were determined on a Perkin-Elmer 341 polarimeter. UV and IR spectra were recorded on a Shimadzu
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115
Fig. 1. Chemical structures of compounds 1–16.
UV-2550 and an Avatar 360 ESP FTIR spectrometer, respectively. CD spectra were measured on a Jasco 810 spectrometer. NMR spectra were recorded on a Bruker AM-400 or DRX-500 instruments; Chemical shifts are expressed in δ (ppm), and referenced to the residual solvent signals. ESI-MS were measured on an Agilent 1100 series mass spectrometer; and HR-ESI-MS were measured on a Bruker Daltonics micrOTOF-QII mass spectrometer. Column chromatography (CC) was performed using silica gel (200–300 mesh, Kang-Bi-Nuo Silysia Chemical Ltd., China), macroporous adsorption resin D101 (0.3–1.25 mm, Cang Zhou Bon Adsorber Technology Ltd., China), MCI gel CHP20P (75–150 μm, Mitsubishi Chemical Industries, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden). Silica gel-precoated plates (GF254, 0.25 mm) were used for TLC detection. Spots were visualized using UV light (254 and/or 366 nm) and by spraying with 15% (v/v) H2SO4–EtOH followed by heating to 120 °C. 2.2. Plant material The leaves of C. formosum ssp. pruniflorum were collected from Ninh Binh, Vietnam, by one of the authors (V.-B. Bui) in July 2011. The plant was identified by Prof. Bao-Kang Huang
(Department of Pharmacognosy, The second Military Medical University, Shanghai, China). A voucher specimen (No. 20110601) was deposited at the Herbarium of the Department of Natural Products Chemistry, School of Pharmacy at Fudan University. 2.3. Extraction and isolation The dried and powdered leaves (7.5 kg) were extracted with 95% EtOH (18 L × 3) at room temperature to give a black crude extract (750 g, semi-dry). The entire EtOH extract was subjected to a D101 macroporus resin column eluted successively by MeOH/H2O (80:20, v/v), EtOH/H2O (95:5, v/v), and acetone (neat). The 95% EtOH fraction (ca. 380 g) was fractionated by CC over silica gel [petroleum ether (PE)–EtOAc, 4:1 → 0:1, v/v] to give five fractions (Frs. 1–5). Fr. 1 (20.9 g) was applied to silica gel CC with PE-acetone (8:1 → 4:1, v/v) followed by gel permeation chromatography (GPC) on Sephadex LH-20 in MeOH to furnish compound 13 (4.8 mg). Fr. 2 (130.5 g) was subjected to silica gel (CH2Cl2/MeOH, 70:1 → 35:1, v/v), yielding Frs. 2.1– 2.5. Purification of Fr. 2.1 by CC over silica gel (CH2Cl2/MeOH, 70:1, v/v) gave compounds 9 (5.6 mg) and 10 (27.8 mg). Accordingly, compound 8 (7.5 mg) was isolated from Fr. 2.2. CC
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of Fr. 2.3 on silica gel (CH2Cl2/MeOH, 50:1, v/v) followed by GPC on Sephadex LH-20 (MeOH) afforded compounds 1 (8.0 mg), 7 (39.8 mg), and 11 (8.5 mg). Fr. 2.4 was subjected to silica gel (CH2Cl2/MeOH, 50:1 → 30:1, v/v), furnishing compounds 2 (40.2 mg), 5 (37.8 mg), and 6 (14.5 mg). Fr. 3 (35.0 g) was chromatographed on silica gel (CH2Cl2/MeOH, 30:1 → 10:1, v/v) to afford compounds 3 (10.3 mg) and 4 (14.1 mg). The acetone fraction (ca. 150 g) was decolorized by a MCI gel column with MeOH/H2O (8:2 → 0:1, v/v), giving three fractions (Frs. 6–8). Compounds 14 (7.0 mg) and 15 (2.6 mg) were obtained from Fr. 6 (32.5 g) by repeated CC over silica gel (PE/acetone 10:1 → 5:1, v/v). Fr. 7 (21.9 g) was applied to silica gel CC (PE/acetone 6:1 → 3:1, v/v) to afford compounds 12 (2.6 mg) and 16 (2.8 mg). Methyl 3-((2R,3S)-5,7-dihydroxy-2-(4-hydroxyphenyl)4-oxochroman-3-yl)-2, 4,6-trihydroxybenzoate (Formosumone A, 1): yellow amorphous powder; [α]20 D −59.0 (c = 0.061, MeOH); UV (MeOH) λmax (log ε) 290 (3.68), 310 (3.51) nm; IR (KBr): 3386 (br), 2964, 2767, 1643, 1463, 1397, and 1128 cm−1; CD (c 3.4 × 10− 4 M, MeOH) λmax (Δε): 284 (+ 23.1), 310 (− 6.7) nm; for 1H and 13C NMR data, see Table 1; ESIMS: m/z 493 [M + K]+, 477 [M + Na]+, 455 [M + H], 453 [M − H]−; HR-ESIMS: m/z 493.0520 [M + K]+ (calcd. for C23H18O10K, 493.0532). 2.4. Measurement of NO production and cell viability in LPS-activated BV-2 cells The mouse microglia BV-2 cell line was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA), and maintained in Dulbecco's modified Eagle's medium containing 1800 mg/L NaHCO3, supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. The anti-neuroinflammatory activity in BV-2 cells was evaluated according to the reported protocol with modification [4,5]. The NO production was quantified by nitrite accumulation in the culture medium using the Griess reaction kit (Beyotime Biotechnology, China) according to the manufacturer's instructions. Briefly, the BV-2 cells were pretreated with different concentrations (3.125, 6.25, 12.5, 25, 50, and 100 μM) of indicated compounds for 4 h, and then stimulated with or without lipopolysaccharide (LPS) (1 μg/mL, Sigma-Aldrich) for 24 h. The isolated supernatants were mixed with an equal volume of Griess reagent. NaNO2 was used to generate a standard curve, and NO production was determined by measuring the optical density at 540 nm by a microplate reader (M200, TECAN, Austria GmbH, Austria). The cell viability was measured using a 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [4,6]. NG-monomethyl-L-arginine (L-NMMA, Beyotime, purity ≥ 99%), a well-known NO synthase inhibitor, was used as the positive control. The IC50 values were determined by GraphPad Prism 5. 2.5. Neuroprotective activity assay in SH-SY5Y cells The cell viability of SH-SY5Y cells was evaluated according to the reported protocol with modification [3]. The cells were high passages from the ATCC maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cells were seeded into 96-well
Table 1 1 H (500 MHz) and
13
C (125 MHz) NMR dataa of compound 1.
No.
δHb (J values in Hz)
δHc (J values in Hz)
δCb
δCc
2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ OCH3 5-OH 7-OH 4′-OH 2″-OH 4″-OH 6″-OH
5.77 (1H, d, 12.0) 4.67 (1H, d, 12.0)
5.89 (1H, d, 12.0) 4.80 (1H, d, 12.0)
5.88 (1H, d, 2.1)
5.96 (1H, d, 2.0)
5.91 (1H, d, 2.1)
5.98 (1H, d, 2.0)
7.14 (1H, d, 8.5) 6.64 (1H, d, 8.5)
7.25 (1H, d, 8.5) 6.72 (1H, d, 8.5)
7.14 (1H, d, 8.5) 6.64 (1H, d, 8.5)
6.72 (1H, d, 8.5) 7.25 (1H, d, 8.5)
5.84 (1H, s)
5.91 (1H, s)
3.85 (3H, s) 12.20 (1H, s) 10.56 (1H, s) 9.27 (1H, s) 10.91 (1H, s) 10.30 (1H, s) 9.80 (1H, s)
4.03 (3H, s) 12.32 (1H, s) ND ND 10.51 (1H, s) ND ND
81.4 47.5 197.3 100.9 163.6 96.0 166.3 95.0 163.0 128.3 128.7 114.7 157.5 114.7 128.7 93.3 162.4 100.9 162.2 95.1 160.3 170.5 52.2
82.7 48.9 198.4 102.9 165.5 96.9 166.9 95.8 164.5 130.2 129.8 115.5 158.5 115.5 129.8 94.0 161.3 102.3 ND 95.9 162.3 171.2 53.1
c
a Assignments were made by a combination of 1D- and 2D-(COSY, HSQC, HMBC) NMR experiments. b Measured in DMSO-d6 (328 K). c Measured in acetone-d6 (298 K); ND: Not detectable.
plates at a density of 2.5 × 105 cells/mL in MEM/F12 medium supplemented with 10% (v/v) FBS. After 24 h, the serum-free MEM/F12 medium was used to substitute the original medium. The test compounds and the positive control (epigallocatechin3-gallate, ECGG) were made to 10−2 M stock solutions with DMSO and then diluted to corresponding concentrations with the cell culture medium. Cells were incubated with test compounds (1 or 10 μM) or ECGG (10 μM, Sigma, purity N 98%) for 2 h prior to treatment with 10 μM Aβ25–35 for another 24 h without changing the culture medium. 10 μL of MTT (5 mg/mL, Sigma, purity: 98%) was then added to each well and incubated at 37 °C for 3 h. The cells were finally lysed with 100 μL of DMSO, and the amount of MTT formazan was measured at 490 nm using a microplate reader (M200, TECAN, Austria GmbH, Austria). Data were evaluated for statistical significance with a one-way ANOVA followed by the Least Significant Difference (LSD) test using a computerized statistical package. 3. Results and discussion The dried leaves of the title plant were extracted with 95% EtOH at room temperature to give a crude extract, which was found to show preliminary anti-neuroinflammatory effect by inhibiting nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine microglia BV-2 cells. From this material, seven flavonoids (1–7), four xanthones (8–11), and five anthranoids (12–16) (Fig. 1) were purified. Comparing their spectroscopic data and physicochemical properties with those
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reported in the literature, the known compounds were identified as naringenin-(3 → 8)5,7-dihydroxychromone (2) [7], GB-1 (3) [8], GB-2 (4) [8], GB-1a (5) [8], GB-2a (6) [8], 2,3-trans-dihydro-kaemferol (7) [9], 2′-hydroxymethyl-3′,3′dimethyl-dihydrofuran-1,5-dihydroxy xanthone (pruniflorone M, 8) [10,11], formoxanthone C (9) [12], 2′-hydroxy-4′,4′dimethyl-pyran-1,5-dihydroxyxanthone (pruniflorone N, 10) [10], toxyloxanthone B (11) [13], vismione D (12) [14,15], vismione B (13) [14,16], parietin (14) [17], vismiaquinone (15) [17], and 11-hydroxy-5-methoxy-2,2,9-trimethyl-2H-anthra [1,2-b]-pyran-7,12-dione (16) [18]. Compound 1 was obtained as a yellow amorphous powder. The molecular formula of 1 was determined to be C23H18O10 based on a pseudo-molecular ion peak at m/z 493.0520 [M + K]+ in its positive mode HR-ESIMS. The IR spectrum exhibited absorption bands (νmax) at 3386 and 1643 cm−1 attributed to hydroxy and conjugated carbonyl groups. The 1H NMR spectrum (Table 1) of 1 displayed signals of a typical 1,4-disubstituted benzene group with an AA′BB′ system [δ 7.14 (2H, d, J = 8.5 Hz, H-2′, 6′); 6.64 (2H, d, J = 8.5 Hz, H-3′, 5′)], a tetrasubstituted aromatic ring with the protons located at the meta-positions [δ 5.88 (1H, d, J = 2.1 Hz, H-6), 5.91 (1H, d, J = 2.1 Hz, H-8)], and two methines at δ 5.77 (1H, d, J = 12.0 Hz, H-2) and 4.67 (1H, d, J = 12.0 Hz, H-3). The above data were characteristic for the presence of a 4′,5,7-trihydroxy flavanone framework, indicating that compound 1 was a naringenin derivative similar to biflavonoids 2–6 [7,8]. The remaining signals including an olefinic proton at δ 5.84 (1H, s, H-5″) and a methoxy group at 3.85 (3H, s) in the 1H NMR spectrum of 1 were thus assigned to the C-3 substituent (Table 1). In addition to the fifteen carbon signals involved in the core structure (naringenin-type nucleus), the 13C NMR spectrum (in DMSO-d6, 328 K) of 1 exhibited eight additional carbon signals including six olefinic carbons [δ 93.3 (qC, C-1″), 162.4 (qC, C-2″), 100.9 (qC, C-3″), 162.2 (qC, C-4″), 95.1 (CH, C-5″), 160.3 (qC, C-6″)], an ester carbonyl at δ 170.5, and a methoxy group at δ 52.2 (Table 1). From the above evidences, a methyl 2,4,6-trihydroxybenzoate unit was proposed as the substitutional moiety bound to C-3 in compound 1 as depicted in Fig. 1. The linkage position between the flavanone moiety and the phloroglucinol unit in 1 was confirmed by the observation of a clear 3J correlation of H-3 (δ 4.67)/C-3″ (δ 100.9) in its HMBC spectrum (Fig. 2). Additionally, HMBC correlations from 2″-OH (δ 10.91) to C-1″ (δ 93.3)/C-3″ (δ 100.9) and from OMe (δ 3.85) to C-7″ (δ 170.5) were also observed. Due
Fig. 2. Observed key HMBC correlations of 1.
117
to the lack of correlations from H-5″ to its neighboring protons (in the NOESY spectrum) and carbons (in the HMBC spectrum), the structure of the C-3 substituent was further secured by comparing its 13C NMR data (Table 1) with those of methyl 2,4,6-trihydroxybenzoate [δ 92.3 (C-1), 164.4 (C-2), 95.3 × 2 (C-3, C-5), 162.1 (C-4), 161.9 (C-6), 169.3 (\CO), 51.8 (\OMe)] [19]. The trans configuration between H-2 and H-3 in 1 can be easily determined by the large coupling constant (J2,3 = 12.0 Hz). The experiment circular dichroism (CD) spectrum of 1 showed a positive cotton effect at 284 nm and a negative one at 310 nm, indicative of a (2R)-configuration [20,21]. Thus, the entire structure of 1 was unambiguously elucidated to be methyl 3-((2R,3S)-5,7-dihydroxy-2-(4-hydroxy-phenyl)-4oxochroman-3-yl)-2,4,6-trihydroxybenzoate (formosumone A). Naturally occurring flavonoids conjugated with a phloroglucinol moiety are quite rare. To our knowledge, only two examples have been previously reported from the skin of Allium cepa [22], and both of which are 2,3-epoxyflavanone derivatives possessing either a phloroglucinoyl or a 2,4,6-trihydroxyphenylglyoxylate unit at C-3. Unlike compound 1, biflavonoids 2–6 showed two sets of signals in their 1H and 13C NMR spectra recorded at room temperature due to their rotameric behavior (atropisomerism) [7,23,24], which resulted from the hindered rotation between the flavanone and the flavanonol moieties around the C-3/C-8″ axis (Fig. 1). The absolute configurations of biflavonoids 2–6 were also determined by comparison of their CD spectra (See Supporting information) with those reported in the literature [21,24]. Undoubtedly, compounds 1–6 have the same (2R,3S)-naringenin scaffold. Among the isolates, pruniflorone M (8), formoxanthone C (9), pruniflorone N (10), vismione D (12), vismiaquinone (15), and 11-hydroxy-5-methoxy-2,2,9-trimethyl-2H-anthra [1,2-b]-pyran-7,12-dione (16) have been previously obtained either from the fruits [10] or from the barks [25] of C. formosum ssp. pruniflorum. Vismione B (13) was firstly isolated from the fruits of C. cochinchinense [26]. The remaining known compounds (i.e., 2–7 and 14) were reported herein for the first time from plants of the Cratoxylum genus. Neuroinflammation has proven to be implicated in the pathogenesis of neurological disorders, such as Alzheimer's
Table 2 Inhibitory effects on NO production in LPS-stimulated BV-2 cells. Compound
IC50a (μM)
Cell viabilityb (%)
1 2 3 4 8 11 12 c L-NMMA
57.3 59.7 71.5 48.8 17.2 12.4 8.3 14.5
100.3 96.6 94.1 98.0 93.0 99.9 91.9 98.4
± ± ± ± ± ± ± ±
0.5 2.4 1.7 1.3 0.6 0.1 0.7 0.3
± ± ± ± ± ± ± ±
5.3 5.0 5.7 5.0 0.5 0.8 5.0 2.2
a IC50 value of each compound was defined as the concentration (μM) of indicated compound that caused 50% inhibition of NO production in LPSstimulated RAW264.7 cells. b Cell viability after treatment with 50 μM of each compound was expressed as a percentage (%) of untreated control cells. The results are averages of three independent experiments. c G L-NMMA (N -monomethyl-L-arginine): Positive control.
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disease (AD) and Parkinson's disease (PD) [27,28]. A potential therapeutic approach for these neurodegenerative (inflammation- or aging-associated) diseases is inhibition of the synthesis or release of inflammatory mediators [e. g. NO] in overactivated microglia [29]. In this study, the 95% EtOH extract from the leaves of the title plant was found to show antineuroinflammatory activity by significantly (P b 0.01) reducing NO production in LPS-stimulated BV-2 cells when compared with the control group treated by LPS only. Therefore, all the purified compounds from this extract were evaluated for their anti-neuroinflammatory effects on NO production in LPS-activated BV-2 cells. The inhibitory activities of the tested compounds on NO production were expressed as 50% inhibition concentration (IC50). As shown in Table 2, four flavonoids (1–4), two xanthones (8, 11) and a prenylated anthranoids (12) could significantly decrease NO production in BV-2 cells without influence on cell viability at concentrations up to 50 μM, while others were inactive (IC50 N 100 μM). Among them, toxyloxanthone B (11) and vismione D (12) demonstrated the most potent NO inhibitory activity (IC50 = 12.4, 8.3 μM, resp.), better than that of the positive control, NG-monomethyl-L-arginine (L-NMMA, IC50 = 14.5 μM). Interestingly, xanthones 8–10 have been previously reported to possess potent inhibitory activity against LPS-induced NO release in macrophages RAW 264.7 cells [10]. All the isolates were also evaluated for their neuroprotective effects against β-amyloid25–35 (Aβ25–35)-induced cell viability decrease in SH-SY5Y neuroblastoma cells [3]. Only toxyloxanthone B (11) was found to show significant neuroprotective effect at 10 μM (22.31% of increase in cell viability) (Fig. 3).
Supporting information The NMR spectra and HR-ESI-MS for compound 1 and the ECD spectra for compounds 1–6 are available as Supporting Information.
Fig. 3. The neuroprotective effect of compound 11 against Aβ25–35-induced neurotoxicity in SH-SY5Y cells. ##P b 0.01 vs. control; **P b 0.01 vs. Aβ25–35 group. ECGG (epigallocatechin-3-gallate) was used as a positive control.
Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments The authors gratefully acknowledge Prof. Bao-Kang Huang (Department of Pharmacognosy at School of Pharmacy, The second Military Medical University, Shanghai, China) for the plant identification. This work was supported by NSFC grants (Nos. 81273401, 81202420), a STCSM grant (No. 11DZ1921203), grants from the Ph.D. Programs Foundation of Ministry of Education (MOE) of China (Nos. 20120071110049, 20120071120049), a MOST grant (No. 2011ZX09307-002-01), and the National Basic Research Program of China (973 Program, Grant No. 2013CB530700). References [1] Na Z. Chemical constituents of volatile oil from leaf of Cratoxylum formosum subsp. pruniflorum in Xishuangbanna of Yunna Province. J Plant Resour Environ 2007;16:75–7. [2] Vo VV. A dictionary of medicinal plants in Vietnam. Ho Chi Minh City: Y Hoc Publisher; 1997 435. [3] Tang Y, Fu Y, Xiong J, Li M, Ma G-L, Yang G-X, et al. Casuarinines A-J, lycodine-type alkaloids from Lycopodiastrum casuarinoides. J Nat Prod 2013;76:1475–84. [4] Wang L-J, Xiong J, Liu S-T, Liu X-H, Hu J-F. Sesquiterpenoids from Chloranthus henryi and their anti-neuroinflammatory activities. Chem Biodivers 2014. http://dx.doi.org/10.1002/cbdv.201300283 (in press). [5] Kim CS, Kim SY, Moon E, Lee MK, Lee KR. Steroidal constituents from the leaves of Hosta longipes and their inhibitory effects on nitric oxide production. Bioorg Med Chem Lett 2013;23:1771–5. [6] Wu SB, Wen Y, Pang F, Zhang H-F, Zhao Z, Hu J-F. Antiproliferative and apoptotic activities of linear furocoumarins from Notopterygium incisum on cancer cells. Planta Med 2010;76:82–5. [7] Kumar V, Brecht V, Frahm AW. Conformational analysis of the biflavonoid GB2 and a polyhydroxylated flavanone–chromone of Cratoxylum neriifolium. Planta Med 2004;70:646–51. [8] Gunatilaka AAL, Sriyani HTB, Sotheeswaran S, Waight ES. 2,5Dihydroxy-1,6-dimethoxyxanthone and biflavonoids of Garcinia thwaitesii. Phytochemistry 1983;22:233–5. [9] Prescott AG, Stamford NPJ, Wheeler G, Firmin JL. In vitro properties of a recombinant flavonol synthase from Arabidopsis thaliana. Phytochemistry 2002;60:589–93. [10] Boonnak N, Khamthip A, Karalai C, Chantrapromma S, Ponglimanont C, Kanjana-Opas A, et al. Nitric oxide inhibitory activity of xanthones from the green fruits of Cratoxylum formosum subsp. pruniflorum. Aust J Chem 2010;63:1550–6. [11] Fun H-K, Chantrapromma S, Boonnak N, Karalai C, Chantrapromma K. Redetermination and absolute configuration of pruniflorone M monohydrate. Acta Crystallogr 2011;E67:o1916–7. [12] Surat L, Wisanu M, Sorwaporn K. Antimalarial and cytotoxic phenolic compounds from Cratoxylum maingayi and Cratoxylum cochinchinense. Molecules 2009;14:1389–95. [13] Ishiguro K, Fukumoto H, Nakajima M, Isoi K. Xanthones in cell suspension cultures of Hypericum paturum. Phytochemistry 1993;33:839–40. [14] Botta B, Delle Monache F, Delle Monache G, Marini Bettolo GB, Oguakwa JU. 3-Geranyloxy-6-methyl-1,8-dihydroxyanthraquinone and vismiones C, D and E from Psorospermum febrifugum. Phytochemistry 1983;22:539–42. [15] Sibanda S, Nyanyira C, Nicoletti M, Galeffi C. Vismiones L and M from Ochna pulchra. Phytochemistry 1993;34:1650–2. [16] Nicoletti M, Marini-Bettolo GB. Keto-enolic tautomerism and spectral data of prenylated anthranoids from Vismia genus. Tetrahedron 1982;38:3679–86. [17] MdeLS Goncalves, Mors WB. Vismiaquinone, a Δ1-isopentenyl substituted anthraquinone from Vismia reichardtiana. Phytochemistry 1981;20:1947–50. [18] Delle Monache F, Ferrari F, Marini-Bettolo GB, Maxfield P, Cerrini S, Fedeli W, et al. Vismiones from Vismia baccifera var. dealbata (H.B.K.): chemistry and X-ray structure determination. Gazz Chim Ital 1979; 109:301–10. [19] Lee YR, Wang X. First concise synthesis of biologically interesting nigrolineabenzopyran A, (±)-blandachromene II, and (±)-daurichromene D. Bull Kor Chem Soc 2007;208:2061–4.
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