Phytochemical study and biological evaluation of chemical constituents of Platanus orientalis and Platanus × acerifolia buds

Phytochemistry xxx (2016) 1e12

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Phytochemical study and biological evaluation of chemical constituents of Platanus orientalis and Platanus
acerifolia buds Quoc Dang Thai a, b, 1, Job Tchoumtchoua a, 1, Maria Makropoulou a, c, Athina Boulaka c, Aggeliki K. Meligova c, Dimitra J. Mitsiou c, Sophia Mitakou c, Sylvie Michel b, Maria Halabalaki a, Michael N. Alexis c, Leandros A. Skaltsounis a, * a b c

Division of Pharmacognosy and Natural Products Chemistry, School of Pharmacy, University of Athens, Panepistimioupoli Zografou, 15771, Athens, Greece Laboratoire de Pharmacognosie de l'Universit
e Paris Descartes, UMR/CNRS 8638, Facult
e de Pharmacie, 4 Avenue de l'Observatoire, F-75006, Paris, France Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, Athens, Greece

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 February 2016 Received in revised form 13 April 2016 Accepted 26 April 2016 Available online xxx

One flavonol glycoside, two O-isoprenylated flavonols, one a,a-dimethylallyl flavonol, one dihydrochalcone, two furanocoumarins and one terpenoid previously undescribed, along with 42 known compounds were isolated from the buds of two European Platanaceae, Platanus orientalis and Platanus
acerifolia. Their chemical structures were elucidated on the basis of spectroscopic analysis, including homonuclear and heteronuclear correlation NMR (COSY, NOESY, HSQC, and HMBC) experiments, as well as HRMS data. The estrogen-like and antiestrogen-like activity of dichloromethane and methanol extracts of P. orientalis and P.
acerifolia buds and isolated compounds was evaluated using estrogen-responsive cell lines. The potency of selected estrogen agonists to regulate gene expression through ERa and/or ERb was compared with their in vitro osteoblastogenic activity. Kaempferol and 8-C(1,1-dimethyl-2-propen-1-yl)-5,7-dihydroxyflavonol displayed osteoblastogenic as well as ERa-mediated estrogenic activity similar to estradiol. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Platanus orientalis Platanus
acerifolia Platanaceae Phytoestrogens Estrogenic activity Flavonoids Coumarins Dihydrochalcones Osteoblast differentiation

1. Introduction The genus Platanus is the unique living member of Platanaceae family and includes seven accepted species and twelve subspecies widespread throughout different regions of the world (The Plant List, 2013). Amongst these, two species, namely Platanus orientalis Linneaus and Platanus
acerifolia (Aiton) Willd are represented in Europe. P. orientalis, the most extended species of the genus Platanus, is traditionally used in Eastern Europe and Near East to treat various ailments including blepharitis, conjunctivitis, and hemorrhage (Nishanbaev et al., 2004), gastro-intestinal disorders, toothache, skin disease, fever, and body pain (Haq et al., 2011; Murad et al., 2011), kidney stones and itching (Polat and Satıl, 2012). Previous phytochemical and pharmacological reports provided

* Corresponding author. E-mail address: [email protected] (L.A. Skaltsounis). 1 These authors contributed equally.

scientific basis for these medicinal uses. Specifically, P. orientalis possesses anti-hepatotoxic, anti-oxidant and cytotoxic activities (El-Alfy et al., 2008), and elicits anti-inflammatory and antinoceptive effects in vivo (Haider et al., 2012; Hajhashemi et al., 2011). As regards to chemical composition, tocopherols derivatives, esters of phytol with fatty acids (Abdullaev et al., 1994) and several polyphenols (El-Alfy et al., 2008; Tantry et al., 2012) were isolated from the leaves; kaempferol derivatives and caffeic acid were isolated from the buds (Dimas et al., 2000; Mitrokotsa et al., 1993); and triterpenoids, proanthocyanidins and proanthocyanidin glycosides were also isolated from the barks (Khan et al., 2013; Nishanbaev et al., 2004, 2010, 2005). Platanus
acerifolia (Aiton) Willd is an interspecific hybrid between Platanus occidentalis Linneaus and P. orientalis growing as a street tree in major cities in North America, Europe, and Asia (Yang et al., 2013). The buds of P.
acerifolia has been reported to contain a number of dihydrochalcones and oxodihydrochalcones (Kaouadji, 1986, 1989; Kaouadji et al., 1986a, 1986b), C- & O-isoprenylated

http://dx.doi.org/10.1016/j.phytochem.2016.04.006 0031-9422/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Thai, Q.D., et al., Phytochemical study and biological evaluation of chemical constituents of Platanus orientalis and Platanus
acerifolia buds, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.04.006

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Q.D. Thai et al. / Phytochemistry xxx (2016) 1e12

flavanones (Kaouadji et al., 1986b), flavonols and flavonol glycosides (Barron et al., 1994; Kaouadji, 1990, 2014; Kaouadji et al., 1992, 1993, 1988) and pyrano-flavanones (Kaouadji et al., 2013). 2styrylchromones and prenylated flavonols were recently isolated from the bark of P.
acerifolia (Yang et al., 2013) as well as three triterpenoids and two flavonoids (Lin et al., 2013). To the best of our knowledge, no pharmacological studies has been performed related to P.
acerifolia extracts and there are only two studies dealing with the bio-activities of isolated components thereof (Yang et al., 2013, 2014). During our ongoing search for novel bioactive metabolites from Platanus species, the dichloromethane and methanol extracts of the buds of P orientalis and P.
acerifolia were selected to be investigated due to their interesting phytochemical and biological profile. Both extracts displayed a rich content in secondary metabolites as revealed by their HPLC-DAD profile as well as strong potential to promote in vitro differentiation of preosteoblasts to mature osteoblasts. In-depth investigation of these extracts was carried out using various chromatographic techniques including HSCCC (highspeed counter current chromatography), MPLC, and column chromatography. This work resulted in the isolation of 50 compounds, including 8 novel compounds and specifically 1 flavonol glycoside, 2 O-isoprenylated flavonols, 1 a,a-dimethylallyl flavonol, 1 dihydrochalcone, 2 furanocoumarins and 1 terpenoid. Their structures were elucidated by direct interpretation of their spectral data, using high resolution mass spectrometry (HRMS), 1D and 2D NMR (COSY, NOESY, HSQC, and HMBC). In addition, in view of the above reports that flavonoids are major constituents of P. orientalis and P.
acerifolia buds and of recent findings that flavonoids induce osteoblast differentiation via estrogen receptor (ER) signaling (Guo et al., 2012), the estrogen-/antiestrogen-like and ER-regulatory activities of bud extracts and isolated compounds were evaluated and active components were assayed for effects on the differentiation of MC3T3-E1 preosteoblasts to osteoblasts. 2. Results and discussion The dichloromethane (CH2Cl2) extracts of the buds of P.
acerifolia and P. orientalis, as well as the methanol (CH3OH) extract of the buds of P. orientalis were subjected to various chromatographic techniques, including either HSCCC or MPLC fractionation, column chromatography including silica gel or Sephadex LH-20, followed by preparative TLC purification. The study of these extracts afforded 50 compounds including 8 previously undescribed (1e8), and 42 (9e50) previously reported compounds. The previously undescribed compounds were unambiguously identified using spectroscopic (NMR) and spectrometric (HRMS) techniques while comparison with literature data (Supplementary information) confirmed the structures of known compounds as described in Fig. 1. Compound 1 was isolated as yellow crystals, and displayed a UV spectrum characteristic of a flavonol with a maxima at 261 nm and 273 nm. Its molecular weight was determined based on its deprotonated molecular ion at m/z 383.1124 [M  H] corresponding to C21H20O7 molecular formula based on ESI()-HRMS analysis; and this was also supported by the NMR spectroscopic data (Table 1). The 1H NMR spectrum displayed four aromatic signals. First, an ABX system consisting of a singlet (H-20 ) at 7.67 ppm and two doublets (H-50 , J ¼ 8.2 Hz) and (H-60 , J ¼ 8.2 Hz) at 7.07 and 7.69 ppm successively was noticed. The carbon atoms of the aforementioned protons were found to resonate at 110.7 ppm (C20 ), 114.8 ppm (C-50 ), and 122.6 ppm (C-60 ), respectively. An additional aromatic signal was observed in the 1H NMR spectrum at 6.30 ppm (H-6), and C-6 resonated at 101.1 ppm. Also, the 1H NMR spectrum provided a signal at 12.15 ppm, characteristic singlet of an

aromatic hydroxyl group closed to a carbonyl. The methoxyl protons were observed as a singlet at 3.97 ppm and the corresponding carbon atom at 56.3 ppm. The HMBC spectrum revealed a correlation of the hydroxyl group with C-5 (d 159.6), and the methoxyl with C-30 (d 146.6) confirming their position. The 1,1-dimethylallyl side chain was established by the appearance of a methine group at 6.49 ppm (H-200 , dd, J ¼ 10.5, 17.5 Hz), which displayed a 1H-1H COSY correlation to the a-proton of the downfield exomethylene at 5.42 ppm (H-300 a, J ¼ 10.5 Hz), and HMBC correlation with the two methyl groups at 1.72 ppm (H-400 and H-500 ). Added to this, the HMBC spectrum revealed that all the protons of this side chain correlated with C-8, specifically H-200 /C-8, H-300 /C-8, H-400 /C8 and H-500 /C-8, thus indicating the position of this chain on the basic skeleton (Table 1). Accordingly, the structure of 1 was determinated as 5,7,40 -trihydroxy-8-(1,1-dimethylallyl)-30 methoxyflavonol. Compound 2 was obtained as an amorphous, yellow solid with UV maxima at 297 nm. This compound was established to have a quasi-molecular formula of C21H19O7 based on the ESI()-HRMS (m/z 383.1145 [M  H]). The NMR data (Table 1) of 2 were typical of a flavonol trisubstituted in the B ring. In the 1H NMR spectrum were signals corresponding to the H-6 (d 6.19, d, J ¼ 1.7 Hz) and H-8 (d 6.28, d, J ¼ 1.7 Hz) protons of the A ring, the signals of the H-20 (d 7.03, s) and H-50 (d 6.76, s) protons of the B ring, and signals of an isoprenyl moiety 5.15 (H-200 , t, J ¼ 7.0 Hz), 3.27 (H-100, d, J ¼ 7.0 Hz), 1.59 (H-400 , s) and 1.51 ppm (H-500 , s). Moreover, the methoxyl group protons were evident at 3.86 ppm, and the position of this group was revealed from the HMBC spectrum by the correlation with C-30 . The HMBC spectrum also helped determine the position of the isoprenyl moiety at C-60 , with correlations of H-100 with C-10, C-50 , and C-60 observed. Hence, the structure of 2 was assigned as 5,7,40 trihydroxy-60 -prenyl-30 -methoxyflavonol. Compound 3 was also obtained as an amorphous, yellow solid. The UV spectrum was alike to that of compound 2 (lmax 294 nm). Its ESI()-HRMS (m/z 383.1146 [M  H]), corresponding to C21H20O7 molecular formula was identical to 2 indicating structural isomers. NMR spectra of compounds 2 and 3 were also similar (Table 1), though a difference resided in the position of the isoprenyl group at the B ring. The cross-peaks COSY correlation between H-50 and H-60 as well as the HMBC correlations of H-100 with C-10 (3J), C-20 (2J), and C-30 (3J) determined the position of the sidechain on B-ring. Therefore, the structure of 3 was established as 5,7,40 -trihydroxy-20 -prenyl-30 -methoxyflavonol. Compound 4 was obtained as amorphous, yellow solid. Its molecular weight was deduced based on its deprotonated molecular ion at m/z 577.1354 [M  H], which corresponded to the molecular formula C30H26O12 as provided by the ESI()-HRMS analysis. According to 1H and 13C NMR data (Table 2), 4 was structurally close to 22, which was previously isolated from the buds of P.
acerifolia (Kaouadji and Morand, 1993). The kaempferol moiety was indicated by two meta-related signals d 6.40 (1H, s, H-8) and d 6.21 (1H, s, H-6); and two groups of ortho-coupled protons d 7.84 (2H, d, J ¼ 8.5 Hz, H-20 , 60 ) and d 6.97 (2H, d, J ¼ 8.5 Hz, H-30 , 50 ). The pcoumaric acid with E configuration was deduced from the two groups of doublets at d 7.48 (2H, d, J ¼ 8.5 Hz, H-2000 , 6000 ) and d 6.82 (2H, d, J ¼ 8.5 Hz, H-3000 , 5000 ) for the aromatic ring, and at d 7.73 (1H, d, J ¼ 15.8 Hz, H-7000 ) and d 6.43 (1H, d, J ¼ 15.8 Hz, H-8000 ) for the side chain. The rhamnose moiety was identified by cross-peaks of the anomeric proton to d 5.47 (1H, d, J ¼ 1.4 Hz, H-100 ), d 5.13 (1H, dd, J ¼ 3.2, 9.9 Hz, H-300 ), d 4.45 (1H, dd, J ¼ 1.4, 2.9 Hz, H-200 ), d 3.62 (1H, t, J ¼ 9.9 Hz, H-400 ), d 3.45 (1H, m, H-500 ), d 0.98 (3H, d, J ¼ 6.4 Hz, H600 ) in the 1H-1H COSY spectrum. Two main differences were observed while comparing the 1H NMR data of 4 and 22, H-200 was shifted up-field (D  1.09 ppm) and H-300 downfield (D þ 1.18 ppm) in 4. Added to this, a HMBC correlation was noticed between H-300 -

Please cite this article in press as: Thai, Q.D., et al., Phytochemical study and biological evaluation of chemical constituents of Platanus orientalis and Platanus
acerifolia buds, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.04.006

Q.D. Thai et al. / Phytochemistry xxx (2016) 1e12

Fig. 1. Structure of compounds 1 50 isolated from P. orientalis and P.
acerifolia.

Please cite this article in press as: Thai, Q.D., et al., Phytochemical study and biological evaluation of chemical constituents of Platanus orientalis and Platanus
acerifolia buds, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.04.006

3

4

Q.D. Thai et al. / Phytochemistry xxx (2016) 1e12

and C-9000 (3J), indicating that the (E)-coumaroyl moiety was located at C-300 position. Thus, the structure of 4 was determined as kaempferol-3-O-a-L-(300 -E-p-coumaroyl)-rhamnoside. Compound 5 was established to have a quasi-molecular ion of C21H23O6 based on the ESI()-HRMS (m/z 371.1499 [M  H]). The dihydrochalcone structure was evidenced by the two typical triplet methylene protons resonating at 2.88 ppm (H2-7, t, J ¼ 7.9 Hz) and 3.34 ppm (H2-8, t, J ¼ 7.9 Hz) in the 1H NMR spectrum and the ketocarbonyl carbon signal at d 206.5 (C-9) in the 13C NMR spectrum (Table 3). The B-ring structure was determined as a parasubstituted phenol by the presence of A2B2-type aromatic proton at 6.70 ppm (H-3/H-5, d, J ¼ 8.4 Hz) and 7.04 ppm (H-2/H-6, d, J ¼ 8.4 Hz), and by the HMBC correlation of H-7 with C-2 and C-6. The 13C spectrum shows that the A-ring was fully substituted as evidenced by three ethylen quaternary O-bound carbon atoms at d 155.1 (C-20 ), d 161.5 (C-40 ) and d 163.8 (C-60 ), as well as three ethylen quaternary carbon atoms at d 106.1(C-10 ), d 100.2 (C-30 ) and d 104.2 (C-50 ). Specifically, this ring bears two hydroxyl groups, a characteristic methyl group [dC 7.7, dH 1.99 (H3-50 , s)], and a C5chain cyclized into a dihydrodimethylpyran ring incorporating a hydroxyl group. This C-5 chain was characterized by two methyl groups [dC 25.7, dH 1.34 (H3-500 , s); dC 21.0, dH 1.27 (H3-600 , s)], one methylene [dC 27.2, dH 2.50 (H-400 a, dd, J ¼ 7.0, 16.5 Hz), dH 2.85 (H400 b, dd, J ¼ 5.5, 16.5 Hz)], one oxygenated methine [dC 69.6, dH 3.74 (H-300 , dd, J ¼ 5.5, 7.0 Hz)], and one oxygenated carbon (dC 79.2, C200 ). The position of these substituents of the A-ring were defined by the HMBC correlations of the proton signal H3-50 with C-40 , C-5΄ & C-60 , the proton signal H-300 with C-30 , C-500 & C-600 , and the proton signal H-4a00 with C-20 , C-30 & C-40 . The orientation of the pyrano ring was confirmed by the 1H-1H NOESY correlation between H-8 and H3-500 /H3-600 . This description of 5 was in conformity with another compound previously isolated from P.
acerifolia, known as 50 ,200 ,200 -trimethyl-40 ,60 ,300 -trihydroxy-dihydropyran (500 ,600 : 30 ,20 ) dihydrochalcone (Kaouadji, 1989). By comparing the NMR and ESIHRMS data of both compounds, the only difference seems to be the supplementary hydroxyl group of B-ring in 5. Thus, the structure of 5 was determined as 4,40 ,60 -trihydroxy-20 ,30 -(2,2-dimethyl-3hydroxy-dihydropyrano)-50 -methyl-dihydrochalcone. Compound 6 exhibited a [M  H] peak in ESI()-HRMS spectrum at m/z 243.0660 in accordance with the quasi-molecular formula C14H11O4. The 1H NMR data (Table 4), with the aid of 1H-1H COSY spectrum, showed a pair of doublet at d 6.23 and 7.58 (d, J ¼ 9.4 Hz each 1H), corresponding to the signals of H-3 and H-4 of a-pyrone ring system, and two singlets at d 7.08 and 6.80 (1H each) suggesting then a 6,7 bi-substituted coumarin skeleton. The side chain of this coumarin structure was elucidated by comparing its 1H and 13C NMR spectra with that of the previously reported compound 5-(40 -methyl-20 ,50 -dihydrofuran-20 -yl) indole (Vougogiannopoulou et al., 2011). The 7-hydroxylation and the presence of the 4-methyl-2,5-dihydrofuran-2-yl chain at C-6 were confirmed through HMBC data. Specifically, a proton (brs) at d 8.65 (7-OH) showed a cross peaks correlation with C-6 (d 123.6), C-7 (d 159.2) and C-8 (d 105.1); while a proton (s) at d 7.08 (H-5) showed correlations with C-4 (d 143.7), C-7 (d 159.2), C-8 (d 105.1), C-9 (d 155.5) and C-20 (d 87.3). Similarly another proton (s) at d 6.80 (H-8) showed cross peaks with C-6 (d 123.6), C-7 (d 159.2), C-9 (d 155.5), C-10 (d 112.3) and C-20 (d 87.3). Hence, 6 could be assigned as 7hydroxy-6-(4-methyl-2-5-dihydrofuran-2-yl) coumarin. Compound 7 showed a quasi-molecular ion peak due to [M þ H]þ, at m/z 259.0977 (C15H15O4) in ESI(þ)-HRMS, 14 mass units higher than 6. Likewise, the NMR data of compound 7 (Table 4) had close resemblance to those of compound 6. The only exception in the 1H NMR spectrum resided in the signal of 7-OH at d 8.65 of compound 6 which was absent in compound 7. Instead, a supplementary singlet was present at d 3.94 (3H), correlated to a

carbon atom (HSQC, 2J) at dC 56.4 indicating that C-7 was substituted with a methoxy group. This substitution was further confirmed by HMBC correlations at d 3.94 (3H, 7-OCH3) with C-6 (d 129.8), C-7 (d 160.7) and C-8 (d 99.3). Added to this, the pseudomolecular formula C15H15O4 of 7 displayed one more methyl group than 6. These assignments concluded that 7 is 7-methoxy-6(4-methyl-2-5-dihydrofuran-2-yl) coumarin. Compound 8 was found to be a derivative of betulinic acid. The 1 H NMR data indicated (Table 5) signals for six triterpenoid methyl groups at d 1.68, 1.04, 1.03, 0.97, 0.95, and 0.90 (s and 3H each), and an exomethylene group at d 4.74 (1H, d, J ¼ 1.5) and 4.61 (1H, t, J ¼ 1.5), which are characteristic of the lup-20(29)-en structure (Menezes-de-Oliveira et al., 2011). The 13C NMR spectrum displayed a total of 30 carbon atoms; one carboxyl, seven quaternary with one oxygenated, five methine with one oxygenated, eleven methylene with one olefinic, and six methyl. This NMR data were very close of those of betulinic acid (Yili et al., 2009), except the presence of the oxygenated quaternary carbon (dC 78.8). In order to define the position of this oxygenated quaternary carbon, the HSQC and HMBC spectra were thoroughly analyzed. In HMBC, the protons at d 1.03 (3H, s, H-23) as well as the protons at d 0.90 (3H, s, H-24) correlated with the quaternary carbon at dC 44.0 (C-4), the oxygenated quaternary carbon at dC 78.8, and the oxygenated methine at dC 74.3 (C-3). In addition, the protons at d 0.97 (3H, s, H25) showed an HMBC correlation with the oxygenated quaternary carbon at dC 78.8, the methylene carbon at dC 32.3 (C-1), and the quaternary carbon at dC 40.8 (C-10). The oxygenated quaternary carbon was thus designated as C-5. Added to the NMR data, the ESI()-HRMS of 8 displayed a [M  H] peak at m/z 471.3485, which corresponded to the deprotonated molecule C30H47O4 vs C30H47O3 for 42; this confirmed the presence of a supplementary oxygen atom in the molecule as compared to compound 42 (betulinic acid). Given these descriptions, 8 was assigned as 3,5dihydroxylup-20(29)-en-28-oic acid. Postmenopausal osteoporosis increases morbidity and mortality rates and imparts a substantial economic burden to society. Current management practices are considered inadequate and novel pharmacological prevention and treatment strategies are warranted (Andreopoulou and Bockman, 2015). The increase of proinflammatory cytokines and estrogen deficiency associated with menopause inhibit proliferation, survival and differentiation of osteoblast lineage, including in vitro differentiation of MC3T3-E1 preosteoblasts to mineralizing osteoblasts (Abdelmagid et al., 2015; Chiang and Pan, 2013; Quarles et al., 1992; Xu et al., 2015). It was recently reported that many inflammation-driven diseases with a dependence on estrogen could be treated by targeting inflammatory NFkappaB signalling with specific ERa and/or ERb ligands (Nwachukwu et al., 2014; Zhao et al., 2015). Chemical constituents of the buds such as caffeic acid, kaempferol and other flavonoids as €ma €la €inen well reportedly display anti-inflammatory properties (Ha et al., 2011; Michaluart et al., 1999; Park et al., 2009; Sala et al., 2003). In addition, several anti-inflammatory flavonoids, including kaempferol, are known to bind and activate both estrogen receptor (ER) subtypes, ERa and ERb (Kuiper et al., 1998; Li et al., 2013). Kaempferol, in particular, has been reported to promote new bone formation when injected into the periosteum of parietal bones of newborn rats (Yang et al., 2010) and to inhibit production of osteoclastogenic cytokines as well as stimulate in vitro differentiation of primary osteoblasts via ER signaling (Guo et al., 2012; Pang et al., 2006). In the light of the above reports the CH2Cl2 and MeOH extracts of P. orientalis and P.
acerifolia buds and 50 compounds (Fig. 1) isolated therefrom were evaluated for estrogen-like and antiestrogen-like activity using estrogen-responsive Ishikawa endometrial adenocarcinoma and MCF7 human breast

Please cite this article in press as: Thai, Q.D., et al., Phytochemical study and biological evaluation of chemical constituents of Platanus orientalis and Platanus
acerifolia buds, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.04.006

Q.D. Thai et al. / Phytochemistry xxx (2016) 1e12

5

Table 1 NMR spectroscopic Data (600 MHz) for compounds 1e3. No

1a

dC 1 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 100 200 300 400 500 600 OCH3-30 OH-5 a b

147.7 135.5 175.6 159.6 101.1 161.8 110.1 155.5 104.9 122.9 110.7 146.4 146.6 114.8 122.6 40.8 149.0 114.1 28.0 28.0 56.2

2b

dH (J in Hz)

6.30 (1H, s)

dC

HMBC

7, 8, 10

7.67 (1H, s)

40 , 60

7.07 (1H, d, 8.2) 7.69 (1H, d, 8.2)

1 0 , 40 2 0 , 40

6.49 5.42 5.50 1.72 1.72

8, 10 0 , 40 0 , 50 0 8, 10 0 , 20 0 , 40 0 , 50 0

(1H, (1H, (1H, (3H, (3H,

dd, 10.5, 17.9) d, 10.5) d, 17.9) s) s)

3.97 (3H, s) 12.15 (1H, s)

149.5 136.1 176.6 161.7 99.2 165.9 94.5 158.4 104.5 122.1 114.5 146.2 150.4 117.4 135.9 32.4 124.3 132.6

3b

dH (J in Hz)

dC

HMBC

6.19 (1H, d, 1.7)

5, 8, 10

6.28 (1H, d, 1.7)

6, 7, 9, 10

7.03 (1H, s)

2, 40 , 60

6.76 (1H, s)

10 , 30 , 100

3.27 (2H, d, 7.0) 5.15 (1H, t, 7.0)

10 ,50 , 60 , 300

151.0 136.2 176.6 162.5 99.1 165.7 94.4 158.2 104.5 123.1 136.7 147.0 152.6 115.0 127.6 26.9 124.0 132.0

dH (J in Hz)

HMBC

6.19 (1H, d, 2.2)

5, 8, 10

6.25 (1H, d, 2.2)

6, 7, 9, 10

6.82 7.08 3.41 5.03

1 0 , 30 , 40 2, 20 , 40 10 , 20 , 30 , 300 400 , 500

(1H, (1H, (2H, (2H,

d, d, d, d,

8.5) 8.5) 6.4) 6.4)

8, 10 0 , 20 0 8, 10 0 , 20 0

25.5 17.5

1.59 (3H, s) 1.51 (3H, s)

200 , 300 , 500 200 , 300 , 400

25.2 17.5

1.48 (3H, s) 1.44 (3H, s)

200 , 300 , 500 200 , 300 , 400

30 5, 6, 10

56.3

3.86 (3H, s)

30

60.7

3.80 (3H, s)

30

Data measured in CDCl3. Data measured in CD3OD.

Table 2 NMR spectroscopic Data (600 MHz, CD3OD) for compound 4. No

dC

4

dН (J in Hz) 1 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60

158.6 135.6 179.2 162.8 99.5 165.2 94.3 157.9 105.6 122.3 131.7 116.2 161.2 116.2 131.7

HMBC

6.21 (1H, s)

5, 7, 8, 10

6.40 (1H, s)

4, 6, 7, 9, 10

7.84 (2H, d, 8,5) 6.97 (2H, d, 8.5)

2, 30 , 40 , 60 1 0 , 40 , 50

6.97 (2H, d, 8.5) 7.84 (2H, d, 8.5)

1 0 , 40 , 30 2, 40 , 20

adenocarcinoma cells. Ishikawa cells are ERa and ERb-positive while MCF7 cells are ERa-positive but ERb-negative. Induction of alkaline phosphatase (AlkP) in Ishikawa cells by steroidal estrogens, phytoestrogens and environmental estrogens is considered a reliable measure of estrogenic activity (Jain et al., 2009; Markiewicz et al., 1993). On the other hand, MCF7 cells are highly responsive to estrogen proliferation-wise and have been extensively used in order to detect synthetic or plant-derived estrogenic chemicals (Orlando et al., 2004; Soto et al., 1995). Estradiol is known to fully induce AlkP expression in Ishikawa cells and proliferation of MCF7 cells whereas the ER-degrader ICI182,780 is known to fully inhibit these hormonal effects (Alexi et al., 2009). However, plant extracts and isolated compounds may display estrogen-like activity despite lack of ER-binding activity or false negative estrogen-like activity and false positive antiestrogen-like activity due to cytotoxicity,

Nο

dC

dН (J in Hz)

HMBC

100 200 300 400 500 600 1000 2000 3000 4000 5000 6000 7000 8000 9000

102.7 69.5 74.7 70.2 72.1 17.3 127.0 130.7 116.6 160.5 116.6 130.7 146.5 115.3 168.5

5.47 4.45 5.13 3.62 3.45 0.98

3, 200 , 300 , 500 100 , 300 , 400 400 , 9000 300 , 500 , 600 100 , 300 , 400 , 600 500

(1H, (1H, (1H, (1H, (1H, (3H,

d, 1.4) dd, 1.4, 2.9) dd, 2.9, 9.9) t, 9.9) m) d, 6.4)

7.48 (2H, d, 8.5) 6.82 (2H, d, 8.5)

3000 , 4000 , 6000 , 7000 1000 , 4000 , 5000

6.82 7.48 7.73 6.43

1000 ,4000 , 3000 2000 , 4000 , 5000 , 7000 1000 , 2000 , 3000 , 9000 1000 , 7000 , 9000

(2H, (2H, (1H, (1H,

d, d, d, d,

8.5) 8.5) 15.8) 15.8)

especially at high concentrations, such as 20 mg/ml for extracts and 10 mM for isolated compounds (Overk et al., 2008). Extracts and isolated compounds were initially assayed at 2 mg/ml and 1 mM, respectively, using on the one hand estrogen-free Ishikawa and MCF7 cells and 0.1 nM estradiol (i.e. postmenopausal level of the hormone) as positive control for estimating estrogen-like activity and on the other hand 0.1 nM estradiol-repleted Ishikawa and MCF7 cells and 0.1 mM ICI182,780 as positive control for assaying antiestrogen-like activity (Table 6). Data of Table 6 shows that the MeOH and CH2Cl2 extracts of P. orientalis displayed full estrogenlike activity in Ishikawa cells, while the MeOH and CH2Cl2 extracts of P.
acerifolia displayed weak and partial estrogen-like activity, respectively; and that none of these extracts displayed antiestrogen-like activity. Table 6 also shows that only 1, 16, 17 and 18 displayed full or partial agonism of induction of AlkP expression,

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Q.D. Thai et al. / Phytochemistry xxx (2016) 1e12

Table 3 NMR spectroscopic Data (600 MHz, CD3OD) for compound 5. No

5

dC 1 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 100 200 300 400 500 600 CH3-50

133.8 130.1 116.1 156.5 116.1 130.1 31.3 46.2 206.5

dH (J in Hz)

HMBC

7.04 (2H, d, 8.4) 6.70 (2H, d, 8.4)

4, 6, 7 1, 4, 5

7.04 6.70 2.88 3.34

(2H, (2H, (2H, (2H,

d, 8.4) d, 8.4) t, 7.9) t, 7.9)

1, 2, 1, 1,

3.74 2.50 2.85 1.34 1.27 1.99

(1H, (1H, (1H, (3H, (3H, (3H,

dd, 5.5, 7.0) dd, 7.0, 16.5) dd, 5.5, 16.5) s) s) s)

30 , 500 , 600 20 , 30 , 40 , 200 , 300

3, 4, 2, 7,

4 7 6, 8, 9 9

106.1 155.1 100.2 161.5 104.2 163.8 79.2 69.6 27.2 25.7 21.0 7.7

200 , 300 , 600 200 , 300 , 500 40 , 50 , 60

with a rank order of AlkP induction efficacies of 18 > 17>1  16; and that 8 (a derivative of betulinic acid) and 42 (betulinic acid) suppressed estradiol-induced AlkP expression, with 42 displaying much higher antiestrogen-like activity compared to 8. In addition, Table 6 shows that all extracts and compounds that displayed estrogen-like activity in Ishikawa cells induced proliferation of MCF7 cells; and that the rank order of proliferation induction efficacies was 18  17  16 > 1. However, none of the two compounds that displayed antiestrogen-like activity in Ishikawa cells suppressed estradiol-induced proliferation of MCF7 cells. Moreover, Table 6 shows that 3, 5, 13, 14, 24, 28, 30, 32, 36, 40 and 44, which were marginally active in Ishikawa cells, induced proliferation of MCF7 cells weakly, with 3 being proliferation-wise more effective than the remainder and therefore more biased towards stimulating

cell proliferation as compared to AlkP expression. Conversely, 5, 11 and 32 suppressed estradiol-dependent proliferation of MCF7 cells weakly but estradiol-induced AlkP expression only marginally. Finally, 32 compounds failed to affect cell proliferation as well as AlkP expression at 1 mM and therefore are considered inactive at pharmacologically relevant concentrations. Table 7 shows the potency (EC25) of induction of MCF7 cell proliferation and Ishikawa AlkP expression by the three more effective compounds in either assay (i.e. 16, 17, 18) and the more biased one (i.e. 3) as assessed following treatment of the cells with increasing concentrations (0.01e10 mM) of these compounds. Induction of cell proliferation and AlkP expression by 3, 16, 17 and 18 was inhibited by ICI182,780, suggesting that it is ER-mediated. The rank order of EC25 of induction of cell proliferation was 18  17  16  3 (t-test), in accordance with the respective efficacies of Table 6. Similarly, the rank order of EC25 of induction of AlkP was 18 < 17<16 < 3 (t-test), in accordance again with the respective efficacies of Table 6. Next the EC25 of induction of estrogen response element (ERE)-dependent gene expression by 3, 16, 17 and 18 through ERa and ERb was assessed using MCF-7:D5L cells, a clone of MCF-7 cells that is stably transfected with an ERE-endowed reporter plasmid, and HEK:ERb cells, a clone of HEK-293 cells that is stably transfected with an ERbexpressing plasmid as well as an ERE-endowed reporter plasmid. Again, induction of luciferase expression by 3, 16, 17 and 18 was inhibited by ICI182,780, suggesting that it is also ER-mediated. Table 7 shows that the rank order of EC25 of induction of luciferase expression through ERa was 18 < 17  16 < 3 (t-test), in accordance with the rank order of potencies of induction of AlkP expression and cell proliferation reported above. However, only 16 displayed high potency of induction of luciferase expression through ERb, while 3, 17 and 18 were totally ineffective up to 10 mM. ERa and ERb bind compounds that possess two hydroxyl groups at a distance of 9.7e12.3 Å (Lambrinidis et al., 2006; Ng et al., 2014). Kaempferol possesses two pairs of OH groups with an OeO distance within this range, 4'- and 5-OH (10.62 Å) and 4'- and 7-OH (10.76 Å). It is therefore expected that kaempferol can bind either ER subtype by way of hydrogen bond formation between the 40 -OH and Glu353/Arg394 on ERa (Glu305/Arg346 on ERb) and the 5-OH or 7-OH and His524 on ERa (His475 on ERb) (Brzozowski et al., 1997; Nettles et al., 2007; Pike et al., 1999). Kaempferol i.e. 5,7,4'-

Table 4 NMR spectroscopic Data (600 MHz, CDCl3) for compound 6 and 7. 6a No 1 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 7-OH 7-OCH3 a b

dC 161.1 113.1 143.7 126.0 123.6 159.2 105.1 155.5 112.3 87.3 122.1 137.6 78.3 12.4

7b

dH (J in Hz)

HMBC

6.23 (1H, d, 9.4) 7.58 (1H, d, 9.4) 7.08 (1H, s)

2, 10 2, 5, 8, 9, 10 4, 7, 8, 9, 20

6.80 (1H, s)

6, 7, 9, 10, 20

6.07 (1H, brs) 5.68 (1H, brs)

20 , 50

4.67 (2H, dddd, 12.6, 5.2)

3 0 , 40

1.84 (3H, s) 8.65 (1H, s)

30 , 40 , 50 6, 7, 8

dC 163.1 113.3 145.9 126.5 129.8 160.7 99.3 156.3 113.6 83.8 123.5 137.3 78.7

dH (J in Hz)

HMBC

6.25 (1H, d, 9.4) 7.89 (1H, d, 9.4) 7.55 (1H, s)

2, 10 2, 5, 8, 9, 10 4, 7, 8, 9, 20

6.96 (1H, s)

6, 7, 9, 10, 20

6.01 (1H, m) 5.60 (1H, m)

2 0 , 50 3 0 , 40

11.6

4.73 (1H, ddm, 12.2, 5.5) 4.62 (1H, dm, 12.2) 1.80 (3H, quint., 1.5)

56.4

3.94 (3H, s)

7, 8

3 0 , 40 , 50

Data measured in CDCl3. Data measured in CD3OD.

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Table 5 NMR spectroscopic Data (600 MHz, CDCl3) for compound 8 and 42. No

8

42

dC

dH (J in Hz)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

32.3 23.7 74.3 44.0 78.8 27.5 27.9 39.7 42.1 40.8 21.1 25.8 38.6 43.2 29.8 32.1 56.4 49.4 47.1 150.5 30.7 37.2 22.8 17.4 19.3 16.3 14.9 180.0 109.9

NI

30

19.5

3.84 (1H, dd, 11.8, 5.3 Hz)

HMBC

4, 23, 24

2.08 (1H, dd, 12.8, 3.4 Hz)

8, 25, 26

1.30 (m) 1.73, 1.08 2.18 (1H, td, 12.6, 3.8 Hz)

NI NI 12, 14, 17, 18, 27

1.60 (1H, m) 2.99 (1H, m)

13, 17, 19, 20, 28 18, 20, 21, 29, 30

1.97, 1.39 1.97, 1.47 1.03 (3H, s) 0.90 (3H, s) 0.97 (3H, s) 0.95 (3H, s) 1.04 (3H, s)

17, 18 18, 19, 28 3, 4, 5, 24 3, 4, 5, 23 1, 5, 10 7, 8, 14 13, 15

4.74 (1H, d, 1.5, 29a) 4.61 (1H, t, 1.5, 29b) 1.68 (3H, s)

19, 30 19, 20, 29

trihydroxy-flavonol (16) was found here to be somewhat less estrogenic than the 8-C-(1,1-dimethyl-2-propen-1-yl) derivatives of 5,7-dihydroxy-flavonol (17) and 5,7,4'-trihydroxy-flavonol (18). This was not expected considering that ER ligands with bulky substituents such as ICI182,780, tamoxifen and raloxifene are estrogen antagonists; their bulky substituents are known to destabilize the coactivator-binding conformation of ER, thus compromising receptor integrity (ICI182,780) and/or agonist activity (Brzozowski et al., 1997; Katzenellenbogen and Katzenellenbogen, 2002; Pike et al., 1999). However, it has been reported that estradiol derivatives with bulky substituents at position 17a can increase the volume of the ligand-binding pocket of ERa and behave as agonists even more potent than estradiol (Nettles et al., 2007). It is therefore possible that the 1,1-dimethyl2-propen-1-yl group at position 8 of 17 and 18 can take advantage of the structural plasticity of ERa and thus endow these flavonols with higher estrogenic activity compared to 16. That 18 was more potent agonist than 17 probably reflects the potential of the 4'hydroxy group of the former to form hydrogen bond with His524. On the other hand, 17 and 18 displayed marginal agonist activity through ERb, suggesting that the induced fit model of enhanced agonism does not apply for ERb. Instead, considering that the volume of the ligand-binding cavity of ERb (390 Å3) is smaller than that of ERa (490 Å3) (Pike et al., 1999), it appears that the 1,1dimethyl-2-propen-1-yl group of 17 and 18 is more likely to sterically clash with residues that line the binding cavity of the former rather than the latter ER subtype, thus preventing 17 and 18 to bind ERb. That 17 and 18 induced AlkP expression in Ishikawa cells as well as proliferation of MCF7 cells while being unable to bind ERb is presumably underlying the dominant role that ERa plays in either of these effects (Fokialakis et al., 2004; Lazennec et al., 1999; Powell and Xu, 2008). We also found that 3, 5, 13, 14, 24, 28, 30, 32, 36, 40

dC

dH (J in Hz)

HMBC

38.8 27.5 79.2 39.0 55.5 18.4 34.5 40.8 50.7 37.4 21.0 25.6 38.5 42.6 29.8 32.3 56.4 49.4 47.1 150.5 30.7 37.2 28.1 15.5 16.3 16.2 14.8 180.6 109.8

NI

NI

3.19 (1H, dd, 11.6, 4.9 Hz)

4, 23, 24

0.68 (1H, br.d, 9.2 Hz)

7, 9, 14, 26

1.27 (s)

NI

NI 1.67, 1.34 2.20 (1H, td, 12.6, 3.8 Hz)

12, 14, 17, 18, 27

1.61 (t, 10.5) 3.00 (1H, td, 10.6, 4.4 Hz))

13, 17, 19, 20, 28 18, 20, 21, 29, 30

1.97, 1.42 1.97 (m) 0.96 (3H, s) 0.75 (3H, s) 0.82 (3H, s) 0.93 (3H, s) 0.97 (3H, s)

18, 19 17, 21, 28 3, 4, 5, 24 3, 4, 5, 23 5, 9 7, 9, 14 13, 14, 15

4.74 (1H, br. s, 29a) 4.61 (1H, br. s, 29b) 1.69 (3H, s)

19, 30

19.5

19, 20, 29

and 44 failed to induce AlkP expression while they stimulated MCF7 cell proliferation, albeit weakly, implying that their weak proliferative effect was not mediated through ERa; and that this is probably also the case with the weak antiproliferative effects of 5, 11 and 32. Finally, concerning the inhibitory effect of 42 on AlkP expression in the absence of any effect on the proliferation of MCF7 cells, it has been reported that betulinic acid can only affect MCF7 cell proliferation at concentrations >20 mM (Kim et al., 2014). Estrogens increase proliferation and survival of the osteoblast lineage and contribute to the maintenance of bone mass (Manolagas et al., 2013). Differentiation of MC3T3-E1 preosteoblasts to osteoblasts is thought to model the key roles that estrogens and ERa play in bone anabolic effects (Khalid et al., 2008). Given the estrogenic potential of CH2Cl2 and MeOH extracts of P. orientalis and P.
acerifolia buds and 3, 16, 17 and 18, their effect on the differentiation of MC3T3-E1 was assessed at 1 mg/ml (extracts) and 1 mM (isolated compounds) using induction of AlkP as differentiation marker. Fig. 2 shows that exposure of MC3T3-E1 preosteoblats to differentiation medium (contains ascorbic acid, bglycerophosphate and postmenopausal levels i.e. ~0.1 nM of estrogen) resulted in 2.5-fold induction of AlkP that was considerably upregulated in the presence of premenopausal levels of estradiol and down-regulated by raloxifene and ICI182,780 whether in the absence or presence of added estradiol. This data shows that MC3T3-E1 cells faithfully model the osteoblastogenic role that estrogens reportedly play in vivo through ERa. In addition, Fig. 2 shows that 16 and 17 promoted differentiation to an extent similar to estradiol, while 3, 18, and the extracts were not effective in this respect but they were not inhibitory either. While the 40 -OH of 16 and 18 are likely able to secure hydrogen bond formation with Glu353/Arg394 on ERa this is not possible for 17. However, the luciferase expression data of Table 7 suggests that 17 can effectively

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Table 6 Effects on cell proliferation and alkaline phosphatase expression.a Compound

Alkaline phosphatase expression (Ishikawa cells) Agonism

Estradiol ICI182,780 P.or.M P.or.D P.ac.M P.ac.D 1 3 5 8 4 13 14 16 17 18 24 28 30 32 36 40 42 44

b

(% of 0.1 nM estradiol)

100 e 67 ± 7 107 ± 8 19 ± 4 36 ± 6 46 ± 1 m m m m m m 37 ± 3 65 ± 5 92 ± 11 m m m m m m m m

Cell proliferation (MCF-7 cells)

Antagonism c (% of 100 nM ICI182,780)

Agonism

e 100 m m m m m m m 14 ± 4 m m m m m m m m m m m m 34 ± 2 m

100 e 57 ± 73 ± 37 ± 58 ± 30 ± 29 ± 13 ± m m 19 ± 12 ± 53 ± 56 ± 61 ± 11 ± 14 ± 14 ± 15 ± 16 ± 14 ± m 13 ±

b

(% of 0.1 nM estradiol)

1 3 4 4 3 6 3

5 1 5 6 6 3 1 3 4 5 4 3

Antagonism c (% of 100 nM ICI182,780) e 100 m m m m m m 11 ± 6 m 14 ± 6 m m m m m m m m 12 ± 4 m m m m

P.or.M ¼ Methanol extract of Platanus orientalis. P.or.D ¼ Dichloromethane extract of Platanus orientalis. P.ac.M ¼ Methanol extract of Platanus
acerifolia. P.ac.D ¼ Dichloromethane extract of Platanus
acerifolia. E2 ¼ Estradiol. a Agonist or antagonist effects were assessed at 1 mM compound or 2 mg/ml extract and were classified as full, partial or weak depending on whether induction or suppression of cell proliferation and alkaline phosphatase expression was 67e100, 34e66 and 10e33% of that of E2 or ICI182,780. Values are Mean ± SEM of three independent experiments carried out in triplicate. Effects <10% and effects that failed to display statistically significant difference from vehicle were classified as marginal (m). b Agonism of E2-induced effects (Ef) was calculated by (Efcompound  Efvehicle)
100/(EfE2  Efvehicle). c Antagonism of E2-induced effects were calculated by (EfE2  EfE2þcompound)
100/(EfE2  EfE2þICI182,780).

Table 7 Potency of induction of cell proliferation and gene expression. Compound Induction of cell proliferationa (MCF-7 cells) EC25b (mM) 3 16 17 18

2.10 0.42 0.24 0.17

± ± ± ±

0.91 0.10 0.06 0.07

Induction of alkaline phosphatasea (Ishikawa cells)

Induction of luciferase expressiona (MCF7:D5L cells)

Induction of luciferase expressiona (HEK:ERb cells)

EC25b (mM)

EC25b (mM)

EC25b (mM)

± ± ± ±

n.a. 0.89 ± 0.24 n.a. n.a.

3.21 1.60 1.03 0.49

± ± ± ±

0.25 0.10 0.07 0.02

1.81 0.61 0.48 0.14

0.15 0.01 0.05 0.01

n.a. ¼ not applicable. a Calculated by: [(Effectcompound e Effectvehicle)
100/(Effect1 nM estradiol e Effectvehicle)]. EC50 values are Mean ± SEM of three independent dose-response experiments carried out in triplicate. b Compound concentration required to achieve 25% of the effect of 1 nM estradiol.

bind ERa by a mechanism that obviates the need of hydrogen bond formation with Glu353/Arg394. It is therefore possible that 17 is capable of promoting differentiation through ERa. That 5- and/or 7hydroxy-flavonols with a bulky substituent at position 8 may display an in vitro osteoblastogenic potential similar to estradiol warrants further investigation.

3. Concluding remarks Platanus orientalis and Platanus
acerifolia, a unique living genus of Platanaceae family was phytochemicaly investigated in the context of the current study. Specifically, the dichloromethane and methanol extracts of the buds of both species were used as starting material. In total, fifty secondary metabolites including

eight previously undescribed ones, were isolated using various and complementary chromatographic methods and identified via spectroscopic (1 & 2D NMR) and spectrometric (HRMS) techniques. The total extracts as well as the purified compounds were evaluated for their potency to stimulate estrogen-responsive cell lines. The most promising constituents being capable to regulate gene expression through ERa and/or ERb were further evaluated for their in vitro osteoblastogenic activity. Kaempferol and 8-C-(1,1dimethyl-2-propen-1-yl)-5,7-dihydroxyflavonol displayed osteoblastogenic as well as ERa-mediated estrogenic activity similar to estradiol.

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Q.D. Thai et al. / Phytochemistry xxx (2016) 1e12

9

Pharmacognosy, University Rouen). The plant materials were dried in the shade at room temperature to a constant weight. 4.3. Extraction and separation

Fig. 2. Effects on the differentiation of MC3T3-E1 cells. Colorimetric assessment of alkaline phosphatase (AlkP) activity of MC3T3-E1 cells growing in the absence (DF) or presence of differentiation factors (þDF) or in combination with vehicle (0.2% DMSO), estradiol (E2, 1 nM), raloxifene (Ral, 100 nM), ICI182,780 (ICI, 100 nM), E2 þ Ral (1 nM þ 100 nM), E2 þ ICI (1 nM þ 100 nM), the methanol or dichloromethane extract of Platanus orientalis (P.or.M or P.or.D, 1 mg/ml) or Platanus
acerifolia (P.ac.M or P.ac.D, 1 mg/ml) or compounds 3, 16, 17 or 18 (1 mM). The AlkP activity of cells treated with DF þ vehicle was taken equal to 100. Values are mean ± SEM. *p < 0.05 vs DF þ vehicle.

4. Experimental section 4.1. General experimental procedures Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 600 MHz spectrometer. The 2D experiments (COSY, HSQC, HMBC, and NOESY) were performed using standard Bruker microprogram. The residual 1H and 13C signals of CD3OD (dH 3.31, dC 49.0, respectively), and CDCl3 (dH 7.26, dC 77.16, respectively) were used as internal standard. ESIHRMS spectra were obtained on a hybrid Thermo Scientific LTQ-Orbitrap Discovery mass spectrometer. HPLC analysis were performed on Thermo Finnigan system equipped with a PDA detector (Spectral System UV6000LP) using a Supelco HS Discovery® C18 column (4.6 mm
250 mm i.d., 5 mm). Preparative hydrostatic CCC fractionations were carried out on a Kromaton FCPC® instrument equipped with a rotor 1000 mL. The solvent was pumped through the system with a preparative pump LabAlliance. The crude extracts were injected into the system through the 30 mL loop and fractions were collected with a Buchi B684 fraction collector. MPLC fractions were obtained using a BÜCHI Labortechnik AG apparatus equipped with a silica column (45
6 cm i.d., 20e40 mm) and a Buchi B-684 fraction collector. Column flash chromatography was performed on silica gel, E. Merk, 40e60 mm. Thin-layer chromatographic (TLC) analysis was performed on Merk precoated silica gel 60 F254 plates. Pre-coated silica gel 60 GF254 preparative plates (20
20, 0.25 mm thick, E. Merk) were used for preparative thin-layer chromatography. Spots were visualized using UV light and vanillineH2SO4 (50/50, v/v) reagent. 4.2. Plant material The buds of P. orientalis were collected in Euboea Island (Greece) in August 2012, authenticated by Dr. Eleftherios Kalpoutzakis at the Division of Pharmacognosy and Natural Products Chemistry of the University of Athens where a voucher specimen is conserved (specimen N 203 ATPH). The buds of P.
acerifolia were identified and collected in Villenomble (Eastern suburds of Paris, France), in October 2012 by Pr. Elizabeth Seguin (Laboratory of

An amount of 348 g of the dried and pulverized buds of P.
acerifolia was sequentially macerated with CH2Cl2 (3 L, 48 h
3), CH3OH (3 L, 48 h
3), and H2O (3 L, 48 h
2) at room temperature. The solvent were further evaporated in vacuo to dry, yielding to three residue extracts: 12.1 g (CH2Cl2), 14.3 g (CH3OH) and 11.2 g (H2O). 8.5 g of the CH2Cl2 extract was subjected to a dualphase HSCCC fractionation using a biphasic mixture of ether petroleum/EtOAc/eCH3OH/eH2O:1/1.5/1/0.5 (v/v/v/v) at a speed of 550 rpm and a flow rate of 10 mL/min 20-mL fractions were collected, combined on the basis of their TLC profile and evaporated in vacuo to give 40 final fractions which were analyzed in HPLCPDA. On the basis of their HPLC profile, 13 out of the 40 fractions tend to contain major constituents of this extract and were firstly targeted. Fraction 7 (149 mg) was subjected to a silica gel column and eluted with CH2Cl2/EtOAc (9/1, v/v) to give 32 (10 mg) and 42 (23.4 mg). Fraction 9 (30 mg) was purified on preparative TLC (CH2Cl2/EtOAc: 96:4) to give 17 (4.9 mg) and 28 (1.2 mg). Fractions 10 and 11 were combined (103.8 mg) and purified on preparative TLC (cyclohexane/EtOAc: 7:3) to yield 12 (1.0 mg), 27 (1.1 mg) and 36 (1.4 mg). Fraction 14 (110.2 mg) was purified on preparative TLC (CH2Cl2/EtOAc: 9:1) to furnish 13 (6.1 mg), 40 (1.2 mg) and 45 (0.9 mg). Fraction 17 (38.6 mg) was applied to a preparative TLC (CH2Cl2/CH3OH: 98:2) to afford 1 (4.0 mg), 18 (1.1 mg) and 41 (0.9 mg). Fractions 19 to 22 were combined (89.2 mg), subjected to a silica gel CC eluted with CH2Cl2/EtOAc: 95:5 (v/v) and further were purified on preparative TLC (CH2Cl2/CH3OH: 96:4) to afford 2 (2.5 mg), 3 (4.5 mg), 5 (10.5 mg) and 41 (2.1 mg). Fractions 25 and 26 were combined (20.6 mg) and purified on preparative TLC (CH2Cl2/CH3OH: 96:4) to yield 11 (1.5 mg), 14 (1.5 mg) and 15 (1.5 mg). Fractions 27 and 28 were combined (26.4 mg) and purified on preparative TLC (CH2Cl2/CH3OH: 85:15) to yield 11 (3.3 mg) and 33 (0.8 mg). Fractions 36 and 37 were pure and identified as compounds 34 (14.2 mg), and 45 (8.6 mg), respectively. The buds (1212 g) of P. orientalis were extracted using the same procedure as in the case of P.
acerifolia with 9 L of solvent in each case. The final extract residues were: 34.4 g (CH2Cl2), 53.1 g (CH3OH) and 42.2 g (H2O). A part of the CH2Cl2 extract (12.6 g) was subjected to an MPLC column for initial fractionation using a gradient solvent system of increasing polarity, cyclohexane/CH2Cl2 (90:10 / 100%, v/v) and CH2Cl2/CH3OH (100% / 50:50, v/v), yielding to 19 major fractions. Amongst these, 3 fractions seemed to contain components different from the above isolated and were prioritized. Fraction 12 (117.3 mg) was eluted on silica gel CC with a solvent of increasing polarity CH2Cl2/EtOAc (100% / 80:20, v/v) giving 46 (3.1 mg) and 48 (1.2 mg) and three subfractions (12A-C). Subfraction 12A (14.3 mg) was purified on preparative TLC (CH2Cl2/ EtOAc: 9:1, v/v) to give 35 (1.3 mg) and 48 (1.2 mg). Fraction 13 (302.9 mg) was eluted on a silica gel CC with CH2Cl2/AcOEt (100% / 80:20, v/v) yielding 49 (1.2 mg), 42 (40 mg) and 34 (2.5 mg). Fraction 15 (279.6 mg) was eluted on a silica gel CC with cyclohexane/EtOAc (9:1 / 8:2, v/v) giving four subfractions (15A-D). Subfraction 15A (15.3 mg) was purified on preparative TLC (cyclohexane/EtOAc: 1:1, v/v) to afford 8 (2.9 mg) and 43 (2.5 mg). Subfraction 15B (13.6 mg) was purified on preparative TLC (cyclohexane/EtOAc: 1:1, v/v) to afford 33 (0.9 mg) and 24 (1.1 mg). Subfraction 15 C (13.8 mg) was purified on preparative TLC (cyclohexane/EtOAc: 3:7, v/v) to yield 38 (1.2 mg). Finally, subfraction 15D (10.9 mg) were purified on preparative TLC (CH2Cl2/ MeOH: 96:4) to yield 50 (4.5 mg). A pre-treatment of 44 g of the CH3OH extract of P. orientalis was

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performed using an Amberlite Xad-7 column in order to isolate the phenolic-enriched fraction (9.5 g), which was subjected to a dual mode HSCCC separation (10 mL/min, 650 rpm) using a biphasic mixture of cyclohexane/EtOAc/CH3OH/H2O: 1/2/0.8/0.9 (v/v/v/v). 30-mL fractions were collected, combined on the basis of their TLC profile and evaporated in vacuo to give 21 final fractions. The first 15 fractions were collected in descending mode while the 6 last fractions were collected in ascending mode. Compounds 20 (138.8 mg) and 23 (43.9 mg), were directly crystallized from fraction 6 (317.6 mg) and purified from fraction 19 (153.8 mg) successively and they seemed to be the predominant constituents of the CH3OH extract. Fraction 4 (405 mg) was subjected to Sephadex LH-20, eluted with MeOH (100%) to give three subfractions (4A-C). Subfraction 4A (35 mg) was purified on prep-TLC (CH2Cl2/MeOH: 80:20, v/v) to afford 21 (7.8 mg) and 19 (7.2 mg), whereas subfraction 4C (20.2 mg) was purified on preparative RP-TLC (CH3OH/ H2O: 50:50, v/v) to afford 20 (5 mg) and 31 (8.2 mg). Fraction 7 (253 mg) was subjected to Sephadex LH-20 eluted with MeOH 100% to give two subfractions (7A & B). Subfraction 7A (66.1 mg) was purified on a silica gel CC and eluted with CH2Cl2/MeOH (100% / 90:10, v/v) yielding 34 (13.8 mg) and 9 (3.6 mg), and subfraction 7B (17.5 mg) was purified on preparative TLC (CH2Cl2/MeOH: 90:10, v/v) to afford 26 (4.4 mg). Fractions 9 and 10 were combined (135.5 mg) and subjected on a silica gel column which was eluted with a solvent of increasing polarity CH2Cl2/MeOH (100% / 90:10) to afford 33 (5.2 mg), 49 (3.7 mg), 39 (1.2 mg), 25 (4.9 mg), 22 (16.1 mg), and 10 (4.5 mg). Fractions 11 and 12 were combined (104.4 mg) and eluted on a silica gel column with a solvent of increasing polarity CH2Cl2/MeOH (100% / 90:10) to afford 25 (5.7 mg), 47 (3.5 mg), 4 (4.7 mg). Fractions 13e15 (122.0 mg) were combined and chromatographied on a silica gel column eluted with a solvent of increasing polarity CH2Cl2/MeOH (100% / 90:10) to afford 25 (1.1 mg), 6 (1.2 mg). Fractions 16 were submitted to a silica gel column eluted with a solvent of increasing polarity CH2Cl2/ MeOH (100% / 90:10) to afford 42 (10.1 mg) and 44 (5.2 mg). Fraction 19 (153.8 mg) was separated on a Sephadex LH-20 column, eluted with MeOH (100%) to give 37 (5.2 mg), 23 (43.9 mg) and a subsfraction 19A (18.5 mg) which was purified on preparative TLC (CH2Cl2-AcOEt: 95:1, v/v) to afford 37 (2.7 mg) and 7 (0.8 mg). Finally, Fraction 20 (42.0 mg) was purified on preparative TLC (CH2Cl2/MeOH: 95:5, v/v) to afford 41 (2.4 mg), 29 (0.8 mg), 30 (2.5 mg), 16 (2.6 mg) and 23 (5.1 mg). 4.4. Compound characterization 4.4.1. 5,7,40 -trihydroxy-8-(1, 1-dimethylallyl)-30 -methoxyflavonol (1) Yellow crystalline; [a]20D þ 10 (c 0.1, MeOH); UV (MeOH) lmax 261, 373 nm; 1H NMR and 13C NMR (CDCl3) see Table 1; ESI-HRMS m/z 383.1124 [M  H] (calcd. for C21H19O7, 383.1125). 4.4.2. 5,7,40 -trihydroxy-60 -prenyl-30 -methoxyflavonol (2) Amorphous, yellow solid; [a]20D þ 20 (c 0.1, MeOH); UV (MeOH) lmax 297, 350 (sh) nm; 1H NMR and 13C NMR (CD3OD) see Table 1; ESI-HRMS m/z 383.1145 [M  H] (calcd. for C21H19O7, 383.1125). 4.4.3. 5,7,40 -trihydroxy-20 -prenyl-30 -methoxyflavonol (3) Amorphous, yellow solid; [a]20D  10 (c 0.1, MeOH); UV (MeOH) lmax 294, 350 (sh) nm; 1H NMR and 13C NMR (CD3OD) see Table 1; ESI-HRMS m/z 383.1146 [M  H] (calcd. for C21H19O7, 383.1125). 4.4.4. Kaempferol-3-O-a-L-(300 -E-p-coumaroyl)-rhamnoside (4) Amorphous, yellow solid; [a]20D  69 (c 0.1, MeOH); UV (MeOH) lmax 241, 266, 314 nm; 1H NMR (CD3OD) see Table 2; ESI-HRMS m/ z 577.1354 [M  H] (calcd. for C30H25O12, 577.1341).

4.4.5. 4,40 ,60 -trihydroxy-20 ,30 -(2,2-dimethyl-3-hydroxydihydropyrano)-50 -methyl dihydrochalcone (5) Amorphous, yellow solid; 1H NMR and 13C NMR (CD3OD) see Table 3; ESI-HRMS m/z 371.1499 [MH] (calcd. for C21H23O6, 371.1489). 4.4.6. 7-hydroxy-6-(4-methyl-2-5-dihydrofuran-2-yl) coumarin (6) Amorphous, white solid; 1H NMR and 13C NMR (CDCl3) see Table 4; ESI-HRMS m/z 243.0660 [M  H] (calcd. for C14H11O4, 243.0651). 4.4.7. 7-methoxy-6-(4-methyl-2-5-dihydrofura-2-yl) coumarin (7) White crystalline; UV (MeOH) lmax 239, 326 nm; 1H NMR and 13 C NMR (CD3OD) see Table 4; ESI-HRMS m/z 259.0977 [M þ H]þ (calcd. for C15H15O4, 259.0964). 4.4.8. 3,5-dihydroxylup-20(29)-en-28-oic acid (8) Amorphous, white solid; 1H NMR and 13C NMR (CDCl3) see Table 5; ESI-HRMS m/z 471.3485 [M  H] (calcd. for C30H47O4, 471.3468). 4.5. Biological study 4.5.1. Tissue culture, biochemicals and reagents Ishikawa human endometrial adenocarcinoma cells were purchased from ECACC. MCF-7 and MC3T3-E1 mouse preosteoblasts were from ATCC. The cells were cultured as recommended by the suppliers. MCF-7:D5L cells and HEK:ERb cells were generated and cultured as already described (Skretas et al., 2007). Unless specified otherwise, cell culture media and hormones were from SigmaAldrich and FBS from Invitrogen. Dextran-coated-charcoal-treated FBS (DCC-FBS) i.e. FBS treated with 10% dextran-coated charcoal to remove endogenous steroids was prepared as previously described (Gritzapis et al., 2003). The ER degrader ICI182,780 was from Tocris Bioscience. 4.5.2. Assessment of cell proliferation Effects on the proliferation of MCF-7 cells were assessed as already described (Katsanou et al., 2007), with modifications. Briefly, cells were plated in 96-flat-bottom-well plates at a density of 4000 cells per well in phenol-red-free MEM supplemented with 1 mg/mL insulin and 5% DCC-FBS. Test compounds (1 mM) and extracts (2 mg/ml) were added 24 h after plating and were allowed to regulate cell proliferation for 96 h in the absence or presence of 0.1 nM estradiol. DMSO (vehicle), the compound diluent, was kept to a final concentration  0.2%. Relative cell numbers were determined using crystal violet (Schafer et al., 1999) and a Safire II microplate reader (Tecan). The difference in optical density at 550 and 690 nm was taken to measure the actual number of cells. Cells exposed to vehicle, 0.1 nM estradiol (know of display full agonism) or estradiol in the presence of 0.1 mM ICI182,780 (know of display full estrogen antagonism) served as controls. Agonist effects on cell proliferation were expressed as % of the effect of estradiol while antagonist effects were expressed as % of the effect of ICI182,780 in the presence of estradiol. Estrogen-agonist and estrogenantagonist effects on cell proliferation were the result of at least three independent experiments carried out in triplicate. In order to assess the potency of selected test compounds to induce cell proliferation, estrogen-free MCF-7 cells were exposed to increasing concentrations (0.01e10 mM) of the selected compounds in the absence or presence of 1 mM ICI182,780. The presence of ICI182,780 served to attest that compound effects on cell proliferation at the concentrations tested were ER-mediated. Cells exposed to 1 nM estradiol served to determine test compound concentrations (EC25)

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capable of producing 25% of the proliferative effect of 1 nM estradiol. 4.5.3. Assessment of ER-regulated gene expression Regulation of AlkP expression of Ishikawa cells was assessed using 96-well plates and 12,000 cells per well in phenol-red-free MEM supplemented with 1 mg/mL insulin and 5% DCC-FBS. 24 h after plating, cells growing in the absence or presence of 0.1 nM estradiol were exposed to test compounds (1 mM) or extracts (2 mg/ ml) for 72 h and compound effects on AlkP activity were assessed spectrophotometrically using the Safire II microplate reader as already described (Alexi et al., 2009; Fokialakis et al., 2004). Regulation of luciferase gene expression through ERa in MCF-7:D5L and through ERb in HEK:ERb cells was assessed as already described (Alexi et al., 2009). Briefly, the cells were plated in 96well plates at a density of 12,000 cells per well in MEM (MCF7:D5L cells) or DMEM (HEK:ERb cells) devoid of phenol-red and supplemented with 1 mg/mL insulin and 5% DCC-FBS. Three days after plating the cells were exposed for 18 h to test compounds (1 mM) or extracts (2 mg/ml) in the absence or presence of 0.1 nM estradiol. Luciferase expression was assessed using the Steady-Glo Luciferase Assay System (Promega) and the Safire II microplate reader. Cells exposed to vehicle, 0.1 nM estradiol or estradiol in the presence of 0.1 mM ICI182,780 served as controls. Agonist effects on AlkP and luciferase expression were expressed as % of the effect of estradiol while antagonist effects were expressed as % of the effect of ICI182,780 in the presence of estradiol. Absolute values were used to express test compound effects on luciferase activity as % of the effect of estradiol or ICI182,780. Agonist and antagonist effects on AlkP and luciferase expression were the result of at least three independent experiments carried out in triplicate. In order to assess the potency of selected test compounds to induce AlkP and luciferase expression, estrogen-free cells were exposed to increasing concentrations (0.01e10 mM) of the selected test compounds in the absence or presence of 1 mM ICI182,780. The presence of ICI182,780 served to attest that test compound effects on AlkP and luciferase expression were ER-mediated at the concentrations tested. Cells exposed to 1 nM estradiol served to determine test compound concentrations (EC25) capable of producing 25% of the AlkP or luciferase-inductive effect of 1 nM estradiol. 4.5.4. Assessment of differentiation of MC3T3-E1 cells to osteoblasts MC3T3-E1 cells were plated in 96-well plates at a density of 3300 cells per well in a-MEM supplemented with 10% FBS. After 24 h, cells were exposed for 6 days to differentiation medium (low glucose DMEM enriched with 3% FBS, 10 mM b-glucerophosphate and 50 mg/ml ascorbic acid) supplemented with test compounds (1 mM), extracts (1 mg/ml) or vehicle only (0.2% DMSO). The medium was replaced after 3 days. AlkP activity was assessed as already described for the AlkP activity of Ishikawa cells (Alexi et al., 2009; Fokialakis et al., 2004). Cells exposed to vehicle, estradiol (1 nM), raloxifene (0.1 mM), ICI182,780 (0.1 mM) or estradiol in the presence of raloxifene or ICI182,780 served as controls. Effects on AlkP expression of MC3T3-E1 cells were the result of at least three independent experiments carried out in triplicate and were expressed as % of the effect of differentiation medium. 4.5.5. Statistical analysis Statistically significant differences were determined using oneway ANOVA and SPSS 13.0 (SPSS, Chicago, IL). Differences were considered significant for values of p  0.05. Acknowledgment The present work was funded by the European Regional

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Development Fund and National Resources - SYNERGASIA 2009 (GAN: OSTEOPRO-1076). Quoc Dang Thai and Job Tchoumtchoua acknowledge the Hellenic State Scholarships Foundation (IKY) for their PhD Scholarships. The authors are grateful to Dr. Eleftherios Kalpoutzakis and Pr. Elizabeth Seguin for the collection of plant material. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2016.04.006. References Abdelmagid, S.M., Barbe, M.F., Safadi, F.F., 2015. Role of inflammation in the aging bones. Life Sci. 123, 25e34. Abdullaev, U.A., Rashkes, Y.V., Khidyrova, N.K., Rashkes, A.M., 1994. Mass-spectrometric Analysis of Phytol Derivatives from Leaves of Platanus Orientalis. Alexi, X., Kasiotis, K.M., Fokialakis, N., Lambrinidis, G., Meligova, A.K., Mikros, E., Haroutounian, S.A., Alexis, M.N., 2009. Differential estrogen receptor subtype modulators: assessment of estrogen receptor subtype-binding selectivity and transcription-regulating properties of new cycloalkyl pyrazoles. J. Steroid Biochem. Mol. Biol. 117, 159e167. Andreopoulou, P., Bockman, R.S., 2015. Management of postmenopausal osteoporosis. Annu. Rev. Med. 66, 329e342. Barron, D., El Aidi, C., Mariotte, A.M., 1994. 13C nuclear magnetic resonance analysis of two prenyl flavonols from Platanus acerifolia buds. Phytochem. Anal. 5, 309e314. € m, O., Brzozowski, A.M., Pike, A.C.W., Dauter, Z., Hubbard, R.E., Bonn, T., Engstro € Ohman, L., Greene, G.L., Gustafsson, J.Å., Carlquist, M., 1997. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389, 753e758. Chiang, S.S., Pan, T.M., 2013. Beneficial effects of phytoestrogens and their metabolites produced by intestinal microflora on bone health. Appl. Microbiol. Biotechnol. 97, 1489e1500. Dimas, K., Demetzos, C., Mitaku, S., Marselos, M., Tzavaras, T., Kokkinopoulos, D., 2000. Cytotoxic activity of kaempferol glycosides against human leukaemic cell lines in vitro. Pharmacol. Res. 41, 85e88. El-Alfy, T.S., El-Gohary, H.M.A., Sokkar, N.M., Sleem, A.A., Al-Mahdy, D.A., 2008. Phenolic constituents of Platanus orientalis L. leaves. Nat. Product. Commun. 3, 199e203. Fokialakis, N., Lambrinidis, G., Mitsiou, D.J., Aligiannis, N., Mitakou, S., Skaltsounis, A.L., Pratsinis, H., Mikros, E., Alexis, M.N., 2004. A new class of phytoestrogens: evaluation of the estrogenic activity of deoxybenzoins. Chem. Biol. 11, 397e406. Gritzapis, A.D., Baxevanis, C.N., Missitzis, I., Katsanou, E.S., Alexis, M.N., Yotis, J., Papamichail, M., 2003. Quantitative fluorescence cytometric measurement of estrogen and progesterone receptors: correlation with the hormone binding assay. Breast Cancer Res. Treat. 80, 1e13. Guo, A.J., Choi, R.C., Zheng, K.Y., Chen, V.P., Dong, T.T., Wang, Z.T., Vollmer, G., Lau, D.T., Tsim, K.W.K., 2012. Kaempferol as a flavonoid induces osteoblastic differentiation via estrogen receptor signaling. Chin. Med. 7. Haider, S., Nazreen, S., Alam, M.M., Hamid, H., Alam, M.S., 2012. Anti-inflammatory and anti-nociceptive activities of Platanus orientalis Linn. and its ulcerogenic risk evaluation. J. Ethnopharmacol. 143, 236e240. Hajhashemi, V., Ghannadi, A., Mousavi, S., 2011. Antinociceptive study of extracts of Platanus orientalis leaves in mice. Res. Pharm. Sci. 6. €ma €l€ Ha ainen, M., Nieminen, R., Asmawi, M.Z., Vuorela, P., Vapaatalo, H., Moilanen, E., 2011. Effects of flavonoids on prostaglandin E2 production and on COX-2 and mPGES-1 expressions in activated macrophages. Planta Medica 77, 1504e1511. Haq, F., Ahmad, H., Alam, M., 2011. Traditional uses of medicinal plants of Nandiar Khuwarr catchment (District Battagram), Pakistan. J. Med. Plants Res. 5, 39e48. Jain, N., Xu, J., Kanojia, R.M., Du, F., Guo, J.Z., Pacia, E., Lai, M.T., Musto, A., Allan, G., Reuman, M., Li, X., Hahn, D., Cousineau, M., Peng, S., Ritchie, D., Russell, R., Lundeen, S., Sui, Z., 2009. Identification and structure - activity relationships of chromene-derived selective estrogen receptor modulators for treatment of postmenopausal symptoms. J. Med. Chem. 52, 7544e7569. Kaouadji, M., 1986. Grenoblone, nouvelle oxodihydrochalcone des bourgeons de Platanus acerifolia. J. Nat. Prod. 49, 500e503. Kaouadji, M., 1989. Two C-methyl-C-prenyldihydrochalcones from Platanus acerifolia. Phytochemistry 28, 3191e3192. Kaouadji, M., 1990. Acylated and non-acylated kaempferol monoglycosides from Platanus acerifolia buds. Phytochemistry 29, 2295e2297. Kaouadji, M., 2014. Further prenylated flavonols from Platanus acerifolia's unripe buds. Tetrahedron Lett. 55, 1285e1288. Kaouadji, M., Morand, J.M., Gilly, C., 1986a. 4-Hydroxygrenoblone, another uncommon C-prenylated flavonoid from Platanus acerifolia buds. J. Nat. Prod. 49, 508e510. Kaouadji, M., Ravanel, P., Mariotte, A.M., 1986b. New prenylated flavanones from Platanus acerifolia buds. J. Nat. Prod. 49, 153e155. Kaouadji, M., Ravanel, P., Tissut, M., Creuzet, S., 1988. Novel methylated flavonols

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Please cite this article in press as: Thai, Q.D., et al., Phytochemical study and biological evaluation of chemical constituents of Platanus orientalis and Platanus
acerifolia buds, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.04.006