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Diverse flavonoids from the roots of Millettia brandisiana
Phytochemistry 162 (2019) 157–164
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Diverse flavonoids from the roots of Millettia brandisiana a
a,b
Phanruethai Pailee , Chulabhorn Mahidol Vilailak Prachyawarakorna,∗ a b c
, Somsak Ruchirawat
a,b,c
,
T
Laboratory of Natural Products, Chulabhorn Research Institute, Kamphaeng Phet 6 Road, Laksi, Bangkok 10210, Thailand Chemical Biology Program, Chulabhorn Graduate Institute, Kamphaeng Phet 6 Road, Laksi, Bangkok 10210, Thailand Center of Excellence on Environmental Health and Toxicology (EHT), CHE, Ministry of Education, Bangkok 10400, Thailand
ARTICLE INFO
ABSTRACT
Keywords: Millettia brandisiana Fabaceae Flavonoids Cytotoxicity Antiaromatase
The phytochemical investigation for the constituents of the roots of Millettia brandisiana, using bioassay guided fractionation, resulted in the isolation of five previously undescribed (namely brandisianones A-E) and twentysix known flavonoids. Their chemical structures were determined using a combination of NMR, MS, IR, optical rotation and CD analysis, as well as comparison with the literature data. The crude extract as well as the isolated compounds were evaluated in various biological assays for their cytotoxicity against a panel of human cancer cell lines, potential inhibitory activity against aromatase, and antioxidant property using the oxygen radical absorbance capacity (ORAC) with an aim to search for leads and develop them to drug candidates in our drug discovery effort, we identified three bioactive flavonoids from M. brandisiana which could be further developed into a potential chemopreventive (antiaromatase) agent against breast cancer.
1. Introduction The genus Millettia (Fabaceae), comprising more than 260 species, is distributed widely in the Africa, Asia, America, and Australia (Banzouzi et al., 2008; Havyarimana et al., 2012). Millettia brandisiana Kurz, also known in Thai as “Kra-Pee-Jun”, is a medium tree growing up to 8–20 m in height while distributing mainly in the northern, central, and northeastern regions of Thailand. The stems of M. brandisiana have been reported for their use in traditional medicine as haematonic (Mahidol University, 2010). Isoflavones and rotenones were isolated from this plant, among which the isoflavones were previously reported to exhibit biological activities to overcome TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) resistance and to exhibit anti-inflammatory activity (Pancharoen et al., 2008; Ishibashi and Ohtsuki, 2008; Kikuchi et al., 2007, 2009). As part of our ongoing program to search for the bioactive compounds, various biological assays on the isolated compounds from the dichloromethane extract of the roots of M. brandisiana were performed. The isolated compounds were evaluated for their cytotoxic activity against five human cancer cell lines, chemoprevention, and inhibition of aromatase, all of which may be useful for the prevention of breast cancer. Thus, we decided to investigate the chemical constituents and isolate the bioactive compounds from this plant. The structure elucidation of five undescribed compounds, their cytotoxicity against a panel of mammalian cancer cell lines, and antioxidant as well
∗
as antiaromatase activities of the isolated compounds were also described. 2. Results and discussion Purification of a dichloromethane extract of the roots of M. brandisiana by (repeated) chromatography provided thirty-one flavonoids (Fig. 1), among which brandisianones A-E (1, 2, 12, 14, and 21) have not been previously reported. The known compounds were first isolated in this plant and identified by comparison of their physical and spectroscopic data with literature values as lanceolatin B (3) (Tanaka et al., 1992; Mbafor et al., 1995; Lee and Morehead, 1995), pongaglabol (4) (Ahmad et al., 1999; Talapatra et al., 1980), pinnatin (5) (Das et al., 1994), 6″,6″-dimethylchromeno-[2″,3′′:7,8]-flavone (6) (Lee and Kim, 2006; Xia and Lee, 2013; Magalhães et al., 1996), candidine (7) (Yadav et al., 2014; Jain and Sharma, 1974), 5-methoxy-6″,6″-dimethylchromeno-[2″,3′′:7,8]-flavone (8) (Andrei et al., 2000), 5-hydroxy-6″,6″dimethylchromeno-[2″,3′′:7,6]-flavone (9) (Jain and Sharma, 1974), 5methoxy-6″,6″-dimethylchromeno-[2″,3′′:7,6]-flavone (10) (Camele et al., 1980), 7-hydroxy-8,4′-dimethoxyisoflavone (11) (http://www. ncbi.nlm.nih.gov/pubmed?term=Puebla%20P%5BAuthor%5D& cauthor=true&cauthor_uid=21076386 Puebla et al., 2010), 7-O-8-bis(3,3-dimethylallyl)-5-hydroxyflavanone (13) (Bohlmann and Misra, 1984), (−)-isolonchocarpin (15) (Rao and Raju, 1979), obovatin (16)
Corresponding author. E-mail address: [email protected] (V. Prachyawarakorn).
https://doi.org/10.1016/j.phytochem.2019.03.013 Received 10 August 2018; Received in revised form 18 March 2019; Accepted 18 March 2019 0031-9422/ © 2019 Published by Elsevier Ltd.
Phytochemistry 162 (2019) 157–164
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Fig. 1. Structures of compounds 1–31.
158
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Table 1 1 H NMR data [δ in ppm, mult, (J in Hz)] of 1, 2, 12, and 14 in CDCl3 (600 MHz). 12a
Position
1
2
2 3
6.79, s
7.12, s
6.90, s
7.92, d (8.7) 7.71, dd (8.7, 0.7)
7.95, 7.57, 7.57, 6.85,
8.23, 7.61, 7.61, 7.34,
5 6 8 2′, 6′ 3′,5′ 4′ 1″ 2″ 4″ 5″ 1‴ 2‴ 4‴ 5‴ 2-OMe 5-OH/OMe 6″eOH a
d (6.8) m m s
1.72, s 1.72, s
12.69, s
5.45, 2.86, 3.00, 7.81, 6.63,
dd (7.8, 1.3) m m d (0.7)
14 dd (12.4, 3.1) dd (16.8, 3.1) dd (16.8, 12.9) d (8.8) d (8.8)
7.48, m 7.42, m 7.36, m 4.60, d (6.6) 5.47, m 1.79, s 1.74, s 3.37, d (7.0) 5.20, m 1.654, s 1.647, s
1.59, s 1.59, s
5.59, s
2.90, d (16.4) 2.97, d (16.4) 6.38, 7.59, 7.45, 7.40, 6.64, 5.60, 1.45, 1.49,
s d (7.3) t (7.2) t (7.2) d (10.0) d (10.0) s s
3.05, s 3.87, s
Recorded at 400 MHz.
(Andrei et al., 2000; Peralta et al., 2011; Arriaga et al., 2009), ovalitenin A (17) (Gupta and Krishnamurti, 1977), lonchocarpine (18) (Lee and Kim, 2006; Lima et al., 2013), 2′-methoxyfurano-[2″,3′′:4′,3′]-dihydrochalcone (19), ovalitenin B (20) (Gupta and Krishnamurti, 1977; Tanaka et al., 1992), pongamol (22) (Lee and Kim, 2006), 2′,6′-dimethoxyfurano-[2″,3′′:4′,3′]-β-hydroxydihydrochalcone (23) (Hishmat et al., 1989), 2′-hydroxy-6″,6″-dimethylchromeno-[2″,3′′:4′,3′]-β-hydroxychalcone (24) [Xia and Lee (2013), 2′-methoxy-6″,6″-dimethylchromeno-[2″,3′′:4′,3′]-β-hydroxychalcone (25) (Magalhães et al., 1996), praecansone B (26) (Tarus et al., 2002), (−)-12α-hydroxyrotenone (27) (Magalhães et al., 1996; Van Puyvelde et al., 1987), (−)-villosinol (28) (Krupadanam et al., 1977), (−)-tephrosin (29) (Ahmad et al., 1999; Belofsky et al., 2014; Lou et al., 2016), (−)-medicarpin (30) (Goel et al., 2012), and (−)-maackiain (31) (Suginome, 1962; Chaudhuri et al., 1995). This represents the first isolation of compound 24 as a natural product (Xia and Lee, 2013). A molecular formula of C20H16O5 was established for compound 1 on the basis of its APCI-TOF MS at m/z 337.1080 [M+H]+ (calcd for C20H17O5, 337.1071). The IR spectrum of 1 indicated the presence of hydroxyl (3400 cm−1), conjugated carbonyl (1662 cm−1) and aromatic (1613, 1587, and 1454 cm−1) groups. The 1H NMR spectrum (Table 1) showed a singlet at δH 12.69 for a hydroxy group (5-OH), which was hydrogen bonded to the carbonyl group (C-4), as well as a doublet at δH 7.95 (2H, J = 6.8 Hz) and a multiplet at δH 7.57 (3H) for the monosubstituted phenyl ring B. A singlet proton at δH 6.79 (H-3) and a conjugated carbonyl carbon at δH 183.2 (C-4) were identified as part of the flavone nucleus (Ayers et al., 2008). The appearance of a hydroxyisopropylfuran group was observed from a singlet of two methyl groups at δH 1.72 (6H) on a carbinol carbon (C-3″) and the other singlet of an olefinic proton at δH 6.85 (1H) on the 1H NMR spectrum. This was in accordance with the correlations from H-1″ to C-2′′ (δC 162.8) as well as CH3-4′′/CH3-5″ to C-3′′ (δC 69.2) and C-2′′ (δC 162.8) in HMBC spectrum. Its location at C-7 and C-8 of the phenyl ring A was also confirmed by HMBC correlation of H-1″ and H-6 to C-7 (δC 149.8) and C-8 (δC 109.3). The remaining singlet at δH 6.90 was assigned as that at the C-6 position based on the correlations from H-6 to C-5 (δC 158.4), C10 (δC 107.5), C-8 (δC 109.3), and C-7 (δC 149.8). On the basis of these spectroscopic data, compound 1 was identified as 5-hydroxy-5′′-(2-hydroxypropan-2-yl)-furano-[2″,3′′:7,8]-flavone, namely brandisianone A. The 1H and 13C NMR spectral data of compound 2 were similar to those of 1, except for the absence of a chelated hydroxyl group at the C-
5 position. Moreover, the appearance of a doublet at δH 7.92 (J = 8.7 Hz) and a doublet of doublet at δH 7.71 (J = 8.7, 0.7 Hz), which exhibited a long range coupling with H-4′′ (δH 7.34, d, J = 0.7 Hz) in the 1H NMR spectrum (Table 1), supported the presence of an ortho-coupled aromatic protons on the ring A in compound 2. The molecular formula of C20H16O4 from the APCI-TOF MS also indicated that compound 2 has one fewer oxygen than 1. Therefore, compound 2 was elucidated as 5′′-(2-hydroxypropan-2-yl)-furano-[2″,3′′:7,8]-flavone, namely brandisianone B. Compound 12 was isolated as a yellow amorphous solid and its molecular formula was established as C25H28O3 by APCI-TOF MS at m/z 377.2110 [M+H]+. The IR spectrum of compound 12 indicated the presence of carbonyl (1683 cm−1) and aromatic (1595 and 1436 cm−1) groups. The 1H NMR spectrum (Table 1) showed the signals of a threeproton spin system at δH 2.86 (dd, J = 16.8, 3.1 Hz), 3.00 (dd, J = 16.8, 12.9 Hz), and 5.45 (dd, J = 12.4, 3.1 Hz) which were characteristic of a C-3 unsubstituted flavanone nucleus (Peralta et al., 2011; Fu et al., 1993). The presence of multiplet signals in 1H NMR data at δH 7.48 (2H, m), 7.42 (2H, m), and 7.36 (1H, m) suggested the appearance of a monosubstituted phenylic ring B. Two doublet signals at δH 6.63 (H-6) and 7.81 (H-5) with the coupling constant of 8.8 Hz in the 1H NMR spectrum supported the presence of an ortho-coupled aromatic protons on the ring A. The remaining signals in the NMR spectra were assigned to two prenyl moieties, the first of which showed signals of a C-prenyl group at δH 3.37 (d, J = 7.0 Hz), 5.20 (m), 1.654 (s), and 1.647 (s), ascribable to an allylic methylene, an olefinic proton, and two methyl groups, respectively. The second prenyl group showed signals for an allylic methylene group at δH 4.60 (d, J = 6.6 Hz), an oleficnic proton at δH 5.47 (m), and two methyl groups at δH 1.79 (s) and 1.74 (s); the low field of this methylene implied that this prenyl group was bonded to an oxygen atom (an O-prenyl group). The correlations observed in the HMBC spectrum from H-5 to C-4 (δC 191.3), C-7 (δC 162.7), and C-9 (δC 160.2), H-6 to C-8 (δC 118.0) and C-10 (δC 115.2), H-1″ to C-7; and H-1‴ to C-7 and C-9 supported the positions of H-5, H-6, O-prenyl group at C-7, and C-prenyl group at C-8. The absolute configuration at C-2 in compound 12 is S-, as determined by the analysis of the CD spectrum where a positive Cotton effect was observed at 334 nm along with a negative Cotton effect at 248–297 nm (Peralta et al., 2011). From these data, the structure of compound 12 was established as (2S)-8-γ,γ-dimethylallyl-7-O-γ,γ-dimethylflavanone, namely brandisianone C. Compound 14 had a molecular formula C22H22O5 as deduced from its APCI-TOF MS. The IR spectrum of 14 displayed characteristic 159
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Table 2 13 C NMR data [δ in ppm] of 1, 2, 12, and 14 in CDCl3 (150 MHz).
Table 3 1 H and 13C NMR data of 19 and 21 in CDCl3 (400 MHz).
Position
1
2
12a
14
2 3 4
163.6 106.6 183.2
161.8 107.1 176.8
79.4 44.4 191.3
104.4 51.2 187.8
5 6 7 8 9 10 1′ 2′, 6′ 3′,5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 1‴ 2‴ 3‴ 4‴ 5‴ 2-OMe 5-OMe
158.4 95.5 149.8 109.3 159.2 107.5 131.1 126.3 129.2 132.0 97.3 162.8 69.2 28.8 28.8
120.2 110.0 149.8 117.8 157.1 118.7 131.0 126.3 129.1 131.7 97.9 166.2 67.6 28.9 28.9
126.1 106.1 162.7 118.0 160.2 115.2 139.4 125.9 128.6 128.3 65.5 119.4 138.0 25.6 18.2
156.7 110.3 160.1 101.4 159.9 109.4 138.9 125.9 128.6 128.8 116.1 128.6 77.8 28.3 28.5
a
22.3 122.0 131.5 25.7 17.7
Position
19 δC
1 2,6 3,5 4 1′ 2′ 3′ 4′ 5′ 6′ CO α
141.7 128.37a 128.41a 125.8 124.9 153.9 118.3 159.0 106.5 126.7 201.2 45.2
β 4″ 5″ 6″ 7″ 8″ 2′-OMe β-OMe
30.6 105.6 144.6
a b
50.8 62.4
60.5
21 δH, mult (J in Hz) 7.27, m 7.27, m 7.20, m
7.21, d (8.7) 7.67, d (8.7) 3.35, t (7.4) 3.05, t (8.1) 6.98, d (2.0) 7.60, d (2.3)
4.11, s
δC 141.6 126.7 128.4 127.6 125.3 156.4 114.7 157.7 112.6 131.0 198.2 50.6 79.9 116.5 130.4 76.8 27.96 28.0 63.1 56.7
δH, mult (J in Hz) 7.36, m 7.36, m 7.28, m
6.60b, d (9.2) 7.50, d (8.5) 3.55, dd (16.3, 8.8) 3.11, dd (16.3, 4.4) 4.84, dd (8.8, 4.4) 6.60b, d (9.2) 5.67, d (10.1) 1.44, 1.44, 3.72, 3.20,
s s s s
Interchangeable signals. Overlapped signals.
and 1He1H COSY experiments revealed the correlation between two triplets of the ethylene unit at δH 3.35 (J = 7.4 Hz) and 3.05 (J = 8.1 Hz). From the data above, compound 19 was deduced as a dihydrochalcone derivative (Gupta and Krishnamurti, 1977; Tanaka et al., 1992). Additionally, the 1H NMR data displayed a singlet of an aromatic methoxy group at δH 4.11 (2′-OMe) and two doublets of furan group at δH 6.98 (J = 2.0 Hz) and 7.60 (J = 2.3 Hz) and their locations were assigned at C-2′ and C-3′/4′, respectively, by the correlations from 2′-OMe to C-2′ (δC 153.9) and from H-6′ (δH 7.67) to C-2′ and C-4′ (δC 159.0). (Gupta and Krishnamurti, 1977). Therefore, compound 19 was identified as 2′-methyoxyfurano-[2″,3″:4′,3′]-dihydrochalcone and has been reported as commercially available albeit with only partially characterized spectroscopic data. Its fully characterized spectroscopic data are first described here. Compound 21 was obtained as a white amorphous powder. Its molecular formula of C22H24O4 was determined by APCI-TOF MS at m/z 353.1749 [M+H]+. 1H and 13C NMR data shown in Table 3 of compound 21 were similar to those of compound 14. A major significant difference was the absence of a singlet aromatic proton and a ketal carbon along with the presence of an ortho-coupled aromatic protons at δH 6.60 (d, J = 9.2 Hz) and 7.50 (d, J = 8.5 Hz) and a three proton coupling system at δH 3.11 (dd, J = 16.3 and 4.4 Hz), 3.55 (dd, J = 16.3 and 8.8 Hz) and 4.84 (dd, J = 8.8, 4.4 Hz) which could be assignable to a structural fragment eCOCH2CHOe. These results, together with the mass spectrum, supported the ring C opening from flavanone 14 to dihydrochalcone skeleton 21. The CD Cotton effects in the region 240–290 and 320–350 nm provide evidence for determining the absolute configurations of β-hydroxyldihydrochalcone (Nel et al., 1999; Muiva et al., 2009). However, the absolute stereochemistry of methoxy group at C-β of compound 21 could not be determined based on the positive Cotton effect at 263 nm and negative Cotton effect at 251 and 274 nm; these results are not in good agreement with those published for a series of β-hydroxydihydrochalcones (Nel et al., 1999; Muiva et al., 2009). However, the stereochemistry at C-β could be established as S- by comparing the optical rotation with that of the elatadihydrochalcone, a β-hydroxydihydrochalcone (Muiva et al., 2009). The elatadihydrochalcone, β-hydroxydihydrochalcones, with S- configuration at β-carbon was reported to exhibit a positive optical rotation ([α]D +34.5°). Compound 21 also showed a positive optical rotation
Recorded at 100 MHz.
absorptions of carbonyl group at 1687 cm−1 and aromatic group at 1601, 1567, and 1468 cm−1. The 1H NMR spectrum (Table 1) exhibited a doublet at δH 7.59 (J = 7.3 Hz) and two triplets at δ H 7.45 and 7.40 (J = 7.2 Hz) as well as two doublets at δH 2.90 and 2.97 (J = 16.4 Hz) representing a monosubstituted aromatic and a nonequivalent methylene group, respectively. From these characteristic data and the lack of a methine proton signal when compared with those of compound 12, compound 14 was assigned as a C-2 substituted flavone. The substituent at the C-2 in ring C was identified as a methoxy group by the existence of the ketal carbon at δC 104.4 (C-2) in 13C NMR spectrum (Table 2) and an additional methoxy proton at δH 3.05 (2-OMe). This was further confirmed by the HMBC correlations from 2-OMe, H-3 and H-2′/6′ to C2 (δC 104.4). In addition, the 1H NMR data displayed the signals of a pentasubstituted aromatic proton at δH 6.38 (H-8), aromatic methoxy proton at δH 3.87 (5-OMe) and 2,2-dimethyl-2H-pyran at δH 6.64 (1H, d, J = 10.0 Hz), 5.60 (1H, d, J = 10.0 Hz), 1.49 (3H, s), and 1.45 (3H, s). The location of all substituents on the ring A was elucidated by the HMBC analysis. The correlations from H-8 to C-4 (δC 187.8) and C-10 (δC 109.4) as well as from 5-OMe and H-1″ to C-5 (δC 156.7) in the HMBC data identified the position of H-8 and the methoxy group at C-5, respectively. In addition, the location of the pyran on the ring B at C-6/ 7 was also indicated by the correlations from H-1″ to C-5 (δC 156.7), C-6 (δC 110.3), and C-7 (δC 160.1). Compound 14 decomposed before an optical rotation and CD experiments could be acquired, and so its absolute configuration was not determined. The structure of compound 14 was therefore identified as 2,5-dimethoxy-6″,6″-dimethylchromeno[2″,3″:7,6]-flavanone, namely brandisianone D. Compound 19 was isolated as a yellow powder and had a molecular formula of C18H16O3 as determined by the APCI-TOF MS. The IR absorption at 1666 cm−1 together with the low field carbon at δC 201.2 suggested the presence of a carbonyl group. The 1H NMR spectra (Table 3) showed two multiplets in the aromatic region at δH 7.27 and 7.20 as well as two doublets at δH 7.21 and 7.67 with a coupling constant of 8.7 Hz corresponding to the monosubstituted aromatic ring and an ortho coupled aromatic system, respectively. Moreover, the 1H NMR 160
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Table 4 Cytotoxicity, antioxidant activity and aromatase inhibition data of pure compounds from the CH2Cl2 extract of M. brandisiana (IC50, μM). Compound
Cell lines (IC50, μM); values are expressed as mean ± S.D. (n = 3) MOLT-3
3 4 5 6 7 8 9 10 12 13 14 15 16 17 18 19 20 21 23 24 25 26 27 28 29 30 31 Doxorubicind Etoposided Ketoconazoled
34.70 ± 3.00 I 22.70 ± 3.10 26.71 ± 1.45 I 20.18 ± 3.62 I 25.51 ± 6.32 I I NDc 51.76 ± 3.27 69.69 ± 28.97 6.62 ± 0.43 20.23 ± 3.07 39.75 ± 7.18 32.55 ± 2.71 22.01 ± 1.96 30.74 ± 2.44 30.62 ± 1.58 6.46 ± 2.89 ND 0.27 ± 0.07 0.61 ± 0.05 19.15 ± 2.59 59.07 ± 5.37 59.65 ± 5.92 ND 0.04 ± 0.01 ND
HepG-2 b
I I 66.20 ± 7.90 58.13 ± 1.91 37.50 ± 0.00 45.90 ± 1.74 37.50 ± 9.38 94.82 ± 22.87 93.09 ± 24.28 I ND 92.58 ± 10.00 100.40 ± 7.17 9.50 ± 1.80 78.89 ± 3.04 101.18 ± 10.32 51.61 ± 3.23 86.56 ± 3.81 103.70 ± 14.91 86.18 ± 6.89 I ND 29.27 ± 8.44 4.53 ± 2.18 4.59 ± 0.61 125.00 ± 27.78 I 0.43 ± 0.01 24.45 ± 2.28 ND
A549
HuCCA-1
HeLa
I I I 59.70 ± 5.33 71.72 ± 1.54 53.35 ± 2.78 I I I I ND 119.28 ± 6.93 113.35 ± 15.37 16.40 ± 3.88 89.80 ± 26.25 I 130.32 ± 2.74 I 132.72 ± 30.56 127.33 ± 21.96 I ND 8.29 ± 2.59 18.45 ± 13.03 48.66 ± 0.51 I I 0.26 ± 0.08 ND ND
I I I 57.57 ± 6.97 I 53.89 ± 4.22 I I I I ND 94.77 ± 4.61 122.67 ± 24.16 1.58 ± 0.30 6.39 ± 15.83 I 42.42 ± 2.96 I 135.80 ± 7.10 105.59 ± 4.38 71.13 ± 3.79 ND 41.46 ± 2.76 19.37 ± 0.82 74.39 ± 1.15 I I 0.26 ± 0.07 ND ND
I I 21.23 ± 2.91 38.16 ± 6.51 I 27.90 ± 1.35 I 140.15 ± 1.23 50.32 ± 3.46 I ND 58.01 ± 5.78 123.32 ± 0.02 8.45 ± 0.13 42.94 ± 0.67 94.11 ± 1.75 I 125.43 ± 0.20 I 108.70 ± 0.00 81.37 ± 0.23 ND 6.71 ± 0.51 0.87 ± 0.08 50.12 ± 2.65 ND I 0.17 ± 0.10 ND ND
Aromatase inhibition (IC50, μM)
Antioxidant activity ORAC (unit)a
0.80 ± 0.10 I I 0.40 ± 0.10 I I 8.70 ± 0.80 I 11.0 ± 2.80 I I 0.20 ± 0.10 3.10 ± 0.40 13.10 ± 1.30 ND 5.60 ± 1.10 I 6.60 ± 0.10 I ND I I I 14.00 ± 0.20 ND I I ND ND 2.40
0.02 0.50 0.20 0.10 0.80 0.70 0.70 0.01 0.20 0.14 0.10 0.10 0.85 0.10 ND 0.10 0.10 ND 2.00 1.50 0.60 0.80 0.00 ND ND 4.60 3.50 ND ND ND
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.10 0.03 0.10 0.20 0.20 0.20 0.01 0.20 0.06 0.20 0.20 0.05 0.10
± 0.10 ± 0.05 ± ± ± ± ±
0.30 0.30 0.02 0.20 0.00
± 0.10 ± 0.50
a Results were expressed as ORAC units, where 1 ORAC unit equals the net protection of β-phycoerythrin produced by 1 μM of 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox). b I = inactive at 50 μg/mL. c ND = not determined. d Etoposide, doxorubicin, and ketoconazole are positive control.
([α]D +42.4°); hence the same configuration was presumed. Thus, the structure of compound 21 was established as 2′-methoxy-6″,6″-dimethylchromeno-[2″,3′′:4′,3′]-(S)-β-methoxydihydrochalcone, namely brandisianone E. The absolute configuration at C-β of ovalitenin B (20) has not been previously reported (Gupta and Krishnamurti, 1977; Tanaka et al., 1992). The CD spectrum of 20 displayed a positive Cotton effect at 262 nm and negative Cotton effect at 246 nm as well as the optical rotation showed the positive value of [α]D +54.3°(c 0.61, CHCl3), similar to that of brandisianone E (21) which also supported the identification of C-β absolute configuration as S-. Some isolated compounds (3–10, 12, 13, 15–21, 23–25, and 27–31) were evaluated for their cytotoxic activities against five human cancer cell lines (MOLT-3, HepG2, A549, HuCCA-1, and HeLa) and their IC50 values are summarized in Table 4. Similar cytotoxicity for other compounds could not be evaluated due to their limited availability. The rotenone analogues (27–29) and chalcones 17 and 18 were found to be the most cytotoxic among the tested flavonoids. (−)-12αHydroxyrotenone (27) showed significant activity against MOLT-3, A549, and HeLa cell lines with the IC50 values of 0.27, 8.29, and 6.71 μM, respectively. (−)-Villosinol (28) exhibited potent cytotoxic activity toward MOLT-3, HepG2, and HeLa cell lines (IC50 values of 0.61, 4.53, and 0.87 μM, respectively), while (−)-tephrosin (29) was selectively cytotoxic against HepG2 cell line with the IC50 value of 4.59 μM. Previously, these rotenones were also reported to exhibit cytotoxic activities (https://www.ncbi.nlm.nih.gov/pubmed/?term= Blatt%20CT%5BAuthor%5D&cauthor=true&cauthor_uid=12112286; Deyou et al., 2017; Lou et al., 2016; https://www.ncbi.nlm.nih.gov/
pubmed/?term=Blatt%20CT%5BAuthor%5D&cauthor=true&cauthor_ uid=12112286 Blatt et al., 2002; Pérez et al., 2013; Ye et al., 2012). This is the first report of these cytotoxic rotenoids evaluated against HuCCA-1, MOLT-3, A549, HepG2, and HeLa cell lines, except that (−)-tephrosin (29) was previously reported to be cytotoxic against HepG2 and A549 cell lines with the IC50 values of 1.41 and 13.27 μM, respectively (Lou et al., 2016; Ye et al., 2012). It should also be noted that the chalcone 17 possessing the α,β-unsaturated ketone was more cytotoxic than the dihydrochalcones 19 and 20. Chalcone 17 showed significant cytotoxicity against MOLT-3, HepG2, A549, HuCCA-1, and HeLa cell lines with the IC50 values of 6.62, 9.50, 16.40, 1.58, and 8.45 μM, respectively. Chalcone 18 exhibited selective cytotoxicity against HuCCA-1 cell line with the IC50 values of 6.39 μM. The intramolecular hydrogen bonding of the hydroxyl group at C-2′ in chalcone 18, and C-β in chalcones 23–25 with the carbonyl group also decreased their cytotoxicity to be less than that of the chalcone 17. Based on these preliminary results, a hydroxy substitution of these chalcone analogues is detrimental for cytotoxicity as imposed by the Michael addition to the α,β-unsaturated ketone (Masi et al., 2017). The antioxidant and aromatase inhibitory activities were also evaluated (Table 4). The ORAC (oxygen radical absorbance capacity) assay was used to determine the antioxidative activity by the application of 2,2′-azobis (2-methylpropionamide) dihydrochloride (AAPH) as free radical initiator to generate the peroxyl radicals. Compounds 23, 24, 30, and 31 exhibited moderate to high antioxidant activity in the ORAC assay with 1.50–4.6 ORAC units. Compounds 3, 6, and 15 possessed relatively low IC50 values of 0.80, 0.40, and 0.20 μM, respectively, with respect to ketoconazole (IC50 = 2.4 μM) used as a positive 161
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control for the anti-aromatase assay; therefore, these three compounds potentially could be considered as candidates for the prevention of breast cancer by acting as an estrogen antagonist.
(85:15) as mobile phase to give compounds 24 (10.9 mg) and 18 (1.3 mg). Compounds 16 (376.5 mg) and 13 (40.7 mg) were obtained after purification of fraction A3 (2.03 g) by preparative TLC using CH2Cl2:hexane (4:6) as mobile phase. Fraction A4 (2.71 g) was chromatographed on RP-18 MPLC with solvents of increasing polarity of 60%–100% MeOH:H2O over 180 min to give 5 fractions (C1eC5). Fraction C2 was separated by HPLC using a C18 reversed phase column eluted with a 70:30 mixture of MeOH:H2O at a flow rate of 7 mL/min to give compounds 17 (9 mg), 19 (29 mg), and 22 (10 mg). Fraction C3 was purified by silica gel column, eluted with a gradient of hexane and acetone as mobile phase to provide compounds 15 (209.6 mg) and 21 (19.5 mg). Fraction C4 was subjected to preparative RP18-HPLC eluted with MeOH:H2O (85:15) as mobile phase at a flow rate of 12 mL/min to provide compounds 7 (231 mg), 9 (95 mg), and 12 (285 mg). Fraction A5 (1.57 g) was then subjected to silica gel column, and eluted with increasing proportions of acetone in hexane (0%–20%) to provide 8 fractions (D1–D8). Fractions D2 and D4 were compounds 25 (118.5 mg) and 20 (84.6 mg), respectively. Compounds 4 (4.0 mg) and 14 (1.9 mg) was obtained after purification of fraction D3 by preparative RP18HPLC with MeOH/H2O (80:20) as eluent at a flow rate of 10 mL/min. Fraction A9 (1.67 g) was chromatographed on silica gel column (gradient from 100% to 0% hexane/acetone) to give 8 fractions (H1eH8). Fraction H3 was subjected to preparative TLC using EtOAc and hexane (20:80) as mobile phase to give compound 26 (3.4 mg). Fraction H5 was purified by preparative RP18-HPLC with MeOH/H2O (75:25), flow rate 12 mL/min as eluent, to give compounds 3 (54 mg) and 6 (129.2 mg). Compounds 30 (6.7 mg) and 31 (9.5 mg) were obtained after purification of fraction H11 by preparative TLC [acetone:CH2Cl2 (1:99)] and preparative RP18-HPLC [MeOH:H2O (60:40), flow rate of 12 mL/min]. Compound 10 (923 mg) was obtained from fraction A10 (1.39 g) after recrystallization with CH2Cl2:MeOH. Fraction A11 (455 mg) was chromatographed on a silica gel column, eluted with a gradient (hexane and EtOAc) to provide 9 fractions (E1-E9). Fraction E3 was purified by preparative TLC using hexane and EtOAc (80:20) as mobile phase to provide compound 23 (19.6 mg). Fraction E6 was subjected to preparative RP18-HPLC with MeOH:H2O (73:27) as eluent at a flow rate of 10 mL/min which gave compounds 1 (3.4 mg), 27 (87.4 mg), 28 (6.2 mg), and 29 (14.4 mg). Combined fractions of A12A14 (756 mg) were subjected to gel permeation over Sephadex LH-20 (3 × 120 cm) with MeOH:CH2Cl2 (1:1) as eluent, to give 4 fractions (F1eF4). Fraction F3 was chromatographed on a silica gel column, eluted with a gradient of mixture CH2Cl2 and MeOH (100%–3%) to provide 8 fractions (G1-G8). Compounds 11 (3.0 mg) and 5 (41.4 mg) were obtained after purification of fraction G5 by preparative HPLC using MeOH and H2O (70:30) as mobile phase at flow rate of 12 mL/ min. Fraction G6 was subjected to preparative TLC using CH2Cl2 and MeOH (99:1) as mobile phase to give compound 2 (4 mg). Compound 8 (236.4 mg) was obtained after purification of fraction A15 (1.44 g) by gel permeation over Sephadex LH-20 CC (3 × 120 cm) [MeOH:CH2Cl2 (1:1)] followed by preparative TLC [MeOH:CH2Cl2 (2:98)]. Brandisianone A (1) White powder; UV (MeOH) λmax (log ε): 202 (4.34), 219 (4.34), 229 (4.33), 260 (4.24), 282 (4.36) nm; IR (ATR) νmax: 3400, 2926, 2853, 1662, 1613, 1587, 1454, 1423, 1348, 1284, 1239, 1148, 1119, 1096, 999, 839, 769, 688 cm−1; 1H and 13C NMR data (see Tables 1 and 2); APCITOFMS m/z 337.1080 [M+H]+ (calcd for C20H17O5, 337.1071). Brandisianone B (2) White powder; UV (MeOH) λmax (log ε): 219 (4.16), 266 (4.06), 298 (3.85) nm; IR (ATR) νmax: 3370, 2924, 1616, 1586, 1406, 1365, 1140, 1073, 882, 770, 682 cm−1; 1H and 13C NMR data (see Tables 1 and 2); APCITOFMS m/z 321.1128 [M+H]+ (calcd for C20H17O4, 321.1121). Brandisianone C (12) Pale yellow amorphous powder; CD (MeOH, c 54.5 μM): λnm (Δε) = 218 (+14.6), 248 (−2.11), 297 (−8.32), 334 (+5.14) nm; UV (MeOH) λmax (log ε): 202 (4.58), 217 (4.52), 287 (4.27) nm; IR (ATR) νmax: 2967, 2914, 1683, 1595, 1436, 1336, 1315,
3. Conclusion A phytochemical study on the dichloromethane extract of the roots of M. brandisiana led to the isolation of a total of 31 flavonoids with diverse skeletons including five previously undescribed flavonoids (1, 2, 3, 14, and 21, namely brandisianones A-E). Among them, compound 14 possessed a rare 2-methoxylated flavonoid skeleton. All twenty-six known flavonoids were first isolated from this plant. The rotenones 27 and 28 and chalcones 17 and 18 are among the most cytotoxic. An exploration of the structure-cytotoxicity relationships of the tested chalcone series revealed that the hydroxyl substitution at C-2′ or C-β position is detrimental for cytotoxicity. In addition, only two pterocapans 30 and 31 exhibited the highest antioxidant activity in the ORAC assay with 4.60 and 3.50 ORAC units while flavones 3 and 6 and flavanone 15 were found to be potent aromatase inhibitors with their IC50 values of 0.8, 0.4, and 0.2 μM, respectively. To our knowledge, this is the first report on the ORAC assay and the inhibitory activity against aromatase of all tested flavonoids, except that medicarpin (30) was previously reported to be inactive in the antiaromatase assay (Le Bail et al., 2000). The results suggested that these bioactive flavonoids could be of value for further studies as potential cytotoxic and cancer chemopreventive agents. 4. Experimental section 4.1. General experimental procedures UV–Vis spectra were obtained from Shimadzu UV-1700 PharmaSpec Spectrophotometer. Optical rotation was measured with sodium D line (590 nm) on JASCO 1020 digital polarimeter. FTIR spectra were recorded with a universal attenuated total reflectance (UATR) attachment on a Perkin–Elmer Spectrum One spectrometer. 1H, 13C and 2D NMR spectra were obtained using a Bruker AM400 spectrometer and a Bruker AVANCE 600 spectrometer with TMS and solvent as internal standard. APCI–TOF–MS were determined using a Bruker MicroTOFLC spectrometer. Column chromatography and preparative TLC were performed with silica gel 60 (70–200 mesh ASTM) and PF254, respectively. Sephadex LH-20 gel (Ambersham Biosciences) was also used for column chromatography. TLC was carried out on silica gel 60 F254 plates (Merck, 0.2 mm). High performance liquid chromatography (HPLC) was performed using Waters Delta 600 and Waters 2998 Photodiode Array Detector. 4.2. Plant material Roots of Millettia brandisiana Kurz (Fabaceae) were collected in April 2011 from the botanical garden at Chulabhorn Research Institute (13°52′N, 100°34′E) in Bangkok. A voucher specimen (CRI-661) has been deposited at the Laboratory of Natural Products, Chulabhorn Research Institute. 4.3. Extraction and isolation The air-dried roots of M. brandisiana (1.2 kg) were extracted with CH2Cl2 (2x5L) at room temperature. The CH2Cl2 layer was evaporated to give a crude extract (21 g), which was subjected to column chromatography on silica gel eluted successively with solvents of increasing polarity (hexane, EtOAc and MeOH) to provide 17 fractions (A1-A17). Fraction A2 (413.3 mg) was purified by preparative TLC using EtOAc:hexane (2:8) as mobile phase to give 3 bands (B1eB3). Band B1 was further purified by preparative RP18-HPLC using acetonitrile: H2O 162
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1267, 1218, 1177, 1112, 1071, 803, 765, 698 cm−1; 1H and 13C NMR data (see Tables 1 and 2); APCITOFMS m/z 377.2110 [M+H]+ (calcd for C25H29O3, 377.2111). Brandisianone D (14) Yellow powder; UV (MeOH) λmax (log ε): 260 (3.45), 326 (2.61) nm; IR (ATR) νmax: 2922, 2851, 1687, 1638, 1601, 1567, 1468, 1370, 1322, 1278, 1148, 1115, 1090, 1043, 987, 889, 767, 702 cm−1; 1H and 13C NMR data (see Tables 1 and 2); APCITOFMS m/z 367.1538 [M+H]+ (calcd for C22H23O5, 367.1540). Chalcone 19 Yellow powder; UV (MeOH) λmax (log ε): 207 (2.53), 235 (2.81), 305 (1.62) nm; IR (ATR) νmax: 2931, 1666, 1584, 1471, 1420, 1357, 1330, 1261, 1239, 1151, 1070, 993, 974, 807, 741, 699 cm−1; 1H and 13C NMR data (see Table 3); APCITOFMS m/z 281.1161 [M+H]+ (calcd for C18H17O3, 281.1172). Brandisianone E (21) Yellow oil; CD (MeOH, c 54.5 μM): λnm (Δε) = 208 (+3.78), 251 (−2.08), 263 (+1.81), 274 (−0.71), 323 (+0.37), 351 (+0.33) nm; [α]25 D +42.42 (c 1.93, CHCl3); UV (MeOH) λmax (log ε): 202 (4.17), 258 (4.26) nm; IR (ATR) νmax: 2977, 2934, 1674, 1591, 1455, 1371, 1314, 1276, 1215, 1190, 1161, 1114, 1075, 985, 890, 738, 702 cm−1; 1H and 13C NMR data (see Table 3); APCITOFMS m/z 353.1749 [M+H]+ (calcd for C22H25O4, 353.1747).
Pretorius, S., Stevenson, D., Thompson, D., Singh, S.B., 2008. Flavones from Struthiola argentea with anthelmintic activity in vitro. Phytochemistry 69, 541–545. Banzouzi, J.T., Prost, A., Rajemiarimirabo, M., Ongoka, P., 2008. Traditional uses of African Millettia species (Fabaceae). Int. J. Bot. 4, 406–420. Belofsky, G., Aronica, M., Foss, E., Diamond, J., Santana, F., Darley, J., Dowd, P.F., Coleman, C.M., Ferreira, D., 2014. Antimicrobial and antiinsectan phenolic metabolites of Dalea searlsiae. J. Nat. Prod. 77, 1140–1149. Blatt, C.T., Chávez, D., Chai, H., Graham, J.G., Cabieses, F., Farnsworth, N.R., Cordell, G.A., Pezzuto, J.M., Kinghorn, A.D., 2002. Cytotoxic flavonoids from the stem bark of Lonchocarpus aff. Fluvialis. Phytother. Res. 16, 320–325. Bohlmann, F., Misra, L.N., 1984. New prenylflavanones and chalcones from Helichrysum rugulosum. Planta Med. 50, 271–272. Camele, G., Delle Monache, F., Delle Monache, G., Bettolo, G.B.M., 1980. Three new flavonoids from Tephrosia praecans. Phytochemistry 19, 707–709. Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D., Mitchell, J.B., 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47, 936–942. Chaudhuri, S.K., Huang, L., Fullas, F., Brown, D.M., Wani, M.C., Wall, M.E., 1995. Isolation and structure identification of an active DNA strand-scission agent, (+)-3,4di-hydroxy-8,9-methylenedioxypterocarpan. J. Nat. Prod. 58, 1966–1969. Das, B., Chakravarty, A.K., Masuda, K., Suzuki, H., Ageta, H., 1994. A diterpenoid from roots of Gelonium multiflorum. Phytochemistry 37, 1363–1366. Deyou, T., Marco, M., Heydenreich, M., Pan, F., Gruhonjic, A., Fitzpatrick, P.A., Koch, A., Derese, S., Pelletier, J., Rissanen, K., Yenesew, A., Erdélyi, M., 2017. Isoflavones and rotenoids from the Leaves of Millettia oblata ssp. teitensis. J. Nat. Prod. 80, 2060–2066. Doyle, A., Griffiths, J.B. (Eds.), 1997. Mammalian Cell Culture: Essential Techniques. John Wiley and Sons, New York. Fu, X., Sévenet, T., Remy, F., Païs, M., Hadi, A.H.A., Zeng, L.M., 1993. Flavanone and chalcone derivatives from Cryptocarya kurzii. J. Nat. Prod. 56, 1153–1163. Gerhäuser, C., Klimo, K., Heiss, E., Neumann, I., Gamal-Eldeen, A., Knauft, J., Liu, G.Y., Sitthimonchai, S., Frank, N., 2003. Mechanism-based in vitro screening of potential cancer chemopreventive agents. Mutat. Res. 523–524, 163–172. Goel, A., Kumar, A., Hemberger, Y., Raghuvanshi, A., Jeet, R., Tiwari, G., Knauer, M., Kureel, J., Singh, A.K., Gautam, A., Trivedi, R., Singh, D., Bringmann, G., 2012. Synthesis, optical resolution, absolute configuration, and osteogenic activity of cispterocarpans. Org. Biomol. Chem. 10, 9583–9592. Gupta, R.K., Krishnamurti, M., 1977. New dibenzoylmethane and chalcone derivatives from Milletia ovalifolia seeds. Phytochemistry 16, 1104–1105. Havyarimana, L., Ndendoung, S.T., de-Dieu, Tamokou J., de Théodore Atchadé, A., Tanyi, J.M., 2012. Chemical constituents of Millettia barteri and their antimicrobial and antioxidant activities. Pharm. Biol. 50, 141–146. Hishmat, O.H., Miky, J.A.A., Saleh, N.M., 1989. Synthesis of some benzofuranylpyridine and pyridone derivatives of possible biological activities. Pharmazie 44, 823–825. Ishibashi, M., Ohtsuki, T., 2008. Studies on search for bioactive natural products targeting TRAIL signaling leading to tumor cell apoptosis. Med. Res. Rev. 28, 688–714. Jain, A.C., Sharma, B.N., 1974. Synthesis of isopentenylated chrysins. J. Org. Chem. 39, 1149–1152. Kikuchi, H., Ohtsuki, T., Koyano, T., Kowithayakorn, T., Sakai, T., Ishibashi, M., 2007. Brandisianins A-F, Isoflavonoids Isolated from Millettia brandisiana in a screening program for death-receptor expression enhancement activity. J. Nat. Prod. 70, 1910–1914. Kikuchi, H., Ohtsuki, T., Koyano, T., Kowithayakorn, T., Sakai, T., Ishibashi, M., 2009. Death receptor 5 targeting activity-guided isolation of isoflavones from Millettia brandisiana and Ardisia colorata and evaluation of ability to induce TRAIL-mediated apoptosis. Bioorg. Med. Chem. 17, 1181–1186. Krupadanam, G.L.D., Sarma, P.N., Srimannarayana, G., Subba Roa, N.V., 1977. New C-6 oxygenated rotenoids from Tephrosia villosa – villosin, villosone, villol, and villinol. Tetrahedron Lett. 24, 2125–2128. Le Bail, J.C., Champavier, Y., Chulia, A.-J., Habrioux, G., 2000. Effects of phytoestrogens on aromatase, 3β and 17β-hydroxysteroid dehydrogenase activities and human breast cancer cells. Life Sci. 66, 1281–1291. Lee, Y.R., Kim, D.H., 2006. A new route to the synthesis of pyranoflavone and pyranochalcone natural products and their derivatives. Synthesis 4, 603–608. Lee, Y.R., Morehead Jr., A.T., 1995. A new route for the synthesis of furanoflavone and furanochalcone natural products. Tetrahedron 51, 4909–4922. Lima, N.M., Andrade, J.I.A., Lima, K.C.S., dos Santos, F.N., Barison, A., Salome, K.S., Matsuura, T., Nunez, C.V., 2013. Chemical profile and biological activities of Deguelia duckeana A.M.G. Azevedo (Fabaceae). Nat. Prod. Res. 27, 425–432. Lou, H.Y., Wu, H.G., Tan, Y.H., Lan, J.J., Ma, X.P., Liang, G.Y., Yi, P., Pan, W.D., 2016. Two new flavonoids from Derris eriocarpa how. Helv. Chim. Acta 99, 302–305. Magalhães, A.F., Tozzi, A.M.G.A., Sales, B.H.L.N., Magalhães, E.G., 1996. Twenty-three flavonoids from Lonchocarpus subglaucescens. Phytochemistry 42, 1459–1471. Masi, M., Meyer, S., Clement, S., Cimmino, A., Cristofaro, M., Evidente, A., 2017. Cochliotoxin, a dihydropyranopyran-4,5-dione, and its analogues produced by Cochliobolus australiensis display phytotoxic activity against Buffelgrass (Cenchrus ciliaris). J. Nat. Prod. 80, 1241–1247. Mbafor, J.T., Atchade, A.T., Nkengfack, A.E., Fomum, Z.T., Sterner, O., 1995. Furanoflavones from root bark of Millettia sanagana. Phytochemistry 40, 949–952. Muiva, L.M., Yenesew, A., Derese, S., Heydenreich, M., Peter, M.G., Akala, H.M., Eyase, F., Waters, N.C., Mutai, C., Keriko, J.M., Walsh, D., 2009. Antiplasmodial β-hydroxydihydrochalcone from seedpods of Tephrosia elata. Phytochem. Lett. 2, 99–102. Nel, R.J.J., Rensburg, H., van Heerden, P.S., Coetzee, J., Ferreir, D., 1999. Stereoselective synthesis of flavonoids. Part 7. Polyoxygenated β-hydroxydihydrochalcone derivatives. Tetrahedron 55, 9727–9736. Pancharoen, O., Athipornchai, A., Panthong, A., Taylor, W.C., 2008. Isoflavones and
4.4. Cytotoxicity assays The cytotoxic activity toward a panel of mammalian cancer cell lines (HepG2, A549, HuCCA-1, HeLa) were tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Carmichael et al., 1987), while the activity toward MOLT-3 cancer cell line was performed by the 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)(2H)-tetrazolium-5-carboxanilide (XTT) assay (Doyle and Griffiths, 1997). Etoposide and doxorubicin were used as positive controls (Table 4). 4.5. Aromatase (CYP19) inhibition assay Inhibition of the aromatase enzyme assay was performed using the method designed by Stresser and co-workers (Stresser et al., 2000). The reference compound, ketoconazole, inhibits CYP19 with an IC50 value of 2.4 μM (Table 4). 4.6. ORAC antioxidant assay The test of the ORAC antioxidant assay was previously described by Gerhäuser and co-workers (Gerhäuser et al., 2003). Acknowledgments We thank Ms. Pakamas Intachote, Ms. Suchada Sengsai, and Ms. Busakorn Saimanee for cytotoxic assays and Dr. Poonsakdi Ploypradith for helpful editing in preparing the manuscript. We thank Mahidol University for partial financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.03.013. References Ahmad, V.U., Ali, Z., Hussaini, S.R., Iqbal, F., Zahid, M., Abbas, M., Saba, N., 1999. Flavonoids of Tephrosia purpurea. Fitoterapia 70 (4), 443–445. Andrei, C.C., Ferreira, D.T., Faccione, M., de Moraes, L.A.B., de Carvalho, M.G., BrazFilho, R., 2000. C-prenylflavonoids from roots of Tephrosia tunicate. Phytochemistry 55, 799–804. Arriaga, A.M.C., Lima, J.Q., Vasconcelos, J.N., de Oliveira, M.C.F., Andrade-Neto, M., Santiago, G.M.P., Uchoa, D.E.A., Malcher, G.T., Mafezoli, J., Braz-Filho, R., 2009. Unequivocal assignments of flavonoids from Tephrosia sp. (Fabaceae). Magn. Reson. Chem. 47, 537–540. Ayers, S., Zink, D.L., Mohn, K., Powell, J.S., Brown, C.M., Murphy, T., Brand, R.,
163
Phytochemistry 162 (2019) 157–164
P. Pailee, et al. rotenoids from the leaves of Millettia brandisiana. Chem. Pharm. Bull. 56, 835–838. Peralta, M.A., Ortega, M.G., Agnese, A.M., Cabrera, J.L., 2011. Prenylated flavanones with anti-tyrosinase activity from Dalea boliviana. J. Nat. Prod. 74, 158–162. Pérez, L.B., Li, J., Lantvit, D.D., Pan, L., Ninh, T.N., Chai, H.B., Soejarto, D.D., Swanson, S.M., Lucas, D.M., Kinghorn, A.D., 2013. Bioactive constituents of Indigofera spicata. J. Nat. Prod. 76, 1498–1504. Puebla, P., Oshima-Franco, Y., Franco, L.M., dos Santos, M.G., Silva, R.V., Rubem-Mauro, L., Feliciano, A.S., 2010. Chemical constituents of the bark of Dipteryx alata vogel, an active species against Bothrops jararacussu venom. Molecules 15, 8193–8204. Rao, E.V., Raju, N.R., 1979. Occurrence of (–)-isolonchocarpin in the roots of Tephrosia purpurea. Phytochemistry 18, 1581–1582. Mahidol University, 2010, 2010. Land of Thai medicinal plant. In: Siri Ruckhachati Nature Park. John Wiley and Sons, New York. http://www.pharmacy.mahidol.ac.th/ siri/index.php?page=search_detail&medicinal_id=107, Accessed date: 2 August 2018. Stresser, D.M., Turner, S.D., McNamara, J., Stocker, P., Miller, V.P., Crespi, C.L., Patten, C.J., 2000. A high-throughput screen to identify inhibitors of aromatase (CYP19). Anal. Biochem. 284, 427–430. Suginome, H., 1962. Oxygen heterocycles. maackiain, a new naturally occurring
chromanocoumaran. Experientia 18, 161–163. Talapatra, S.K., Mallik, A.K., Talapatra, B., 1980. Pongaglabol, a new hydroxyfuranoflavone, and aurantiamide acetate, a dipeptide, from the flowers of pongamia glabra. Phytochemistry 19, 1199–1202. Tanaka, T., Iinuma, M., Yuki, K., Fujii, Y., Mizuno, M., 1992. Flavonoids in root bark of Pongamia pinnata. Phytochemistry 31, 993–998. Tarus, P.K., Machocho, A.K., Lang'at-Thoruwa, C.C., Chhabra, S.C., 2002. Flavonoids from Tephrosia aequilata. Phytochemistry 60, 375–379. Van Puyvelde, L., De Kimpe, N., Mudaheranwa, J.P., Gasiga, A., Schamp, N., Declercq, J.P., Van Meerssche, M., 1987. Isolation and structural elucidation of potentially insecticidal and acaricidal isoflavone-type compounds from Neorautanenia mitis. J. Nat. Prod. 50, 349–356. Xia, L., Lee, Y.R., 2013. New synthetic approaches to naturally occurring and unnatural pyranoflavones. Helv. Chim. Acta 96, 644–650. Yadav, A., Singh, V., Singh, P., Singh, J.P., 2014. A new flavone and glycosides of T. Purpurea. J. Chem. Biol. Phys. Sci. 4, 3048–3052. Ye, H., Fu, A., Wu, W., Li, Y., Wang, G., Tang, M., Li, S., He, S., Zhong, S., Lai, H., Yang, J., Xiang, M., Peng, A., Chen, L., 2012. Cytotoxic and apoptotic effects of constituents from Millettia pachycarpa Benth. Fitoterapia 83, 1402–1408.
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Diverse flavonoids from the roots of Millettia brandisiana a
a,b
Phanruethai Pailee , Chulabhorn Mahidol Vilailak Prachyawarakorna,∗ a b c
, Somsak Ruchirawat
a,b,c
,
T
Laboratory of Natural Products, Chulabhorn Research Institute, Kamphaeng Phet 6 Road, Laksi, Bangkok 10210, Thailand Chemical Biology Program, Chulabhorn Graduate Institute, Kamphaeng Phet 6 Road, Laksi, Bangkok 10210, Thailand Center of Excellence on Environmental Health and Toxicology (EHT), CHE, Ministry of Education, Bangkok 10400, Thailand
ARTICLE INFO
ABSTRACT
Keywords: Millettia brandisiana Fabaceae Flavonoids Cytotoxicity Antiaromatase
The phytochemical investigation for the constituents of the roots of Millettia brandisiana, using bioassay guided fractionation, resulted in the isolation of five previously undescribed (namely brandisianones A-E) and twentysix known flavonoids. Their chemical structures were determined using a combination of NMR, MS, IR, optical rotation and CD analysis, as well as comparison with the literature data. The crude extract as well as the isolated compounds were evaluated in various biological assays for their cytotoxicity against a panel of human cancer cell lines, potential inhibitory activity against aromatase, and antioxidant property using the oxygen radical absorbance capacity (ORAC) with an aim to search for leads and develop them to drug candidates in our drug discovery effort, we identified three bioactive flavonoids from M. brandisiana which could be further developed into a potential chemopreventive (antiaromatase) agent against breast cancer.
1. Introduction The genus Millettia (Fabaceae), comprising more than 260 species, is distributed widely in the Africa, Asia, America, and Australia (Banzouzi et al., 2008; Havyarimana et al., 2012). Millettia brandisiana Kurz, also known in Thai as “Kra-Pee-Jun”, is a medium tree growing up to 8–20 m in height while distributing mainly in the northern, central, and northeastern regions of Thailand. The stems of M. brandisiana have been reported for their use in traditional medicine as haematonic (Mahidol University, 2010). Isoflavones and rotenones were isolated from this plant, among which the isoflavones were previously reported to exhibit biological activities to overcome TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) resistance and to exhibit anti-inflammatory activity (Pancharoen et al., 2008; Ishibashi and Ohtsuki, 2008; Kikuchi et al., 2007, 2009). As part of our ongoing program to search for the bioactive compounds, various biological assays on the isolated compounds from the dichloromethane extract of the roots of M. brandisiana were performed. The isolated compounds were evaluated for their cytotoxic activity against five human cancer cell lines, chemoprevention, and inhibition of aromatase, all of which may be useful for the prevention of breast cancer. Thus, we decided to investigate the chemical constituents and isolate the bioactive compounds from this plant. The structure elucidation of five undescribed compounds, their cytotoxicity against a panel of mammalian cancer cell lines, and antioxidant as well
∗
as antiaromatase activities of the isolated compounds were also described. 2. Results and discussion Purification of a dichloromethane extract of the roots of M. brandisiana by (repeated) chromatography provided thirty-one flavonoids (Fig. 1), among which brandisianones A-E (1, 2, 12, 14, and 21) have not been previously reported. The known compounds were first isolated in this plant and identified by comparison of their physical and spectroscopic data with literature values as lanceolatin B (3) (Tanaka et al., 1992; Mbafor et al., 1995; Lee and Morehead, 1995), pongaglabol (4) (Ahmad et al., 1999; Talapatra et al., 1980), pinnatin (5) (Das et al., 1994), 6″,6″-dimethylchromeno-[2″,3′′:7,8]-flavone (6) (Lee and Kim, 2006; Xia and Lee, 2013; Magalhães et al., 1996), candidine (7) (Yadav et al., 2014; Jain and Sharma, 1974), 5-methoxy-6″,6″-dimethylchromeno-[2″,3′′:7,8]-flavone (8) (Andrei et al., 2000), 5-hydroxy-6″,6″dimethylchromeno-[2″,3′′:7,6]-flavone (9) (Jain and Sharma, 1974), 5methoxy-6″,6″-dimethylchromeno-[2″,3′′:7,6]-flavone (10) (Camele et al., 1980), 7-hydroxy-8,4′-dimethoxyisoflavone (11) (http://www. ncbi.nlm.nih.gov/pubmed?term=Puebla%20P%5BAuthor%5D& cauthor=true&cauthor_uid=21076386 Puebla et al., 2010), 7-O-8-bis(3,3-dimethylallyl)-5-hydroxyflavanone (13) (Bohlmann and Misra, 1984), (−)-isolonchocarpin (15) (Rao and Raju, 1979), obovatin (16)
Corresponding author. E-mail address: [email protected] (V. Prachyawarakorn).
https://doi.org/10.1016/j.phytochem.2019.03.013 Received 10 August 2018; Received in revised form 18 March 2019; Accepted 18 March 2019 0031-9422/ © 2019 Published by Elsevier Ltd.
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Fig. 1. Structures of compounds 1–31.
158
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Table 1 1 H NMR data [δ in ppm, mult, (J in Hz)] of 1, 2, 12, and 14 in CDCl3 (600 MHz). 12a
Position
1
2
2 3
6.79, s
7.12, s
6.90, s
7.92, d (8.7) 7.71, dd (8.7, 0.7)
7.95, 7.57, 7.57, 6.85,
8.23, 7.61, 7.61, 7.34,
5 6 8 2′, 6′ 3′,5′ 4′ 1″ 2″ 4″ 5″ 1‴ 2‴ 4‴ 5‴ 2-OMe 5-OH/OMe 6″eOH a
d (6.8) m m s
1.72, s 1.72, s
12.69, s
5.45, 2.86, 3.00, 7.81, 6.63,
dd (7.8, 1.3) m m d (0.7)
14 dd (12.4, 3.1) dd (16.8, 3.1) dd (16.8, 12.9) d (8.8) d (8.8)
7.48, m 7.42, m 7.36, m 4.60, d (6.6) 5.47, m 1.79, s 1.74, s 3.37, d (7.0) 5.20, m 1.654, s 1.647, s
1.59, s 1.59, s
5.59, s
2.90, d (16.4) 2.97, d (16.4) 6.38, 7.59, 7.45, 7.40, 6.64, 5.60, 1.45, 1.49,
s d (7.3) t (7.2) t (7.2) d (10.0) d (10.0) s s
3.05, s 3.87, s
Recorded at 400 MHz.
(Andrei et al., 2000; Peralta et al., 2011; Arriaga et al., 2009), ovalitenin A (17) (Gupta and Krishnamurti, 1977), lonchocarpine (18) (Lee and Kim, 2006; Lima et al., 2013), 2′-methoxyfurano-[2″,3′′:4′,3′]-dihydrochalcone (19), ovalitenin B (20) (Gupta and Krishnamurti, 1977; Tanaka et al., 1992), pongamol (22) (Lee and Kim, 2006), 2′,6′-dimethoxyfurano-[2″,3′′:4′,3′]-β-hydroxydihydrochalcone (23) (Hishmat et al., 1989), 2′-hydroxy-6″,6″-dimethylchromeno-[2″,3′′:4′,3′]-β-hydroxychalcone (24) [Xia and Lee (2013), 2′-methoxy-6″,6″-dimethylchromeno-[2″,3′′:4′,3′]-β-hydroxychalcone (25) (Magalhães et al., 1996), praecansone B (26) (Tarus et al., 2002), (−)-12α-hydroxyrotenone (27) (Magalhães et al., 1996; Van Puyvelde et al., 1987), (−)-villosinol (28) (Krupadanam et al., 1977), (−)-tephrosin (29) (Ahmad et al., 1999; Belofsky et al., 2014; Lou et al., 2016), (−)-medicarpin (30) (Goel et al., 2012), and (−)-maackiain (31) (Suginome, 1962; Chaudhuri et al., 1995). This represents the first isolation of compound 24 as a natural product (Xia and Lee, 2013). A molecular formula of C20H16O5 was established for compound 1 on the basis of its APCI-TOF MS at m/z 337.1080 [M+H]+ (calcd for C20H17O5, 337.1071). The IR spectrum of 1 indicated the presence of hydroxyl (3400 cm−1), conjugated carbonyl (1662 cm−1) and aromatic (1613, 1587, and 1454 cm−1) groups. The 1H NMR spectrum (Table 1) showed a singlet at δH 12.69 for a hydroxy group (5-OH), which was hydrogen bonded to the carbonyl group (C-4), as well as a doublet at δH 7.95 (2H, J = 6.8 Hz) and a multiplet at δH 7.57 (3H) for the monosubstituted phenyl ring B. A singlet proton at δH 6.79 (H-3) and a conjugated carbonyl carbon at δH 183.2 (C-4) were identified as part of the flavone nucleus (Ayers et al., 2008). The appearance of a hydroxyisopropylfuran group was observed from a singlet of two methyl groups at δH 1.72 (6H) on a carbinol carbon (C-3″) and the other singlet of an olefinic proton at δH 6.85 (1H) on the 1H NMR spectrum. This was in accordance with the correlations from H-1″ to C-2′′ (δC 162.8) as well as CH3-4′′/CH3-5″ to C-3′′ (δC 69.2) and C-2′′ (δC 162.8) in HMBC spectrum. Its location at C-7 and C-8 of the phenyl ring A was also confirmed by HMBC correlation of H-1″ and H-6 to C-7 (δC 149.8) and C-8 (δC 109.3). The remaining singlet at δH 6.90 was assigned as that at the C-6 position based on the correlations from H-6 to C-5 (δC 158.4), C10 (δC 107.5), C-8 (δC 109.3), and C-7 (δC 149.8). On the basis of these spectroscopic data, compound 1 was identified as 5-hydroxy-5′′-(2-hydroxypropan-2-yl)-furano-[2″,3′′:7,8]-flavone, namely brandisianone A. The 1H and 13C NMR spectral data of compound 2 were similar to those of 1, except for the absence of a chelated hydroxyl group at the C-
5 position. Moreover, the appearance of a doublet at δH 7.92 (J = 8.7 Hz) and a doublet of doublet at δH 7.71 (J = 8.7, 0.7 Hz), which exhibited a long range coupling with H-4′′ (δH 7.34, d, J = 0.7 Hz) in the 1H NMR spectrum (Table 1), supported the presence of an ortho-coupled aromatic protons on the ring A in compound 2. The molecular formula of C20H16O4 from the APCI-TOF MS also indicated that compound 2 has one fewer oxygen than 1. Therefore, compound 2 was elucidated as 5′′-(2-hydroxypropan-2-yl)-furano-[2″,3′′:7,8]-flavone, namely brandisianone B. Compound 12 was isolated as a yellow amorphous solid and its molecular formula was established as C25H28O3 by APCI-TOF MS at m/z 377.2110 [M+H]+. The IR spectrum of compound 12 indicated the presence of carbonyl (1683 cm−1) and aromatic (1595 and 1436 cm−1) groups. The 1H NMR spectrum (Table 1) showed the signals of a threeproton spin system at δH 2.86 (dd, J = 16.8, 3.1 Hz), 3.00 (dd, J = 16.8, 12.9 Hz), and 5.45 (dd, J = 12.4, 3.1 Hz) which were characteristic of a C-3 unsubstituted flavanone nucleus (Peralta et al., 2011; Fu et al., 1993). The presence of multiplet signals in 1H NMR data at δH 7.48 (2H, m), 7.42 (2H, m), and 7.36 (1H, m) suggested the appearance of a monosubstituted phenylic ring B. Two doublet signals at δH 6.63 (H-6) and 7.81 (H-5) with the coupling constant of 8.8 Hz in the 1H NMR spectrum supported the presence of an ortho-coupled aromatic protons on the ring A. The remaining signals in the NMR spectra were assigned to two prenyl moieties, the first of which showed signals of a C-prenyl group at δH 3.37 (d, J = 7.0 Hz), 5.20 (m), 1.654 (s), and 1.647 (s), ascribable to an allylic methylene, an olefinic proton, and two methyl groups, respectively. The second prenyl group showed signals for an allylic methylene group at δH 4.60 (d, J = 6.6 Hz), an oleficnic proton at δH 5.47 (m), and two methyl groups at δH 1.79 (s) and 1.74 (s); the low field of this methylene implied that this prenyl group was bonded to an oxygen atom (an O-prenyl group). The correlations observed in the HMBC spectrum from H-5 to C-4 (δC 191.3), C-7 (δC 162.7), and C-9 (δC 160.2), H-6 to C-8 (δC 118.0) and C-10 (δC 115.2), H-1″ to C-7; and H-1‴ to C-7 and C-9 supported the positions of H-5, H-6, O-prenyl group at C-7, and C-prenyl group at C-8. The absolute configuration at C-2 in compound 12 is S-, as determined by the analysis of the CD spectrum where a positive Cotton effect was observed at 334 nm along with a negative Cotton effect at 248–297 nm (Peralta et al., 2011). From these data, the structure of compound 12 was established as (2S)-8-γ,γ-dimethylallyl-7-O-γ,γ-dimethylflavanone, namely brandisianone C. Compound 14 had a molecular formula C22H22O5 as deduced from its APCI-TOF MS. The IR spectrum of 14 displayed characteristic 159
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Table 2 13 C NMR data [δ in ppm] of 1, 2, 12, and 14 in CDCl3 (150 MHz).
Table 3 1 H and 13C NMR data of 19 and 21 in CDCl3 (400 MHz).
Position
1
2
12a
14
2 3 4
163.6 106.6 183.2
161.8 107.1 176.8
79.4 44.4 191.3
104.4 51.2 187.8
5 6 7 8 9 10 1′ 2′, 6′ 3′,5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 1‴ 2‴ 3‴ 4‴ 5‴ 2-OMe 5-OMe
158.4 95.5 149.8 109.3 159.2 107.5 131.1 126.3 129.2 132.0 97.3 162.8 69.2 28.8 28.8
120.2 110.0 149.8 117.8 157.1 118.7 131.0 126.3 129.1 131.7 97.9 166.2 67.6 28.9 28.9
126.1 106.1 162.7 118.0 160.2 115.2 139.4 125.9 128.6 128.3 65.5 119.4 138.0 25.6 18.2
156.7 110.3 160.1 101.4 159.9 109.4 138.9 125.9 128.6 128.8 116.1 128.6 77.8 28.3 28.5
a
22.3 122.0 131.5 25.7 17.7
Position
19 δC
1 2,6 3,5 4 1′ 2′ 3′ 4′ 5′ 6′ CO α
141.7 128.37a 128.41a 125.8 124.9 153.9 118.3 159.0 106.5 126.7 201.2 45.2
β 4″ 5″ 6″ 7″ 8″ 2′-OMe β-OMe
30.6 105.6 144.6
a b
50.8 62.4
60.5
21 δH, mult (J in Hz) 7.27, m 7.27, m 7.20, m
7.21, d (8.7) 7.67, d (8.7) 3.35, t (7.4) 3.05, t (8.1) 6.98, d (2.0) 7.60, d (2.3)
4.11, s
δC 141.6 126.7 128.4 127.6 125.3 156.4 114.7 157.7 112.6 131.0 198.2 50.6 79.9 116.5 130.4 76.8 27.96 28.0 63.1 56.7
δH, mult (J in Hz) 7.36, m 7.36, m 7.28, m
6.60b, d (9.2) 7.50, d (8.5) 3.55, dd (16.3, 8.8) 3.11, dd (16.3, 4.4) 4.84, dd (8.8, 4.4) 6.60b, d (9.2) 5.67, d (10.1) 1.44, 1.44, 3.72, 3.20,
s s s s
Interchangeable signals. Overlapped signals.
and 1He1H COSY experiments revealed the correlation between two triplets of the ethylene unit at δH 3.35 (J = 7.4 Hz) and 3.05 (J = 8.1 Hz). From the data above, compound 19 was deduced as a dihydrochalcone derivative (Gupta and Krishnamurti, 1977; Tanaka et al., 1992). Additionally, the 1H NMR data displayed a singlet of an aromatic methoxy group at δH 4.11 (2′-OMe) and two doublets of furan group at δH 6.98 (J = 2.0 Hz) and 7.60 (J = 2.3 Hz) and their locations were assigned at C-2′ and C-3′/4′, respectively, by the correlations from 2′-OMe to C-2′ (δC 153.9) and from H-6′ (δH 7.67) to C-2′ and C-4′ (δC 159.0). (Gupta and Krishnamurti, 1977). Therefore, compound 19 was identified as 2′-methyoxyfurano-[2″,3″:4′,3′]-dihydrochalcone and has been reported as commercially available albeit with only partially characterized spectroscopic data. Its fully characterized spectroscopic data are first described here. Compound 21 was obtained as a white amorphous powder. Its molecular formula of C22H24O4 was determined by APCI-TOF MS at m/z 353.1749 [M+H]+. 1H and 13C NMR data shown in Table 3 of compound 21 were similar to those of compound 14. A major significant difference was the absence of a singlet aromatic proton and a ketal carbon along with the presence of an ortho-coupled aromatic protons at δH 6.60 (d, J = 9.2 Hz) and 7.50 (d, J = 8.5 Hz) and a three proton coupling system at δH 3.11 (dd, J = 16.3 and 4.4 Hz), 3.55 (dd, J = 16.3 and 8.8 Hz) and 4.84 (dd, J = 8.8, 4.4 Hz) which could be assignable to a structural fragment eCOCH2CHOe. These results, together with the mass spectrum, supported the ring C opening from flavanone 14 to dihydrochalcone skeleton 21. The CD Cotton effects in the region 240–290 and 320–350 nm provide evidence for determining the absolute configurations of β-hydroxyldihydrochalcone (Nel et al., 1999; Muiva et al., 2009). However, the absolute stereochemistry of methoxy group at C-β of compound 21 could not be determined based on the positive Cotton effect at 263 nm and negative Cotton effect at 251 and 274 nm; these results are not in good agreement with those published for a series of β-hydroxydihydrochalcones (Nel et al., 1999; Muiva et al., 2009). However, the stereochemistry at C-β could be established as S- by comparing the optical rotation with that of the elatadihydrochalcone, a β-hydroxydihydrochalcone (Muiva et al., 2009). The elatadihydrochalcone, β-hydroxydihydrochalcones, with S- configuration at β-carbon was reported to exhibit a positive optical rotation ([α]D +34.5°). Compound 21 also showed a positive optical rotation
Recorded at 100 MHz.
absorptions of carbonyl group at 1687 cm−1 and aromatic group at 1601, 1567, and 1468 cm−1. The 1H NMR spectrum (Table 1) exhibited a doublet at δH 7.59 (J = 7.3 Hz) and two triplets at δ H 7.45 and 7.40 (J = 7.2 Hz) as well as two doublets at δH 2.90 and 2.97 (J = 16.4 Hz) representing a monosubstituted aromatic and a nonequivalent methylene group, respectively. From these characteristic data and the lack of a methine proton signal when compared with those of compound 12, compound 14 was assigned as a C-2 substituted flavone. The substituent at the C-2 in ring C was identified as a methoxy group by the existence of the ketal carbon at δC 104.4 (C-2) in 13C NMR spectrum (Table 2) and an additional methoxy proton at δH 3.05 (2-OMe). This was further confirmed by the HMBC correlations from 2-OMe, H-3 and H-2′/6′ to C2 (δC 104.4). In addition, the 1H NMR data displayed the signals of a pentasubstituted aromatic proton at δH 6.38 (H-8), aromatic methoxy proton at δH 3.87 (5-OMe) and 2,2-dimethyl-2H-pyran at δH 6.64 (1H, d, J = 10.0 Hz), 5.60 (1H, d, J = 10.0 Hz), 1.49 (3H, s), and 1.45 (3H, s). The location of all substituents on the ring A was elucidated by the HMBC analysis. The correlations from H-8 to C-4 (δC 187.8) and C-10 (δC 109.4) as well as from 5-OMe and H-1″ to C-5 (δC 156.7) in the HMBC data identified the position of H-8 and the methoxy group at C-5, respectively. In addition, the location of the pyran on the ring B at C-6/ 7 was also indicated by the correlations from H-1″ to C-5 (δC 156.7), C-6 (δC 110.3), and C-7 (δC 160.1). Compound 14 decomposed before an optical rotation and CD experiments could be acquired, and so its absolute configuration was not determined. The structure of compound 14 was therefore identified as 2,5-dimethoxy-6″,6″-dimethylchromeno[2″,3″:7,6]-flavanone, namely brandisianone D. Compound 19 was isolated as a yellow powder and had a molecular formula of C18H16O3 as determined by the APCI-TOF MS. The IR absorption at 1666 cm−1 together with the low field carbon at δC 201.2 suggested the presence of a carbonyl group. The 1H NMR spectra (Table 3) showed two multiplets in the aromatic region at δH 7.27 and 7.20 as well as two doublets at δH 7.21 and 7.67 with a coupling constant of 8.7 Hz corresponding to the monosubstituted aromatic ring and an ortho coupled aromatic system, respectively. Moreover, the 1H NMR 160
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Table 4 Cytotoxicity, antioxidant activity and aromatase inhibition data of pure compounds from the CH2Cl2 extract of M. brandisiana (IC50, μM). Compound
Cell lines (IC50, μM); values are expressed as mean ± S.D. (n = 3) MOLT-3
3 4 5 6 7 8 9 10 12 13 14 15 16 17 18 19 20 21 23 24 25 26 27 28 29 30 31 Doxorubicind Etoposided Ketoconazoled
34.70 ± 3.00 I 22.70 ± 3.10 26.71 ± 1.45 I 20.18 ± 3.62 I 25.51 ± 6.32 I I NDc 51.76 ± 3.27 69.69 ± 28.97 6.62 ± 0.43 20.23 ± 3.07 39.75 ± 7.18 32.55 ± 2.71 22.01 ± 1.96 30.74 ± 2.44 30.62 ± 1.58 6.46 ± 2.89 ND 0.27 ± 0.07 0.61 ± 0.05 19.15 ± 2.59 59.07 ± 5.37 59.65 ± 5.92 ND 0.04 ± 0.01 ND
HepG-2 b
I I 66.20 ± 7.90 58.13 ± 1.91 37.50 ± 0.00 45.90 ± 1.74 37.50 ± 9.38 94.82 ± 22.87 93.09 ± 24.28 I ND 92.58 ± 10.00 100.40 ± 7.17 9.50 ± 1.80 78.89 ± 3.04 101.18 ± 10.32 51.61 ± 3.23 86.56 ± 3.81 103.70 ± 14.91 86.18 ± 6.89 I ND 29.27 ± 8.44 4.53 ± 2.18 4.59 ± 0.61 125.00 ± 27.78 I 0.43 ± 0.01 24.45 ± 2.28 ND
A549
HuCCA-1
HeLa
I I I 59.70 ± 5.33 71.72 ± 1.54 53.35 ± 2.78 I I I I ND 119.28 ± 6.93 113.35 ± 15.37 16.40 ± 3.88 89.80 ± 26.25 I 130.32 ± 2.74 I 132.72 ± 30.56 127.33 ± 21.96 I ND 8.29 ± 2.59 18.45 ± 13.03 48.66 ± 0.51 I I 0.26 ± 0.08 ND ND
I I I 57.57 ± 6.97 I 53.89 ± 4.22 I I I I ND 94.77 ± 4.61 122.67 ± 24.16 1.58 ± 0.30 6.39 ± 15.83 I 42.42 ± 2.96 I 135.80 ± 7.10 105.59 ± 4.38 71.13 ± 3.79 ND 41.46 ± 2.76 19.37 ± 0.82 74.39 ± 1.15 I I 0.26 ± 0.07 ND ND
I I 21.23 ± 2.91 38.16 ± 6.51 I 27.90 ± 1.35 I 140.15 ± 1.23 50.32 ± 3.46 I ND 58.01 ± 5.78 123.32 ± 0.02 8.45 ± 0.13 42.94 ± 0.67 94.11 ± 1.75 I 125.43 ± 0.20 I 108.70 ± 0.00 81.37 ± 0.23 ND 6.71 ± 0.51 0.87 ± 0.08 50.12 ± 2.65 ND I 0.17 ± 0.10 ND ND
Aromatase inhibition (IC50, μM)
Antioxidant activity ORAC (unit)a
0.80 ± 0.10 I I 0.40 ± 0.10 I I 8.70 ± 0.80 I 11.0 ± 2.80 I I 0.20 ± 0.10 3.10 ± 0.40 13.10 ± 1.30 ND 5.60 ± 1.10 I 6.60 ± 0.10 I ND I I I 14.00 ± 0.20 ND I I ND ND 2.40
0.02 0.50 0.20 0.10 0.80 0.70 0.70 0.01 0.20 0.14 0.10 0.10 0.85 0.10 ND 0.10 0.10 ND 2.00 1.50 0.60 0.80 0.00 ND ND 4.60 3.50 ND ND ND
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.10 0.03 0.10 0.20 0.20 0.20 0.01 0.20 0.06 0.20 0.20 0.05 0.10
± 0.10 ± 0.05 ± ± ± ± ±
0.30 0.30 0.02 0.20 0.00
± 0.10 ± 0.50
a Results were expressed as ORAC units, where 1 ORAC unit equals the net protection of β-phycoerythrin produced by 1 μM of 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox). b I = inactive at 50 μg/mL. c ND = not determined. d Etoposide, doxorubicin, and ketoconazole are positive control.
([α]D +42.4°); hence the same configuration was presumed. Thus, the structure of compound 21 was established as 2′-methoxy-6″,6″-dimethylchromeno-[2″,3′′:4′,3′]-(S)-β-methoxydihydrochalcone, namely brandisianone E. The absolute configuration at C-β of ovalitenin B (20) has not been previously reported (Gupta and Krishnamurti, 1977; Tanaka et al., 1992). The CD spectrum of 20 displayed a positive Cotton effect at 262 nm and negative Cotton effect at 246 nm as well as the optical rotation showed the positive value of [α]D +54.3°(c 0.61, CHCl3), similar to that of brandisianone E (21) which also supported the identification of C-β absolute configuration as S-. Some isolated compounds (3–10, 12, 13, 15–21, 23–25, and 27–31) were evaluated for their cytotoxic activities against five human cancer cell lines (MOLT-3, HepG2, A549, HuCCA-1, and HeLa) and their IC50 values are summarized in Table 4. Similar cytotoxicity for other compounds could not be evaluated due to their limited availability. The rotenone analogues (27–29) and chalcones 17 and 18 were found to be the most cytotoxic among the tested flavonoids. (−)-12αHydroxyrotenone (27) showed significant activity against MOLT-3, A549, and HeLa cell lines with the IC50 values of 0.27, 8.29, and 6.71 μM, respectively. (−)-Villosinol (28) exhibited potent cytotoxic activity toward MOLT-3, HepG2, and HeLa cell lines (IC50 values of 0.61, 4.53, and 0.87 μM, respectively), while (−)-tephrosin (29) was selectively cytotoxic against HepG2 cell line with the IC50 value of 4.59 μM. Previously, these rotenones were also reported to exhibit cytotoxic activities (https://www.ncbi.nlm.nih.gov/pubmed/?term= Blatt%20CT%5BAuthor%5D&cauthor=true&cauthor_uid=12112286; Deyou et al., 2017; Lou et al., 2016; https://www.ncbi.nlm.nih.gov/
pubmed/?term=Blatt%20CT%5BAuthor%5D&cauthor=true&cauthor_ uid=12112286 Blatt et al., 2002; Pérez et al., 2013; Ye et al., 2012). This is the first report of these cytotoxic rotenoids evaluated against HuCCA-1, MOLT-3, A549, HepG2, and HeLa cell lines, except that (−)-tephrosin (29) was previously reported to be cytotoxic against HepG2 and A549 cell lines with the IC50 values of 1.41 and 13.27 μM, respectively (Lou et al., 2016; Ye et al., 2012). It should also be noted that the chalcone 17 possessing the α,β-unsaturated ketone was more cytotoxic than the dihydrochalcones 19 and 20. Chalcone 17 showed significant cytotoxicity against MOLT-3, HepG2, A549, HuCCA-1, and HeLa cell lines with the IC50 values of 6.62, 9.50, 16.40, 1.58, and 8.45 μM, respectively. Chalcone 18 exhibited selective cytotoxicity against HuCCA-1 cell line with the IC50 values of 6.39 μM. The intramolecular hydrogen bonding of the hydroxyl group at C-2′ in chalcone 18, and C-β in chalcones 23–25 with the carbonyl group also decreased their cytotoxicity to be less than that of the chalcone 17. Based on these preliminary results, a hydroxy substitution of these chalcone analogues is detrimental for cytotoxicity as imposed by the Michael addition to the α,β-unsaturated ketone (Masi et al., 2017). The antioxidant and aromatase inhibitory activities were also evaluated (Table 4). The ORAC (oxygen radical absorbance capacity) assay was used to determine the antioxidative activity by the application of 2,2′-azobis (2-methylpropionamide) dihydrochloride (AAPH) as free radical initiator to generate the peroxyl radicals. Compounds 23, 24, 30, and 31 exhibited moderate to high antioxidant activity in the ORAC assay with 1.50–4.6 ORAC units. Compounds 3, 6, and 15 possessed relatively low IC50 values of 0.80, 0.40, and 0.20 μM, respectively, with respect to ketoconazole (IC50 = 2.4 μM) used as a positive 161
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control for the anti-aromatase assay; therefore, these three compounds potentially could be considered as candidates for the prevention of breast cancer by acting as an estrogen antagonist.
(85:15) as mobile phase to give compounds 24 (10.9 mg) and 18 (1.3 mg). Compounds 16 (376.5 mg) and 13 (40.7 mg) were obtained after purification of fraction A3 (2.03 g) by preparative TLC using CH2Cl2:hexane (4:6) as mobile phase. Fraction A4 (2.71 g) was chromatographed on RP-18 MPLC with solvents of increasing polarity of 60%–100% MeOH:H2O over 180 min to give 5 fractions (C1eC5). Fraction C2 was separated by HPLC using a C18 reversed phase column eluted with a 70:30 mixture of MeOH:H2O at a flow rate of 7 mL/min to give compounds 17 (9 mg), 19 (29 mg), and 22 (10 mg). Fraction C3 was purified by silica gel column, eluted with a gradient of hexane and acetone as mobile phase to provide compounds 15 (209.6 mg) and 21 (19.5 mg). Fraction C4 was subjected to preparative RP18-HPLC eluted with MeOH:H2O (85:15) as mobile phase at a flow rate of 12 mL/min to provide compounds 7 (231 mg), 9 (95 mg), and 12 (285 mg). Fraction A5 (1.57 g) was then subjected to silica gel column, and eluted with increasing proportions of acetone in hexane (0%–20%) to provide 8 fractions (D1–D8). Fractions D2 and D4 were compounds 25 (118.5 mg) and 20 (84.6 mg), respectively. Compounds 4 (4.0 mg) and 14 (1.9 mg) was obtained after purification of fraction D3 by preparative RP18HPLC with MeOH/H2O (80:20) as eluent at a flow rate of 10 mL/min. Fraction A9 (1.67 g) was chromatographed on silica gel column (gradient from 100% to 0% hexane/acetone) to give 8 fractions (H1eH8). Fraction H3 was subjected to preparative TLC using EtOAc and hexane (20:80) as mobile phase to give compound 26 (3.4 mg). Fraction H5 was purified by preparative RP18-HPLC with MeOH/H2O (75:25), flow rate 12 mL/min as eluent, to give compounds 3 (54 mg) and 6 (129.2 mg). Compounds 30 (6.7 mg) and 31 (9.5 mg) were obtained after purification of fraction H11 by preparative TLC [acetone:CH2Cl2 (1:99)] and preparative RP18-HPLC [MeOH:H2O (60:40), flow rate of 12 mL/min]. Compound 10 (923 mg) was obtained from fraction A10 (1.39 g) after recrystallization with CH2Cl2:MeOH. Fraction A11 (455 mg) was chromatographed on a silica gel column, eluted with a gradient (hexane and EtOAc) to provide 9 fractions (E1-E9). Fraction E3 was purified by preparative TLC using hexane and EtOAc (80:20) as mobile phase to provide compound 23 (19.6 mg). Fraction E6 was subjected to preparative RP18-HPLC with MeOH:H2O (73:27) as eluent at a flow rate of 10 mL/min which gave compounds 1 (3.4 mg), 27 (87.4 mg), 28 (6.2 mg), and 29 (14.4 mg). Combined fractions of A12A14 (756 mg) were subjected to gel permeation over Sephadex LH-20 (3 × 120 cm) with MeOH:CH2Cl2 (1:1) as eluent, to give 4 fractions (F1eF4). Fraction F3 was chromatographed on a silica gel column, eluted with a gradient of mixture CH2Cl2 and MeOH (100%–3%) to provide 8 fractions (G1-G8). Compounds 11 (3.0 mg) and 5 (41.4 mg) were obtained after purification of fraction G5 by preparative HPLC using MeOH and H2O (70:30) as mobile phase at flow rate of 12 mL/ min. Fraction G6 was subjected to preparative TLC using CH2Cl2 and MeOH (99:1) as mobile phase to give compound 2 (4 mg). Compound 8 (236.4 mg) was obtained after purification of fraction A15 (1.44 g) by gel permeation over Sephadex LH-20 CC (3 × 120 cm) [MeOH:CH2Cl2 (1:1)] followed by preparative TLC [MeOH:CH2Cl2 (2:98)]. Brandisianone A (1) White powder; UV (MeOH) λmax (log ε): 202 (4.34), 219 (4.34), 229 (4.33), 260 (4.24), 282 (4.36) nm; IR (ATR) νmax: 3400, 2926, 2853, 1662, 1613, 1587, 1454, 1423, 1348, 1284, 1239, 1148, 1119, 1096, 999, 839, 769, 688 cm−1; 1H and 13C NMR data (see Tables 1 and 2); APCITOFMS m/z 337.1080 [M+H]+ (calcd for C20H17O5, 337.1071). Brandisianone B (2) White powder; UV (MeOH) λmax (log ε): 219 (4.16), 266 (4.06), 298 (3.85) nm; IR (ATR) νmax: 3370, 2924, 1616, 1586, 1406, 1365, 1140, 1073, 882, 770, 682 cm−1; 1H and 13C NMR data (see Tables 1 and 2); APCITOFMS m/z 321.1128 [M+H]+ (calcd for C20H17O4, 321.1121). Brandisianone C (12) Pale yellow amorphous powder; CD (MeOH, c 54.5 μM): λnm (Δε) = 218 (+14.6), 248 (−2.11), 297 (−8.32), 334 (+5.14) nm; UV (MeOH) λmax (log ε): 202 (4.58), 217 (4.52), 287 (4.27) nm; IR (ATR) νmax: 2967, 2914, 1683, 1595, 1436, 1336, 1315,
3. Conclusion A phytochemical study on the dichloromethane extract of the roots of M. brandisiana led to the isolation of a total of 31 flavonoids with diverse skeletons including five previously undescribed flavonoids (1, 2, 3, 14, and 21, namely brandisianones A-E). Among them, compound 14 possessed a rare 2-methoxylated flavonoid skeleton. All twenty-six known flavonoids were first isolated from this plant. The rotenones 27 and 28 and chalcones 17 and 18 are among the most cytotoxic. An exploration of the structure-cytotoxicity relationships of the tested chalcone series revealed that the hydroxyl substitution at C-2′ or C-β position is detrimental for cytotoxicity. In addition, only two pterocapans 30 and 31 exhibited the highest antioxidant activity in the ORAC assay with 4.60 and 3.50 ORAC units while flavones 3 and 6 and flavanone 15 were found to be potent aromatase inhibitors with their IC50 values of 0.8, 0.4, and 0.2 μM, respectively. To our knowledge, this is the first report on the ORAC assay and the inhibitory activity against aromatase of all tested flavonoids, except that medicarpin (30) was previously reported to be inactive in the antiaromatase assay (Le Bail et al., 2000). The results suggested that these bioactive flavonoids could be of value for further studies as potential cytotoxic and cancer chemopreventive agents. 4. Experimental section 4.1. General experimental procedures UV–Vis spectra were obtained from Shimadzu UV-1700 PharmaSpec Spectrophotometer. Optical rotation was measured with sodium D line (590 nm) on JASCO 1020 digital polarimeter. FTIR spectra were recorded with a universal attenuated total reflectance (UATR) attachment on a Perkin–Elmer Spectrum One spectrometer. 1H, 13C and 2D NMR spectra were obtained using a Bruker AM400 spectrometer and a Bruker AVANCE 600 spectrometer with TMS and solvent as internal standard. APCI–TOF–MS were determined using a Bruker MicroTOFLC spectrometer. Column chromatography and preparative TLC were performed with silica gel 60 (70–200 mesh ASTM) and PF254, respectively. Sephadex LH-20 gel (Ambersham Biosciences) was also used for column chromatography. TLC was carried out on silica gel 60 F254 plates (Merck, 0.2 mm). High performance liquid chromatography (HPLC) was performed using Waters Delta 600 and Waters 2998 Photodiode Array Detector. 4.2. Plant material Roots of Millettia brandisiana Kurz (Fabaceae) were collected in April 2011 from the botanical garden at Chulabhorn Research Institute (13°52′N, 100°34′E) in Bangkok. A voucher specimen (CRI-661) has been deposited at the Laboratory of Natural Products, Chulabhorn Research Institute. 4.3. Extraction and isolation The air-dried roots of M. brandisiana (1.2 kg) were extracted with CH2Cl2 (2x5L) at room temperature. The CH2Cl2 layer was evaporated to give a crude extract (21 g), which was subjected to column chromatography on silica gel eluted successively with solvents of increasing polarity (hexane, EtOAc and MeOH) to provide 17 fractions (A1-A17). Fraction A2 (413.3 mg) was purified by preparative TLC using EtOAc:hexane (2:8) as mobile phase to give 3 bands (B1eB3). Band B1 was further purified by preparative RP18-HPLC using acetonitrile: H2O 162
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1267, 1218, 1177, 1112, 1071, 803, 765, 698 cm−1; 1H and 13C NMR data (see Tables 1 and 2); APCITOFMS m/z 377.2110 [M+H]+ (calcd for C25H29O3, 377.2111). Brandisianone D (14) Yellow powder; UV (MeOH) λmax (log ε): 260 (3.45), 326 (2.61) nm; IR (ATR) νmax: 2922, 2851, 1687, 1638, 1601, 1567, 1468, 1370, 1322, 1278, 1148, 1115, 1090, 1043, 987, 889, 767, 702 cm−1; 1H and 13C NMR data (see Tables 1 and 2); APCITOFMS m/z 367.1538 [M+H]+ (calcd for C22H23O5, 367.1540). Chalcone 19 Yellow powder; UV (MeOH) λmax (log ε): 207 (2.53), 235 (2.81), 305 (1.62) nm; IR (ATR) νmax: 2931, 1666, 1584, 1471, 1420, 1357, 1330, 1261, 1239, 1151, 1070, 993, 974, 807, 741, 699 cm−1; 1H and 13C NMR data (see Table 3); APCITOFMS m/z 281.1161 [M+H]+ (calcd for C18H17O3, 281.1172). Brandisianone E (21) Yellow oil; CD (MeOH, c 54.5 μM): λnm (Δε) = 208 (+3.78), 251 (−2.08), 263 (+1.81), 274 (−0.71), 323 (+0.37), 351 (+0.33) nm; [α]25 D +42.42 (c 1.93, CHCl3); UV (MeOH) λmax (log ε): 202 (4.17), 258 (4.26) nm; IR (ATR) νmax: 2977, 2934, 1674, 1591, 1455, 1371, 1314, 1276, 1215, 1190, 1161, 1114, 1075, 985, 890, 738, 702 cm−1; 1H and 13C NMR data (see Table 3); APCITOFMS m/z 353.1749 [M+H]+ (calcd for C22H25O4, 353.1747).
Pretorius, S., Stevenson, D., Thompson, D., Singh, S.B., 2008. Flavones from Struthiola argentea with anthelmintic activity in vitro. Phytochemistry 69, 541–545. Banzouzi, J.T., Prost, A., Rajemiarimirabo, M., Ongoka, P., 2008. Traditional uses of African Millettia species (Fabaceae). Int. J. Bot. 4, 406–420. Belofsky, G., Aronica, M., Foss, E., Diamond, J., Santana, F., Darley, J., Dowd, P.F., Coleman, C.M., Ferreira, D., 2014. Antimicrobial and antiinsectan phenolic metabolites of Dalea searlsiae. J. Nat. Prod. 77, 1140–1149. Blatt, C.T., Chávez, D., Chai, H., Graham, J.G., Cabieses, F., Farnsworth, N.R., Cordell, G.A., Pezzuto, J.M., Kinghorn, A.D., 2002. Cytotoxic flavonoids from the stem bark of Lonchocarpus aff. Fluvialis. Phytother. Res. 16, 320–325. Bohlmann, F., Misra, L.N., 1984. New prenylflavanones and chalcones from Helichrysum rugulosum. Planta Med. 50, 271–272. Camele, G., Delle Monache, F., Delle Monache, G., Bettolo, G.B.M., 1980. Three new flavonoids from Tephrosia praecans. Phytochemistry 19, 707–709. Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D., Mitchell, J.B., 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47, 936–942. Chaudhuri, S.K., Huang, L., Fullas, F., Brown, D.M., Wani, M.C., Wall, M.E., 1995. Isolation and structure identification of an active DNA strand-scission agent, (+)-3,4di-hydroxy-8,9-methylenedioxypterocarpan. J. Nat. Prod. 58, 1966–1969. Das, B., Chakravarty, A.K., Masuda, K., Suzuki, H., Ageta, H., 1994. A diterpenoid from roots of Gelonium multiflorum. Phytochemistry 37, 1363–1366. Deyou, T., Marco, M., Heydenreich, M., Pan, F., Gruhonjic, A., Fitzpatrick, P.A., Koch, A., Derese, S., Pelletier, J., Rissanen, K., Yenesew, A., Erdélyi, M., 2017. Isoflavones and rotenoids from the Leaves of Millettia oblata ssp. teitensis. J. Nat. Prod. 80, 2060–2066. Doyle, A., Griffiths, J.B. (Eds.), 1997. Mammalian Cell Culture: Essential Techniques. John Wiley and Sons, New York. Fu, X., Sévenet, T., Remy, F., Païs, M., Hadi, A.H.A., Zeng, L.M., 1993. Flavanone and chalcone derivatives from Cryptocarya kurzii. J. Nat. Prod. 56, 1153–1163. Gerhäuser, C., Klimo, K., Heiss, E., Neumann, I., Gamal-Eldeen, A., Knauft, J., Liu, G.Y., Sitthimonchai, S., Frank, N., 2003. Mechanism-based in vitro screening of potential cancer chemopreventive agents. Mutat. Res. 523–524, 163–172. Goel, A., Kumar, A., Hemberger, Y., Raghuvanshi, A., Jeet, R., Tiwari, G., Knauer, M., Kureel, J., Singh, A.K., Gautam, A., Trivedi, R., Singh, D., Bringmann, G., 2012. Synthesis, optical resolution, absolute configuration, and osteogenic activity of cispterocarpans. Org. Biomol. Chem. 10, 9583–9592. Gupta, R.K., Krishnamurti, M., 1977. New dibenzoylmethane and chalcone derivatives from Milletia ovalifolia seeds. Phytochemistry 16, 1104–1105. Havyarimana, L., Ndendoung, S.T., de-Dieu, Tamokou J., de Théodore Atchadé, A., Tanyi, J.M., 2012. Chemical constituents of Millettia barteri and their antimicrobial and antioxidant activities. Pharm. Biol. 50, 141–146. Hishmat, O.H., Miky, J.A.A., Saleh, N.M., 1989. Synthesis of some benzofuranylpyridine and pyridone derivatives of possible biological activities. Pharmazie 44, 823–825. Ishibashi, M., Ohtsuki, T., 2008. Studies on search for bioactive natural products targeting TRAIL signaling leading to tumor cell apoptosis. Med. Res. Rev. 28, 688–714. Jain, A.C., Sharma, B.N., 1974. Synthesis of isopentenylated chrysins. J. Org. Chem. 39, 1149–1152. Kikuchi, H., Ohtsuki, T., Koyano, T., Kowithayakorn, T., Sakai, T., Ishibashi, M., 2007. Brandisianins A-F, Isoflavonoids Isolated from Millettia brandisiana in a screening program for death-receptor expression enhancement activity. J. Nat. Prod. 70, 1910–1914. Kikuchi, H., Ohtsuki, T., Koyano, T., Kowithayakorn, T., Sakai, T., Ishibashi, M., 2009. Death receptor 5 targeting activity-guided isolation of isoflavones from Millettia brandisiana and Ardisia colorata and evaluation of ability to induce TRAIL-mediated apoptosis. Bioorg. Med. Chem. 17, 1181–1186. Krupadanam, G.L.D., Sarma, P.N., Srimannarayana, G., Subba Roa, N.V., 1977. New C-6 oxygenated rotenoids from Tephrosia villosa – villosin, villosone, villol, and villinol. Tetrahedron Lett. 24, 2125–2128. Le Bail, J.C., Champavier, Y., Chulia, A.-J., Habrioux, G., 2000. Effects of phytoestrogens on aromatase, 3β and 17β-hydroxysteroid dehydrogenase activities and human breast cancer cells. Life Sci. 66, 1281–1291. Lee, Y.R., Kim, D.H., 2006. A new route to the synthesis of pyranoflavone and pyranochalcone natural products and their derivatives. Synthesis 4, 603–608. Lee, Y.R., Morehead Jr., A.T., 1995. A new route for the synthesis of furanoflavone and furanochalcone natural products. Tetrahedron 51, 4909–4922. Lima, N.M., Andrade, J.I.A., Lima, K.C.S., dos Santos, F.N., Barison, A., Salome, K.S., Matsuura, T., Nunez, C.V., 2013. Chemical profile and biological activities of Deguelia duckeana A.M.G. Azevedo (Fabaceae). Nat. Prod. Res. 27, 425–432. Lou, H.Y., Wu, H.G., Tan, Y.H., Lan, J.J., Ma, X.P., Liang, G.Y., Yi, P., Pan, W.D., 2016. Two new flavonoids from Derris eriocarpa how. Helv. Chim. Acta 99, 302–305. Magalhães, A.F., Tozzi, A.M.G.A., Sales, B.H.L.N., Magalhães, E.G., 1996. Twenty-three flavonoids from Lonchocarpus subglaucescens. Phytochemistry 42, 1459–1471. Masi, M., Meyer, S., Clement, S., Cimmino, A., Cristofaro, M., Evidente, A., 2017. Cochliotoxin, a dihydropyranopyran-4,5-dione, and its analogues produced by Cochliobolus australiensis display phytotoxic activity against Buffelgrass (Cenchrus ciliaris). J. Nat. Prod. 80, 1241–1247. Mbafor, J.T., Atchade, A.T., Nkengfack, A.E., Fomum, Z.T., Sterner, O., 1995. Furanoflavones from root bark of Millettia sanagana. Phytochemistry 40, 949–952. Muiva, L.M., Yenesew, A., Derese, S., Heydenreich, M., Peter, M.G., Akala, H.M., Eyase, F., Waters, N.C., Mutai, C., Keriko, J.M., Walsh, D., 2009. Antiplasmodial β-hydroxydihydrochalcone from seedpods of Tephrosia elata. Phytochem. Lett. 2, 99–102. Nel, R.J.J., Rensburg, H., van Heerden, P.S., Coetzee, J., Ferreir, D., 1999. Stereoselective synthesis of flavonoids. Part 7. Polyoxygenated β-hydroxydihydrochalcone derivatives. Tetrahedron 55, 9727–9736. Pancharoen, O., Athipornchai, A., Panthong, A., Taylor, W.C., 2008. Isoflavones and
4.4. Cytotoxicity assays The cytotoxic activity toward a panel of mammalian cancer cell lines (HepG2, A549, HuCCA-1, HeLa) were tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Carmichael et al., 1987), while the activity toward MOLT-3 cancer cell line was performed by the 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)(2H)-tetrazolium-5-carboxanilide (XTT) assay (Doyle and Griffiths, 1997). Etoposide and doxorubicin were used as positive controls (Table 4). 4.5. Aromatase (CYP19) inhibition assay Inhibition of the aromatase enzyme assay was performed using the method designed by Stresser and co-workers (Stresser et al., 2000). The reference compound, ketoconazole, inhibits CYP19 with an IC50 value of 2.4 μM (Table 4). 4.6. ORAC antioxidant assay The test of the ORAC antioxidant assay was previously described by Gerhäuser and co-workers (Gerhäuser et al., 2003). Acknowledgments We thank Ms. Pakamas Intachote, Ms. Suchada Sengsai, and Ms. Busakorn Saimanee for cytotoxic assays and Dr. Poonsakdi Ploypradith for helpful editing in preparing the manuscript. We thank Mahidol University for partial financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.03.013. References Ahmad, V.U., Ali, Z., Hussaini, S.R., Iqbal, F., Zahid, M., Abbas, M., Saba, N., 1999. Flavonoids of Tephrosia purpurea. Fitoterapia 70 (4), 443–445. Andrei, C.C., Ferreira, D.T., Faccione, M., de Moraes, L.A.B., de Carvalho, M.G., BrazFilho, R., 2000. C-prenylflavonoids from roots of Tephrosia tunicate. Phytochemistry 55, 799–804. Arriaga, A.M.C., Lima, J.Q., Vasconcelos, J.N., de Oliveira, M.C.F., Andrade-Neto, M., Santiago, G.M.P., Uchoa, D.E.A., Malcher, G.T., Mafezoli, J., Braz-Filho, R., 2009. Unequivocal assignments of flavonoids from Tephrosia sp. (Fabaceae). Magn. Reson. Chem. 47, 537–540. Ayers, S., Zink, D.L., Mohn, K., Powell, J.S., Brown, C.M., Murphy, T., Brand, R.,
163
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P. Pailee, et al. rotenoids from the leaves of Millettia brandisiana. Chem. Pharm. Bull. 56, 835–838. Peralta, M.A., Ortega, M.G., Agnese, A.M., Cabrera, J.L., 2011. Prenylated flavanones with anti-tyrosinase activity from Dalea boliviana. J. Nat. Prod. 74, 158–162. Pérez, L.B., Li, J., Lantvit, D.D., Pan, L., Ninh, T.N., Chai, H.B., Soejarto, D.D., Swanson, S.M., Lucas, D.M., Kinghorn, A.D., 2013. Bioactive constituents of Indigofera spicata. J. Nat. Prod. 76, 1498–1504. Puebla, P., Oshima-Franco, Y., Franco, L.M., dos Santos, M.G., Silva, R.V., Rubem-Mauro, L., Feliciano, A.S., 2010. Chemical constituents of the bark of Dipteryx alata vogel, an active species against Bothrops jararacussu venom. Molecules 15, 8193–8204. Rao, E.V., Raju, N.R., 1979. Occurrence of (–)-isolonchocarpin in the roots of Tephrosia purpurea. Phytochemistry 18, 1581–1582. Mahidol University, 2010, 2010. Land of Thai medicinal plant. In: Siri Ruckhachati Nature Park. John Wiley and Sons, New York. http://www.pharmacy.mahidol.ac.th/ siri/index.php?page=search_detail&medicinal_id=107, Accessed date: 2 August 2018. Stresser, D.M., Turner, S.D., McNamara, J., Stocker, P., Miller, V.P., Crespi, C.L., Patten, C.J., 2000. A high-throughput screen to identify inhibitors of aromatase (CYP19). Anal. Biochem. 284, 427–430. Suginome, H., 1962. Oxygen heterocycles. maackiain, a new naturally occurring
chromanocoumaran. Experientia 18, 161–163. Talapatra, S.K., Mallik, A.K., Talapatra, B., 1980. Pongaglabol, a new hydroxyfuranoflavone, and aurantiamide acetate, a dipeptide, from the flowers of pongamia glabra. Phytochemistry 19, 1199–1202. Tanaka, T., Iinuma, M., Yuki, K., Fujii, Y., Mizuno, M., 1992. Flavonoids in root bark of Pongamia pinnata. Phytochemistry 31, 993–998. Tarus, P.K., Machocho, A.K., Lang'at-Thoruwa, C.C., Chhabra, S.C., 2002. Flavonoids from Tephrosia aequilata. Phytochemistry 60, 375–379. Van Puyvelde, L., De Kimpe, N., Mudaheranwa, J.P., Gasiga, A., Schamp, N., Declercq, J.P., Van Meerssche, M., 1987. Isolation and structural elucidation of potentially insecticidal and acaricidal isoflavone-type compounds from Neorautanenia mitis. J. Nat. Prod. 50, 349–356. Xia, L., Lee, Y.R., 2013. New synthetic approaches to naturally occurring and unnatural pyranoflavones. Helv. Chim. Acta 96, 644–650. Yadav, A., Singh, V., Singh, P., Singh, J.P., 2014. A new flavone and glycosides of T. Purpurea. J. Chem. Biol. Phys. Sci. 4, 3048–3052. Ye, H., Fu, A., Wu, W., Li, Y., Wang, G., Tang, M., Li, S., He, S., Zhong, S., Lai, H., Yang, J., Xiang, M., Peng, A., Chen, L., 2012. Cytotoxic and apoptotic effects of constituents from Millettia pachycarpa Benth. Fitoterapia 83, 1402–1408.
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