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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Two new flavanonols from the bark of Akschindlium godefroyanum a
a
Nattapong Chaipukdee , Somdej Kanokmedhakul , Ratsami a
Lekphrom & Kwanjai Kanokmedhakul
a
a
Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand Published online: 20 Dec 2013.
To cite this article: Nattapong Chaipukdee, Somdej Kanokmedhakul, Ratsami Lekphrom & Kwanjai Kanokmedhakul (2014) Two new flavanonols from the bark of Akschindlium godefroyanum, Natural Product Research: Formerly Natural Product Letters, 28:3, 191-195, DOI: 10.1080/14786419.2013.866113 To link to this article: http://dx.doi.org/10.1080/14786419.2013.866113
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 3, 191–195, http://dx.doi.org/10.1080/14786419.2013.866113
Two new flavanonols from the bark of Akschindlium godefroyanum
Downloaded by [University of Newcastle (Australia)] at 21:37 03 October 2014
Nattapong Chaipukdee, Somdej Kanokmedhakul, Ratsami Lekphrom and Kwanjai Kanokmedhakul* Natural Products Research Unit, Department of Chemistry, Center of Excellence for Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand (Received 26 July 2013; final version received 4 November 2013) The first phytochemical investigation of the bark of Akschindlium godefroyanum (Kuntze) H. Ohashi (Fabaceae), a Thai herbal medicine, resulted in the isolation of two new flavanonols, 7,30 ,50 -trihydroxy-5-methoxyflavanonol (1) and 7,40 -dihydroxy-5,30 dimethoxyflavanonol (2), and eight known compounds comprising one flavonol, geraldol (3); three flavanonols, (þ )-taxifolin (4), (þ )-fustin (5) and aromadendrin 5-methyl ether (6); one catechin, ( – )-epigallocatechin (7); one triterpenoid, lupeol (8); one steroid, stigmasterol (9) and one steroid glycoside, stigmasterol-3-O-b-D -glucoside (10). Their structures were identified by spectroscopic methods. Keywords: Akschindlium godefroyanum; flavanonol; flavonol; catechin
1. Introduction Akschindlium godefroyanum (Kuntze) H. Ohashi (Fabaceae) is a shrub, 1.5 – 3 m in height, widely found in the lower zone of the northern, northeastern and southern parts of Thailand. It is called ‘Chai Hin’ in Thai and it is used as antiemetic in Thai herbal medicine (Chuakul et al. 2000). However, phytochemical study of the genus Akschindlium has not been reported. We report herein the first isolation and identification of the chemical constituents from the bark of A. godefroyanum. 2. Results and discussion Three crude extracts, crude n-hexane, crude EtOAc and crude MeOH, were obtained from the dried bark of A. godefroyanum. Chromatographic separation of these extracts gave two new flavanonols, 7,30 ,50 -trihydroxy-5-methoxyflavanonol (1) and 7,40 -dihydroxy-5,30 -dimethoxyflavanonol (2), and eight known compounds. The structures of the isolated compounds were identified using physical and spectroscopic data measurements ([a ]D, 1H NMR, 13C NMR and 2D NMR) as well as comparing the data with those reported in the literature as geraldol (3) (Lee et al. 2008), (þ )-taxifolin (4) (Lee et al. 2011), (þ )-fustin (5) (Park et al. 2004), aromadendrin 5-methyl ether (6) (Bilia et al. 1993), ( –)-epigallocatechin (7) (Kumar & Rajapaksha 2005; Alimova et al. 2007), lupeol (8) (Jamal et al. 2008), stigmasterol (9) (Kamboj & Saluja 2011) and stigmasterol-3-O-b-D -glucoside (10) (Kojima et al. 1990) (Figure 1). Compound 1 was obtained as a pale yellow powder and the molecular formula (C16H14O7) was deduced from the molecular ion peak at m/z 341.0629 [M þ Na]þ by HR-ESI-TOF-MS (calcd for C16H14O7 þ Na, 341.0637), indicating 10 degrees of unsaturation. The UV spectrum showed absorption maxima at 286 and 362 nm as those of the flavanonol skeleton (Tsimogiannis
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
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R3 5'
6'
R2
8 7 6
9
10
5
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R1
1 2 4 5 6
1'
C
A
B
2
O
3 4
OH R4
4' 3'
2'
HO
O
OH
OMe
R5
O
HO
OH
OH
3
OH OH
O
OH
7
O
R1
R2
OMe OMe HO H OMe
OH OH OH OH OH
R3 OH H H H H
R4 H OH OH OH OH
R5 OH OMe OH OH H
H
H H
H H
H HO
H
H
R 8
9 R = OH 10 R = O–β–D–glucosy
Figure 1. Structures of isolated compounds 1 –10.
et al. 2007). The IR spectrum showed the presence of hydroxyl (3447 cm21), conjugated ketone (1652 cm21) and aromatic (1589 cm21) groups. The 1H NMR spectrum of 1 showed signals for two oxygenated methines of ring C at d 4.85 (1H, d, J ¼ 11.2 Hz, H-2) and 4.22 (1H, d, J ¼ 11.2 Hz, H-3), two meta aromatic protons of ring A at d 6.06 (1H, d, J ¼ 1.6 Hz, H-6) and 5.91 (1H, d, J ¼ 1.6 Hz, H-8). Consistent with the above 1H NMR analysis, 13C NMR and HMQC spectra of 1 displayed the carbon signals for two oxygenated methine carbons at d 82.9 (C-2) and 73.0 (C-3), an aromatic ring A at d 162.6 (C-5), 93.8 (C-6), 165.2 (C-7), 95.9 (C-8), 164.2 (C-9) and 102.9 (C-10) which agreed well with those reported for the related compound 7,30 ,40 -trihydroxy-5-methoxy-dihydroflavanonol (Foo 1987). The two aromatic signals of ring B showed at dH 6.84 (1H, brs, H-20 ) and d 6.71 (2H, brs, H-40 and H-60 ) indicating 1,3,5trisubstituted aromatic, and the three protons were in meta positions. While its 13C NMR spectrum showed signals at dC 128.8 (C-10 ), 115.6 (C-20 ), 146.1 (C-30 ), 115.6 (C-40 ), 145.4 (C-50 ) and 119.7 (C-60 ). The HMBC spectrum of 1 exhibited the correlations of H-2 with carbonyl (C4), C-3, C-9, C-10 , C-20 and C-60 ; H-3 with C-2; H-6 with C-5, C-7, C-8 and C-10; H-8 with C-6, C-7, C-9 and C-10; H-20 with C-2, C-10 , C-30 , C-40 and C-60 ; H-40 with C-20 , C-30 , C-50 and C-60 ; H-60 with C-2, C-10 , C-20 , C-40 and C-50 ; and methoxy protons at d 3.73 with C-5 confirming the structure of 1 (Figure S1). On comparison of the NMR data of ring B in 1 with those reported for the analogue 5,7,30 ,50 -tetrahydroxyflavanonol (Tang et al. 2012), the two aromatic proton signals of three protons of ring B in the report were at dH 6.74 (H-20 and H-60 ) and 6.87 (H-40 ), while those of our assignment were at dH 6.71 (H-40 and H-60 ) and 6.84 (H-20 ). However, the assignment of the three corresponding carbons at dC 115.6 (C-20 and C-40 ) and dC 119.7 (C-60 ) agreed well with those of the reported values. Finally, the configurations of C-2 and C-3 were assigned as 2R and 3R resulting from a large coupling constants between H-2 and H-3 (J ¼ 11.2 Hz) and comparison of its specific rotation (þ 38) with the available specific rotation values reported for the related compounds, 5,7,30 ,40 ,50 -pentahydroxyflavanonol, (þ 41) (Tung et al. 2008) and taxifolin (þ 44) (Markham & Mabry 1968). On the basis of the above data, 1 was deduced as a new flavanonol, 7,30 ,50 -trihydroxy-5-methoxyflavanonol. Compound 2 was obtained as a white amorphous solid and the molecular formula (C17H16O7) was deduced from the molecular ion peak at m/z 355.0796 [M þ Na]þ by HR-ESITOF-MS (calcd for C16H14O7 þ Na, 355.0794), indicating 10 degrees of unsaturation. The UV spectrum showed absorption maxima at 286 nm. The IR spectrum showed the presence of hydroxyl (3499 cm21), conjugated ketone (1658 cm21) and aromatic (1592 cm21) groups as in
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1. The 1H and 13C NMR spectra of 2 were similar to those of the analogue 7,40 ,30 -trihydroxy-5methoxyflavanonol reported in the literature (Foo 1987), except that the hydroxyl group at C-30 was displaced by a methoxy group. The 1H NMR spectrum of ring B showed 1,3,4-trisubstituted aromatic at dH 7.05 (1H, d, J ¼ 1.5 Hz, H-20 ), 6.87 (1H, dd, J ¼ 1.5, 8.0 Hz, H-60 ) and d 6.76 (1H, d, J ¼ 8.0 Hz, H-50 ), and methoxy group at dH 3.75 (s). The 13C NMR and HSQC spectra indicated the resonance signals of aromatic ring B at dC 128.8 (C-10 ), 112.6 (C-20 ), 147.7 (C-30 ), 147.3 (C-40 ), 115.4 (C-50 ) and 121.4 (C-60 ). The HMBC spectrum showed correlations of H-20 with C-2, C-10 , C-40 and C-60 ; H-50 with C-30 and C-10 ; H-60 with C-2, C-20 and C-40 ; and methoxy protons at d 3.75 with C-30 confirming the substituted groups on ring B (Figure S2). The configurations of C-2 and C-3 were 2R and 3R resulting from large coupling constants between H-2 and H-3 (J ¼ 11.4 Hz) and comparing its specific rotation (þ 41) with those of the related compounds, 5,7,30 ,40 ,50 -pentahydroxyflavanonol, (þ 41) (Tung et al. 2008) and taxifolin (þ 44) (Markham & Mabry 1968). On the basis of the above data, 2 was deduced as a new flavanonol, 7,40 -dihydroxy-5,30 -dimethoxyflavanonol. 3. Experimental 3.1. General experimental procedures Melting points were determined using Electrothermal IA9200 digital melting point apparatus (Bibby Scientific Limited, Staffordshire, UK). Optical rotations were measured on a JASCO DIP-1000 digital polarimeter (JASCO Inc., USA) and UV spectra were recorded using an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). IR spectra were obtained using a Bruker Tenser 27 spectrophotometer (Bruker, Germany). NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer using (Varian Inc., USA) CDCl3, CD3OD and DMSO-d6 as solvents. The internal standards were referenced from the residue of those solvents. The HR-ESI-TOF-MS were recorded on a Bruker micrOTOF mass spectrometer (Brucker, Germany). Column chromatography was carried out on MERCK silica gel 60 (230 – 400 mesh) (Merck, Darmstadt, Germany). Thin-layer chromatography was carried out with pre-coated MERCK silica gel 60 PF254 (Merck, Darmstadt, Germany); the spots were visualised under UV light (254 and 365 nm) and further by spraying with anisaldehyde and then heating until charred. 3.2. Plant material The bark of A. Godefroyanum was collected from Ubolratana district, Khon Kaen province, Thailand, in April 2012 and was identified by Prof. Pranom Chantaranothai, Department of Biology, Khon Kaen University, Thailand, where a voucher specimen (voucher number S. Kanokmedhakul-13) has been deposited. 3.3. Extraction and isolation Air-dried bark of A. godefroyanum (4.36 kg) was ground and extracted successively with n-hexane (3 £ 15.5 L), EtOAc (3 £ 15.5 L) and MeOH (3 £ 15.5 L) at room temperature. Removal of solvents from each extract under reduced pressure gave crude n-hexane (19.5 g, 0.45%), EtOAc (29.1 g, 0.67%) and MeOH (40.1 g, 0.92%) extracts, respectively. The n-hexane extract (19.5 g) was dissolved with n-hexane and the precipitate was filtered out to obtain a white powder of lupeol (8) (197.0 mg, 1.01%). The filtrate was evaporated and the residue (19.1 g) was then separated on a silica gel flash column chromatography (FCC) gradient eluting with n-hexane, EtOAc – n-hexane, EtOAc, MeOH – EtOAc and MeOH by increasing polarity to give five fractions, HF1 – HF5. Fraction HF4 was subjected to silica gel FCC, gradually
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eluted with EtOAc – n-hexane (5:95) by increasing polarity to afford colourless needles of stigmasterol (9) (136.1 mg, 0.70%). The EtOAc extract (29.1 g) was subjected to silica gel FCC, eluted with a gradient system of n-hexane, EtOAc – n-hexane, EtOAc, MeOH –EtOAc and MeOH, to provide nine fractions, EF1 – EF9. Fraction EF3 was then separated on silica gel FCC, eluted with a gradient system of n-hexane, EtOAc – n-hexane, EtOAc, MeOH –EtOAc and MeOH, to give four subfrations, EF3.1 – EF3.4. Subfraction EF3.2 was purified by silica FCC using EtOAc – n-hexane (55:45) as eluent to afford a yellow amorphous powder of geraldol (3) (17.0 mg, 0.06%). Subfraction EF3.3 was recrystallised with EtOAc–n-hexane (50:50) to give a yellow amorphous powder of (þ)-taxifolin (4) (131.2 mg, 0.45%). Subfraction EF3.4 was further separated by silica FCC using EtOAc–nhexane (65:35) as an eluent to provide a white amorphous solid of (þ)-fustin (5) (25.6 mg, 0.09%). Fraction EF5 was purified on silica gel FCC, gradually eluted with EtOAc–n-hexane and MeOH to give a white solid of aromadendrin 5-methyl ether (6) (10.0 mg, 0.03%) and a white solid of 7,40 dihydroxy-5,30 -dimethoxyflavanonol (2) (28.3 mg, 0.10%). Fraction EF7 was purified on silica gel FCC, eluted with EtOAc–n-hexane, EtOAc, MeOH–EtOAc and MeOH to give a white amorphous solid of stigmasterol-3-O-b-D -glucoside (10) (12 mg, 0.04%). Fraction EF9 gave a pale yellow amorphous powder of 7,30 ,50 -trihydroxy-5-methoxyflavanonol (1) (10.0 mg, 0.03%). The MeOH extract (40.1 g) was separated on silica gel FCC gradient eluting with n-hexane, EtOAc – n-hexane, EtOAc, MeOH – EtOAc and MeOH by increasing polarity, to give seven fractions, MF1 – MF7. Fraction MF3 afforded a yellow brown amorphous solid of (– )-epigallocatechin (7) (21.1 mg, 0.05%). 7,30 ,50 -Trihydroxy-5-methoxyflavanonol (1): pale yellow amorphous powder; m.p. ¼ 246– 2478C; [a ]25 D þ 38 (c 0.1, MeOH); Rf: 0.38 (CHCl3 – MeOH, 9:1); UV lmax (log 1): 286 (4.01), 362 (0.99); IR (neat) nmax (cm21): 3447, 2975, 2933, 1652 (shoulder), 1627, 1589, 1514, 1463, 1383, 1352, 1286, 1244, 1215, 1165, 1107, 997, 808, 774, 665; HR-ESI-TOF-MS: m/z 341.0629 [M þ Na]þ (calcd for C16H14O7 þ Na, 341.0637); 1H NMR (400 MHz, DMSO-d6): d 6.84 (1H, brs, H-20 ), 6.71 (2H, brs, H-40 , 60 ), 6.06 (1H, d, J ¼ 1.6 Hz, H-6), 5.91 (1H, d, J ¼ 1.6 Hz, H-8), 5.19 (1H, brs, OH-3), 4.85 (1H, d, J ¼ 11.2 Hz, H-2), 4.22 (1H, d, J ¼ 11.2 Hz, H-3), 3.73 (3H, s, OCH3-5); 13C NMR (100 MHz DMSO-d6) d 190.3 (C-4), 165.2 (C-7), 164.2 (C-9), 162.6 (C-5), 146.1 (C-30 ), 145.4 (C-50 ), 128.8 (C-10 ), 119.7 (C-60 ), 115.6 (C-20 ), 115.6 (C-40 ), 102.9 (C-10), 95.9 (C-8), 93.8 (C-6), 82.9 (C-2), 73.0 (C-3), 56.1 (OCH3-5). 7,40 -Dihydroxy-5,30 -dimethoxyflavanonol (2): white amorphous solid; m.p. ¼ 239 –2408C; 25 [a ]D þ 41 (c 0.1, MeOH); Rf: 0.51 (CHCl3 –MeOH, 9:1); UV lmax (log 1): 286 (4.46); IR (neat) nmax (cm21): 3499, 3183, 2944, 2852, 1658, 1592, 1515, 1462, 1346, 1270, 1237, 1205, 1161, 1132, 1105, 1029, 991, 831, 774, 663, 645; HR-ESI-TOF-MS: m/z 355.0796 [M þ Na]þ (calcd for C16H14O7 þ Na, 355.0794); 1H NMR (400 MHz, DMSO-d6): d 7.05 (1H, d, J ¼ 1.5 Hz, H20 ), 6.87 (1H, dd, J ¼ 1.5, 8.0 Hz, H-60 ), 6.76 (1H, d, J ¼ 8.0 Hz, H-50 ), 6.06 (1H, d, J ¼ 1.9 Hz, H-6), 5.91 (1H, d, J ¼ 1.9 Hz, H-8), 5.17 (1H, d, J ¼ 4.2 Hz, OH-3), 4.91 (1H, d, J ¼ 11.4 Hz, H-2), 4.36 (1H, dd, J ¼ 4.2, 11.4 Hz, H-3), 3.75 (3H, s, OCH3-30 ), 3.74 (3H, s, OCH3-5); 13C NMR (100 MHz DMSO-d6) d 190.4 (C-4), 165.1 (C-7), 164.2 (C-9), 162.5 (C-5), 147.7 (C-30 ), 147.3 (C-40 ), 128.8 (C-10 ), 121.4 (C-60 ), 115.4 (C-50 ), 112.6 (C-20 ), 102.9 (C-10), 95.9 (C-8), 93.8 (C-6), 83.1 (C-2), 72.8 (C-3), 56.1 (OCH3-5,30 ). 4. Conclusions The isolation of chemical constituents from the bark of ‘Chai Hin’ or A. Godefroyanum, a Thai herbal medicine, is reported for the first time. Two triterpenoids, lupeol (8) and stigmasterol (9), were isolated from the non-polar extract, while seven flavonoids and one sterol glucoside were isolated from the more polar extract. Among these, lupeol (8) (1.01%) was a major constituent while a flavanonol, (þ )-taxifolin (4) (0.45%), was a major constituent of all flavonoids isolated.
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In addition, 7,30 ,50 -trihydroxy-5-methoxyflavanonol (1) and 7,40 -dihydroxy-5,30 -dimethoxyflavanonol (2) were isolated as two new flavanonols. Supplementary material Supplementary Figures S1 –S20 are available online.
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Acknowledgements We are grateful for the financial support from the Development and Promotion of Science and Technology Talents Project (DPST), the Center for Innovation in Chemistry (PERCH-CIC) and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Advanced Functional Materials Cluster of Khon Kaen University.
References Alimova DF, Kuliev ZA, Vdovin AD. 2007. Catechins and proanthocyanidines from Alhagi pseudoalhagi. Chem Nat Compd. 43(3):326–327. Bilia AR, Catalano S, Pistelli L, Morelli I. 1993. Flavonoids from Pyracantha coccinea roots. Phytochemistry. 33 (6):1449–1452. Chuakul W, Saralamp P, Prathanturarug S. 2000. Siam Pi Saj Cha Ya Pruek. 3rd ed. Bangkok: Amarin Printing. p. 208. Foo LY. 1987. Configuration and conformation of dihydroflavonols from Acacia melanoxylon. Phytochemistry. 26:823–827. Jamal AK, Yaacob WA, Din LB. 2008. A chemical study on Phyllanthus reticulatus. J Phys Sci. 19(2):45–50. Kamboj A, Saluja AK. 2011. Isolation of stigmasterol and b-sitosterol from petroleum ether extract of aerial parts of Ageratum Conyzoides (ASTERACEAE). Int J Pharm Pharm Sci. 3(1):94–96. Kojima H, Sato N, Hatano A, Ogura H. 1990. Sterol glucosides from Prunella vulgaris. Phytochemistry. 29:2351– 2355. Kumar NS, Rajapaksha M. 2005. Separation of catechin constituents from five tea cultivars using high-speed countercurrent chromatography. J Chromatogr A. 1083(1–2):223– 228. Lee E, Moon B, Park Y, Hong S, Lee S, Lee Y, Lim Y. 2008. Effects of hydroxy and methoxy substituents on NMR data in flavonols. Bull Korean Chem Soc. 29(2):507–510. Lee I, Bae J, Kim T, Kwon OJ, Kim TH. 2011. Polyphenolic constituents from the aerial parts of Thymus quinquecostatus var. japonica collected on Ulleung Island. J Korean Soc Appl Biol Chem. 54(5):811–816. Markham KR, Mabry TJ. 1968. Structure and stereochemistry of two new dihydroflavonol glycosides. Tetrahedron. 24:823–827. Park K, Jung G, Lee K, Choi J, Choi M, Kim G, Jung H, Park H. 2004. Antimutagenic activity of flavonoids from the heartwood of Rhus verniciflua. J Ethnopharmacol. 90(1):73–79. Tang R, Qu X, Guan S, Xu P, Shi Y, Guo D. 2012. Chemical constituents of Spatholobus suberectus. Chin J Nat Med. 10(1):32–35. Tsimogiannis D, Samiotaki M, Panayotou G, Oreopoulou V. 2007. Characterization of flavonoid subgroups and hydroxy substitution by HPLC-MS/MS. Molecules. 12:593–606. Tung NH, Ding Y, Kim SK, Bae K, Kim YH. 2008. Total peroxyl radical-scavenging capacity of the chemical components from the stems of Acer tegmentosum Maxim. J Agric Food Chem. 26:10510– 10514.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Two new flavanonols from the bark of Akschindlium godefroyanum a
a
Nattapong Chaipukdee , Somdej Kanokmedhakul , Ratsami a
Lekphrom & Kwanjai Kanokmedhakul
a
a
Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand Published online: 20 Dec 2013.
To cite this article: Nattapong Chaipukdee, Somdej Kanokmedhakul, Ratsami Lekphrom & Kwanjai Kanokmedhakul (2014) Two new flavanonols from the bark of Akschindlium godefroyanum, Natural Product Research: Formerly Natural Product Letters, 28:3, 191-195, DOI: 10.1080/14786419.2013.866113 To link to this article: http://dx.doi.org/10.1080/14786419.2013.866113
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 3, 191–195, http://dx.doi.org/10.1080/14786419.2013.866113
Two new flavanonols from the bark of Akschindlium godefroyanum
Downloaded by [University of Newcastle (Australia)] at 21:37 03 October 2014
Nattapong Chaipukdee, Somdej Kanokmedhakul, Ratsami Lekphrom and Kwanjai Kanokmedhakul* Natural Products Research Unit, Department of Chemistry, Center of Excellence for Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand (Received 26 July 2013; final version received 4 November 2013) The first phytochemical investigation of the bark of Akschindlium godefroyanum (Kuntze) H. Ohashi (Fabaceae), a Thai herbal medicine, resulted in the isolation of two new flavanonols, 7,30 ,50 -trihydroxy-5-methoxyflavanonol (1) and 7,40 -dihydroxy-5,30 dimethoxyflavanonol (2), and eight known compounds comprising one flavonol, geraldol (3); three flavanonols, (þ )-taxifolin (4), (þ )-fustin (5) and aromadendrin 5-methyl ether (6); one catechin, ( – )-epigallocatechin (7); one triterpenoid, lupeol (8); one steroid, stigmasterol (9) and one steroid glycoside, stigmasterol-3-O-b-D -glucoside (10). Their structures were identified by spectroscopic methods. Keywords: Akschindlium godefroyanum; flavanonol; flavonol; catechin
1. Introduction Akschindlium godefroyanum (Kuntze) H. Ohashi (Fabaceae) is a shrub, 1.5 – 3 m in height, widely found in the lower zone of the northern, northeastern and southern parts of Thailand. It is called ‘Chai Hin’ in Thai and it is used as antiemetic in Thai herbal medicine (Chuakul et al. 2000). However, phytochemical study of the genus Akschindlium has not been reported. We report herein the first isolation and identification of the chemical constituents from the bark of A. godefroyanum. 2. Results and discussion Three crude extracts, crude n-hexane, crude EtOAc and crude MeOH, were obtained from the dried bark of A. godefroyanum. Chromatographic separation of these extracts gave two new flavanonols, 7,30 ,50 -trihydroxy-5-methoxyflavanonol (1) and 7,40 -dihydroxy-5,30 -dimethoxyflavanonol (2), and eight known compounds. The structures of the isolated compounds were identified using physical and spectroscopic data measurements ([a ]D, 1H NMR, 13C NMR and 2D NMR) as well as comparing the data with those reported in the literature as geraldol (3) (Lee et al. 2008), (þ )-taxifolin (4) (Lee et al. 2011), (þ )-fustin (5) (Park et al. 2004), aromadendrin 5-methyl ether (6) (Bilia et al. 1993), ( –)-epigallocatechin (7) (Kumar & Rajapaksha 2005; Alimova et al. 2007), lupeol (8) (Jamal et al. 2008), stigmasterol (9) (Kamboj & Saluja 2011) and stigmasterol-3-O-b-D -glucoside (10) (Kojima et al. 1990) (Figure 1). Compound 1 was obtained as a pale yellow powder and the molecular formula (C16H14O7) was deduced from the molecular ion peak at m/z 341.0629 [M þ Na]þ by HR-ESI-TOF-MS (calcd for C16H14O7 þ Na, 341.0637), indicating 10 degrees of unsaturation. The UV spectrum showed absorption maxima at 286 and 362 nm as those of the flavanonol skeleton (Tsimogiannis
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
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R3 5'
6'
R2
8 7 6
9
10
5
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R1
1 2 4 5 6
1'
C
A
B
2
O
3 4
OH R4
4' 3'
2'
HO
O
OH
OMe
R5
O
HO
OH
OH
3
OH OH
O
OH
7
O
R1
R2
OMe OMe HO H OMe
OH OH OH OH OH
R3 OH H H H H
R4 H OH OH OH OH
R5 OH OMe OH OH H
H
H H
H H
H HO
H
H
R 8
9 R = OH 10 R = O–β–D–glucosy
Figure 1. Structures of isolated compounds 1 –10.
et al. 2007). The IR spectrum showed the presence of hydroxyl (3447 cm21), conjugated ketone (1652 cm21) and aromatic (1589 cm21) groups. The 1H NMR spectrum of 1 showed signals for two oxygenated methines of ring C at d 4.85 (1H, d, J ¼ 11.2 Hz, H-2) and 4.22 (1H, d, J ¼ 11.2 Hz, H-3), two meta aromatic protons of ring A at d 6.06 (1H, d, J ¼ 1.6 Hz, H-6) and 5.91 (1H, d, J ¼ 1.6 Hz, H-8). Consistent with the above 1H NMR analysis, 13C NMR and HMQC spectra of 1 displayed the carbon signals for two oxygenated methine carbons at d 82.9 (C-2) and 73.0 (C-3), an aromatic ring A at d 162.6 (C-5), 93.8 (C-6), 165.2 (C-7), 95.9 (C-8), 164.2 (C-9) and 102.9 (C-10) which agreed well with those reported for the related compound 7,30 ,40 -trihydroxy-5-methoxy-dihydroflavanonol (Foo 1987). The two aromatic signals of ring B showed at dH 6.84 (1H, brs, H-20 ) and d 6.71 (2H, brs, H-40 and H-60 ) indicating 1,3,5trisubstituted aromatic, and the three protons were in meta positions. While its 13C NMR spectrum showed signals at dC 128.8 (C-10 ), 115.6 (C-20 ), 146.1 (C-30 ), 115.6 (C-40 ), 145.4 (C-50 ) and 119.7 (C-60 ). The HMBC spectrum of 1 exhibited the correlations of H-2 with carbonyl (C4), C-3, C-9, C-10 , C-20 and C-60 ; H-3 with C-2; H-6 with C-5, C-7, C-8 and C-10; H-8 with C-6, C-7, C-9 and C-10; H-20 with C-2, C-10 , C-30 , C-40 and C-60 ; H-40 with C-20 , C-30 , C-50 and C-60 ; H-60 with C-2, C-10 , C-20 , C-40 and C-50 ; and methoxy protons at d 3.73 with C-5 confirming the structure of 1 (Figure S1). On comparison of the NMR data of ring B in 1 with those reported for the analogue 5,7,30 ,50 -tetrahydroxyflavanonol (Tang et al. 2012), the two aromatic proton signals of three protons of ring B in the report were at dH 6.74 (H-20 and H-60 ) and 6.87 (H-40 ), while those of our assignment were at dH 6.71 (H-40 and H-60 ) and 6.84 (H-20 ). However, the assignment of the three corresponding carbons at dC 115.6 (C-20 and C-40 ) and dC 119.7 (C-60 ) agreed well with those of the reported values. Finally, the configurations of C-2 and C-3 were assigned as 2R and 3R resulting from a large coupling constants between H-2 and H-3 (J ¼ 11.2 Hz) and comparison of its specific rotation (þ 38) with the available specific rotation values reported for the related compounds, 5,7,30 ,40 ,50 -pentahydroxyflavanonol, (þ 41) (Tung et al. 2008) and taxifolin (þ 44) (Markham & Mabry 1968). On the basis of the above data, 1 was deduced as a new flavanonol, 7,30 ,50 -trihydroxy-5-methoxyflavanonol. Compound 2 was obtained as a white amorphous solid and the molecular formula (C17H16O7) was deduced from the molecular ion peak at m/z 355.0796 [M þ Na]þ by HR-ESITOF-MS (calcd for C16H14O7 þ Na, 355.0794), indicating 10 degrees of unsaturation. The UV spectrum showed absorption maxima at 286 nm. The IR spectrum showed the presence of hydroxyl (3499 cm21), conjugated ketone (1658 cm21) and aromatic (1592 cm21) groups as in
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1. The 1H and 13C NMR spectra of 2 were similar to those of the analogue 7,40 ,30 -trihydroxy-5methoxyflavanonol reported in the literature (Foo 1987), except that the hydroxyl group at C-30 was displaced by a methoxy group. The 1H NMR spectrum of ring B showed 1,3,4-trisubstituted aromatic at dH 7.05 (1H, d, J ¼ 1.5 Hz, H-20 ), 6.87 (1H, dd, J ¼ 1.5, 8.0 Hz, H-60 ) and d 6.76 (1H, d, J ¼ 8.0 Hz, H-50 ), and methoxy group at dH 3.75 (s). The 13C NMR and HSQC spectra indicated the resonance signals of aromatic ring B at dC 128.8 (C-10 ), 112.6 (C-20 ), 147.7 (C-30 ), 147.3 (C-40 ), 115.4 (C-50 ) and 121.4 (C-60 ). The HMBC spectrum showed correlations of H-20 with C-2, C-10 , C-40 and C-60 ; H-50 with C-30 and C-10 ; H-60 with C-2, C-20 and C-40 ; and methoxy protons at d 3.75 with C-30 confirming the substituted groups on ring B (Figure S2). The configurations of C-2 and C-3 were 2R and 3R resulting from large coupling constants between H-2 and H-3 (J ¼ 11.4 Hz) and comparing its specific rotation (þ 41) with those of the related compounds, 5,7,30 ,40 ,50 -pentahydroxyflavanonol, (þ 41) (Tung et al. 2008) and taxifolin (þ 44) (Markham & Mabry 1968). On the basis of the above data, 2 was deduced as a new flavanonol, 7,40 -dihydroxy-5,30 -dimethoxyflavanonol. 3. Experimental 3.1. General experimental procedures Melting points were determined using Electrothermal IA9200 digital melting point apparatus (Bibby Scientific Limited, Staffordshire, UK). Optical rotations were measured on a JASCO DIP-1000 digital polarimeter (JASCO Inc., USA) and UV spectra were recorded using an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). IR spectra were obtained using a Bruker Tenser 27 spectrophotometer (Bruker, Germany). NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer using (Varian Inc., USA) CDCl3, CD3OD and DMSO-d6 as solvents. The internal standards were referenced from the residue of those solvents. The HR-ESI-TOF-MS were recorded on a Bruker micrOTOF mass spectrometer (Brucker, Germany). Column chromatography was carried out on MERCK silica gel 60 (230 – 400 mesh) (Merck, Darmstadt, Germany). Thin-layer chromatography was carried out with pre-coated MERCK silica gel 60 PF254 (Merck, Darmstadt, Germany); the spots were visualised under UV light (254 and 365 nm) and further by spraying with anisaldehyde and then heating until charred. 3.2. Plant material The bark of A. Godefroyanum was collected from Ubolratana district, Khon Kaen province, Thailand, in April 2012 and was identified by Prof. Pranom Chantaranothai, Department of Biology, Khon Kaen University, Thailand, where a voucher specimen (voucher number S. Kanokmedhakul-13) has been deposited. 3.3. Extraction and isolation Air-dried bark of A. godefroyanum (4.36 kg) was ground and extracted successively with n-hexane (3 £ 15.5 L), EtOAc (3 £ 15.5 L) and MeOH (3 £ 15.5 L) at room temperature. Removal of solvents from each extract under reduced pressure gave crude n-hexane (19.5 g, 0.45%), EtOAc (29.1 g, 0.67%) and MeOH (40.1 g, 0.92%) extracts, respectively. The n-hexane extract (19.5 g) was dissolved with n-hexane and the precipitate was filtered out to obtain a white powder of lupeol (8) (197.0 mg, 1.01%). The filtrate was evaporated and the residue (19.1 g) was then separated on a silica gel flash column chromatography (FCC) gradient eluting with n-hexane, EtOAc – n-hexane, EtOAc, MeOH – EtOAc and MeOH by increasing polarity to give five fractions, HF1 – HF5. Fraction HF4 was subjected to silica gel FCC, gradually
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eluted with EtOAc – n-hexane (5:95) by increasing polarity to afford colourless needles of stigmasterol (9) (136.1 mg, 0.70%). The EtOAc extract (29.1 g) was subjected to silica gel FCC, eluted with a gradient system of n-hexane, EtOAc – n-hexane, EtOAc, MeOH –EtOAc and MeOH, to provide nine fractions, EF1 – EF9. Fraction EF3 was then separated on silica gel FCC, eluted with a gradient system of n-hexane, EtOAc – n-hexane, EtOAc, MeOH –EtOAc and MeOH, to give four subfrations, EF3.1 – EF3.4. Subfraction EF3.2 was purified by silica FCC using EtOAc – n-hexane (55:45) as eluent to afford a yellow amorphous powder of geraldol (3) (17.0 mg, 0.06%). Subfraction EF3.3 was recrystallised with EtOAc–n-hexane (50:50) to give a yellow amorphous powder of (þ)-taxifolin (4) (131.2 mg, 0.45%). Subfraction EF3.4 was further separated by silica FCC using EtOAc–nhexane (65:35) as an eluent to provide a white amorphous solid of (þ)-fustin (5) (25.6 mg, 0.09%). Fraction EF5 was purified on silica gel FCC, gradually eluted with EtOAc–n-hexane and MeOH to give a white solid of aromadendrin 5-methyl ether (6) (10.0 mg, 0.03%) and a white solid of 7,40 dihydroxy-5,30 -dimethoxyflavanonol (2) (28.3 mg, 0.10%). Fraction EF7 was purified on silica gel FCC, eluted with EtOAc–n-hexane, EtOAc, MeOH–EtOAc and MeOH to give a white amorphous solid of stigmasterol-3-O-b-D -glucoside (10) (12 mg, 0.04%). Fraction EF9 gave a pale yellow amorphous powder of 7,30 ,50 -trihydroxy-5-methoxyflavanonol (1) (10.0 mg, 0.03%). The MeOH extract (40.1 g) was separated on silica gel FCC gradient eluting with n-hexane, EtOAc – n-hexane, EtOAc, MeOH – EtOAc and MeOH by increasing polarity, to give seven fractions, MF1 – MF7. Fraction MF3 afforded a yellow brown amorphous solid of (– )-epigallocatechin (7) (21.1 mg, 0.05%). 7,30 ,50 -Trihydroxy-5-methoxyflavanonol (1): pale yellow amorphous powder; m.p. ¼ 246– 2478C; [a ]25 D þ 38 (c 0.1, MeOH); Rf: 0.38 (CHCl3 – MeOH, 9:1); UV lmax (log 1): 286 (4.01), 362 (0.99); IR (neat) nmax (cm21): 3447, 2975, 2933, 1652 (shoulder), 1627, 1589, 1514, 1463, 1383, 1352, 1286, 1244, 1215, 1165, 1107, 997, 808, 774, 665; HR-ESI-TOF-MS: m/z 341.0629 [M þ Na]þ (calcd for C16H14O7 þ Na, 341.0637); 1H NMR (400 MHz, DMSO-d6): d 6.84 (1H, brs, H-20 ), 6.71 (2H, brs, H-40 , 60 ), 6.06 (1H, d, J ¼ 1.6 Hz, H-6), 5.91 (1H, d, J ¼ 1.6 Hz, H-8), 5.19 (1H, brs, OH-3), 4.85 (1H, d, J ¼ 11.2 Hz, H-2), 4.22 (1H, d, J ¼ 11.2 Hz, H-3), 3.73 (3H, s, OCH3-5); 13C NMR (100 MHz DMSO-d6) d 190.3 (C-4), 165.2 (C-7), 164.2 (C-9), 162.6 (C-5), 146.1 (C-30 ), 145.4 (C-50 ), 128.8 (C-10 ), 119.7 (C-60 ), 115.6 (C-20 ), 115.6 (C-40 ), 102.9 (C-10), 95.9 (C-8), 93.8 (C-6), 82.9 (C-2), 73.0 (C-3), 56.1 (OCH3-5). 7,40 -Dihydroxy-5,30 -dimethoxyflavanonol (2): white amorphous solid; m.p. ¼ 239 –2408C; 25 [a ]D þ 41 (c 0.1, MeOH); Rf: 0.51 (CHCl3 –MeOH, 9:1); UV lmax (log 1): 286 (4.46); IR (neat) nmax (cm21): 3499, 3183, 2944, 2852, 1658, 1592, 1515, 1462, 1346, 1270, 1237, 1205, 1161, 1132, 1105, 1029, 991, 831, 774, 663, 645; HR-ESI-TOF-MS: m/z 355.0796 [M þ Na]þ (calcd for C16H14O7 þ Na, 355.0794); 1H NMR (400 MHz, DMSO-d6): d 7.05 (1H, d, J ¼ 1.5 Hz, H20 ), 6.87 (1H, dd, J ¼ 1.5, 8.0 Hz, H-60 ), 6.76 (1H, d, J ¼ 8.0 Hz, H-50 ), 6.06 (1H, d, J ¼ 1.9 Hz, H-6), 5.91 (1H, d, J ¼ 1.9 Hz, H-8), 5.17 (1H, d, J ¼ 4.2 Hz, OH-3), 4.91 (1H, d, J ¼ 11.4 Hz, H-2), 4.36 (1H, dd, J ¼ 4.2, 11.4 Hz, H-3), 3.75 (3H, s, OCH3-30 ), 3.74 (3H, s, OCH3-5); 13C NMR (100 MHz DMSO-d6) d 190.4 (C-4), 165.1 (C-7), 164.2 (C-9), 162.5 (C-5), 147.7 (C-30 ), 147.3 (C-40 ), 128.8 (C-10 ), 121.4 (C-60 ), 115.4 (C-50 ), 112.6 (C-20 ), 102.9 (C-10), 95.9 (C-8), 93.8 (C-6), 83.1 (C-2), 72.8 (C-3), 56.1 (OCH3-5,30 ). 4. Conclusions The isolation of chemical constituents from the bark of ‘Chai Hin’ or A. Godefroyanum, a Thai herbal medicine, is reported for the first time. Two triterpenoids, lupeol (8) and stigmasterol (9), were isolated from the non-polar extract, while seven flavonoids and one sterol glucoside were isolated from the more polar extract. Among these, lupeol (8) (1.01%) was a major constituent while a flavanonol, (þ )-taxifolin (4) (0.45%), was a major constituent of all flavonoids isolated.
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In addition, 7,30 ,50 -trihydroxy-5-methoxyflavanonol (1) and 7,40 -dihydroxy-5,30 -dimethoxyflavanonol (2) were isolated as two new flavanonols. Supplementary material Supplementary Figures S1 –S20 are available online.
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Acknowledgements We are grateful for the financial support from the Development and Promotion of Science and Technology Talents Project (DPST), the Center for Innovation in Chemistry (PERCH-CIC) and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Advanced Functional Materials Cluster of Khon Kaen University.
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