This article was downloaded by: [Umeå University Library] On: 18 November 2014, At: 20:18 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20

A new flavonoid from the aerial parts of Andrographis paniculata ab

ab

ab

ab

Li-Xia Chen , Hao He , Gui-Yang Xia , Kai-Lan Zhou Qiu

& Feng

ab

a

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China b

Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China Published online: 18 Nov 2013.

To cite this article: Li-Xia Chen, Hao He, Gui-Yang Xia, Kai-Lan Zhou & Feng Qiu (2014) A new flavonoid from the aerial parts of Andrographis paniculata, Natural Product Research: Formerly Natural Product Letters, 28:3, 138-143, DOI: 10.1080/14786419.2013.856907 To link to this article: http://dx.doi.org/10.1080/14786419.2013.856907

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 &

Downloaded by [Umeå University Library] at 20:18 18 November 2014

Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions

Natural Product Research, 2014 Vol. 28, No. 3, 138–143, http://dx.doi.org/10.1080/14786419.2013.856907

A new flavonoid from the aerial parts of Andrographis paniculata Li-Xia Chena,b, Hao Hea,b, Gui-Yang Xiaa,b, Kai-Lan Zhoua,b and Feng Qiua,b* a School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China; bKey Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China

Downloaded by [Umeå University Library] at 20:18 18 November 2014

(Received 11 August 2013; final version received 15 October 2013) A new flavonoid, 7,8-dimethoxy-20 -hydroxy-5-O-b-D -glucopyranosyloxyflavone (1), along with 15 known flavonoids (2 – 16), was isolated from the aerial parts of Andrographis paniculata Nees. Their structures were elucidated on the basis of chemical and spectroscopic analyses. Most of them have uncommon O-substitution patterns involving 5-, 7-, 8-, 20 -, 30 -, 40 - and 50 -O-substituents. The antiproliferative effects of these flavonoids against human leukaemia HL-60 cells were investigated. Among them, 13 was the most active, displaying potent antiproliferative activity with IC50 of 3.50 mM. The structure – activity relationships of these isolated compounds were discussed. Keywords: Andrographis paniculata; flavonoids; antiproliferative effects; structure – activity relationships

1. Introduction Andrographis paniculata Nees. (Acanthaceae) is an erect herb widely distributed in southeast of China. The entire plant has been used as an anti-inflammatory and antipyretic agent for the treatment of fever, cold, laryngitis, diarrhea and inflammatory disease (Chang & But 1987). The crude extract of A. paniculata and its major constituents were reported to display a number of biological activities such as antiviral (Yao et al. 1992), anti-bacterial (George & Pandalai 1949), immunostimulatory (Puri et al. 1993), hepatoprotective (Zeha & Rawal 2001) and anti-cancer effects (Matsuda et al. 1994). Phytochemical investigations have shown that ent-labdane diterpenoid lactones (Matsuda et al. 1994; Chen et al. 2006; Chen et al. 2008) and flavonoids (Jalal et al. 1979; Gupta et al. 1983; Kuroyanagi et al. 1987; Gupta et al. 1996) are the main components in A. paniculata. Our previous research found that ent-labdane diterpenoid lactones from A. paniculata exhibited antiproliferative activity against human leukaemia HL-60 cells (Chen et al. 2008). As the continuous study on bioactive components of this plant, we investigated the other type of major components, flavonoids and their antiproliferative effects against human leukaemia HL-60 cells. In this work, one new flavonoid (1), along with 15 known ones (2–16), was obtained from the aerial parts of this plant, and some of them showed antiproliferative activity against HL-60 cells.

2. Results and discussion Compound 1 was isolated as pale yellow solid. The maximal absorptions at 263 and 339 nm of the UV spectrum and a positive Molisch test revealed a flavone glycoside. The positive

*Corresponding author. Email: [email protected] q 2013 Taylor & Francis

Downloaded by [Umeå University Library] at 20:18 18 November 2014

Natural Product Research

139

HR-ESI-MS analysis (found 477.1389 [M þ H]þ, calc. 477.1391) established a molecular formula of C23H24O11, in combination with the 1H and 13C NMR data. Compound 1 has one hexose according to the positive-ion ESI-MS data at m/z 499 [M þ Na]þ and 337 [M þ Na2 162]þ. A sharp one-proton singlet at d 7.05 in the 1H NMR spectrum was the characteristic H-3 signal of a 20 -oxygenated flavone (Tanaka et al. 1986). The other sharp one-proton singlet at d 7.17 was attributed to H-6, which was further substantiated by the HMBC correlations observed from H-6 to C-5 (d 153.3) and C-8 (d 132.3). The 1H NMR spectrum exhibited two methoxyl singlets at d 3.93 and 3.87, which were determined to be linked with C-7 and C-8, on the basis of the HMBC correlations of the two methoxyl signals with C-7 (d 156.8) and C-8 (d 132.3), respectively. The NOE correlation between the methoxyl signal at d 3.93 and H-6 (d 7.17) in the NOESY spectrum further corroborated the above assignments. After hydrolysis with b-D -glucosidase (Waridel et al. 2004), a sugar and an aglycone were obtained from compound 1, in combination with the coupling constant of the anomeric proton at d 4.73 (1H, d, J ¼ 7.8 Hz), indicated that b-D -glucose was present in compound 1. The HMBC correlation of anomeric proton at d 4.73 with C-5 (d 153.3) and the NOE correlation between anomeric proton at d 4.73 and H-6 (d 7.17) suggested that the sugar unit was linked to C-5. The 1 H NMR spectrum also showed four B-ring aromatic protons at d 7.07 (1H, dd, J ¼ 8.4, 1.8 Hz), 7.41 (1H, ddd, J ¼ 8.4, 7.8, 1.8 Hz), 7.03 (1H, dd, J ¼ 7.8, 1.8 Hz) and 7.88 (1H, dd, J ¼ 7.8, 1.8 Hz), which are the characteristic signals of a 20 -oxygenated flavone (Gupta et al. 1983). According to the molecular formula of C23H24O11 and the above determined groups, one hydroxyl moiety was remained, which should be attached to C-20 . The signal assignments in B-ring were confirmed by the HMBC and NOESY spectra. Based on these above parameters, the structure of compound 1 was elucidated as 7,8-dimethoxy-20 -hydroxy-5-O-b-D -glucopyranosyloxyflavone as shown in Figure 1. Fifteen known flavonoids were identified as 7-O-methylwogonin (2) (Kuroyanagi et al. 1987), 7-O-methyl-dihydrowogonin (3) (Gupta et al. 1983), 5-hydroxy-7, 8,20 ,50 tetramethoxyflavone (4) (Vijaya et al. 2003), skullcapflavone-20 -methoxylether (5) (Kuroyanagi et al. 1987), 5-hydroxy-7,8, 20 ,30 -tetramethoxyflavone (6) (Kuroyanagi et al. 1987), 5,40 -dihydroxy-7,8, 20 ,30 -tetramethoxyflavone (7) (Kuroyanagi et al. 1987), 5,40 dihydroxy-7-methoxy-8-O-b-D -glucopyranosyloxyflavone (8) (Saxena & Jain 2003), 5,7,8trimethoxydihydroflavone (9) (Kuroyanagi et al. 1987), andrographidine A (10) (Kuroyanagi et al. 1987), andrographidine C (11) (Kuroyanagi et al. 1987), apigenin (12) (Deng et al. 2006), luteolin (13) (Ma et al. 2006), 7,8,20 ,50 -tetramethoxy-5-O-b-D -glucopyranosyloxyflavone (14) (Li et al. 2007), 5,40 -dihydroxy-7-O-b-D -pyranglycuronate butyl ester (15) (Zheng et al. 2006) and 5,40 -dihydroxy-7-O-b-D -glucopyranosyloxyflavone (16) (Ren & Yang 2001), respectively, by comparison of their spectroscopic data (NMR and MS) with those reported in literatures. To the best of our knowledge, compound 11 is reported for the first time in the genus Andrographis. The antiproliferative effects of the flavonoids isolated were determined against human leukaemia HL-60 cells by the methods reported previously (Wang et al. 2006). However, the activities of compounds 4, 6 and 7 were not determined due to their poor solubility that probably resulted from the presence of four methoxyl groups which decreased the polarities of these compounds. The antiproliferative activities of other 13 compounds were shown in Table 1. Among these tested flavonoids, compound 13 was the most active with an IC50 of 3.50 mM, which indicated that the presence of one hydroxyl group substitution at C-5 in A-ring and two hydroxyl group substitutions at C-30 and C-40 in B-ring might play an important role in improving the antiproliferative activities. Compounds 2, 5, 12 and 15 showed modestly antiproliferative effects with the IC50 of 10-20 mM. This result further confirmed that the hydroxyl group substitution at C-5 in A-ring was necessary for inhibiting HL-60 cell growth. Since compounds 2 and 3 with a skeleton of flavone and flavonone,

Downloaded by [Umeå University Library] at 20:18 18 November 2014

140

L.-X. Chen et al.

Figure 1. Structures of compounds 1 – 16 isolated from Andrographis paniculata.

respectively, showed similar activities, the skeleton of flavone or flavonone probably was not a major influence on the activity. The activities of compounds 10, 11 and 16 with a sugar moiety in their structures were decreased in comparison with those of their aglycones 3, 2 and 12, respectively. The formation of glycuronate ester could, however, increase the antiproliferative effects of flavonoid glycosides, just as the activity difference showed between 15 and 16. Table 1. The antiproliferative effects against HL-60 cells of flavonoids from Andrographis paniculata.a – c Compound 1 2 3 5 8 9 10 a b c

IC50 ^ SE (mM)

Compound

IC50 ^ SE (mM)

38.34 ^ 1.15 17.62 ^ 0.38 23.22 ^ 0.37 10.34 ^ 0.55 34.29 ^ 1.38 20.57 ^ 0.43 50.04 ^ 0.76

11 12 13 14 15 16 Adriamycin

47.34 ^ 1.31 10.63 ^ 0.37 3.50 ^ 0.25 52.01 ^ 0.85 14.79 ^ 1.11 45.78 ^ 0.63 0.018 ^ 0.46

The data shown are the mean ^ SE of triplicate analyses. HL-60 cells were treated with the indicated compound at different concentrations for 3 days. Adriamycin was used as positive control.

Natural Product Research

141

Downloaded by [Umeå University Library] at 20:18 18 November 2014

3. Experimental 3.1. General The UV spectra were taken on a UV-1201 Shimadzu spectrometer. IR spectra were measured with a Bruker IFS 55 spectrometer. The NMR spectra were recorded on a Bruker ARX-600 spectrometer (600 MHz for 1H and 150 MHz for 13C) in DMSO-d6 with TMS as an internal standard. HR-ESI-MS were obtained on a Bruker APEX II mass spectrometer, and ESI-MS were recorded on an Agilent Series 1100 SL mass spectrometer. Preparative HPLC system consisted of a LC-6AD intelligent prep. pump, SPD-20A intelligent UV/vis detector and YMCPark ODS-A column (5 mm, f 20 £ 250 mm, YMC Co. Ltd., Kyoto, Japan). Analytical HPLC system consisted of a Waters-600 controller pump, YMC column, waters-2400 and 996 photodiode array detectors. All the reagents were HPLC grade or analytical grade and purchased from Shenyang Chemical Company (Shenyang, China). Silica gel G60 and GF254 for thin-layer chromatography were obtained from Qingdao Marine Chemical Factory (Qingdao, China). Sephadex LH-20 was purchased from Pharmacia (Peapack, NJ, USA) and was used for molecular exclusion chromatography.

3.2. Plant material Dried aerial parts of A. paniculata Nees. were collected from Fujian Province, China. The voucher specimen (AP-2003-824) was identified by Professor Qishi Sun and has been deposited in the Department of Natural Products Chemistry, Shenyang Pharmaceutical University, China.

3.3. Extraction and isolation The plant material (10 kg) was cut into small pieces and extracted successively with 85% ethanol (80 L) for three times under reflux to yield an ethanol extract. The ethanol extract was concentrated in vacuo, suspended in water, and then partitioned thoroughly with cyclohexane, ethyl acetate and n-butanol, respectively. The ethyl acetate extract (295 g) was subjected to silica gel chromatography (10 £ 120 cm) using a gradient mixture of CHCl3 – MeOH as eluent. It yielded eight fractions. The eight fractions (E1 –E8) were subjected to a Sephadex LH-20 column (3 £ 70 cm) eluted with CHCl3 –MeOH (1:1) to remove the chlorophyll. Then, fraction E1 yielded compounds 2 (46.5 mg), 3 (23.8 mg), 4 (25.6 mg), and 5 (8.7 mg) after purification with a Si gel column (6 £ 80 cm) eluted with the mixture of cyclohexane and ethyl acetate. Fraction E2 yielded compounds 6 (12.5 mg) and 7 (9.6 mg) after a series of Si gel chromatographic separations (6 £ 80 cm, 3 £ 40 cm) with the mixtures of cyclohexane and ethyl acetate as eluent. Fraction E3 was subjected to silica gel column chromatography (Si gel CC) (8 £ 100 cm) with elution in step gradients with mixtures of cyclohexane and acetone to give compounds 9 (10.8 mg), 11 (3.5 mg) and 12 (10.3 mg). Fraction E6 was purified with repeated Si gel CC (6 £ 80 cm, 3 £ 40 cm) using CHCl3 –MeOH in step gradients and Prep-HPLC eluted with MeOH –H2O (55:45) to afford compounds 14 (19.2 mg), 10 (6.7 mg) and 13 (12.6 mg). Fraction E8 was subjected to Si gel CC (6 £ 80 cm) eluting with CHCl3 – MeOH in step gradients, and then purified with Prep-HPLC eluted with MeOH – H2O (40:60) to yield compound 8 (15.7 mg). The n-butanol extract (195 g) was subjected to silica gel chromatography (8 £ 100 cm) using a gradient mixture of CHCl3 – CH3OH as the eluent to obtain five fractions (B1 – B5). Fraction B3 (15.2 g) was fractionated by chromatography on Sephadex LH-20 (3 £ 80 cm) with MeOH – H2O (50:50) as eluent and further purified by Prep-HPLC using MeOH –H2O (35:65) to give compounds 15 (4.0 mg), 16 (11.3 mg) and 1 (11.5 mg).

142

L.-X. Chen et al.

Downloaded by [Umeå University Library] at 20:18 18 November 2014

7,8-Dimethoxy-20 -hydroxy-5-O-b-D -glucopyranosyloxyflavone (1): a pale yellow solid 21 (MeOH). UV lmax (MeOH) nm (log 1): 263 (4.28), 339 (4.02); IR nKBr max cm : 3434, 1650, 1611, 1 1575; H NMR (600 MHz, DMSO-d6): d 7.05 (1H, s, H-3), 7.17 (1H, s, H-6), 7.07 (1H, dd, J ¼ 8.4, 1.8 Hz, H-30 ), 7.41 (1H, ddd, J ¼ 8.4, 7.8, 1.8 Hz, H-40 ), 7.03 (1H, dd, J ¼ 7.8, 1.8 Hz, H-50 ), 7.88 (1H, dd, J ¼ 7.8, 1.8 Hz, H-60 ), 3.93 (3H, s, OCH3-7), 3.87 (3H, s, OCH3-8), 4.73 (1H, d, J ¼ 7.8 Hz, H-100 ), 3.35 (1H, m, H-200 ), 3.29 (1H, m, H-300 ), 3.14 (1H, m, H-400 ), 3.40 (1H, m, H-500 ), 3.75 (1H, dd, J ¼ 12.0, 6.6 Hz, H-600 a), 3.50 (1H, m, H-600 b); 13C NMR (150 MHz, DMSO-d6): d 159.0 (C-2), 111.6 (C-3), 177.6 (C-4), 153.3 (C-5), 101.2 (C-6), 156.8 (C-7), 132.3 (C-8), 150.5 (C-9), 109.2 (C-10), 117.1 (C-10 ), 156.3 (C-20 ), 117.3 (C-30 ), 132.6 (C-40 ), 119.5 (C-50 ), 128.1(C-60 ), 56.3 (OCH3-7), 61.1 (OCH3-8), 104.6 (C-100 ), 73.5 (C-200 ), 76.0 (C-300 ), 70.2 (C-400 ), 77.7 (C-500 ), 61.1 (C-600 ); ESI-MS m/z: 499 [M þ Na]þ, 337 [M þ Na 2 162]þ; HR-ESI-MS m/z: 477.1389 [M þ H]þ (calc. C23H25O11, 477.1391). 3.4. Hydrolysis of compound 1 Compound 1 (2 mg) was dissolved in acetate buffer at pH 5.5 (1 mL) and incubated for 24 h at 378C with the addition of 4 mg of b-D -glucosidase purchased from Sigma (St Louis, MO, USA). The reaction solution was extracted with ethyl acetate (5 mL) and the aglycone of compound 1 was obtained after solvent evaporation. 3.5. Antiproliferative assay Human leukaemia HL-60 cells were cultured in RPMI-1640 medium supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, 1 mM L -glutamine and 10% faetal bovine serum. Cells in logarithmic growth were seeded at 8 £ 104 cells/mL into 24-well plates and were treated with the various compounds for 3 days. The total cell number of each well was determined with the aid of a hemocytometer. A half growth inhibitory concentration (IC50) was obtained by regression analysis of the concentration response data. Studies were performed in triplicate. 4. Conclusions In summary, 16 flavonoids including 1 new compound were isolated from the aerial parts of A. paniculata Nees. Most of them have uncommon O-substitution patterns involving 5-, 7-, 8-, 20 -, 30 -, 40 -, and 50 -O-substituents, which is the characteristics of flavonoids isolated from A. paniculata (Kuroyanagi et al. 1987). The present work, along with our previous research results (Chen et al. 2008) explained somewhat the anti-cancer effects of crude extract of A. paniculata, and revealed that the major pharmacologically active compounds of A. paniculata were perhaps the aglycones of ent-labdane diterpenoid lactones and flavonoids. These active ingredients have the potential to be developed into effective anti-cancer agents. Supplementary material Spectral data relating to this article are available online. Acknowledgements The authors are grateful to the Project of National Natural Science Foundation of China (No. 81202426), Shenyang Planning Project of Science and Technology (No. F10-205-1-38) and Program for Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative Research Team in the University for the financial support.

Natural Product Research

143

Downloaded by [Umeå University Library] at 20:18 18 November 2014

References Chang HM, But PPH. 1987. Pharmacology and application of Chinese material medica. Vol. 1. Singapore: World Scientific. p. 918–928. Chen LX, Qiu F, Qu GX, Yao XS. 2006. Nine new ent-labdane diterpenoid lactones and their derivatives from the aerial parts of Andrographis paniculata. Helv Chim Acta. 89:2654–2664. Chen LX, Zhu HJ, Wang R, Zhou KL, Jing YK, Qiu F. 2008. ent-Labdane diterpenoid lactone stereoisomers from Andrographis paniculata. J Nat Prod. 71:852–855. Deng YL, Xu ZH, Yang B, Liu Y, Dai RJ, Yu YH. 2006. Isolation and structure identification of chemical constituents from the root of Crataegus. Beijing Ligong Daxue Xuebao. 26:464–467. George M, Pandalai KM. 1949. Investigations on plant antibiotics, Part IV. Further search for antibiotics substances in Indian medicinal plants. Indian J Med Res. 37:169–181. Gupta KK, Taneja SC, Dhar KL. 1996. Flavonoid glycoside of Andrographis paniculata. Indian J Chem. 35B:512–513. Gupta KK, Taneja SC, Dhar KL, Atal CK. 1983. Flavonoids of Andrographis paniculata. Phytochemistry. 22:314–315. Jalal MAF, Overton KH, Rycroft DS. 1979. Formation of three new flavones by differentiating callus cultures of Andrographis paniculata. Phytochemistry. 18:149–151. Kuroyanagi M, Sato M, Ueno A, Nishi K. 1987. Flavonoids from Andrographis paniculata. Chem Pharm Bull. 35:4429–4435. Li WK, Xu XD, Zhang HJ, Ma CY, Fong H, Breemen VR, Fitzloff J. 2007. Secondary Metabolites from Andrographis paniculata. Chem Pharm Bull. 55:455–458. Ma SC, Liu Y, Pui-Hay PB, Yang Y, Eng-Chun VO, Lee SHS, Lee SF, Lu J, Lin RC. 2006. Antiviral activities of flavonoids isolated from Lonicera japonica Thunb. Yaowu Fenxi Zazhi. 26:426– 430. Matsuda T, Kuroyanagi M, Sugiyama S, Umehara K, Ueno A, Nishi K. 1994. Cell differentiation-inducing diterpenes from Andrographis paniculata Nees. Chem Pharm Bull. 42:1216–1225. Puri A, Saxena R, Saxena RP, Srivastava V, Tandon JS. 1993. Immunostimulant agents from Andrographis paniculata. J Nat Prod. 56:995–999. Ren YL, Yang JS. 2001. Study on chemical constituents of Saussurea tridac-tyla Sch Bip II. Zhongguo Zhongyao Zazhi. 36:590–593. Saxena S, Jain DC. 2003. Phytochemicals from Andrographis paniculata. Indian J Chem. 42B:3159–3163. Tanaka T, Iinuma M, Mizuno M. 1986. Spectral properties of 20 -oxygenated flavones. Chem Pharm Bull. 34:1667–1671. Vijaya M, Hari P, Venkata C, Gunasekar D. 2003. New 20 -oxygenated flavonoids from Andrographis paniculata. J Nat Prod. 66:295–297. Wang F, Hua HM, Pei YH, Chen D, Jing YK. 2006. Triterpenoids from the resin of Styrax tonkinensis and their antiproliferative and differentiation effects in human leukemia HL-60 cells. J Nat Prod. 69:807–810. Waridel P, Wolfende JL, Lachavanne JB, Hostettmann K. 2004. ent-Labdane glycosides from the aquatic plant Potamogeton lucens and analytical evaluation of the lipophilic extract constituents of various Potamogeton species. Phytochemistry. 65:945–954. Yao XJ, Wainberg MA, Parniak MA. 1992. Mechanism of inhibition of HIV-1 infection in vitro by purified extract of Prunella vulgaris. Virology. 187:56–62. Zeha PT, Rawal UM. 2001. Hepatoprotective and antioxidant property of Andrographis paniculata (Nees) in BHC induced liver damage in mice. Indian J Exp Biol. 39:41–46. Zheng J, Gao HY, Chen GT, Yang XK, Wu B, Wu LJ. 2006. Chemical constituents of the active parts of Mentha spicata L.(II). Shenyang Yaoke Daxue Xuebao. 23:212–215.

A new flavonoid from the aerial parts of Andrographis paniculata.

A new flavonoid, 7,8-dimethoxy-2'-hydroxy-5-O-β-d-glucopyranosyloxyflavone (1), along with 15 known flavonoids (2-16), was isolated from the aerial pa...
161KB Sizes 0 Downloads 0 Views