- Email: [email protected]
New flavone and eudesmane derivatives from Lawsonia inermis and their inhibitory activity against NO production
Phytochemistry Letters 21 (2017) 123–127
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
New flavone and eudesmane derivatives from Lawsonia inermis and their inhibitory activity against NO production
MARK
Chang-Syun Yanga,1, Jih-Jung Chenb,c,1, Hui-Chi Huanga, Guan-Jhong Huanga,1, ⁎ Sheng-Yang Wangd,e, Louise-Kuoping Chaof, Chin Hsug, Yueh-Hsiung Kuoa,h, a
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan Faculty of Pharmacy, School of Pharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan c Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan d Department of Forestry, National Chung-Hsing University, Taichung 402, Taiwan e Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan f Department of Cosmeceutics, China Medical University Hospital, China Medical University, Taichung 404, Taiwan g Department of Exercise Health Science, National Taiwan University of Physical Eucation and Sport, Taichung 404, Taiwan h Department of Biotechnology, Asia University, Taichung 413, Taiwan b
A R T I C L E I N F O
A B S T R A C T
Keywords: Keywords: Lawsonia inermis Henna 7-Hydroxy-3,5-dimethoxy-6,8-dimethylflavone Eudesmane-4β,7α-diol Inhibitory activities against NO production
A new flavone derivative, 7-hydroxy-3,5-dimethoxy-6,8-dimethylflavone (1) and a new eudesmane derivative, eudesmane-4β,7α-diol (2), have been isolated from the aerial part of Lawsonia inermis, together with ten known compounds (3–12). The structures of two new compounds were determined through spectroscopic and MS analyses. All compounds were evaluated for their inhibitory effects on Nitric Oxide production in LPS-stimulated RAW264.7 cells and compounds 3, 4, 6, 7, 9, and 10 showed inhibition with IC50 values of 8.11, 2.32, 1.87, 7.72, 2.18, and 6.34 μg/mL, respectively.
1. Introduction
the isolates are described herein.
Lawsonia inermis Linn (Lythraceae) is an important medicinal plant, distributed in northern Africa, western and southern Asia, and northern Australasia (Chen and Qian, 2007). The leaves are used as a prophylactic against skin diseases (Lin et al., 2003). The plants of the family Lythraceae are rich in flavonoids (Yang et al., 2016; Ahmed et al., 2000), isocoumarins (Yang et al., 2016), coumarins (Ahmed et al., 2000), quinoids (Ahmed et al., 2000), triterpenoids (Ahmed et al., 2000), naphthalene derivatives (Ahmed et al., 2000) as the major constituents, some of them have demonstrated anti-inflammatory (Yang et al., 2016; Liou et al., 2013), antimycotic, antifungal, antibacterial, and antiparasitic activities (Babu and Subhasree, 2009). In our continuing studies on the anti-inflammatory constituents of native Formosan plants, many species have been screened for in vitro inhibitory activity on macrophage pro-inflammatory responses, and L. inermis has been found to be an active species. The current phytochemical investigation of the aerial part of this plant has led to the isolation of two new compounds, 7-hydroxy-3,5-dimethoxy-6,8-dimethylflavone (1) and eudesmane-4β,7α-diol (2), along with 10 known compounds. The structural elucidation of 1 and 2 and the anti-inflammatory activity of
2. Results and discussion
⁎
1
Chromatographic purification of the EtOAc-soluble fraction of a MeOH extract of aerial part of L. inermis on a silica gel column and HPLC afforded two new (1 and 2) and ten known compounds (3–12) (Fig. 1). Compound 1 was isolated as yellow needle crystal with molecular formula C19H18O5 as determined by HR-EI-MS m/z 326.1147 [M]+ (calcd for C19H18O5, 326.1150). The IR absorption bands implied the presence of an OH (3447 cm−1) and a conjugated carbony (1622 cm−1) groups. The 1H NMR spectrum of 1 showed the presence of five mutually coupling aromatic protons [δ 7.50 (3H, m, H-3ʹ, 4ʹ, 5ʹ), 8.11 (2H, dd, J = 8.5, 1.5 Hz, H-2ʹ, 6ʹ)], two methoxy groups [δ 3.87 (3H, s, OMe-3), 3.89 (3H, s, OMe-5], two methyl group protons [δ 2.26 (3H, s, Me-6), 2.40 (3H, s, Me-8)], and a hydroxyl group [δ 5.62 (1H, br s, D2O exchangeable, OH-7)]. The 1H NMR data of 1 were similar to those of 7hydroxy-5-methoxy-6,8-dimethylflavone (1a) (Dao et al., 2010), except that an additional 3-methoxy group [δ 3.87 (3H, s)] of 1 replaced H-3 of 7-hydroxy-5-methoxy-6,8-dimethylflavone (1a) (Dao et al., 2010). This
Corresponding author at: Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan. E-mail address: [email protected] (Y.-H. Kuo). Authors have contributed equally in this manuscript.
http://dx.doi.org/10.1016/j.phytol.2017.06.012 Received 9 May 2017; Received in revised form 14 June 2017; Accepted 16 June 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 21 (2017) 123–127
C.-S. Yang et al.
3'
HO
O 8
2'
4'
1'
5'
6'
Fig. 1. The chemical structures of compounds 1–12 from the aerial part of Lawsonia inermis.
R
14
1
1
9
7
9
2
2
10
6
10
3
3
5
5
OMe
4
8 7 6
4
R HO
O 1 R = OMe 1a R = H
12
OH 15
2 R=H 2a R = OH
R1
11
13
R2 R1
R2
O
O
R3
O 9 R1 = OH, R2 = OMe, R3 = H 10 R1 = OMe, R2 = OH, R3 = OMe R1
R2 R4
O
R3 OH
11 R1 = H, R2 = H2 12 R1 = OH, R2 = O
O
3 4 5 6 7 8
R1 = R2 = R3 = H, R4 = OH R1 = OH, R2 = R3 = CH3, R4 = OH R1 = H, R2 = R3 = CH3, R4 = OH R1 = OH, R2 = R3 = CH3, R4 =OMe R1 = H, R2 = H, R3 = OMe, R4 = OH R1 = H, R2 = OMe, R3 = H, R4 = OH Fig. 2. Key NOESY ( correlations of 1.
) and HMBC (
)
NMR spectrum of 2 showed an isopropyl group [δ 0.92 (3H, d, J = 6.8 Hz, H-12), 0.94 (3H, d, J = 6.8 Hz, H-13), 1.57 (1H. m, H-11)], two methyl groups [δ 0.98 (3H, s, Me-10), 1.14 (3H, s, Me-4)], twelve methylene protons [δ 1.10 (1H, m, H-9ax), 1.13 (1H, m, H-1ax), 1.34 (1H, m, H-6ax), 1.38 (2H, m, H-2ax and H-9eq), 1.44 (2H, m, H-3ax and H-8ax), 1.52 (1H. m, H-8eq), 1.59 (1H, m, H-6eq), 1.60 (1H, m, H-1eq), 1.62 (1H, m, H-3eq), and 1.78 (1H, m, H-2eq)], and a methine proton [δ 1.43 (1H, m, H-5)]. The 1H NMR data of 2 were similar to those of lβ,4β,7α-trihydroxyeudesmane (2a) (Sung et al., 1992), except that H1eq (δ 1.60) of 2 replaced 1β-hydroxy group of lβ,4β,7α-trihydroxyeudesmane (2a) (Sung et al., 1992). This was supported by (1) NOESY correlations observed between Me-10 (δH 0.96) and H-1eq (δH 1.60),
was supported by (1) NOESY correlations observed between OMe-3 (δH 3.87) and H-6ʹ (δH 8.11), (2) HMBC correlations observed between OMe-3 (δH 3.87) and C-3 (δC 174.1), and (3) the chemical shifts of H-2ʹ and H-6ʹ were shifted to lower field (δH 8.11) when compared to those of 1a. The full assignment of 1H and 13C NMR resonances was confirmed by NOESY (Fig. 2), HMBC (Fig. 2), 1H–1H COSY, DEPT, and HSQC techniques. According to the evidence above, the structure of 1 was elucidated as 7-hydroxy-3,5-dimethoxy-6,8-dimethylflavone. Compound 2 was obtained as yellow gum. Its molecular formula, C15H28O2, was determined on the basis of the positive HR-EI-MS m/z 240.2096 [M]+ (calcd for C15H28O2, 240.2090). The IR absorption bands implied the presence of the hydroxy group (3476 cm−1). The 1H 124
Phytochemistry Letters 21 (2017) 123–127
C.-S. Yang et al.
14
H OH H
H
H
H
H
H
1 2
H H H
H
H
Fig. 3. Key NOESY (
8
5
7 6
4
H OH
HO
) correlations of 2.
9 10
3
) and HMBC (
12
OH
13
15
could be drawn: (a) The high cell viability (> 83%) indicated that the inhibitory activities of compounds 3, 4, 6, 7, 9, and 10 on LPS-induced NO production did not result from their cytotoxicities. (b) Among the flavonoids with 6,8-dimethyl-5,7-dioxygenated substituents (1, 4–6), syzalterin (4) and sideroxyline (6) with 4′-hydroxy group exhibited more effective inhibition than its analogues (1 and 5) against LPS-induced NO generation. (c) Compounds 4, 6, and 9 exhibited inhibitory effects on lipopolysaccharides (LPS)-induced nitric oxide production in RAW 264.7 cells with IC50 values of 2.32 ± 0.21, 1.87 ± 1.43, and 2.18 ± 0.26 μg/mL, respectively. (d) Sideroxyline (6) is the most effective among the isolated compounds, with IC50 = 1.87 ± 0.15 μg/ mL, against LPS-induced NO generation. Thus, our study suggests L. inermis and its isolates (especially 4, 6, and 9) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases. Meanwhile, this research extended the chemical diversity of Lythraceae and may serve as chemotaxonomic mark of L. inermis.
and (2) HMBC correlations observed between Me-10 (δH 0.96)/C-1 (δC 41.2) and H-1eq (δH 1.60)/C-3 (δC 41.5). The full assignment of 1H and 13 C NMR resonances was confirmed by 1H–1H COSY, NOESY (Fig. 3), DEPT, HSQC, and HMBC (Fig. 3) techniques. According to the evidence above, the structure of 2 was elucidated as eudesmane-4β,7α-diol. The known isolates were readily identified by comparison of their physical and spectroscopic data (UV, IR, 1H NMR, [α]D, and MS) with those of the corresponding authentic samples or literature values. They include six flavonoids, chrysin (3) (Liu et al., 2010), syzalterin (4) (Youssef et al., 1998), 5,7-dihydroxy-6,8-dimethylflavone (5) (Kuo and Chu, 2002), sideroxyline (6) (Junio et al., 2011), oroxylin a (7) (Marques et al., 2010), and wogonin (8) (Cao et al., 2012), two coumarins, isoscopoletin (9) (Gao et al., 2013) and isofraxidin (10) (Gao et al., 2013), and two sesquiterpenes: α-ionone (11) (Wang and Lugtenburg, 2004) and dehydrovomifoliol (12) (Kai et al., 2007). 2.1. Inhibitory activity against nitric oxide production
3. Experimental
Nitric oxide (NO) is derived from the oxidation of L-arginine by NO synthase (NOS) and is a mediator in the inflammatory response involved in host defense (Geller and Billiar, 1998). In inflammation and carcinogenesis conditions, there is an increased production of NO by inducible NO synthase (iNOS) (Moncada et al., 1991). The anti-inflammatory effects of the compounds isolated from Lawsonia inermis were evaluated for suppression of lipopolysaccharide (LPS)-induced NO generation in murine macrophage. In this study, the inhibitory activity toward NO production of two new (1and 2) and ten known compounds (3–12) was evaluated by measurement of nitrite/nitrate in LPS-stimulated RAW 264.7 cells. To search for the appropriate concentrations for the above assay, these 12 compounds were first tested for their cytotoxic activity against the RAW 264.7 cells, and no significant cytotoxic activities were observed under all tested concentrations. From the results of our anti-inflammatory tests (Table 1), the following conclusions
3.1. General experimental procedures Optical rotations were measured using a Jasco DIP-370 polarimeter in CHCl3. Ultraviolet (UV) spectra were obtained with a Shimadzu Pharmaspec-1700 UV–vis spectrophotometer (Kyoto, Japan). Infrared (IR) spectra (neat or KBr) were recorded on a Shimadzu IR prestige-21 Fourier transform infrared spectrophotometer (Kyoto, Japan). Nuclear magnetic resonance (NMR) spectra, including correlation spectroscopy (COSY), nuclear Overhauser effect spectrometry (NOESY), heteronuclear multiplebond correlation (HMBC), heteronuclear single-quantum coherence (HSQC) experiments, were recorded on Bruker DRX-500 and Bruker DRX400 FT-NMR (Bruker, Bremen, Germany) operating at 500 (400) MHz (1H) and 125 (100) MHz (13C), respectively, with chemical shifts given in ppm (δ) using tetramethylsilane (TMS) as an internal standard. Mass spectrometric (EI-MS and HR-EI-MS) data were generated at the Mass Spectrometry Laboratory of the Chung Hsing University (Taichung, Taiwan). HPLC chromatograms were obtained with an LC-6A instrument and an IOTA-2 RI-detector (Shimadzu, Kyoto, Japan). Semi-preparative NP-HPLC column chromatography was performed using MonoChrom Si gel (250 mm × 10 mm i.d., 5 μm, MetaChem, Torrance, CA, USA). Column chromatography was performed using LiChroCART Si gel (5 μM; Merck, Darmstadt, Germany) and Sephadex® LH-20 (25–100 μm; Merck, Darmstadt, Germany). Silica gel 60 F-254 (Merck, Darmstadt, Germany) was used for thin-layer chromatography (TLC) and preparative thin-layer chromatography (PTLC).
Table 1 Inhibitory effect of compounds 1–12 on overproduction of nitric oxide in LPS-stimulated RAW 264.7 cells. Compounds
IC50 (μg/ml)a
7-Hydroxy-3,5-dimethoxy-6,8-dimethylflavone (1) Eudesmane-4β,7α-diol (2) Chrysin (3) Syzalterin (4) 5,7-Dihydroxy-6,8-dimethylflavone (5) Sideroxyline (6) Oroxylin a (7) Wogonin (8) Isoscopoletin (9) Isofraxidin (10) α-Ionone (11) Dehydrovomifoliol (12) Indomethacinb
18.98 ± 1.62*** > 20 8.11 ± 2.36** 2.32 ± 0.21*** 11.67 ± 3.48** 1.87 ± 0.15*** 7.72 ± 3.34*** 12.91 ± 3.31** 2.18 ± 0.26*** 6.34 ± 3.34*** 17.42 ± 3.47** 12.50 ± 1.20*** 57.39 ± 1.28
11
3.2. Plant material Lawsonia inermis was collected from Neipu Township, Pingtung, Taiwan, in February 2009 and identified by I.-S. Chen (Emeritus Professor, School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Taiwan). A voucher specimen (CMU-LIY-090711) was deposited at the School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources.
**
p < 0.01 and ***p < 0.001 compared with indomethacin. a The IC50 values were calculated from the slope equation of the dose-response curves.Values are expressed as average ± SEM (n = 3). b Indomethacin was used as a positive control.
125
Phytochemistry Letters 21 (2017) 123–127
C.-S. Yang et al.
3.3. Extraction and isolation
3.5. Cell viability
The dried aerial part (5.0 kg) of Lawsonia inermis were pulverized and extracted three times with MeOH (50 L each) for 7 days. After MeOH extracts filtration, the combined extract was concentrated under reduced at 25 °C pressure to give a brown extract (440.0 g). The methanol extract (280.0 g) was partitioned between H2O (2 L × 3) and ethyl acetate (2 L × 3) to yield ethyl acetate layer (fraction A, 132.5 g) and a H2O layer (fraction B, 147.5 g). The obtained fraction A (132.5 g) was subjected to silica gel Column Chromatography using mixtures of n-hexane − ethyl acetate − methanol with increasing polarity as eluants to afford 14 fractions (fraction A1–14). Fraction A2 (32.80 g) was chromatographed further on silica gel (230–400 mesh, 205 g) eluting with n-hexane/EtOAc (30:1–0:1) to give 6 fractions (each 1 L, A2-1–A2-6). Part (206 mg) of fraction A2-1 was purified by preparative TLC (silica gel, n-hexane/acetone, 10:1) to afford 11 (23.4 mg) (Rf = 0.42) and 12 (34.5 mg) (Rf = 0.56). Part (460 mg) of fraction A2-4 was re-separated by semi-preparative normal phase HPLC (n-hexane: acetone = 7:1) to afford pure compounds 2 (24.5 mg), 9 (32.7 mg), and 10 (6.9 mg). Fraction A8 (22.4 g) was reseparated by Sephadex LH 20 column chromatography (25–100 μm, 250 g) (chloroform: methanol = 3:7) to give 10 fractions (each 2.0 L, A8-1–A8-10). Part (460 mg) of fraction A8-3 was purified by preparative TLC (silica gel, n-hexane/acetone, 4:1) to afford 7 (68.6 mg) (Rf = 0.36) and 8 (34.2 mg) (Rf = 0.48). Part (326 mg) of Fraction A86 was re-separated by semi-preparative normal phase HPLC (chloroform: acetone = 6:1) to afford pure compounds 1 (18.6 mg), 3 (5.7 mg), 4 (14.6 mg), 5 (24.8 mg), and 6 (32.4 mg). 7-Hydroxy-3,5-dimethoxy-6,8-dimethylflavone (1): yellow needle crystal; UV (MeOH): λmax (log ε) 263 (4.32), 315 (3.91) nm; IR (KBr) νmax: 3447, 1622, 1597, 1493, 1478, 1445 cm−1; 1H NMR (CDCl3, 400 MHz): δ 2.26 (3H, s, CH3-6), 2.40 (3H, s, CH3-8), 3.87 (3H, s, OMe3), 3.89 (3H, s, OMe-5), 5.62 (1H, br s, OH-7), 7.50 (3H. m, H-3′, 4′, 5′), 8.11 (2H. dd, J = 8.5, 1.5 Hz, H-2′ and H-6′); 13C NMR (CDCl3, 100 MHz): δ 8.3 (Me-6), 8.4 (Me-8), 60.1 (OMe-3), 60.7 (OMe-5), 106.9 (C-8), 112.6 (C-10), 115.1 (C-6), 128.2 (C-3′, 5′), 128.6 (C-2′, 6′), 130.4 (C-4′), 131.3 (C-1′), 141.5 (C-3), 153.0 (C-2), 154.0 (C-7), 155.8 (C-5), 156.8 (C-9), 174.1 (C-4); EI-MS m/z 326 [M]+; HR-EI-MS m/z 326.1147 [M]+ (calcd for C19H18O5, 326.1150). Eudesmane-4β,7α-diol (2): pale yellow gum; [α]D20–108° (c 0.2, CHCl3); IR (KBr) νmax: 3476, 2936, 1463, 1378 cm−1; 1H NMR (CDCl3, 500 MHz): δ 0.92 (3H, d, J = 6.8 Hz, H-12), 0.94 (3H, d, J = 6.8 Hz, H13), 0.98 (3H, s, H-14), 1.10 (1H, m, H-9ax), 1.13 (1H, m, H-1ax) 1.14 (3H, s, H-15), 1.34 (1H, m, H-6ax), 1.38 (2H, m, H-2ax, H-9eq), 1.43 (1H, m, H-5), 1.44 (2H, m, H-3ax and H-8ax), 1.52 (1H. m, H-8eq), 1.57 (1H. m, H-11), 1.59 (1H, m, H-6eq), 1.60 (1H, m, H-1eq), 1.62 (1H, m, H-3eq), 1.78 (1H, m, H-2eq); 13C NMR (CDCl3, 125 MHz): δ 16.8 (C-13), 16.9 (C-12), 17.7 (C-14), 18.0 (C-2), 29.3 (C-6), 29.5 (C-8), 30.0 (C-15), 33.6 (C-10), 39.1 (C-9), 39.2 (C-11), 41.2 (C-1), 41.5 (C-3), 46.0 (C-5), 72.1 (C-4), 74.4 (C-7); EI-MS m/z 240 [M]+; HR-EI-MS m/z 240.2096 [M]+ (calcd for C15H28O2, 240.2090).
Cells (2 × 105)/well were cultured in 96-well plate containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then cells were cultured with compounds 1–12 (each 20 μg/mL) for 24 h. After that, the cells were incubated with 100 μL of 0.5 mg/mL MTT for 4 h at 37 °C testing for cell viability. The medium was then discarded and 100 μL dimethyl sulfoxide (DMSO) was added. After 30min incubation, absorbance at 570 nm was read using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). 3.6. Measurement of nitric Oxide/Nitrite NO production was indirectly assessed by measuring the nitrite levels in the cultured media and serum determined by a colorimetric method based on the Griess reaction. The cells were incubated with different concentration of samples in the presence of LPS (100 ng/mL) at 37 °C for 24 h. Take the supernatant in another 96-well plate and add an equal amount of mixed Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 5% phosphoric acid) and incubated at room temperature for 10 min, the absorbance was measured at 540 nm with a Micro-Reader (Molecular Devices). By using sodium nitrite to generate a standard curve, the concentration of nitrite was measured from absorbance at 540 nm. 3.7. Statistical analysis The data are expressed as means ± standard errors (SE). The IC50 values were calculated from the dose curves using a non-linear regression algorithm (SigmaPlot 8.0; SPSS Inc., Chicago, IL, USA, 2002). Statistical evaluation was carried out by one-way analysis of variance (ANOVA followed by Scheffe’smultiple range tests). Acknowledgements This research was supported from CMU under the Aim for Top University Plan of the Ministry of Education, Taiwan (CHM 106-5-2), and Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW106- TDU-B-212-113104). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.06.012. References Ahmed, S., Rahman, A., Alam, A., Saleem, M., Athar, M., Sultana, S., 2000. Evaluation of the efficacy of Lawsonia alba in the alleviation of carbon tetrachloride-induced oxidative stress. J. Ethnopharmacol. 69, 157–164. Babu, P.D., Subhasree, R.S., 2009. Antimicrobial activities of Lawsonia inermis–a review. Acad. J. Plant Sci. 2, 231–232. Cao, X.D., Ding, Z.S., Jiang, F.S., Ding, X.H., Chen, J.Z., Chen, S.H., Lu, G.Y., 2012. Antitumor constituents from the leaves of Carya cathayensis. Nat. Prod. Res. 22, 2089–2094. Chen, H.Y., Qian, C., 2007. Flora of China. Editorial Committee of the Flora of China, Beijing, China, pp. 274–288. Dao, T.T., Tung, B.T., Nguyen, P.H., Thuong, P.T., Yoo, S.S., Kim, E.H., Kim, S.K., Oh, W.K., 2010. C-methylated flavonoids from Cleistocalyx operculatus and their inhibitory effects on novel influenza a (H1N1) neuraminidase. J. Nat. Prod. 73, 1636–1642. Gao, W., Li, Q., Chen, J., Wang, Z., Hua, C., 2013. Total synthesis of six 3,4-unsubstituted coumarins. Molecules 18, 15613–15623. Geller, D.A., Billiar, T.R., 1998. Molecular biology of nitric oxide synthases. Cancer Metastasis Rev. 17, 7–23. Junio, H.A., Sy-Cordero, A.A., Ettefagh, K.A., Burns, J.T., Micko, K.T., Graf, T.N., Richter, S.J., Cannon, R.E., Oberlies, N.H., Cech, N.B., 2011. Synergy-directed fractionation of botanical medicines: a case study with goldenseal (Hydrastis canadensis). J. Nat. Prod. 74, 1621–1629. Kai, H., Baba, M., Okuyama, T., 2007. Two new megastigmanes from the leaves of Cucumis sativus. Chem. Pharm. Bull. 55, 133–136.
3.4. Cell culture A murine macrophage cell line RAW 264.7 (BCRC No. 60001) was purchased from the Bioresources Collection and Research Center (BCRC, Hsinchu, Taiwan) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in plastic dishes containing Dulbecco′s Modified Eagle Medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma) in a CO2 incubator (5% CO2 in air) at 37 °C and subcultured every 3 days at a dilution of 1:5 using 0.05% trypsin-0.02% EDTA in Ca2+-, Mg2+-free phosphate-buffered saline (DPBS).
126
Phytochemistry Letters 21 (2017) 123–127
C.-S. Yang et al.
Moncada, S., Palmer, R.M., Higgs, E.A., 1991. Nitric oxide physiology, pathophysiology, and pharmacology. Pharmcol. Rev. 43, 109–142. Sung, T.V., Steffan, B., Steglich, W., Klebet, G., Adam, G., 1992. Sesquiterpenoids from the roots of Homalomena Aromatica. Phytdwnistry 31, 3515–3520. Wang, Y., Lugtenburg, J., 2004. 4,5-Didehydro-9-demethyl-9-halo-5, 6-dihydroretinals and their 9-Cyclopropyl and 9-Isopropyl derivatives—simple preparation of α-Ionone derivatives and pure (all-E)-, (9Z)- and (11Z)-α-retinals. Eur. J. Org. Chem. 16, 3497–3510. Yang, C.S., Huang, H.C., Wang, S.Y., Sung, P.J., Huang, G.J., Chen, J.J., Kuo, Y.H., 2016. New diphenol and isocoumarins from the aerial part of Lawsonia inermis and their inhibitory activities against NO production. Molecules 21, 1299. Youssef, D.T.A., Ramadan, M.A., Khalifa, A.A., 1998. Acetophenones, a chalcone, a chromone and flavonoids form Pancratium maritimum. Phytochemistry 49, 2579–2583.
Kuo, Y.H., Chu, P.H., 2002. Studies on the constituents from the bark of Bauhinia purpurea. J. Chin. Chem. Soc. 49, 269–274. Lin, Y.X., Chang, Y.S., Chen, I.S., Ou, J.C., 2003. The Catalogue of Medicinal Plant Resources in Taiwan. Committee on Chinese Medicine and Pharmacy, Taipei, Taiwan. Liou, J.R., Mohamed, E.S., Du, Y.C., Tseng, C.N., Hwang, T.L., Chuang, Y.L., Hsu, Y.M., Hsieh, P.W., Wu, C.C., Chen, S.L., Hou, M.F., Chang, F.R., Wu, Y.C., 2013. 1,5Diphenylpent-3-en-1-ynes and methyl naphthalene carboxylates from Lawsonia inermis and their anti-inflammatory activity. Phytochemistry 88, 67–73. Liu, H., Mou, Y., Zhao, J., Wang, J., Zhou, L., Wang, M., Wang, D., Han, J., Yu, Z., Yang, F., 2010. Flavonoids from halostachys caspica and their antimicrobialand antioxidant activities. Molecules 15, 7933–7945. Marques, M.R., Stüker, C., Kichik, N., Tarragó, T., Giralt, E., Morel, A.F., Dalcol, I.I., 2010. Flavonoids with prolyl oligopeptidase inhibitory activity isolated from Scutellaria racemosa Pers. Fitoterapia 81, 552–556.
127
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
New flavone and eudesmane derivatives from Lawsonia inermis and their inhibitory activity against NO production
MARK
Chang-Syun Yanga,1, Jih-Jung Chenb,c,1, Hui-Chi Huanga, Guan-Jhong Huanga,1, ⁎ Sheng-Yang Wangd,e, Louise-Kuoping Chaof, Chin Hsug, Yueh-Hsiung Kuoa,h, a
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan Faculty of Pharmacy, School of Pharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan c Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan d Department of Forestry, National Chung-Hsing University, Taichung 402, Taiwan e Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan f Department of Cosmeceutics, China Medical University Hospital, China Medical University, Taichung 404, Taiwan g Department of Exercise Health Science, National Taiwan University of Physical Eucation and Sport, Taichung 404, Taiwan h Department of Biotechnology, Asia University, Taichung 413, Taiwan b
A R T I C L E I N F O
A B S T R A C T
Keywords: Keywords: Lawsonia inermis Henna 7-Hydroxy-3,5-dimethoxy-6,8-dimethylflavone Eudesmane-4β,7α-diol Inhibitory activities against NO production
A new flavone derivative, 7-hydroxy-3,5-dimethoxy-6,8-dimethylflavone (1) and a new eudesmane derivative, eudesmane-4β,7α-diol (2), have been isolated from the aerial part of Lawsonia inermis, together with ten known compounds (3–12). The structures of two new compounds were determined through spectroscopic and MS analyses. All compounds were evaluated for their inhibitory effects on Nitric Oxide production in LPS-stimulated RAW264.7 cells and compounds 3, 4, 6, 7, 9, and 10 showed inhibition with IC50 values of 8.11, 2.32, 1.87, 7.72, 2.18, and 6.34 μg/mL, respectively.
1. Introduction
the isolates are described herein.
Lawsonia inermis Linn (Lythraceae) is an important medicinal plant, distributed in northern Africa, western and southern Asia, and northern Australasia (Chen and Qian, 2007). The leaves are used as a prophylactic against skin diseases (Lin et al., 2003). The plants of the family Lythraceae are rich in flavonoids (Yang et al., 2016; Ahmed et al., 2000), isocoumarins (Yang et al., 2016), coumarins (Ahmed et al., 2000), quinoids (Ahmed et al., 2000), triterpenoids (Ahmed et al., 2000), naphthalene derivatives (Ahmed et al., 2000) as the major constituents, some of them have demonstrated anti-inflammatory (Yang et al., 2016; Liou et al., 2013), antimycotic, antifungal, antibacterial, and antiparasitic activities (Babu and Subhasree, 2009). In our continuing studies on the anti-inflammatory constituents of native Formosan plants, many species have been screened for in vitro inhibitory activity on macrophage pro-inflammatory responses, and L. inermis has been found to be an active species. The current phytochemical investigation of the aerial part of this plant has led to the isolation of two new compounds, 7-hydroxy-3,5-dimethoxy-6,8-dimethylflavone (1) and eudesmane-4β,7α-diol (2), along with 10 known compounds. The structural elucidation of 1 and 2 and the anti-inflammatory activity of
2. Results and discussion
⁎
1
Chromatographic purification of the EtOAc-soluble fraction of a MeOH extract of aerial part of L. inermis on a silica gel column and HPLC afforded two new (1 and 2) and ten known compounds (3–12) (Fig. 1). Compound 1 was isolated as yellow needle crystal with molecular formula C19H18O5 as determined by HR-EI-MS m/z 326.1147 [M]+ (calcd for C19H18O5, 326.1150). The IR absorption bands implied the presence of an OH (3447 cm−1) and a conjugated carbony (1622 cm−1) groups. The 1H NMR spectrum of 1 showed the presence of five mutually coupling aromatic protons [δ 7.50 (3H, m, H-3ʹ, 4ʹ, 5ʹ), 8.11 (2H, dd, J = 8.5, 1.5 Hz, H-2ʹ, 6ʹ)], two methoxy groups [δ 3.87 (3H, s, OMe-3), 3.89 (3H, s, OMe-5], two methyl group protons [δ 2.26 (3H, s, Me-6), 2.40 (3H, s, Me-8)], and a hydroxyl group [δ 5.62 (1H, br s, D2O exchangeable, OH-7)]. The 1H NMR data of 1 were similar to those of 7hydroxy-5-methoxy-6,8-dimethylflavone (1a) (Dao et al., 2010), except that an additional 3-methoxy group [δ 3.87 (3H, s)] of 1 replaced H-3 of 7-hydroxy-5-methoxy-6,8-dimethylflavone (1a) (Dao et al., 2010). This
Corresponding author at: Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan. E-mail address: [email protected] (Y.-H. Kuo). Authors have contributed equally in this manuscript.
http://dx.doi.org/10.1016/j.phytol.2017.06.012 Received 9 May 2017; Received in revised form 14 June 2017; Accepted 16 June 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 21 (2017) 123–127
C.-S. Yang et al.
3'
HO
O 8
2'
4'
1'
5'
6'
Fig. 1. The chemical structures of compounds 1–12 from the aerial part of Lawsonia inermis.
R
14
1
1
9
7
9
2
2
10
6
10
3
3
5
5
OMe
4
8 7 6
4
R HO
O 1 R = OMe 1a R = H
12
OH 15
2 R=H 2a R = OH
R1
11
13
R2 R1
R2
O
O
R3
O 9 R1 = OH, R2 = OMe, R3 = H 10 R1 = OMe, R2 = OH, R3 = OMe R1
R2 R4
O
R3 OH
11 R1 = H, R2 = H2 12 R1 = OH, R2 = O
O
3 4 5 6 7 8
R1 = R2 = R3 = H, R4 = OH R1 = OH, R2 = R3 = CH3, R4 = OH R1 = H, R2 = R3 = CH3, R4 = OH R1 = OH, R2 = R3 = CH3, R4 =OMe R1 = H, R2 = H, R3 = OMe, R4 = OH R1 = H, R2 = OMe, R3 = H, R4 = OH Fig. 2. Key NOESY ( correlations of 1.
) and HMBC (
)
NMR spectrum of 2 showed an isopropyl group [δ 0.92 (3H, d, J = 6.8 Hz, H-12), 0.94 (3H, d, J = 6.8 Hz, H-13), 1.57 (1H. m, H-11)], two methyl groups [δ 0.98 (3H, s, Me-10), 1.14 (3H, s, Me-4)], twelve methylene protons [δ 1.10 (1H, m, H-9ax), 1.13 (1H, m, H-1ax), 1.34 (1H, m, H-6ax), 1.38 (2H, m, H-2ax and H-9eq), 1.44 (2H, m, H-3ax and H-8ax), 1.52 (1H. m, H-8eq), 1.59 (1H, m, H-6eq), 1.60 (1H, m, H-1eq), 1.62 (1H, m, H-3eq), and 1.78 (1H, m, H-2eq)], and a methine proton [δ 1.43 (1H, m, H-5)]. The 1H NMR data of 2 were similar to those of lβ,4β,7α-trihydroxyeudesmane (2a) (Sung et al., 1992), except that H1eq (δ 1.60) of 2 replaced 1β-hydroxy group of lβ,4β,7α-trihydroxyeudesmane (2a) (Sung et al., 1992). This was supported by (1) NOESY correlations observed between Me-10 (δH 0.96) and H-1eq (δH 1.60),
was supported by (1) NOESY correlations observed between OMe-3 (δH 3.87) and H-6ʹ (δH 8.11), (2) HMBC correlations observed between OMe-3 (δH 3.87) and C-3 (δC 174.1), and (3) the chemical shifts of H-2ʹ and H-6ʹ were shifted to lower field (δH 8.11) when compared to those of 1a. The full assignment of 1H and 13C NMR resonances was confirmed by NOESY (Fig. 2), HMBC (Fig. 2), 1H–1H COSY, DEPT, and HSQC techniques. According to the evidence above, the structure of 1 was elucidated as 7-hydroxy-3,5-dimethoxy-6,8-dimethylflavone. Compound 2 was obtained as yellow gum. Its molecular formula, C15H28O2, was determined on the basis of the positive HR-EI-MS m/z 240.2096 [M]+ (calcd for C15H28O2, 240.2090). The IR absorption bands implied the presence of the hydroxy group (3476 cm−1). The 1H 124
Phytochemistry Letters 21 (2017) 123–127
C.-S. Yang et al.
14
H OH H
H
H
H
H
H
1 2
H H H
H
H
Fig. 3. Key NOESY (
8
5
7 6
4
H OH
HO
) correlations of 2.
9 10
3
) and HMBC (
12
OH
13
15
could be drawn: (a) The high cell viability (> 83%) indicated that the inhibitory activities of compounds 3, 4, 6, 7, 9, and 10 on LPS-induced NO production did not result from their cytotoxicities. (b) Among the flavonoids with 6,8-dimethyl-5,7-dioxygenated substituents (1, 4–6), syzalterin (4) and sideroxyline (6) with 4′-hydroxy group exhibited more effective inhibition than its analogues (1 and 5) against LPS-induced NO generation. (c) Compounds 4, 6, and 9 exhibited inhibitory effects on lipopolysaccharides (LPS)-induced nitric oxide production in RAW 264.7 cells with IC50 values of 2.32 ± 0.21, 1.87 ± 1.43, and 2.18 ± 0.26 μg/mL, respectively. (d) Sideroxyline (6) is the most effective among the isolated compounds, with IC50 = 1.87 ± 0.15 μg/ mL, against LPS-induced NO generation. Thus, our study suggests L. inermis and its isolates (especially 4, 6, and 9) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases. Meanwhile, this research extended the chemical diversity of Lythraceae and may serve as chemotaxonomic mark of L. inermis.
and (2) HMBC correlations observed between Me-10 (δH 0.96)/C-1 (δC 41.2) and H-1eq (δH 1.60)/C-3 (δC 41.5). The full assignment of 1H and 13 C NMR resonances was confirmed by 1H–1H COSY, NOESY (Fig. 3), DEPT, HSQC, and HMBC (Fig. 3) techniques. According to the evidence above, the structure of 2 was elucidated as eudesmane-4β,7α-diol. The known isolates were readily identified by comparison of their physical and spectroscopic data (UV, IR, 1H NMR, [α]D, and MS) with those of the corresponding authentic samples or literature values. They include six flavonoids, chrysin (3) (Liu et al., 2010), syzalterin (4) (Youssef et al., 1998), 5,7-dihydroxy-6,8-dimethylflavone (5) (Kuo and Chu, 2002), sideroxyline (6) (Junio et al., 2011), oroxylin a (7) (Marques et al., 2010), and wogonin (8) (Cao et al., 2012), two coumarins, isoscopoletin (9) (Gao et al., 2013) and isofraxidin (10) (Gao et al., 2013), and two sesquiterpenes: α-ionone (11) (Wang and Lugtenburg, 2004) and dehydrovomifoliol (12) (Kai et al., 2007). 2.1. Inhibitory activity against nitric oxide production
3. Experimental
Nitric oxide (NO) is derived from the oxidation of L-arginine by NO synthase (NOS) and is a mediator in the inflammatory response involved in host defense (Geller and Billiar, 1998). In inflammation and carcinogenesis conditions, there is an increased production of NO by inducible NO synthase (iNOS) (Moncada et al., 1991). The anti-inflammatory effects of the compounds isolated from Lawsonia inermis were evaluated for suppression of lipopolysaccharide (LPS)-induced NO generation in murine macrophage. In this study, the inhibitory activity toward NO production of two new (1and 2) and ten known compounds (3–12) was evaluated by measurement of nitrite/nitrate in LPS-stimulated RAW 264.7 cells. To search for the appropriate concentrations for the above assay, these 12 compounds were first tested for their cytotoxic activity against the RAW 264.7 cells, and no significant cytotoxic activities were observed under all tested concentrations. From the results of our anti-inflammatory tests (Table 1), the following conclusions
3.1. General experimental procedures Optical rotations were measured using a Jasco DIP-370 polarimeter in CHCl3. Ultraviolet (UV) spectra were obtained with a Shimadzu Pharmaspec-1700 UV–vis spectrophotometer (Kyoto, Japan). Infrared (IR) spectra (neat or KBr) were recorded on a Shimadzu IR prestige-21 Fourier transform infrared spectrophotometer (Kyoto, Japan). Nuclear magnetic resonance (NMR) spectra, including correlation spectroscopy (COSY), nuclear Overhauser effect spectrometry (NOESY), heteronuclear multiplebond correlation (HMBC), heteronuclear single-quantum coherence (HSQC) experiments, were recorded on Bruker DRX-500 and Bruker DRX400 FT-NMR (Bruker, Bremen, Germany) operating at 500 (400) MHz (1H) and 125 (100) MHz (13C), respectively, with chemical shifts given in ppm (δ) using tetramethylsilane (TMS) as an internal standard. Mass spectrometric (EI-MS and HR-EI-MS) data were generated at the Mass Spectrometry Laboratory of the Chung Hsing University (Taichung, Taiwan). HPLC chromatograms were obtained with an LC-6A instrument and an IOTA-2 RI-detector (Shimadzu, Kyoto, Japan). Semi-preparative NP-HPLC column chromatography was performed using MonoChrom Si gel (250 mm × 10 mm i.d., 5 μm, MetaChem, Torrance, CA, USA). Column chromatography was performed using LiChroCART Si gel (5 μM; Merck, Darmstadt, Germany) and Sephadex® LH-20 (25–100 μm; Merck, Darmstadt, Germany). Silica gel 60 F-254 (Merck, Darmstadt, Germany) was used for thin-layer chromatography (TLC) and preparative thin-layer chromatography (PTLC).
Table 1 Inhibitory effect of compounds 1–12 on overproduction of nitric oxide in LPS-stimulated RAW 264.7 cells. Compounds
IC50 (μg/ml)a
7-Hydroxy-3,5-dimethoxy-6,8-dimethylflavone (1) Eudesmane-4β,7α-diol (2) Chrysin (3) Syzalterin (4) 5,7-Dihydroxy-6,8-dimethylflavone (5) Sideroxyline (6) Oroxylin a (7) Wogonin (8) Isoscopoletin (9) Isofraxidin (10) α-Ionone (11) Dehydrovomifoliol (12) Indomethacinb
18.98 ± 1.62*** > 20 8.11 ± 2.36** 2.32 ± 0.21*** 11.67 ± 3.48** 1.87 ± 0.15*** 7.72 ± 3.34*** 12.91 ± 3.31** 2.18 ± 0.26*** 6.34 ± 3.34*** 17.42 ± 3.47** 12.50 ± 1.20*** 57.39 ± 1.28
11
3.2. Plant material Lawsonia inermis was collected from Neipu Township, Pingtung, Taiwan, in February 2009 and identified by I.-S. Chen (Emeritus Professor, School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Taiwan). A voucher specimen (CMU-LIY-090711) was deposited at the School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources.
**
p < 0.01 and ***p < 0.001 compared with indomethacin. a The IC50 values were calculated from the slope equation of the dose-response curves.Values are expressed as average ± SEM (n = 3). b Indomethacin was used as a positive control.
125
Phytochemistry Letters 21 (2017) 123–127
C.-S. Yang et al.
3.3. Extraction and isolation
3.5. Cell viability
The dried aerial part (5.0 kg) of Lawsonia inermis were pulverized and extracted three times with MeOH (50 L each) for 7 days. After MeOH extracts filtration, the combined extract was concentrated under reduced at 25 °C pressure to give a brown extract (440.0 g). The methanol extract (280.0 g) was partitioned between H2O (2 L × 3) and ethyl acetate (2 L × 3) to yield ethyl acetate layer (fraction A, 132.5 g) and a H2O layer (fraction B, 147.5 g). The obtained fraction A (132.5 g) was subjected to silica gel Column Chromatography using mixtures of n-hexane − ethyl acetate − methanol with increasing polarity as eluants to afford 14 fractions (fraction A1–14). Fraction A2 (32.80 g) was chromatographed further on silica gel (230–400 mesh, 205 g) eluting with n-hexane/EtOAc (30:1–0:1) to give 6 fractions (each 1 L, A2-1–A2-6). Part (206 mg) of fraction A2-1 was purified by preparative TLC (silica gel, n-hexane/acetone, 10:1) to afford 11 (23.4 mg) (Rf = 0.42) and 12 (34.5 mg) (Rf = 0.56). Part (460 mg) of fraction A2-4 was re-separated by semi-preparative normal phase HPLC (n-hexane: acetone = 7:1) to afford pure compounds 2 (24.5 mg), 9 (32.7 mg), and 10 (6.9 mg). Fraction A8 (22.4 g) was reseparated by Sephadex LH 20 column chromatography (25–100 μm, 250 g) (chloroform: methanol = 3:7) to give 10 fractions (each 2.0 L, A8-1–A8-10). Part (460 mg) of fraction A8-3 was purified by preparative TLC (silica gel, n-hexane/acetone, 4:1) to afford 7 (68.6 mg) (Rf = 0.36) and 8 (34.2 mg) (Rf = 0.48). Part (326 mg) of Fraction A86 was re-separated by semi-preparative normal phase HPLC (chloroform: acetone = 6:1) to afford pure compounds 1 (18.6 mg), 3 (5.7 mg), 4 (14.6 mg), 5 (24.8 mg), and 6 (32.4 mg). 7-Hydroxy-3,5-dimethoxy-6,8-dimethylflavone (1): yellow needle crystal; UV (MeOH): λmax (log ε) 263 (4.32), 315 (3.91) nm; IR (KBr) νmax: 3447, 1622, 1597, 1493, 1478, 1445 cm−1; 1H NMR (CDCl3, 400 MHz): δ 2.26 (3H, s, CH3-6), 2.40 (3H, s, CH3-8), 3.87 (3H, s, OMe3), 3.89 (3H, s, OMe-5), 5.62 (1H, br s, OH-7), 7.50 (3H. m, H-3′, 4′, 5′), 8.11 (2H. dd, J = 8.5, 1.5 Hz, H-2′ and H-6′); 13C NMR (CDCl3, 100 MHz): δ 8.3 (Me-6), 8.4 (Me-8), 60.1 (OMe-3), 60.7 (OMe-5), 106.9 (C-8), 112.6 (C-10), 115.1 (C-6), 128.2 (C-3′, 5′), 128.6 (C-2′, 6′), 130.4 (C-4′), 131.3 (C-1′), 141.5 (C-3), 153.0 (C-2), 154.0 (C-7), 155.8 (C-5), 156.8 (C-9), 174.1 (C-4); EI-MS m/z 326 [M]+; HR-EI-MS m/z 326.1147 [M]+ (calcd for C19H18O5, 326.1150). Eudesmane-4β,7α-diol (2): pale yellow gum; [α]D20–108° (c 0.2, CHCl3); IR (KBr) νmax: 3476, 2936, 1463, 1378 cm−1; 1H NMR (CDCl3, 500 MHz): δ 0.92 (3H, d, J = 6.8 Hz, H-12), 0.94 (3H, d, J = 6.8 Hz, H13), 0.98 (3H, s, H-14), 1.10 (1H, m, H-9ax), 1.13 (1H, m, H-1ax) 1.14 (3H, s, H-15), 1.34 (1H, m, H-6ax), 1.38 (2H, m, H-2ax, H-9eq), 1.43 (1H, m, H-5), 1.44 (2H, m, H-3ax and H-8ax), 1.52 (1H. m, H-8eq), 1.57 (1H. m, H-11), 1.59 (1H, m, H-6eq), 1.60 (1H, m, H-1eq), 1.62 (1H, m, H-3eq), 1.78 (1H, m, H-2eq); 13C NMR (CDCl3, 125 MHz): δ 16.8 (C-13), 16.9 (C-12), 17.7 (C-14), 18.0 (C-2), 29.3 (C-6), 29.5 (C-8), 30.0 (C-15), 33.6 (C-10), 39.1 (C-9), 39.2 (C-11), 41.2 (C-1), 41.5 (C-3), 46.0 (C-5), 72.1 (C-4), 74.4 (C-7); EI-MS m/z 240 [M]+; HR-EI-MS m/z 240.2096 [M]+ (calcd for C15H28O2, 240.2090).
Cells (2 × 105)/well were cultured in 96-well plate containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then cells were cultured with compounds 1–12 (each 20 μg/mL) for 24 h. After that, the cells were incubated with 100 μL of 0.5 mg/mL MTT for 4 h at 37 °C testing for cell viability. The medium was then discarded and 100 μL dimethyl sulfoxide (DMSO) was added. After 30min incubation, absorbance at 570 nm was read using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). 3.6. Measurement of nitric Oxide/Nitrite NO production was indirectly assessed by measuring the nitrite levels in the cultured media and serum determined by a colorimetric method based on the Griess reaction. The cells were incubated with different concentration of samples in the presence of LPS (100 ng/mL) at 37 °C for 24 h. Take the supernatant in another 96-well plate and add an equal amount of mixed Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 5% phosphoric acid) and incubated at room temperature for 10 min, the absorbance was measured at 540 nm with a Micro-Reader (Molecular Devices). By using sodium nitrite to generate a standard curve, the concentration of nitrite was measured from absorbance at 540 nm. 3.7. Statistical analysis The data are expressed as means ± standard errors (SE). The IC50 values were calculated from the dose curves using a non-linear regression algorithm (SigmaPlot 8.0; SPSS Inc., Chicago, IL, USA, 2002). Statistical evaluation was carried out by one-way analysis of variance (ANOVA followed by Scheffe’smultiple range tests). Acknowledgements This research was supported from CMU under the Aim for Top University Plan of the Ministry of Education, Taiwan (CHM 106-5-2), and Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW106- TDU-B-212-113104). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.06.012. References Ahmed, S., Rahman, A., Alam, A., Saleem, M., Athar, M., Sultana, S., 2000. Evaluation of the efficacy of Lawsonia alba in the alleviation of carbon tetrachloride-induced oxidative stress. J. Ethnopharmacol. 69, 157–164. Babu, P.D., Subhasree, R.S., 2009. Antimicrobial activities of Lawsonia inermis–a review. Acad. J. Plant Sci. 2, 231–232. Cao, X.D., Ding, Z.S., Jiang, F.S., Ding, X.H., Chen, J.Z., Chen, S.H., Lu, G.Y., 2012. Antitumor constituents from the leaves of Carya cathayensis. Nat. Prod. Res. 22, 2089–2094. Chen, H.Y., Qian, C., 2007. Flora of China. Editorial Committee of the Flora of China, Beijing, China, pp. 274–288. Dao, T.T., Tung, B.T., Nguyen, P.H., Thuong, P.T., Yoo, S.S., Kim, E.H., Kim, S.K., Oh, W.K., 2010. C-methylated flavonoids from Cleistocalyx operculatus and their inhibitory effects on novel influenza a (H1N1) neuraminidase. J. Nat. Prod. 73, 1636–1642. Gao, W., Li, Q., Chen, J., Wang, Z., Hua, C., 2013. Total synthesis of six 3,4-unsubstituted coumarins. Molecules 18, 15613–15623. Geller, D.A., Billiar, T.R., 1998. Molecular biology of nitric oxide synthases. Cancer Metastasis Rev. 17, 7–23. Junio, H.A., Sy-Cordero, A.A., Ettefagh, K.A., Burns, J.T., Micko, K.T., Graf, T.N., Richter, S.J., Cannon, R.E., Oberlies, N.H., Cech, N.B., 2011. Synergy-directed fractionation of botanical medicines: a case study with goldenseal (Hydrastis canadensis). J. Nat. Prod. 74, 1621–1629. Kai, H., Baba, M., Okuyama, T., 2007. Two new megastigmanes from the leaves of Cucumis sativus. Chem. Pharm. Bull. 55, 133–136.
3.4. Cell culture A murine macrophage cell line RAW 264.7 (BCRC No. 60001) was purchased from the Bioresources Collection and Research Center (BCRC, Hsinchu, Taiwan) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in plastic dishes containing Dulbecco′s Modified Eagle Medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma) in a CO2 incubator (5% CO2 in air) at 37 °C and subcultured every 3 days at a dilution of 1:5 using 0.05% trypsin-0.02% EDTA in Ca2+-, Mg2+-free phosphate-buffered saline (DPBS).
126
Phytochemistry Letters 21 (2017) 123–127
C.-S. Yang et al.
Moncada, S., Palmer, R.M., Higgs, E.A., 1991. Nitric oxide physiology, pathophysiology, and pharmacology. Pharmcol. Rev. 43, 109–142. Sung, T.V., Steffan, B., Steglich, W., Klebet, G., Adam, G., 1992. Sesquiterpenoids from the roots of Homalomena Aromatica. Phytdwnistry 31, 3515–3520. Wang, Y., Lugtenburg, J., 2004. 4,5-Didehydro-9-demethyl-9-halo-5, 6-dihydroretinals and their 9-Cyclopropyl and 9-Isopropyl derivatives—simple preparation of α-Ionone derivatives and pure (all-E)-, (9Z)- and (11Z)-α-retinals. Eur. J. Org. Chem. 16, 3497–3510. Yang, C.S., Huang, H.C., Wang, S.Y., Sung, P.J., Huang, G.J., Chen, J.J., Kuo, Y.H., 2016. New diphenol and isocoumarins from the aerial part of Lawsonia inermis and their inhibitory activities against NO production. Molecules 21, 1299. Youssef, D.T.A., Ramadan, M.A., Khalifa, A.A., 1998. Acetophenones, a chalcone, a chromone and flavonoids form Pancratium maritimum. Phytochemistry 49, 2579–2583.
Kuo, Y.H., Chu, P.H., 2002. Studies on the constituents from the bark of Bauhinia purpurea. J. Chin. Chem. Soc. 49, 269–274. Lin, Y.X., Chang, Y.S., Chen, I.S., Ou, J.C., 2003. The Catalogue of Medicinal Plant Resources in Taiwan. Committee on Chinese Medicine and Pharmacy, Taipei, Taiwan. Liou, J.R., Mohamed, E.S., Du, Y.C., Tseng, C.N., Hwang, T.L., Chuang, Y.L., Hsu, Y.M., Hsieh, P.W., Wu, C.C., Chen, S.L., Hou, M.F., Chang, F.R., Wu, Y.C., 2013. 1,5Diphenylpent-3-en-1-ynes and methyl naphthalene carboxylates from Lawsonia inermis and their anti-inflammatory activity. Phytochemistry 88, 67–73. Liu, H., Mou, Y., Zhao, J., Wang, J., Zhou, L., Wang, M., Wang, D., Han, J., Yu, Z., Yang, F., 2010. Flavonoids from halostachys caspica and their antimicrobialand antioxidant activities. Molecules 15, 7933–7945. Marques, M.R., Stüker, C., Kichik, N., Tarragó, T., Giralt, E., Morel, A.F., Dalcol, I.I., 2010. Flavonoids with prolyl oligopeptidase inhibitory activity isolated from Scutellaria racemosa Pers. Fitoterapia 81, 552–556.
127