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Secondary metabolites from the root of Lindera reflexa Hemsl
Fitoterapia 105 (2015) 222–227
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Secondary metabolites from the root of Lindera reflexa Hemsl Suiqing Chen, Lili Wang ⁎, Wanqing Zhang, Yalei Wei School of Pharmacy, Henan University of Traditional Chinese Medicine, BoXue Road, Jinshui District, ZhengZhou, 450046, China
a r t i c l e
i n f o
Article history: Received 22 April 2015 Received in revised form 2 July 2015 Accepted 3 July 2015 Available online 12 July 2015 Keywords: Lindera reflexa Hemsl New skeleton Stilbene Antitumor
a b s t r a c t Fourteen compounds were isolated from the root of Lindera reflexa Hemsl. Among these compounds, reflexan A (compound 1) was discovered to possess a novel skeleton and two stilbenes (compounds 2 and 3) were newly discovered compounds. Eleven known compounds (4–14) were also isolated, which included three alkaloids, one coumarin, four stilbenes, two flavonoids and katsumadain. The unusual structure of compounds 1, 2 and 3, along with their absolute configurations, was determined from UV, IR, HR ESI-MS, and 1D and 2D NMR data and by the comparison of experimental and calculated electronic circular dichroism (ECD) spectra. All of the isolates were tested with antitumor and anti-inflammatory assays. The results revealed that compounds 1, 2, and 3 all showed inhibitory activity toward three tumor cell lines, A549 (human lung carcinoma), BEL-7402 (human liver carcinoma) and HCT-8 (human colon carcinoma). None of the compounds exhibited anti-inflammatory activity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Lindera reflexa Hemsl is a type of shrub or vine in the family of Lauraceae, mainly distributed in the southern region of the Yangtze River in China. The root of L. reflexa Hemsl, which is used for the treatment of gastritis and peptic ulcers, was a newly discovered herbal drug and was listed in the Dictionary of Chinese Medicine. The root was the main ingredient of a Chinese patent medicine for treating peptic ulcers and has been used in clinical practice for the past few years. Previous phytochemical studies showed that the root contains volatile oil [1]; alkaloids of launobine, lindcarpine and laurolitsine [2,3]; flavonoids of pinostrobin, pinocembrin, sakuranetin; and 3, 5-didroxystilbene [4,5]. To further study this herbal medicine, our lab has conducted a great deal of research on the volatile oil as well as on water, petroleum ether, ethyl acetate and n-butanol extracts. Additionally, pharmacological evaluations of the anti-inflammatory, analgesic, and anti-ulcer activities of these extracts have been performed. Ultimately, the ethyl acetate and n-butanol extracts were found to have significant anti-ulcer and antiinflammatory activities. In this study, the ethyl acetate and n-butanol extracts were analyzed. At the same time, 14 compounds were obtained, among which compounds 1, 2 and 3 were newly discovered. Compound 1, named reflexan A, was found to possess a novel skeleton. Compound 2 and compound 3, named reflexanbene I and reflexanbene II, respectively, were newly discovered compounds fused to stilbene, and the structure of the new compounds is shown in Fig. 1. Their
⁎ Corresponding author at: Henan University of Traditional Chinese Medicine, BoXue Road, Jinshui District, ZhengZhou 450046, China. E-mail addresses: [email protected] (S. Chen), [email protected] (L. Wang).
http://dx.doi.org/10.1016/j.fitote.2015.07.005 0367-326X/© 2015 Elsevier B.V. All rights reserved.
antitumor activities and anti-inflammatory potencies have been investigated in various systems to date. 2. Results and discussion Reflexan A (compound 1) was obtained as colorless needles. mp.164–167 °C; [α]20 D = − 2.2° (c 0.09, CHCl3);and was soluble in chloroform and slightly soluble in methanol. Upon addition of the test solution of ferric chloride–potassium ferricyanide, the compound turned blue, which suggested that the compound containing phenol groups. The molecular formula was determined to be C27H34O5 by HR ESI-MS at m/z: 439.2490[M + H]+ (calcd 439.2484), which indicated the compound contained 11 degrees of unsaturation. The UV spectrum showed the maximum absorption wavelength at 298.4 nm. IR absorptions indicated the existence of a hydroxyl group (3433 cm−1), ketone (1713 cm−1, 922 cm− 1), olefin (1660 cm−1), and an aromatic ring (1575 cm−1, 1496 cm−1, 1455 cm−1). The 1H-NMR spectrum (Table 1) displayed five protons signals at δ7.13–7.27, δ7.15 (3H,t,J = 7.2,7.0Hz) and 7.24 (2H,m), which are typical mono-substituted benzene ring proton signals. The signals at δ5.94 (1H,s) and δ4.98 (1H,s) were proton signals of a double bond, and each of the two carbons were substituted; the signals at δ1.55 (3H,s), 1.93 (3H,s), 0.69 (3H,d), and 0.79 (3H,d) corresponded to four methyl groups. The 13C-NMR spectrum (Table 1) displayed fourteen carbon signals, including a benzene ring, two carboxyls, and two double bond carbons. The remaining two carbon signals were indicative of sp2-hybridized quaternary carbons (C-3,C-4). Thirteen carbon signals were observed downfield, among which δ70.6 was located upfield and suggested a carbon bonded to an oxygen atom. Together with the DEPT135 spectrum,
S. Chen et al. / Fitoterapia 105 (2015) 222–227
223
Fig. 1. New compounds from Lindera reflexa Hemsl.
the results indicated the presence of five methylenes, four methines and four methyls. The HMBC spectrum (Table 2) showed correlations of δ0.69 (H-10′) and 0.79 (H-9′), with δ28.4 (C-8′), and 39.9 (C-4′) and δ1.55 (H-7′), with δ132.2 (C-1′), 125.2 (C-2′), and 30.5 (C-6′), and indicated that compound 1 might be a C-3-substituted menthene derivative, which was also confirmed by the 1H–1H COSY spectrum. δ5.94 (H-5) correlated to δ104.2 (C-3) and 159.4 (C-6), and δ3.37 (H-3′) exhibited long-range correlations with δ104.2 (C-3), 163.5 (C-4) and 165.3 (C-2). These correlations, combined with the monosubstituted benzene ring proton signals in the 1H NMR spectrum, suggested that a pyran-2-one group existed in the compound. These data were similar to those of the pyran-2-one moiety of katsumadain [6], and C-3′ linked to C-3. The 1H-1H COSY spectrum showed the correlations of H-7 with H-8, H-8 with H-9, and H-9 with H-10, and the HMBC spectrum showed the correlations of δ4.96 (H-8) with δ169.9 (C-17), and δ2.60 (H-10) with δ70.6 (C-8), which indicated the existence of a \\CH2CH2CH2OCOCH3 moiety [7]. In contrast to katsumadain, compound 1 had no alkenyl group at C-7 and contained a\\CH2CH2CH2OCOCH3 moiety.
δ2.60 (H-10) exhibited long-range correlations with δ141.3 (C-11), 128.4 (C-12, 16), 70.6 (C-8) and 35.1 (C-9), indicating a benzene ring linked to C-10, δ2.65 (H-7), which had long-range correlation with 159.4 (C-6) and indicated C-7 was connected to C-6 of the pyran-2one moiety. The NOE spectrum (Fig. 2) showed that C-3′ and C-4′ represented a trans-oriented structure because H-3′ and H-4′ had no NOE correlations. The absolute configuration was determined by comparing the experimental and calculated CD spectra. The latter was performed using time-dependent density functional theory. Optimized geometries were obtained by systematic conformational analysis using the MMFF94 force field, and ECD spectra were calculated in methanol solution using the B3LYP/6–31 g (d) level of theory with the CPCM model. The results showed that the experimental and calculated spectra were in good agreement (Fig. 3). Thus, the absolute configuration of 1 was determined to be 3′S,4′S. In summary, the compound was determined to be 4-hydroxy-3-[(3′ R,4′R)-1′-p-mentheneyl]-6-(4-phenyl-2-acetoxy group-n-butyl)-2Hpyran-2-one.
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Table 1 1 H-NMR and 13C-NMR data for compound 1 (in DMSO-d6). Table 2. HMBC list of compound 1, compound 2 and compound 3. No.
δC
2 3 4 5 6 7 8 9 10 11 12, 16 14 13, 15 17 18 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′
165.3 104.2 163.5 101.3 159.4 37.7 70.6 35.1 31.0 141.3 128.4 126.1 128.5 169.9 20.9 132.2 125.2 35.2 39.9 22.7 30.5 23.4 28.4 21.4 16.6
δH
5.94 (1H, s) 2.70 (1H, d, J = 4.8 Hz) 2.65 (1H, m) 4.96 (1H, m) 1.86 (2H, m) 2.60 (2H, m) 7.15 (3H, t, J = 7.2, 7.0 Hz) 7.24 (2H, m) 1.93 (3H, s) 4.98 (1H, s) 3.37 (1H, m) 1.96 (1H, m) 1.66 (1H, m), 1.23 (1H, m) 1.95 (1H, m), 1.85 (1H, m) 1.55 (3H, s) 1.44 (1H, m) 0.79 (3H, d, J = 6.8 Hz) 0.69 (3H, d, J = 6.8 Hz)
HNMR (400 MHz) (δC in ppm, J in Hz), 13CNMR(100 MHz).
Reflexanbene I (compound 2) was obtained as a reddish-brown powder, mp.164–167 °C; [α]20 D = −2.2° (c 0.09, CHCl3); and was soluble in methanol, and spraying the compound with an anisyl aldehyde/ sulfuric acid reagent caused it to turn purple. Upon addition of the ferric chloride–potassium ferricyanide test solution, the color turned blue, which suggested the compound contained phenol groups. HR ESI-MS m/z: 371.1987[M + Na]+(calcd 371.1987), molecular formula was determined to be C24H28O2, with 11 degrees of unsaturation. The Table 2 HMBC list of compound 1, compound 2 and compound 3. Compound Compound 1
HMBC data
Fig. 2. Key HMBC, NOESY correlations of compound 1.
UV spectrum showed the maximum absorption wavelength at 300.8 nm. IR absorptions indicated the existence of a hydroxyl group (3415 cm− 1) and an aromatic ring (1610 cm− 1, 1446 cm− 1, 1585 cm− 1, 1496 cm− 1). The 1H-NMR spectrum (Table 3) showed nine aromatic proton signals; δ6.77 (1H, d, J = 16.1 Hz) and 7.73 (1H, d, J = 16.0 Hz) were typical trans double bond proton signals; δ7.16–7.40 displayed five proton signals; δ7.37 (2H, d, J = 7.6 Hz), 7.29 (2H, t, J = 7.4, 7.8 Hz), and 7.19 (1H, t, J = 7.3, 7.2 Hz) were typical mono-substituted benzene ring proton signals; and δ6.24 (1H, d, J = 2.4 Hz), 6.56 (1H, d, J = 1.8 Hz) were meta-substituted proton signals of a benzene ring; the signals at δ1.73 (3H, s), 0.80 (3H, d, J = 6.4 Hz), 0.74 (3H, d, J = 6.9 Hz) corresponded to three methyl groups, and the 13CNMR spectrum (Table 3) showed twenty-four carbon signals, of which fourteen corresponded to aromatic carbons. These data suggested two benzene rings in the compound, and after comparing these data with data from the reference compound [8,9], the compound was determined to be a stilbene derivative. In addition, DEPT-135 and 13C-NMR spectra showed that the remaining seven carbons included two methylenes (δ24.2, 32.4), four methines (δ129.4, 37.7, 46.9, 28.9), and one quaternary carbon (δ133.5). The HMBC spectrum (Table. 2) showed cross-peaks of δ0.80 (Me-9″), with δ16.9 (C-10″); δ0.74 (Me-10″) with δ22.0 (C-9″); δ1.44–1.51 (H-8″), with δ22.0 (C-9″) and 16.9 (C-10″); and δ0.80, 0.74 and δ1.44–1.51, with 46.9 (C-4″), which suggested an isopropyl moiety linked to δ46.9. The 1H-1H COSY spectrum showed that H-3″, H-4″, H-
Compound 2
Compound 3
Fig. 3. Experimental and calculated CDs of compound 1 in MeOH.
S. Chen et al. / Fitoterapia 105 (2015) 222–227 Table 3 1 H-NMR and 13C-NMR data of compound 2 (in MeOD). NO.
δC
1 2 3 4 5 6 α β 1′ 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4″ 5″″ 6″ 7″ 8″ 9″ 10″
139.9 122.3 156.9 102.7 158.1 105.5 130.0 128.3 139.5 127.2 129.6 128.4 133.5 129.4 37.7 46.9 24.2 32.4 23.9 28.9 22.0 16.9
225
Table 4 1 H-NMR and 13C-NMR data of compound 3 (in MeOD). δH
6.24 (1H, d, J = 2.4 Hz) 6.56 (1H, d, J = 1.8 Hz) 7.77 (1H, d, J = 15.2 Hz) 6.70 (1H, d, J = 16.1 Hz) 7.38 (2H, d, J = 7.6 Hz) 7.29 (2H, t, J = 7.4, 7.8 Hz) 7.19 (1H, t, J = 7.3, 7.2 Hz) 5.36 (1H, s) 4.06 (1H, t J = 1.56, 1.50 Hz) 1.76 (1H, m) 1.73 (1H, s), 1.41 (1H, m) 2.14 (2H, m) 1.73 (3H, s) 1.44–1.51 (1H, m) 0.80 (3H, d, J = 6.4 Hz) 0.74 (3H, d, J = 6.9 Hz)
NO.
δC
1 2 3 4 5 6 α β 1′ 2′, 6′ 4′ 3′, 5′ 1″ 2″ 3″ 4″ 5″ 6″
135.6 126.3 161.9 98.9 158.7 103.7 127.8 130.0 138.7 127.4 128.6 129.7 70.0 92.0 41.9 47.7 18.5 35.8
7″ 8″ 9″ 10″
1
H-NMR (400 MHz) (δC in ppm, J in Hz), 13C NMR (100 MHz).
28.1 27.4 22.1 16.4
δH
6.23 (1H, s) 6.67 (1H, s) 7.13 (1H, d, J = 16.0 Hz) 7.04 (1H, d, J = 16.0 Hz) 7.49 (2H, d, J = 7.5 Hz) 7.22 (1H, t, J = 7.0, 7.0 Hz) 7.32 (2H, t, J = 7.5, 7.5 Hz) 4.03 (1H, d, J = 4.3 Hz) 3.18 (1H, brs.) 1.02 (1H, t, J = 11.5, 11.5 Hz) 1.51 (1H, m), 1.36 (1H, m) 1.74 (1H, d, J = 13.4 Hz), 1.58–1.64 (1H, m) 1.38 (3H, s) 1.87 (1H, t, J = 6.0, 6.0 Hz) 0.92 (3H, d, J = 6.5 Hz) 0.73 (3H, d, J = 7.0 Hz)
1
H-NMR (500 MHz) (δC in ppm, J in Hz), 13CNMR(125 MHz).
5″, and H-6″ were connected in sequence and that δ5.36 (H-2″) and 1.76 (H-4″) had a cross-peak with δ122.3 (C-2), which indicated that C-3″ was linked to C-2. These data suggested the compound contained a menthen moiety linked to C-3 [10]. H-3″ and H-4″ had no NOE correlations, suggesting the two protons was ipso-lateral, C-3″ and C-4″ was determined to possess a trans structure by NOESY spectrum (Fig. 4). The absolute configuration was also determined using the same method used for compound 1, and the configuration was determined to be 3″R 4″R. Based on the above analysis, compound 2 was determined to be 3,5dihydroxy-2-[(3″R4″R)-L-menthene]-trans-stilbene. The compound named reflexanbene I had never been reported and was confirmed to be a newly discovered compound. Reflexanbene II (compound 3) was obtained as a reddish-brown powder that was soluble in methanol and chloroform. Spraying the compound with an anisyl aldehyde/sulfuric acid reagent caused it to turn purple. Upon addition of the ferric chloride–potassium ferricyanide test solution, the color turned blue, which suggested the compound contained phenol groups. From the ESI-MS m/z: 387.1938 [M + H]+ (calcd 387.1931),the molecular formula was determined to be C 24 H28O 3 , with 11 degrees of unsaturation. UV (MeOH) λ max at 311.50 nm. IR absorptions at 1122 cm−1 and 975 cm−1 were assigned to an epoxy (C\\O) moiety and a trans double bond, respectively.
The data for compound 3 were similar to those of compound 2, except for a deletion of the C-2″and C-3″ double bond and the existence of an epoxy quaternary carbon and an epoxy methane. The 13C-NMR spectrum and 1H-NMR spectrum are shown in Table 4. C-1″, C-2″, C-3″, and C-4″ were chiral carbons and H-4″ and H-2″ had no NOE correlations; there were no NOE correlations between H-3″ and H-2″ or between H-3″and 1″-CH3, suggesting that H-4″ and H-2″, H-3″, and 1′-CH3 were trans-oriented. The absolute configuration was also determined using the same method as was used for compound 1, and the configuration was 1″ S2″R3″S4″R. In summary, the compound was determined to be 3,5dihydroxy-2-[(1″S2″R3″S4″R)- L -(1″,2″-epoxy)-phmentheneyl]trans-stilbene, which was named reflexanbene II and was also a newly discovered compound. In addition to the new compounds, three alkaloids, launobine [11], laetanine [12], and norbracteoline [13]; one scoplin coumarin [14]; four pinosylvin stilbenes [15]; cis-3,5-didroxystilbene [16], 3methoxy-5-hydroxy-trans-stilbene [17]; β,β′-pinosylvin diglucoside [4]; two pinostrobin flavonoids [15]; pinocembrin [15]; and katsumadain [6] were elucidated. Two different assays, an in vitro antitumor activity assay of three tumor cell lines, A549 (human lung carcinoma), Bel-7402 (human liver carcinoma) and HCT-8 (human colon carcinoma), and an inhibitory assay on TNF-α, IL-6 and NO production from RAW264.7 stimulated
Fig. 4. Key HMBC, NOESY correlations of compound 2.
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by LPS, were carried out to evaluate the bioactivities of the isolated compounds. The new compounds (1,2,3) showed antitumor effects against BEL-7402, HCT-8 and A549; as shown in Table 5, the results provided some evidence for antitumor activities of stilbenes. None of the compounds exhibited significant anti-inflammatory activity toward any of the three inflammatory cell lines. As for reflexan A, it was considered to be a new skeleton; the structure of this compound has many similarities to those compounds isolated from the Linderae genus of the Lauraceae family. The p-menthene group was similar to the p-menthene substituents of linderatin, linderation, linderachalcone, and methylinderatone, which were isolated from Lindera umbellata THUNB.var.membranacea (MAXIM.) MOMIYAMA [18,19]. The \\CH2CH2CH2OCO-group was similar to the \\CH2CH2CH2OCOmoiety of (2S,3S)-2,3-bis[(4-hydroxy-3,5-dimethoxyphenyl)methyl]1,4-butanediol 1,4-diferulate, which was isolated from the stems and twigs of L. umbellata [20]. The pyran-2-one group was similar to the pyran-2-one moiety of katsumadain B from Alpinia katsumadai [21]. In Chinese traditional medicines, the seeds of Alpinia katsumadai and the root of L. reflexa were considered to have identical pharmacological effects for treating stomach diseases; thus, the two plants were thought to have similar chemical constituents such as essential oils, stilbenes and flavonoids of pinocembrin [22]. 3. Exprimental 3.1. General experimental procedure All of the melting points were measured using a Kofler micromelting apparatus and were uncorrected. UV spectra were recorded on an Agilent 8453 spectrometer in MeOH. IR spectra were obtained on a Shimadzu FTIR-8201 infrared spectrophotometer. The 1H and 13C spectra were obtained in MeOD on Bruker Avance DPX-400 (400 MHz for 1H-NMR and 100 MHz for 13C-NMR) and Bruker Avance III 500 (500 MHz for 1H-NMR and 125 MHz for 13C-NMR) spectrometers, using TMS as an internal standard. APEX II FT-ICR mass spectrometry was used. Column chromatography (CC) was performed using Toyopearl HW-40 (Tosoh, Japan) resins and Pharmadex LH-20 (Amersham Life Science, Shanghai). Preparative thin-layer chromatography (TLC) was conducted with silica gel (Qingdao Haiyang Chemical Co.). 3.2. Plant materials The root of the L. reflexa Hemsl. was collected in August 2010 at Xin Xian county, Henan province. The voucher specimen was deposited in the pharmacognosy lab of the Pharmacy College of Henan University of Traditional Chinese Medicine.
Table 5 Cytotoxic activity of compounds by MTT method. Compound
BEL-7402 Inhibition (%)
1 2 3 4 5 6 8 9 11 12 14 13 5-Fu
4.96 70.38 72.14 5.74 27.70 14.16 18.05 9.23 4.99 11.49 3.68 32.27 75.31
HCT-8 IC50 (μg/mL) 10.90 10.48
3.93
A549
Inhibition (%)
IC50 (μg/mL)
Inhibition (%)
IC50 (μg/mL)
63.42 62.86 11.39 0.27 6.52 11.67 −4.02 0.49 −2.66 −7.11 19.21 −1.09 80.59
11.96 11.14
75.07 68.90 15.12 9.30 1.79 24.55 10.59 27.08 −0.77 21.52 22.77 4.44 54.47
10.53 11.75
7.51
16.19
3.3. Extraction and isolation Dried root (8.0 kg) was extracted with 95% ethanol by refluxing three times for 2 h each, and the extract was concentrated to dryness (624.5 g). The residue was dissolved in water and extracted with petroleum ether, ethyl acetate, and n-butyl alcohol sequentially. A portion of the active EtOAc-soluble fraction (256 g) was subjected to silica gel CC and eluted with chloroform and petroleum ether mixtures of various proportions to obtain Fr.1 to Fr.3, eluted with chloroform to obtain Fr.4, and eluted with different proportions of chloroform and MeOH to obtain Fr.5 to Fr. 10. Fr.1 was subjected to silica gel CC and recrystallization to obtain compound 5 (2080 mg). Fr.2 also contained compound 5 (840 mg). Fr.3 was subjected to silica gel CC eluted with different proportions of petroleum ether and EtOAc; then, the fractions was subjected to Toyopearl HW-40 and Pharmadex LH20 resin gel CC to obtain compounds 1 (350 mg), 2 (23 mg), 4 (52 mg), 6 (370 mg), 7 (45 mg), and 10 (4.5 mg). Fr.4, Fr.5, Fr.6 and Fr.10 were subjected to silica gel CC eluted with different proportions of petroleum ether and EtOAc, then the fractions were subjected to Toyopearl HW-40 and Pharmadex LH-20 resin gel CC to obtain compounds 8 (2840 mg), 8 (6870 mg), 8 (320 mg), and 9 (220 mg). A portion of the active n-butyl alcohol-soluble fraction (113 g) was subjected to silica gel CC and eluted with different proportions of a methanol and dichloromethane mixture to obtain Fr.11 and Fr.12. Fr.11 was subjected to silica gel CC and recrystallization to obtain compound 11 (300 mg); then, the fractions were eluted with petroleum: EtOAc (5:1) to obtain compound 3 (48 mg). Fr.12 was subjected to silica gel CC and recrystallization and then eluted with chloroform-MeOHH2O (9:1:0.1) to obtain compound 12 (1360 mg) and compound 13 (108 mg); elution with chloroform-MeOH (100:5) gave compound 14 (23 mg). Compound 1: colorless pin needless (MeOH-CHCl3), mp.164–167 °C; [α]20 D − 2.2° (c 0.09, CHCl 3 ); UV λ max (log ε) 298.4 nm; IR(KBr) νmax cm − 1 3433, 2953, 1745, 1660, 1615, and 1405. For 1 H and 13 C NMR data, see Table 1; supplementary data; HR ESI-MS m/z: 439.2490[M + H]+(calcd for C27H34O5, 439.2484). Compound 2: red brown powder (MeOH); UV λmax nm: 300.8; IR(KBr) cm− 1 3415,2956, 1610, 1446; for 1H and 13C NMR data, see Table 3; supplementary data; HR ESIMS m/z: 371.1987[M + Na] + (calcd for C24H28O2, 371.1987). Compound 3: red brown powder (MeOH); [α]20 D + 20° (c 0.70, CHCl3); UV (MeOH) λmax (log ε) 311.50 nm; IR (KBr) νmax cm− 1 3277, 1614, 1589, 1122, 756, and 741; for 1H and 13C NMR data, see Table 4; supplementary data; HRESIMS m/z: 387.1938 [M + H]+ (calcd for C24H28O3, 387.1931). TNF-α, IL-6 and NO production from RAW264.7 stimulated by LPS: RAW264.7 cells (5 × 104 per well) were suspended in 100 μL of 1640 supplemented with 10% fetal calf serum, penicillin (100 units/mL), and streptomycin (100 μg/mL) and were pre-cultured in 96-well microplates at 37 °C 5% CO2 in air for 12 h. RAW264.7 cells were added by LPS (final concentration 5 μg/mL). The final concentrations of the test compounds in the assay were 5 μg/mL. After 12 h of incubation, the amount of TNF-α and IL-6 production in each well was determined by analyzing the culture medium using the ELISA method. NO production in the culture medium was detected by the Griess assay. The cytotoxicity was assessed by the CCK-8 colorimetric assay. All of the tests were performed in duplicate. Inhibition (%) was calculated using the formula: Inhibition (%) = (A − B)/A × 100, where A − B = TNF-α or IL-6 or NO concentration (pg/mL); A = LPS (+), sample (−); B = LPS (+), sample (+). In vitro antitumor activity assay: All of the isolates were tested for cytotoxicity against A549 (human lung carcinoma), BEL-7402 (human liver carcinoma), and HCT-8 (human colon carcinoma) cells by means of the MTT assay. These cells were plated in 96-well plates and cultured for 24 h. The appropriate test compounds and positive control were added into triplicate wells at concentrations of 20, 10, 2.5, and
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1.25 μg/mL and incubated for 4 days at 37 °C. MTT solution (10 μL, 5 mg/mL) was added into each well, and the plate was incubated for another 4 h. The resulting formazan crystals were dissolved in DMSO (100 μL), and the UV/vis absorbance (optical density: OD) was determined with a microplate spectrophotometer at 570 nm. The reference compound, 5-fluorouracil, exhibited activity toward the A549, Bel-7402 and HCT-8 cell lines with an IC50 range of 0.2–0.7 μg/mL. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by National Science and Technology Major Project of the Ministry of Science and Technology of China (Grant No. 2012ZX09103201-024) and Innovation Scientists and Technicians Troop Construction Projects of Henan Province (Grant No. 114200510014). We thank the National Center of Pharmaceutical Screening, Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College, for cytotoxic tests. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2015.07.005. References [1] Y.M. Luo, L.Q. Huang, P. Wang, Q. Li, Study on the volatile oil in aerial part and underground part of Linderae reflexae Hemsl, Chin. Pharm. J. 39 (4) (2004) 307–309. [2] C.F. Zhang, Z.T. Wang, An advance in the study on the medicinal plant of Lindera, J. Shenyang Pharma. Univ. 17 (3) (2000) 230–234. [3] J.Z. Zhang, Q.C. Fang, Study on the chemical constituents of Montane Spicebush (Lindera reflexa), Chin. Tradit. Herb. Drugs 25 (11) (1994) 565–568. [4] J.Z. Zhang, Q.C. Fang, Application of high speed counter-current chromatography to the separation of stilbene derivatives from the roots of Lindera reflexa, Planta Med. 60 (1994) 190–191.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Secondary metabolites from the root of Lindera reflexa Hemsl Suiqing Chen, Lili Wang ⁎, Wanqing Zhang, Yalei Wei School of Pharmacy, Henan University of Traditional Chinese Medicine, BoXue Road, Jinshui District, ZhengZhou, 450046, China
a r t i c l e
i n f o
Article history: Received 22 April 2015 Received in revised form 2 July 2015 Accepted 3 July 2015 Available online 12 July 2015 Keywords: Lindera reflexa Hemsl New skeleton Stilbene Antitumor
a b s t r a c t Fourteen compounds were isolated from the root of Lindera reflexa Hemsl. Among these compounds, reflexan A (compound 1) was discovered to possess a novel skeleton and two stilbenes (compounds 2 and 3) were newly discovered compounds. Eleven known compounds (4–14) were also isolated, which included three alkaloids, one coumarin, four stilbenes, two flavonoids and katsumadain. The unusual structure of compounds 1, 2 and 3, along with their absolute configurations, was determined from UV, IR, HR ESI-MS, and 1D and 2D NMR data and by the comparison of experimental and calculated electronic circular dichroism (ECD) spectra. All of the isolates were tested with antitumor and anti-inflammatory assays. The results revealed that compounds 1, 2, and 3 all showed inhibitory activity toward three tumor cell lines, A549 (human lung carcinoma), BEL-7402 (human liver carcinoma) and HCT-8 (human colon carcinoma). None of the compounds exhibited anti-inflammatory activity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Lindera reflexa Hemsl is a type of shrub or vine in the family of Lauraceae, mainly distributed in the southern region of the Yangtze River in China. The root of L. reflexa Hemsl, which is used for the treatment of gastritis and peptic ulcers, was a newly discovered herbal drug and was listed in the Dictionary of Chinese Medicine. The root was the main ingredient of a Chinese patent medicine for treating peptic ulcers and has been used in clinical practice for the past few years. Previous phytochemical studies showed that the root contains volatile oil [1]; alkaloids of launobine, lindcarpine and laurolitsine [2,3]; flavonoids of pinostrobin, pinocembrin, sakuranetin; and 3, 5-didroxystilbene [4,5]. To further study this herbal medicine, our lab has conducted a great deal of research on the volatile oil as well as on water, petroleum ether, ethyl acetate and n-butanol extracts. Additionally, pharmacological evaluations of the anti-inflammatory, analgesic, and anti-ulcer activities of these extracts have been performed. Ultimately, the ethyl acetate and n-butanol extracts were found to have significant anti-ulcer and antiinflammatory activities. In this study, the ethyl acetate and n-butanol extracts were analyzed. At the same time, 14 compounds were obtained, among which compounds 1, 2 and 3 were newly discovered. Compound 1, named reflexan A, was found to possess a novel skeleton. Compound 2 and compound 3, named reflexanbene I and reflexanbene II, respectively, were newly discovered compounds fused to stilbene, and the structure of the new compounds is shown in Fig. 1. Their
⁎ Corresponding author at: Henan University of Traditional Chinese Medicine, BoXue Road, Jinshui District, ZhengZhou 450046, China. E-mail addresses: [email protected] (S. Chen), [email protected] (L. Wang).
http://dx.doi.org/10.1016/j.fitote.2015.07.005 0367-326X/© 2015 Elsevier B.V. All rights reserved.
antitumor activities and anti-inflammatory potencies have been investigated in various systems to date. 2. Results and discussion Reflexan A (compound 1) was obtained as colorless needles. mp.164–167 °C; [α]20 D = − 2.2° (c 0.09, CHCl3);and was soluble in chloroform and slightly soluble in methanol. Upon addition of the test solution of ferric chloride–potassium ferricyanide, the compound turned blue, which suggested that the compound containing phenol groups. The molecular formula was determined to be C27H34O5 by HR ESI-MS at m/z: 439.2490[M + H]+ (calcd 439.2484), which indicated the compound contained 11 degrees of unsaturation. The UV spectrum showed the maximum absorption wavelength at 298.4 nm. IR absorptions indicated the existence of a hydroxyl group (3433 cm−1), ketone (1713 cm−1, 922 cm− 1), olefin (1660 cm−1), and an aromatic ring (1575 cm−1, 1496 cm−1, 1455 cm−1). The 1H-NMR spectrum (Table 1) displayed five protons signals at δ7.13–7.27, δ7.15 (3H,t,J = 7.2,7.0Hz) and 7.24 (2H,m), which are typical mono-substituted benzene ring proton signals. The signals at δ5.94 (1H,s) and δ4.98 (1H,s) were proton signals of a double bond, and each of the two carbons were substituted; the signals at δ1.55 (3H,s), 1.93 (3H,s), 0.69 (3H,d), and 0.79 (3H,d) corresponded to four methyl groups. The 13C-NMR spectrum (Table 1) displayed fourteen carbon signals, including a benzene ring, two carboxyls, and two double bond carbons. The remaining two carbon signals were indicative of sp2-hybridized quaternary carbons (C-3,C-4). Thirteen carbon signals were observed downfield, among which δ70.6 was located upfield and suggested a carbon bonded to an oxygen atom. Together with the DEPT135 spectrum,
S. Chen et al. / Fitoterapia 105 (2015) 222–227
223
Fig. 1. New compounds from Lindera reflexa Hemsl.
the results indicated the presence of five methylenes, four methines and four methyls. The HMBC spectrum (Table 2) showed correlations of δ0.69 (H-10′) and 0.79 (H-9′), with δ28.4 (C-8′), and 39.9 (C-4′) and δ1.55 (H-7′), with δ132.2 (C-1′), 125.2 (C-2′), and 30.5 (C-6′), and indicated that compound 1 might be a C-3-substituted menthene derivative, which was also confirmed by the 1H–1H COSY spectrum. δ5.94 (H-5) correlated to δ104.2 (C-3) and 159.4 (C-6), and δ3.37 (H-3′) exhibited long-range correlations with δ104.2 (C-3), 163.5 (C-4) and 165.3 (C-2). These correlations, combined with the monosubstituted benzene ring proton signals in the 1H NMR spectrum, suggested that a pyran-2-one group existed in the compound. These data were similar to those of the pyran-2-one moiety of katsumadain [6], and C-3′ linked to C-3. The 1H-1H COSY spectrum showed the correlations of H-7 with H-8, H-8 with H-9, and H-9 with H-10, and the HMBC spectrum showed the correlations of δ4.96 (H-8) with δ169.9 (C-17), and δ2.60 (H-10) with δ70.6 (C-8), which indicated the existence of a \\CH2CH2CH2OCOCH3 moiety [7]. In contrast to katsumadain, compound 1 had no alkenyl group at C-7 and contained a\\CH2CH2CH2OCOCH3 moiety.
δ2.60 (H-10) exhibited long-range correlations with δ141.3 (C-11), 128.4 (C-12, 16), 70.6 (C-8) and 35.1 (C-9), indicating a benzene ring linked to C-10, δ2.65 (H-7), which had long-range correlation with 159.4 (C-6) and indicated C-7 was connected to C-6 of the pyran-2one moiety. The NOE spectrum (Fig. 2) showed that C-3′ and C-4′ represented a trans-oriented structure because H-3′ and H-4′ had no NOE correlations. The absolute configuration was determined by comparing the experimental and calculated CD spectra. The latter was performed using time-dependent density functional theory. Optimized geometries were obtained by systematic conformational analysis using the MMFF94 force field, and ECD spectra were calculated in methanol solution using the B3LYP/6–31 g (d) level of theory with the CPCM model. The results showed that the experimental and calculated spectra were in good agreement (Fig. 3). Thus, the absolute configuration of 1 was determined to be 3′S,4′S. In summary, the compound was determined to be 4-hydroxy-3-[(3′ R,4′R)-1′-p-mentheneyl]-6-(4-phenyl-2-acetoxy group-n-butyl)-2Hpyran-2-one.
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Table 1 1 H-NMR and 13C-NMR data for compound 1 (in DMSO-d6). Table 2. HMBC list of compound 1, compound 2 and compound 3. No.
δC
2 3 4 5 6 7 8 9 10 11 12, 16 14 13, 15 17 18 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′
165.3 104.2 163.5 101.3 159.4 37.7 70.6 35.1 31.0 141.3 128.4 126.1 128.5 169.9 20.9 132.2 125.2 35.2 39.9 22.7 30.5 23.4 28.4 21.4 16.6
δH
5.94 (1H, s) 2.70 (1H, d, J = 4.8 Hz) 2.65 (1H, m) 4.96 (1H, m) 1.86 (2H, m) 2.60 (2H, m) 7.15 (3H, t, J = 7.2, 7.0 Hz) 7.24 (2H, m) 1.93 (3H, s) 4.98 (1H, s) 3.37 (1H, m) 1.96 (1H, m) 1.66 (1H, m), 1.23 (1H, m) 1.95 (1H, m), 1.85 (1H, m) 1.55 (3H, s) 1.44 (1H, m) 0.79 (3H, d, J = 6.8 Hz) 0.69 (3H, d, J = 6.8 Hz)
HNMR (400 MHz) (δC in ppm, J in Hz), 13CNMR(100 MHz).
Reflexanbene I (compound 2) was obtained as a reddish-brown powder, mp.164–167 °C; [α]20 D = −2.2° (c 0.09, CHCl3); and was soluble in methanol, and spraying the compound with an anisyl aldehyde/ sulfuric acid reagent caused it to turn purple. Upon addition of the ferric chloride–potassium ferricyanide test solution, the color turned blue, which suggested the compound contained phenol groups. HR ESI-MS m/z: 371.1987[M + Na]+(calcd 371.1987), molecular formula was determined to be C24H28O2, with 11 degrees of unsaturation. The Table 2 HMBC list of compound 1, compound 2 and compound 3. Compound Compound 1
HMBC data
Fig. 2. Key HMBC, NOESY correlations of compound 1.
UV spectrum showed the maximum absorption wavelength at 300.8 nm. IR absorptions indicated the existence of a hydroxyl group (3415 cm− 1) and an aromatic ring (1610 cm− 1, 1446 cm− 1, 1585 cm− 1, 1496 cm− 1). The 1H-NMR spectrum (Table 3) showed nine aromatic proton signals; δ6.77 (1H, d, J = 16.1 Hz) and 7.73 (1H, d, J = 16.0 Hz) were typical trans double bond proton signals; δ7.16–7.40 displayed five proton signals; δ7.37 (2H, d, J = 7.6 Hz), 7.29 (2H, t, J = 7.4, 7.8 Hz), and 7.19 (1H, t, J = 7.3, 7.2 Hz) were typical mono-substituted benzene ring proton signals; and δ6.24 (1H, d, J = 2.4 Hz), 6.56 (1H, d, J = 1.8 Hz) were meta-substituted proton signals of a benzene ring; the signals at δ1.73 (3H, s), 0.80 (3H, d, J = 6.4 Hz), 0.74 (3H, d, J = 6.9 Hz) corresponded to three methyl groups, and the 13CNMR spectrum (Table 3) showed twenty-four carbon signals, of which fourteen corresponded to aromatic carbons. These data suggested two benzene rings in the compound, and after comparing these data with data from the reference compound [8,9], the compound was determined to be a stilbene derivative. In addition, DEPT-135 and 13C-NMR spectra showed that the remaining seven carbons included two methylenes (δ24.2, 32.4), four methines (δ129.4, 37.7, 46.9, 28.9), and one quaternary carbon (δ133.5). The HMBC spectrum (Table. 2) showed cross-peaks of δ0.80 (Me-9″), with δ16.9 (C-10″); δ0.74 (Me-10″) with δ22.0 (C-9″); δ1.44–1.51 (H-8″), with δ22.0 (C-9″) and 16.9 (C-10″); and δ0.80, 0.74 and δ1.44–1.51, with 46.9 (C-4″), which suggested an isopropyl moiety linked to δ46.9. The 1H-1H COSY spectrum showed that H-3″, H-4″, H-
Compound 2
Compound 3
Fig. 3. Experimental and calculated CDs of compound 1 in MeOH.
S. Chen et al. / Fitoterapia 105 (2015) 222–227 Table 3 1 H-NMR and 13C-NMR data of compound 2 (in MeOD). NO.
δC
1 2 3 4 5 6 α β 1′ 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4″ 5″″ 6″ 7″ 8″ 9″ 10″
139.9 122.3 156.9 102.7 158.1 105.5 130.0 128.3 139.5 127.2 129.6 128.4 133.5 129.4 37.7 46.9 24.2 32.4 23.9 28.9 22.0 16.9
225
Table 4 1 H-NMR and 13C-NMR data of compound 3 (in MeOD). δH
6.24 (1H, d, J = 2.4 Hz) 6.56 (1H, d, J = 1.8 Hz) 7.77 (1H, d, J = 15.2 Hz) 6.70 (1H, d, J = 16.1 Hz) 7.38 (2H, d, J = 7.6 Hz) 7.29 (2H, t, J = 7.4, 7.8 Hz) 7.19 (1H, t, J = 7.3, 7.2 Hz) 5.36 (1H, s) 4.06 (1H, t J = 1.56, 1.50 Hz) 1.76 (1H, m) 1.73 (1H, s), 1.41 (1H, m) 2.14 (2H, m) 1.73 (3H, s) 1.44–1.51 (1H, m) 0.80 (3H, d, J = 6.4 Hz) 0.74 (3H, d, J = 6.9 Hz)
NO.
δC
1 2 3 4 5 6 α β 1′ 2′, 6′ 4′ 3′, 5′ 1″ 2″ 3″ 4″ 5″ 6″
135.6 126.3 161.9 98.9 158.7 103.7 127.8 130.0 138.7 127.4 128.6 129.7 70.0 92.0 41.9 47.7 18.5 35.8
7″ 8″ 9″ 10″
1
H-NMR (400 MHz) (δC in ppm, J in Hz), 13C NMR (100 MHz).
28.1 27.4 22.1 16.4
δH
6.23 (1H, s) 6.67 (1H, s) 7.13 (1H, d, J = 16.0 Hz) 7.04 (1H, d, J = 16.0 Hz) 7.49 (2H, d, J = 7.5 Hz) 7.22 (1H, t, J = 7.0, 7.0 Hz) 7.32 (2H, t, J = 7.5, 7.5 Hz) 4.03 (1H, d, J = 4.3 Hz) 3.18 (1H, brs.) 1.02 (1H, t, J = 11.5, 11.5 Hz) 1.51 (1H, m), 1.36 (1H, m) 1.74 (1H, d, J = 13.4 Hz), 1.58–1.64 (1H, m) 1.38 (3H, s) 1.87 (1H, t, J = 6.0, 6.0 Hz) 0.92 (3H, d, J = 6.5 Hz) 0.73 (3H, d, J = 7.0 Hz)
1
H-NMR (500 MHz) (δC in ppm, J in Hz), 13CNMR(125 MHz).
5″, and H-6″ were connected in sequence and that δ5.36 (H-2″) and 1.76 (H-4″) had a cross-peak with δ122.3 (C-2), which indicated that C-3″ was linked to C-2. These data suggested the compound contained a menthen moiety linked to C-3 [10]. H-3″ and H-4″ had no NOE correlations, suggesting the two protons was ipso-lateral, C-3″ and C-4″ was determined to possess a trans structure by NOESY spectrum (Fig. 4). The absolute configuration was also determined using the same method used for compound 1, and the configuration was determined to be 3″R 4″R. Based on the above analysis, compound 2 was determined to be 3,5dihydroxy-2-[(3″R4″R)-L-menthene]-trans-stilbene. The compound named reflexanbene I had never been reported and was confirmed to be a newly discovered compound. Reflexanbene II (compound 3) was obtained as a reddish-brown powder that was soluble in methanol and chloroform. Spraying the compound with an anisyl aldehyde/sulfuric acid reagent caused it to turn purple. Upon addition of the ferric chloride–potassium ferricyanide test solution, the color turned blue, which suggested the compound contained phenol groups. From the ESI-MS m/z: 387.1938 [M + H]+ (calcd 387.1931),the molecular formula was determined to be C 24 H28O 3 , with 11 degrees of unsaturation. UV (MeOH) λ max at 311.50 nm. IR absorptions at 1122 cm−1 and 975 cm−1 were assigned to an epoxy (C\\O) moiety and a trans double bond, respectively.
The data for compound 3 were similar to those of compound 2, except for a deletion of the C-2″and C-3″ double bond and the existence of an epoxy quaternary carbon and an epoxy methane. The 13C-NMR spectrum and 1H-NMR spectrum are shown in Table 4. C-1″, C-2″, C-3″, and C-4″ were chiral carbons and H-4″ and H-2″ had no NOE correlations; there were no NOE correlations between H-3″ and H-2″ or between H-3″and 1″-CH3, suggesting that H-4″ and H-2″, H-3″, and 1′-CH3 were trans-oriented. The absolute configuration was also determined using the same method as was used for compound 1, and the configuration was 1″ S2″R3″S4″R. In summary, the compound was determined to be 3,5dihydroxy-2-[(1″S2″R3″S4″R)- L -(1″,2″-epoxy)-phmentheneyl]trans-stilbene, which was named reflexanbene II and was also a newly discovered compound. In addition to the new compounds, three alkaloids, launobine [11], laetanine [12], and norbracteoline [13]; one scoplin coumarin [14]; four pinosylvin stilbenes [15]; cis-3,5-didroxystilbene [16], 3methoxy-5-hydroxy-trans-stilbene [17]; β,β′-pinosylvin diglucoside [4]; two pinostrobin flavonoids [15]; pinocembrin [15]; and katsumadain [6] were elucidated. Two different assays, an in vitro antitumor activity assay of three tumor cell lines, A549 (human lung carcinoma), Bel-7402 (human liver carcinoma) and HCT-8 (human colon carcinoma), and an inhibitory assay on TNF-α, IL-6 and NO production from RAW264.7 stimulated
Fig. 4. Key HMBC, NOESY correlations of compound 2.
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by LPS, were carried out to evaluate the bioactivities of the isolated compounds. The new compounds (1,2,3) showed antitumor effects against BEL-7402, HCT-8 and A549; as shown in Table 5, the results provided some evidence for antitumor activities of stilbenes. None of the compounds exhibited significant anti-inflammatory activity toward any of the three inflammatory cell lines. As for reflexan A, it was considered to be a new skeleton; the structure of this compound has many similarities to those compounds isolated from the Linderae genus of the Lauraceae family. The p-menthene group was similar to the p-menthene substituents of linderatin, linderation, linderachalcone, and methylinderatone, which were isolated from Lindera umbellata THUNB.var.membranacea (MAXIM.) MOMIYAMA [18,19]. The \\CH2CH2CH2OCO-group was similar to the \\CH2CH2CH2OCOmoiety of (2S,3S)-2,3-bis[(4-hydroxy-3,5-dimethoxyphenyl)methyl]1,4-butanediol 1,4-diferulate, which was isolated from the stems and twigs of L. umbellata [20]. The pyran-2-one group was similar to the pyran-2-one moiety of katsumadain B from Alpinia katsumadai [21]. In Chinese traditional medicines, the seeds of Alpinia katsumadai and the root of L. reflexa were considered to have identical pharmacological effects for treating stomach diseases; thus, the two plants were thought to have similar chemical constituents such as essential oils, stilbenes and flavonoids of pinocembrin [22]. 3. Exprimental 3.1. General experimental procedure All of the melting points were measured using a Kofler micromelting apparatus and were uncorrected. UV spectra were recorded on an Agilent 8453 spectrometer in MeOH. IR spectra were obtained on a Shimadzu FTIR-8201 infrared spectrophotometer. The 1H and 13C spectra were obtained in MeOD on Bruker Avance DPX-400 (400 MHz for 1H-NMR and 100 MHz for 13C-NMR) and Bruker Avance III 500 (500 MHz for 1H-NMR and 125 MHz for 13C-NMR) spectrometers, using TMS as an internal standard. APEX II FT-ICR mass spectrometry was used. Column chromatography (CC) was performed using Toyopearl HW-40 (Tosoh, Japan) resins and Pharmadex LH-20 (Amersham Life Science, Shanghai). Preparative thin-layer chromatography (TLC) was conducted with silica gel (Qingdao Haiyang Chemical Co.). 3.2. Plant materials The root of the L. reflexa Hemsl. was collected in August 2010 at Xin Xian county, Henan province. The voucher specimen was deposited in the pharmacognosy lab of the Pharmacy College of Henan University of Traditional Chinese Medicine.
Table 5 Cytotoxic activity of compounds by MTT method. Compound
BEL-7402 Inhibition (%)
1 2 3 4 5 6 8 9 11 12 14 13 5-Fu
4.96 70.38 72.14 5.74 27.70 14.16 18.05 9.23 4.99 11.49 3.68 32.27 75.31
HCT-8 IC50 (μg/mL) 10.90 10.48
3.93
A549
Inhibition (%)
IC50 (μg/mL)
Inhibition (%)
IC50 (μg/mL)
63.42 62.86 11.39 0.27 6.52 11.67 −4.02 0.49 −2.66 −7.11 19.21 −1.09 80.59
11.96 11.14
75.07 68.90 15.12 9.30 1.79 24.55 10.59 27.08 −0.77 21.52 22.77 4.44 54.47
10.53 11.75
7.51
16.19
3.3. Extraction and isolation Dried root (8.0 kg) was extracted with 95% ethanol by refluxing three times for 2 h each, and the extract was concentrated to dryness (624.5 g). The residue was dissolved in water and extracted with petroleum ether, ethyl acetate, and n-butyl alcohol sequentially. A portion of the active EtOAc-soluble fraction (256 g) was subjected to silica gel CC and eluted with chloroform and petroleum ether mixtures of various proportions to obtain Fr.1 to Fr.3, eluted with chloroform to obtain Fr.4, and eluted with different proportions of chloroform and MeOH to obtain Fr.5 to Fr. 10. Fr.1 was subjected to silica gel CC and recrystallization to obtain compound 5 (2080 mg). Fr.2 also contained compound 5 (840 mg). Fr.3 was subjected to silica gel CC eluted with different proportions of petroleum ether and EtOAc; then, the fractions was subjected to Toyopearl HW-40 and Pharmadex LH20 resin gel CC to obtain compounds 1 (350 mg), 2 (23 mg), 4 (52 mg), 6 (370 mg), 7 (45 mg), and 10 (4.5 mg). Fr.4, Fr.5, Fr.6 and Fr.10 were subjected to silica gel CC eluted with different proportions of petroleum ether and EtOAc, then the fractions were subjected to Toyopearl HW-40 and Pharmadex LH-20 resin gel CC to obtain compounds 8 (2840 mg), 8 (6870 mg), 8 (320 mg), and 9 (220 mg). A portion of the active n-butyl alcohol-soluble fraction (113 g) was subjected to silica gel CC and eluted with different proportions of a methanol and dichloromethane mixture to obtain Fr.11 and Fr.12. Fr.11 was subjected to silica gel CC and recrystallization to obtain compound 11 (300 mg); then, the fractions were eluted with petroleum: EtOAc (5:1) to obtain compound 3 (48 mg). Fr.12 was subjected to silica gel CC and recrystallization and then eluted with chloroform-MeOHH2O (9:1:0.1) to obtain compound 12 (1360 mg) and compound 13 (108 mg); elution with chloroform-MeOH (100:5) gave compound 14 (23 mg). Compound 1: colorless pin needless (MeOH-CHCl3), mp.164–167 °C; [α]20 D − 2.2° (c 0.09, CHCl 3 ); UV λ max (log ε) 298.4 nm; IR(KBr) νmax cm − 1 3433, 2953, 1745, 1660, 1615, and 1405. For 1 H and 13 C NMR data, see Table 1; supplementary data; HR ESI-MS m/z: 439.2490[M + H]+(calcd for C27H34O5, 439.2484). Compound 2: red brown powder (MeOH); UV λmax nm: 300.8; IR(KBr) cm− 1 3415,2956, 1610, 1446; for 1H and 13C NMR data, see Table 3; supplementary data; HR ESIMS m/z: 371.1987[M + Na] + (calcd for C24H28O2, 371.1987). Compound 3: red brown powder (MeOH); [α]20 D + 20° (c 0.70, CHCl3); UV (MeOH) λmax (log ε) 311.50 nm; IR (KBr) νmax cm− 1 3277, 1614, 1589, 1122, 756, and 741; for 1H and 13C NMR data, see Table 4; supplementary data; HRESIMS m/z: 387.1938 [M + H]+ (calcd for C24H28O3, 387.1931). TNF-α, IL-6 and NO production from RAW264.7 stimulated by LPS: RAW264.7 cells (5 × 104 per well) were suspended in 100 μL of 1640 supplemented with 10% fetal calf serum, penicillin (100 units/mL), and streptomycin (100 μg/mL) and were pre-cultured in 96-well microplates at 37 °C 5% CO2 in air for 12 h. RAW264.7 cells were added by LPS (final concentration 5 μg/mL). The final concentrations of the test compounds in the assay were 5 μg/mL. After 12 h of incubation, the amount of TNF-α and IL-6 production in each well was determined by analyzing the culture medium using the ELISA method. NO production in the culture medium was detected by the Griess assay. The cytotoxicity was assessed by the CCK-8 colorimetric assay. All of the tests were performed in duplicate. Inhibition (%) was calculated using the formula: Inhibition (%) = (A − B)/A × 100, where A − B = TNF-α or IL-6 or NO concentration (pg/mL); A = LPS (+), sample (−); B = LPS (+), sample (+). In vitro antitumor activity assay: All of the isolates were tested for cytotoxicity against A549 (human lung carcinoma), BEL-7402 (human liver carcinoma), and HCT-8 (human colon carcinoma) cells by means of the MTT assay. These cells were plated in 96-well plates and cultured for 24 h. The appropriate test compounds and positive control were added into triplicate wells at concentrations of 20, 10, 2.5, and
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1.25 μg/mL and incubated for 4 days at 37 °C. MTT solution (10 μL, 5 mg/mL) was added into each well, and the plate was incubated for another 4 h. The resulting formazan crystals were dissolved in DMSO (100 μL), and the UV/vis absorbance (optical density: OD) was determined with a microplate spectrophotometer at 570 nm. The reference compound, 5-fluorouracil, exhibited activity toward the A549, Bel-7402 and HCT-8 cell lines with an IC50 range of 0.2–0.7 μg/mL. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by National Science and Technology Major Project of the Ministry of Science and Technology of China (Grant No. 2012ZX09103201-024) and Innovation Scientists and Technicians Troop Construction Projects of Henan Province (Grant No. 114200510014). We thank the National Center of Pharmaceutical Screening, Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College, for cytotoxic tests. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2015.07.005. References [1] Y.M. Luo, L.Q. Huang, P. Wang, Q. Li, Study on the volatile oil in aerial part and underground part of Linderae reflexae Hemsl, Chin. Pharm. J. 39 (4) (2004) 307–309. [2] C.F. Zhang, Z.T. Wang, An advance in the study on the medicinal plant of Lindera, J. Shenyang Pharma. Univ. 17 (3) (2000) 230–234. [3] J.Z. Zhang, Q.C. Fang, Study on the chemical constituents of Montane Spicebush (Lindera reflexa), Chin. Tradit. Herb. Drugs 25 (11) (1994) 565–568. [4] J.Z. Zhang, Q.C. Fang, Application of high speed counter-current chromatography to the separation of stilbene derivatives from the roots of Lindera reflexa, Planta Med. 60 (1994) 190–191.
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