2016 Volume 64 Issue 11 Pages 1641-1646
Six polyphenolic acids (1–6), including the three new compounds citriodolic acids A, B, and C (1–3), were isolated from the leaves of Eucalyptus citriodora. Their structures were elucidated by spectroscopic methods including one dimensional (1D)- and 2D-NMR, high-resolution electrospray ionization (HR-ESI)-MS, and circular dichroism (CD). The potential antivirus activity against respiratory syncytial virus (RSV) of all the isolated compounds was evaluated.
Viruses are the leading cause of respiratory infections in children and adults and are a major cause of morbidity and mortality worldwide.1) The screening of plants for viral growth inhibitors in vitro and the use of the ethnopharmacological approach enhance the probability of identifying new bioactive compounds.2) The extracts from the Eucalyptus citriodora have been reported to possess antivirus, anti-inflammation, and antioxidant activities.3,4) Previously, we reported several flavonoid glycosides from the leaves of Eucalyptus citriodora and evaluation of their respiratory syncytial virus (RSV) inhibitory activity.5,6) In continuation of our search for additional novel bioactive substances from this medicine plant, which has been proven to possess antivirus activity, three new polyphenolic acids, citriodolic acids A (1), B (2), and C (3), were isolated from the leaves of Eucalyptus citriodora by using various chromatographic methods, with three known phenolic compounds (4–6). The latter were identified with rosmarinic acid (4), ferulic acid (5), and gallic acid (6) by detailed analysis of their spectroscopic data and comparison with literature values7–9) (Fig. 1). In this study, we report the isolation, structure elucidation, and antivirus activity of these polyphenolic acids from this medicinal plant.
The phytochemical study of 95% aqueous ethanol extract obtained from the leaves of Eucalyptus citriodora afforded six polyphenolic acids. The structures of novel compounds, citriodolic acid A (1), citriodolic acid B (2), and citriodolic acid C (3) were determined by the one dimensional (1D)- and 2D-NMR elucidations, and mass spectral analysis.
Citriodolic acid A (1) was isolated as a brown amorphous powder and had a molecular formula C43H42O14, which was derived from its positive-ion high-resolution electrospray ionization (HR-ESI)-MS (m/z 805.2468 [M+Na]+) and 13C-NMR data. The 1H-NMR spectrum indicated that the presence of two β-(3,4-dihydroxyphenyl) lactic acid moieties [δH 6.76 (1H, d, J=2.0 Hz, H-2), 6.64 (1H, d, J=8.0 Hz, H-5), 6.73 (1H, dd, J=2.0, 8.0 Hz, H-6), 5.16 (1H, d, J=3.2 Hz, H-7), 4.71 (1H, d, J=3.2 Hz, H-8); and δH 6.74 (1H, d, J=2.0 Hz, H-2″), 6.68 (1H, d, J=8.0 Hz, H-5″), 6.56 (1H, dd, J=2.0, 8.0 Hz, H-6″), 2.88 (1H, dd, J=9.2, 14.0 Hz, H-7″a), 3.01 (1H, dd, J=3.4, 14.0 Hz, H-7″b), 5.03 (1H, d, J=9.2, 3.4 Hz, H-8″)], and a (E)-caffeoyl unit [δH 7.27 (1H, d, J=2.0 Hz, H-2′), 6.89 (1H, d, J=8.0 Hz, H-5′), 7.16 (1H, dd, J=2.0, 8.0 Hz, H-6′), 7.48 (1H, d, J=15.9 Hz, H-7′), 6.37 (1H, d, J=15.9 Hz, H-8′)], suggesting that 1 was a polyphenolic acid.9) Meantime, the 1H- and 13C-NMR spectra of 1 also showed typical signals of an allyl [δH 3.39 (2H, td, J=1.6, 1.6, 6.4 Hz), 5.91 (1H, ddd, J=6.4, 12.0, 16.0 Hz), 4.87 (1H, ddd, J=1.6, 2.4, 16.0 Hz), and 5.08 (1H, ddd, J=1.6, 2.4, 12.0 Hz); δC 37.1, 137.2, 115.1], a prenyl [δH 3.45 (2H, d, J=6.6 Hz), 5.47 (1H, t, J=6.6 Hz), 1.68 (3H, s), and 1.79 (3H, s); δC 25.4, 122.9, 136.1, 17.8, 25.7], a penta-substituted phenyl [δH 6.50 (1H, s); δC 123.8, 147.4, 126.1, 136.6, 108.4, 148.1], and two methoxy groups [δH 3.86 (3H, s), 3.83 (3H, s); δC 58.6, 55.8]. Comparison of the 1H- and 13C-NMR data of 1 with those of salvianolic acid J (SAJ) and 4-allyl-2,6-dimethoxy-3-prenyl phenol (ADPP) showed that the signals were substantially coincident.9,10) All the above evidence combined with the detailed 2D-NMR analysis of 1H–1H correlated spectroscopy (1H–1H COSY) and heteronuclear multiple bond correlation (HMBC) confirmed that 1 was composed of SAJ unit and ADPP unit (Figs. 1, 2). Moreover, the C-9 carboxyl group of the SAJ moiety was linked to the C-1‴ hydroxy group of the ADPP component. That is to say, the structure of 1 is an ester dimer of SAJ and ADPP between the hydroxyl group at C-1‴ and the carboxylic acid group at C-9. The suggestion was in accord with the observation of the chemical shift of C-9 signal upfield shifted from δ 169.6 in SAJ to δ 167.2 in 1 and the chemical shift of C-1‴ signal upfield shifted from δ 128.8 in ADPP to 123.8 in 1. This was further confirmed by the rotating frame Overhauser effect spectroscopy (ROESY) correlation of 2‴-OCH3 with H-2 (Fig. 3) and acid hydrolysis of citriodolic acid A with 11 N HCl gave SAJ, which was confirmed by HPLC analysis. Three chirality centers (7R, 8R, 8″R) in 1 were suggested by circular dichroism (CD) spectrum in ethanol with three positive bands at 238, 260, and 284 nm and a negative band at 327 nm.11) A coupling constant of 3.2 Hz indicated that the H-7 and H-8 were cis oriented,9) which was further confirmed by the ROESY correlation of H-7 with H-8. Consequently, the structure of 1, which was established as shown in 1, is a new natural compound, which we named citriodolic acid A.
The molecular formula of citriodolic acid B (2) was determined as C38H34O14 by a quasi-molecular ion peak at m/z 737.1844 [M+Na]+ in the HR-ESI-MS. The existence of SAJ was determined by hydrolysis of 2 with 11 M HCl and HPLC analysis. The 1H- and 13C-NMR data of 2 were similar to those of 1, except for the presence of a 4‴-allyl-2‴,6‴-dimethoxy phenolic group [allyl: δH 3.35 (2H, td, J=1.4, 1.4, 6.8 Hz), 5.99 (1H, ddd, J=6.8, 12.0, 16.0 Hz), 4.91 (1H, ddd, J=1.4, 2.1, 16.0 Hz), 5.12 (1H, ddd, J=1.4, 2.1, 12.0 Hz), and δC 40.5, 137.6, 116.1; two methoxy groups: δH 3.77 (6H, s) and δC 56.5 (2×C); tetra-substituted phenyl: δH 6.42 (2H, s) and δC 124.2, 148.6 (2×C), 137.2, 107.1 (2×C)] at C-9 (δC 167.3) in 2,12) instead of a 4‴-allyl-2‴,6‴-dimethoxy-3‴-prenyl phenolic group in 1. This was revealed by the 1H–1H correlations of the spin system H-1″″/H-2″″/H-3″″ as well as the HMBC correlations from H-1″″ to C-2″″, C-3″″, C-3‴, C-4‴ and C-5‴, H-2″″ to C-1″″, C-3″″ and C-4‴, H-3″″ to C-1″″ and C-2″″, H-3‴ to C-1‴, C-2‴, C-4‴, C-5‴ and C-1″″, H-5‴ to C-1‴, C-3‴, C-4‴, C-6‴ and C-1″″, 2‴-MeO to C-2‴, and 6‴-MeO to C-6‴ (Fig. 2). The relative configuration of 2 was assigned by CD and ROESY experiments. Similar as in case of 1, three positive bands at 238, 260, and 284 nm and a negative band at 327 nm were observed in the CD spectrum of 2 suggesting for three chirality centers (7R, 8R, 8″R) in 2.9) Thus, the structure of 2, which was established as shown in 2, is a new natural compound, which we named citriodolic acid B.
Citriodolic acid C (3) was isolated as a brown amorphous powder and its elemental composition was determined to be C42H40O13 by HR-ESI-MS. Acid hydrolysis of 3 also gave SAJ by HPLC analysis. The proton and carbon signals in the 1H- and 13C-NMR spectra of 3 were very similar to those of 1. However, preliminary inspection of the 1H-NMR spectrum of 3 revealed the absence of one of the two methoxys in 1. Moreover, comparison of the 13C- and distortionless enhancement by polarization transfer (DEPT) NMR data for 1 and 3 indicated that the 4‴-allyl-2‴,6‴-dimethoxy-3‴-prenyl phenolic group of 1 was replaced by a 4‴-allyl-2‴-methoxy-6‴-prenyl phenolic group in 3. The 1H-NMR spectrum of 3 revealed the presence of two aromatic proton signals [δH 6.68 (1H, d, J=2.0 Hz), 6.59 (1H, d, J=2.0 Hz)], a methoxy signal [δH 3.87 (3H, s)], one allyl signal [δH 3.34 (2H, td, J=1.4, 1.4, 6.6 Hz), 5.96 (1H, ddd, J=6.6, 12.0, 16.0 Hz), 5.08 (1H, ddd, J=1.4, 2.2, 12.0 Hz), 4.99 (1H, ddd, J=1.4, 2.2, 16.0 Hz)], and a prenyl signal [δH 3.41 (2H, d, J=6.8 Hz), 5.33 (1H, t, J=6.8 Hz)], suggesting that 3 has a 4‴-allyl-2‴-methoxy-6‴-prenyl phenolic group.10) This was further confirmed by the 1H–1H correlations of the spin system H-1″″/H-2″″ and H-1‴″/H-2‴″/H-3‴″, as well as the HMBC correlations from H-3‴ to C-1‴, C-2‴, C-4‴, C-5‴ and C-1‴″, H-5‴ to C-1‴, C-3‴, C-4‴, C-6‴, C-1‴″ and C-1″″, H-1″″ to C-2″″, C-3″″, C-1‴, C-5‴ and C-6‴, H-2″″ to C-1″″, C-3″″, C-4″″, C-5″″and C-6‴, H-1‴″ to C-2‴″, C-3‴″, C-3‴, C-4‴ and C-5‴, H-2‴″ to C-1‴″, C-3‴″, C-3‴ and C-4‴, H-3‴″ to C-1‴″ and C-2‴″, and 2‴-MeO to C-2‴ (Fig. 2). The CD spectrum of 3 showed a negative cotton effect at 327 nm and positive cotton effects at 238, 260, and 284 nm, indicating that 3 has three chirality centers (7R, 8R, 8″R).9) On the basis of the above data, the structure of 3, which was established as shown in 3, is a new natural compound, which we named citriodolic acid C.
| No. | 1 | 2 | 3 | |||
|---|---|---|---|---|---|---|
| δH | δC | δH | δC | δH | δC | |
| 1 | 128.3 | 128.1 | 128.1 | |||
| 2 | 6.76 d (2.0) | 114.1 | 6.78 d (2.0) | 114.3 | 6.76 d (2.0) | 114.5 |
| 3 | 145.5 | 145.3 | 145.6 | |||
| 4 | 145.8 | 145.7 | 145.9 | |||
| 5 | 6.64 d (8.0) | 115.9 | 6.64 d (8.0) | 115.9 | 6.66 d (8.0) | 115.7 |
| 6 | 6.73 dd (2.0, 8.0) | 118.2 | 6.76 dd (2.0, 8.0) | 118.2 | 6.73 dd (2.0, 8.0) | 118.3 |
| 7 | 5.16 d (3.2) | 73.4 | 5.18 d (3.0) | 73.4 | 5.15 d (3.0) | 73.7 |
| 8 | 4.71 d (3.2) | 71.3 | 4.77 d (3.0) | 71.1 | 4.73 d (3.0) | 71.3 |
| 9 | 167.2 | 167.3 | 167.6 | |||
| 1′ | 128.3 | 128.3 | 128.4 | |||
| 2′ | 7.27 d (2.0) | 116.1 | 7.28 d (2.0) | 116.1 | 7.28 d (2.0) | 116.3 |
| 3′ | 143.7 | 143.8 | 143.8 | |||
| 4′ | 143.1 | 143.1 | 142.9 | |||
| 5′ | 6.89 d (8.0) | 117.5 | 6.88 d (8.0) | 117.6 | 6.86 d (8.0) | 117.9 |
| 6′ | 7.16 dd (2.0, 8.0) | 122.9 | 7.16 dd (2.0, 8.0) | 122.8 | 7.14 dd (2.0, 8.0) | 122.8 |
| 7′ | 7.48 d (15.9) | 145.0 | 7.48 d (16.0) | 145.2 | 7.44 d (16.0) | 145.4 |
| 8′ | 6.37 d (15.9) | 115.7 | 6.38 d (16.0) | 115.5 | 6.40 d (16.0) | 115.8 |
| 9′ | 166.1 | 166.1 | 166.3 | |||
| 1″ | 127.8 | 127.4 | 127.7 | |||
| 2″ | 6.74 d (2.0) | 116.8 | 6.76 d (2.0) | 116.8 | 6.73 d (2.0) | 116.9 |
| 3″ | 145.3 | 145.3 | 145.5 | |||
| 4″ | 145.2 | 145.2 | 145.3 | |||
| 5″ | 6.68 d (8.0) | 116.8 | 6.68 d (8.0) | 116.7 | 6.66 d (8.0) | 116.7 |
| 6″ | 6.56 dd (2.0, 8.0) | 120.3 | 6.58 dd (2.0, 8.0) | 120.1 | 6.56 dd (2.0, 8.0) | 119.9 |
| 7″ | 2.88 dd (9.2, 14.0) | 36.8 | 2.91 dd (9.0, 14.0) | 36.6 | 2.89 dd (9.4, 14.0) | 36.8 |
| 3.01 dd (3.4, 14.0) | 3.04 dd (3.5, 14.0) | 3.03 dd (3.5, 14.0) | ||||
| 8″ | 5.03 dd (3.4, 9.2) | 73.8 | 5.05 dd (3.5, 9.0) | 73.8 | 5.01 dd (3.5, 9.4) | 73.7 |
| 9″ | 171.6 | 171.6 | 171.8 | |||
| 1‴ | 123.8 | 124.2 | 141.3 | |||
| 2‴ | 147.4 | 148.6 | 150.6 | |||
| 3‴ | 126.1 | 6.42 d (2.0) | 107.1 | 6.68 d (2.0) | 108.9 | |
| 4‴ | 136.6 | 137.2 | 138.4 | |||
| 5‴ | 6.50 s | 108.4 | 6.42 d (2.0) | 107.1 | 6.59 d (2.0) | 122.1 |
| 6‴ | 148.1 | 148.6 | 136.7 | |||
| 1″″ | 3.45 d (6.6) | 25.4 | 3.35 td (1.4, 1.4, 6.8) | 40.5 | 3.41 d (6.8) | 28.6 |
| 2″″ | 5.47 t (6.6) | 122.9 | 5.99 ddd (6.8, 12.0, 16.0) | 137.6 | 5.33 t (6.8) | 121.9 |
| 3″″ | 136.1 | 4.91 ddd (1.4, 2.1, 16.0) | 116.1 | 132.3 | ||
| 5.12 ddd (1.4, 2.1, 12.0) | ||||||
| 4″″ | 1.68 s | 17.8 | 1.74 s | 17.6 | ||
| 5″″ | 1.79 s | 25.7 | 1.77 s | 25.7 | ||
| 1″‴ | 3.39 td (1.6, 1.6, 6.4) | 37.1 | 3.34 td (1.4, 1.4, 6.6) | 39.9 | ||
| 2″‴ | 5.91 ddd (6.4, 12.0, 16.0) | 137.2 | 5.96 ddd (6.6, 12.0, 16.0) | 137.4 | ||
| 3″‴ | 4.87 ddd (1.6, 2.4,16.0) | 115.1 | 4.99 ddd (1.4, 2.2, 16.0) | 115.1 | ||
| 5.08 ddd (1.6, 2.4, 12.0) | 5.08 ddd (1.4, 2.2, 12.0) | |||||
| 2‴-OMe | 3.86 s | 58.6 | 3.77 s | 56.5 | 3.87 s | 56.1 |
| 6‴-OMe | 3.83 s | 55.8 | 3.77 s | 56.5 | ||
Compounds 1–6 were isolated for the first time from the Eucalyptus citriodora in the present study. They were tested for their antiviral activity against RSV (Table 2). Among these compounds, compounds 1–3 showed significant antiviral activity against RSV with IC50 values of 2.3, 4.8, and 1.8 µg/mL, comparable to that of ribavirin, an approved drug for the treatment of RSV infections in humans. The other compounds exhibited moderate or weak antiviral activity against RSV.
| Compounds | IC50 (µg/mL)a) | CC50 (µg/mL)b) | SIc) |
|---|---|---|---|
| 1 | 2.3 | 99.7 | 43.3 |
| 2 | 4.8 | 113.2 | 23.6 |
| 3 | 1.8 | 124.6 | 69.2 |
| 4 | 51.5 | 229.1 | 4.4 |
| 5 | 87.1 | 246.3 | 2.8 |
| 6 | 127.3 | 274.4 | 2.2 |
| Ribavirin | 2.6 | 62.5 | 24.0 |
a) IC50 is the concentration of the sample required to inhibit virus-induced CPE 50%. b) CC50 is the concentration of the 50% cytotoxic effect. c) SI, CC50/IC50.
UV spectra were recorded on a Hewlett-Packard HP-845 UV-VIS spectrophotometer (Palo Alto, U.S.A.). CD spectra were recorded with a Jasco J-815 spectropolarimeter (Maryland, U.S.A.). Optical rotations were measured using a JASCO P-1010 digital polarimeter (Japan) and IR spectra were obtained from a PerkinElmer, Inc. Spectrum One FT-IR spectrometer (U.K.). The HPLC system (Dionex, Germany) consisted of a pump (LPG 3X00), column oven, and diode array UV/Vis detector [DAD-3000(RS)]. For separation of sample, a hypersil C18 (5 µm, 250 mm×4.6 mm) was used and the detection UV wavelength was set at 286 nm. MS on a Finnigan LCQ Advantage Spectrometer (Thermo Scientific, U.S.A.) and a Shimadzu GC-MS model QP2010 Plus spectrophotometer (Japan), respectively. NMR spectra were recorded on 400 MHz FT-NMR spectrometer (Varian Inova AS 400, U.S.A.), and deuterium solvents for NMR were purchased from Merck Co., Ltd. Chemical shifts are given as δ values with reference to tetramethylsilane (TMS) as internal standard. Column chromatography separations were carried out on silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, P. R. China), ODS (50 mesh, AA12S50, YMC), MCI-gel CHP20P (35–75 µm, Japan) and Diaion HP-20 (Pharmacia, Peapack, NJ, U.S.A.). The TLC analysis was carried out using a Kiesel gel 60 F254 and RP-18 F254S plates (Merck), and UV lamp (Spectroline Model ENF-240 C/F, Spectronics Corporation, U.S.A.) and 10% H2SO4 solution were used for detection. All other chemicals and reagents of analytical grade were obtained from Sigma-Aldrich, unless indicated otherwise.
Plant MaterialsThe leaves of Eucalyptus citriodora were collected in Zhanjiang, Guangdong Province of China in September 2012, and were identified by Professor Wen-Qing Yin. A voucher specimen (No. 20120903) has been deposited in the authors’ laboratory.
Extraction and IsolationThe dry leaves of Eucalyptus citriodora (5 kg) were extracted three times under reflux with 95% aqueous ethanol (EtOH) (150 L×2 h). After removing the solvent under reduced pressure, the residue (1.1 kg) was suspended in water (0.5 L) and then sequentially extracted with petroleum ether (PE), chloroform (CHCl3), ethyl acetate (EtOAc), and n-butyl alcohol (n-BuOH). The extract solutions were concentrated to produce the residues of the PE fraction (48 g), the CHCl3 fraction (33 g), the EtOAc fraction (144 g), the n-BuOH fraction (98 g), and the H2O fraction (718 g). The n-BuOH fraction (98 g) was subjected to silica gel column chromatography (CC) using CHCl3–MeOH–H2O mixtures (9 : 1 : 0.1 to 7 : 3 : 0.5, 60 L of each). The eluting solutions were monitored by TLC to produce 6 fractions (Frs. 1–6). Fraction 2 (2.8 g) was applied to a MCI-gel CHP20P CC and eluted with MeOH–H2O (0 : 1–1 : 0) to yield five fractions (Frs. 2.1–2.5). Fraction 2.5 (0.75 g) was subjected to CC on silica gel (CHCl3–MeOH–H2O, 9 : 1 : 0.1 to 7 : 3 : 0.5) and sephadex LH-20, eluting with H2O–MeOH (1 : 0 to 0 : 1), to afford compound 5 (16 mg). Repeated CC on sephadex LH-20, MCI-gel CHP20P and octadecylsilanized silica gel (ODS)-A, eluting with H2O–MeOH (1 : 0 to 0 : 1), finally with 50% aqueous acetone, respectively, gave 1 (19 mg), 2 (16 mg), 3 (25 mg), and 4 (38 mg) from Fr. 5 (21 g). Fraction 6 (17 g) was separated by CC on sephadex LH-20 (H2O–MeOH, 1 : 0–0 : 1) and silica gel (CHCl3–MeOH–H2O, 9 : 1 : 0.1 to 7 : 3 : 0.5) to yield 6 (41 mg).
Citriodolic Acid A (1)Brown amorphous powder, [α]D25 +28.6 (c=0.05, EtOH); IR νmax (KBr): 3435, 2941, 1606, 1523, 1448, 1364, 1288, 1193, 1111, and 1063 cm−1. UV (MeOH) λmax (log ε): 322 (4.23), 288 (4.26), and 222 (4.55) nm; HR-ESI-MS m/z: 805.2468 [M+Na]+ (Calcd for C43H56O25Na, 805.2474). 1H-NMR (400 MHz, dimethyl sulfoxide (DMSO)-d6) and 13C-NMR (100 MHz, DMSO-d6) (Table 1).
Citriodolic Acid B (2)Brown amorphous powder, [α]D25 +25.1 (c=0.05, EtOH); IR νmax (KBr): 3436, 2938, 1606, 1522, 1446, 1362, 1288, 1190, 1113, and 1066 cm−1. UV (MeOH) λmax (log ε): 322 (4.21), 288 (4.25), and 222 (4.54) nm; HR-ESI-MS m/z: 737.1844 [M+Na]+ (Calcd for C38H34O14Na, 737.1848). 1H-NMR (400 MHz, DMSO-d6) and 13C-NMR (100 MHz, DMSO-d6) (Table 1).
Citriodolic Acid C (3)Brown amorphous powder, [α]D25 +31.8 (c=0.05, EtOH); IR νmax (KBr): 3433, 2939, 1609, 1526, 1446, 1364, 1289, 1193, 1113, and 1066 cm−1. UV (MeOH) λmax (log ε): 322 (4.22), 288 (4.26), and 222 (4.54) nm; HR-ESI-MS m/z: 775.2366 [M+Na]+ (Calcd for C42H40O13Na, 775.2368). 1H-NMR (400 MHz, DMSO-d6) and 13C-NMR (100 MHz, DMSO-d6) (Table 1).
Acid Hydrolysis of Citriodolic Acids A–CA solution (3 mg each) of 1–3 in 11 N HCl (1.5 mL) was heated at 100°C for 5 min under an N2 atmosphere. After cooling, the solution was removed by blowing with N2. The residue was dissolved in the solution of methanol, stirred at 40°C for 5 min. The methanol solution was analyzed by HPLC-diode array (DAD) using Hypersil C18 (250×4.6 mm). The HPLC linear gradient profile was as follows: water (containing 0.5% phosphoric acid), acetonitrile (containing 0.5% phosphoric acid) 57 : 43 v/v (0–15 min), 57 : 43 to 20 : 80 (15–18 min), and 20 : 80 (18–26 min) at a flow-rate of 0.8 mL/min. The volume injected was 20 µL. The separation was carried out at 35°C. The detector wavelength was set according to the maximum absorption wavelength in the UV spectrum: compounds were analyzed 286 nm. The peak identity of each component was confirmed by comparison of the retention time and UV spectrum. The mixed standard solution was separated by using the developed HPLC method. Retention times of SAJ, citriodolic acid A, citriodolic acid B and citriodolic acid C were 14.55, 17.58, 16.58, and 17.54 min.
Cells and VirusRSV (Long strain) and Hep-2 cells were provided by American Type Culture Collection. Hep-2 cell was grown in Dulbecco’s modified Eagle’s medium (DMEM) containing Eagle’s balanced salt solution supplemented with 10% fetal bovine serum (FBS), 100 U of penicillin per mL, 25 µg of gentamicin per mL, and 2 mM L-glutamine (growth medium). RSV-infected cells were maintained in DMEM supplemented with 1% FBS.
Cytotoxicity AssayHep-2 cell cultures were prepared in 96-well plastic plates. After 2 d of incubations at 37°C in a CO2 incubator. When the cell cultures were confluent, culture medium was removed from each well and replenished with 0.1 mL of maintenance medium. To test for cytotoxicity, 0.1 mL of maintenance medium containing serial 2-fold dilutions of the tested compounds was added to the wells. For the cell control, 0.1 mL maintenance medium without the compound was added. All cultures were incubated at 37°C, for 2–5 d. The morphology of the cells was inspected daily and observed for microscopically detectable alterations, e.g. loss of monolayer, rounding, shrinking of the cells, granulation, and vacuolization in the cytoplasm. The cytopathogenic effect (CPE) was scored (scores, 0=0% CPE; 1=0–25% CPE; 2=25–50% CPE; 3=50–75% CPE; 4=75–100% CPE). The 50% cytotoxic concentration (CC50), the concentration required to cause visible changes in 50% of intact cells, was estimated from graphic plots. The maximal non-cytotoxic concentration (MNCC) was determined as the maximal concentration of the natural products that did not exert toxic effect detected by microscopic monitoring.13,14)
Antiviral Activity AssayThe antiviral activities of 1–6 against viruses were measured by the CPE inhibition assay. Twofold serial dilutions of 1–6 were seeded into cell monolayers cultivated in 96-well culture plates, using MNCC as the highest concentration. An infection control was made in the absence of test compounds. An equal volume of virus suspension was added to the cell monolayers. The plates were incubated at 37°C in a humidified CO2 atmosphere (5% CO2) for 2–5 d. Subsequently, CPE was observed. The virus-induced CPE was scored as described above in the cytotoxicity assay. The reduction of virus multiplication was calculated as percent of virus control (% virus control=CPEexp/CPEvirus control×100). The concentration reducing CPE by 50% with respect to virus control was estimated from graphic plots and was defined as IC50 expressed in µg/mL. The selective index (SI) was calculated from the ratio CC50/IC50.13,14)
This study was supported by the Foundation for Distinguished Young Teachers in Higher Education of Guangdong, China (YQ2015109) and the China Spark Program (2015GA780041).
The authors declare no conflict of interest.
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