Three new polyphenolic acids from the leaves of Eucalyptus citriodora with antivirus activity

Article abstract of DOI:10.1248/cpb.c16-00362

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.

Full text of DOI:10.1248/cpb.c16-00362

Vol. 64, No. 11
Chem. Pharm. Bull. 64, 1641–1646 (2016)
1641
Note
Three New Polyphenolic Acids from the Leaves of Eucalyptus citriodora
with Antivirus Activity
,a
San-qing Lin,a Zhong-liu Zhou,* and Wen-Qing Yinb
a School of Chemistry and Chemical Engineering, Lingnan Normal University; 29 Cunjin Road, Zhanjiang 524048,
b
China: and Ministry of Education Key Laboratory of Chemistry and Molecular Engineering of Medicinal Resource,
Guangxi Normal University; 15 Yucai Road, Guilin 541004, China.
Received April 28, 2016; accepted August 7, 2016; advance publication released online August 13, 2016
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.
Key words Eucalyptus citriodora; citriodolic acid A; citriodolic acid B; citriodolic acid C; antivirus activity
Viruses are the leading cause of respiratory infections in 2.88 (1H, dd, J=9.2, 14.0Hz, H-7″a), 3.01 (1H, dd, J=3.4,
children and adults and are a major cause of morbidity and 14.0Hz, H-7″b), 5.03 (1H, d, J=9.2, 3.4Hz, H-8″)], and a (E)-
mortality worldwide.1) The screening of plants for viral growth caffeoyl unit [δH 7.27 (1H, d, J=2.0Hz, H-2′), 6.89 (1H, d,
inhibitors in vitro and the use of the ethnopharmacological J=8.0Hz, H-5′), 7.16 (1H, dd, J=2.0, 8.0Hz, H-6′), 7.48 (1H,
approach enhance the probability of identifying new bioac- d, J=15.9Hz, H-7′), 6.37 (1H, d, J=15.9Hz, H-8′)], suggest-
1
tive compounds.2) The extracts from the Eucalyptus citriodora ing that 1 was a polyphenolic acid.9) Meantime, the H- and
have been reported to possess antivirus, anti-inflammation, 13C-NMR spectra of 1 also showed typical signals of an allyl
and antioxidant activities.3,4) Previously, we reported several [δH 3.39 (2H, td, J=1.6, 1.6, 6.4Hz), 5.91 (1H, ddd, J=6.4,
flavonoid glycosides from the leaves of Eucalyptus citriodora 12.0, 16.0Hz), 4.87 (1H, ddd, J=1.6, 2.4, 16.0Hz), and 5.08
and evaluation of their respiratory syncytial virus (RSV) (1H, ddd, J=1.6, 2.4, 12.0Hz); δC 37.1, 137.2, 115.1], a prenyl
inhibitory activity.5,6) In continuation of our search for ad- [δH 3.45 (2H, d, J=6.6Hz), 5.47 (1H, t, J=6.6Hz), 1.68 (3H,
ditional novel bioactive substances from this medicine plant, s), and 1.79 (3H, s); δC 25.4, 122.9, 136.1, 17.8, 25.7], a penta-
which has been proven to possess antivirus activity, three substituted phenyl [δH 6.50 (1H, s); δC 123.8, 147.4, 126.1,
new polyphenolic acids, citriodolic acids A (1), B (2), and C 136.6, 108.4, 148.1], and two methoxy groups [δH 3.86 (3H,
1
(3), were isolated from the leaves of Eucalyptus citriodora by s), 3.83 (3H, s); δC 58.6, 55.8]. Comparison of the H- and
using various chromatographic methods, with three known 13C-NMR data of 1 with those of salvianolic acid J (SAJ) and
phenolic compounds (4–6). The latter were identified with 4-allyl-2,6-dimethoxy-3-prenyl phenol (ADPP) showed that the
rosmarinic acid (4), ferulic acid (5), and gallic acid (6) by signals were substantially coincident.9,10) All the above evi-
1
detailed analysis of their spectroscopic data and comparison dence combined with the detailed 2D-NMR analysis of H–1H
with literature values7–9) (Fig. 1). In this study, we report the correlated spectroscopy (1H–1H COSY) and heteronuclear
isolation, structure elucidation, and antivirus activity of these multiple bond correlation (HMBC) confirmed that 1 was com-
polyphenolic acids from this medicinal plant.
posed 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
Results and Discussion
The phytochemical study of 95% aqueous ethanol extract structure of 1 is an ester dimer of SAJ and ADPP between
obtained from the leaves of Eucalyptus citriodora afforded the hydroxyl group at C-1‴ and the carboxylic acid group at
six polyphenolic acids. The structures of novel compounds, C-9. The suggestion was in accord with the observation of
citriodolic acid A (1), citriodolic acid B (2), and citriodolic the chemical shift of C-9 signal upfield shifted from δ 169.6
acid C (3) were determined by the one dimensional (1D)- and in SAJ to δ 167.2 in 1 and the chemical shift of C-1‴ signal
2D-NMR elucidations, and mass spectral analysis.
upfield shifted from δ 128.8 in ADPP to 123.8 in 1. This was
Citriodolic acid A (1) was isolated as a brown amorphous further confirmed by the rotating frame Overhauser effect
powder and had a molecular formula C43H42O14, which was spectroscopy (ROESY) correlation of 2‴-OCH3 with H-2 (Fig.
derived from its positive-ion high-resolution electrospray ion- 3) and acid hydrolysis of citriodolic acid A with 11N HCl gave
ization (HR-ESI)-MS (m/z 805.2468 [M+Na]+) and 13C-NMR SAJ, which was confirmed by HPLC analysis. Three chirality
1
data. The H-NMR spectrum indicated that the presence of centers (7R, 8R, 8″R) in 1 were suggested by circular dichro-
two β-(3,4-dihydroxyphenyl) lactic acid moieties [δH 6.76 (1H, ism (CD) spectrum in ethanol with three positive bands at
d, J=2.0Hz, H-2), 6.64 (1H, d, J=8.0Hz, H-5), 6.73 (1H, dd, 238, 260, and 284nm and a negative band at 327nm.11) A cou-
J=2.0, 8.0Hz, H-6), 5.16 (1H, d, J=3.2Hz, H-7), 4.71 (1H, pling constant of 3.2Hz indicated that the H-7 and H-8 were
d, J=3.2Hz, H-8); and δH 6.74 (1H, d, J=2.0Hz, H-2″), 6.68 cis oriented,9) which was further confirmed by the ROESY
(1H, d, J=8.0Hz, H-5″), 6.56 (1H, dd, J=2.0, 8.0Hz, H-6″), correlation of H-7 with H-8. Consequently, the structure of 1,
*To whom correspondence should be addressed. e-mail: zlzhou@hotmail.com
© 2016 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 64, No. 11 (2016)
Fig. 1. Chemical Structures of Compounds 1–6 Isolated from the Leaves of Eucalyptus citriodora
which was established as shown in 1, is a new natural com- was established as shown in 2, is a new natural compound,
pound, which we named citriodolic acid A.
which we named citriodolic acid B.
The molecular formula of citriodolic acid B (2) was de-
Citriodolic acid C (3) was isolated as a brown amorphous
termined as C38H34O14 by a quasi-molecular ion peak at m/z powder and its elemental composition was determined to be
737.1844 [M+Na]+ in the HR-ESI-MS. The existence of C42H40O13 by HR-ESI-MS. Acid hydrolysis of 3 also gave
SAJ was determined by hydrolysis of 2 with 11M HCl and SAJ by HPLC analysis. The proton and carbon signals in the
1
HPLC analysis. The H- and 13C-NMR data of 2 were simi- 1H- and 13C-NMR spectra of 3 were very similar to those of
1
lar to those of 1, except for the presence of a 4‴-allyl-2‴,6‴- 1. However, preliminary inspection of the H-NMR spectrum
dimethoxy phenolic group [allyl: δH 3.35 (2H, td, J=1.4, 1.4, of 3 revealed the absence of one of the two methoxys in 1.
6.8Hz), 5.99 (1H, ddd, J=6.8, 12.0, 16.0Hz), 4.91 (1H, ddd, Moreover, comparison of the 13C- and distortionless enhance-
J=1.4, 2.1, 16.0Hz), 5.12 (1H, ddd, J=1.4, 2.1, 12.0Hz), and ment by polarization transfer (DEPT) NMR data for 1 and 3
δC 40.5, 137.6, 116.1; two methoxy groups: δH 3.77 (6H, s) and indicated that the 4‴-allyl-2‴,6‴-dimethoxy-3‴-prenyl phenolic
δC 56.5 (2×C); tetra-substituted phenyl: δH 6.42 (2H, s) and δC group of 1 was replaced by a 4‴-allyl-2‴-methoxy-6‴-prenyl
1
124.2, 148.6 (2×C), 137.2, 107.1 (2×C)] at C-9 (δC 167.3) in phenolic group in 3. The H-NMR spectrum of 3 revealed
2,12) instead of a 4‴-allyl-2‴,6‴-dimethoxy-3‴-prenyl phenolic the presence of two aromatic proton signals [δH 6.68 (1H, d,
1
group in 1. This was revealed by the H–1H correlations of the J=2.0Hz), 6.59 (1H, d, J=2.0Hz)], a methoxy signal [δH 3.87
spin system H-1⁗/H-2⁗/H-3⁗ as well as the HMBC correla- (3H, s)], one allyl signal [δH 3.34 (2H, td, J=1.4, 1.4, 6.6Hz),
tions from H-1⁗ to C-2⁗, C-3⁗, C-3‴, C-4‴ and C-5‴, H-2⁗ to 5.96 (1H, ddd, J=6.6, 12.0, 16.0Hz), 5.08 (1H, ddd, J=1.4,
C-1⁗, C-3⁗ and C-4‴, H-3⁗ to C-1⁗ and C-2⁗, H-3‴ to C-1‴, 2.2, 12.0Hz), 4.99 (1H, ddd, J=1.4, 2.2, 16.0Hz)], and a prenyl
C-2‴, C-4‴, C-5‴ and C-1⁗, H-5‴ to C-1‴, C-3‴, C-4‴, C-6‴ signal [δH 3.41 (2H, d, J=6.8Hz), 5.33 (1H, t, J=6.8Hz)], sug-
and C-1⁗, 2‴-MeO to C-2‴, and 6‴-MeO to C-6‴ (Fig. 2). The gesting that 3 has a 4‴-allyl-2‴-methoxy-6‴-prenyl phenolic
1
relative configuration of 2 was assigned by CD and ROESY group.10) This was further confirmed by the H–1H correla-
experiments. Similar as in case of 1, three positive bands at tions of the spin system H-1⁗/H-2⁗ and H-1‴″/H-2‴″/H-3‴″,
238, 260, and 284nm and a negative band at 327nm were ob- as well as the HMBC correlations from H-3‴ to C-1‴, C-2‴,
served in the CD spectrum of 2 suggesting for three chirality C-4‴, C-5‴ and C-1‴″, H-5‴ to C-1‴, C-3‴, C-4‴, C-6‴, C-1‴″
centers (7R, 8R, 8″R) in 2.9) Thus, the structure of 2, which and C-1⁗, H-1⁗ to C-2⁗, C-3⁗, C-1‴, C-5‴ and C-6‴, H-2⁗

Vol. 64, No. 11 (2016)
Chem. Pharm. Bull.
1643
1
Fig. 2. Key HMBC and H–1H COSY Correlations of Compounds 1–3
Fig. 3. Key ROESY Correlations of Compounds 1–3

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Chem. Pharm. Bull.
Vol. 64, No. 11 (2016)
to C-1⁗, C-3⁗, C-4⁗, C-5⁗and C-6‴, H-1‴″ to C-2‴″, C-3‴″, Eucalyptus citriodora in the present study. They were tested
C-3‴, C-4‴ and C-5‴, H-2‴″ to C-1‴″, C-3‴″, C-3‴ and C-4‴, for their antiviral activity against RSV (Table 2). Among
H-3‴″ to C-1‴″ and C-2‴″, and 2‴-MeO to C-2‴ (Fig. 2). The these compounds, compounds 1–3 showed significant antiviral
CD spectrum of 3 showed a negative cotton effect at 327nm activity against RSV with IC50 values of 2.3, 4.8, and 1.8µg/
and positive cotton effects at 238, 260, and 284nm, indicating mL, comparable to that of ribavirin, an approved drug for the
that 3 has three chirality centers (7R, 8R, 8″R).9) On the basis treatment of RSV infections in humans. The other compounds
of the above data, the structure of 3, which was established exhibited moderate or weak antiviral activity against RSV.
as shown in 3, is a new natural compound, which we named
citriodolic acid C.
Experimental
Compounds 1–6 were isolated for the first time from the
General UV spectra were recorded on a Hewlett-Packard
Table 1. 1H-NMR (400MHz) and 13C-NMR (100MHz) Data of Compounds 1–3 (δ, ppm: J, Hz)
1
2
3
No.
δH
δC
δH
δC
δH
δC
1
128.3
114.1
145.5
145.8
115.9
118.2
73.4
128.1
114.3
145.3
145.7
115.9
118.2
73.4
128.1
114.5
145.6
145.9
115.7
118.3
73.7
2
6.76 d (2.0)
6.78 d (2.0)
6.76 d (2.0)
3
4
5
6.64 d (8.0)
6.64 d (8.0)
6.66 d (8.0)
6
6.73 dd (2.0, 8.0)
5.16 d (3.2)
6.76 dd (2.0, 8.0)
5.18 d (3.0)
6.73 dd (2.0, 8.0)
5.15 d (3.0)
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
128.3
116.1
143.7
143.1
117.5
122.9
145.0
115.7
166.1
127.8
116.8
145.3
145.2
116.8
120.3
36.8
167.3
128.3
116.1
143.8
143.1
117.6
122.8
145.2
115.5
166.1
127.4
116.8
145.3
145.2
116.7
120.1
36.6
167.6
128.4
116.3
143.8
142.9
117.9
122.8
145.4
115.8
166.3
127.7
116.9
145.5
145.3
116.7
119.9
36.8
1′
2′
3′
4′
5′
6′
7′
8′
9′
1″
2″
3″
4″
5″
6″
7″
7.27 d (2.0)
7.28 d (2.0)
7.28 d (2.0)
6.89 d (8.0)
6.88 d (8.0)
6.86 d (8.0)
7.16 dd (2.0, 8.0)
7.48 d (15.9)
6.37 d (15.9)
7.16 dd (2.0, 8.0)
7.48 d (16.0)
6.38 d (16.0)
7.14 dd (2.0, 8.0)
7.44 d (16.0)
6.40 d (16.0)
6.74 d (2.0)
6.76 d (2.0)
6.73 d (2.0)
6.68 d (8.0)
6.68 d (8.0)
6.66 d (8.0)
6.56 dd (2.0, 8.0)
2.88 dd (9.2, 14.0)
3.01 dd (3.4, 14.0)
5.03 dd (3.4, 9.2)
6.58 dd (2.0, 8.0)
2.91 dd (9.0, 14.0)
3.04 dd (3.5, 14.0)
5.05 dd (3.5, 9.0)
6.56 dd (2.0, 8.0)
2.89 dd (9.4, 14.0)
3.03 dd (3.5, 14.0)
5.01 dd (3.5, 9.4)
8″
9″
73.8
171.6
123.8
147.4
126.1
136.6
108.4
148.1
25.4
73.8
171.6
124.2
148.6
107.1
137.2
107.1
148.6
40.5
73.7
171.8
141.3
150.6
108.9
138.4
122.1
136.7
28.6
1‴
2‴
3‴
4‴
5‴
6‴
1⁗
2⁗
3⁗
6.42 d (2.0)
6.42 d (2.0)
6.68 d (2.0)
6.59 d (2.0)
6.50 s
3.45 d (6.6)
5.47 t (6.6)
3.35 td (1.4, 1.4, 6.8)
3.41 d (6.8)
5.33 t (6.8)
122.9
136.1
5.99 ddd (6.8, 12.0, 16.0)
4.91 ddd (1.4, 2.1, 16.0)
5.12 ddd (1.4, 2.1, 12.0)
137.6
116.1
121.9
132.3
4⁗
5⁗
1″‴
2″‴
3″‴
1.68 s
17.8
25.7
1.74 s
17.6
25.7
1.79 s
1.77 s
3.39 td (1.6, 1.6, 6.4)
5.91 ddd (6.4, 12.0, 16.0)
4.87 ddd (1.6, 2.4,16.0)
5.08 ddd (1.6, 2.4, 12.0)
3.86 s
37.1
3.34 td (1.4, 1.4, 6.6)
5.96 ddd (6.6, 12.0, 16.0)
4.99 ddd (1.4, 2.2, 16.0)
5.08 ddd (1.4, 2.2, 12.0)
3.87 s
39.9
137.2
115.1
137.4
115.1
2‴-OMe
6‴-OMe
58.6
55.8
3.77 s
3.77 s
56.5
56.5
56.1
3.83 s

Vol. 64, No. 11 (2016)
Chem. Pharm. Bull.
1645
Table 2. Antiviral Activity of Compounds 1–6
2.1–2.5). Fraction 2.5 (0.75g) 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 com-
pound 5 (16mg). 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 (19mg), 2 (16mg), 3 (25mg), and
4 (38mg) from Fr. 5 (21g). Fraction 6 (17g) 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 (41mg).
Citriodolic Acid A (1)
Compounds
IC50 (µg/mL)a)
CC50 (µg/mL)b)
SIc)
1
2.3
4.8
99.7
113.2
124.6
229.1
246.3
274.4
62.5
43.3
23.6
69.2
4.4
2
3
1.8
4
51.5
87.1
127.3
2.6
5
2.8
6
2.2
Ribavirin
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
.
Brown amorphous powder, [α]D25 +28.6 (c=0.05, EtOH); IR
νmax (KBr): 3435, 2941, 1606, 1523, 1448, 1364, 1288, 1193,
HP-845 UV-VIS spectrophotometer (Palo Alto, U.S.A.). CD 1111, and 1063 cm−1. UV (MeOH) λmax (logε): 322 (4.23),
spectra were recorded with a Jasco J-815 spectropolarimeter 288 (4.26), and 222 (4.55) nm; HR-ESI-MS m/z: 805.2468
(Maryland, U.S.A.). Optical rotations were measured using a [M+Na]+ (Calcd for C43H56O25Na, 805.2474). 1H-NMR
JASCO P-1010 digital polarimeter (Japan) and IR spectra were (400MHz, dimethyl sulfoxide (DMSO)-d6) and 13C-NMR
obtained from a PerkinElmer, Inc. Spectrum One FT-IR spec- (100MHz, DMSO-d6) (Table 1).
trometer (U.K.). The HPLC system (Dionex, Germany) con-
sisted of a pump (LPG 3X00), column oven, and diode array
Citriodolic Acid B (2)
Brown amorphous powder, [α]D25 +25.1 (c=0.05, EtOH); IR
UV/Vis detector [DAD-3000(RS)]. For separation of sample, νmax (KBr): 3436, 2938, 1606, 1522, 1446, 1362, 1288, 1190,
a hypersil C18 (5 µm, 250mm×4.6mm) was used and the de- 1113, and 1066cm−1. UV (MeOH) λmax (logε): 322 (4.21),
tection UV wavelength was set at 286nm. MS on a Finnigan 288 (4.25), and 222 (4.54) nm; HR-ESI-MS m/z: 737.1844
LCQ Advantage Spectrometer (Thermo Scientific, U.S.A.) and [M+Na]+ (Calcd for C38H34O14Na, 737.1848). 1H-NMR
a Shimadzu GC-MS model QP2010 Plus spectrophotometer (400MHz, DMSO-d6) and 13C-NMR (100MHz, DMSO-d6)
(Japan), respectively. NMR spectra were recorded on 400MHz (Table 1).
FT-NMR spectrometer (Varian Inova AS 400, U.S.A.), and
deuterium solvents for NMR were purchased from Merck
Citriodolic Acid C (3)
Brown amorphous powder, [α]D25 +31.8 (c=0.05, EtOH); IR
Co., Ltd. Chemical shifts are given as δ values with refer- νmax (KBr): 3433, 2939, 1609, 1526, 1446, 1364, 1289, 1193,
ence to tetramethylsilane (TMS) as internal standard. Column 1113, and 1066cm−1. UV (MeOH) λmax (logε): 322 (4.22),
chromatography separations were carried out on silica gel 288 (4.26), and 222 (4.54) nm; HR-ESI-MS m/z: 775.2366
(200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qing- [M+Na]+ (Calcd for C42H40O13Na, 775.2368). 1H-NMR
dao, P. R. China), ODS (50 mesh, AA12S50, YMC), MCI-gel (400MHz, DMSO-d6) and 13C-NMR (100MHz, DMSO-d6)
CHP20P (35–75µm, Japan) and Diaion HP-20 (Pharmacia, (Table 1).
Peapack, NJ, U.S.A.). The TLC analysis was carried out using
Acid Hydrolysis of Citriodolic Acids A–C A solution
a Kiesel gel 60F254 and RP-18F254S plates (Merck), and UV (3mg each) of 1–3 in 11N HCl (1.5mL) was heated at 100°C
lamp (Spectroline Model ENF-240 C/F, Spectronics Corpora- for 5min under an N2 atmosphere. After cooling, the solu-
tion, U.S.A.) and 10% H2SO4 solution were used for detection. tion was removed by blowing with N2. The residue was dis-
All other chemicals and reagents of analytical grade were ob- solved in the solution of methanol, stirred at 40°C for 5min.
tained from Sigma-Aldrich, unless indicated otherwise.
The methanol solution was analyzed by HPLC-diode array
Plant Materials The leaves of Eucalyptus citriodora were (DAD) using Hypersil C18 (250×4.6mm). The HPLC linear
collected in Zhanjiang, Guangdong Province of China in Sep- gradient profile was as follows: water (containing 0.5% phos-
tember 2012, and were identified by Professor Wen-Qing Yin. phoric acid), acetonitrile (containing 0.5% phosphoric acid)
A voucher specimen (No. 20120903) has been deposited in the 57:43 v/v (0–15min), 57:43 to 20:80 (15–18min), and 20:80
authors’ laboratory.
(18–26min) at a flow-rate of 0.8mL/min. The volume injected
Extraction and Isolation The dry leaves of Eucalyptus was 20µL. The separation was carried out at 35°C. The detec-
citriodora (5kg) were extracted three times under reflux with tor wavelength was set according to the maximum absorption
95% aqueous ethanol (EtOH) (150L×2h). After removing wavelength in the UV spectrum: compounds were analyzed
the solvent under reduced pressure, the residue (1.1kg) was 286nm. The peak identity of each component was confirmed
suspended in water (0.5L) and then sequentially extracted by comparison of the retention time and UV spectrum. The
with petroleum ether (PE), chloroform (CHCl3), ethyl acetate mixed standard solution was separated by using the developed
(EtOAc), and n-butyl alcohol (n-BuOH). The extract solutions HPLC method. Retention times of SAJ, citriodolic acid A,
were concentrated to produce the residues of the PE fraction citriodolic acid B and citriodolic acid C were 14.55, 17.58,
(48g), the CHCl3 fraction (33g), the EtOAc fraction (144g), 16.58, and 17.54min.
the n-BuOH fraction (98g), and the H2O fraction (718g). The
Cells and Virus RSV (Long strain) and Hep-2 cells were
n-BuOH fraction (98g) was subjected to silica gel column provided by American Type Culture Collection. Hep-2 cell
chromatography (CC) using CHCl3–MeOH–H2O mixtures was grown in Dulbecco’s modified Eagle’s medium (DMEM)
(9:1:0.1 to 7:3:0.5, 60L of each). The eluting solutions containing Eagle’s balanced salt solution supplemented with
were monitored by TLC to produce 6 fractions (Frs. 1–6). 10% fetal bovine serum (FBS), 100U of penicillin per mL,
Fraction 2 (2.8g) was applied to a MCI-gel CHP20P CC and 25µg of gentamicin per mL, and 2mM L-glutamine (growth
eluted with MeOH–H2O (0:1–1:0) to yield five fractions (Frs. medium). RSV-infected cells were maintained in DMEM

1646
Chem. Pharm. Bull.
Vol. 64, No. 11 (2016)
supplemented with 1% FBS.
Acknowledgments This study was supported by the
Cytotoxicity Assay Hep-2 cell cultures were prepared Foundation for Distinguished Young Teachers in Higher
in 96-well plastic plates. After 2d of incubations at 37°C in Education of Guangdong, China (YQ2015109) and the China
a CO2 incubator. When the cell cultures were confluent, cul- Spark Program (2015GA780041).
ture medium was removed from each well and replenished
with 0.1mL of maintenance medium. To test for cytotoxic-
Conflict of Interest The authors declare no conflict of
ity, 0.1mL of maintenance medium containing serial 2-fold interest.
dilutions of the tested compounds was added to the wells.
For the cell control, 0.1mL maintenance medium without the
Supplementary Materials The online version of this ar-
compound was added. All cultures were incubated at 37°C, ticle contains supplementary materials.
for 2–5d. The morphology of the cells was inspected daily
and observed for microscopically detectable alterations, e.g.
loss of monolayer, rounding, shrinking of the cells, granula-
tion, 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 re-
quired 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 con-
centration of the natural products that did not exert toxic
effect detected by microscopic monitoring.13,14)
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