Oleanane triterpenes from the flowers of camellia japonica inhibit porcine epidemic diarrhea virus (PEDV) replication

Article abstract of DOI:10.1021/jm501567f

Porcine epidemic diarrhea virus (PEDV) infections have resulted in a severe economic loss in the swine industry in many countries due to no effective treatment approach. Fifteen oleanane triterpenes (1-15), including nine new ones (1-4 and 10-14), were isolated from the flowers of Camellia japonica, and their molecular structures were determined by extensive spectroscopic methods. These compounds were evaluated for their antiviral activity against PEDV replication, and the structure-activity relationships (SARs) were discussed. Compounds 6, 9, 11, and 13 showed most potent inhibitory effects on PEDV replication. They were found to inhibit PEDV genes encoding GP6 nucleocapsid, GP2 spike, and GP5 membrane protein synthesis based on RT-PCR data. Western blot analysis also demonstrated their inhibitory effects on PEDV GP6 nucleocapsid and GP2 spike protein synthesis during viral replication. The present study suggested the potential of compounds 6, 9, 11, and 13 as promising scaffolds for treating PEDV infection via inhibiting viral replication.

Full text of DOI:10.1021/jm501567f

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Article
Oleanane Triterpenes from the Flowers of Camellia japonica
Inhibit Porcine Epidemic Diarrhea Virus (PEDV) Replication
Jun-Li Yang, Thi Kim Quy Ha, Basanta Dhodary, Euisun Pyo, Ngoc
Hieu Nguyen, Hyomoon Cho, Eunhee Kim, and Won Keun Oh
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501567f • Publication Date (Web): 08 Jan 2015
Downloaded from http://pubs.acs.org on January 27, 2015
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Oleanane Triterpenes from the Flowers of Camellia japonica
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Inhibit Porcine Epidemic Diarrhea Virus (PEDV) Replication
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,
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JunꢀLi Yang,† § ThiꢀKimꢀQuy Ha,† § Basanta Dhodary,† Euisun Pyo,† Ngoc Hieu Nguyen,†
Hyomoon Cho,† Eunhee Kim,ꢀ and Won Keun Oh*,
†
†Korea Bioactive Natural Material Bank, Research Institute of Pharmaceutical Sciences,
College of Pharmacy, Seoul National University, Seoul 151ꢀ742, Republic of Korea
ꢀChoong Ang Vaccine Laboratory, 59ꢀ3 Hwaamꢀdong, Yuseongꢀgu, Daejeon 305ꢀ348,
Republic of Korea
§These authors contributed equally to this work.
*To whom correspondence should be addressed. Tel and Fax: +82ꢀ02ꢀ880ꢀ7872. Eꢀmail:
wkoh1@snu.ac.kr.
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ABSTRACT: Porcine epidemic diarrhea virus (PEDV) infections have resulted in a severe
economic loss in swine industry in many countries due to no effective treatment approach.
Fifteen oleanane triterpenes (1−15), including nine new (1−4 and 10−14), were isolated from
the flowers of Camellia japonica, and their molecular structures were determined by
extensive spectroscopic methods. These compounds were evaluated for their antiviral activity
against PEDV replication and the structureꢀactivity relationships (SARs) were discussed.
Compounds 6, 9, 11, and 13 showed most potent inhibitory effects on PEDV replication.
They were found to inhibit PEDV genes encoding GP6 nucleocapsid, GP2 spike, and GP5
membrane protein synthesis based on RTꢀPCR data. Western blot analysis also demonstrated
their inhibitory effects on PEDV GP6 nucleocapsid and GP2 spike protein synthesis during
viral replication. The present study suggested the potential of compounds 6, 9, 11, and 13 as
promising scaffolds for treating PEDV infection via inhibiting viral replication.
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INTRODUCTION
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Coronaviruses (CoVs), first identified in the 1960s, are one type of enveloped viruses with
positive strand RNA genome.1 They contain the largest genome to date, ranging from
approximately 26 to 32 kilobases.2 During CoVs replication, genes encoding the viral
structural proteins, including nucleocapsid (N), membrane (M), spike (S), and envelope (E),
are key elements for viral integrity. CoVs infections are a major cause of enteric, respiratory,
and central nervous system diseases in humans. Between November 2002 and July 2003, an
outbreak of SARSꢀCoV infections in Hong Kong caused severe acute respiratory syndrome
(SARS) of more than 8,000 individuals and 774 fatalities worldwide.3 CoVs are also able to
cause a range of seasonal or local epidemics of gastrointestinal and respiratory diseases in
farm animals, which are fatal in young animals. There are four common CoVs infecting
animals, including porcine epidemic diarrhea virus (PEDV), canine coronavirus (CCV), avian
infectious bronchitis virus (IBV), and transmissible gastroenteritis coronavirus (TGEV).4
Porcine epidemic diarrhea virus (PEDV), an enveloped and singleꢀstranded RNA virus
from the genus Alphacoronavirus and family Coronaviridae, can spread via vertical
transmission through lactation or the fecalꢀoral route.5 By targeting the epithelial cells of the
small intestine PEDV can cause severe mucosal atrophy and malabsorption, and result in
acute and lethal diarrhea in piglets.6 PEDV infection can cause a high mortality in piglets and
fattening swine with almost 100% for oneꢀweek old piglets, and these diseases have resulted
in a considerable economic loss at swine industry in swineꢀproducing countries.7 PEDV
infection is becoming increasingly problematic over the world, including Europe, Asia, and
America. Since 2010, PEDV infections have swept China causing more than 1 million
fatalities of newborn piglets.8 In U. S. A., with the first detection of PEDV in Ohio in 16 April
of 2013, this lethal virus is spreading to 17 states with 40−50 new cases diagnosed each week,
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which could cause an epidemic with loss of millions of dollars.7,9 In concerning about the
control and eradication of PEDV infection, generous efforts have been made with major focus
on preventive therapy by vaccination. Basically, live attenuated virus and the gene containing
spike protein sequences are being tested as vaccines.10 Pꢀ5V, a commercial attenuated virus
vaccine has been developed in Japan. However, not all sows treated with Pꢀ5V developed
stable lactationꢀinduced immunity.11 Another example is that DR13, an oral vaccine for
preventing PEDV infections in South Korea, is still not able to significantly alter the duration
of virus shedding, indicating the immune protection in challenged piglets.12 Thus, a noble
effective control strategy may be the benefit to prevent such infections.
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There are three key steps throughout the virus life cycle: viral entry, viral replication,
and viral assembly and budding. Among these, viral replication is the core of viral life cycle,
and most current antiviral agents target this stage,13 which make it an exciting and promising
target for antiviral research. Compounds inhibiting viral replications are virusꢀspecific and
chemically diverse. These properties match current effects to discover specific antiviral
agents.10
Natural products have been regarded as a rich source of novel chemical entities with
unique pharmacological activities. Of them, oleanane saponins have been reported to show
phytotoxicity,14a antiꢀleishmanial activity,14b,14c the inhibition on HCV entry,14d and
membrane permeabilizing effects,14e as well as the haemolytic,14f antiꢀinflammatory,14g and
HIV inhibitory activities.14g Structureꢀactivity relationship (SAR) studies on these bioactive
saponins indicated that hydroxylation at Cꢀ3 positively influence the phytotoxicity,14a while
hydroxylation at Cꢀ16 would increase the antiꢀleishmanial activity,14b the inhibition on HCV
entry,14d and the membrane permeabilizing effects.14e Furthermore, the glycosylation at OHꢀ3
and COOHꢀ28 also contribute to the antiꢀleishmanial activity14b and inhibition on HCV
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entry,14d respectively. However, coꢀglycosylation at OHꢀ3 and COOHꢀ28 would be another
approach for enhancing the potency of such compounds for membrane permeabilizing.14e
We have screened thousands of plant extracts for antiviral activity against PEDV. During
this process, it was found that a 70% ethanol extract of Camellia japonica flowers showed
potential inhibitory effects on PEDV replication. Camellia japonica L. (Theaceae) is an
evergreen tree widely distributed in Korea, China, and Japan.15 Flowers of C. japonica,
known as Camellia flowers, have long been used as a traditional medicine for tonic, antiꢀ
inflammatory, and stomatic, as well as treating hematemesis and bleeding due to internal and
external injury.16 Bioassayꢀguided fractionation of this active extract afforded nine previously
undescribed oleanane triterpenes (1−4 and 10−14), as well as six previously reported but no
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antiviral studied oleanane triterpenes (5−9 and 15). Herein, the antiviral activities, including
action of mechanisms, against PEDV of all isolates were evaluated, and a brief structureꢀ
activity relationship was also discussed.
RESULTS AND DISCUSSION
Isolation and Structure Elucidation. The flowers of C. japonica were extracted with
70% EtOH using sonication. By means of diverse chromatographic methods, including silica
gel, RPꢀC18, LHꢀ20, and HPLC, nine new (1−4 and 10−14) and six known (5−9, and 15)
oleanane triterpenes (Figure 1) were purified and obtained.
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Compound 1 was obtained as a white powder with [
α]
–50.0 (c 0.1, MeOH). Its
molecular formula C35H54O9 was deduced from HRFABMS of m/z 641.3648 [M + Na]+
(calcd 641.3666), indicating nine degrees of unsaturation. The IR spectrum showed
absorptions for hydroxy (3381 cm−1), carbonyl (1706 cm−1), and olefin (1664 cm−1)
functionalities. Acid hydrolysis of 1 with 5% aqueous H2SO4ꢀ1,4ꢀdioxane afforded
Dꢀ
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glucuronic acid, based on gas chromatography analysis following treatment with Lꢀcysteine
methyl ester hydrochloride and TMS derivatization,17 and coupling pattern of the anomeric
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proton (d, J = 7.7 Hz) indicated a
β
configuration for the glucuronopyranosyl unit.18 Besides,
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1
the H and 13C NMR spectroscopic data (Table 1) revealed one oxymethine signal (δH 3.17,
1H, dd, J = 11.6, 4.0 Hz; δC 91.3), a triꢀsubstituted olefin system (δH 5.39, 1H, br t, J = 3.6
Hz; δC 143.1, 125.7), one oxygenated quaternary carbon (δC 77.9), and one ketone carbon (δC
217.3), as well as seven tertiary methyls (δH 1.14, 1.10, 1.06, 0.99, 0.96, 0.86, 0.85). The
above observations displayed signals characteristic of an oleanane triterpene glycoside.
Further analysis on HSQC and HMBC experiments (Figure 2) indicated that compound 1
possessed the same aglycone part as camellioside A,19 which was connected with a
glucuronopyranosyl moiety at Cꢀ3, proven by HMBC correlations as follows: from Hꢀ3 (δH
3.17, 1H, dd, J = 11.6, 4.0 Hz) to Cꢀ1′ ( C 107.3); and Hꢀ1′ ( H 4.37, 1H, d, J = 7.7 Hz) to Cꢀ3
βꢀDꢀ
δ
δ
(
δC 91.3). The coupling pattern of Hꢀ3 (dd, J = 11.6, 4.0 Hz) suggested the
αꢀorientation of
this proton,16 which was proven by the ROESY correlation from Hꢀ3 (
δH 3.17) to Hꢀ5 (δH
0.80). The chemical shifts of Cꢀ16, Cꢀ17, Cꢀ18, and Cꢀ22 of 1 (δCꢀ16 214.7, δCꢀ17 76.0, δCꢀ18
52.3,
δCꢀ22 31.2, in pyridineꢀd5), similar to those of camellioside A (δCꢀ16 214.9, δCꢀ17 76.3, δCꢀ
18 52.7,
δ
Cꢀ22 31.5, in pyridineꢀd5),19 were used to support the
β
ꢀorientation of OHꢀ17. Finally,
compound 1 was determined as camellenodiol 3ꢀOꢀ
β
ꢀDꢀglucuronopyranoside.
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The molecular formula of compound 2, a white powder with [
α]
–31.7 (c 0.1, MeOH),
was analyzed for C36H56O9 by HRFABMS (m/z 655.3820 [M + Na]+, calcd 655.3822). A
βꢀDꢀ
glucuronopyranosyl unit was present in the structure of 2 based on GC analysis and coupling
pattern of the acid hydrolysis product.17,18 Compound 2 demonstrated almost superimposable
1H and 13C NMR data (Table 1) with those for 1, and the major difference was the presence of
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one additional oxymethyl group (δH 3.76, 3H, s; δC 52.8) in 2. An HMBC experiment
revealed that this oxymethyl group showed a correlation to Cꢀ6′ (δC 171.5), which suggested
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βꢀDꢀglucuronopyranosyl moiety.
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Consequently, compound 2 was elucidated as camellenodiol 3ꢀOꢀ6′ꢀmethoxyꢀβꢀDꢀ
glucuronopyranoside.
Compound 3, compared with 2, had closely similar NMR pattern and showed 14 more
mass units by HRESIMS (m/z 669.3990 [M + Na]+, calcd 669.3979). Same as 1 and 2, acid
hydrolysis of 3 released
D
ꢀglucuronic acid,17 and the coupling constant J = 7.8 Hz indicated a
1
β
configuration of this glucuronopyranosyl moiety.18 Analysis of the H and 13C NMR data
(Table 1) of 3 suggested the occurrence of one ethoxyl group (
3H, t, J = 6.8 Hz; C 63.2, 20.0), which showed a HMBC correlation to Cꢀ6′ (
ꢀglucuronopyranosyl unit. Thus, compound 3 was determined as camellenodiol 3ꢀOꢀ6′ꢀ
ethoxyꢀ ꢀglucuronopyranoside. Compound 3 was proven to be an artifact due to its absence
δH 4.21, 2H, q, J = 6.8 Hz; 1.28,
δ
δC 171.8) of the
β
ꢀD
β
ꢀD
in a later crude extract based on LCꢀMS and NMR analysis.
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Compound 4, with [
α]
–27.0 (c 0.1, MeOH), was assigned a molecular formula of
C47H74O19 by HRFABMS (m/z 965.4744 [M + Na]+, calcd 965.4722). Its NMR data (Table 2)
was closely similar to those of 1 except for the values of the sugar part. Acid hydrolysis of 4
afforded
D
ꢀglucuronic acid,
configurations for the glucuronopyranosyl (J = 7.8 Hz), galactopyranosyl
Dꢀgalactose, and D
ꢀglucose.17 Coupling constants of anomeric
protons indicated
β
(J = 7.6 Hz), and glucopyranosyl (J = 7.6 Hz) units.18 On the basis of HSQC and HMBC
experiments (Figure 2), together with comparing the NMR data with those of camelliosides E
and F,16a the linkage pattern of three sugar units was constructed as in Figure 1. Finally,
compound 4 was determined as camellenodiol 3ꢀOꢀ[βꢀDꢀglucopyranosyl(1→2)ꢀβꢀDꢀ
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galactopyranosyl(1→3)]ꢀ
β
ꢀDꢀglucuronopyranoside.
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Compound 10, a white powder with [
α]
–20.7 (c 0.1, MeOH), was shown to have a
molecular formula C36H54O8 by HRFABMS (m/z 615.3899 [M + H]+, calcd 615.3897). The
IR absorptions at 1731 cm−1 (C=O) and 1653 cm−1 (C=C) and UV peak at λmax 298 nm
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indicated the existence of an extended conjugated α,β
ꢀunsaturated ketone moiety,19,20 which
was supported by characteristic 13C NMR resonances at δC 200.4, 146.8, 139.2, 129.0, and
126.8. This moiety was determined as 12,17ꢀdienꢀ16ꢀone based on HMBC correlations as
follows: from Hꢀ12 (δH 6.12) to Cꢀ9 (δC 46.1), Cꢀ13 (δC 139.2), Cꢀ14 (δC 44.9), and Cꢀ18 (δC
146.8); and from Hꢀ15 (δH 2.60, 2.14) to Cꢀ13 (δC 139.2), Cꢀ16 (δC 200.4), and Cꢀ17 (δC
129.0). Further analysis of the NMR data implied that compound 10 possessed the same
aglycone part as that of camellioside C with the difference in sugar moiety.19
A βꢀDꢀ
glucuronopyranosyl unit was identified in 10,17,18 and it was attached to Cꢀ3 based on HMBC
correlations from Hꢀ3 (δH 3.18) to Cꢀ1′ (δC 105.0) and Hꢀ1′ (δH 4.39) to Cꢀ3 (δC 90.0). In
addition, one oxymethyl group (δH 3.81; δC 52.8) showed HMBC correlation to Cꢀ6′ (δC
170.0), implying methyl esterification of the carboxylic group of the βꢀDꢀglucuronopyranosyl
unit. The
αꢀorientation of Hꢀ3 was determined based on its coupling pattern (dd, J = 11.5, 4.3
Hz)16 and ROESY correlation between Hꢀ3 (δH 3.18) and Hꢀ5 (δH 0.79). Finally, compound
10 was determined as 3βꢀhydroxyꢀ28ꢀnoroleanꢀ12,17ꢀdienꢀ16ꢀone 3ꢀOꢀ6′ꢀmethoxyꢀβꢀDꢀ
glucuronopyranoside.
Compound 11 showed the same molecular formula C36H54O8 as that of 10 by HRFABMS
(m/z 615.3884 [M + H]+, calcd 615.3897). The IR, UV, and NMR spectra exhibited that 11
possessed the same aglycone part as that of 10, which was confirmed by HSQC and HMBC
experiments, with the clear different signals ascribe to sugar units. Acid hydrolysis of 11
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released
suggested a
acid methyl ester occurred at Cꢀ6′ based on a HMBC correlation from an oxymethyl group
δH 3.82) to Cꢀ6′ (δC 170.9). Consequently, compound 11 was elucidated as 3βꢀhydroxyꢀ28ꢀ
D
ꢀglucuronic acid, and the coupling constant of anomeric proton (J = 4.0 Hz)
α
configuration for this D
ꢀglucuronopyranosyl unit.18 In addition, a carboxylic
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(
noroleanꢀ12,17ꢀdienꢀ16ꢀone 3ꢀOꢀ6′ꢀmethoxyꢀ
α
ꢀ
D
ꢀglucuronopyranoside.
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Compound 12 was isolated as a white powder with [
α]
–10.0 (c 0.1, MeOH), and was
assigned a molecular formula C29H40O4 by HRFABMS (m/z 453.3035 [M + H]+, calcd
453.3005). Its IR absorptions at 1643, 1645, and 1702 cm−1 and UV peak at λmax 311 nm,
together with NMR data analysis (Table 1), suggested the existence of a 12,17ꢀdienꢀ16ꢀone
moiety, 19,20 which was confirmed by HMBC experiments (Figure 2): from Hꢀ12 (δH 6.19) to
Cꢀ9 (δC 61.2), Cꢀ13 (δC 158.1), Cꢀ14 (δC 46.9), and Cꢀ17 (δC 138.6); and from H2ꢀ15 (δH 2.68,
2.37) to Cꢀ13 (δC 158.1), Cꢀ16 (δC 198.0), Cꢀ17 (δC 138.6), and Cꢀ18 (δC 144.0). However,
difference in the chemical shift and coupling pattern of Hꢀ12 between 12 (δH 6.19, s) and 11
(
δH 6.10, br s) indicated that compound 12 possessed one more ketone group at Cꢀ11 (δC
202.3). This proposal was supported by HMBC correlations from Hꢀ9 ( H 2.66) and Hꢀ12 (δH
6.19) to Cꢀ11 (δC 202.3). Further analysis of HMBC spectrum revealed three important
correlations from Hꢀ5 ( H 1.43), H3ꢀ23 ( H 1.09), and H3ꢀ24 ( H 1.07) to a carbon at C 214.8,
δ
δ
δ
δ
δ
which indicated a ketone group attached at Cꢀ3. Moreover, an oxymethine proton at δH 3.90
(dd, J = 7.9, 2.8 Hz) showing HMBC correlations to Cꢀ2 (δC 43.5), Cꢀ3 (δC 214.8), Cꢀ10 (δC
42.7) and Cꢀ25 (δC 12.9), together with the established molecular formula, supported the
occurrence of an OHꢀ1 group. The coupling pattern of Hꢀ1 (dd, J = 8.0, 2.8 Hz), which was
similar to that of the known compound glochidonol (Hꢀ1: dd, J = 8.1, 3.4 Hz), was used to
determine the
β
ꢀorientation of OHꢀ1.21 Therefore, compound 12 was established as 1
βꢀ
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hydroxyꢀ28ꢀnoroleanꢀ12,17ꢀdienꢀ3,11,16ꢀtrione.
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Compound 13, a white powder with [
α]
–46.4 (c 0.1, MeOH), exhibited a molecular
8
9
formula of C36H56O9 on the basis of HRESIMS (m/z 655.3810 [M + Na]+, calcd 655.3822).
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Its IR peaks were characteristic for hydroxy (3410 cm−1), olefin (1657 cm−1), and carbonyl
1
(1702 cm−1) functionalities. Detailed analysis of H and 13C NMR data demonstrated that
compound 13 possessed the same aglycone part with that of camellioside F,16 which was
proven by HSQC and HMBC experiments (Figure 2). The major difference was that there
was only one sugar unit in 13. After acid hydrolysis, D
ꢀglucuronic acid was released,17 and a
β
configuration was determined based on the coupling constant of anomeric proton (J = 7.8
Hz).18 This
βꢀDꢀglucuronopyranosyl unit was attached at Cꢀ3, analyzed by HMBC
correlations from Hꢀ3 (δH 3.18, dd, J = 10.6, 3.9 Hz) to Cꢀ1′ (δC 106.8) and Hꢀ1′ (δH 4.36, d, J
= 7.8 Hz) to Cꢀ3 (
δC 90.8). The coupling pattern of Hꢀ3 (dd, J = 10.6, 3.9 Hz) and Hꢀ16 (br s),
which were similar to those of camellioside D (Hꢀ3: dd, J = 11.9, 4.3 Hz; Hꢀ16: br s),19 were
used to identify the
α
ꢀorientation of Hꢀ3 and
β
ꢀorientation of Hꢀ16, respectively. Similarity of
C: 73.1, 52.0, 41.3, and 27.5,
C: 73.1, 51.4, 40.9, and 27.3, respectively,
chemical shifts of Cꢀ16, Cꢀ17, Cꢀ18, and Cꢀ22 of compound 13 (
δ
respectively, in pyridineꢀd5) and camellioside F (
δ
in pyridineꢀd5)16a indicated the
β
ꢀorientation of the formyl group at Cꢀ17. These conclusions
were further confirmed by ROESY correlations from Hꢀ3 (δH 3.18) and Hꢀ5 (δH 0.77), and
from Hꢀ18 ( H 2.66) to Hꢀ16 ( H 4.27) and Hꢀ28 ( H 9.21). So, compound 13 was determined
as 3β,16αꢀdihydroxyoleanꢀ12ꢀenꢀ28ꢀal 3ꢀOꢀ ꢀglucuronopyranoside.
The molecular formula of compound 14, a white powder with [
δ
δ
δ
β
ꢀD
25
D
α]
–18.3 (c 0.1, MeOH),
was determined as C54H86O25 based on HRFABMS (m/z 1157.5317 [M + Na]+, calcd
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1157.5356). Compound 14 exhibited very similar H and 13C NMR data to those of
camellioside F,16a with the replacement of a formyl carbon (δC 205.8 in pyridineꢀd5) in
camellioside F by a carboxyl carbon (δC 180.6 in methanolꢀd4) in 14. This observation
suggested that compound 14 had a carboxylic group at Cꢀ17, which was confirmed by HMBC
correlations from Hꢀ16 (δH 3.79), Hꢀ18 (δH 2.19), and H2ꢀ22 (δH 2.02, 1.57) to this carboxyl
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carbon at δC 180.6. Acid hydrolysis of 14 released
galactose,17 and
Dꢀglucuronic acid, Dꢀglucose, and Dꢀ
β
configurations were determined for the three sugar units based on the
coupling patterns of anomeric protons (J = 7.8 Hz). The
αꢀorientation of Hꢀ3 was determined
by its coupling pattern (dd, J = 11.0, 3.6 Hz)19 and ROESY correlation from Hꢀ3 (δH 3.19)
and Hꢀ5 (
δH 0.78). The Hꢀ16 was analyzed as
β
ꢀorientation on the basis of its coupling pattern
(br s)19 and ROESY correlation from Hꢀ16 (
δ
H 3.79) and Hꢀ18 ( H 2.19). The chemical shifts
δ
of Cꢀ16, Cꢀ17, Cꢀ18, and Cꢀ22 (δC: 74.9, 49.5, 42.3, and 33.5, respectively, in pyridineꢀd5),
which were closely similar to those of codonoposide (δC: 74.2, 50.0, 41.7, and 33.7,
respectively, in pyridineꢀd5),17c were used to determine the
β
ꢀorientation of the carboxylic
group at Cꢀ17. Therefore, compound 14 was assigned as echinocystic acid 3ꢀOꢀ[βꢀDꢀ
galactopyranosyl(1→2)]ꢀ[βꢀDꢀglucopyranosyl(1→2)ꢀβꢀDꢀgalactopyranosyl(1→3)]ꢀβꢀDꢀ
glucuronopyranoside.
Based on spectroscopic and optical rotation data as well as literature values, other
compounds were determined as camelliosides A (5),19 schimperinone (6),22 primulagenin A
(8),22
3β Dꢀ
ꢀhydroxyꢀ28ꢀnoroleanaꢀ12,17ꢀdienꢀ16ꢀone (9),23 and echinocystic acid 3ꢀOꢀβꢀ
glucuronopyranoside (15).23 In addition, compound 7 was discovered from nature for the first
time in the present study, which was obtained previously by a chemical reaction.24
Inhibitory Effects of Camellia Triterpenes on PEDV Replication Analyzed by CPE
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Assay. The purified fifteen oleananeꢀtype triterpenes (1−15) were evaluated for their
inhibitory effects on PEDV replication with azauridin as positive control. Vero cells were
incubated with test compounds at different concentrations after inoculation with PEDV for 2
h. As shown in Table 3, compounds 6−9, 11, and 13 exhibited potent inhibitory effects on
PEDV replication, with EC50 values at submicromolar range (0.06 ± 0.02 to 0.93 ± 0.22 ꢀM),
which was stronger than that of positive control (EC50 3.37 ± 0.71 ꢀM). In addition,
compounds 6, 9, and 11 gave higher selective index (SI) values in this assay (44.54 ± 8.34,
32.72 ± 6.22, and 14.75 ± 1.62, respectively), compared to azauridin (14.30 ± 1.24), while
compounds 7 and 13 showed lower SI values (7.99 ± 0.28 and 6.68 ± 0.14, respectively).
Even though compound 8 gave considerable SI value (12.98 ± 2.34), it demonstrated strong
cytotoxicity (0.81 ± 0.07 ꢀM) in this assay. Compounds 2, 3, 10, and 15 also showed
promising EC50 values at low micromolar range (1.09 ± 0.22 to 3.70 ± 0.68 ꢀM). However,
there were no obvious antiviral activity observed for compounds 1, 4, 5, 12, and 14 in this
assay. Moreover, the inhibition on viral replication of compound 9 was further supported by a
timeꢀcourse study, the infectivity of PEDV particles assay, and cell protection assay for PEDV
infection (Supporting Information).
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Structure-Activity Relationships (SARs) of Camellia Triterpenes on PEDV
Replication Based on CPE Assay. To discuss the SARs of Camellia triterpenes, the oleanane
triterpenes (1−15) obtained in this investigation were divided into four groups based on the
substitution pattern at the Cꢀ17 position: (1) substituted by OH (1−5); (2) substituted by
CH2OH (6−8); (3) occurrence of an olefinic double bond between Cꢀ17 and Cꢀ18 (9−12); (4)
substituted by CHO or COOH (13−15). Biological data of group 1 (1−5) (Table 3) indicated
that the monoglycosylation by glucuronopyranosyl moiety with esterification at Cꢀ6′ could
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improve the antiviral property (comparing 2 and 3 with 1, 4, and 5). Group 2 (6−8)
demonstrated promising EC50 values in the CPE assay (Table 3). In this group, the
replacement of a hydroxy group by a ketone group at Cꢀ16 (comparing 6 with 8) or
monoglycosylation at Cꢀ3 (comparing 7 with 8) induced lower cytotoxicity with keeping
considerable antiviral effects. The antiviral profile of group 3 (9−12) (Table 3) indicated that
monoglycosylation at Cꢀ3 could reduce cytotoxicity with keeping considerable antiviral
activity (comparing 9 with 10 and 11), and this trend depends on the type of attached sugar
moieties. However, based on the activity data of compounds 2 and 10 and compounds 6 and
9, the existence of an olefinic double bond between Cꢀ17 and Cꢀ18 slightly decreased the SI
value. From the antiviral effects of compounds 13−15 (Table 3), the substitution of a formyl
group at Cꢀ17 increased antiꢀPEDV inhibition, and also increased the cytotoxicity (comparing
13 with 15). However multiple glycosylation at Cꢀ3 would significantly decrease the antiviral
activity (comparing 14 with 15), and this observation was also supported by the data of
compounds 4 and 5. In addition, based on the results of compounds 7 and 15 (Table 3), the
replacement of a carboxylic group by a hydroxymethyl group at Cꢀ17 improved the antiviral
property. Although chemical diversity of Camellia triterpenes, the above observations allowed
the outline of SARs for this compound class: (1) the coexistence of a ketone group at Cꢀ16
and a hydroxymethyl group at Cꢀ17 positively contribute to the antiꢀPEDV property of
Camellia triterpenes, which is supported by the comparison of biological data of compounds
6, 8, and 9, as well as the highest SI value of compound 6; (2) the multiple glycosylation at Cꢀ
3 significantly decrease the antiꢀPEDV property of Camellia oleanane saponins, which is
supported by the biological data of compounds 4, 5, and 14.
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Inhibitory Effects of Camellia Triterpenes on Key Gene and Protein Synthesis
during PEDV Replication. During the PEDV life cycle, three key structural proteins,
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including spike, membrane, and nucleocapsid proteins, play a pivotal role.25 The spike protein
functions as a key regulator in viral entry step.26 The membrane protein mainly regulates the
viral assembly process,27 and the nucleocapsid protein, which binds to viral RNA, is a basic
protein associated with the genome.28 Their biological functions place the three proteins as
potential and promising targets for antiꢀPEDV research.
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Considering CC50, EC50 and SI values as analyzed in CPE assay (Table 3), four
structurally representative oleananeꢀtriterpenes 6, 9, 11, and 13 were selected and evaluated
for their biological effects on several key genes and proteins required for PEDV replication.
First, we measured the levels of intracellular viral RNA, encoding PEDV nucleocapsid, spike,
and membrane protein synthesis. Vero cells, which were infected with PEDV, were treated
with test compounds at different concentrations, at which compounds showed the strongest
antiviral property. After 24 h, total RNA was isolated from cells and analyzed by realꢀtime
PCR, which was performed using selective primers for PEDV (Supporting Information). As
shown in Figures 3A, 3B, and 3C, compounds 6, 9, 11, and 13 were found to significantly
reduce the RNA levels, associated with GP6 nucleocapsid, GP2 spike, and GP5 membrane
protein, at concentrations of 2 ꢀM, 2 ꢀM, 8 ꢀM, and 1 ꢀM, respectively. All of them (6, 9, 11,
and 13) demonstrated comparable or stronger inhibitory effects on RNA expression,
compared with azauridin that was used as positive control in this assay. The inhibitory effects
of compounds 6, 9, 11, and 13 on RNA expression were further evaluated at the same
concentration of 0.25 ꢀM (Supporting Information). Furthermore, compound 9 was measured
in detail and it was found to inhibit PEDV RNA expression, encoding nucleocapsid, spike,
and membrane protein, in a doseꢀdependent manner at concentrations of 2.0, 1.0, 0.5, and
0.25 ꢀM (Figures 3D, 3E, and 3F). In addition, compounds 1, 4, and 5 having no activity on
PEDV RNA expression encoding GP6 nucleocapsid protein synthesis were further confirmed
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by RTꢀPCR analysis (Supporting Information).
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9
Based on the above observations, compounds 6, 9, 11, and 13 were further evaluated for
their inhibitory effects on GP6 nucleocapsid and GP2 spike protein synthesis during PEDV
replication. Based on western blot analysis (Figures 4A and 4B), compound 11 showed
significant inhibitory effects on PEDV nucleocapsid and spike protein synthesis, which was
even stronger than that of azauridin. Compound 13 demonstrated considerable inhibitory
effects, at the corresponding concentration to that in RTꢀPCR analysis. Moreover, compound
11 was found to inhibit nucleocapsid protein synthesis in a doseꢀdependent manner at
concentrations of 8.0, 4.0, 2.0, and 1.0 ꢀM (Figure 4C).
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Based on promising EC50, SI value, and the potent inhibition on PEDV RNA expression,
compound 6 was further evaluated for its inhibition on PEDV replication, analyzed by an
immunofluorescence assay. As shown in Figure 5, virusꢀinfected cells showed green
fluorescence, but not in mockꢀtreated cells. The result implied that compound 6 showed
marked inhibition on PEDV replication in a doseꢀdependent manner at concentrations of 4.0,
2.0, and 1.0 ꢀM. It was found that compound 6 showed a comparable effect at a concentration
of 4 ꢀM, compared with azauridin at 10 ꢀM.
CONCLUSION
An acetone extract of the pericarp and leaves of C. japonica has been reported to show
inhibitory effects on HIV type 1 protease.29 In the present study, bioassayꢀguided
fractionation of a 70% ethanol extract of C. japonica flowers yielded fifteen oleanane
triterpenes (1−15). A CPE assay, RTꢀPCR analysis, and western blot analysis, and
immunofluorescence assay indicated that four representative triterpenes 6, 9, 11, and 13
showed potent and promising inhibitory effects on PEDV replication by targeting key
structural protein synthesis and relevant gene expression. This study provided a new class of
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natural scaffolds that may be able to be developed as potential antiꢀPEDV agents via
inhibiting viral replication. The clear SARs outlined will facilitate the further structure
optimization of this compound class for developing novel antiviral agents. Furthermore,
human corona viruses, like SARS, from the same Coronaviridae family shares some similar
replication mechanism with PEDV, and the development of SARS vaccine and PEDV vaccine
shared similar theory.30 Therefore, the antiꢀPEDV molecules obtained in this study could also
be the noteworthy candidates for further investigation against the fatal human coronaviruses,
including SARS.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were analyzed by a JASCO Pꢀ
2000 polarimeter (JASCO International Co. Ltd., Tokyo, Japan). UV data were determined
using an Optizen 3220 UV spectrophotometer (Mekasys Co. Ltd., Daejon, Korea). IR spectra
were recorded on a Nicolet 6700 FTꢀIR (Thermo Electron Corp., Waltham, MA, U. S. A.).
NMR spectra were checked by Varian Unity Inova 500 and 600 MHz spectrometers, at the
College of Pharmacy, Seoul National University, Korea. HRFABMS and HRESIMS were
recorded on a JEOL JMS 700 (JEOL, Ltd., Tokyo, Japan) and Agilent 6530 QꢀTOF (Agilent
Technologies, Inc., Santa Clara, CA, U. S. A.) spectrometer, respectively. Column
chromatography (CC) was performed with silica gel (63−200
ꢀm particle size) and RPꢀC18
(40–63 m particle size) obtained from Merck (Darmstadt, Germany), as well as Sephadex
ꢀ
LHꢀ20 (SigmaꢀAldrich Corp., St. Louis, Missouri, U. S. A.). TLC profiles were checked on
RPꢀC18 F254 and silica gel 60 F254 plates. HPLC was performed on a Gilson System with an
Optima Pak C18 column (10 ꢀm particle size, 10 × 250 mm; RS Tech, Seoul, Korea) and a
UV detector. All solvents used for extraction and isolation are of analytical grade. The purities
of all compounds were confirmed to be ≥ 95% by HPLC/MS analysis (see Supporting
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Information for details).
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Plant Material. Dried flowers of C. japonica were purchased from Gangwon Herbs,
Kangwonꢀdo, Republic of Korea, on May, 2013. The sample was botanically identified by
Prof. W. K. Oh, and a voucher specimen (SNUꢀ2013ꢀ0012) was deposited at the College of
Pharmacy, Seoul National University, Korea.
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Extraction and Isolation. The dried C. japonica flowers (3.0 kg) was extracted with
70% EtOH (3 × 15 L, each time for 2 days) at room temperature, which gave a black residue
(490 g) after drying. Part of this extract (400 g) was suspended in H2O (4.0 L), and
partitioning against nꢀhexane and nꢀBuOH. The nꢀBuOH portion was selected for further
fractionation, due to a screening indicating the antiꢀPEDV activity in nꢀBuOH portion, as
analyzed in a CPE assay, which also guided further separation and purification. The nꢀBuOH
part (100.2 g) was first subjected to a silica gel CC (15 × 60 cm) eluting with a solvent system
of nꢀhexane/EtOAc/nꢀBuOH (from 10:1:0.1 to 0:0:1) to give twenty subfractions (CJ1−CJ20).
Fraction CJ12 (10.3 g) was further separated by a RPꢀC18 column (2.0 × 30 cm) eluted with
MeOH:H2O (from 1:3 to 5:1) to give twelve subfractions (CJ12.1−CJ12.12). Fraction
CJ12.10 (506 mg) was purified on a Gilson HPLC system [mobile phase: MeOH in H2O
containing 0.1% HCO2H (0ꢀ10 min: 75%; 11ꢀ40 min: 85%); flow rate: 2 mL/min; UV
detection at 205 and 254 nm] giving compounds 3 (6.5 mg; tR = 15.1 min; 2.65 × 10−6% dry
weight of C. japonica flowers) and 12 (5.1 mg; tR = 22.2 min; 2.08 × 10−6% dry weight).
Fraction CJ13 (16.0 g) was chromatographied over a RPꢀC18 column (2.0 × 50 cm), eluting
with MeOH:H2O (from 1:10 to 1:0), to yield fifteen subfractions (CJ13.1−CJ13.15).
Subfraction CJ13.8 (1.1 g) was further separated by Sephadex LHꢀ20 eluting with EtOH to
give compound 8 (26.1 mg; 1.07 × 10−5% dry weight), 9 (200.6 mg; 8.19 × 10−5% dry weight),
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and an impurity fraction (661.2 mg), which was purified by HPLC [mobile phase: MeOH in
H2O containing 0.1% HCO2H (0−20 min: 75%; 21−35 min: 83%); flow rate: 2 mL/min; UV
detection at 205 and 254 nm] yielding compounds 6 (10.5 mg; tR = 34.1 min; 4.29 × 10−6%
dry weight), 10 (4.8 mg; tR = 29.5 min; 1.96 × 10−6% dry weight), 11 (9.6 mg; tR = 32.2 min;
3.92 × 10−6% dry weight), and 13 (4.6 mg; tR = 28.6 min; 1.88 × 10−6% dry weight). Fraction
CJ13.9 (931.6 mg) was purified by HPLC [mobile phase: MeOH in H2O containing 0.1%
HCO2H (0−15 min: 60%; 16−45 min: 78%); flow rate: 2 mL/min; UV detection at 205 and
254 nm] to afford compounds 1 (80.3 mg; tR = 32.6 min; 3.28 × 10−5% dry weight), 2 (50.2
mg; tR = 41.5 min; 2.05 × 10−5% dry weight), 7 (5.8 mg; tR = 31.2 min; 2.37 × 10−6% dry
weight), and 15 (30.5 mg; tR = 26.1 min; 1.25 × 10−5% dry weight). Fraction CJ14 (6.1 g) was
repeatedly purified by RPꢀC18 and Sephadex LHꢀ20 CC, eluted mixtures of MeOH and H2O,
to give compounds 4 (16.3 mg; 6.66 × 10−6% dry weight), 5 (9.9 mg; 4.04 × 10−6% dry
weight), and 14 (10.8 mg; 4.41 × 10−6% dry weight). The purities of compounds 1ꢀ15 (≥
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95%) were checked by HPLC/MS with two different UV detection conditions (see Supporting
Information for details).
25
D
Camellenodiol 3-O-
MeOH). IR (KBr)
500 MHz): H 5.39 (1H, br t, J = 3.6 Hz, Hꢀ12), 4.37 (1H, d, J = 7.7 Hz, Hꢀ1′), 3.76 (1H, d, J
β
-D-glucuronopyranoside (1). White powder; [
α]
–50.0 (c 0.1,
ν
max 3381, 2891, 1706, 1664, 1456, 1024, 617 cm−1. 1H NMR (methanolꢀd4,
δ
= 8.9 Hz, Hꢀ5′), 3.50 (1H, t, J = 8.9 Hz, Hꢀ4′), 3.36 (1H, t, J = 8.9 Hz, Hꢀ3′), 3.30 (1H, d, J =
11.6 Hz, Hꢀ15a), 3.24 (1H, dd, J = 8.9, 7.7 Hz, Hꢀ2′), 3.17 (1H, dd, J = 11.6, 4.0 Hz, Hꢀ3),
2.78 (1H, dd, J = 11.3, 3.8 Hz, Hꢀ18), 2.03 (1H, m, Hꢀ22a), 1.73 (1H, overlap, Hꢀ9), 1.64 (1H,
d, J = 11.6 Hz, Hꢀ15b), 1.42 (1H, m, Hꢀ22b), 1.14, 1.10, 1.06, 0.99, 0.96, 0.86, 0.85 (each 3H,
all s, H3ꢀ27, 26, 23, 25, 30, 24, 29), 0.80 (1H, br d, J = 11.0 Hz, Hꢀ5). 13C NMR (methanolꢀd4,
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125 MHz), Table 1. HRFABMS m/z 641.3648 [M + Na]+ (calcd for C35H54O9 Na, 641.3666).
6
7
25
D
Camellenodiol 3-O-6′-methoxy-
β
-D-glucuronopyranoside (2). White powder; [
α]
–
8
9
1
31.7 (c 0.1, MeOH). IR (KBr) νmax 3408, 2911, 1712, 1672, 1497, 1035, 605 cm−1. H NMR
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(methanolꢀd4, 500 MHz): δH 5.39 (1H, br t, J = 3.4 Hz, Hꢀ12), 4.37 (1H, d, J = 7.7 Hz, Hꢀ1′),
3.81 (1H, d, J = 9.6 Hz, Hꢀ5′), 3.76 (3H, s, OMe), 3.50 (1H, dd, J = 9.6, 9.2 Hz, Hꢀ4′), 3.35
(1H, t, J = 9.2 Hz, Hꢀ3′), 3.22 (1H, dd, J = 9.2, 7.7 Hz, Hꢀ2′), 3.29 (1H, d, J = 12.8 Hz, Hꢀ
15a), 3.15 (1H, dd, J = 12.5, 4.6 Hz, Hꢀ3), 2.78 (1H, dd, J = 10.8, 4.0 Hz, Hꢀ18), 2.01 (1H, m,
Hꢀ22a), 1.79 (1H, overlap, Hꢀ9), 1.63 (1H, d, J = 12.8 Hz, Hꢀ15b), 1.40 (1H, m, Hꢀ22b), 1.14,
1.10, 1.05, 0.98, 0.96, 0.85, 0.85 (each 3H, all s, H3ꢀ27, 26, 23, 25, 30, 24, 29), 0.80 (1H, br d,
J = 10.3 Hz, Hꢀ5). 13C NMR (methanolꢀd4, 125 MHz), Table 1. HRFABMS m/z 655.3820 [M
+ Na]+ (calcd for C36H56O9Na, 655.3822).
25
D
Camellenodiol 3-O-6′-ethoxy-
β
-D-glucuronopyranoside (3). White powder; [
α]
–
1
24.1 (c 0.1, MeOH). IR (KBr) νmax 3410, 2928, 1712, 1652, 1480, 1015, 621 cm−1. H NMR
(methanolꢀd4, 500 MHz): δH 5.39 (1H, br t, J = 3.1 Hz, Hꢀ12), 4.37 (1H, d, J = 7.8 Hz, Hꢀ1′),
4.21 (2H, q, J = 6.8 Hz, OEt), 3.79 (1H, d, J = 9.6 Hz, Hꢀ5′), 3.51 (1H, dd, J = 9.6, 9.3 Hz, Hꢀ
4′), 3.35 (1H, t, J = 9.3 Hz, Hꢀ3′), 3.31 (1H, d, J = 12.0 Hz, Hꢀ15a), 3.22 (1H, dd, J = 9.3, 7.8
Hz, Hꢀ2′), 3.16 (1H, dd, J = 12.0, 4.1 Hz, Hꢀ3), 2.78 (1H, dd, J = 11.8, 3.6 Hz, Hꢀ18), 2.01
(1H, m, Hꢀ22a), 1.76 (1H, overlap, Hꢀ9), 1.61 (1H, d, J = 12.0 Hz, Hꢀ15b), 1.40 (1H, m, Hꢀ
22b), 1.28 (3H, t, J = 6.8 Hz, OEt), 1.14, 1.10, 1.05, 0.98, 0.96, 0.86, 0.86 (each 3H, all s, H3ꢀ
27, 26, 23, 25, 30, 24, 29), 0.80 (1H, br d, J = 11.2 Hz, Hꢀ5). 13C NMR (methanolꢀd4, 125
MHz), Table 1. HRESIMS m/z 669.3990 [M + Na]+ (calcd for C37H58O9Na, 669.3979).
Camellenodiol
3-O-[
β
-D-glucopyranosyl(1→2)-β-D-galactopyranosyl(1→3)]-β-D-
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D
glucuronopyranoside (4). White powder; [
α]
–27.0 (c 0.1, MeOH). IR (KBr) νmax 3411,
6
7
8
2902, 1708, 1661, 1461, 1010, 625 cm−1. 1H NMR (pyridineꢀd5, 500 MHz):
δH 5.46 (1H, br s,
9
Hꢀ12), 5.09 (1H, d, J = 7.6 Hz, Hꢀ1″″), 5.06 (1H, d, J = 7.6 Hz, Hꢀ1′″), 5.01 (1H, d, J = 7.8
Hz, Hꢀ1′), 4.63 (1H, d, J = 8.9 Hz, Hꢀ5′), 3.72 (1H, d, J = 12.8 Hz, Hꢀ15a), 3.32 (1H, dd, J =
11.2, 4.0 Hz, Hꢀ3), 3.12 (1H, br d, J = 11.6 Hz, Hꢀ18), 2.52 (1H, br d, J = 9.8 Hz, Hꢀ22a),
1.96 (1H, d, J = 12.8 Hz, Hꢀ15b), 1.91 (1H, m, Hꢀ22b), 1.54 (1H, t, J = 8.1 Hz, Hꢀ9), 1.37,
1.29, 1.26, 1.12, 0.92, 0.84, 0.80 (each 3H, all s, H3ꢀ27, 23, 26, 24, 30, 29, 25), 0.75 (1H, br d,
J = 11.6 Hz, Hꢀ5). 13C NMR (pyridineꢀd5, 125 MHz), Table 2. HRFABMS m/z 965.4744 [M
+ Na]+ (calcd for C47H74O19Na, 965.4722).
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3β-Hydroxy-olean-12,17-dien-16-one 3-O-6′-methoxy-
β
-D-glucuronopyranoside (10).
25
D
White powder; [
α]
–20.7 (c 0.1, MeOH). UV (MeOH) λmax (log ɛ) 298 (4.02) nm. IR (KBr)
1
νmax 3401, 2935, 1731, 1653, 1410, 1136, 612 cm−1. H NMR (CDCl3, 400 MHz): δH 6.12
(1H, br s, Hꢀ12), 4.39 (1H, d, J = 7.6 Hz, Hꢀ1′), 3.86 (1H, d, J = 9.6 Hz, Hꢀ5′), 3.81 (1H, s,
OMe), 3.77 (1H, dd, J = 9.6, 8.8 Hz, Hꢀ4′), 3.61 (1H, t, J = 8.8 Hz, Hꢀ3′), 3.50 (1H, dd, J =
8.8, 7.6 Hz, Hꢀ2′), 3.18 (1H, dd, J = 11.5, 4.3 Hz, Hꢀ3), 2.60 (1H, d, J = 15.1 Hz, Hꢀ15a), 2.45
(1H, m, Hꢀ22a), 2.14 (1H, d, J = 15.1 Hz, Hꢀ15b), 2.01 (1H, m, Hꢀ22b), 1.56 (1H, m, Hꢀ9),
1.10, 1.01, 0.98, 0.94, 0.94, 0.92, 0.85 (each 3H, all s, H3ꢀ27, 23, 25, 30, 26, 29, 24), 0.79 (1H,
br d, J = 11.6 Hz, Hꢀ5). 13C NMR (CDCl3, 100 MHz), Table 1. HRFABMS m/z 615.3899 [M
+ H]+ (calcd for C36H55O8, 615.3897).
3β-Hydroxy-28-norolean-12,17-dien-16-one
3-O-6′-methoxy-α-D-
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D
glucuronopyranoside (11). White powder; [α]
–15.7 (c 0.1, MeOH). UV (MeOH) λmax
(log ɛ) 296 (4.03) nm. IR (KBr)
ν
max 3434, 2921, 1739, 1658, 1454, 1032, 616 cm−1. 1H NMR
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(CDCl3, 500 MHz):
δH 6.10 (1H, br s, Hꢀ12), 5.13 (1H, d, J = 4.0 Hz, Hꢀ1′), 4.29 (1H, d, J =
6
7
8
9
9.1 Hz, Hꢀ5′), 3.82 (1H, s, OMe), 3.78~3.72 (2H, overlap, Hꢀ3′ and Hꢀ4′), 3.58 (1H, br d, J =
8.9 Hz, Hꢀ2′), 3.34 (1H, dd, J = 11.8, 4.3 Hz, Hꢀ3), 2.60 (1H, d, J = 15.5 Hz, Hꢀ15a), 2.43
(1H, m, Hꢀ22a), 2.14 (1H, d, J = 15.5 Hz, Hꢀ15b), 2.02 (1H, m, Hꢀ22b), 1.57 (1H, m, Hꢀ9),
1.09, 1.04, 0.98, 0.94, 0.93, 0.90, 0.84 (each 3H, all s, H3ꢀ27, 23, 25, 30, 26, 29, 24), 0.81 (1H,
br d, J = 10.2 Hz, Hꢀ5). 13C NMR (CDCl3, 125 MHz), Table 1. HRFABMS m/z 615.3884 [M
+ H]+ (calcd for C36H55O8, 615.3897).
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D
1β
-Hydroxy-olean-12,17-dien-3,12,16-trione (12). White powder; [
α]
–10.0 (c 0.1,
MeOH). UV (MeOH) λmax (log ɛ) 311 (4.10) nm. IR (KBr) νmax 3420, 2910, 1702, 1645,
1
1643, 1142, 606 cm−1. H NMR (CDCl3, 500 MHz): δH 6.19 (1H, s, Hꢀ12), 5.68 (1H, br
s,OHꢀ1), 3.90 (1H, dd, J = 8.0, 2.8 Hz, Hꢀ1), 2.91 (1H, dd, J = 14.2, 7.9 Hz, Hꢀ2a), 2.68 (1H,
d, J = 15.6 Hz, Hꢀ15a), 2.66 (1H, s, Hꢀ9), 2.56 (1H, m, Hꢀ22a), 2.38 (1H, dd, J = 14.2, 2.8 Hz,
Hꢀ2b), 2.37 (1H, d, J = 15.6 Hz, Hꢀ15b), 2.33 (1H, m, Hꢀ22b), 1.34, 1.13, 1.09, 1.09, 1.07,
0.96, 0.94 (each 3H, all s, H3ꢀ27, 26, 23, 25, 24, 30, 29), 1.43 (1H, overlap, Hꢀ5). 13C NMR
(CDCl3, 100 MHz), Table 1. HRFABMS m/z 453.3035 [M + H]+ (calcd for C29H41O4,
453.3005).
3β,16α-Dihydroxy-olean-12-en-28-al 3-O-
β
-D
-glucuronopyranoside (13). White
max 3410, 2910, 1702, 1657, 1441, 1001, 685
δH 9.21 (1H, s, Hꢀ28), 5.40 (1H, br t, J = 3.5 Hz, Hꢀ
25
powder; [
α]
–46.4 (c 0.1, MeOH); IR (KBr)
ν
D
cm−1. 1H NMR (methanolꢀd4, 600 MHz):
12), 4.36 (1H, d, J = 7.8 Hz, Hꢀ1′), 4.27 (1H, br s, Hꢀ16), 3.18 (1H, dd, J = 10.6, 3.9 Hz, Hꢀ3),
2.66 (1H, dd, J = 10.8, 2.3 Hz, Hꢀ18), 1.91 (1H, m, Hꢀ22a), 1.66 (1H, m, Hꢀ22b), 1.63 (1H, t,
J = 8.7 Hz, Hꢀ9), 1.52 (1H, m, Hꢀ15a), 1.38, 1.05, 0.96, 0.95, 0.91, 0.85, 0.73 (each 3H, all s,
H3ꢀ27, 23, 30, 25, 29, 24, 26), 1.26 (1H, m, Hꢀ15b), 0.77 (1H, br d, J = 11.5 Hz, Hꢀ5). 13C
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NMR (methanolꢀd4, 150 MHz), Table 1. HRESIMS m/z 655.3810 [M + Na]+ (calcd for
6
7
C36H56O9Na, 655.3822).
8
9
Echinocystic acid 3-O-[
β
-D
-galactopyranosyl(1→2)]-[
β
-D
-glucopyranosyl(1→2)-β-D-
10
11
12
13
14
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galactopyranosyl(1→3)]-
β
-D
-glucuronopyranoside (14). White powder; [
α]
–18.3 (c 0.1,
D
MeOH). IR (KBr) νmax 3401, 2901, 1711, 1642, 1438, 1023, 1010, 612 cm−1. H NMR
1
(methanolꢀd4, 600 MHz): δH 5.25 (1H, br t, J = 2.4 Hz, Hꢀ12), 5.10 (1H, d, J = 7.8 Hz, Hꢀ1″′),
4.99 (1H, d, J = 7.8 Hz, Hꢀ1″), 4.66 (1H, d, J = 7.8 Hz, Hꢀ1″″), 4.45 (1H, d, J = 7.8 Hz, Hꢀ1′),
3.79 (1H, br s, Hꢀ16), 3.19 (1H, dd, J = 11.0, 3.6 Hz, Hꢀ3), 2.19 (1H, br d, J = 10.0 Hz, Hꢀ18),
2.02 (1H, m, Hꢀ22a), 1.93 (1H, m, Hꢀ15a), 1.62 (1H, m, Hꢀ9), 1.57 (1H, m, Hꢀ22b), 1.34,
1.09, 1.02, 0.97, 0.94, 0.89, 0.88 (each 3H, all s, H3ꢀ27, 23, 30, 25, 26, 24, 29), 1.21 (1H, m,
Hꢀ15b), 0.78 (1H, br d, J = 12.0 Hz, Hꢀ5). 13C NMR (methanolꢀd4, 150 MHz), Table 2.
HRFABMS m/z 1157.5317 [M + Na]+ (calcd for C54H86O25Na, 1157.5356).
Determination of the Absolute Configuration of Sugar Components. Each glycoside
(2 mg) was hydrolyzed with 1 M HCl (H2O/ethylene oxide, 1:1, 2 mL) under reflux at 100 oC
for 2 h. After dried in vacuo, the residue was subjected to partitioning between H2O and
EtOAc. The H2O layer was concentrated to give a monosaccharide residue. This residue was
dissolved in pyridine (1 mL) and 2 mg of Lꢀcysteine methyl ester hydrochloride was added.
This solution was kept at 60 oC for 2 h, and then trimethylsilylimidazole (0.2 mL) was added.
o
The mixture was maintained at 60 C for another 2 h. After drying the solution, the residue
was partitioned between H2O (2 mL) and nꢀhexane (2 mL). The nꢀhexane layer was analyzed
by GC (column: DBꢀ5MS, 30 m × 0.32 mm × 0.25 ꢀm; detector: FID; detector temperature:
o
o
o
280 C; injected temperature: 250 C; column temperature: from 100 to 280 C with 10
oC/min, then to 300 C with 20 C/min, held for 15 min; carrier gas: He). The sugar
o
o
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derivatives showed retention time of 10.37, 13.29, and 19.35 min, identical to the
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7
8
9
trimethylsilylꢀLꢀcysteine derivatives of authentic
acid, respectively.
Dꢀgalactose, Dꢀglucose, and Dꢀglucuronic
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Cell Culture and Virus Stock. Vero cells (African green monkey kidney cell line; ATCC
CCRꢀ81) were provided by American Type Culture Collection (ATCC, Manassas, VA, U. S.
A.) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 100 U/mL
penicillin, 100 ꢀg/mL streptomycin, and 10% fetal bovine serum (FBS). PEDV was obtained
from Choong Ang Vaccine Laboratory, Korea. Virus stock was kept at −80 °C before use.
Cytotoxicity Assay. The cell viability was calculated using a MTT (3ꢀ(4,5ꢀdimethylꢀ2ꢀ
thiazolyl)ꢀ2,5ꢀdiphenylꢀ2Hꢀtetrazolium bromide) assay. Vero cells were grown in 96ꢀwell
plates at 1 × 105 cells per well and adhered for 24 h before treatment. The cells were treated
with different concentrations of compounds and incubated for 48 h. To avoid solvent toxicity,
the final concentration of DMSO was maintained at 0.05% (v/v) in the culture medium. Then,
20 µL of the 2 mg/mL MTT solution was added to each well and incubated for 4 h. After
removing supernatant, 100 µL DMSO was added to solubilize formazan crystals.
Consequently, the absorbance was measured at 550 nm. Percentage cell viability is
determined as the absorbance in the experiment well compared to that in the control wells,
and toxicities of the compounds were defined as the percentage cell viability. Regression
analysis was used to calculate the 50% cytotoxic concentration (CC50).
Cytopathic Effect (CPE) Inhibition Assay. Vero cells were seeded onto 96ꢀwell plates at
1 × 105 cells per well. One day later, medium was removed and then washed with phosphate
buffered saline (PBS). PEDV at 0.01 MOI was inoculated onto near confluent Vero cell
monolayers for 2 h. The media was replaced by DMEM with several compounds at different
concentrations. After incubation of 72 h at 37 °C under 5% CO2 atmosphere, cells were
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replaced with DMEM and 20 µL of 2 mg/mL MTT to each well and incubated for 4 h at
37 °C. The 50% effective concentration (EC50) was calculated using regression analysis, and
the formula SI = CC50/EC50 was applied to determine the selective index (SI).
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Quantitative Real-Time PCR. Vero cells were grown to about 90% confluence in 6ꢀwell
plates, infected with PEDV at 0.01 MOI and incubated for 2 h. Then, media was replaced by
DMEM and cultured with compounds of interest at various concentrations. After 24 h, total
RNA was isolated from the cells by following TRIzol method, and reverse transcribed using
random primer (iNtRON Biotechnology, Inc, Seongnam, Korea) based on manufacturer′s
instruction. Realꢀtime PCR was performed using selective primers for PEDV, which was
listed in Table 2 (Supporting Information) and conducted using 2 µL of cDNA and Maxima
SYBR Green qPCR master mix 2X (Thermo sci., Rockford, IL, U. S. A.). Cycling conditions
for realꢀtime PCR were follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s,
and 60 °C for 1 min. Realꢀtime PCR was conducted using the StepOnePlusTM RealꢀTime PCR
System (Applied Biosystems, Inc., Foster City, CA, U. S. A.). The data was analyzed with
StepOne software v2.3.
Western Blot Analysis. The cultures were prepared using similar methods to quantitative
realꢀtime PCR. After 24 h, the cells were washed with cold PBS and kept at −80 °C. For
whole cell lysate, the cells were lysed on ice in 100 µL lysis buffer [50 mM NaF, 0.5% NPꢀ40,
1 mM EDTA, 120 mM NaCl, 50 mM TrisꢀHCl (pH 7.6)] and centrifuged at 12,000 rpm for
20 min. Protein concentrations were calculated using protein assay kit (BioꢀRad Laboratories,
Inc., Hercules, CA, U. S. A.). After boiled for 5 min, aliquots of lysates were electrophoresed
on 10% or 12% SDSꢀpolyacrylamide gels. Protein in the gels were electrotransferred to
PVDF membranes (PVDF 0.45 µm, ImmobilonꢀP, U. S. A.). Membranes were incubated with
primary antibodies spike (S) protein, nucleocapsid (N) (AbFrontier Co., Ltd., Seoul, Korea)
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or mouse monoclonal actin antibody, and further incubated with secondary antibodies. Finally,
they were detected using enhanced chemiluminescence Western blotting dectection kit
(Thermo Sci., Rockford, IL, U. S. A.).
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Immunofluorescence Assay. Vero cells were grown on 8ꢀwell chamber slides (LABꢀTEK,
NUNC, Thermo Fisher Scientific, Waltham, MA, U. S. A.) and PEDV at 0.01 MOI were
injected to the cell monolayers for 2 h. Then, the solution was replaced by DMEM and treated
with compounds of interest. After incubation at 37 °C under 5% CO2 atmosphere for 24 h,
cells were washed with PBS (pH 7.4) three times and fixed with a 4% paraformaldehyde
solution for 30 min at room temperature. After blocking with 1% BSA for 1 h, the cells were
incubated overnight with monoclonal antibody against N protein of PEDV (AbFrontier Co.,
Ltd., Seoul, Korea) diluted 1:50 in PBS (pH 7.4). Then, the cells were incubated with FITCꢀ
conjugated goat antiꢀmouse lgG antibody (Jackson ImmunoResearch, Inc., West Grove, PA,
U. S. A.) for 1 h after washing with PBS (pH 7.4). After washing three times with PBS (pH
7.4), the cells were stained with 500 nM DAPI solution for 10 min at room temperature and
washed with PBS (pH 8.0) three times. Slides were mounted with mounting reagent for
fluorescence (Vectashield, Vector Laboratories Inc., Burlingame, CA, U. S. A.) and observed
by fluorescence microscopy (Olympus ix70 Fluorescence Microscope, Olympus Corporation,
Tokyo, Japan).
Statistical Analysis. The results are expressed as the means ± SD of three independent
experiments. Statistical analysis was conducted on Sigma Plot Statistical Analysis software,
and differences between group mean values were determined by oneꢀway analysis of variance
followed by a twoꢀtailed Student’s tꢀtest for unpaired samples, assuming equal variances.
Statistical significance was accepted at p < 0.05.
ASSOCIATED CONTENT
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Supporting Information
6
7
8
9
Purity check of compounds 1−15 by HPLC/MS analysis, characterization data of compounds
6 and 9, 1D (1H and 13C) and 2D (HSQC and HMBC) NMR spectra for new compounds 1−4
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and 10−14, a timeꢀcourse study, the infectivity of PEDV particles assay, and cell protection
assay for compound 9, inhibitory effects of compounds 6, 9, 11, and 13 on PEDV RNA
expression by RTꢀPCR at the same concentration of 0.25 ꢀM, inhibitory effects of compounds
1, 4, 5, and 9 on PEDV RNA expression encoding GP6 nucleocapsid protein synthesis by RTꢀ
PCR analysis, and the table for primers used for realꢀtime PCR. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Tel and Fax: +82ꢀ02ꢀ880ꢀ7872. Eꢀmail: wkoh1@snu.ac.kr.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported in part by grants from the National Research Foundation of Korea
(NRF) (NRFꢀ2012R1A2A2A01009417) and from the Procurement and Development of
Foreign Biological Resources (2012ꢀK1A1A3307871) funded by the Korean government.
ABBREVIATIONS USED
PEDV, porcine epidemic diarrhea virus; GP, glycoprotein; RTꢀPCR, real timeꢀPCR; CoVs,
coronaviruses; N, nucleocapsid; M, membrane; S, spike; E, envelope; SARS, severe acute
respiratory syndrome; CCV, canine coronavirus; IBV, infectious bronchitis virus; TGEV,
transmissible gastroenteritis coronavirus; EtOH, ethanol; RPꢀC18, reverse phaseꢀC18; MeOH,
methanol; H2SO4, sulfuric acid; CPE, cytopathic effect; SI, selective index; CC50, 50%
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cytotoxic concentration; CC, column chromatography; nꢀBuOH, nꢀbutanol; EtOAc, ethyl
acetate; HCO2H, formic acid; KBr, potassium bromide; CDCl3, deuterated chloroform; HCl,
hydrochloric acid; He, helium; ATCC, American Type Culture Collection; DMEM,
Dulbecco’s Modified Eagle’s Medium; FBS, fetal bovine serum; MTT, 3ꢀ(4,5ꢀdimethylꢀ2ꢀ
thiazolyl)ꢀ2,5ꢀdiphenylꢀ2Hꢀtetrazolium bromide; MOI, multiplicity of infection; CO2, carbon
dioxide; qPCR, quantitative PCR, NaF, sodium fluoride; NPꢀ40, Nonidet Pꢀ40; NaCl, sodium
chloride; PVDF, polyvinylidene difluoride; FITC, fluorescein isothiocyanate; DAPI, 4',6ꢀ
diamidinoꢀ2ꢀphenylindole; SD, standard deviation.
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