4
K.B. Kang et al. / Phytochemistry xxx (2015) xxx–xxx
Table 3
Marfey’s reagent (D-FDLA and L-FDLA) and GITC were purchased
Inhibitory effects on PEDV replication of compounds 1–3, and 6.
from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). The (S)-
(+)-Phenylglycine methyl ester hydrochloride [(S)-(+)-PGME]
reagent was purchased from Sigma–Aldrich (St. Louis, MO, USA).
The absolute configuration of amino acids was analyzed using Agi-
lent 6120 quadruple MSD consisting of the 1260 Infinity pump,
1260 Infinity autosampler, 1260 Infinity DAD detector (Agilent
Technologies, Santa Clara, CA, USA), a Phenomenex column (Luna
Compound
CC50
(
l
M)
EC50
NA
13.41 1.13
4.49 0.67
6.17 0.50
5.58 0.53
(
l
M)
SI
1
2
3
6
>400
>400
211.26 29.64
165.30 16.49
44.47 6.11
>30.04 2.74
47.11 0.49
26.75 0.54
7.98 0.37
6-Azauridine
5
l
C18 (2) 100 Å New Column, 4.6 mm ꢀ 100 mm, i.d.; 5
lm,
Sungmoon Systech Co. Ltd., Seoul, Korea), and an Openlab ChemS-
convenience, compound 8 is named mucronine K, after the plant
name in which this compound was first isolated.
tation (Agilent) for data acquisition and processing.
Pocrine epidemic diarrhea virus (PEDV) infection in pigs causes
epidemic diarrhea, dehydration, and vomiting. Most newborn pig-
lets infected by PEDV would be dying with almost 100% mortality
and pigs of all ages are also affected. Infection with this virus has
been become a serious economic loss in the swine industry and
recent outbreaks led to serious economic losses in many swine
producing countries. Only compounds 1–3, and 6 were screened
for their antiviral potential against PEDV, due to the scarce
amounts of other compounds that were obtained. Compounds 2,
3, and 6 showed potent inhibitory effects on PEDV infected Vero
cells, as shown in Table 3. Compounds 3 and 6 showed EC50 values
4.2. Plant materials
Z. jujuba roots were collected in JinJu, Korea, in 2012. The plant
was identified by Prof. Eun Ju Jeong (Gyeongnam National Univer-
sity of Science and Technology, JinJu, Republic of Korea). A voucher
specimen (SUPH-1204-01) is deposited in the Herbarium in the
Medicinal Plant Garden, College of Pharmacy at Seoul National
University in Korea.
4.3. Extraction and isolation
at the micromolar range (EC50 4.49 0.67 and 6.17 0.50
respectively), which was potent as compared to the positive con-
trol 6-azauridine (5.58 0.53 M). However, both compounds
showed much lower cytotoxicity (CC50 211.26 29.64 and
165.30 16.49 M, respectively) compared to the positive control
(44.47 6.11 M), so they had higher selective index (SI) values
(47.11 0.49 and 26.75 0.54, respectively) than 6-azauridine
(7.98 0.37). Compound 2 exhibited lower potency than 3 and 6
(EC50 13.41 1.13 lM), but 2 also demonstrated high SI values
due to the very low cytotoxicity (CC50 > 400 lM).
lM,
Powdered dried roots of Z. jujuba (14.5 kg) were extracted
through maceration with MeOH (2 ꢀ 60 L, for one week each) at
rt. A crude extract (0.5 kg) resulted from extraction solvent
removal. The extract was suspended in H2O and acidified with
1 N HCl to pH 3–4. The acidic solution was firstly extracted with
EtOAc to yield the EtOAc fraction (186.2 g). The aqueous residue
was basified with NaOH to pH 9 and extracted with CHCl3 to pro-
vide the alkaloid fraction (1.7 g), with the latter subjected to silica
CC eluted with increasingly polar CHCl3–MeOH combinations
(30:1, 10:1, 5:1, 3:1 and 1:1) to yield four subfractions, A1–4.
Subfraction A3 (348.5 mg) was separated by Sephadex LH-20 CC
eluted with CH2Cl2–MeOH (3:1) to give five fractions, A3a–A3e.
A3c was purified by a preparative HPLC (0.1% NH4Ac in H2O–MeCN,
7:3, 4 mL/min) to yield 1 (7.8 mg), 2 (53.1 mg), 3 (6.9 mg) and 6
(13.5 mg). A2 (207.8 mg) was subjected to Sephadex LH-20 CC
eluted with CH2Cl2–MeOH (3:1) to give four subfractions, A2a–
A2d. A2b was separated by preparative HPLC (0.1% NH4Ac in
H2O–MeCN, 3:2 to 1:4, 4 mL/min) to yield 4 (2.7 mg), 5 (2.0 mg),
7 (1.6 mg), and 8 (2.0 mg).
l
l
l
3. Conclusion
In the phytochemical investigation herein of the Z. jujuba roots,
five new Ib-type cyclopeptide alkaloids were isolated with three
known cyclopeptide alkaloids. Among the isolated compounds,
three new and one known alkaloids were subjected to in vitro
antiviral assays. Three compounds showed potent inhibitory activ-
ities against a swine-infecting coronavirus, PEDV. To our knowl-
edge, this is the first report on the antiviral activities of plant-
derived cyclopeptide alkaloids.
4.3.1. Jubanine F (1)
White amorphous powder; UV (MeOH) kmax (log
323 (3.87) nm; CD (c 0.10, MeOH) kmax (
e) 276 (3.47),
4. Experimental
D
e
) 321 (ꢁ2.2), 294
(ꢁ0.9), 265 (ꢁ4.4), 234 (ꢁ1.8), 213 (ꢁ5.9) nm; [
a]
20 = ꢁ309.8
D
4.1. General experimental procedures
(MeOH); IR mmax 2966, 2352, 2317, 1674, 1645, 1514, 1566,
1222 cmꢁ1; See Tables 1 and 2 for 1H and 13C NMR spectroscopic
data; ESI-qTOF-MS (positive ion mode) m/z 544.3112 [M+H]+
(calcd. for C28H42N5O6, 544.3135).
Column chromatography (CC) was carried out with Kieselgel 60
silica gel (40–60
l
m, 230–400 mesh, Merck, Darmstadt, Germany)
and Sephadex LH-20 (25–100
l
m, Pharmacia, Piscataway, NJ, USA).
TLC was performed on the Kieselgel 50 F254 coated normal silica
gel (Merck). The preparative HPLC system consisted of a G-321
pump (Gilson, Middleton, WI, USA), a G-151 UV detector (Gilson),
4.3.2. Jubanine G (2)
White amorphous powder; UV (MeOH) kmax (log
323 (3.06) nm; CD (c 0.10, MeOH) kmax (
e) 273 (3.22),
D
e
) 321 (ꢁ2.5), 294
and a XBridge Prep C18 column (250 mm ꢀ 10 mm i.d.; 5
l
m,
(ꢁ0.9), 264 (ꢁ4.9), 231 (ꢁ0.5), 215 (ꢁ3.7) nm; [
a]
20 = ꢁ247.8
D
Waters, Milford, MA, USA). All solvents (analytical grade) were
purchased from Daejung Chemicals & Metals Co. Ltd. (Si-heung,
Korea). NMR spectra were recorded on Bruker AMX 500 and 600
spectrometers (Bruker, Billerica, MA, USA). High resolution ESI-
qTOF-MS were obtained by a Waters Xevo G2 qTOF mass spec-
trometer (Waters MS Technologies, Manchester, UK). UV and CD
spectra were obtained using a Chirascan and CD spectrometer
(Applied photophysics, Surrey, UK). Optical rotations were taken
on a JASCO P-2000 polarimeter (JASCO, Easton, MD, USA). IR spec-
tra were recorded on JASCO FT/IR-4200 spectrometer. Advanced
(MeOH); IR mmax 3332, 2964, 1643, 1513, 1446, 1222, 1186, 1038,
1027 cmꢁ1; See Tables 1 and 2 for 1H and 13C NMR spectroscopic
data; ESI-qTOF-MS (positive ion mode) m/z 558.3296 [M+H]+
(calcd. for C29H44N5O6, 558.3292).
4.3.3. Jubanine H (3)
White amorphous powder; UV (MeOH) kmax (log
323 (3.50) nm; CD (c 0.10, MeOH) kmax (
(ꢁ1.7), 262 (ꢁ8.9), 231 (ꢁ0.8), 215 (ꢁ6.2) nm; [
e) 271 (3.65),
D
e
) 321 (ꢁ4.2), 295
a]
20 = ꢁ280.9
D
(MeOH); IR mmax 3337, 2966, 1678, 1643, 1513, 1432, 1222, 1031,
Please cite this article in press as: Kang, K.B., et al. Jubanines F–J, cyclopeptide alkaloids from the roots of Ziziphus jujuba. Phytochemistry (2015), http://dx.