Improved Biological Activities of Isoepoxypteryxin by Biotransformation

Article abstract of DOI:10.1002/cbdv.201600049

Isoepoxypteryxin is the major coumarin of a Japanese medicinal plant Angelica shikokiana. This research was designed to study the effect of structural changes through fungal biotransformation on the reported biological activities of isoepoxypteryxin. Among the tested microorganisms, only Cordyceps sinensis had enzymes that could catalyze the ester hydrolysis and the reductive cleavage of the epoxide ring of isoepoxypteryxin, separately, to give two more polar metabolites (+)-cis-khellactone (P1) and a new coumarin derivative (+)-cis-3′-[(2-methyl-3-hydroxybutanoyl)oxy]-4′-acetoxy-3′,4′-dihydroseselin (P2), respectively. The polar metabolite P2 showed stronger cytotoxicity and higher selectivity than isoepoxypteryxin. On the molecular level, P2 showed more in vitro inhibition of both tubulin polymerization and histone deacetylase 8 (HDAC8). Similarly, P2 showed more neuroprotection against amyloid beta fragment 1 – 42 (Aβ_{1 – 42})-induced neurotoxicity in human neuroblastoma cells (SH-SY5Y) and exhibited more inhibition of the in vitro aggregation of Aβ_{1 – 42}. Both metabolites showed stronger antiplatelet aggregation by increased inhibition of thromboxane-A2 synthase (TXS) activity and thromboxane-A2 (TXA2) production. This study is the first to describe the improved cytotoxic, neuroprotective, and antiplatelet aggregation activities of isoepoxypteryxin through its biotransformation by C. sinensis.

Full text of DOI:10.1002/cbdv.201600049

Received Date: 18-Feb-2016
Revised Date: 10-Apr-2016
Accepted Date: 18-Apr-2016
Article Type: Full Paper
Improved Biological Activities of Isoepoxypteryxin by Biotransformation
by Amira Miraa), Wael Alkhiaryb), Qinchang Zhuc), Toshinori Nakagawac), Hai-Bang Tranc),
Yhiya M. Amena, c) and Kuniyoshi Shimizuc*)
a) Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516,
b
Egypt. ) Department of Clinical Pathology, Faculty of Medicine, Mansoura University,
Mansoura 35516, Egypt.
c) Division of Systematic Forest and Forest Products Sciences, Department of Agro-
Environmental Sciences, Faculty of Agriculture, Graduate School of Kyushu University 812-
8581, Japan (Tel / Fax: +81-92-642-3002; e-mail: shimizu@agr.kyushu-u.ac.jp).
Isoepoxypteryxin is the major coumarin of a Japanese medicinal plant; Angelica shikokiana. This
research was designed to study the effect of the structure change through fungal
biotransformation on the reported biological activities of isoepoxypteryxin. Among the tested
microorganisms, only Cordyceps sinensis (C. sinensis) had enzymes that could catalyze the ester
hydrolysis and the reductive cleavage of the epoxide ring of isoepoxypteryxin, separately, to
This article has been accepted for publication and undergone full peer review but has not been
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10.1002/cbdv.201600049
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give two more polar metabolites; (+) cis-khellactone (P1) and a new coumarin derivative; (+)-
cis-3'-[2''-methyl-3''-hydroxy-butanoyloxy]-4'-acetoxy-3', 4'-dihydroseselin (P2), respectively.
P2 showed stronger cytotoxicity and higher selectivity than isoepoxypteryxin. On the molecular
level, P2 showed more in vitro inhibition of both tubulin polymerization and histone deacetylase
8 (HDAC8). Similarly, P2 showed more neuroprotection against amyloid beta fragment 1-42
(Aβ1-42)-induced neurotoxicity in human neuroblastoma cells (SH-SY5Y) and exhibited more
inhibition of the in vitro aggregation of Aβ1-42. Both metabolites showed stronger anti-platelet
aggregation through by more strongly inhibiting thromboxane-A2 synthase (TXS) activity and
thromboxane-A2 (TXA2) production. This study is the first to describe the improved cytotoxic,
neuroprotective and anti-platelet aggregation activities of isoepoxypteryxin through its
biotransformation by C. sinensis.
Keywords:
Antiplatelet, Cordyceps sinensis, Cytotoxic, Isoepoxypteryxin, Neuroprotection.
1. Introduction.-Microbial biotransformation is an efficient experimental method of
modifying the structures of both natural and synthetic compounds. It is used to either improve
the biological activities of the parent compound or to decrease the compound’s toxicity [1].
Isoepoxypteryxin is the major coumarin compound (13.29 mg/g methanol extract of the aerial
part) of a Japanese medicinal plant; Angelica shikokiana [2]. It was reported to have cytotoxic
and hepatoprotective activities [3]. Furthermore, in our recent study, isoepoxypteryxin showed
neuroprotection against amyloid β25-35−induced neurotoxicity in mouse neuroblastoma cells [2].
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However, it did not show any activity against platelet aggregation [4]. The present study was,
therefore, designed to modify the structure of isoepoxypteryxin by microbial biotransformation
and to estimate the effect of structural changes on the cytotoxic, neuroprotective and antiplatelet
aggregation activities. The possible molecular targets of the studied activities were also
investigated.
2. Results and Discussion. – Biotransformation and Structural Elucidation of the two
metabolites. Isoepoxypteryxin was screened for the first time for biotransformation using 18
filamentous fungi. Only C. sinensis, after an incubation of 12 days, gave two more polar
metabolites (Fig. 2) P1; 300 mg and P2; 60 mg with % yields of 60 and 12%, respectively.
Structures of the metabolites were confirmed by 1H-NMR, 13C-NMR, DEPT-135, HSQC, HMBC
and HREIMS.13C-NMR spectrum of P1 was similar to that of isoepoxypteryxin in the 14 signals
assigned to the 2',2'-dimethyl-angular pyranocoumarin part. The difference was observed in the
1
disappearance of the 7 carbon signals of the ester groups at C3' and C4'. Similarly, H-NMR
spectrum of P1 did not show the corresponding signals of the ester groups at C3' and C4'. Based
on these findings and by comparing the spectral NMR data with previously reported data [5], P1
was identified as cis-khellactone. This data suggested that C. sinensis has enzyme that could
catalyze the hydrolysis of the ester bonds at the 3' and 4' positions of the parent compound,
possibly the carboxyl esterase enzyme [6]. 1H- and 13C-NMR spectra of P2 were similar to those
of the parent compound except for the signals of the two carbons of the epoxy moiety of the
13
parent compound. In the C-NMR spectrum, the signal at δ 47.4 assigned to C2'', was more
upfield-shifted (-13.9 ppm) than the corresponding signal of the parent compound and correlated
in the HSQC spectrum with a methine proton at δ 2.47 (quintet) that did not appear in the
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corresponding spectrum of the parent compound. The other carbon signal at δ 69.4 attributed to
C3'', was more downfield-shifted (+7.1 ppm) compared to the parent compound, and correlated
with the other downfield-shifted (+ 0.82 ppm) methine (quintet). Furthermore, a doublet signal
(J= 6.6 Hz) integrated for six protons, assigned to H-4'' & CH3-C2'' was correlated to two
different carbon signals [δ 12.7 (C4'') and δ 21.2 (CH3-C2'')] in the HSQC spectrum. All those
investigations suggested that C. sinensis has enzyme or set of enzymes that could catalyze the
reductive cleavage of the epoxy ring at the α-β carbons of ester group at the C3ʹ of the parent
compound (a possible pathway is shown in Fig. 2). The structure was finally confirmed by
HMBC correlations (important correlations of the 3ʹ-acyl group are shown in Fig. 2). Such a
possible pathway was previously reported through chemical catalysis using samarium (II) iodide
[7] however, to the best of our knowledge, this is the first report about the enzyme (from fungi)
catalysis of the reductive cleavage of the epoxide ring.
Biological evaluations. Cytotoxic activity. Cytotoxicity was estimated by measuring the
IC50 values of isoepoxypteryxin and its metabolites against three cancer cell lines: hepatocellular
carcinoma (HepG2), human epithelioma (Hep2), and human breast adenocarcinoma (MCF7).
Selectivity was determined by measuring the effect on the cell viability for human lung and
dermal fibroblasts (WI-38 and NHDF) as normal cell lines (Table 1 and Supplementary file Table
1). The results showed that one of the metabolites; P2, had potent cytotoxicity and higher
selectivity (from the calculated selectivity index using WI-38) against cancer cell lines than the
parent compound. The HepG2 and MCF7 were the most susceptible cell lines to the cytotoxicity
of P2. P2 activity was comparable to that of the positive control 5-FU, and even with higher
selectivity. On the other hand, P1 showed lower cytotoxicity and selectivity than the parent
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compound. It was observed that isoepoxypteryxin and the biotransformed products did not show
any cytotoxicity to NHDF cells (Supplementary file Table 1). To gain some insights into the
mechanism underlying the cytotoxicity, the in vitro activities of the parent compound and the
metabolites on two targets of cytotoxicity; tubulin polymerization [8] and histone deacetylase 8
[9] were investigated. Table 2 shows the IC50 values of the inhibition of tubulin polymerization
and HDAC8. Isoepoxypteryxin and the biotransformed products inhibited tubulin polymerization.
The results matched those obtained for cytotoxic activities. The inhibitory activities of P2 on
tubulin polymerization and HDAC8 were nearly three times higher than those of
isoepoxypteryxin while P1 showed lower activities than isoepoxypteryxin.The results confirmed
that the presence of 3ʹ- and 4ʹ- acyl groups contributes to better cytotoxicity and the presence of a
free hydroxyl group in the 3ʹ- acyl side chain is essential for potent activity.
Neuroprotection against Aβ1-42 induced neurotoxicity in SH-SY5Y cells. Our previous
study [2] showed that the parent compound had neuroprotective activity against Aβ25-35 in mouse
neuroblastoma (neuro-2A) cells. In this study we investigated the neuroprotective activities of
the parent compound and the biotransformed products against the full length and the major
component of the cerebrovascular amyloid deposits fragment [10]; Aβ1-42, in the SH-SY5Y;
human neuronal cell line. P1 did not show any significant neuroprotection, while P2 exhibited
stronger (nearly by 7.3 1.71 and 8.9 3.20 % at 50 and 100 μM concentration, respectively)
dose-dependent neuroprotection than the parent compound (Fig. 3). The abilities of the parent
compound and the biotransformed products to inhibit the in vitro aggregation of Aβ1-42 as a
neuroprotective mechanism [11] were investigated using the Th-T assay. IC50 values of
fibrillation inhibition are shown in Table 3. The results are consistent with the level of
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neuroprotection of the compounds against Aβ1-42induced-neurotoxicity in SH-SY5Y cells. P2
exhibited stronger (nearly by one and half fold increase) inhibition of Aβ1-42 fibrillation than
isoepoxypteryxin while cis-khellactone (P1) did not show any activity. It could be observed; as
in the cytotoxic activity; that substitutions with acyl groups at 3' and 4' are mandatory for the
neuroprotective activity and that the presence of the hydroxyl group at the 3' acyl side chain
increases the activity.
Antiplatelet aggregation activity. Antiplatelet activity was determined by evaluating the
inhibitory activity against arachidonic acid (AA) and thrombin-induced platelet aggregation. The
results (Table 4) showed that isoepoxypteryxin, did not exhibit any inhibitory activity on either
AA or thrombin-induced aggregation, while the biotransformed products P1 and P2 showed
concentration-dependent increase in the inhibitory activity against both agonist-induced platelet
aggregation. The inhibitory activity of cis-khellactone (P1) was nearly two and three times the
activity of P2 on thrombin- and AA-induced aggregation at the 5 and 10 μM concentrations,
respectively.
The effect of structure modification on the activity was deeply investigated by evaluating
the activities of the parent compound and the biotransformed products on TXS activity (which
converts prostaglandin H2 into thromboxane A2, a potent vasoconstrictor and platelet aggregator
[12]) and TXA2 production (shown in TXB2, the more stable form [13]) (Table 5).
Isoepoxypteryxin did not inhibit TXS at any of the tested concentrations while P1 and P2
inhibited TXS and reduced the amount of TXB2 production in a dose-dependent manner. These
results suggested that P1 and P2 exhibited strong antiplatelet activity by inhibiting TXS, which
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causes a consequent reduction in TXA2 production [14]. Through a deep observation of these
results, it could be inferred that acylation at 3' and 4' decreases the activity and the presence of a
hydroxyl group in the 3'- acyl group causes a marked increase in activity.
3. Conclusion. – The present study is the first to describe the production of more biologically
active metabolites of isoepoxypteryxin by biotransformation. Among the tested microorganisms,
only C. sinensis could biotransform isoepoxypteryxin to (+) cis khellactone (P1) (indicating the
presence of enzyme that could catalyze the ester hydrolysis at C-3' and C-4' of the parent
compound) and a new coumarin derivative; (+)-cis-3'-[2''-methyl-3''-hydroxy-butanoyloxy]-4'-
acetoxy-3', 4'-dihydroseselin (P2), (indicating the presence of enzyme or set of enzymes that
could catalyze the reductive cleavage of the epoxide ring). The new coumarin derivative; P2
exhibited more cytotoxic activity as well as a higher selectivity on cancer cell lines than the
parent compound through the greater inhibition of tubulin polymerization and HDAC8 activity.
Furthermore, P2 showed more neuroprotection against Aβ1-42−induced neurotoxicity in SH-
SY5Y cells and exhibited stronger inhibition of the in vitro aggregation of Aβ1-42 than the parent
compound. P1 and P2 displayed a higher antiplatelet aggregation activity through the inhibition
of the AA pathway by inhibiting TXA2 synthase and decreasing TXA2 production. Future
isolation and identification of the catalytic enzymes involved in isoepoxypteryxin
biotransformation are warranted.
Acknowledgments: We would like to thank Dr. Ohga Shoj, Division of Systematic Forest
and Forest Products Sciences, Department of Agro-Environmental Sciences, Faculty of
Agriculture, Graduate School of Kyushu University for supplying C. sinensis to our experiment,
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Joy Plus (Y. Tateyama), of Fukuoka, Japan, for providing the plant powder used for isolation of
isoepoxypteryxin, and the Evaluation Center of Materials Properties and Functions for the NMR
analysis of isolated compounds.
Experimental Part
List of Abbreviations. C. sinensis: Cordyceps sinensis, HDAC8: Histone deacetylase 8,
DMEM: Dulbecco's Modified Eagle's medium, EMEM: Eagle’s Minimum Essential Medium,
FBS: Fetal bovine serum, HepG2: Hepatocellular carcinoma, Hep2: Human epithelioma, MCF7:
Human breast adenocarcinoma, WI-38: Human lung fibroblasts, NHDF: Human dermal
fibroblasts, SH-SY5Y: Human neuroblastoma cells, Aβ1-42:Amyloid beta peptide fragment 1-
42, TXS: Thromboxane-A2 synthase, TXA2: Thromboxane-A2.
Substrate: Isoepoxypteryxin was isolated from the methanol extract of the aerial part of A.
shikokiana (voucher specimen No. Y-1) as previously described [2].
Microorganisms: The following filamentous fungal species were screened for their
ability to biotransform isoepoxypteryxin: Fomitopsis annosa (NBRC6769), Daedalea malicola
(NBRC4978), Gloeophyllum sepiarium
(NBRC6267), Stereum roseum
(NBRC6514),
Punctularia atropurpurascens (NBRC31076), Aleurodiscus disciformis (NBRC6280),
Bjerkandera adusta (NBRC104974) (Biological Resource Center, Tokyo, Japan), Isarias
Sinclairii
(ATCC24400), Merulius tremellosus
(ATCC32558), Hericium coralloides
(ATCC52796), Phanerochaete chrysosporium (ATCC20696), Coriolus versicolar (ATCC48424),
Ceriporia spissa (ATCC62024), Cordyceps ophioglossoides
(ATCC36865), Cordyceps
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memorabilis (ATCC36743), Cordyceps brongniartii (ATCC66779), Cordyceps gracilis
(ATCC34498) (American Type Culture Collection, ATCC, Tokyo, Japan) and Cordyceps sinensis
(was a kind gift of Dr. Ohga Shoj, Division of Systematic Forest and Forest Products Sciences,
Department of Agro-Environmental Sciences, Faculty of Agriculture, Graduate School of
Kyushu University).
Cell lines HepG2 (hepatocellular carcinoma), SH-SY5Y (human neuroblastoma), and
NHDF (human dermal fibroblasts) were cultured in Dulbecco's Modified Eagle's medium
(DMEM) (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine
serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin (GibcoBRL, Tokyo, Japan). Hep2
(human epithelioma), MCF7 (human breast adenocarcinoma) and WI-38 (human lung
fibroblasts) were cultured in Eagle’s Minimum Essential Medium (EMEM) (Nissui
Pharmaceutical, Tokyo, Japan) under the same conditions as with DMEM medium. All cell lines
were obtained from Riken Bioresource Center of Japan (Ibaraki, Japan).
General experimental material: A tubulin polymerization assay kit was purchased from
Cytoskeleton (Denver, CO, USA). HDAC8 Deacetylase Fluorometric kit was obtained from
CycLex (Nagano, Japan). Lipase enzyme, thioflavin T (Th-T), amyloid β1-42 (Aβ1-42), and
positive controls (morin, vinblastine, paclitaxel, 5-fluorouracil (5-FU), and acetylsalicylic acid)
were purchased from Sigma (St. Louis, MO, USA). WST-1, thiazolyl blue tetrazolium bromide
(MTT), and dexamethasone were purchased from Wako Pure Chemical Industries (Osaka, Japan).
Thromboxane B2 (TXB2) and thromboxane-A2 synthase (TXS) ELISA kits were purchased
from MyBioSource, Inc. (CA, USA). The SensoLyte® Thioflavin T β-Amyloid (1 - 42)
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Aggregation Kit was purchased from Funakoshi (Tokyo, Japan). Arachidonic acid (AA) and
thrombin were purchased from Chrono-log (Chicago, IL, USA). All other chemicals were
purchased from Sigma (St. Louis, MO, USA).
Microorganism screening, scaled-up production, and isolation of the metabolites:
Microbial screening was carried out using two-stage fermentation protocol as previously
described [15,16]. TLC silica gel 60 F254 normal phase and RP-18 F254 plates (Merck, Darmstadt,
Germany) were used for screening the metabolites production using several solvent systems
ratios of n- hexane- EtOAc and MeOH-H2O, respectively. Isoepoxypteryxin was only
biotransformed by C. sinensis to two more polar metabolites after incubation period of 12 days.
So, C. sinensis was used for the scaled-up production of the metabolites.
Filaments and spores of C. sinensis cultured on slanted agar plates were suspended at
25 °C with shaking at 120 rpm in a medium (adjusted to pH 7.0 before being autoclaved for 15
min at 121 °C) containing 2% glucose, 0.5% peptone, 0.5% yeast extract, 0.5% NaCl, and 0.5%
K2HPO4. After 72 h, four ml of the suspended filaments were transferred to 25 ml media
contained in 150-ml flasks. After 24 h, isoepoxypteryxin (500 mg, dissolved in DMSO; at a final
concentration 0.5 mg/ml) was added to the cultures and incubated (25 °C, 120 rpm) for 12 days.
Culture control consisted of fermentation blank in which C. sinensis was suspended without
isoepoxypteryxin addition, was kept along the biotransformation experiment. Similarly, substrate
control composed of a sterile medium to which isoepoxypteryxin was added and incubated
without C. sinensis was kept as well. The cultures were collected and extracted with EtOAc (1
liter × 5) and dried over anhydrous Na2SO4. The combined dried extracts were concentrated
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using a rotary evaporator at 45 °C to yield 455 mg of a brown residue. RP-18 F254 TLC
developed with (H2O-MeOH, 3: 7) showed the production of two more polar metabolites (Rf =
0.22 and 0.35).
The residue was separated and purified using reversed-phase preparative HPLC (Inertsil
ODS-3, 20 × 250 mm, H2O-MeOH, 3 : 7, isocratic, flow rate 5 ml/min; injection volume 1 ml,
75 mg/ml, λ = 280, 356 nm) (Fig. 1) to yield P1 (white powder, 300 mg) and P2 (white powder,
1
60 mg). The structures of the metabolites were identified by H-, 13C- and 2D-NMR
(Supplementary material) experiments which were recorded using a BrukerDRX 600 NMR
spectrometer (Bruker Daltonics, Billerica, MA, USA). HR-EIMS spectra were recorded with a
JMS-70 mass spectrometer (JEOL, Tokyo, Japan) at 70 eV using a direct inlet system. Melting
points were determined on an Electrothermal 9100 (Bibby Scientific, NJ, USA) apparatus and
were uncorrected. The optical rotation was measured on a polarimeter (Jasco international,
Tokyo, Japan) using a sodium lamp (589 nm) at room temperature. Melting point, specific
rotation and 1H-, 13C-NMR of isoepoxypteryxin were also mentioned for comparison.
o
Isoepoxypteryxin: White crystals; mp: 101.5-103 C; [α]D: -12.6o(c, 0.03, CD3OD).1H-
NMR (600 MHz, CD3OD) δ:6.27 (1H, d, J =9.6, H-3), 7.88 (1H, d, J =9.6, H-4), 7.55 (1H, d, J
=8.4, H-5), 6.88 (1H, d, J =8.4, H-6), 5.40 (1H, d, J =4.8, , H-3'), 6.56 (1H, d, J=4.8, H-4'), 1.49
and 1.45 (3H each, both s, 2'-gem-CH3), 3.12 (1H, d, J =5.4, H-3''), 1.35 (3H, d, J =5.4, H-4''),
1.54 (3H, s, CH3-C2''), 2.11 (3H, s, H-2''').13C-NMR (150 MHz, CD3OD) δ: 162.0 (C2), 113.7
(C3), 145.7 (C4), 131.1 (C5), 115.7 (C6), 157.9 (C7), 107.9 (C8), 155.2 (C9), 114.2 (C10), 78.6
(C2'), 72.9 (C3'), 61.0 (C4'), 22.9 and 25.5 (C2' gem-CH3), 171.7 (C1''), 61.3 (C2''), 62.3 (C3''),
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14.3 (C4''), 19.5 (C2''-CH3), 170.6 (C1'''), 20.8 (C2'''). HREIMS m/z: 402.1315 [M]+ (calculated
402.1315, C21H22O8).
o
Metabolite 1 [(+)-cis-khellactone: 300 mg, P1]: White powder mp: 160.5-163 C. [α]D:
+57.5o (c, 1, CDCl3) (lit. [5] +57 oC, c, 1, CDCl3). 1H-NMR (600 MHz, CD3OD) δ: 6.24 (1H, d,
J =9.6, H-3), 7.87 (1H, d, J =9.6, H-4), 7.46 (1H, d, J =8.4, H-5), 6.78 (1H, d, J =8.4, H-6), 5.01
(1H, d, J = 4.8, H-3'), 6.56 (1H, d, J=4.8, H-4'), 1.43 and 1.42 (3H each, both s, 2'-gem-CH3).
13C-NMR (150 MHz, CD3OD) δ: 163.4 (C2), 112.7 (C3), 146.2 (C4), 130.1 (C5), 115.8 (C6),
157.9 (C7), 112.9 (C8), 155.8 (C9), 113.7 (C10), 80.3 (C2'), 73.1 (C3'), 62.1 (C4'), 21.5 and 26.9
(C2' gem-CH3). EI-MS m/z (%): 262 (27), 244 (53), 213 (52), 201 (79), 191 (100), 173 (22), 162
(23), 134 (21). HREIMS m/z: 262.0841 [M]+ (calculated 262.0841, C14H14O5).
Metabolite
2
[(+)-cis-3'-[2''-methyl-3''-hydroxy-butanoyloxy]-4'-acetoxy-3',4'-
1
dihydroseselin, P2]: white powder mp: 110.5-112oC. [α]D: +97o (c, 0.03, CD3OD). H-NMR
(600 MHz, CD3OD) δ: 6.24 (1H, d, J =9.6, H-3), 7.87 (1H, d, J =9.6, H-4), 7.54 (1H, d, J =8.4,
H-5), 6.86 (1H, d, J =8.4, H-6), 5.34 (1H, d, J =4.8, H-3'), 6.53 (1H, d, J=4.8, H-4'), 1.46 and
1.43 (3H each, both s, 2'-gem-CH3), 2.47 (1H, quintet, J =7.2, 6.6, H-2''), 3.94 (1H, quintet, J =
6.0, 6.6, H-3''), 1.94 (3H, d, J = 6.6, H-4''), 1.18 (6H, d, J = 5.4, CH3-C2'' and CH3-C4''), 2.01
(3H, s, H-2''').13C-NMR (150 MHz, CD3OD) δ: 162.0 (C2), 113.5 (C3), 145.7 (C4), 130.9 (C5),
115.6 (C6), 158.1 (C7), 107.9 (C8), 155.1 (C9), 114.1 (C10), 78.8 (C2'), 71.4 (C3'), 62.4 (C4'),
23.0 and 25.2 (C2' gem-CH3), 175.4 (C1''), 47.4 (C2''), 69.4 (C3''), 12.7 (C4''), 21.2 (CH3-C2''),
171.7 (C1'''), 20.7 (C2'''). EI-MS m/z (%): 404 (37), 300 (13), 286 (14), 244 (57), 229 (100), 227
(14), 191 (19). HREIMS m/z: 404.1472 (calculated 404.1472, C21H24O8).
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Measurement of cell viability: The viability of cells was determined using WST-1 reagent
as follows: 10 μL of WST-1 reagent was added to each well, followed by 4 h incubation at 37 °C,
after which the absorbance was measured at 450 nm using a microplate reader (Biotek, Winooski,
VT, USA).
Cytotoxic activity: Cytotoxicity to cancer and normal cell lines: HepG2, Hep2, MCF7,
and WI-38 were cultured in 96-well plates at densities of 1 x 104, 0.5 x 104 , 2.5 x 104, and 2 x
104 cells/well, respectively, in a humidified atmosphere of 5% (v/v) CO2 and 95% (v/v) air at
37 °C. After 24 h, the cells were treated with five different concentrations of samples (6.25, 12.5,
25, 50, and 100 μM, dissolved in DMSO). 5-fluorouracil (5-FU) [17] was used as a positive
control. After 48 h, cell viability was determined using WST-1 reagent.
Tubulin polymerization assay: The abilities of the samples to either inhibit or enhance
tubulin polymerization were tested according to the manufacturer’s protocols. Vinblastine (3 μM)
was used as positive control [18] for the inhibition and enhancement of tubulin polymerization,
respectively. Tubulin polymerization was determined by measuring the fluorescence emission at
λ= 420 nm (excitation λ= 360 nm) for 1 h at 1 min intervals at 37 °C.
Histone Deacetylase 8 (HDAC8) inhibitory assay: The inhibitory effects of the samples
and trichostatin A (TSA, positive control [19]) on HDAC8 were examined according to the
manufacturer’s protocols. HDAC8 activity was measured by monitoring the fluorescence
emission at λ= 440 nm (excitation λ= 340 nm) for 1 h at 1 min intervals at 37 °C.
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Neuroprotective activity: Neuroprotection against Aβ 1-42−induced neurotoxicity in SH-
SY5Y cells. The concentration of Aβ1-42 that decreased cell viability to about 50% was
determined as previously described [2]. Aβ1-42 at a concentration of 20 μM caused the desired
cytotoxicity. To estimate the neuroprotective activity, SH-SY5Y cells were cultured in 96-well
plate at a density of 2 x 104 cells/well in a serum-free DMEM and a humidified atmosphere of
5% (v/v) CO2 and 95% (v/v) air at 37 °C. After 24 h, cells were treated with different
concentrations (25, 50, 100 μM) of samples and morin, (as a positive control [20]) that showed
no significant cytotoxicity. After 4 h incubation, 20 μM of Aβ1-42, freshly dissolved in PBS (50
mM, pH = 7.4) and previously incubated at 37 °C for 48 h to induce aggregation [21], was added
to the cells. Cell viability was determined using WST-1 reagent.
Thioflavin T assay (Th-T): The abilities of the samples to inhibit Aβ1-42 aggregation in
vitro were estimated by the SensoLyte® Thioflavin T β-Amyloid (1- 42) Aggregation Kit
according to the manufacturer’s protocol. Fluorescence emission was monitored at Ex/Em= 440/
484 nm at 37oC every 5 min for 180 min with 15 sec of shaking between reads.
Antiplatelet aggregation activity: In vitro antiplatelet aggregation: All the following
procedures were in accordance with the guidelines of the British Committee for Standards in
Hematology (BCSH) on the laboratory aspects of assays used in hemostasis and thrombosis [22]
and on the laboratory investigation of blood platelets function [23]. Blood samples were
collected from 50 voluntary healthy resting blood donors (aged of 20–28 years), who provided
signed informed consent before participating in the research. 1.8 mL of venous blood was
delivered into the collection system’s sterile Vacutainer tube containing 200 µL sodium citrate
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(3.2%) in a 9:1 (v/v) ratio (0.129 M) solution as an anticoagulant (9 parts of blood to 1 part of
anticoagulant). From the citrated blood samples, platelet-rich plasma (PRP) and platelet-poor
plasma (PPP) were prepared. PRP was prepared by slow-speed centrifugation (MPW 350R,
MPW Med. Instruments, Warsaw, Poland) at 170 g for 10 min at room temperature (25 ºC)
without application of the brake. PPP was prepared after the collection of PRP, by centrifugation
of the remaining blood at 2000 g for a further 15 min at 25 ºC and stored at -70ºC.
Platelet aggregation was investigated using the light transmittance platelet aggregometry
method. First, platelets were induced for aggregation using thrombin and arachidonic acid (AA),
resulting in decreased turbidity and increased light transmittance [24]. Briefly, PRP was freshly
prepared for every experiment. The platelet count was performed on the PRP to ensure that the
platelet concentration was in the range of 2.5×105/μL. PRP (480 µL) was incubated for 5 min at
37 ºC in the aggregation cuvette with different concentrations (1, 5, and 10 μM) of samples
dissolved in PBS and DMSO; the final concentration of DMSO was less than 1%). An
aggregation agonist, 5 μL of AA or thrombin (the final concentration was 0.5 mM or 0.5 U/ml,
respectively) was added to the mixtures. Aspirin (with a final concentration of 10 µg/ml) and
PBS containing 1% DMSO were used as positive [25] and negative controls, respectively. The
resulting aggregation was monitored and recorded for 6 min using a dual-channel Lumi-
aggregometer (Model Chronolog 540-VS Platelet Aggregometer; Chrono-log) with constant
stirring at 1,000 rpm, and was expressed as a percentage change in light transmission. The results
are expressed as the percentage of inhibition of agonist-induced platelet aggregation, which was
calculated as follows: percentage inhibition (%) [1 - (Platelet aggregation of sample/platelet
aggregation of control)] × 100%.
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Measurement of TXB2 generation and both TXS and Thromboxane-A2 synthase
activities: Thromboxane A2 (TXA2) is an activator of platelet aggregation. It is unstable, with a
very short half-life (37 sec). Its production is usually estimated by the quantitative evaluation of
another stable thromboxane, TXB2, which is produced by a non-enzymatic hydration of
TXA2[26]. Freshly prepared PRP (as previously described) was pre-incubated at 37 ºC with the
same concentrations (as in the aggregation assay) of samples, aspirin as a positive control, and
the sample solvent vehicle for 5 min. Aggregation was induced by the addition of AA (final
concentration0.5 mM) with stirring (1000 rpm). The plasma samples were transferred into the
kits and determined according to the manufacturer's instructions. The amount (ng/L) of TXB2
production and the inhibitory activity (expressed as percentage of inhibition) on TXS was
determined according to the manufacturer's instructions.
Statistical analysis: IC50 (the concentration of sample showed 50% of the activity) values
were calculated using Probit Analysis (Version 16.0 for Windows; SPSS, Chicago, IL, USA) for
five different concentrations in three independent experiments. Cell viability was expressed as a
percentage of WST-1 or MTT reduction, assuming 100% viability of the cells treated with 1%
DMSO as a negative control. Figures were built in Microsoft Excel 2010. All values are the
means SDs of three independent experiments. Data were analyzed for statistical significance
using one-way ANOVA, followed by Dunnett’s test as a post-hoc test with Prism 5.0 software for
Windows (GraphPad, San Diego, CA, USA). All the fluorescence readings for tubulin
polymerization, HDAC 8, and thioflavin T assays are expressed in relative fluorescence units
(RFU). From the mean values, percentage inhibition was calculated using the following
equation:
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% Inhibition = RFU control - RFU sample x 100
RFU control
Conflict of interest: All authors have no conflict of interest to disclose.
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Table 1: Cytotoxicity and selectivity of isoepoxypteryxin and the biotransformed products.
IC50 (μM)
Compound
HepG2
Hep2
MCF7
WI-38
5-FUa
10.4 2.80 (6.1 b)
20.7 3.71 (4.8 b)
50.6 1.57 (3.4 b)
10.6 3.26 (9.3 b)
9.3 0.61 (6.8 b)
36.4 1.86 (2.7 b)
70.3 2.43 (1.9 b)
20.3 1.24 (4.9 b)
3.1 2.36 (20.5 b)
25.1 1.90 (3.9 b)
85.6 0.71 (2.8 b)
9.8 2.40 (10.1b)
63.4 4.52
99.6 7.59
70.9 6.21
89.9 6.73
Isoepoxypteryxin
P1
P2
aused as a positive control.
bselectivity index obtained by dividing IC50 value for WI-38 (normal cell line)/IC50 of each cancer cell line.
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Table 2. Inhibition of tubulin polymerization and HDAC8 activity.
IC50 (μM)
Compound
Tubulin polymerizationa HDAC8 activityb
Isoepoxypteryxin
P1
22.6 2.98
43.3 1.91
7.1 2.70
1.1 1.20 μM
--------
43.52 4.91
53.86 1.43
15.47 4.77
--------
P2
Vinblastinea
Trichostatin A (TSA) a
a positive controls.
2.3 1.20
Table 3: In vitro inhibition of Aβ1-42 fibrillation.
Compound
Morina
IC50 (μM)
46.3 5.73
72.4 2.61
˃ 100 μM
49.8 1.87
Isoepoxypteryxin
P1
P2
a Positive control
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Table 4: % Inhibition of Thrombin- and AA-induced platelet aggregationa
1 (µM)
Thrombin
5 (µM)
Thrombin
10 (µM)
Thrombin
Compound
AA
AA
2.4
AA
3.8
Isoepoxyptryxin
1.4 0.62
15.9 1.20
10.4 1.34
-7.5 1.48
2.6 0.90
57.8 0.51
22.6 0.97
2.7 1.21
83.9 0.76
38.2 1.35
0.51
77.8
3.60
17.8
0.78
2.57
93.9
0.61
25.7
1.43
28.8
0.91
12.8
0.83
P1
P2
a Aspirin at final conc. 10 µg/ml was used as a positive control and had 60.2 3.2 and 93.2 4.5% inhibition
against thrombin- and AA-induced platelet aggregation, respectively.
Table 5: Inhibition of TXB2 production and TXS activity.a
1 (µM)
5 (µM)
10 (µM)
TXB2
Inhibition
rate of TXS
(%)
TXB2
Inhibition
rate of TXS
(%)
TXB2
Inhibition
rate of TXS
(%)
Compound
Production
(ng/L)
Production
(ng/L)
Production
(ng/L)
Isoepoxyptryxin
63.6 2.37
28.6 1.51b
40.4 3.30 b
-
59.8 0.90
17.8 2.61 b
35.3 1.91 b
-
59.8 1.32
13.6 1.41b
23.2 1.77 b
3.6 0.37
73.9 2.7
45.7 2.5
P1
P2
18.8 3.48
10.5 2.12
27.8 2.87
15.2 0.71
a Data are expressed as means SDs (n=3), aspirin as a positive control at 10 µg/ml conc. showed TXB2
production of 10.8 0.6 ng/L and TXS inhibition by 89.3 4.1%. TXB2 production of negative control was
65.8 2.73 ng/L. b Significant difference from negative control (p <0.05).
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Figure Captions:
Fig. 1: HPLC chromatogram of the isolation of the metabolites using Inertsil ODS-3, 20 × 250
mm, H2O-MeOH, 3 : 7, isocratic, flow rate 5 ml/min; injection volume, 1 ml, 75 mg/ ml, λ = 280,
356 nm).
Fig. 2: Possible pathways of the biotransformation of isoepoxypteryxin by C. sinensis and the
important HMBC correlations of P2.
Fig. 3: Neuroprotection of isoepoxypteryxin and biotransformed products againstt Aβ1-42 (20 µM)
-induced neurotoxicity in SH-SY5Y cells.. All values are means SDs (n = 5). * and ** indicate
significant differences from the cell viability of Aβ1-42 (20 µM) treatment at p < 00.05 and p < 0.01,
respectively.
Fig. 1
P1
P2
A
U
Time (min.)
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Fig. 2:
O
O
O
H+
.
.
CH
C
C
O
O
OH
_
_
O
OH
O
_
H+
C
C
O
OH
_
OH
OH
O
4
5
8
10
9
Possible mechanism
6
7
3
2
O
O
O
2'''
4'
2'
1'''
3'
O
R
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s
i
e
O
s
d
u
c
y
l
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o
4''
3''
C
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r
t
i
d
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y
e
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c
l
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r
e
t
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g
e
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9
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8
9
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2'
4'
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3'
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4'
2'
O
3'
OH
O
4''
H
C
2''
H
C
C
OH
1''
3''
P2
P1
O
OH
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Fig. 3:
**
**
**
**
*
**
**
*
*
*
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