Halogenated Compounds from Directed Fermentation of Penicillium concentricum, an Endophytic Fungus of the Liverwort Trichocolea tomentella

Article abstract of DOI:10.1021/acs.jnatprod.6b01069

One new chlorinated xanthone, 6-chloro-3,8-dihydroxy-1-methylxanthone (1), a new 2-bromo-gentisyl alcohol (2), and a mixture of 6-epimers of 6-dehydroxy-6-bromogabosine C (3a and 3b), together with 19 previously identified compounds, epoxydon (4), norlichexanthone (5), 2-chlorogentisyl alcohol (6), hydroxychlorogentisyl quinone (7), 6-dehydroxy-6α-chlorogabosine C (8a), 6-dehydroxy-6β-chlorogabosine C (8b), gentisyl alcohol (9), gentisyl quinone (10), (R,S)-1-phenyl-1,2-ethanediol (11), dehydrodechlorogriseofulvin (12), dechlorogriseofulvin (13), dehydrogriseofulvin (14), griseofulvin (15), ethylene glycol benzoate (16), alternariol (17), griseoxanthone C (18), drimiopsin H (19), griseophenone C (20), and griseophenone B (21), were isolated from cultures of Penicillium concentricum, a fungal endophyte of the liverwort Trichocolea tomentella. The structures of the new compounds (1, 2, 3a, and 3b) were elucidated by interpretation of spectroscopic data including one- and two-dimensional NMR techniques. Among these, compounds 2-4 displayed modest cytotoxicity to the MCF-7 hormone-dependent breast cancer cell line with IC₅₀ values of 8.4, 9.7, and 5.7 μM, respectively, whereas compound 9 exhibited selective cytotoxicity against the HT-29 colon cancer cell line with an IC₅₀ value of 6.4 μM. During this study we confirmed that the brominated gentisyl alcohol (2) was formed by chemical conversion of 4 during bromide salt addition to culture media.

Full text of DOI:10.1021/acs.jnatprod.6b01069

Article
pubs.acs.org/jnp
Halogenated Compounds from Directed Fermentation of Penicillium
concentricum, an Endophytic Fungus of the Liverwort Trichocolea
tomentella
Tehane Ali,† Masanori Inagaki,† Hee-byung Chai,† Thomas Wieboldt,‡ Chad Rapplye,§
and L. Harinantenaina Rakotondraibe*,†
§
†Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, and Department of Microbiology, The Ohio State
University, Columbus, Ohio 43210, United States
‡Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States
S
* Supporting Information
ABSTRACT: One new chlorinated xanthone, 6-chloro-3,8-
dihydroxy-1-methylxanthone (1), a new 2-bromo-gentisyl
alcohol (2), and a mixture of 6-epimers of 6-dehydroxy-6-
bromogabosine C (3a and 3b), together with 19 previously
identified compounds, epoxydon (4), norlichexanthone (5), 2-
chlorogentisyl alcohol (6), hydroxychlorogentisyl quinone (7),
6-dehydroxy-6α-chlorogabosine C (8a), 6-dehydroxy-6β-chlorogabosine C (8b), gentisyl alcohol (9), gentisyl quinone (10),
(R,S)-1-phenyl-1,2-ethanediol (11), dehydrodechlorogriseofulvin (12), dechlorogriseofulvin (13), dehydrogriseofulvin (14),
griseofulvin (15), ethylene glycol benzoate (16), alternariol (17), griseoxanthone C (18), drimiopsin H (19), griseophenone C
(20), and griseophenone B (21), were isolated from cultures of Penicillium concentricum, a fungal endophyte of the liverwort
Trichocolea tomentella. The structures of the new compounds (1, 2, 3a, and 3b) were elucidated by interpretation of
spectroscopic data including one- and two-dimensional NMR techniques. Among these, compounds 2−4 displayed modest
cytotoxicity to the MCF-7 hormone-dependent breast cancer cell line with IC50 values of 8.4, 9.7, and 5.7 μM, respectively,
whereas compound 9 exhibited selective cytotoxicity against the HT-29 colon cancer cell line with an IC50 value of 6.4 μM.
During this study we confirmed that the brominated gentisyl alcohol (2) was formed by chemical conversion of 4 during bromide
salt addition to culture media.
tomentella (Trichocoleaceae). The genus Penicillium is well-
iverworts are one of the three phyla (mosses: about 14 000
Lspecies, liverworts: about 6000 species, and hornworts: known to produce a variety of bioactive compounds including
the clinically used antibiotic penicillin and the antifungal agent
griseofulvin (15). The ethyl acetate extract from a fermentation
of the fungal strain on potato dextrose agar (PDA) showed
significant antiproliferative activity against HT-29 cells with an
IC50 value of 2.2 μg/mL. Scale-up fermentation on PDA led to
the isolation of epoxydon (4) as the major bioactive compound.
To isolate epoxydon-related and unrelated new and bioactive
compounds, P. concentricum was cultured on rice and on
halogen salt (KCl or KBr)-supplemented rice media for 14
days. Successive normal, reversed-phase open, and flash silica
gel (C18) and high-performance liquid chromatography
(HPLC) separation of the ethyl acetate fraction of the defatted
methanol extract of P. concentricum fermented on rice medium
afforded a new chlorinated xanthone, 6-chloro-3,8-dihydroxy-1-
methylxanthone (1), together with 15 known compounds,
identified as epoxydon (4),3−7 norlichexanthone (5),8−10
gentisyl alcohol (9),11 gentisyl quinone (10),12 (R,S)-1-
phenyl-1,2-ethanediol (11),13 dehydrodechlorogriseofulvin
(12),9 dechlorogriseofulvin (13),9 dehydrogriseofulvin (14),9
about 300 species) of bryophytes (Hepaticeae), which are
taxonomically located between algae and pteridophytes (ferns).
Our interest in the chemistry of liverworts started with the
isolation of bioactive compounds from Marchantiales.1 Liver-
worts grow in contact with microbial-rich environments such as
soil, rotten logs, and wet rocks. There is thus a high chance that
liverworts are exposed to microorganisms and host them as
epiphytes and endophytes. In fact our previous study showed
that HM-1 (22), a cuparene-type sesquiterpene isolated from
the Basidiomycoteous fungus Helicobasidium mompa, has also
been isolated from Bazzania, a liverwort collected from
Madagascar.2 This showed that epiphytic and endophytic
microorganisms of liverworts play a very important role not
only in the biosynthesis of plant secondary metabolites but also
in the protection of their host plant. Most liverworts are not
consumed by insects, worms, and other predators. This is
obviously linked to their ability to produce bioactive
compounds.
In the course of our ongoing systematic search of microbial
endophytes of liverworts that contribute to the production of
bioactive compounds, we isolated an endophytic fungus,
Penicillium concentricum, from the liverwort Trochocolea
Received: November 18, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b01069
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Chart 1
griseofulvin (15),9 ethylene glycol benzoate (16),14 alternariol
(17),15,16 griseoxanthone C (18),10,17 drimiopsin H (19),8
griseophenone C (20),9 and griseophenone B (21).9 Ethyl
acetate extracts of the same strain fermented on KBr and KCl
salt supplemented rice media afforded 2-bromogentisyl alcohol
(2), 2-chlorogentisyl alcohol (6),18,19 an epimeric mixture of
cis- and trans-bromohydrins (3a, 3b), and trans- and cis-
chlorohydrins (8a, 8b),19,20 together with the four known
compounds identified as 4, 5, 13, and 15. Herein, we report the
isolation, structure elucidation, and antiproliferative activity
evaluation of these compounds of P. concentricum and discuss
the in media synthesis of halogenated compounds in halogen
salt-rich media.
Hz, H-5), 6.50 (d, J = 2.7 Hz, H-4), and 6.51 (d, J = 2.7 Hz, H-
2) and one methyl group attached to an aromatic ring (δ 2.75,
s, 3H, CH3-10). Inspection of the 13C NMR and HSQC spectra
revealed the presence of signals for four aromatic methines (δ
94.8, C-5; 99.4, C-7; 102.2, C-4; 118.6, C-2), four oxygen-
bearing aromatic carbons (δ 164.8, C-8; 164.9, C-3; 161.2, C-
4a; 158.8, C-5a), four additional aromatic carbons (δ 144.2, C-
1; 111.7, C-1a; 103.4, C-6 and C-8a), and one polyconjugated
ketone carbonyl (δ 183.2) of a xanthone and suggested that
compound 1 was a 1,3,6,8-tetrasubstituted methylxanthone.
The presence of a hydroxy group at C-8 was deduced based on
observation of the downfield hydrogen-bonded 1H NMR
(DMSO-d6) signal at δ 14.1 ppm. The aromatic methyl group
was attached to C-1 due to a long-range correlation observed
from the methyl group at δ 2.75 to C-1, C-1a, C-2, C-4, C-4a,
and C-9. Moreover, the cross-peak from the doublet aromatic
proton at δ 6.51 ppm (H-2) to CH3-10 (δ 23.7), C-3 (δ 164.9),
C-4 (δ 102.2), and C-1a (δ 111.7) and the splitting pattern and
coupling constant of H-2 and H-4 (both d, J = 2.7 Hz)
confirmed the location of the hydroxy group at C-3. The
second set of meta-coupled aromatic methines at H-5 and H-7
were assigned based on the observation of HMBC cross-peaks
from the proton signal at δ 6.05 (H-7) to C-8, C-6 (δ 103.4),
and C-5. In addition, comparison of the 13C NMR
spectroscopic data of 1 with those of 3,6,8-trihydroxy-1-
methylxanthone (5) revealed that the downfield aromatic
carbon signal at C-6 in δ 165.4 was replaced by a chlorine-
bearing carbon signal at δ 103.4. From this evidence, the
structure of 1 was concluded to be 6-chloro-3,8-dihydroxy-1-
methylxanthone.
RESULTS AND DISCUSSION
■
Structure Elucidation of the New Compound 1
Isolated from P. concentricum Cultured on Rice Medium.
Silica gel column chromatography and HPLC on octadecyl silyl
silica gel (ODS) of the P. concentricum rice medium
fermentation extract led to the isolation of one new chlorinated
methylxanthone (1). Negative high-resolution electrospray
ionization (HRESI) mass analysis of compound 1 exhibited
two deprotonated molecular ion peaks at m/z 275.0119 [M −
H]− (100%) and m/z 277.0086 [M − H]− (32%), with the
ratio 3:1, indicating the presence of one chlorine atom in the
molecule and corresponding to a molecular formula of
−
C14H8ClO4 . The IR spectrum showed conjugated ketone
stretching at νmax 1616 cm−1 and absorption bands correspond-
ing to aromatic (νmax 1586, 1424 cm−1) and hydroxy (νmax 3402
cm−1) functions. Its UV maxima (λmax 240 and 310 nm) were
very similar to those of methylxanthones previously isolated
Structure Elucidation of Compounds Isolated from
P. concentricum Cultured on Halogen Salt-Supple-
mented Rice Medium. Bioactive halogenated secondary
metabolites such as griseofulvin, sclerotioramine (23),24 and
from Penicillium species.7,8,21−23 The H NMR spectroscopic
1
data of 1 displayed resonances due to two sets of meta-coupled
aromatic protons at δ 6.05 (d, J = 2.2 Hz, H-7), 6.15 (d, J = 2.2
B
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brominated. Therefore, the structure of 2 was deduced to be 2-
bromogentisyl alcohol.
Compounds 3a and 3b had the molecular formula C7H9BrO4
as determined by positive HRESIMS, which showed typical
brominated molecular ion peak patterns at m/z 258.9583
(100%) and m/z 260.9562 (97%), corresponding to [M + Na]
and [M + 2 + Na]+, respectively. Their structures were
elucidated to be 6-dehydroxy-6α-bromogabosine C (3a, major)
and 6-dehydroxy-6β-bromogabosine C (3b, minor) by analyses
of their 1D- (including 1H and TOCSY) and 2D-NMR
spectroscopic data together with comparison of the data of the
Figure 1. Key HMBC correlations observed in compound 1.
Figure 2. Key HMBC correlations observed in compound 2.
known chlorohydrins reported in the literature.19,20 The H
1
signals of the major compound (3a) was assigned by the
irradiation of the proton signal of H-6 (δ 4.33) in the 1D-
TOCSY experiment, which led to the identification of the spin
network H-3, H-4, H-5, and H-6. The corresponding carbon
signals were identified by HSQC experiment. The attachment
1
of the bromine group at C-6 was deduced by the H and 13C
bromoroquefortine C (24)25 have been reported from
Penicillium species. The isolation of the two chlorinated
compounds (1, 15) from the Penicillium fermented in rice
medium suggested that the present strain produces a
halogenase capable of introducing halogens in the secondary
metabolites. In the aim to produce more halogenated
compounds, we carried out fermentations in media using two
halogen salts (KBr and KCl, respectively). From the culture
containing KCl, we isolated the known chlorogentisyl alcohol
(6) together with an epimeric mixture of chlorohydrin
derivatives of epoxydon (8a, 8b). A mixture of two
bromohydrin derivatives, 6-dehydroxy-6α-bromogabosine C
(3a, major) and 6-dehydroxy-6β-bromogabosine C (3b,
minor), was isolated from KBr-supplemented medium along
with the new bromogentisyl alcohol (2). Interestingly,
griseofulvin was not produced when KBr was used (Figure
S21, Supporting Information). This suggested that KBr could
have inhibited the biosynthesis of 15. In addition, mass
spectroscopy analysis of the ethyl acetate extract obtained from
the KBr salt-supplemented fermentation did not show any
bromo derivatives of griseofulvin (data not shown).
chemical shift values of H-6 (δH 4.33) and C-6 (δC 49.9). The
HMBC correlation from the H-7 methylene group at δ 4.21
(dt, J = 2.69, 3.96 Hz, H-7) to the olefinic methine at δC143.0
(C-3), the carbonyl at C-1 (δC192.1), and the olefinic carbon at
C-2 (δC137.3) confirmed the proposed planar structure. The
assignment of the minor epimer was deduced by the same
manner. The alpha (trans) and beta (cis) orientations of the
bromine in 3a and 3b, respectively, were deduced from the
coupling constant values of the proton resonances at C-6. As
observed in the previously reported halohydrins,20 the trans
epimer (3a) displayed a wider coupling constant (4.9 Hz) than
the cis counterpart (2.2 Hz).
There is an increased interest in in culture halogenation of
microbial secondary metabolites. Recently, chlorinated epidi-
thiodiketopiperazine (25) has been isolated from a marine-
derived Trichoderma sp. TPU199 cultured in various concen-
trations of seawater media, while the brominated analogue (26)
was detected during the utilization of NaBr salt dissolved in
fresh water in the culture broth of TPU199.26 It is obvious that
the presence of the chemically reactive epoxide ring in the
structure of the parent compound (27) facilitated non-
enzymatic (pure chemical) halogenations. Interestingly, a
similar chlorination pattern has been observed in the
chlorinated epidithiodiketopiperazines penicisulfuranols A and
D produced by the mangrove endophytic fungus Penicillium
janthinellum HDN13-309 cultured in liquid medium containing
seawater. This suggested that an epoxide-bearing molecule (not
isolated during the study) could have facilitated the
chlorination of one of the epoxide-bearing carbons.27
Furthermore, Nabeta and co-workers reported that the
formation of chlorinated gentisyl alcohol is a nonenzymatic
process.19 Similar processes have been performed during the
present study to confirm the nonenzymatic production of 2, 3a,
3b, 8a, and 8b (Scheme S1, Supporting Information). Although
formation of halohydrins has been observed during the present
chemical conversion study of epoxydon, regeneration of 4 via
an intramolecular SN2 attack by the nonbonding electron pair
on the bromohydrin oxygen to the adjacent electrophilic
carbon has been observed (Scheme S2, Supporting Informa-
tion). Excess KBr in the solution prevented the regeneration of
epoxydon. Halogen-initiated epoxide ring opening has been
reported to be a stereoselective process that yields the
corresponding trans-halohydrin as the major product.28−30
During the present study, the favored trans chloro- and trans-
bromohydrins were detected as major stereoisomers of the
Negative HRESI-MS data of compound 2 displayed two
deprotonated molecular ion peaks at m/z 216.9503 [M − H]−
(100%) and 218.9482 [M − H]− (97%), corresponding to a
−
molecular formula of C7H6BrO3 . The IR spectrum exhibited
absorption bands characteristic for hydroxy groups (3389
cm−1) and an aromatic ring (1591, 1446, 1107, 1022 cm−1),
while the UV spectrum of 2 showed the presence of a
substituted aromatic ring. The 1H NMR spectrum of 2
exhibited signals due to two meta-coupled aromatic protons
at δ 6.75 (d, J = 2.9 Hz, H-5) and δ 6.82 (d, J = 2.9 Hz, H-3)
and an isolated oxygen-bearing methylene signal at δ 4.62 (s,
H-7ab). The 13C NMR spectrum displayed resonances due to
two aromatic methines at δ 115.2 (C-5) and 118.6 (C-3), four
aromatic carbons at δ 111.8 (C-2), 132.1 (C-6), 145.5 (C-1),
and 152.3 (C-4), and an oxygen-bearing methylene at δ 61.7
(C-7). The 1H and 13C NMR data of 2 are very similar to those
of gentisyl alcohol (9) except for the presence of an AB
aromatic system instead of the ABX system in 9. The
hydroxymethylene, bromine, and the two hydroxylated carbons
were assigned to be at C-6, C-2, C-1, and C-4, respectively, due
to the HMBC long-range correlations from the aromatic
methine proton at δ 6.75 (H-5) to C-1, C-3, C-4, and C-7
together with the cross-peaks from δ 6.82 (H-3) to C-1, C-2, C-
4, and C-5. Moreover, the upfield shift of the chemical shift of
the aromatic carbon at δ 111.8 (C-2) confirmed that C-2 was
C
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Chart 2
Table 1. NMR Spectroscopic Data for Compounds 1 and Norlichexanthone (5)
a
b
1
5
position
δC, type
δH
(J in Hz)
HMBC
δC, type
δH
(J in Hz)
1
144.2, C
111.6, C
118.6, CH
164.9, C
102.2, CH
161.2, C
94.8, CH
158.8, C
103.4, C
99.4, CH
164.8, C
103.4, C
183.2, C
23.7, CH3
144.8
112.6
117.6
164.2
101.8
161.0
94.5
1a
2
6.51
6.50
6.15
d (2.7)
d (2.7)
d (2.2)
1a, 4, 4a, 9, 10
1a, 2, 3, 4a, 9, 10
5a, 7, 8, 8a, 9
6.59
6.59
6.22
br
3
4
br
4a
5
d (2.0)
5a
158.7
165.4
99.1
6
7
6.05
d (2.2)
5, 6, 8
6.11
d (2.0)
8
164.9
103.9
183.5
23.7
8a
9
10-Me
2.75
s
1, 1a, 2, 4, 4a, 9
2.77
s
a
b
Measured in methanol-d4 (1H: 700 MHz and 13C: 175 MHz). Measured in acetone-d6 (1H: 500 MHz and 13C: 125 MHz).
chemical conversion, confirming the reported favored non-
enzymatic reaction.30 Similarly, abiotic synthesis of anthrasesa-
mone C (28) from its epoxide-containing precursor 2,3-
epoxyanthrasesamone B (29) via a nucleophilic addition
reaction of chloride anion has been previously studied by
Furumoto and Hoshikuma.31 This pathway was in agreement
with the previously reported nonenzymatic synthesis of
chlorogentisyl alcohol from epoxydon using chloride ion-rich
media and the present results on brominated gentisyl alcohol
(2).19
Halogen salts have been widely used in the fermentation of
microorganisms in order to obtain halogenated compounds. It
is worth noting that attention should be paid when the strain
has been known to produce epoxide-containing compounds or
the halogen in the isolated metabolite is alpha-attached to an
oxygen-bearing carbon. All of the halogen groups present in the
compounds isolated during the halogen incorporation26,27 are
attached at one of the carbons that bears the epoxide.
Most of the isolated compounds in this study (except 7, 10,
and 14) have been evaluated for cytotoxicity against HT-29
(colon) and/or MCF-7 (breast) cancer cell lines (Table 3).
Among the compounds tested, epoxydon (4) was the most
active compound against MCF-7 cells (IC50 5.7 μM), while
gentisyl alcohol (9) showed moderate and weak antiprolifer-
ative activities against HT-29 and MCF-7, with IC50 values of
6.4 and 17.1 μM, respectively. Brominated gentisyl alcohol (2)
Table 2. NMR Spectroscopic Data for Compound 2
position
δC, type
δH
(J in Hz)
HMBC
1
2
3
4
5
6
7
145.5, C
111.8, C
118.6, CH
152.3, C
115.2, CH
132.1, C
61.7, CH2
6.82
6.75
4.62
d (2.9)
d (2.9)
s
1, 2, 4, 5
1, 3, 4, 7
1, 5, 6
a
Measured in methanol-d4 (1H: 400 MHz and 13C: 100 MHz).
was more active against the MCF-7 cell line (IC50 8.4 μM) than
its chlorine counterpart (6, IC50 14.9 μM).
EXPERIMENTAL SECTION
■
1
General Experimental Procedures. H and 13C NMR spectra
were recorded at 25 °C with a Bruker Avance III 400 HD NMR
spectrometer and Bruker Avance III HD Ascend 700 MHz. High-
resolution mass spectra were acquired with a Thermo LTQ Orbitrap
(analyzer: ITMS and FTMS, mass range: 50−4000 m/z, resolution:
7500−100 000). High-performance liquid chromatography was
performed with a Hitachi Primaide HPLC (Hitachi High-Technolo-
gies Corporation, Tokyo, Japan) equipped with a Primaide 1430 diode
array detector, Primaide 1210 autosampler, and Primaide 1110 pump
with a degasser and a semipreparative C18 column (Dynamax C18
HPLC column 10 × 250 mm). A Shimadzu HPLC system with an LC-
D
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The fungal culture was then soaked with 1 L of EtOAc and left for 1
day at room temperature. The procedure was repeated three times,
yielding about 3 L of EtOAc extract. The resulting EtOAc extract was
evaporated under vacuum using a rotary evaporator to yield a
brownish residue (3.5 g). A portion of the concentrated extract was
suspended in methanol and partitioned with hexane (3 × 200 mL) to
afford 1.6 g of hexanes and 589 mg of methanol fractions. The
methanol fraction exhibited an ED50 value of 7 μg/mL against the
human breast adenocarcinoma cell line (MCF-7). This bioactive
MeOH fraction was subsequently fractionated on reversed-phase silica
gel liquid chromatography and eluted with 70% aqueous methanol (2
× 150 mL) and MeOH (2 × 150 mL) to yield four fractions, F-1
(360.85 mg), F-2 (31.71 mg), F-3 (56.41 mg), and F-4 (7.47 mg), of
which two fractions (F-2 and F-3) were active against MCF-7 cells
with ED50 values of 17.5 and 15.1 μg/mL, respectively. F-1 was
subjected to silica gel column chromatography [2.2 cm × 28 cm;
solvent system: hexane−EtOAc (3:2)] to give seven subfractions (F-1a
through F-1g). Cytotoxicity evaluation of these fractions revealed that
F-1c and F-1g were active, with ED50 values of 11 and 12.1 μg/mL
against MCF-7 cells, respectively. Fraction F-1c (43.63 mg) was
subjected to a semipreparative RP-18 HPLC on a Dynamax C18 HPLC
column (10 × 250 mm; flow rate: 2 mL/min) with a gradient mixture
of H2O−MeOH from 60:40 to 0:100 for 42 min to give compounds 1
(1.8 mg), 17 (2.4 mg), 20 (6.8 mg), and 21 (4.9 mg). HPLC using an
octadecyl silyl silica gel column (ADV5013 C18, 5 μm 4.6 × 250 cm;
flow rate: 1 mL/min) with a H2O−MeOH gradient (from 90:10 to
0:100 for 35 min) of the bioactive fraction F-1f (ED50 15.4 μg/mL,
MCF-7) led to the isolation of gentisyl alcohol (9) (3.8 mg), gentisyl
quinone (10) (0.8 mg), and (R,S)-1-phenyl-1,2-ethanediol (11) (2.2
mg). Silica gel column chromatography of F-1g [182.8 mg, column
size: 1.5 cm × 25 cm; solvent system: EtOAc−hexane (3:2)] yielded
five fractions (F-1g-I to F-1g-V). HPLC analysis of the five fractions
showed that fraction F-1g-III contained four compounds (12−15),
identified as dehydro-dechlorogriseofulvin (12) (2.6 mg), dechlor-
ogriseofulvin (13) (4.1 mg), dehydrogriseofulvin (14), and
griseofulvin (15). Compounds 14 and 15 were obtained as a mixture
(6.8 mg, 14/15 in 1:4 ratio) exhibiting an IC50 value of 4.9 μg/mL
against MCF-7 cells. To get some idea about the activity of
dehydrogriseofulvin (14), the standard griseofulvin (15) was tested
against the two cell lines. HPLC of fraction F-2 yielded compound 5
(norlichexanthone, 4.4 mg) and 16 (ethylene glycol benzoate, 3.7 mg).
Fraction F-3 was subjected to semipreparative RP-18 HPLC
(Dynamax C18 HPLC column 10 × 250 mm; flow rate: 2 mL/
min) using a H2O−MeOH gradient (from 50:50 to 0:100 for 40 min)
to afford drimiopsin H (19, 8.4 mg). HPLC on RP C18 of the hexanes-
soluble fraction gave compounds 4 (0.7 mg), 5 (1.7 mg), 9 (0.9 mg),
12 (2.3 mg), 13 (1.2 mg), 14, 15 (7.2 mg), 19 (1.9 mg), 20 (1.3 mg),
and 21 (1.1 mg).
Table 3. Cytotoxicity Data of the Compounds Isolated from
Penicillium concentricum on Human Breast (MCF-7) and
Colon (HT-29) Cancer Cell Lines
a
a
compound
MCF-7
HT-29
b
1
>20
8.4
NT
2
NT
NT
14.1
>20
NT
NT
NT
6.4
3
9.7
4
5.7
5
>20
14.9
NT
>20
17.1
NT
>20
>20
>20
NT
13.9
>20
>20
>20
>20
>20
>20
0.073
NT
6
7
8
9
10
NT
>20
>20
>20
NT
>20
>20
>20
>20
>20
>20
>20
NT
11
12
13
14
15
16
17
18
19
20
21
camptotecin
paclitaxel
0.0014
a
b
IC50 in μM. NT: Not tested.
10AD vp oump, an SCL-10A vp system controller, an SPD- M10A vp
UV/vis detector, and a DGU-14A degasser were used.
Fungal Source. The endophytic fungus (strain 4E-4) was isolated
from the healthy liverwort Trichocolea tomentella collected in Newport,
Virginia (N 37.27279; W 80.52425). Leaves of T. tomentella were
washed with running tap water and investigated microscopically for
surface damage and disease symptoms. Ten healthy leaves were
selected for surface disinfection by stepwise soaking in 2% sodium
hypochlorite solution for 1 min, followed by 70% ethanol for 30 s, and
two rinses with sterile distilled water. To confirm successful surface
disinfection, disinfected leaves were pressed onto PDA plates, the final
rinse was plated onto the same medium, and the plates were examined
for growth after incubation at room temperature for 3, 7, and 14 days.
Surface-disinfected leaves were cut into small pieces (about 1 mm × 1
mm) with a sterilized blade and placed on PDA plates. Incubation was
carried out at room temperature for 3 to 14 days to allow growth of
microbial endophytes. Penicllium (which appeared on the seventh day
of incubation) was purified by streaking single colonies onto PDA agar.
The fungus was identified by morphological inspection (data not
shown) and sequencing of the ITS regions of the rRNA locus. The
ITS sequences of the liverwort-derived fungus showed 100% identity
to that of P. concentricum (GenBank accession KM023345.1 and
DQ339561.1) and P. coprophilium (GenBank accession AJ608952.1).
The isolated fungus also showed high similarity to P. griseofulvum
(99% identity to GenBank accession KR703615.1) and P. chrysogenum
(99% identity to GenBank accession KU925907.1). A voucher
specimen (4E-4) was deposited in the Division of Medicinal
Chemistry and Pharmacognosy of the College of Pharmacy, OSU.
Fermentation, Extraction, and Isolation of Secondary
Metabolites from Rice Culture Medium. P. concentricum was
first cultured on PDA medium for 5 days at room temperature, and
then two 0.5 × 0.5 cm of agar containing fungal hyphae were added to
a sterilized 500 mL Erlenmeyer flask containing potato dextrose broth
for 3 days in a shaker (150 rpm) at room temperature. One milliliter of
the fungal culture was then seeded to a 500 mL Erlenmeyer flask
containing serilized rice medium. The cultivation was left static at
room temperature for another 14 days and stopped by adding EtOAc.
6-Chloro-3,8-dihydroxy-1-methylxanthone (1): yellow, amor-
phous semisolid; UV (MeOH) λmax (log ε) 242 (4.26), 310 (3.97)
1
nm; IR (film) νmax 3402, 1616, 1586, 1424 cm−1; H and 13C NMR
data, see Table 1; HRESIMS m/z 275.0119 and 277.0086 (calcd for
−
C14H8ClO4 [M − H]−, 275.0116 and 277.0087).
Fermentation, Extraction, and Isolation of Chlorinated
Compounds from KCl-Supplemented Rice Culture Medium.
The production of chlorinated compounds and other secondary
metabolites was accomplished by incubation of two 0.5 × 0.5 cm of
agar containing P. concentricum hyphae in a 500 mL Erlenmeyer flasks
containing rice medium (250 mL) and KCl at rt for 14 days. The
whole rice culture of the fungal strain was extracted three times with
EtOAc, and the solution was evaporated under reduced pressure. The
obtained crude ethyl acetate extract (2.1 g) was subjected to HPLC
analysis using a Dynamax C18 HPLC column (10 × 250 mm; flow
rate: 2 mL/min) with a gradient of H2O−MeOH (from 95:5 to 0:100
for 38 min) as eluting system to afford 4 (0.9 mg), 5 (2.1 mg), 6 (3.2
mg), 8a and 8b mixture (1.2 mg), 13 (1.9 mg), and 15 (4.9 mg)
(Figure S19).
Fermentation, Extraction, and Isolation of Brominated
Compounds from KBr-Supplemented Rice Culture Medium.
The fermentation of the fungal strain with KBr was carried out in a
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similar manner to the fermentation described with KCl. The
fermented rice material with KBr was extracted repeatedly with
EtOAc, and the organic solvent was evaporated to dryness under
vacuum to afford the crude extract (1.7 g). The extract was separated
by semipreparative C18 reversed-phase HPLC with H2O−MeOH
gradient (from 95:5 to 0:100 for 38 min) to yield four compounds,
determined as: 2 (5.6 mg), 3a and 3b mixture (3.8 mg), 4 (1.1 mg),
and 13 (0.8 mg) (Figure S21).
authors (L.H.R) and Grant IRG-67-003-50 from the American
Cancer Society. We also wish to thank the Libyan government
for providing a scholarship to T. Ali. We thank The Ohio State
University, College of Pharmacy, instrumentation facility and
the Campus Chemical Instrument Center (CCIC, OSU) for
the acquisition of the NMR and mass spectra.
2-Bromogentisyl alcohol (2): brown, amorphous solid powder; UV
(MeOH) λmax (log ε) 298 (3.42) nm; IR (film) νmax 3389, 1591, 1446,
1107, 1022 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z
216.9503 and 218.9482 (calcd for C7H6BrO3−, 216.9505 and
218.9485).
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AUTHOR INFORMATION
Corresponding Author
*Tel: +1-614-292-4733. Fax: +1-614-292-4733. E-mail:
Rakotondraibe.1@osu.edu.
■
ORCID
L. Harinantenaina Rakotondraibe: 0000-0003-2166-4992
Notes
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ACKNOWLEDGMENTS
This work was partially supported by The Ohio State
Comprehensive Cancer Center Seed Grant to one of the
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