(+)-Altholactone exhibits broad spectrum immune modulating activity by inhibiting the activation of pro-inflammatory cytokines in RAW 264.7 cell lines

Article abstract of DOI:10.1016/j.bmc.2013.04.055

An evaluation of Indonesian plants to identify compounds with immune modulating activity revealed that the methanolic extract of an Alphonsea javanica Scheff specimen possessed selective anti-inflammatory activity in a nuclear factor-kappa B (NF-κB) lucif

Full text of DOI:10.1016/j.bmc.2013.04.055

Bioorganic & Medicinal Chemistry 21 (2013) 4358–4364
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
(+)-Altholactone exhibits broad spectrum immune modulating
activity by inhibiting the activation of pro-inflammatory cytokines
in RAW 264.7 cell lines
Tyler A. Johnson a,b, , Johann Sohn a, , Aidan E. Ward b, Tanya L. Cohen b, Nicholas D. Lorig-Roach b,
Haixia Chen a, Ronaldo A. Pilli c, Elizabeth A. Widjaja d, Muhammad Hanafi e, Leonardus B. S. Kardono e,
Puspa D. Lotulung e, Kyria Boundy-Mills f, Leonard F. Bjeldanes a,
⇑
a Department of Nutritional Sciences & Toxicology, University of California, Berkeley, CA 94720, USA
b Department of Chemistry & Biochemistry, University of California, Santa Cruz, CA 95064, USA
c Departamento de Quimica Organica, Instituto de Quimica, UNICAMP, CP 6154, 13083-970 Campinas, SP, Brazil
d Research Center for Biology, Indonesian Institute of Science (LIPI), Cibinong 16911, Indonesia
e Research Center for Chemistry, Indonesian Institute of Science (LIPI), Serpong, Tangerang 15310, Indonesia
f Phaff Yeast Culture Collection, Food Science and Technology, University of California Davis, Davis, CA 95616, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An evaluation of Indonesian plants to identify compounds with immune modulating activity revealed
Received 21 February 2013
Revised 12 April 2013
Accepted 22 April 2013
Available online 28 April 2013
that the methanolic extract of an Alphonsea javanica Scheff specimen possessed selective anti-inflamma-
tory activity in a nuclear factor-kappa B (NF-
immune (Raw264.7) cells. A high-throughput LC/MS-ELSD based library approach of the extract in com-
bination with the NF- B and MTT assays revealed the styryl lactone (+)-altholactone (2) was responsible
jB) luciferase and MTT assay using transfected macrophage
j
for the activity. Compound 2, its acetylated derivate (+)-3-O-acetylaltholactone (3), and the major com-
pound of this class, (+)-goniothalmin (1), were further evaluated to determine their anti-inflammatory
Keywords:
Styryl lactones
(+)-Altholactone
(+)-Goniothalamin
Anti-inflammatory
NF-kB
qPCR
NO
IjBa
iNOS
COX-2
IP-10
IL-1b
MCP-1
GCS-F
IL-6
potential in the NF-
NF- B based anti-inflammatory activity. Compound 2 reduced the LPS-induced NO production, phos-
phorylation of I , and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-
2 (COX-2) using Western blot analysis. Further studies using qPCR indicated 2 reduced the expression
of eight pro-inflammatory cytokines/enzymes (0.8–5.0 M) which included: COX-2, iNOS, IP-10, IL-1b,
MCP-1, GCS-F, IL-6 and IFN-b. These results indicated that 2 displays broad spectrum immune modulat-
ing activity by functioning as an anti-inflammatory agent against LPS-induced NF- B signaling. Con-
versely the selective cytotoxicity and in vivo anti-tumor and anti-inflammatory activity previously
jB assay. Concentration–response studies of 1–3 indicated that only 2 possessed
j
jBa
l
j
reported for 1 do not appear to arise from a mechanism that is linked to the NF-jB immune mediated
pathway.
Ó 2013 Published by Elsevier Ltd.
IFN-b
1. Introduction
This has motivated a number of research groups to engage in the
total synthesis of this class of compounds.5 The majority of the re-
search involving styryl lactones has revolved around the structure
of (+)-goniothalmin (1) with over 100 derivatives reported in the
primary literature isolated as natural products or derived from
synthetic or semi-synthetic experiments.1–6 Compound 1 has been
reported to induce selective cytotoxicity to a variety of cancer cell
lines,1,4,6,7 and has been repeatedly isolated from the genus Gonio-
thalamus which has a long history of use in folk medicine through-
out South East Asia.2,8 The extracts and leaves of Annonaceae
plants have also traditionally been used for the treatment of
rheumatism and edema.5 Recent work has shown that racemic 1
Styryl lactones are an important group of plant derived natural
products from the family Annonaceae that exhibit a wide variety of
biological activities. Selected examples, particularly from the genus
Goniothalamus, include selectivity to cancer cell lines and against
neglected tropical diseases (Chagas), as well as antifungal, antimi-
crobial, anti-inflammatory and immunosuppressive activity.1–4
⇑
Corresponding author. Tel.: +1 510 642 1601.
E-mail address: lbjel@berkeley.edu (L.F. Bjeldanes).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.04.055

T. A. Johnson et al. / Bioorg. Med. Chem. 21 (2013) 4358–4364
4359
tion-dependent evaluations of the styryl lactones 1–3, using our
NF- B and MTT assays, to investigate their anti-inflammatory and
cytotoxic activities side by side. Our results indicated that only 2
deserved further investigation to: (a) confirm its anti-inflammatory
activity by nitrite production and Western blotting analysis, (b)
demonstrates encouraging antiproliferative activity in vivo using
the Ehrlich solid tumor experimental model in mice with no re-
ported toxicity after single or repeated doses.9 A mechanism for
the observed antiproliferative activity may be due to its ability to
serve as an inhibitor of nucleo-cytoplasmic transport via blocking
of CRM-1 mediated nuclear export.4 Anti-edematogenic activity
has also been observed using racemic 1 in a carrageenan edema
model in vivo, which suggested a relationship between the anti-tu-
mor and anti-inflammatory activities with the anti-inflammatory
activity favoring the antiproliferative activity.9 These results indi-
cate further in vivo evaluations of 1 are warranted as a preclinical
lead for the treatment of cancer and that its apparent anti-inflam-
matory activity could also be responsible for its anti-tumor activ-
ity. The immune modulating effects of 1 have been linked to its
ability to inhibit the expression of both intercellular adhesion mol-
ecule and vascular adhesion molecule-1 (ICAM and VCAM-1) on
the surface of murine endothelial cells (F-2).10 However, the con-
j
evaluate its activity by qPCR to probe its target(s) in the NF-jB
pathway, and (c) examine its selective cytotoxicity against breast
and prostate cancer cell lines. The results of our experiments out-
lined above are reported here in.
2. Results and discussion
To rapidly identify the metabolite(s) responsible for the ob-
served anti-inflammatory activity from the A. javanica extract (coll.
no. UHA 105 M), we used our previously reported high throughput
LC–MS/ELSD based peak-library approach in conjunction with an
NF-
and indicated three fractions (H27, H28 and H31), obtained from
the crude extract, displayed potency in the NF- B luciferase assay.
j
B luciferase and MTT assay.21 This data set is shown in Figure 1
centration reported for activity of 70
lM does not suggest it con-
tains potent anti-inflammatory activity.
j
Several studies have also reported the selective in vitro cytotox-
icty of (+)-altholactone (2) and its derivatives against a variety of
human tumor cell lines.11–13 Mechanistic research has shown that
2 may function to inhibit human HL-60 or CEM leukemia cells by
inducing apoptosis through either oxidative stress, based on its
ability to serve as a mitochondria complex I inhibitor14 or syner-
gism with tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL).13 Also noteworthy is that acetylated 2, (+)-3-O-acetylal-
tholactone (3), was reported to have five fold greater NADH oxi-
dase inhibitory activity compared to 2.15 The above reports
suggested that evaluating 1–3 for the first time in the nuclear fac-
Also noteworthy was that each of these fractions showed weak
cytotoxicity to macrophage (RAW 264.7) immune cells in the
MTT assay (data not shown) indicating selectivity. Analytical LC-
MS/ELSD data indicated these fractions possessed m/z ions which
corresponded to a pure major metabolite H27 (233 m/z), and what
turned out to be an inseparable mixture of metabolites in H28
(233, 282 and 235 m/z) and H31 (205, 249, 233 and 367 m/z) as
seen in Figures S1–S3 in the Supplementary data. Scale up reverse
phase HPLC of the parent methanol extract (100.0 mg) was needed
to provide greater quantities of the major metabolite and minor
components for further structural and bioassay analysis. Fraction
H27 (13.2 mg) contained pure (+)-altholactone (2) (syn. goniot-
halenol)2,23 and was de-replicated based on the molecular formula
of C13H12O4Na [M+Na]+ established from HRESITOFMS along with
1H and 13C NMR data (see Figs. S4–S5) that matched previous
reports.23
tor-kappa B (NF-
anti-inflammatory activities could prove noteworthy because dys-
regulation of NF- B has been implicated in many inflammatory
jB) mediated immune pathway to examine their
j
diseases including rheumatoid arthritis and cancer.16–18 Our
hypothesis was that an assessment of their immune modulating
activity could provide insights as to whether their selective cyto-
toxicity to cancer cell lines was in part due to their anti-inflamma-
tory activity based on the close links observed between
inflammation and tumorigenesis.19,20 We have recently observed
this occurrence from our work with the bengamides, where we dis-
Based on the distinct anti-inflammatory activity we observed
for the fraction containing (+)-altholactone (2), we decided to
investigate more thoroughly its effects on key targets in the im-
mune pathway, including NF-jB signaling, nitrite production,
and expression of a series of pro-inflammatory cytokines. It also
seemed logical to obtain or generate and further evaluate the
anti-inflammatory activity of (+)-goniothalamin (1) and (+)-3-O-
acetylaltholactone (3), versus 2 due to the wide variety of biologi-
cal activities observed for 11 and the fact that 3 was reported to
possess NADH oxidase inhibitory activity five times greater than
2.15 The first step involved determining the IC50 inhibitory values
observed for 1–3 shown in Table 1, on lipopolysacharide (LPS)
covered their potent anti-inflammatory activity involving NF-jB
appears to be responsible at least partially for their significant
anti-cancer activity.21
mediated induction of the NF-
with their cytotoxicity in RAW 264.7 macrophage immune cells
in the NF-
B luciferase and MTT assays.21 Compound 1 displayed
moderate NF- B inhibitory activity (IC50 = 27.4 M) with compara-
ble cytotoxicity in the MTT assay (IC50 = 34.2 M). Alternatively,
(+)-altholactone (2), exhibited selective NF- B inhibitory activity
(IC50 = 6.7 M) with weak cytotoxicity in the MTT assay
(IC50 = 43.4 M). Further analysis indicated compound also
inhibited nitrite production (IC50 = 0.8 M). Interestingly, com-
pound exhibited much weaker NF- inhibitory activity
(IC50 = 34.3 M) versus 2 and had comparable cytotoxicity in the
MTT assay (IC50 = 36.1 M). Overall, these data indicated that only
2 possessed potential anti-inflammatory activity in our assays and
was on par with other immune modulating plant-derived natural
products reported by others.24
The next step required using Western blotting analysis, shown
in Figure 2, to measure the effects of (+)-altholactone (2) in the
jB reporter gene when compared
(+)-goniothalamin (1)
(+)-altholactone R = OH (2)
j
(+)-3-O-acetylaltholactone (3) R = OAc
j
l
l
j
During the course of our International Cooperative Biodiversity
Group (ICBG) collaboration to screen 114 Indonesian plants for their
immune modulating and cytotoxic activity against tumorigenic and
nontumorigenic cell lines,22 we identified an extract from the fam-
ily Annonaceae (Alphonsea javanica Scheff, coll. no. UHA 105) that
had noticeable anti-inflammatory activity. Using an LC/MS-ELSD
based library approach on the extract in combination with our
l
l
2
l
3
jB
l
l
NF-jB luciferase and MTT cytotoxicity assays to define anti-inflam-
matory leads from plants,18 we discovered that (+)-altholactone (2)
was responsible for the activity. A perusal of the styryl lactone liter-
ature indicated that our discovery was the first example to report 2
from specimens of A. javanica.1–4 We then conducted concentra-
presence of LPS, on the phosphorylation of IjBa, and the expres-

4360
T. A. Johnson et al. / Bioorg. Med. Chem. 21 (2013) 4358–4364
Figure 1. Observed LC–MS/ELSD library trace of coll. no. UHA 105 M including: (a) UV data at 254 nm; (b) ELSD data with selected annotations including m/z ions and (c) NF-
B luciferase assay data stimulated by the LPS for the standard celastrol (250 nM), the MeOH crude extract (20 g/mL).
g/mL) and LC library fractions (H5-H48, ꢀ10
j
l
l
Table 1
Comparative IC50 values of compounds 1–3
Compound
Raw 264.7a
IC50 (lM)
NF-kBb
NOc
1
2
3
34.2
43.4
36.1
27.4
6.7
34.3
nt
0.8
nt
a
Cytotoxicity measured against murine macrophage (RAW 264.7) cell lines
ꢁ18 h.
b
NF-kB inhibition measured using an LPS-induced NF-kB luciferase assay with
RAW 264.7 cells ꢁ18 h.
Figure 2. Western blot analysis of (+)-altholactone (2) at varying concentrations
and its inhibition of LPS-induced phosphorylation of IjBa, and expression of iNOS
and COX-2 with b actin as a control.
c
Nitric oxide (NO) production. Not tested (nt). The values (IC50) represent the
mean SE for n = 3.
sion of inducible nitric oxide synthase (iNOS) and cyclooxygenase-
2 (COX-2) compared to b-actin as a positive control. With increas-
inflammatory cytokines could provide insights into the upstream
targets responsible for this observed anti-inflammatory activity.
In an effort to locate the specific immune modulating target(s)
of (+)-altholactone (2) we examined its inhibition of LPS-induced
mRNA expression of eight pro-inflammatory cytokines/enzymes
measured using qPCR analysis and these results are shown in Fig-
ure 3. The inhibition of 2 on the expression of iNOS and COX-2
shown using Western blotting in Figure 2 was further substanti-
ated by its ability to inhibit the mRNA expression of these pro-
inflammatory cytokines as shown in Figure 3a (iNOS,
ing concentrations of 2 (1.5–12.0
the observed phosphorylation of IjBa
l
M), there is a clear decrease in
, which has been established
B mediated pathway.25
as a true indicator of inhibition of the NF-
There was also a noticeable decrease in the expression of iNOS and
COX-2 at 5.0 M. This data set indicated that 2 exhibits potential
immune modulating activity by inhibiting several key downstream
targets in the immune pathway (NF- B, iNOS, COX-2) and that fur-
ther investigations of 2 using qPCR analysis on a series of pro-
j
l
j

T. A. Johnson et al. / Bioorg. Med. Chem. 21 (2013) 4358–4364
4361
Figure 3. Effects of (+)-altholactone (2) on the inhibition of LPS-induced mRNA expression of pro-inflammatory cytokines and enzymes measured by qPCR including: (a)
inducible nitrous oxide synthase (iNOS); (b) cyclo-oxygenase-2 (COX-2); (c) interferon gamma-induced protein 10 (IP-10); (d) interleukin-1 beta (IL-1b); (e) monocyte
chemotactic protein-1 (MCP-1); (f) granulocyte colony-stimulating factor (G-CSF); (g) interleukin-6 (IL-6) and (h) interferon beta (IFN-b). LPS (100 ng/mL) is used as a positive
control and added to (0.7–12.5 l
M) samples. Data are presented as mean SD of three independent experiments. ⁄⁄p <0.05, ⁄⁄⁄p <0.001.

4362
T. A. Johnson et al. / Bioorg. Med. Chem. 21 (2013) 4358–4364
IC50 = 2.0
l
M) and Figure 3b (COX-2, IC50 = 3.0
l
M). Similar results
detector (ELSD), and an Applied Biosystems Mariner electrospray
ionization time of flight (ESI-TOF) mass spectrometer. Optical rota-
tion was performed on a Jasco P-2000 Polarimeter. All NMR exper-
iments were run on a Varian Unity spectrometer (500 and
125 MHz for 1H and 13C, respectively).
were observed for 2 in Figures 3c–f regarding its inhibition of inter-
feron gamma-induced protein 10 (IP-10, IC50 = 5.0 M), interleu-
kin-1 beta (IL-1b, IC50 = 3.0 M), monocyte chemotactic protein-1
(MCP-1, IC50 = 4.0 M) and granulocyte colony-stimulating factor
(G-CSF, IC50 = 4.0 M). The greatest potency was observed against
interleukin-6 (IL-6, IC50 = 0.9 M) and interferon beta (IFN-b,
IC50 = 0.8 M). Although we were unable to determine a specific
l
l
l
l
l
3.2. Biological material, collection and identification
l
target fully responsible for the immune modulating activity, our
results indicate 2 functions as a broad spectrum anti-inflammatory
Specimens of the Alphonsea javanica Scheff. (coll. no. UHA 105)
were collected at 978 m (S 03.63951, E 121.15631) from the Me-
kongga mountainous regions South East Sulawesi province,
Indonesia.
agent by inhibiting multiple targets in the NF-
ated pathway.
jB immune medi-
Based on the selective anti-inflammatory activity we observed
for (+)-altholactone (2), and previous reports indicating that it dis-
plays cytotoxcity to several tumor cell lines,1,11–13 we decided to
evaluate its cytotoxicity against both non-tumorigenic breast
(MCF-10A) and tumorigenic breast (MCF-7, MDA-MB-231) and
prostate (PC3, LNCaP) cell lines. Our screening results of 2 against
these cell lines are shown in Figure S6 in the Supplementary data.
3.3. Extraction, LC–MS/ELSD library fraction collection and
isolation
Specimens of A. javanica (coll. no. UHA 105) were dried
(200.0 g) and extracted first using methanol and subsequently with
hexanes to generate 7.40 and 1.57 grams of oil, respectively. A por-
tion of the methanol extract (187.3 mg) and hexanes extract
(193.6 mg) was provided to UC Berkeley to include in a 96-well
plate crude extract library that was initially screened in the NF-
No inhibitory effects were seen at concentrations up to 100 lM
against any of the cell lines and these results are consistent with
one report that 2 does not display potency against tumorigenic
breast cancer cell lines.13 Alternatively, 2 has shown between six
and 10-fold selectivity to human colorectal colon (Sw480), lung
carcinoma (COR-L23) and leukemic T-lymphocytes (CEM) cells ver-
sus human non-tumorigenic (HT1080, MRC-5, and MCF-12) cell
lines.11–13 Its structural analogs, including 3, have also reportedly
jB luciferase and MTT assays. The active methanol extract (sample
coded UHA 105 M) was further prepared as a [15.0 mg/150 L]
l
sample in methanol to undergo a standard LC–MS/ELSD library
for bioassay to pinpoint the active component(s) using methods re-
ported previously.22 Gradient conditions involved using
H2O:CH3CN (with 0.1% formic acid) consisting of 10:90 ? 100%
exhibited low micromolar activity (2.0–20.0 lM) against mouse
leukemia (P-388, L1210), human lung (A549), colon (HT-29), epi-
dermoid carcinoma (KB) and (COR-L23) cancer cell lines.1,12
In summary our results indicate that (+)-altholactone (2) exhib-
its broad spectrum immune modulating activity and functions as
an anti-inflammatory agent by inhibiting a wide variety of targets
over 30 min at 2 mL/min into a 5 l RP-HPLC C-18 column—
250 ꢂ 10 mm (Phenomenex, Inc., Torrance, CA) that was then frac-
tionated into a 96-well plate. The well fractions were monitored
using a Waters model 996 photodiode array (PDA) UV detector.
The elution was subsequently split (1:1) between a S.E.D.E.R.E.
model 55 evaporative light scattering detector (ELSD), and an Ap-
plied Biosystems Mariner electrospray ionization time of flight
(ESI-TOF) mass spectrometer. A duplicate 96-well plate library
was then made using a 12-channel pipette creating an exact copy
and counter balance for analytical reference and centrifugal drying.
Plates were dried and concentrated using a Savant AES2010 Speed-
Vac. Scale up RP-HPLC of (+)-altholactone (2) involved repeated
automated injections using the same conditions reported for the
LC–MS/ELSD library with fractions instead being partitioned into
20 mL scintillation vials. The vials were dried and concentrated
using a Savant AES2010 SpeedVac. A total of 100.0 mg of UHA
in the NF-jB immune mediated pathway. Compound 2 was not
cytotoxic against tumorigenic breast and prostate cancer cell lines
in our assays however its selective cytotoxicty against other cell
lines11–13 may arise from its anti-inflammatory activity due to
the close link that exists between inflammation and tumorigene-
sis.19,20 NF-
jB has been shown to be a key protein favoring cell
proliferation and inhibiting apoptosis in various cell types.26 To
the best of our knowledge this is the first report describing 2 or
any of the other hundreds of reported sytryl lactones as demon-
strating definitive NF-jB based immune modulating activity. Alter-
natively, (+)-goniothalmin (1), the parent styryl lactone in this
class, along with the acetylated derivative of 2 (+)-3-O-acetylaltho-
lactone (3), do not exhibit NF-kB based anti-inflammatory activity.
These results suggest the modes of action of 1 and 3 involving their
cytotoxicity against tumor cell lines appear to arise from mecha-
105 M was used to make ten injections ([10.0 mg/150 lL]) that
were purified using the aforementioned conditions which resulted
in 48 fractions. The HPLC fraction H27 (13.2 mg) contained pure
(+)-altholactone (2). Compound 2 was used to provide (+)-3-O-
acetylaltholactone (3) by semi-synthesis. A sample of 2 (5.0 mg,
nisms independent of targets in the NF-
pathway.
jB immune mediated
isolated from A. javanica) was dissolved in dry pyridine (300
lL)
and acetic anyhydride (1000 L). The mixture was stirred for
l
24 h and the reaction was quenched with water and extracted with
CH2Cl2. The organic layer was then dried with NaSO4 and yielded
pure (+)-3-O-acetylaltholactone (3, 4.2 mg). (+)-Goniothalaman
(1) was prepared using synthetic methods described previously.1
3. Experimental section
3.1. General experimental procedures
Analytical LC–MS analysis was performed on samples at a con-
centration of approximately 5 mg/mL, using a reversed-phase
3.4. NF-jB luciferase assay
150 ꢂ 4.60 mm 5
lm C18 Phenomenex Luna column in conjunction
Extracts and pure compounds were tested in an NF-
ase reporter assay in mouse macrophage (RAW264.7) immune
cells to determine NF- B activity. The RAW264.7 cells were kindly
provided by Dr. Greg Barton (Dept. of MCB, UCB) along with the
jB lucifer-
with a 4.0 ꢂ 3.0 mm C18 (Octadecyl) guard column and cartridge
(Holder part number: KJ0-4282, Cartridge part number: AJ0-
4287, (Phenomenex, Inc., Torrance, CA). Samples were injected
j
onto the column using a volume of 15 lL, with a flow rate of
NF-jB luciferase reporter construct which has been described else-
where.27 Stably transfected RAW 264.7 cells with the NF-kB repor-
ter gene were plated in 96-well plates (5 x 104 cells per well).
Following a 24 h recovery period, the cells were treated with the
1 mL/min that was monitored using a Waters model 996 photodi-
ode array (PDA) UV detector. The elution was subsequently split
(1:1) between a S.E.D.E.R.E. model 55 evaporative light scattering

T. A. Johnson et al. / Bioorg. Med. Chem. 21 (2013) 4358–4364
4363
extract(s) or compound(s) for an additional 18 h in the presence of
LPS (100 ng/mL). To check NF- B luciferase activity, the Luciferase
Reporter Assay System purchased from Promega (Madison, WI)
were used. Cell lysates (15 L) from treated RAW264.7 cells were
placed in opaque 96-well plates. Luciferase Assay Reagent (50 L)
was injected and read by a fluorometer (LMAX 2, Molecular de-
vices). The values (IC50) represent the mean SE for n = 3. The
anti-inflammatory standard compound celastrol was obtained
from Cayman Chemical Company, Ann Arbor, MI, USA.
formed according to the manufacturer protocol with the iTaq™
Universal SYBRÒ Green Supermix (BioRad). Quantitative real-time
PCR was performed on Applied BiosystemsÒ 7500 Real-Time PCR
System (Applied Biosystems, Foster City, CA, USA). mRNA expres-
sion for each gene was normalized using the expression of GAPDH
as a control housekeeping gene.
j
l
l
3.8. MTT cytotoxicity assay
The methanol crude extract LC-MS/ELSD library well fractions
3.5. Determination of phosphorylation of IkB
analysis
a
by Western blot
were tested at 20 lg/mL, respectively based on assumed weights
of 0.15 mg/well estimated from a 15 mg injection divided into
two library plates (7.5 mg each) equally fractionated/50-well
ꢀ0.15 mg/well. (+)-Altholactone (2) was tested using concentra-
RAW264.7 cells were cultured in 6-well plates to near conflu-
ency, treated with increasing concentrations of compound in the
presence of LPS and incubated at 37 °C for 4 h. Cells were immedi-
tions of 6.25, 12.5, 25, 50 and 100 lM, respectively against the
non tumorigenic murine macrophage (RAW264.7) and breast
(MCF-10A) cell lines as well as against tumorigenic breast (MCF-
7, MDA-MB-231) and prostate (PC3, LNCaP) cell lines. Cells in 96-
well plates in the required growth medium were treated with ex-
tracts dissolved in DMSO for 20 h. After incubation, MTT solution
was added to wells and incubated for another 2 h. Media were re-
moved and DMSO was added to dissolve purple precipitates. Then
ately harvested in 200 lL of loading buffer (10% glycerol, 5% 2-
mercaptoethanol, 10% SDS, 0.125 M Tris–HCl pH 6.7, 0.15% bromo-
phenol blue), sonicated for 15 s, heated to 99.9 °C for 5 min and
fractionated by electrophoresis on 4–15% polyacrylamide, 0.1%
SDS resolving gels (BioRad). Proteins were electrically transferred
to Immobilon-II membranes (Millipore, Billerica, MA), and blocked
at room temperature for 1 h with 5% non-fat dry milk in wash buf-
fer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20). Blots
were subsequently incubated with antibodies against mouse phos-
plates were read at 570 nm using a plate reader. The values (IC50
)
represent the mean SE for n = 3. The cytotoxic standard control
compound doxorubicin was obtained from Cayman Chemical Com-
pany, Ann Arbor, MI, USA.
phor-IjBa and IjBa (Cell Signaling, 1:1000). Immunoreactive pro-
teins were detected after 1 h of incubation at room temperature
with appropriate horseradish peroxidase-conjugated secondary
antibodies (Santa Cruz Biotechnology, 1:3000 dilution). Blots were
incubated with Western Lighting Chemiluminecense Reagent Plus
(PerkinElmer Life Sciences) and chemiluminescence was detected
using CL-X Posure clear blue X-ray film (Thermo Scientific).
3.8.1. (+)-Goniothalamin (1)
LRESITOFMS m/z 201.1 [M+H]+ see Figure S10 in the Supple-
mentary data. This compound contained spectroscopy data equiv-
alent to previous reports.1
3.8.2. (+)-Altholactone (2)
3.6. Griess (nitrite oxide) assay
Brown oil: ½a 2D0
þ 127:5 (c 0.2, CHCl3); LRESITOFMS m/z 233.1
ꢃ
[M+H]+; HRMS [M+Na]+: C13H12O4Na calcd 255.06278, found:
255.06575. 1H NMR (CDCl3, 500 MHz): d 7.35 (5H, m), 7.01 (1H,
dd, J = 5.0, 10.0), 6.23 (1H, d, J = 9.5), 4.93 (1H, dd, J = 2.0, 5.0),
4.75 (1H, d, J = 6.0), 4.64 (1H, t, J = 5.0), 4.45 (1H, dd, J = 2.5, 6.0);
13C NMR (CDCl3, 125 MHz): d 161.6, 140.6, 138.2, 128.8,128.8,
128.5, 126.3,126.3 123.8, 86.6, 86.1, 83.8 and 68.3. Additional 1H
and 13C NMR data is provided in Figures S4–S5 (in the Supplemen-
tary data) and is equivalent to spectroscopic data reported
previously.23
Murine macrophage cells (RAW264.7) were plated in a 96-well
plate in RPMI1640 and incubated at 37 °C for 24 h. The medium
was changed, allowing cell induction by addition of compound
and lipopolysaccharide (LPS) from Escherichia coli 055:B5 (LPS; Sig-
ma–Aldrich) in the medium. After incubation, 50
natant was removed and incubated with the Griess reagent
[150 L; 0.5% sulfanilamide, 0.05% (N-1-naphthyl) ethylenedia-
lL of the super-
l
mine dihydrochloride, 2.5% H3PO4 and 97% H2O by weight] for
30 min at room temperature in the dark. The absorbance is mea-
sured at 530 nm on a Dynex MRX II microplate spectrophotometer
and calibrated using a standard curve constructed with sodium ni-
trite to yield NOꢁ2 concentration.
3.8.3. (+)-3-O-Acetylaltholactone (3)
Clear oil: ½a 2D0
þ 36:7 (c 0.12, EtOH; LRESITOFMS m/z 275.1
ꢃ
[M+Na]+; HRMS [M+Na]+: C15H14O5Na calcd. 297.0733, found:
297.0727. 1H NMR (CDCl3, 500 MHz): 7.35 (5H, m), 7.04 (1H, dd,
J = 5.5 9.5), 6.27 (1H, d, J = 9.5), 5.39 (1H, dd, J = 1.5, 3.5), d 4.97
(1H, d, J = 3.5, H-2), 4.95 (1H, d, J = 1.0, 4.0), 4.93 (1H, dd, J = 2,
5 Hz), 4.62 (1H, dd, J = 4.0, 5.5), 2.14 (3H, s); 13C NMR (CDCl3,
125 MHz): d 169.5, 160.6, 139.3, 137.6, 128.8, 128.8, 128.6, 126.3,
126.3, 124.8, 86.2, 83.7, 70.7, 69.2 and 20.9. Additional 1H and
13C NMR data is provided in Figures S8–S9 (in the Supplamentary
data) and is equivalent to spectroscopic data reported previously.15
3.7. In vitro quantitative real-time PCR analysis
(+)-Altholactone (2) was evaluated in triplicate (n = 3) for its ef-
fects on inducible nitrous oxide synthase (iNOS), interleukin-6 (IL-
6), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1b), interferon
c
inducible protein 10 (IP-10), interferon beta (IFN-b), granulocyte
colony-stimulating factor (GCS-F) and monocyte chemotactic pro-
tein-1 (MCP-1). The results are presented as means standard
deviation of the mean (STD). Differences in mean values between
groups were analyzed by a one-way analysis of variance (ANOVA)
using GraphPad Prism software (version 3.02 for Windows, Graph-
Pad Software Inc., La Jolla, CA, USA). The statistical significance of
mean differences was based on a p value of <0.05. A total RNA of
Acknowledgments
The project described was supported by Grant Number
U01TW008160 from the Fogarty International Center, the Office
of Dietary Supplements, the National Science Foundation and the
Department of Energy. This project was supported by the USDA
Agricultural Food Research Initiative of the National Institute of
Food and Agriculture, USDA, Grant #35621-04750. The content is
solely the responsibility of the authors and does not necessarily
represent the official views of the Fogarty International Center or
0.5 lg from RAW264.7 cells after treatment was exposed to DNase
I treatment (Aurum™ Total RNA Mini Kit, BioRad, Hercules, CA,
USA). cDNA was synthesized from DNase-treated RNA by reverse
transcription (iScript™ RT Supermix, BioRad). cDNAs from each
experimental condition were pooled and qPCR analysis was per-

4364
T. A. Johnson et al. / Bioorg. Med. Chem. 21 (2013) 4358–4364
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or the Department of Agriculture. Additional funding was provided
by NIH-MARC GM007910-31 (T.L.C.).
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Supplementary data (ten figures are provided and include: (a)
LC–MS/ELSD data of library fractions, (+)-goniothalamin (1), (+)-
altholactone (2) and (+)-3-O-acetylaltholactone (3), (b) 1H and
13C NMR data of 2–3 and (c) the MTT cytotoxicity assay data
against one non tumorigenic and four tumorigenic cell lines for
(2). These data sets associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.bmc.2013.04.055.
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