Antioxidant, anti-adipocyte differentiation, antitumor activity and anthelmintic activities against

13 Pages·2013·2.18 MB·English

Linetal.BMCComplementaryandAlternativeMedicine2013,13:237
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RESEARCH ARTICLE Open Access
Antioxidant, anti-adipocyte differentiation,
antitumor activity and anthelmintic activities
against Anisakis simplex and Hymenolepis nana
of yakuchinone A from Alpinia oxyphylla
Rong-Jyh Lin1, Chuan-Min Yen1, Tzung-Han Chou2, Feng-Yu Chiang3, Guey-Horng Wang4, Ya-Ping Tseng5,
Lin Wang6, Ting-Wei Huang7, Hui-Chuan Wang8, Leong-Perng Chan3,9*, Hsiou-Yu Ding8* and Chia-Hua Liang4*
Abstract
Background: Alpinia oxyphylla is a common remedy intraditional Chinese medicine. Yakuchinone A is a major
constituent of A. oxyphylla and exhibitsanti-inflammatory, antitumor,antibacterial, and gastric protective activities.
Methods: Antioxidant and antitumor characteristics ofyakuchinone A inskin cancer cells as well as novel
mechanisms for theinhibition ofadipocytedifferentiation, cestocidal activities against Hymenolepis nana adults, and
nematocidal activitiesagainst Anisakis simplex larvae are investigated.
Results: YakuchinoneApresentstheabilityoftheremovalofDPPH·andABTS+freeradicalsandinhibitionoflipid
peroxidation.YakuchinoneAsuppressesintracellularlipidaccumulationduringadipocytedifferentiationin3T3-L1cells
andtheexpressionsofleptinandperoxisomeproliferator-activatedreceptorγ(PPARγ).YakuchinoneAinducesapoptosis
andinhibitscellproliferationinskincancercells.TheinhibitionofcellgrowthbyyakuchinoneAismoresignificantfor
non-melanomaskincancer(NMSC)cellsthanformelanoma(A375andB16)andnoncancerous(HaCaTandBNLCL2)cells.
TreatmentBCCcellswithyakuchinoneAshowsdown-regulationofBcl-2,up-regulationofBax,andanincreasein
cleavagepoly(ADP-ribose)polymerase(PARP).ThissuggeststhatyakuchinoneAinducesBCCcellsapoptosisthroughthe
Bcl-2-mediatedsignalingpathway.TheanthelminticactivitiesofyakuchinoneAforA.simplexarebetterthanforH.nana.
Conclusions:Inthiswork,yakuchinoneAexhibitsantioxidativeproperties,anti-adipocytedifferentiation,antitumor
activity,andanthelminticactivitiesagainstA.simplexandH.nana.
Keywords:YakuchinoneA,Antioxidant,Adipogenesis,Apoptosis,Hymenolepisnana,Anisakissimplex
Background established general mechanism for cell as well as tissue
Free radicals include superoxide anion (O -), hydroxyl injury [1,2]. ROS are strongly associated with lipid per-
2
(HO·), peroxyl (ROO·), alkoxyl (RO·) and nitric oxide, oxidation, which leads to the deterioration of the food,
which are oxygen-centered free radicals occasionally and are also involved in a variety of diseases including
known as reactive oxygen species (ROS). Cellular oxida- cellar aging, mutagenesis, carcinogenesis, coronary heart
tive damage that is caused primarily by ROS is a well- disease,diabetesmellitus,andneurodegeneration [2].
Obesity has become a global health problem due to its
association with various metabolic disorders such as
*Correspondence:[email protected];[email protected];
type-II diabetes, cardiovascular disease, hypertension,
[email protected]
3DepartmentofOtolaryngology-HeadandNeckSurgery,KaohsiungMedical and non-alcoholicfatty liver disease [3,4]. Synthetic anti-
UniversityHospital,KaohsiungMedicalUniversity,Kaohsiung,Taiwan obesity drugs have been reported to be costly, and some
8InstituteofCosmeticScience,ChiaNanUniversityofPharmacyandScience,
of them also beset with undesirable side effects. There-
Tainan,Taiwan
4DepartmentofCosmeticScience,ChiaNanUniversityofPharmacyand fore, developing drugs to directly modulate energy
Science,Tainan,Taiwan
Fulllistofauthorinformationisavailableattheendofthearticle
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metabolism without affecting the central nervous system musculature depends on environmental conditions and/or
hascaused substantialattention[4,5]. the species of parasite and fish condition [13,14]. AsL3 are
Natural/herbalcompoundsincludingberberine,resvera- repeatedly transferred between fish and fish through the
trol, and curcumin are known to modulate obesity either food chain. Therefore, piscivorous fish accumulate large
through increasing energy expenditure or inhibiting adi- numbers of AsL3 [14]. Finally, the ingestion of infected
pocyte differentiation [6-8]. Presently the focus is to de- fish or squid by a marine mammal (i.e. the final host)
velopnaturalcompoundsasantioxidantsthatarepossibly leads to the development of fourth-stage larvae and
used to reduce damage caused by oxidative stress, age- then adults. Humans may be accidental hosts by con-
dependentdiseases,andobesity[9]. suming undercooked and/or raw second intermediate
Hymenolepis nana is a general occasion of cestode in- hosts that contain AsL3. A. simplex rarely develop fur-
fections, and is found worldwide. In human adults, the ther within the human gastrointestinal tract, instead,
tapeworm is more of a nuisance than a health problem, by means of proteolytic enzymes, but they typically
but in small children, H. nana is dangerous. It is often embed in the gastric or intestinal mucosa and die or
seen in children in countries with inadequate sanitation invasion the muscular layers of the stomach and intes-
and hygiene. H. nana infections are typically asymptom- tine to induce allergic reactions and a variety of ab-
atic but heavy infections also cause headaches, anorexia, dominal symptoms that are characterized as anisakiasis
weakness, abdominal pain, and diarrhea [10]. H. nana is or anisakidosis [15]. The four main clinical syndromes
the only cestode without any intermediate hosts in its in humans who experience symptomatic anisakidosis
life cycle [11]. H. nana infection is typically acquired include gastric, intestinal, extra-gastrointestinal, and
from eggs in the feces from another infected individual, allergic diseases. Anisakidosis is globally recognized as
which are transferred by contaminated food. Eggs hatch a public health problem, which is relative to Asia and
in the duodenum, releasing oncospheres that penetrate Europe [16,17]. The prevalence of anisakidosis has in-
the mucosa and enter the lymph channels of the villi. creased unusually because of the increasing popularity of
Then, oncospheres develop into a cysticercoid, which Japanese cuisine,suchas “sushi” and “sashimi”.Theavail-
has a tail and a well formed scolex. About five to six ability of an anthelmintic compound against A. simplex
days cysticercoids migrate into the lumen of the small has the potential to shorten the clinical course and pre-
intestine and attach before maturing. Eggs of H. nana ventmechanicalinvasionthatcausefromendoscopicpro-
infect when passed with stool and transfer in contami- cedures. Because few effective studies for anthelmintic
nated food. Eggs are ingested by an arthropod inter- drugs and nature compounds against A. simplex, the ef-
mediate host and hatch in the duodenum, releasing fectiveness of treatment with anthelmintic agents, anti-
oncospheres, and develop into cysticercoid larvae. Upon biotics, anticholinergics, and/or corticosteroids against
rupture of the villus, the cysticercoids return to the in- A. simplex remains controversial [18].
testinal lumen, evaginate their scoleces, attach to the Alpinia oxyphylla is an important traditional Chinese
intestinal mucosa, and mature into adults that reside medicinal herb whose fruits are widely used as a tonic,
in the ileal portion of the small intestine, producing aphrodisiac, anti-salivation, anti-polyuria, and anti-
gravid proglottids. The eggs are then passed in stools diarrhea [19]. The extracts from A. oxyphylla possess
when released from the proglottids or disintegration of neuroprotective activity, anti-tumor, anti-anaphylactic,
proglottids in the small intestine. An alternate mode of and inhibition of nitric oxide production [19,20].
infection consists of internal autoinfection without Yakuchinone A [1-(4′-hydroxy-3′-methoxyphenyl)-7-
passing through the external environment. The short phenyl-3-heptanone], a major pungent ingredient de-
life span and rapid course of development also facili- rived from A. oxyphylla exhibits anti-inflammatory,
tates the spread and ready availability of this worm, antitumor,antibacterial,antiviral,andgastricprotectiveac-
but internal autoinfection allows the infection to con- tivities [21]. Yakuchinone A has been reported to be a
tinue for years [11,12]. strong inhibitor of prostaglandin biosynthesis in vitro[22].
Anisakis simplex adult worms mature and release eggs Moreover, yakuchinone A can act as an anti-tumor pro-
from the primary host. The eggs pass from stool into moter as determined by the ability to suppress phorbol
seawater and are embryonated to form A. simplex first- ester-induced activation of ornithine decarboxylase (ODC)
stagelarvae(AsL1)andsubsequentlymoultedtoA.simplex and inhibits the promotion of papilloma formation in
second-stage larvae (AsL2). When larvae are ingested by mouse skin [23]. 12-O-tetradecanoylphorbol-13-acetate
smallcrustaceanfirstintermediatehosts,theAsL2matures (TPA)-stimulatedsuperoxidegenerationandtumornecro-
into A. simplex third-stage larvae (AsL3) that are subse- sis factor-α (TNF-α) or interleukin-1α production in hu-
quently consumed by second intermediate hosts such as man promyelocytic leukemia (HL-60) cells as well as on
marine fish or squid. The AsL3 migrate into the viscera DNAbindingofactivatorprotein1(AP-1)inmousefibro-
andperitonealcavity.Thedegreeofmigrationintothefish blast(NIH3T3)cellsarealsosuppressedbyyakuchinoneA
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[23,24]. Furthermore, yakuchinone A induces apoptotic (OCH ),44.6(C-2),42.9(C-4),35.7(C-7),30.9(C-6),29.5
3
deathinHL-60cellsaccountfortheantiproliferativeactiv- (C-1), 23.3 (C-5). These data were compared with litera-
ity[23].However,thebiochemicalmechanismsunderlying turevalues[25].ThechemicalstructureofyakuchinoneA
the antioxidant, anti-obesity, anti-skin cancer effects of was shown in Figure 1A. The purity of yakuchinone A is
yakuchinone A and its cestocidal effects on H. nana and 99.2%. The solubility of yakuchinone A was 100 mM in
larvicidal effects on A. simplex remain unclear. This dimethylsulfoxide(DMSO).
study confirms the antioxidant and antitumor effects
of yakuchinone A and elucidates the novel mechanisms Assayforfreeradicalscavengingabilityagainst
for its inhibition of adipocyte differentiation as well as its DPPH·andABTS+
anthelminticactivitiesagainstH.nanaandA.simplex. The radical scavenging activities of yakuchinone A
against DPPH·and ABTS·+ radicals were measured by
Methods using the method as previously reported [26]. For
Materials DPPH·radical scavenging activity analysis, 5, 10, 20, 30,
1,1-Diphenyl-2-picrylhydrazyl (DPPH•), 2,2′-azinobis(3- 40, 50, and 100 μM yakuchinone A (10 μl of solution)
ethylbenzothiazoline-6-sulfonic acid) diammonium salt was mixed with 90 μl of DMSO and 900 μl of ethanolic
(ABTS•+), 2,5,7,8-tetramethylchroman carboxylic acid DPPH·solution (0.1 mM). After incubation in darkness
(trolox), trichloracetic acid (TCA), 2-thiobarbituric acid at 25°C for 30 min, the absorbance (A) was determined
(TBA)and3-isobutyl-1-methylxanthine(IBMX)werepur- at 517 nm (Hitachi U-2001, Japan). For ABTS•+ radical
chasedformSigmaChemicalCo.(Sigma,St.Louis,MO). scavenging activity analysis, ABTS·+ was dissolve in
water to 7 mM. ABTS·+ radical was produced by
Extractionandisolation reacting ABTS·+ stock solution with 2.45 mM potas-
The “Yizhiren”, A. oxyphylla, was supplied from Kwong- sium persulfate, and the mixture stood in the dark at
Te Co., Kaohsiung,Taiwan and was identified by profes- room temperature for 12–16 h. The ABTS·+ radical so-
sor Hang-Ching Lin of the National Defense Medicinal lution was diluted to an absorbance of 0.70±0.02 at
Center,whereavoucherspecimenwasdeposited(CNUPS 734 nm at 30°C. Each agent (0.1 ml) reacted with 2.9 ml
No.970801).ThedrypowderofA.oxyphylla seed(6.0kg) of diluted ABTS·+ radical solution for 20 min at 30°C,
was extracted with 95% ethanol at room temperature. and then the absorbance was measured at 734 nm
After removal of the solvent by evaporation, the residue (Hitachi U-2001, Japan). The TEAC (trolox equivalent
(559.0 g) was dissolved in methanol–water (9.5:0.5) and antioxidant capacity) of the reagent was calculated by
partitioned with n-hexane. The methanol (95%) was comparing their reactivities to the standard antioxidant,
removed by evaporation and the residue was then trolox. Ethanol or distilled water was used as negative
suspended in water and partitioned with ethyl acetate controls. Trolox was used as a standard antioxidant. The
(359.0 g). The ethyl acetate layer was subjected to LH- scavenging ability of yakuchinone A or trolox in DPPH·
20 Sephadex and eluted with methanol. Each fraction and ABTS·+ was calculated using the following equation:
collected from the column was monitored by thin-layer radical scavengingability (%)= (1−A /A )×100.
sample control
chromatography and the similar fractions were combined EC values were estimated from the percent inhibition
50
toproduce4fractions.Thefraction3wasfurtherpurified versus concentration plot derived from the percentage
bya silicagel and elutedwithn-hexane-ethylacetate(9:1, scavenging activity. This data was shown as mean
7.5:2.5, 1:1, 2.5:7.5), ethyl acetate, ethyl acetate-methanol values±standard deviation (n=3).
(9:1),methanoltoisolateyakuchinoneA(276.1mg).Their
structures were confirmed by NMR and mass spectra Determinationofantioxidanteffectonliposome
analysis. peroxidation
Yakuchinone A: slightly yellow oil; EI/MS m/z (rel. The effect on liposome peroxidation was assayed by
int.%): 312(80, [M]+), 194 (6), 179 (45), 161 (14), 151 measuring concentrations of thiobarbituric acid reactive
(33), 137 (100), 119 (23); 1H-NMR (CDCl , 500 MHz) substances (TBARS). Liposomes were prepared according
3
δ: 1.60 (4H, m, H-5,6), 2.40 (2H, t, J =7.0 Hz, H-4), to the method of Chou et al. [27]. In brief, the liposomes
2.60 (2H, t, J =7.0 Hz, H-7), 2.68 (2H, t, J =7.6 Hz, H-2), were obtained by dispersing lipids in demineralized water
2.82(2H,t,J=7.6Hz,H-1),3.86(3H,s,OCH ),6.66(1H, (1:10).Fortheassay,32μlofsuspensionofliposomeswas
3
dd, J =8.0, 2.0 Hz, H-6’), 6.68 (1H, d, J =2.0 Hz, H-2’), incubated together with 11 μl of 10 mM FeSO , 11 μl of
4
6.83 (1H, d, J =2.0 Hz, H-5), 7.15~7.20 (3H, m, H-3”, 10mMascorbicacidandappropriateamountsofdifferent
4”, 5”), 7.26~7.29 (2H, m, H-2”, 6”); 13C-NMR (CDCl , concentrations (5, 10, 20, 30, 40, 50 and 100 μM) of
3
125MHz)δ:210.3(C-3),146.3(C-3′),143.8(C-4′),142.1 yakuchinone A, trolox and rutin in 1.515 ml of 50 mM
(C-1″), 133.0 (C-1′), 128.2 (c-2″, 6″), 128.3 (C-3″, 5″), Na HPO -NaH PO buffer, pH 7.4 (2.5 ml final solution)
2 4 2 4
125.7(C-4″),120.7(C-6′),114.3(C-5′),111.0(C-2′),55.8 at 37°C for 1 h. Lipid peroxidation was terminated by the
Linetal.BMCComplementaryandAlternativeMedicine2013,13:237 Page4of13
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A) B)
120
Control
5 M * * *
) 100 10 M
% 20 M
y of al ( 80 3400 MM *
tivitadic 5100 0 MM * * *
g acee r 60 *
nr *
eniH f 40 *
rePP * *
ScD
20
0
Control YA Trolox
C) D)
120 120
Control
Control
) 100 51 0 MM * * %) 100 ***** 512 00 MMM
Screening activity of ABTS+ free radical (% 24680000 2345100000 0 MMMMM** * * * ** * ** Inhibition of lipid eroxidation formation ( 24680000 * ****** ******* 34510000 0 MMMM
p *
0 0
Control YA Trolox Control YA Trolox Rutin
Figure1AntioxidantactivityofyakuchinoneA.A)ChemicalstructureofyakuchinoneAfromAlpiniaoxyphyllaMiq.M.W.=312.B)DPPH·and
C)ABTS·+freeradicalscavengingactivitiesofyakuchinoneAandtrolox(5,10,20,30,40,50,and100μM).D)Inhibitionoflipidperoxidationby
yakuchinoneA,trolox,andrutin(5,10,20,30,40,50,and100μM)usingliposomeasanoxidizablesubstrate.Dataarepresentedasmean±SDfrom
threeindependentexperiments;*p<0.05indicatessignificantdifferencefromvehicle-treatedcells.YakuchinoneA;YA.
reaction of 0.8 ml of 1% TBA and 10% TCA and 106 μl Celllines
of 0.1 M ethylene diamine-tetraacetic acid disodium Human epidermoid carcinoma A431, human oral squa-
salt dehydrate at 100°C for 20 min. After cooling and mouscellcarcinomaSCC25.humanskinmalignantmel-
centrifugation (2600g for 10 min), the malonaldehyde anoma A375, mouse melanoma B16, mouse leukemic
(MDA)-TBA complex was determined by measuring monocyte macrophage RAW 264.7, mouse normal em-
the absorbance (A) at 532 nm. A control with DMSO bryonic liver BNLCL2 cells, and 3 T3-L1 preadipocytes
instead of sample was also analyzed and expressed no were purchased from the American Type Culture Col-
activity. Trolox and rutin were utilized as standards. lection (Rockville, MD). Human basal cell carcinoma
The percentage inhibition was calculated using the fol- BCC and human premalignant keratinocytic HaCaT
lowing equation: Inhinition of lipid peroxidation (%)= cells were kindly donated by Prof. Hamm-Ming Sheu
(1−A /A )×100. EC values were estimated (NationalChengKungUniversityMedicalCollege,Tainan,
sample control 50
from the percentage inhibition versus concentration Taiwan). Cells were cultured in medium supplemented
plot. This data was shown as mean values±standard with 10% fetal bovine serum (Hazelton Product, Denver,
deviation (n=3). PA) and 1% penicillin-streptomycin at 37°C in 5% CO
2
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humidified atmosphere; specifically, A431, A375, B16, werewashed by PBSandfixed with4%paraformaldehyde
HaCaT, RAW 264.7, BNLCL2, and 3 T3-L1 cells were andstainedwithHoechst33342(0.1μg/ml)(Sigma)at37°C
maintained in DMEM medium (GIBCO, Grand Island, for 10 min in the dark. The nuclear morphology
NY),BCC cellsinRPMImedium,andSCC25inDMEM/ changes were viewed under a fluorescent microscope
F12mediumsupplementedwith0.4μg/mlhydrocortisone (Nikon,TE2000-U, Japan).
(Sigma,St.Louis,MO).
RNAisolationandreversetranscription-polymerasechain
Adipocytedifferentiation reaction(RT-PCR)analysis
Cultivation of 3 T3-L1 cells and their conversion to adi- 3 T3-L1 cells were treated with vehicle control (DMSO)
pocytes were carried out according to the method as de- or yakuchinone A (5 μM) during the differentiation
scribed previously [28]. To induce differentiation, four process.BCCcells(1×105cells/ml)weretreatedwithve-
day postconfluent 3 T3-L1 preadipocytes were stimu- hicle control (DMSO) or yakuchinone A (20 μM) for 24
lated for 72 h in 10% FBS/DMEM with containing the and 48 h. Total RNA was prepared from cells using the
MDI hormone mixture (0.5 mM IBMX, 1 μM dexa- Trizolreagent(Invitrogen,Carlsbad,CA,USA),andaRT-
methasone, and 10 μg/ml of insulin) in six-well plates. PCR was conducted using 3 μg of total RNA and the
After four days, the medium was replaced with 10% Superscript cDNA Preamplification System (Weiterstadt,
FBS/DMEM medium containing 10 μg/ml of insulin. Germany) according to the manufacturers’ instructions.
The medium was replaced with fresh medium (10% Thefollowingprimerswereutilized:rightprimer5′-GCT
FBS/DMEM, 10 μg/ml of insulin) every two days until CTA GAC GTG ACA ATC TGT CTG AGG TCT GTC
analysis on day eight. Yakuchinone A (5 μM) was added AT-3′ and left primer 5′-CGG CAT CCG TTG TCG
duringthedifferentiationprocess. GTT TCA CAA ATG CCT TGC AGT G-3′ for PPAR γ
(870 bp), right primer 5′-CAT CTG CTG GCC TTC
OilRedOstaining TCC AA-3′ and left primer 5′-ATC CAG GCT CTC
Differentiated3T3-L1cellswerestainedusingtheOilRed TGG CTT CTG-3′ for leptin (71 bp), right primer 5′-
O method [29] for adipocyte lipid accumulation. At day AGA TGT CCA GCC AGC TGC ACC TGA C-3′ and
eightofdifferentiation,thecellswerewashedwithPBSand left primer 5′-AGA TAG GCA CCC AGG GTG ATG
fixed with 10% formaldehyde for 2 h. The fixed cells were CAA GCT-3′ for bcl-2 (367 bp), right primer 5′-AAG
washed with 60% isopropanol, and stained with 0.2% Oil CTGAGCGAGTGTCTCAAGCGC-3′andleftprimer
RedOfor10min.Theplateswererinsedthreetimeswith 5′-TCC CGC CAC AAA GAT GGT CAC G-3′ for bax
water and examined under a phase contrast inverted light (366bp),andrightprimer5′-ACCCAC ACTGTG CCC
microscope (Nikon, TE2000-U, Japan). After thorough ATC TA-3′ and left primer 5′-CGG AAC CGC TCA
washing with water and evaporation of excess water, Oil TTG CC-3′ for β-actin (286 bp). The amplified RT-PCR
Red O was extracted in isopropyl alcohol and the absorb- products were analysed in 2% agarose gels, visualized by
ancewasmonitoredat520nm(BioTek,Synergy™2). ethidiumbromidestainingandphotographedunderultra-
violetlight.
Cellviability
Cells (1×105 cells/ml) were plated in 100 μl of 96-well Westernblotting
multidishes and treated with a series of concentrations Cells (1×105 cells/ml) were treated with vehicle con-
(5, 10, 20, 30, 40, and 50 μM) of yakuchinone A or ve- trol (DMSO) or yakuchinone A (20 μM) for 72 h.
hicle control (DMSO) for 72 h. The control groups were Then, cells were washed with PBS, and lysed in lysis
treated with DMSO, and the final DMSO concentration buffer [50 mM Tris–HCl, pH 7.5, 1% Triton X-100,
did not exceed 0.1%. The cell viability was measured by 5 mM EGTA (ethylene glycol-bis(2-aminoethylether)-
performing the MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5- N,N,N’,N’-tetraacetic acid), 150 mM NaCl and 1 mM
diphenyl-tetrazolium bromide] assay [30]. The IC phenylmethylsulfonyl fluoride (PMSF)]. After centrifuga-
50
values were calculated from the agent concentrations tion (10,000g, 10 min), supernatants were collected. The
thatyielded acellviabilityof50%. cell lysates containing 40 μg of solubilized protein were
subjected to 12% sodium dodecyl sulfate-polyacrylamide
Cellmorphologicalchanges gel electrophoresis (SDS-PAGE) and electrophoretically
Cells (1×105cells/ml) wereplated in24-wellplates then transferred to nitrocellulose membranes. The membranes
treated with vehicle control (DMSO) or yakuchinone A were blocked in 5% skim milk. Blots were incubated with
(20 μM) for 72 h. Cells in each well were washed once theantibodiesagainstBcl-2,Bax,PARPandβ-actin(Santa
with 1× PBS, and analysis was performed using a phase Cruz, CA). The membranes were incubated with the ap-
contrast inverted light microscope (Nikon, TE2000-U, propriatesecondaryantibodyconjugatedwithhorseradish
Japan). To assess specific apoptosis, after incubation, cells peroxidase (Bio-Rad, Hercules, CA). Blotted antibodies
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were visualized by chemiluminescence method (ECL kit, and washed several times. The majority of the larvae
Amersham). were encysted, but they quickly became excysted upon
washing in NaCl solution. They were individually ob-
PreparationofH.nanaadultworms served under an inverted microscope, with subsequent
H. nana adult worms were obtained from each part of discarding of those that exhibited internal or external
the intestines of wild type mice, purchased from Lin’s damage. The larvae were then identified by morpho-
farm in Fengshan, Kaohsiung,Taiwan.These parts of the logical features, divided into groups and placed in 24-
intestine were duodenum, jejunum, ileum, colon and well plates contained cultivated media RPMI-1640 plus
rectum. TheH. nana had an average length of 5–50 mm 20% FBS, pH 4.0, in an atmosphere of 95% O /5% CO ,
2 2
and was collected using a needle with a blunt tip, before 37°C. These showed culture conditions demonstrated to
being placed in Petri dishes with 0.9% NaCl and provide for the maximum development and survival
gentamycin (10 mg/ml). They were then washed several of A [18,32].
times.The adult worms wereindividually observed under
an inverted microscope, with subsequent discarding of AssayofnematocidalactivityonA.simplex
those that exhibited internal or external damage. The TheaboveAsL3cultivatedmediaweresupplementedwith
adult worms were then identified by their morphological L-glutamine (2mM),penicillin (100IU/ml),streptomycin
features, divided into groups and placed in 24-well plates (100 mg/ml) and amphotericin B (0.25 μg/ml), and tested
contained cultivated media RPMI-1640 plus 20% FBS, of yakuchinone A for 10, 100, and 200 μM. The survival
pH7.4,inanatmosphereof95%O /5%CO ,37°C.These and mobility of the larvae were assessed at 2, 4, 8, 12, 24,
2 2
culture conditions have been shown to maximize the de- 48 and 72 h using a stereomicroscope. Two investigators
velopmentandsurvivalofH.nana. blindly scored the larvae as dead, with poor mobility or
with normal mobility. The percentage losses of spontan-
Assayofcestocidalactivityofoscillationandperistalsis eousmotionduring3minperiodsimmediatelyafterincu-
testonH.nana bation and complete standstill were determined by
The above H. nana cultivated media were supplemented stimulation 4–5 h later (defined as death). The mortality
withL-glutamine (2mM),penicillin (100IU/ml), strepto- was recorded after ascertaining that the worms neither
mycin (100mg/ml) and amphotericin B(0.25μg/ml), and movedwhenshakenvigorouslynorwhendippedinwarm
thentheeffectsofyakuchinoneAatconcentrationsof10, medium.Thenematocidalactivitywasmodifiedaccording
50 and 100 μM were tested. The survival and mobility of to a scoring system that was developed by Kiuchi et al.
the adult worm were assessed at 2, 4, 6, 12, 24, 48, and [33]andLinetal.[18].
72 h using a stereomicroscope. They were observed for
theirspontaneousmotilityandevokedresponsesat2,4,6, Statisticalanalysis
12,24,48,and72husingastereomicroscope.Theoscilla- The results are expressed as mean±standard deviation
tion and peristalsis states of adult worms were scored (SD). Statistical differences were estimated by one-way
blindly by two investigators. Cestode activity was scored analysis of variance (ANOVA) followed by Dunnett’s test
by monitoring both oscillation and peristalsis. Oscillation or the Tukey-Kramer test. A p value of 0.05 was
was scored of movement at scolex and neck for each sec- regarded as significant. The data were analyzed and the
ond for 30 seconds, and then the highest score was 30. figures plotted using software (SigmaPlot Version 8.0
Peristalsis was record the contraction real times at scolex andSigmaStatVersion2.03,Chicago,IL).
andneck.Alldatawerecomparedwiththeinitialtimebe-
fore the test compounds had been added. Worms death Results and discussion
andcompletestandstillasdeterminedbynoneanyoscilla- FreeradicalscavengingactivityofyakuchinoneA
tionandperistalsischangesfor30secondswereidentified. The DPPH·and ABTS·+ radical has been widely used
The mortality was recorded after ascertaining that the for assessment of radical scavenging because of the easy
worms neither moved when shaken vigorously nor when and convenient consideration [34]. The soluble free rad-
dippedinwarmmedium[31]. ical DPPH·is well known as a good hydrogen abstractor
that yields DPPH-H as a by-product. Thus, the scaven-
A.simplexlarvaepreparation ging of DPPH radicals by phenols is effective. The anti-
The AsL3 were obtained from the muscle and periton- oxidant activity of yakuchinone A and trolox (a positive
eum of freshTrichiurus lepturuss (largehead hairtail, At- control) was measured based on scavenging activities for
lantic cutlassfish) that were purchased from the fish stable DPPH radical as presented in Figure 1B. With in-
market of Kaohsiung, Taiwan. The AsL3 had an average creasing doses from 5 to 100 μM of yakuchinone A and
length of 20–22 mm, and were collected using a needle trolox, the values of DPPH·scavenging activity were
with a blunt tip, placed in Petri dishes with 0.9% NaCl 9.6%, 29.2%, 34.5%, 44.6%, 60.0%, 64.5%, and 70.7% for
Linetal.BMCComplementaryandAlternativeMedicine2013,13:237 Page7of13
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yakuchinone A and 14.5%, 27.8%, 55.9%, 77.7%, 95.0%, InhibitionoflipidaccumulationbyyakuchinoneAin
96.3%, and 97.4% for trolox, respectively. The EC 3T3-L1adipocytes
50
values of yakuchinone A for the scavenging of DPPH· Numerousstudiesshow that obesity may inducesystemic
radicals were 33.5 (yakuchinone A) and 17.9 μM (trolox). oxidative stress, and the increase in ROS in adipocytes
Thegenerationof ABTS·+ involves the direct production contributestoderegulatedexpressionofinflammatorycy-
of the blue/green ABTS·+ chromophore through the re- tokines suchas tumornecrosis factor-α,whichmaybean
actionofpotassiumpersulfateandABTS.Theadditionof early instigator of the obesity-associated diabetes and
hydrogen-donating antioxidants to the preformed radical cardiovascular disease [37,38]. This work demonstrates
reduces it to ABTS [35]. Figure 1C shows the scavenging that yakuchinone A exhibits anti-oxidation activities,
activityofyakuchinoneAtowardsABTS·+.As increasing suggesting yakuchinone A has an inhibitory effect on
dosesof5,10,20, 30,40, 50,and 100μMofyakuchinone adipogenesis. 3 T3-L1 adipogenic differentiation re-
A and trolox, the values of ABTS·+ scavenging capacity quires a network of adipogenic markers [3]. We exam-
were 5.7%, 11.7%, 22.5%, 31.0%, 49.6%, 63.6%, and 70.6% ined the ability of the yakuchinone A to prevent
foryakuchinoneAand21.9%,27.3%,47.6%,49.6%,75.1%, adipogenesis in 3 T3-L1 adipocytes. The amount of ac-
92.2%, and 98.0% for trolox, respectively. The EC cumulated intracellular lipid droplets were compared
50
values for the scavenging of ABTS·+-radicals were in differentiated 3 T3-L1 cells after treatment in a
40.2 (yakuchinone A) and 30.1 μM (trolox). The extent MDI mixture and differentiated cells. The amount of
of decolorization as percentage inhibition of the ABTS·+ intracellular lipid droplets increased in differentiated
radicalcationwasproportionaltotheconcentrationofan- 3 T3- L1 cells, as shown by the Oil Red O staining
tioxidantsandcalculatedrelativetothereactivityoftrolox (Figure 2A). However, incubation of differentiated cells
as a standard (TEAC). TheTEAC value derived from the with low concentration of yakuchinone A (5 μM) de-
dose–response curve for yakuchinone A was 3.4 mM of creased MDI-induced lipid accumulation. This result
trolox/g. These results suggest that yakuchinone A ex- was further supported by quantitative spectrophotometric
hibits an antioxidant capacity to scavenge DPPH·and analysis of cellular neutral lipid content. Figure 2B shows
ABTS·+freeradicals. lipid accumulation was significantly inhibited in the pres-
enceof5μMyakuchinoneA.Theleveloflipidaccumula-
PotentialofyakuchinoneAtoinhibitlipidperoxidation tion over eight days was 19.2% of the MDI-treated
The antioxidant action is assessed by inhibiting the positive control cells. Adipocytokines are adipocyte-
damage caused by free radicals and the mechanisms derived hormones, such as leptin and adiponectin, which
involved in many human diseases such as hepatotoxic- modulated hepatic and peripheral lipid and glucose me-
ities, hepatocarcinogenesis,diabetes,malaria,acutemyocar- tabolism[4].Theamountofleptinsecretedintheadipose
dialinfarction,andskincancertoincludelipidperoxidation tissue is positively correlated with the lipid content and
asamainsourceofmembranedamage[9]. Lipidperoxida- adipocyte size [4]. Furthermore, previous research has
tion in biological systems has been thought to be a toxico- established that adenosine 5′-phosphate-activated protein
logical phenomenon that leads to various pathological kinase (AMPK) and peroxisome proliferator-activated re-
consequences. MDA formed from lipid peroxidation of un- ceptor γ (PPARγ) appears to be involved in adipocyte dif-
saturated phospholipid reacts withTBA to produce a pink ferentiation and maturation. This can be potential drug
MDA-TBA adducts. MDA is reactive and active in cross- targets for the treatment of obesity [3]. We evaluated the
linkingwithDNAandproteinsanddamageslivercells[36]. yakuchinone A-induced changes in the expression of adi-
Phospholipidsarebelievedtobepresentinhighamountsin pose tissue genes associated with adipogenesis through
cellmembranes[37].Thephospholipidpreparedasalipo- RT-PCR analyses. As shown in Figure 2C, addition of
somewasusedtoevaluatetheeffectofyakuchinoneAon yakuchinone A (5 μM) suppressed the expression of
liposome peroxidation to investigate yakuchinone A in a leptin and PPARγ significantly as revealed by RT-PCR.
biological system. Figure 1D presents the inhibition of These results suggest that yakuchinone A inhibits and
lipidperoxidationbyyakuchinoneA(5,10,20,30,40,50, adipogenesis due in part to the inhibition of angiogen-
and 100 μM) depended on dose. The EC values of the esis. These events may be mediated, in part, through
50
inhibition of lipid peroxidation efficiency by yakuchinone antioxidative properties of yakuchinone A responsible
A, trolox and rutin were 10.3, 14.3 and 6.2 μM, re- for inhibition of angiogenesis.
spectively. Although the inhibition of lipid peroxida-
tion activity by yakuchinone A was weaker than by EffectofyakuchinoneAoncellviabilityandskincancer
rutin, theinhibitionefficiencyofyakuchinoneAexceeded cellapoptosis
trolox. The MDA lowering effect of yakuchinone A indi- Previous report have demonstrated that yakuchinone A
cates a protective action against lipid peroxidation of un- exhibits no cytotoxicity against human lung adenocar-
saturatedphospholipids. cinoma A549 cells, human colorectal carcinoma HT-29
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A) 0 d 1 d 2 d 3 d 4 d
5 d 6 d 7 d 8 d
B) 120 C)
)
% 100
(
n
o
i 80
t
a
l
u
m 60
u
c
c
a 40
d
i
p
Li 20
0
0 1 2 3 4 5 6 7 8
Treatment (days)
Figure2YakuchinoneAinhibitsadipocytedifferentiation.A)PhotomicrographofOilRedOstaineddifferentiating3T3-L1cells.Cellswere
treatedwithyakuchinoneA(5μM)foreightdays.LipidaccumulationwasmeasuredbyOilRedOstaining.B)Percentagelipidaccumulationwas
analyzedbyquantitativeanalysisofOilRedOstaining.Dataarepresentedasmean±SDfromthreeindependentexperiments;*p<0.05indicates
significantdifferencefromvehicle-treatedcells.C)Thegeneexpressionsofleptin,PPARγ,andβ-actinweredeterminedbyRT-PCR.
cells,andhumangastriccancerSGC-7901cellsatacon- monocyte macrophage RAW 264.7 cells were 22.2, 32.2,
centration of 10 μg/ml [39], but yakuchinone A induces and 46.4 μM, respectively (Figure 4). Yakuchinone A
apoptotic death in HL-60 cells [23]. Nevertheless, cyto- appeared to have a more potent inhibitory effect on
toxic effects of yakuchinone A on skin cancer cells re- non-melanoma skin cancer (NMSC) cells (A431, BCC,
main poorly understood. In this work, the inhibition and SCC25) and cell viability than in melanoma cells
potential of yakuchinone A on human skin cancer cells (A375andB16),noncancerouscells(HaCaTandBNLCL2),
(epidermoid carcinoma A431 cells, basal cell carcinoma and RAW 264.7 cells. Previous studies have demonstrated
BCC cells, squamous cell carcinoma SCC25 cells and that yakuchinone A has a phenolic diarylheptanoid
malignant melanoma A375 cells) and mouse melanoma moiety with a carbonyl functional group to suggest
B16 cells was determined by MTT assay and morpho- that yakuchinone A is anticipated to exhibit potential
logical change. Treatment these cells with yakuchinone cancer chemopreventive activities [39]. These experi-
A (5, 10, 20, 30, 40, and 50 μM) for 72 h resulted in a mental data further suggest that yakuchinone A has an
dose-dependent significant cell death (Figure 3). The antioxidant affect that exhibits less toxic to noncancerous
IC values of yakuchinone A were 13.3, 11.3, 18.7, 23.8, cellsandselectivecytotoxicitytoNMSCcells.
50
and 40.0 μM for A431, BCC, SCC25, A375, and B16 The cell death induction by yakuchinone A was fur-
cells, respectively. Moreover, after 72 h treatment with ther confirmed by cellular morphological examination.
yakuchinone A (5, 10, 20, 30, 40, and 50 μM), the IC After exposure of 20 μM yakuchinone A to BCC cells at
50
values of yakuchinone A against noncancerous cells (hu- 72 h, distinct cytoplasmic shrinkage, cell bodies became
man premalignant keratinocytic HaCaTcells and mouse rounded and detached from the surface under phase-
embryonic liver BNLCL2 cells) and mouse leukemic contrast-inverted microscopic examination (Figure 5A).
Linetal.BMCComplementaryandAlternativeMedicine2013,13:237 Page9of13
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120 120 120
A431 BCC SCC25
Control Control Control
100 5 M 100 5 M 100 5 M
%) 10 M %) 10 M %) * 10 M
20 M 20 M 20 M
( ( (
y 80 * 30 M y 80 30 M y 80 30 M
bilit * 4500 MM bilit * 4500 MM bilit * 4500 MM
a 60 a 60 * a 60
vi vi vi
ell 40 * ell 40 * ell 40
C * C C *
* *
20 * 20 20
* * * *
0 0 0
Control YA Control YA Control YA
120 A375 120 B16
Control Control
5 M 5 M
) 100 10 M )100 10 M
% * * 20 M % 20 M
bility ( 80 * 345000 MMM bility ( 80 * 345000 MMM
a 60 a 60 *
vi vi
ell 40 * ell 40 *
C * C
20 20
*
0 0
Control YA Control YA
Figure3EffectofyakuchinoneAoncellviabilityinskincancercells.CellviabilityofyakuchinoneA(5,10,20,30,40,and50μM)toskin
cancer(A431,BCC,SCC25,A375,andB16)cellsfor72h,andassessedbyMTTassay.Eachvalueispresentedasmean±SDofthreeindividual
experiments;*p<0.05indicatesasignificantdifferencefromvehiclecontrol(DMSO)-treatedcells.YakuchinoneA;YA.
Treatment of BCC cells with yakuchinone A showed mitochondrial functions, expression levels of anti-
chromatin condensation and nuclear fragmentation by apoptotic protein Bcl-2, and pro-apoptotic protein Bax
Hoechst 33342 staining under a fluorescent microscope, were determined. Bcl-2 expression was time-dependent
indicating apoptosis (Figure 5A). Bcl-2 family members decreased; whereas, bax was increased and investigated
are major apoptosis-regulating proteins [40]. Given that by RT-PCR following the exposure of BCC cells to
the Bcl-2 family proteins are known mediators of yakuchinone A (20 μM) for 24 and 48 h (Figure 5B).
HaCaT RAW 264.7 BNLCL2
120 Control 120 Control 120 Control
5 M 5 M 5 M
10 M 10 M 10 M
%) 100 2300 MM %) 100 2300 MM %) 100 * 2300 MM
bility ( 80 * 4500 MM bility ( 80 * * 4500 MM bility ( 80 * 4500 MM
via 60 * via 60 via 60 *
Cell 40 * * Cell 40 * Cell 40
*
20 20 20
0 0 0
Control YA Control YA Control YA
Figure4EffectofyakuchinoneAoncellviabilityinnoncancerouscells(HaCaTandBNLCL2)andRAW264.7cells.Cellsweretreated
withyakuchinoneA(5,10,20,30,40,and50μM)for72h.CellviabilitywasevaluatedwithMTTassay.Eachvalueispresentedasmean±SDof
threeindividualexperiments;*p<0.05indicatesasignificantdifferencefromvehiclecontrol(DMSO)-treatedcells.YakuchinoneA;YA.
Linetal.BMCComplementaryandAlternativeMedicine2013,13:237 Page10of13
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B)
Yakuchinone A
A)
Yakuchinone A 0 24 48 h
st
a
r
nt
o
c
se C) Yakuchinone A
a
h
P
e
c
n
e
c
s
e
r
o
u
Fl
Figure5ExpressionofBcl-2andbax-dependentapoptoticpathwayinBCCcellsafteryakuchinoneAtreatment.A)Morphological
changesinducedbyyakuchinoneAinBCCcells.BCCcellsweretreatedwithyakuchinoneA(20μM)andvehiclecontrol(DMSO)for72h,and
thenthenuclearwasstainedwithHoechst33342.Apoptoticcells(arrows)werecharacterizedbycellularshrinkageandroundedcellbodies
(phase-contrast-invertedmicroscopic,200×).Underafluorescentmicroscope,apoptoticcells(arrows)werecharacterizedbymarkednuclear
condensation,shrinkingandfragmentation(200×).B)EffectofyakuchinoneAonbcl-2andbaxexpressions.BCCcellsweretreatedwith
yakuchinoneA(20μM)andvehiclecontrol(DMSO)for24and48h,andthebcl-2,baxandβ-actinexpressionsweredeterminedbyRT-PCR.
C)ExpressionsofBcl-2,BaxandPARPoncellsafteryakuchinoneAtreatment.BCCcellsweretreatedwith(+)orwithout(−)yakuchinoneA
(20μM)for72h,andtheBcl-2,Bax,PARPandβ-actinexpressionwereassessedbyWesternblotting.
These experimental results are consistent with the effectof27%ofH.nana.Treatment with yakuchinone A
yakuchinone A (20 μM) applied for 72 h by Western (a concentration of 50, 100 μM but not 10 μM) for 48
blotting (Figure 5C). Cleavage of the poly (ADP-ribose) and 72 h reduced the oscillation up to 21% and 31% or
polymerase (PARP) in BCC cells after yakuchinone A 47% and 73%, respectively. Yakuchinone A slowly re-
treatment gave further evidence that apoptosis happened duced oscillation from 2 to 72 h but did not cause
becausetheactiveformofPARP,aproteinassociatedwith death. Yakuchinone A reduced the oscillation activity
DNA repair, is considered as a hallmark of apoptosis. of H. nana in a time- and dose-dependent manner for
These results suggest that yakuchinone A-induced cell 24 to 72 h (Figure 6A).
deathismainlyduetoapoptosis. The effect of yakuchinone A over time of the peristal-
sis activity of H. nana was investigated (Figure 6B). For
CestocidalactivityagainstH.nana peristalsis activity assay, a dose- and time-dependent ef-
Figure 6 plots the time course of oscillation and peristal- fect for 24 to 72 h was also observed by treatment with
sis during yakuchinone A treatment. In oscillation activ- yakuchinone A. Treatment for 48 h with 50 and 100 μM
ity assay, the percentage of oscillation for the vehicle yakuchinone A stopped peristalsis in more than approxi-
control (0.1% DMSO) decreased by about 18% from mately 21 to 25% of worms. Yakuchinone A at 50 and
72 h cultivation (Figure 6A). However, in the peristalsis 100μM slowly reduced peristalsis from 2 to 72 h. Treat-
activity assay, the percentage of peristalsis for the vehicle ment with 10 μM yakuchinone A for 72 h reduced peri-
control (0.1% DMSO) decreased by 31% from 72 h culti- stalsis to 22% (Figure 6B). This effect on peristalsis is
vation (Figure 6B). The change of peristalsis of H. nana stronger than on oscillation activity. The above perfor-
wasmoresensitivethanthatofoscillationviatreatmentof mances were the same for other concentrations of
vehicle. Treatment with 10, 50, and 100 μM yakuchinone yakuchinone Ainperistalsisactivity.
Ahasagreatereffectonperistalsisthanoscillationfor24,
48,and72h.Peristalsisactivitydisappearedbeforeoscilla- NematocidalactivityagainstA.simplex
tion activity was lost when H. nana was dead. In fact, H. In the first series of experiments, the larvicidal effects
nanahasnoperistalsisoroscillationeffectwhendead. were used to study the ability of yakuchinone A to alter
In the oscillation activity assay (Figure 6A), exposure survival of AsL3. The time course of the yakuchinone
to100μMyakuchinoneAfor72hcausedthemaximum A-induced loss of mobility on AsL3 was also studied.