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Antioxidant, anti-adipocyte differentiation, antitumor activity and anthelminticactivities against Anisakis simplex and Hymenolepis nana ofyakuchinone A from Alpinia oxyphylla
BMC Complementary and Alternative Medicinevolume 13, Article number: 237 (2013)
Alpinia oxyphylla is a common remedy in traditional Chinese medicine.Yakuchinone A is a major constituent of A. oxyphylla andexhibits anti-inflammatory, antitumor, antibacterial, and gastric protectiveactivities.
Antioxidant and antitumor characteristics of yakuchinone A in skin cancer cells aswell as novel mechanisms for the inhibition of adipocyte differentiation,cestocidal activities against Hymenolepis nana adults, and nematocidalactivities against Anisakis simplex larvae are investigated.
Yakuchinone A presents the ability of the removal of DPPH·andABTS+ free radicals and inhibition of lipid peroxidation.Yakuchinone A suppresses intracellular lipid accumulation during adipocytedifferentiation in 3 T3-L1 cells and the expressions of leptin andperoxisome proliferator-activated receptor γ(PPAR γ). Yakuchinone A induces apoptosis and inhibits cellproliferation in skin cancer cells. The inhibition of cell growth by yakuchinone Ais more significant for non-melanoma skin cancer (NMSC) cells than for melanoma(A375 and B16) and noncancerous (HaCaT and BNLCL2) cells. Treatment BCC cells withyakuchinone A shows down-regulation of Bcl-2, up-regulation of Bax, and anincrease in cleavage poly (ADP-ribose) polymerase (PARP). This suggests thatyakuchinone A induces BCC cells apoptosis through the Bcl-2-mediated signalingpathway. The anthelmintic activities of yakuchinone A for A. simplex arebetter than for H. nana.
In this work, yakuchinone A exhibits antioxidative properties, anti-adipocytedifferentiation, antitumor activity, and anthelmintic activities against A.simplex and H. nana.
Free radicals include superoxide anion (O2 -), hydroxyl (HO·),peroxyl (ROO·), alkoxyl (RO·) and nitric oxide, which are oxygen-centered freeradicals occasionally known as reactive oxygen species (ROS). Cellular oxidative damagethat is caused primarily by ROS is a well-established general mechanism for cell as wellas tissue injury [1, 2]. ROS are strongly associated with lipid peroxidation, which leads to thedeterioration of the food, and are also involved in a variety of diseases includingcellar aging, mutagenesis, carcinogenesis, coronary heart disease, diabetes mellitus,and neurodegeneration .
Obesity has become a global health problem due to its association with various metabolicdisorders such as type-II diabetes, cardiovascular disease, hypertension, andnon-alcoholic fatty liver disease [3, 4]. Synthetic anti-obesity drugs have been reported to be costly, and some ofthem also beset with undesirable side effects. Therefore, developing drugs to directlymodulate energy metabolism without affecting the central nervous system has causedsubstantial attention [4, 5].
Natural/herbal compounds including berberine, resveratrol, and curcumin are known tomodulate obesity either through increasing energy expenditure or inhibiting adipocytedifferentiation [6–8]. Presently the focus is to develop natural compounds as antioxidants that arepossibly used to reduce damage caused by oxidative stress, age-dependent diseases, andobesity .
Hymenolepis nana is a general occasion of cestode infections, and is foundworldwide. In human adults, the tapeworm is more of a nuisance than a health problem,but in small children, H. nana is dangerous. It is often seen in children incountries with inadequate sanitation and hygiene. H. nana infections aretypically asymptomatic but heavy infections also cause headaches, anorexia, weakness,abdominal pain, and diarrhea . H. nana is the only cestode without any intermediate hosts in itslife cycle . H. nana infection is typically acquired from eggs in the feces fromanother infected individual, which are transferred by contaminated food. Eggs hatch inthe duodenum, releasing oncospheres that penetrate the mucosa and enter the lymphchannels of the villi. Then, oncospheres develop into a cysticercoid, which has a tailand a well formed scolex. About five to six days cysticercoids migrate into the lumen ofthe small intestine and attach before maturing. Eggs of H. nana infect whenpassed with stool and transfer in contaminated food. Eggs are ingested by an arthropodintermediate host and hatch in the duodenum, releasing oncospheres, and develop intocysticercoid larvae. Upon rupture of the villus, the cysticercoids return to theintestinal lumen, evaginate their scoleces, attach to the intestinal mucosa, and matureinto adults that reside in the ileal portion of the small intestine, producing gravidproglottids. The eggs are then passed in stools when released from the proglottids ordisintegration of proglottids in the small intestine. An alternate mode of infectionconsists of internal autoinfection without passing through the external environment. Theshort life span and rapid course of development also facilitates the spread and readyavailability of this worm, but internal autoinfection allows the infection to continuefor years [11, 12].
Anisakis simplex adult worms mature and release eggs from the primary host. Theeggs pass from stool into seawater and are embryonated to form A. simplexfirst-stage larvae (AsL1) and subsequently moulted to A. simplex second-stagelarvae (AsL2). When larvae are ingested by small crustacean first intermediate hosts,the AsL2 matures into A. simplex third-stage larvae (AsL3) that aresubsequently consumed by second intermediate hosts such as marine fish or squid. TheAsL3 migrate into the viscera and peritoneal cavity. The degree of migration into thefish musculature depends on environmental conditions and/or the species of parasite andfish condition [13, 14]. AsL3 are repeatedly transferred between fish and fish through the foodchain. Therefore, piscivorous fish accumulate large numbers of AsL3 . Finally, the ingestion of infected fish or squid by a marine mammal (i.e.the final host) leads to the development of fourth-stage larvae and then adults. Humansmay be accidental hosts by consuming undercooked and/or raw second intermediate hoststhat contain AsL3. A. simplex rarely develop further within the humangastrointestinal tract, instead, by means of proteolytic enzymes, but they typicallyembed in the gastric or intestinal mucosa and die or invasion the muscular layers of thestomach and intestine to induce allergic reactions and a variety of abdominal symptomsthat are characterized as anisakiasis or anisakidosis . The four main clinical syndromes in humans who experience symptomaticanisakidosis include gastric, intestinal, extra-gastrointestinal, and allergic diseases.Anisakidosis is globally recognized as a public health problem, which is relative toAsia and Europe [16, 17]. The prevalence of anisakidosis has increased unusually because of theincreasing popularity of Japanese cuisine, such as “sushi” and“sashimi”. The availability of an anthelmintic compound against A.simplex has the potential to shorten the clinical course and prevent mechanicalinvasion that cause from endoscopic procedures. Because few effective studies foranthelmintic drugs and nature compounds against A. simplex, the effectivenessof treatment with anthelmintic agents, antibiotics, anticholinergics, and/orcorticosteroids against A. simplex remains controversial .
Alpinia oxyphylla is an important traditional Chinese medicinal herb whosefruits are widely used as a tonic, aphrodisiac, anti-salivation, anti-polyuria, andanti-diarrhea . The extracts from A. oxyphylla possess neuroprotective activity,anti-tumor, anti-anaphylactic, and inhibition of nitric oxide production [19, 20]. Yakuchinone A[1-(4′-hydroxy-3′-methoxyphenyl)-7-phenyl-3-heptanone], a major pungentingredient derived from A. oxyphylla exhibits anti-inflammatory,antitumor, antibacterial, antiviral, and gastric protective activities . Yakuchinone A has been reported to be a strong inhibitor of prostaglandinbiosynthesis in vitro. Moreover, yakuchinone A can act as an anti-tumor promoter as determined bythe ability to suppress phorbol ester-induced activation of ornithine decarboxylase(ODC) and inhibits the promotion of papilloma formation in mouse skin . 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated superoxidegeneration and tumor necrosis factor-α (TNF-α) or interleukin-1αproduction in human promyelocytic leukemia (HL-60) cells as well as on DNA binding ofactivator protein 1 (AP-1) in mouse fibroblast (NIH3T3) cells are also suppressed byyakuchinone A [23, 24]. Furthermore, yakuchinone A induces apoptotic death in HL-60 cells accountfor the antiproliferative activity . However, the biochemical mechanisms underlying the antioxidant,anti-obesity, anti-skin cancer effects of yakuchinone A and its cestocidal effects onH. nana and larvicidal effects on A. simplex remain unclear. Thisstudy confirms the antioxidant and antitumor effects of yakuchinone A and elucidates thenovel mechanisms for its inhibition of adipocyte differentiation as well as itsanthelmintic activities against H. nana and A. simplex.
1,1-Diphenyl-2-picrylhydrazyl (DPPH•),2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt(ABTS•+), 2,5,7,8-tetramethylchroman carboxylic acid (trolox),trichloracetic acid (TCA), 2-thiobarbituric acid (TBA) and3-isobutyl-1-methylxanthine (IBMX) were purchased form Sigma Chemical Co. (Sigma, St.Louis, MO).
Extraction and isolation
The “Yizhiren”, A. oxyphylla, was supplied from Kwong-Te Co.,Kaohsiung, Taiwan and was identified by professor Hang-Ching Lin of the NationalDefense Medicinal Center, where a voucher specimen was deposited (CNUPS No.970801).The dry powder of A. oxyphylla seed (6.0 kg) was extracted with 95%ethanol at room temperature. After removal of the solvent by evaporation, the residue(559.0 g) was dissolved in methanol–water (9.5:0.5) and partitioned withn-hexane. The methanol (95%) was removed by evaporation and the residuewas then suspended in water and partitioned with ethyl acetate (359.0 g). Theethyl acetate layer was subjected to LH-20 Sephadex and eluted with methanol. Eachfraction collected from the column was monitored by thin-layer chromatography and thesimilar fractions were combined to produce 4 fractions. The fraction 3 was furtherpurified by a silica gel and eluted with n-hexane-ethyl acetate (9:1,7.5:2.5, 1:1, 2.5:7.5), ethyl acetate, ethyl acetate-methanol (9:1), methanol toisolate yakuchinone A (276.1 mg). Their structures were confirmed by NMR andmass spectra analysis.
Yakuchinone A: slightly yellow oil; EI/MS m/z (rel. int.%): 312(80,[M]+), 194 (6), 179 (45), 161 (14), 151 (33), 137 (100), 119 (23);1H-NMR (CDCl3, 500 MHz) δ: 1.60 (4H, m, H-5,6),2.40 (2H, t, J =7.0 Hz, H-4), 2.60 (2H, t, J =7.0 Hz,H-7), 2.68 (2H, t, J =7.6 Hz, H-2), 2.82 (2H, t, J=7.6 Hz, H-1), 3.86 (3H, s, OCH3), 6.66 (1H, dd, J =8.0,2.0 Hz, H-6’), 6.68 (1H, d, J =2.0 Hz, H-2’), 6.83(1H, d, J =2.0 Hz, H-5), 7.15 ~ 7.20 (3H, m, H-3”,4”, 5”), 7.26 ~ 7.29 (2H, m, H-2”, 6”);13C-NMR (CDCl3, 125 MHz) δ: 210.3 (C-3), 146.3(C-3′), 143.8 (C-4′), 142.1 (C-1″), 133.0 (C-1′), 128.2(c-2″, 6″), 128.3 (C-3″, 5″), 125.7 (C-4″), 120.7(C-6′), 114.3 (C-5′), 111.0 (C-2′), 55.8 (OCH3), 44.6(C-2), 42.9 (C-4), 35.7 (C-7), 30.9 (C-6), 29.5 (C-1), 23.3 (C-5). These data werecompared with literature values . The chemical structure of yakuchinone A was shown in Figure 1A. The purity of yakuchinone A is 99.2%. The solubility ofyakuchinone A was 100 mM in dimethylsulfoxide (DMSO).
Assay for free radical scavenging ability against DPPH· andABTS+
The radical scavenging activities of yakuchinone A againstDPPH · and ABTS · + radicals weremeasured by using the method as previously reported . For DPPH · radical scavenging activity analysis, 5, 10,20, 30, 40, 50, and 100 μM yakuchinone A (10 μl of solution) wasmixed with 90 μl of DMSO and 900 μl of ethanolicDPPH · solution (0.1 mM). After incubation in darkness at25°C for 30 min, the absorbance (A) was determined at 517 nm(Hitachi U-2001, Japan). For ABTS•+ radical scavenging activityanalysis, ABTS · + was dissolve in water to 7 mM.ABTS · + radical was produced by reactingABTS · + stock solution with 2.45 mM potassiumpersulfate, and the mixture stood in the dark at room temperature for12–16 h. The ABTS · + radical solution wasdiluted to an absorbance of 0.70 ± 0.02 at 734 nm at 30°C.Each agent (0.1 ml) reacted with 2.9 ml of dilutedABTS · + radical solution for 20 min at30°C, and then the absorbance was measured at 734 nm (Hitachi U-2001,Japan). The TEAC (trolox equivalent antioxidant capacity) of the reagent wascalculated by comparing their reactivities to the standard antioxidant, trolox.Ethanol or distilled water was used as negative controls. Trolox was used as astandard antioxidant. The scavenging ability of yakuchinone A or trolox inDPPH · and ABTS · + was calculated usingthe following equation:radical scavengingability (%) = (1 - A sample/A control) × 100.EC50 values were estimated from the percent inhibition versusconcentration plot derived from the percentage scavenging activity. This data wasshown as mean values ± standard deviation(n = 3).
Determination of antioxidant effect on liposome peroxidation
The effect on liposome peroxidation was assayed by measuring concentrations ofthiobarbituric acid reactive substances (TBARS). Liposomes were prepared according tothe method of Chou et al. . In brief, the liposomes were obtained by dispersing lipids indemineralized water (1:10). For the assay, 32 μl of suspension of liposomeswas incubated together with 11 μl of 10 mM FeSO4,11 μl of 10 mM ascorbic acid and appropriate amounts of differentconcentrations (5, 10, 20, 30, 40, 50 and 100 μM) of yakuchinone A, troloxand rutin in 1.515 ml of 50 mMNa2HPO4-NaH2PO4 buffer, pH 7.4(2.5 ml final solution) at 37°C for 1 h. Lipid peroxidation wasterminated by the reaction of 0.8 ml of 1% TBA and 10% TCA and 106 μlof 0.1 M ethylene diamine-tetraacetic acid disodium salt dehydrate at 100°Cfor 20 min. After cooling and centrifugation (2600 g for10 min), the malonaldehyde (MDA)-TBA complex was determined by measuring theabsorbance (A) at 532 nm. A control with DMSO instead of sample wasalso analyzed and expressed no activity. Trolox and rutin were utilized as standards.The percentage inhibition was calculated using the following equation:Inhinition of lipid peroxidation(%) = (1 - A sample/A control) × 100.EC50 values were estimated from the percentage inhibition versusconcentration plot. This data was shown as mean values ± standarddeviation (n = 3).
Human epidermoid carcinoma A431, human oral squamous cell carcinoma SCC25. human skinmalignant melanoma A375, mouse melanoma B16, mouse leukemic monocyte macrophage RAW264.7, mouse normal embryonic liver BNLCL2 cells, and 3 T3-L1 preadipocytes werepurchased from the American Type Culture Collection (Rockville, MD). Human basal cellcarcinoma BCC and human premalignant keratinocytic HaCaT cells were kindly donated byProf. Hamm-Ming Sheu (National Cheng Kung University Medical College, Tainan,Taiwan). Cells were cultured in medium supplemented with 10% fetal bovine serum(Hazelton Product, Denver, PA) and 1% penicillin-streptomycin at 37°C in 5%CO2 humidified atmosphere; specifically, A431, A375, B16, HaCaT, RAW264.7, BNLCL2, and 3 T3-L1 cells were maintained in DMEM medium (GIBCO, GrandIsland, NY), BCC cells in RPMI medium, and SCC25 in DMEM/F12 medium supplemented with0.4 μg/ml hydrocortisone (Sigma, St. Louis, MO).
Cultivation of 3 T3-L1 cells and their conversion to adipocytes were carried outaccording to the method as described previously . To induce differentiation, four day postconfluent 3 T3-L1preadipocytes were stimulated for 72 h in 10% FBS/DMEM with containing the MDIhormone mixture (0.5 mM IBMX, 1 μM dexamethasone, and10 μg/ml of insulin) in six-well plates. After four days, the medium wasreplaced with 10% FBS/DMEM medium containing 10 μg/ml of insulin. Themedium was replaced with fresh medium (10% FBS/DMEM, 10 μg/ml of insulin)every two days until analysis on day eight. Yakuchinone A (5 μM) was addedduring the differentiation process.
Oil Red O staining
Differentiated 3 T3-L1 cells were stained using the Oil Red O method  for adipocyte lipid accumulation. At day eight of differentiation, thecells were washed with PBS and fixed with 10% formaldehyde for 2 h. The fixedcells were washed with 60% isopropanol, and stained with 0.2% Oil Red O for10 min. The plates were rinsed three times with water and examined under a phasecontrast inverted light microscope (Nikon, TE2000-U, Japan). After thorough washingwith water and evaporation of excess water, Oil Red O was extracted in isopropylalcohol and the absorbance was monitored at 520 nm (BioTek,Synergy™2).
Cells (1 × 105 cells/ml) were plated in 100 μlof 96-well multidishes and treated with a series of concentrations (5, 10, 20, 30,40, and 50 μM) of yakuchinone A or vehicle control (DMSO) for 72 h.The control groups were treated with DMSO, and the final DMSO concentration did notexceed 0.1%. The cell viability was measured by performing the MTT[3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay . The IC50 values were calculated from the agent concentrationsthat yielded a cell viability of 50%.
Cell morphological changes
Cells (1 × 105 cells/ml) were plated in 24-well platesthen treated with vehicle control (DMSO) or yakuchinone A (20 μM) for72 h. Cells in each well were washed once with 1× PBS, and analysis wasperformed using a phase contrast inverted light microscope (Nikon, TE2000-U, Japan).To assess specific apoptosis, after incubation, cells were washed by PBS and fixedwith 4% paraformaldehyde and stained with Hoechst 33342 (0.1 μg/ml) (Sigma)at 37°C for 10 min in the dark. The nuclear morphology changes were viewedunder a fluorescent microscope (Nikon, TE2000-U, Japan).
RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)analysis
3 T3-L1 cells were treated with vehicle control (DMSO) or yakuchinone A(5 μM) during the differentiation process. BCC cells(1 × 105 cells/ml) were treated with vehicle control(DMSO) or yakuchinone A (20 μM) for 24 and 48 h. Total RNA wasprepared from cells using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), and aRT-PCR was conducted using 3 μg of total RNA and the Superscript cDNAPreamplification System (Weiterstadt, Germany) according to the manufacturers’instructions. The following primers were utilized: right primer 5′-GCT CTA GACGTG ACA ATC TGT CTG AGG TCT GTC AT-3′ and left primer 5′-CGG CAT CCG TTGTCG GTT TCA CAA ATG CCT TGC AGT G-3′ for PPAR γ (870 bp), rightprimer 5′-CAT CTG CTG GCC TTC TCC AA-3′ and left primer 5′-ATC CAGGCT CTC TGG CTT CTG-3′ for leptin (71 bp), right primer 5′-AGA TGTCCA GCC AGC TGC ACC TGA C-3′ and left primer 5′-AGA TAG GCA CCC AGG GTGATG CAA GCT-3′ for bcl-2 (367 bp), right primer 5′-AAG CTG AGC GAGTGT CTC AAG CGC-3′ and left primer 5′-TCC CGC CAC AAA GAT GGT CACG-3′ for bax (366 bp), and right primer 5′-ACC CAC ACT GTG CCC ATCTA-3′ and left primer 5′-CGG AAC CGC TCA TTG CC-3′ for β-actin(286 bp). The amplified RT-PCR products were analysed in 2% agarose gels,visualized by ethidium bromide staining and photographed under ultraviolet light.
Cells (1 × 105 cells/ml) were treated with vehiclecontrol (DMSO) or yakuchinone A (20 μM) for 72 h. Then, cells werewashed with PBS, and lysed in lysis buffer [50 mM Tris–HCl, pH 7.5,1% Triton X-100, 5 mM EGTA (ethyleneglycol-bis(2-aminoethylether)-N,N,N’,N’-tetraaceticacid), 150 mM NaCl and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. Aftercentrifugation (10,000 g, 10 min), supernatants were collected.The cell lysates containing 40 μg of solubilized protein were subjected to12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andelectrophoretically transferred to nitrocellulose membranes. The membranes wereblocked in 5% skim milk. Blots were incubated with the antibodies against Bcl-2, Bax,PARP and β-actin (Santa Cruz, CA). The membranes were incubated with theappropriate secondary antibody conjugated with horseradish peroxidase (Bio-Rad,Hercules, CA). Blotted antibodies were visualized by chemiluminescence method (ECLkit, Amersham).
Preparation of H. nana adult worms
H. nana adult worms were obtained from each part of the intestines of wildtype mice, purchased from Lin’s farm in Fengshan, Kaohsiung, Taiwan. Theseparts of the intestine were duodenum, jejunum, ileum, colon and rectum. The H.nana had an average length of 5–50 mm and was collected using aneedle with a blunt tip, before being placed in Petri dishes with 0.9% NaCl andgentamycin (10 mg/ml). They were then washed several times. The adult worms wereindividually observed under an inverted microscope, with subsequent discarding ofthose that exhibited internal or external damage. The adult worms were thenidentified by their morphological features, divided into groups and placed in 24-wellplates contained cultivated media RPMI-1640 plus 20% FBS, pH 7.4, in anatmosphere of 95% O2/5% CO2, 37°C. These cultureconditions have been shown to maximize the development and survival of H.nana.
Assay of cestocidal activity of oscillation and peristalsis test on H.nana
The above H. nana cultivated media were supplemented with L-glutamine(2 mM), penicillin (100 IU/ml), streptomycin (100 mg/ml) andamphotericin B (0.25 μg/ml), and then the effects of yakuchinone A atconcentrations of 10, 50 and 100 μM were tested. The survival and mobilityof the adult worm were assessed at 2, 4, 6, 12, 24, 48, and 72 h using astereomicroscope. They were observed for their spontaneous motility and evokedresponses at 2, 4, 6, 12, 24, 48, and 72 h using a stereomicroscope. Theoscillation and peristalsis states of adult worms were scored blindly by twoinvestigators. Cestode activity was scored by monitoring both oscillation andperistalsis. Oscillation was scored of movement at scolex and neck for each secondfor 30 seconds, and then the highest score was 30. Peristalsis was record thecontraction real times at scolex and neck. All data were compared with the initialtime before the test compounds had been added. Worms death and complete standstill asdetermined by none any oscillation and peristalsis changes for 30 seconds wereidentified. The mortality was recorded after ascertaining that the worms neithermoved when shaken vigorously nor when dipped in warm medium .
A. simplex larvae preparation
The AsL3 were obtained from the muscle and peritoneum of fresh Trichiuruslepturus s (largehead hairtail, Atlantic cutlassfish) that were purchased fromthe fish market of Kaohsiung, Taiwan. The AsL3 had an average length of20–22 mm, and were collected using a needle with a blunt tip, placed inPetri dishes with 0.9% NaCl and washed several times. The majority of the larvae wereencysted, but they quickly became excysted upon washing in NaCl solution. They wereindividually observed under an inverted microscope, with subsequent discarding ofthose that exhibited internal or external damage. The larvae were then identified bymorphological features, divided into groups and placed in 24-well plates containedcultivated media RPMI-1640 plus 20% FBS, pH 4.0, in an atmosphere of 95%O2/5% CO2, 37°C. These showed culture conditionsdemonstrated to provide for the maximum development and survival of A[18, 32].
Assay of nematocidal activity on A. simplex
The above AsL3 cultivated media were supplemented with L-glutamine (2 mM),penicillin (100 IU/ml), streptomycin (100 mg/ml) and amphotericin B(0.25 μg/ml), and tested of yakuchinone A for 10, 100, and200 μM. The survival and mobility of the larvae were assessed at 2, 4, 8,12, 24, 48 and 72 h using a stereomicroscope. Two investigators blindly scoredthe larvae as dead, with poor mobility or with normal mobility. The percentage lossesof spontaneous motion during 3 min periods immediately after incubation andcomplete standstill were determined by stimulation 4–5 h later (defined asdeath). The mortality was recorded after ascertaining that the worms neither movedwhen shaken vigorously nor when dipped in warm medium. The nematocidal activity wasmodified according to a scoring system that was developed by Kiuchi et al.  and Lin et al. .
The results are expressed as mean ± standard deviation (SD).Statistical differences were estimated by one-way analysis of variance (ANOVA)followed by Dunnett’s test or the Tukey-Kramer test. A p value of 0.05was regarded as significant. The data were analyzed and the figures plotted usingsoftware (SigmaPlot Version 8.0 and SigmaStat Version 2.03, Chicago, IL).
Results and discussion
Free radical scavenging activity of yakuchinone A
The DPPH · and ABTS · + radical has beenwidely used for assessment of radical scavenging because of the easy and convenientconsideration . The soluble free radical DPPH · is well known as a goodhydrogen abstractor that yields DPPH-H as a by-product. Thus, the scavenging of DPPHradicals by phenols is effective. The antioxidant activity of yakuchinone A andtrolox (a positive control) was measured based on scavenging activities for stableDPPH radical as presented in Figure 1B. With increasingdoses from 5 to 100 μM of yakuchinone A and trolox, the values ofDPPH · scavenging activity were 9.6%, 29.2%, 34.5%, 44.6%, 60.0%,64.5%, and 70.7% for yakuchinone A and 14.5%, 27.8%, 55.9%, 77.7%, 95.0%, 96.3%, and97.4% for trolox, respectively. The EC50 values of yakuchinone A for thescavenging of DPPH · radicals were 33.5 (yakuchinone A) and17.9 μM (trolox). The generation of ABTS · +involves the direct production of the blue/green ABTS · +chromophore through the reaction of potassium persulfate and ABTS. The addition ofhydrogen-donating antioxidants to the preformed radical reduces it to ABTS . Figure 1C shows the scavenging activity ofyakuchinone A towards ABTS · +. As increasing doses of 5,10, 20, 30, 40, 50, and 100 μM of yakuchinone A and trolox, the values ofABTS · + scavenging capacity were 5.7%, 11.7%, 22.5%,31.0%, 49.6%, 63.6%, and 70.6% for yakuchinone A and 21.9%, 27.3%, 47.6%, 49.6%,75.1%, 92.2%, and 98.0% for trolox, respectively. The EC50 values for thescavenging of ABTS · +-radicals were 40.2 (yakuchinone A)and 30.1 μM (trolox). The extent of decolorization as percentage inhibitionof the ABTS · + radical cation was proportional to theconcentration of antioxidants and calculated relative to the reactivity of trolox asa standard (TEAC). The TEAC value derived from the dose–response curve foryakuchinone A was 3.4 mM of trolox/g. These results suggest that yakuchinone Aexhibits an antioxidant capacity to scavenge DPPH · andABTS · + free radicals.
Potential of yakuchinone A to inhibit lipid peroxidation
The antioxidant action is assessed by inhibiting the damage caused by free radicalsand the mechanisms involved in many human diseases such as hepatotoxicities,hepatocarcinogenesis, diabetes, malaria, acute myocardial infarction, and skin cancerto include lipid peroxidation as a main source of membrane damage . Lipid peroxidation in biological systems has been thought to be atoxicological phenomenon that leads to various pathological consequences. MDA formedfrom lipid peroxidation of unsaturated phospholipid reacts with TBA to produce a pinkMDA-TBA adducts. MDA is reactive and active in cross-linking with DNA and proteinsand damages liver cells . Phospholipids are believed to be present in high amounts in cellmembranes . The phospholipid prepared as a liposome was used to evaluate the effectof yakuchinone A on liposome peroxidation to investigate yakuchinone A in abiological system. Figure 1D presents the inhibition oflipid peroxidation by yakuchinone A (5, 10, 20, 30, 40, 50, and 100 μM)depended on dose. The EC50 values of the inhibition of lipid peroxidationefficiency by yakuchinone A, trolox and rutin were 10.3, 14.3 and 6.2 μM,respectively. Although the inhibition of lipid peroxidation activity by yakuchinone Awas weaker than by rutin, the inhibition efficiency of yakuchinone A exceeded trolox.The MDA lowering effect of yakuchinone A indicates a protective action against lipidperoxidation of unsaturated phospholipids.
Inhibition of lipid accumulation by yakuchinone A in 3 T3-L1 adipocytes
Numerous studies show that obesity may induce systemic oxidative stress, and theincrease in ROS in adipocytes contributes to deregulated expression of inflammatorycytokines such as tumor necrosis factor-α, which may be an early instigator ofthe obesity-associated diabetes and cardiovascular disease [37, 38]. This work demonstrates that yakuchinone A exhibits anti-oxidationactivities, suggesting yakuchinone A has an inhibitory effect on adipogenesis.3 T3-L1 adipogenic differentiation requires a network of adipogenic markers . We examined the ability of the yakuchinone A to prevent adipogenesis in3 T3-L1 adipocytes. The amount of accumulated intracellular lipid droplets werecompared in differentiated 3 T3-L1 cells after treatment in a MDI mixture anddifferentiated cells. The amount of intracellular lipid droplets increased indifferentiated 3 T3- L1 cells, as shown by the Oil Red O staining(Figure 2A). However, incubation of differentiatedcells with low concentration of yakuchinone A (5 μM) decreased MDI-inducedlipid accumulation. This result was further supported by quantitativespectrophotometric analysis of cellular neutral lipid content. Figure 2B shows lipid accumulation was significantly inhibited in thepresence of 5 μM yakuchinone A. The level of lipid accumulation over eightdays was 19.2% of the MDI-treated positive control cells. Adipocytokines areadipocyte-derived hormones, such as leptin and adiponectin, which modulated hepaticand peripheral lipid and glucose metabolism . The amount of leptin secreted in the adipose tissue is positivelycorrelated with the lipid content and adipocyte size . Furthermore, previous research has established that adenosine5′-phosphate-activated protein kinase (AMPK) and peroxisomeproliferator-activated receptor γ (PPARγ) appears to be involved inadipocyte differentiation and maturation. This can be potential drug targets for thetreatment of obesity . We evaluated the yakuchinone A-induced changes in the expression ofadipose tissue genes associated with adipogenesis through RT-PCR analyses. As shownin Figure 2C, addition of yakuchinone A (5 μM)suppressed the expression of leptin and PPAR γ significantly asrevealed by RT-PCR. These results suggest that yakuchinone A inhibits andadipogenesis due in part to the inhibition of angiogenesis. These events may bemediated, in part, through antioxidative properties of yakuchinone A responsible forinhibition of angiogenesis.
Effect of yakuchinone A on cell viability and skin cancer cell apoptosis
Previous report have demonstrated that yakuchinone A exhibits no cytotoxicity againsthuman lung adenocarcinoma A549 cells, human colorectal carcinoma HT-29 cells, andhuman gastric cancer SGC-7901 cells at a concentration of 10 μg/ml , but yakuchinone A induces apoptotic death in HL-60 cells . Nevertheless, cytotoxic effects of yakuchinone A on skin cancer cellsremain poorly understood. In this work, the inhibition potential of yakuchinone A onhuman skin cancer cells (epidermoid carcinoma A431 cells, basal cell carcinoma BCCcells, squamous cell carcinoma SCC25 cells and malignant melanoma A375 cells) andmouse melanoma B16 cells was determined by MTT assay and morphological change.Treatment these cells with yakuchinone A (5, 10, 20, 30, 40, and 50 μM) for72 h resulted in a dose-dependent significant cell death (Figure 3). The IC50 values of yakuchinone A were 13.3, 11.3,18.7, 23.8, and 40.0 μM for A431, BCC, SCC25, A375, and B16 cells,respectively. Moreover, after 72 h treatment with yakuchinone A (5, 10, 20, 30,40, and 50 μM), the IC50 values of yakuchinone A againstnoncancerous cells (human premalignant keratinocytic HaCaT cells and mouse embryonicliver BNLCL2 cells) and mouse leukemic monocyte macrophage RAW 264.7 cells were 22.2,32.2, and 46.4 μM, respectively (Figure 4).Yakuchinone A appeared to have a more potent inhibitory effect on non-melanoma skincancer (NMSC) cells (A431, BCC, and SCC25) and cell viability than in melanoma cells(A375 and B16), noncancerous cells (HaCaT and BNLCL2), and RAW 264.7 cells. Previousstudies have demonstrated that yakuchinone A has a phenolic diarylheptanoid moietywith a carbonyl functional group to suggest that yakuchinone A is anticipated toexhibit potential cancer chemopreventive activities . These experimental data further suggest that yakuchinone A has anantioxidant affect that exhibits less toxic to noncancerous cells and selectivecytotoxicity to NMSC cells.
The cell death induction by yakuchinone A was further confirmed by cellularmorphological examination. After exposure of 20 μM yakuchinone A to BCCcells at 72 h, distinct cytoplasmic shrinkage, cell bodies became rounded anddetached from the surface under phase-contrast-inverted microscopic examination(Figure 5A). Treatment of BCC cells with yakuchinone Ashowed chromatin condensation and nuclear fragmentation by Hoechst 33342 stainingunder a fluorescent microscope, indicating apoptosis (Figure 5A). Bcl-2 family members are major apoptosis-regulating proteins . Given that the Bcl-2 family proteins are known mediators of mitochondrialfunctions, expression levels of anti-apoptotic protein Bcl-2, and pro-apoptoticprotein Bax were determined. Bcl-2 expression was time-dependent decreased; whereas,bax was increased and investigated by RT-PCR following the exposure of BCC cells toyakuchinone A (20 μM) for 24 and 48 h (Figure 5B). These experimental results are consistent with the yakuchinone A(20 μM) applied for 72 h by Western blotting (Figure 5C). Cleavage of the poly (ADP-ribose) polymerase (PARP) in BCCcells after yakuchinone A treatment gave further evidence that apoptosis happenedbecause the active form of PARP, a protein associated with DNA repair, is consideredas a hallmark of apoptosis. These results suggest that yakuchinone A-induced celldeath is mainly due to apoptosis.
Cestocidal activity against H. nana
Figure 6 plots the time course of oscillation andperistalsis during yakuchinone A treatment. In oscillation activity assay, thepercentage of oscillation for the vehicle control (0.1% DMSO) decreased by about 18%from 72 h cultivation (Figure 6A). However, in theperistalsis activity assay, the percentage of peristalsis for the vehicle control(0.1% DMSO) decreased by 31% from 72 h cultivation (Figure 6B). The change of peristalsis of H. nana was more sensitive thanthat of oscillation via treatment of vehicle. Treatment with 10, 50, and100 μM yakuchinone A has a greater effect on peristalsis than oscillationfor 24, 48, and 72 h. Peristalsis activity disappeared before oscillationactivity was lost when H. nana was dead. In fact, H. nana has noperistalsis or oscillation effect when dead.
In the oscillation activity assay (Figure 6A), exposure to100 μM yakuchinone A for 72 h caused the maximum effect of 27% ofH. nana. Treatment with yakuchinone A (a concentration of 50,100 μM but not 10 μM) for 48 and 72 h reduced theoscillation up to 21% and 31% or 47% and 73%, respectively. Yakuchinone A slowlyreduced oscillation from 2 to 72 h but did not cause death. Yakuchinone Areduced the oscillation activity of H. nana in a time- and dose-dependentmanner for 24 to 72 h (Figure 6A).
The effect of yakuchinone A over time of the peristalsis activity of H. nanawas investigated (Figure 6B). For peristalsisactivity assay, a dose- and time-dependent effect for 24 to 72 h was alsoobserved by treatment with yakuchinone A. Treatment for 48 h with 50 and100 μM yakuchinone A stopped peristalsis in more than approximately 21 to25% of worms. Yakuchinone A at 50 and 100 μ M slowly reducedperistalsis from 2 to 72 h. Treatment with 10 μM yakuchinone A for72 h reduced peristalsis to 22% (Figure 6B). Thiseffect on peristalsis is stronger than on oscillation activity. The aboveperformances were the same for other concentrations of yakuchinone A in peristalsisactivity.
Nematocidal activity against A. simplex
In the first series of experiments, the larvicidal effects were used to study theability of yakuchinone A to alter survival of AsL3. The time course of theyakuchinone A-induced loss of mobility on AsL3 was also studied. Figure 7A shows more than 20% of the worms had stopped moving at72 h of treatment with 10, 100 and 200 μM yakuchinone A, whereas up to10% of the larvae ceased movement activity at 12 h of treatment with200 μM. Additionally, the maximum loss of spontaneous movement occurred ata concentration of 200 μM. Yakuchinone A caused a dose- and time-dependentloss of spontaneous movement. However, the vehicle (0.1% DMSO) had no effect on AsL3.Approximately, up to 20% of the larvae were dead at 48 h at 10, 100 and200 μM yakuchinone A (Figure 7B), and up to 35%and 40% of the larvae were dead at 48 and 72 h, respectively, including 100 and200 μM. Figure 7B shows A. simplexmortality was observed to be up 40% at 48 h after exposure to 100 and200 μM yakuchinone A, which showed more lethal efficacy than against H.nana (Figure 6).
Yakuchinone A isolated from A. oxyphylla scavenges radicals ofbiological interest and preventes damage to oxidative stress. The study suggests thatyakuchinone A inhibits adipocyte differentiation in 3 T3-L1 cells. Treatment withyakuchinone A reduces the intracellular accumulation of neutral lipids and suppressesthe induction of leptin and PPAR γ. Moreover, theses experimentalresults suggest that inhibition of cell growth by yakuchinone A is more significant forNMSC than for melanoma and noncancerous cells. Following incubation with yakuchinone Ain BCC cells increases apoptotic body formation as well as down-regulated Bcl-2,up-regulated Bax, and increased cleavage PARP. Additionally, previous studies have shownthat yakuchinone A has a stronger nematocidal activity of A. simplex thancestocidal activity of H. nana. These results support the development ofselective and efficient natural anthelmintic compounds against helmineth or cestode(Additional file 1). Previous evidence has established thatlarvicide activity toward A. simplex does not depend on scavenging activity,and that free radicals can be harmful to A. simplex, for which the scavengingof these free radicals permits larvae to survive. However, this report is the first toverify that yakuchinone A has the cestocidal activity against H. nana, thescavenging activity against DPPH · andABTS · + radicals, and the elimination effect on thespontaneous movement of AsL3. Therefore, the radical scavenging activity of yakuchinoneA does not reduce its ability to stop the spontaneous movement of AsL3 or its cestocidalactivity on H. nana. Further investigations must be conducted to elucidate theanthelmintic mechanisms of yakuchinone A against A. simplex and H.nana as well as its ability to eliminate the spontaneous movement of A.simplex and H. nana including their relationships to free radicalscavenging activities.
Adenosine 5′-phosphate-activated protein kinase
Activator protein 1
Non-melanoma skin cancer
Poly (ADP-ribose) polymerase
Peroxisomeproliferator-activated receptor γ
Reactive oxygen species
Thiobarbituric acid reactive substances
Tumor necrosis factor-α
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The authors would like to thank the National Science Council of the Republic ofChina, Taiwan, for financially supporting this research under Contract No. NSC100-2320-B-041-003-MY3 and NSC 98-2320-B-037-014-MY3.
The authors declare that they have no competing interests.
RJL, LPC, HYD, CHL, acquisition of data; analysis and interpretation of data;statistical analysis; drafting of the manuscript; obtained funding; study supervision.All authors read and approved the final manuscript. CMY, FYC, administrative support;study supervision. THC, GHW: review of the manuscript. YPT, LW, TWH, HCW, acquisition ofdata.
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