selaginella doederleinii hieron in vitro and in...

Research ArticleAntitumor Activities of Ethyl Acetate Extracts fromSelaginella doederleinii Hieron In Vitro and In Vivoand Its Possible Mechanism

Jia-zhi Wang Juan Li Ping Zhao Wen-tao Ma Xie-he Feng and Ke-li Chen

Hubei University of Traditional Chinese Medicine Key Laboratory of TCM Resource and TCM CompoundCo-Constructed by Hubei Province and Ministry of Education Wuhan 430065 China

Correspondence should be addressed to Juan Li lz198207126com and Ke-li Chen kelichen126com

Received 28 July 2014 Accepted 30 November 2014

Academic Editor Senthamil R Selvan

Copyright copy 2015 Jia-zhi Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The antitumor activities of ethyl acetate extracts from Selaginella doederleinii Hieron (SD extracts) in vitro and in vivo and itspossible mechanism were investigated HPLC method was developed for chemical analysis SD extracts were submitted to 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT) assay on different cells flow cytometry and RT-PCR analysis usingHepG2 cell and antitumor activity in vivo using H-22 xenograft tumor mice Six biflavonoids from SD extracts were submitted tomolecular docking assay The results showed that SD extracts had considerable antitumor activity in vitro and in vivo withoutobvious toxicity on normal cells and could induce cell apoptosis The mechanisms of tumorigenesis and cell apoptosis induced bySD extracts may be associated with decreasing the ratio of bcl-2 and bax mRNA level activating caspase-3 suppressing survivinand decreasing the gene expression of COX-2 5-LOX FLAP and 12-LOX mRNA The main active component in SD extracts isbiflavonoids and some exhibited strong interactions with COX-2 5-LOX 12-LOX and 15-LOX These results offering evidence ofpossible mechanisms of SD extracts suppress cell proliferation and promote apoptosis and provide the molecular theoretical basisof clinical application of S doederleinii for cancer therapy

1 Introduction

Selaginella doederleinii Hieron is a traditional Chinese folkherb which belongs to the family Selaginellaceae and isabundant in South and Southwestern China at low altitude[1] It has been used as folk medicine for the therapy ofsore throat rheumatoid arthritis and different tumors witha long history especially for nasopharyngeal carcinomachoriocarcinoma lung cancer and cervical cancer [2ndash4]

Lian et al reported that the ethanol extract of S doeder-leinii can induce mitochondria-related apoptosis in humannasopharyngeal carcinoma CNE cells [5 6] Also researcheson the cytotoxic activity against HCT NCI-H358 K562 andCNE cells of S doederleinii have been reported [1 6 7]Compounds from this herb such as several biflavonoidslignans and alkaloids have been reported [7ndash9] Howeverethyl acetate extracts had stronger antitumor activities thanethanol extract for their abundant biflavonoids such as amentoflavone robustaflavone 210158401015840310158401015840-dihydro-310158403101584010158401015840-biapigenin

310158403101584010158401015840-binaringenin heveaflavone and 7410158407101584010158404101584010158401015840-tetra-O-methyl-amentoflavone [10 11]

Aberrant arachidonic acid (AA) metabolism is involvedin the inflammatory and carcinogenic processes The effectsof biflavonoidmixture from S doederleinii on cyclooxygenase(COX) and lipoxygenase- (LOX-) dependent AA expressionin hepatocellular carcinoma were investigated and theireffects on cell proliferation and apoptosis were also studied

2 Materials and Methods

21 Plant Materials The Chinese herbal S doederleinii wascollected from Nanning (Guangxi China) Identificationof specimen was confirmed by Dr Dingrong Wan South-Central University for Nationalities (Wuhan China) and avoucher specimen was deposited in the herbarium of HubeiUniversity of Chinese Medicine China

SD extract was obtained by the previously describedmethod [8] Briefly the air-dried and powdered samples were

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 865714 9 pageshttpdxdoiorg1011552015865714

2 Evidence-Based Complementary and Alternative Medicine

extracted twice with petroleum ether and then were filteredThe residues were extracted twice with ethyl acetate and thenwere filtered Then the solution was dried using a rotaryevaporator and it was lyophilized and transformed into apower before dissolved in DMSO (dimethyl sulfoxide) Thepurity of SD extract was about 93

22 HPLC Analysis The detailed method of HPLC analysiscould be seen in the literature [11 12] Briefly HPLC analysiswas performed on a Dionex HPLC system with P680 Pumpa DiamonsilTM C18 column (250mm times 56mm 5120583m) anda UVD 170U variable wavelength UV-Vis detector Datawere collected and processed using ldquoChromeleon version 60rdquosoftware The mobile phase consisted of acetonitrile (A) andwater (B) The gradient program was as follows 25 A in0ndash5min 25ndash35 A in 5ndash12min 35ndash45 A in 12ndash17min45ndash50 A in 17ndash25min 50ndash55 A in 25ndash40min 55ndash70A in 40ndash45min 70ndash100 A in 45ndash50min 100 A in 50ndash55min and 100ndash25 A in 55ndash60min The flow rate was10mLmin and column temperature wasmaintained at 30∘CThe injection volume was 10 120583L The detector was set at330 nm for acquiring chromatograms

23 Reagents and Cell Culture MTT Trizol andDMSOwereobtained from Sigma (St LouisMOUSA) DMEMmediumtrypsin penicillin and streptomycin were purchased fromGibco (USA) FBS (fetal bovine serum) was bought fromHyclone (USA) All chemicals and reagents were of analyticalreagent grade

HepG2 (hepatocellular carcinoma) Hela (cervical carci-noma) A549 (lung cancer) DU145 (prostatic carcinoma)PC12 (pheochromocytoma) and Vero (African green mon-key kidney) cells were obtained from China Center forType Culture Collection of Wuhan University The cells wereincubated in a humidified atmosphere of 95 air and 5CO

2

at 37∘C and maintained in DMEM culture medium with 10FBS plus 100UmL streptomycin and 100UmL penicillinThe cellswere subculturedwith 025 trypsinwhen theywere80 confluent Then the cells in exponential growth phasewere collected for the following experiments

24 MTT Assay Evaluation of antitumor activity in vitrowas determined with MTT assay which was performed asdescribed before [13] Briefly the cells were seeded in 96-well culture plates at a density of 2 times 103 cells per well andthen allowed to attach for 24 h before treated with varyingconcentrations of SD extracts (0 125 25 50 75 100 and200120583gmL) for 72 h Subsequently 50 120583L MTT of 1mgmLwas added to each well to react 4 h The absorbance wasdetermined using the 96-well microplate reader at 570 nmafter the formed purple formazan crystals dissolved in 100 120583LDMSO The growth inhibitory ratio was calculated by thefollowing formula rate of growth inhibition () = (1 minusODtreatedODcontrol) times 100 [14] The IC

50value (concentra-

tion of 50 inhibition) was obtained from the dose-responseplots of three independent repetitive trials

25 Morphology Observation HepG2 cells were seeded into24-well plates at a density of 104 cells per well and then

exposed to different concentrations of SD extracts for 48 hThe morphological changes of cells were observed and pho-tographed using inverted light microscopy (IX70 OlympusOptical Co Tokyo Japan)

26 Annexin V-FITCPI Double Staining Assay Exponen-tially growing HepG2 cells were placed down in 6-well plateand cultured as above Then the cells were treated withSD extracts at different concentrations for 24 h After thedrug incubation time all cells were harvested with trypsinand washed twice with PBS followed by resuspended in400 120583L Annexin V binding bufferThen the cells were stainedwith 5 120583L Annexin V-FITC for 15 minutes and 10 120583L PIfor 5 minutes at 4∘C This assay was performed exactlyas the manufacturersrsquo instruction of the Annexin V-FITCcell apoptosis detection kit (BestBio china) A FACSCaliburflow cytometer was used to detected fluorescence and thepercentage of apoptotic cells was calculated by the internalsoftware system of the FACSCalibur Approximately 104 cellswere analyzed for each trail

27 RNA Extraction and Real-Time PCR ThemRNA levels ofCOX-2 FLAP 5-LOX 12-LOX 15-LOX bcl-2 bax caspase-3and survivinwere quantified by real-timeRT-PCRassaysThe120573-actin was internal reference gene HepG2 cells were placeddown in 6-well plate at a concentration of 5 times 105 cellsmLAfter 60 confluency the cells were exposed to SD extracts atthe concentration of IC

50for different times (0 15 3 6 9 and

12 h) Then the cells were harvested to SD extract total RNAusing Trizol reagent A UV spectrophotometer was used toestimate RNA concentration at 260 nm The purity of RNAwas assessed by the ratio of absorbance at 260 and 280 nm(A260A280 between 18 and 20) After quantification 2 120583gRNA was used for the synthesis of cDNA in each reversetranscription reaction via TIANScript RT Kit We conductedPCR to amplify the target genes with reagents and protocolsfrom the SYBR Green PCR Master Mix kit by Invitrogen(Carlsbad CA USA) The 20 120583L reaction system contained1 120583L generated cDNA template 1120583L of specific sense primer(Table 1) 1 120583L of specific antisense primer 10 120583L 2 times SYBRGreen PCR Master Mix and 7 120583L double distilled water Thethermal cycling conditions were 95∘C for 15min followed by40 cycles of 95∘C for 10 s 59ndash63∘C (annealing temperature)for 20 s and 72∘C for 30 s and a final incubation at 72∘C for5minThe relative expression of each gene was normalized tothe amount of 120573-actin in the same dosage of cDNA and therelative quantification method was 2minusΔΔCT method [15] AllRT-PCR reactions for each sample were done in triplicate

28 Molecular Docking We performed a series of moleculardocking experiments to estimate the binding affinity of somebiflavonoids with COX-2 LOX-5 LOX-12 and LOX-15

281 Preparing Protein and Ligands The three-dimensionalstructure of biflavonoids was downloaded from PubChem(httppubchemncbinlmnihgov) database [16] and refinedwith the help of Discovery Studio Visualizer (httpaccelryscomproductsdiscovery-studiovisualizationhtml)Thenthese ligands were converted to MOL2 format using

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primer list

Gene Forward primer (51015840 to 31015840) Reverse primer (51015840 to 31015840)5-LOX GCCTCCCTGTGCTTTCC ACCTGGTCGCCCTCGTAFLAP GCTGCGTTTGCTGGACTGATGTA TAGAGGGGAGATGGTGGTGGAGAT12-LOX CTTCCCGTGCTACCGCTG TGGGGTTGGCACCATTGAG15-LOX CTGGAGCCTTCCTAACCT GTGACAAAGTGGCAAACCCOX-2 TATGAGTGTGGGATTTGACCAG TCAGCATTGTAAGTTGGTGGACbcl-2 GTGGAGGAGCTCTTCAGGGA AGGCACCCAGGGTGATGCAAbax GGCCCACCAGCTCTGAGCAGA GCCACGTGGGCGTCCCAAAGTcaspase-3 ATGGAGAACACTGAAAACTCAGT TTAGTGATAAAAATAGAGTTCTTTTGTsurvivin ATGGGTGCCCCGACGTTGCCCCCT TCAATCCATGGCAGCCAGCTGCTCG120573-actin TGACGTGGACATCCGCAAAG CTGGAAGGTGGACAGCGAGG

this software Finally these mol2 files are optimized byldquoPrepare Ligandsrdquo in Autodock Tools [17] and converted toPDBQTfilesThe3D structure ofCOX-2 (PDBID 1CX2) [18]LOX-5 (PDBID 2Q7M) [19] LOX-12 (PDBID 3RDE) [20]and LOX-15 (PDBID 1LOX) [21] was obtained from ProteinData Bank based on their resolution and chemical similaritybetween bioflavonoids of S doederleinii and the cocrystalledligands Protein structure was imported to Autodock ToolsAfter deleting duplicated chains removing water and otherligands and adding hydrogen the preprocessed structurewas assigned with AMBER force field [22] and converted toPDBQT files automatically

282 Docking Methodology Docking procedure can bedivided into two parts grid generation and docking In theAutoGrid procedure the target enzyme was embedded ona three-dimensional grid point [17] The energy of interac-tion of each atom in the ligand was encountered In thefollowing phase Autodock 42 was employed in this studyThis software uses Lamarckian genetic algorithm (LGA) toperform ligand conformational searching This algorithmfirst builds a population of individuals (genes) and then eachindividual ismutated to acquire a slightly different translationand rotation The individuals with the low resulting energyafter energy minimization process are transferred to thenext generation and the process is repeated to obtain thebest scored one For each binding energy of each pose wascalculated using AutoDock 42 scoring functions [23]

In the present study three-dimensional affinity grids ofsize 60 times 60 times 60 A with 06 A spacing which centered onthe geometric center of cocrystalled ligand were calculatedfor each of the following atom types HD C A N OA andSA which represent all possible atom types in a protein andligand Additionally an electrostatic map and a desolvationmap were also calculated Rapid energy evaluation wasachieved by precalculating atomic affinity potentials for eachatom in the ligand molecule We have selected importantdocking parameters for the LGA as follows population sizeof 150 individuals 5 million energy evaluations maximumof 30000 generations number of top individuals to automat-ically survive to next generation of 1 mutation rate of 002crossover rate of 08 10 docking runs and random initialpositions and conformations The probability of performing

local search on an individual in the population was set to006 Top-scored conformation of each ligand was analyzedby AutoDock Tools and Pymol

29 Antitumor Activity of SD Extracts In Vivo Kunmingmice (male 20 plusmn 2 g) were procured from the Center ofExperimental Animals in Wuhan University Wuhan ChinaIn order to evaluate the antitumor effect of SD extracts invivo the mice were injected with H-22 (mice hepatoma)cells in the armpit for subcutaneous xenograft tumormodelsrespectively except the normal group mice Then the tumorbearing mice were divided into five groups randomly ofwhich each group has 12 mice The negative and normalcontrol group mice received only 09 normal saline 5-Fuintraperitoneal injection was taken as positive control Thelow-dosemedial-dose and high-dose drug groupwere orallyadministrated with 4 8 and 16 gkgd SD extracts separatelyAll samples were administrated once every day for 10 daysexcept positive group mice which administrated every otherday (10mgkg2d) On the 11th day all mice were sacrificedand tumors were excised and weighed for evaluating thetumor growth inhibition The spleen and thymus were alsosegregated and weighed to calculate the spleen index andthymus index

210 Statistical Analysis Thestatistical analysis was evaluatedby Studentrsquos 119905-test All data were expressed as mean plusmn SDVariance of 119875 values obtained was calculated by means of asingle-factor ANOVA testThe values of 119875 less than 005 wereconsidered to be significantly different from each control

3 Results

31 Fingerprints Analysis HPLC chromatogram was appliedfor examining biflavonoids from ethyl acetate extracts Asreported in literature [10] the peaks of amentoflavonerobustaflavone 210158401015840310158401015840-dihydro-310158403101584010158401015840-biapigenin 310158403101584010158401015840-binaringenin heveaflavone and 7410158407101584010158404101584010158401015840-tetra-O-methyl-amentoflavone were marked in Figure 1

32 Growth Inhibitory Effects of SD Extract on Different CellsIt was widely assumed that MTT has become one of themost widely used methods for measuring cell proliferation

4 Evidence-Based Complementary and Alternative Medicine(m

AU)

(min)

160

125

100

75

50

25

minus2000 100 200 300 400 500 600

1

2

34

5

6WVL 330nm

Figure 1 HPLC chromatogram of ethyl acetate extracts 1 amen-toflavone 2 robustaflavone 3 210158401015840310158401015840-dihydro-310158403101584010158401015840-biapigenin 4310158403101584010158401015840-binaringenin 5 heveaflavone 6 7410158407101584010158404101584010158401015840-tetra-O-meth-ylamentoflavone

Table 2 IC50 values for SD extracts on the proliferation of differentcells (120583gmL)

DU145 HeLa A549 HepG2 PC12 VeroSDextracts 705 plusmn 26 761 plusmn 19 519 plusmn 15 658 plusmn 44 gt150 gt150

Values are means plusmn SD (119899 = 5)

and viability In our study inhibitory activities against fivedifferent tumor cells (DU145 HepG2 HeLa A549 and PC12)and one normal cell (Vero) were evaluated by MTT methodThe data from Table 2 suggested that extracts had definitecytotoxic effect on various cancer cells with a close IC

50value

except for PC12 which have similar structure and function tothe nerve cell and (but) a higher chance of surviving so it isoften used as a cell model to study the nervous system [24]For this reason the higher IC

50value of SD extracts against

PC12 indicated that SD extracts (with little damage to) hurtthe central nervous system little and that was also revealed inFigure 2

As shown in Figure 2 SD extracts caused the tumor cellsdeath in a dose-dependent manner and exhibited apparentcytotoxicity to cancer cells In addition for normal cellsVero SD extracts lead to a growth inhibition rate less than30 even at the concentration of 100 120583gmL The level ofcytotoxicity of SD extracts against Vero cells was muchlower than cancer cells (Figure 2) The results identified thatSD extracts had considerable antitumor activity and lowcytotoxicity on normal cells

33 Morphological Changes of HepG2 Cells Induced by SDExtracts under Inverted Microscope As shown in Figure 3morphological changes of HepG2 cells which were treatedwith SD extracts at different concentrations (25 50 and100 120583gmL) were observed as compared with the untreatedcontrol cells Untreated HepG2 cells attached closely on theculture surface in polygon or rotundity with a good refraction

0

20

40

60

80

100

0 20 40 60 80 100

Inhi

bito

ry ra

te (

)

Du145HepG2

HeLa

A549

PC12Vero

Concentrations (120583gmL)

Figure 2 Inhibitory effect of SD extracts on the proliferation ofDu145 HepG2 HeLa A549 PC12 and Vero cells Cells were treatedwith different concentrations of SD extracts for 72 h Values aremeans plusmn SD (119899 = 5)

and some of them contacted each other to form colonies(Figure 3) However after treatment with SD extracts thecells lost their surface morphology significantly and becomesmall and roundmade fewer cellular contacts reduced in sizeand number It also can be seen that cell number is depressedobviously Also these cell morphological changes were in adose- and time-dependent manner

34 Apoptosis Analysis by Flow Cytometry To determinewhether SD extracts-induced cell death was related to apop-tosis Annexin V-FITCPI staining assay was conducted Dis-play of phosphatidylserine on the surface of cells consideredas the hallmark of apoptosis in early phase was determinedby PIAnnexin V staining assayWhen cells were treated with25 to 200120583gmL SD extracts for 24 h cell populations in lateapoptotic phases increased from 576 to 1429 comparedwith 28 of apoptotic cells in the control (Figure 4)

35 SD Extracts Changed mRNA Expression of Proinflam-matory Factors and Apoptosis-Related Genes We preformedRT-PCR assay to determine whether SD extracts treatmentchanged expression of several proinflammatory genes andapoptosis-related genes which have been verified relevantto cell proliferation apoptosis invasion metastasis andangiogenesis [25] As seen in Figure 5 the level of COX-25-LOX FLAP and 12-LOX mRNA was markedly decreasedby SD extracts in HepG2 cells compared with untreated cellswhich implied SD extracts might restrain proinflammatorycytokines production at gene level In the meanwhile themRNA expression of 15-LOX which was generally thought topresent tumor inhibition effect increased significantly up to


(a) (b)

(c) (d)

Figure 3 Morphological changes of HepG2 cells induced by different concentration of SD extracts Changes of cellular morphology wereexamined at 24 h with 400 magnification (a) Control (b) 25120583gmL (c) 50 120583gmL (d) 100 120583gmL

551 times higher than control group (data was not shown)In addition the expression of COX-2 5-LOX FLAP 12-LOXand 15-LOX mRNA levels changes regularly with the actiontime of SD extracts extending in 12 h The treatment of SDextracts on HepG2 cells for 12 h inhibited COX-2 5-LOXFLAP and 12-LOX mRNA production up to 7422 671713 and 8303 respectively These results demonstratedthat SD extracts treated for indicated time would resulted inregression of inflammatory in a time-dependentmanner andits effect of inhibiting cell proliferation and promoting cellapoptosis were related with down-regulation of COX-2 5-LOX FLAP and 12-LOX and up-regulation of 15-LOX

In another aspect bcl-2 and bax are a pair of momentousapoptosis genes the ratio of bax to bcl-2 determines whethera cell undergoes apoptosis [26] and survivin can preventand attenuate cell apoptosismarkedly [27]mRNAexpressionlevels of antiapoptotic genes bcl-2 and survivin proapop-totic gene bax and caspase-3 were also determined by RT-PCR Survivin and bcl-2 mRNA expression were decreasedwhereas that of bax and caspase-3 were increased in time-dependent manner after SD extracts treatment (Figure 6) Asshown in Figure 6 bax and caspase-3 genes were observed tobe induced by about 192- and 262-fold respectively whereasbcl-2 and survivin were repressed by about 739 and 718after SD extracts treatment compared to untreated cellsThese results demonstrated that one of the possible mecha-nisms of SD extracts induced apoptosis was associated withexpression changes of bax bcl-2 caspase-3 and survivin

36 Molecular Docking The docking process was accom-plished by AutoDock (version 42) and the docking resultswere quantified by AutoDock 42 scoring functions Thedocking score for biflavonoids could be seen in Figure 7310158403101584010158401015840-binaringenin exhibited strong interactions with COX-2 all compounds showed good interactions with LOX-5and amentoflavone 210158401015840310158401015840-dihydro-310158403101584010158401015840-biapigenin 310158403101584010158401015840-binaringenin and heveaflavone might have potential betterbinding ability with LOX-12 while all compounds showedweak interactions with LOX-15 However robustaflavoneexhibited no interactions with COX-2 and LOX-15 and7410158407101584010158404101584010158401015840-tetra-O-methylamentoflavone exhibited no inter-actions with LOX-12 and LOX-15The binding mode analysisof amentoflavone with COX-2 could be seen in Figure 8

37 Antitumor Activity In Vivo Due to the good antitumoractivities in vitro of SD extracts they were examined foranimal studies As daily observation no mice dead whentreated with SD extracts The effects of SD extracts onmice transplanted with H-22 were presented in Table 3 Theresults revealed that SD extracts significantly decreased thetumor weights of H-22 tumor-bearing mice The inhibitoryrates were 238 398 and 577 at the dosages of 4 8and 16 gkgday respectively Furthermore SD extracts coulddecrease the spleen and thymus index of the tumor-bearingmice (Table 3)


101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625017

28

186

11

(a)

101

102

103

104

100 101 102 103 104100

FL2

-HFL1-H

20140625019

558

576076

(b)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625015

132 712

513

(c)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625014

674

816300

(d)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625010

503

1268114

(e)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625009

144 1429

544

(f)

Figure 4 Apoptosis induction by SD extracts in HepG2 cells Phosphatidylserine translocation of HepG2 cells after SD extracts treatmentfor 24 h The population in the region of PIminusAnnexin Vminus was considered to be normal cells the population in the region of PIminusAnnexin V+was considered to be early apoptotic cells while that of PI+Annexin V+ was considered to be late apoptotic cells (a) Control (b) 25 120583gmL(c) 50 120583gmL (d) 75 120583gmL (e) 100120583gmL (f) 150 120583gmL

4 Discussion

In the present study we investigated the anticancer effectsof SD extracts on tumor cells and the possible mechanismsMTT assay proved that SD extracts significantly exhibitedantiproliferation activity on various carcinoma cells with alow IC

50value and had little damage on Vero and PC12

cells demonstrating its selective antitumor action to somedegree and potential practical valuableness in the therapyfor cancer However more in-depth research on the detailedmechanisms of SD extracts killing tumor cells rather thannormal cells is needed

Several proinflammatory mediators have been confirmedto play a significant role in inhibition of angiogenesisapoptosis proliferation and metastasis [28] Both COX andLOX pathway function as a crucial mediator of cell survivaland apoptosis [29ndash31] COX-2 has been implicated in thegrowth and progression of a variety of human cancers 5-LOX and FLAP play a vital role in tumor cells growth relatedsignal transduction and can stimulate oncogene expression

and 12-LOX may be responsible for the adhesion invasionand metastasis of cancer cells and also can promote tumorangiogenesis [30] However 15-LOX is now considered tobe a tumor-inhibiting factor and mainly inhibit carcinomacells growth Our RT-PCR results indicated that the mRNAexpression of COX-2 5-LOX FLAP and 12-LOX decreasedand 15-LOX increased in HepG2 cells after SD extractstreatment The susceptibility to apoptosis by SD extracts isassociated with the level of COX-2 and LOXs in HepG2cells which present high COX-2 expression spontaneouslyTherefore SD extracts as agents which bring the geneexpression of COX-2 5-LOX FLAP and 12-LOX mRNAdown should play an inhibitory role in tumorigenesis andmetastasis and induce carcinoma cells apoptosis

Themain active component in SD extracts is biflavonoidsand different biflavonoids exhibited interactions with COX-2 5-LOX 12-LOX and 15-LOX in varying degrees Thisstudy just analyzed six biflavonoids and there were othercompounds in SD extracts and more works are needed


0

50

100

Time (h)

mRN

A ex

pres

sion

( o

f0h

actio

n)

0 15 3 6 9 12

COX-25-LOX

FLAP12-LOX

Figure 5 Effects of SD extracts on COX-2 5-LOX FLAP and 12-LOX mRNA expression HepG2 cells were treated with SD extracts(658 120583gmL) for 0 15 3 6 9 and 12 h COX-2 5-LOX FLAP and 12-LOX mRNA levels were determined by RT-PCR Values are meansplusmn SD (119899 = 5)

0

100

200

300

Caspase-3Survivin

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(a)

0

100

200

BaxBcl-2

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(b)

Figure 6 Effects of SD extracts on mRNA expression of apoptosis-related genes (a) mRNA expression of caspase-3 and survivin genes inHepG2 cells treated with SD extracts (658 120583gmL) for 0 15 3 6 9 and 12 h Values are means plusmn SD (119899 = 5) (b) mRNA expression of baxand bcl-2 genes in HepG2 cells treated with SD extracts for 0 15 3 6 9 and 12 h Values are means plusmn SD (119899 = 5)

Table 3 Antitumor effects of SD extracts against tumor growth on H-22 tumor-bearing mice

Tumor weight (g) Tumor growth inhibition () Spleen index (mg10 g) Thymus index (mg10 g)

H22

Control 146 plusmn 030 562 plusmn 083 202 plusmn 04216 gkg 062 plusmn 027lowastlowast 577 496 plusmn 072 164 plusmn 024lowast

8 gkg 088 plusmn 028lowast 398 530 plusmn 102 179 plusmn 0384 gkg 112 plusmn 040 233 555 plusmn 095 190 plusmn 0325-Fu 060 plusmn 015lowastlowast 589 476 plusmn 072lowast 142 plusmn 02lowastlowast

Values represent mean plusmn SE lowast119875 lt 005 compared to the control group lowastlowast119875 lt 001 compared to the control group


0

Com

poun

d 1

Com

poun

d 2

Com

poun

d 3

Com

poun

d 4

Com

poun

d 5

Com

poun

d 6

Docking result

COX-2LOX-5

LOX-12LOX-15

minus14

minus12

minus10

minus8

minus6

minus4

minus2

Figure 7 Quantify the interaction between biflavonoids and COX-2 LOX-5 LOX-12 and LOX-15 by AutoDock program 1 amen-toflavone 2 robustaflavone 3 210158401015840310158401015840-dihydro-310158403101584010158401015840-biapigenin4 310158403101584010158401015840-binaringenin 5 heveaflavone 6 7410158407101584010158404101584010158401015840-tetra-O-meth-ylamentoflavone

Figure 8The bindingmode analysis of amentoflavonewith COX-2The interactions were analyzed by Pymol

We also utilized an RT-PCR method to quantitate theexpression of both bcl-2 bax caspase-3 and survivin mRNAexpressionThe results showed that one of themechanisms ofcell apoptosis induced by SD extracts may decrease the ratioof bcl-2 and baxmRNA level activate caspase-3 and suppresssurvivin to promote cell apoptosis

5 Conclusion

In summary SD extracts had considerable antitumor activityin vitro and in vivo without obvious toxicity on normalcells and could induce cell apoptosis The mechanisms oftumorigenesis and carcinoma cell apoptosis induced by SDextracts may be associated with decreasing the ratio of bcl-2 and bax mRNA level activating caspase-3 suppressingsurvivin and decreasing the gene expression of COX-2 5-LOX FLAP and 12-LOXmRNAThemain active componentin SD extracts is biflavonoids and some exhibited stronginteractions with COX-2 5-LOX 12-LOX and 15-LOX

Abbreviations

AA Aberrant arachidonic acidIC50 Concentration of 50 inhibition

COX CyclooxygenaseDMSO Dimethyl sulfoxideFBS Fetal bovine serumHEp-2 Human laryngeal carcinoma cellsLOX LipoxygenaseMTT 3-(45-Dimethylthiazol-2-yl)-25-diphenyl

tetrazolium bromidePI Propidium iodideSD extracts Ethyl acetate extract of Selaginella

doederleinii

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Jia-zhi Wang and Juan Li contributed equally to the work

Acknowledgment

This work was supported by the Scientific Research Founda-tion Project of the Education Department of Hubei Province(Q20142007)

References

[1] R C Lin A L Skaltsounis E Seguin F Tillequin and MKoch ldquoPhenolic constituents of Selaginella doederleiniirdquo PlantaMedica vol 60 no 2 pp 168ndash170 1994

[2] TC ZhouXD Lin X L Song et al ldquoLong term treatment out-come of radiation combined with the Chinese herbal Selaginellafor terminal Nasopharyngeal Carcinomardquo China Journal ofHospital Pharmacy vol 28 no 24 pp 2122ndash2123 2008

[3] T C Zhou X D Lin X L Song et al ldquoClinical observation ofSelaginella combined with radiotherapy for terminal nasopha-ryngeal carcinomardquo Chinese Journal of Cancer Prevention andTreatment vol 13 no 10 pp 772ndash773 2006

[4] H Zhong and C Ben Editorial Committee of Chinese MateriaMedica Shanghai Science and Technology Press ShanghaiChina 1999

[5] R Lian J Li H M Ma et al ldquoEffect of ethanol extract ofSelaginella doederleiniiHieron on the proliferation of nasopha-ryngeal carcinoma CNE-1 and C666-1 cellsrdquo African Journal ofTraditional Complementary and Alternative Medicines vol 10no 6 pp 490ndash493 2013

[6] H Liu H Peng Z Ji et al ldquoReactive oxygen species-mediatedmitochondrial dysfunction is involved in apoptosis in humannasopharyngeal carcinoma CNE cells induced by Selaginelladoederleinii extractrdquo Journal of Ethnopharmacology vol 138 no1 pp 184ndash191 2011

[7] N-Y Lee H-Y Min J Lee et al ldquoIdentification of a newcytotoxic biflavanone from Selaginella doederleiniirdquo Chemicaland Pharmaceutical Bulletin vol 56 no 9 pp 1360ndash1361 2008


[8] S Li R Zhu M Zhong et al ldquoEffects of ultrasonic-assistantextraction parameters on total flavones yield of Selaginelladoederleinii and its antioxidant activityrdquo Journal of MedicinalPlants Research vol 4 no 17 pp 1743ndash1750 2010

[9] S Li H Yao M Zhao Y Li L Huang and X Lin ldquoDeter-mination of seven biflavones of Selaginella doederleinii by highperformance liquid chromatographyrdquoAnalytical Letters vol 46no 18 pp 2835ndash2845 2013

[10] S Li M Zhao Y Li et al ldquoPreparative isolation of six anti-tumour biflavonoids from Selaginella Doederleinii Hieron byhigh-speed counter-current chromatographyrdquo PhytochemicalAnalysis vol 25 no 2 pp 127ndash133 2014

[11] J Li X Lei K L Chen et al ldquoComparison of cytotoxic activitiesof extracts from Selaginella speciesrdquo Pharmacognosy Magazinevol 10 no 40 pp 529ndash535 2014

[12] X L Fan D R Wan and K L Chen ldquoStudies on HPLC finger-print characteristics from Selaginella plantsrdquo Zhongguo ZhongYao Za Zhi vol 32 no 11 pp 1094ndash1095 2007

[13] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[14] F Liu J GWang S YWang Y Li Y PWu and SM Xi ldquoAnti-tumor effect and mechanism of Gecko on human esophagealcarcinoma cell lines in vitro and xenografted sarcoma 180 inKunming micerdquo World Journal of Gastroenterology vol 14 no25 pp 3990ndash3996 2008

[15] M L Wong and J F Medrano ldquoReal-time PCR for mRNAquantitationrdquo BioTechniques vol 39 no 1 pp 75ndash85 2005

[16] D L Wheeler T Barrett D A Benson et al ldquoDatabase resour-ces of the National Center for Biotechnology InformationrdquoNucleic Acids Research vol 35 no 1 pp D5ndashD12 2007

[17] G M Morris H Ruth W Lindstrom et al ldquoAutoDock4 andAutoDockTools4 automated docking with selective receptorflexibilityrdquo Journal of Computational Chemistry vol 30 no 16pp 2785ndash2791 2009

[18] R G Kurumbail A M Stevens J K Gierse et al ldquoStructuralbasis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agentsrdquo Nature vol 384 no 6610 pp 644ndash6481996

[19] A D Ferguson B M McKeever S Xu et al ldquoCrystal structureof inhibitor-bound human 5-lipoxygenase-activating proteinrdquoScience vol 317 no 5837 pp 510ndash512 2007

[20] S Xu T C Mueser L J Marnett and M O Funk Jr ldquoCrystalstructure of 12-Lipoxygenase catalytic-domain-inhibitor com-plex identifies a substrate-binding channel for catalysisrdquo Struc-ture vol 20 no 9 pp 1490ndash1497 2012

[21] S A Gillmor A Villasenor R Fletterick E Sigal and M FBrowner ldquoThe structure of mammalian 15-lipoxygenase revealssimilarity to the lipases and the determinants of substratespecificityrdquo Nature Structural Biology vol 4 no 12 pp 1003ndash1009 1997

[22] E Darian and P M Gannett ldquoApplication of molecular dynam-ics simulations to spin-labeled oligonucleotidesrdquo Journal ofBiomolecular Structure and Dynamics vol 22 no 5 pp 579ndash593 2005

[23] RHuey GMMorris A J Olson andD S Goodsell ldquoSoftwarenews and update a semiempirical free energy force field withcharge-based desolvationrdquo Journal of Computational Chemistryvol 28 no 6 pp 1145ndash1152 2007

[24] T J Shafer and W D Atchison ldquoIon channel and receptorproperties of pheochromocytoma (PC12) cells a model forneurotoxicological studiesrdquo Neurotoxicology vol 12 no 3 pp473ndash492 1990

[25] D G Tang Y Q Chen and K V Honn ldquoArachidonate lipoxy-genases as essential regulators of cell survival and apoptosisrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 93 no 11 pp 5241ndash5246 1996

[26] H-K Chang M-S Shin H-Y Yang et al ldquoAmygdalin inducesapoptosis through regulation of Bax and Bcl-2 expressions inhuman DU145 and LNCaP prostate cancer cellsrdquo Biological andPharmaceutical Bulletin vol 29 no 8 pp 1597ndash1602 2006

[27] N Zaffaroni andMGDaidone ldquoSurvivin expression and resis-tance to anticancer treatments perspectives for new therapeuticinterventionsrdquoDrug Resistance Updates vol 5 no 2 pp 65ndash722002

[28] J K Kundu and Y-J Surh ldquoEmerging avenues linking inflam-mation and cancerrdquo Free Radical Biology and Medicine vol 52no 9 pp 2013ndash2037 2012

[29] G P Pidgeon J Lysaght S Krishnamoorthy et al ldquoLipoxy-genase metabolism roles in tumor progression and survivalrdquoCancer and Metastasis Reviews vol 26 no 3-4 pp 503ndash5242007

[30] B B Aggarwal S Shishodia S K Sandur M K Pandey andG Sethi ldquoInflammation and cancer how hot is the linkrdquoBiochemical Pharmacology vol 72 no 11 pp 1605ndash1621 2006

[31] K Hasegawa Y Ohashi K Ishikawa et al ldquoExpression ofcyclooxygenase-2 in uterine endometrial cancer and anti-tumor effects of a selective COX-2 inhibitorrdquo InternationalJournal of Oncology vol 26 no 5 pp 1419ndash1428 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


extracted twice with petroleum ether and then were filteredThe residues were extracted twice with ethyl acetate and thenwere filtered Then the solution was dried using a rotaryevaporator and it was lyophilized and transformed into apower before dissolved in DMSO (dimethyl sulfoxide) Thepurity of SD extract was about 93

22 HPLC Analysis The detailed method of HPLC analysiscould be seen in the literature [11 12] Briefly HPLC analysiswas performed on a Dionex HPLC system with P680 Pumpa DiamonsilTM C18 column (250mm times 56mm 5120583m) anda UVD 170U variable wavelength UV-Vis detector Datawere collected and processed using ldquoChromeleon version 60rdquosoftware The mobile phase consisted of acetonitrile (A) andwater (B) The gradient program was as follows 25 A in0ndash5min 25ndash35 A in 5ndash12min 35ndash45 A in 12ndash17min45ndash50 A in 17ndash25min 50ndash55 A in 25ndash40min 55ndash70A in 40ndash45min 70ndash100 A in 45ndash50min 100 A in 50ndash55min and 100ndash25 A in 55ndash60min The flow rate was10mLmin and column temperature wasmaintained at 30∘CThe injection volume was 10 120583L The detector was set at330 nm for acquiring chromatograms

23 Reagents and Cell Culture MTT Trizol andDMSOwereobtained from Sigma (St LouisMOUSA) DMEMmediumtrypsin penicillin and streptomycin were purchased fromGibco (USA) FBS (fetal bovine serum) was bought fromHyclone (USA) All chemicals and reagents were of analyticalreagent grade

HepG2 (hepatocellular carcinoma) Hela (cervical carci-noma) A549 (lung cancer) DU145 (prostatic carcinoma)PC12 (pheochromocytoma) and Vero (African green mon-key kidney) cells were obtained from China Center forType Culture Collection of Wuhan University The cells wereincubated in a humidified atmosphere of 95 air and 5CO

2

at 37∘C and maintained in DMEM culture medium with 10FBS plus 100UmL streptomycin and 100UmL penicillinThe cellswere subculturedwith 025 trypsinwhen theywere80 confluent Then the cells in exponential growth phasewere collected for the following experiments

24 MTT Assay Evaluation of antitumor activity in vitrowas determined with MTT assay which was performed asdescribed before [13] Briefly the cells were seeded in 96-well culture plates at a density of 2 times 103 cells per well andthen allowed to attach for 24 h before treated with varyingconcentrations of SD extracts (0 125 25 50 75 100 and200120583gmL) for 72 h Subsequently 50 120583L MTT of 1mgmLwas added to each well to react 4 h The absorbance wasdetermined using the 96-well microplate reader at 570 nmafter the formed purple formazan crystals dissolved in 100 120583LDMSO The growth inhibitory ratio was calculated by thefollowing formula rate of growth inhibition () = (1 minusODtreatedODcontrol) times 100 [14] The IC

50value (concentra-

tion of 50 inhibition) was obtained from the dose-responseplots of three independent repetitive trials

25 Morphology Observation HepG2 cells were seeded into24-well plates at a density of 104 cells per well and then

exposed to different concentrations of SD extracts for 48 hThe morphological changes of cells were observed and pho-tographed using inverted light microscopy (IX70 OlympusOptical Co Tokyo Japan)

26 Annexin V-FITCPI Double Staining Assay Exponen-tially growing HepG2 cells were placed down in 6-well plateand cultured as above Then the cells were treated withSD extracts at different concentrations for 24 h After thedrug incubation time all cells were harvested with trypsinand washed twice with PBS followed by resuspended in400 120583L Annexin V binding bufferThen the cells were stainedwith 5 120583L Annexin V-FITC for 15 minutes and 10 120583L PIfor 5 minutes at 4∘C This assay was performed exactlyas the manufacturersrsquo instruction of the Annexin V-FITCcell apoptosis detection kit (BestBio china) A FACSCaliburflow cytometer was used to detected fluorescence and thepercentage of apoptotic cells was calculated by the internalsoftware system of the FACSCalibur Approximately 104 cellswere analyzed for each trail

27 RNA Extraction and Real-Time PCR ThemRNA levels ofCOX-2 FLAP 5-LOX 12-LOX 15-LOX bcl-2 bax caspase-3and survivinwere quantified by real-timeRT-PCRassaysThe120573-actin was internal reference gene HepG2 cells were placeddown in 6-well plate at a concentration of 5 times 105 cellsmLAfter 60 confluency the cells were exposed to SD extracts atthe concentration of IC

50for different times (0 15 3 6 9 and

12 h) Then the cells were harvested to SD extract total RNAusing Trizol reagent A UV spectrophotometer was used toestimate RNA concentration at 260 nm The purity of RNAwas assessed by the ratio of absorbance at 260 and 280 nm(A260A280 between 18 and 20) After quantification 2 120583gRNA was used for the synthesis of cDNA in each reversetranscription reaction via TIANScript RT Kit We conductedPCR to amplify the target genes with reagents and protocolsfrom the SYBR Green PCR Master Mix kit by Invitrogen(Carlsbad CA USA) The 20 120583L reaction system contained1 120583L generated cDNA template 1120583L of specific sense primer(Table 1) 1 120583L of specific antisense primer 10 120583L 2 times SYBRGreen PCR Master Mix and 7 120583L double distilled water Thethermal cycling conditions were 95∘C for 15min followed by40 cycles of 95∘C for 10 s 59ndash63∘C (annealing temperature)for 20 s and 72∘C for 30 s and a final incubation at 72∘C for5minThe relative expression of each gene was normalized tothe amount of 120573-actin in the same dosage of cDNA and therelative quantification method was 2minusΔΔCT method [15] AllRT-PCR reactions for each sample were done in triplicate

28 Molecular Docking We performed a series of moleculardocking experiments to estimate the binding affinity of somebiflavonoids with COX-2 LOX-5 LOX-12 and LOX-15

281 Preparing Protein and Ligands The three-dimensionalstructure of biflavonoids was downloaded from PubChem(httppubchemncbinlmnihgov) database [16] and refinedwith the help of Discovery Studio Visualizer (httpaccelryscomproductsdiscovery-studiovisualizationhtml)Thenthese ligands were converted to MOL2 format using


Table 1 Primer list








3 Results




AU)

(min)

160

125

100

75

50

25

minus2000 100 200 300 400 500 600

1

2

34

5

6WVL 330nm






50value






0

20

40

60

80

100

0 20 40 60 80 100

Inhi

bito

ry ra

te (

)

Du145HepG2

HeLa

A549

PC12Vero







(a) (b)

(c) (d)







101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625017

28

186

11

(a)

101

102

103

104

100 101 102 103 104100

FL2

-HFL1-H

20140625019

558

576076

(b)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625015

132 712

513

(c)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625014

674

816300

(d)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625010

503

1268114

(e)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625009

144 1429

544

(f)


4 Discussion








0

50

100

Time (h)

mRN

A ex

pres

sion

( o

f0h

actio

n)

0 15 3 6 9 12

COX-25-LOX

FLAP12-LOX


0

100

200

300

Caspase-3Survivin

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(a)

0

100

200

BaxBcl-2

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(b)




H22





0

Com

poun

d 1

Com

poun

d 2

Com

poun

d 3

Com

poun

d 4

Com

poun

d 5

Com

poun

d 6

Docking result

COX-2LOX-5

LOX-12LOX-15

minus14

minus12

minus10

minus8

minus6

minus4

minus2




5 Conclusion


Abbreviations




doederleinii





Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 1 Primer list








3 Results




AU)

(min)

160

125

100

75

50

25

minus2000 100 200 300 400 500 600

1

2

34

5

6WVL 330nm






50value






0

20

40

60

80

100

0 20 40 60 80 100

Inhi

bito

ry ra

te (

)

Du145HepG2

HeLa

A549

PC12Vero







(a) (b)

(c) (d)







101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625017

28

186

11

(a)

101

102

103

104

100 101 102 103 104100

FL2

-HFL1-H

20140625019

558

576076

(b)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625015

132 712

513

(c)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625014

674

816300

(d)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625010

503

1268114

(e)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625009

144 1429

544

(f)


4 Discussion








0

50

100

Time (h)

mRN

A ex

pres

sion

( o

f0h

actio

n)

0 15 3 6 9 12

COX-25-LOX

FLAP12-LOX


0

100

200

300

Caspase-3Survivin

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(a)

0

100

200

BaxBcl-2

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(b)




H22





0

Com

poun

d 1

Com

poun

d 2

Com

poun

d 3

Com

poun

d 4

Com

poun

d 5

Com

poun

d 6

Docking result

COX-2LOX-5

LOX-12LOX-15

minus14

minus12

minus10

minus8

minus6

minus4

minus2




5 Conclusion


Abbreviations




doederleinii





Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















AU)

(min)

160

125

100

75

50

25

minus2000 100 200 300 400 500 600

1

2

34

5

6WVL 330nm






50value






0

20

40

60

80

100

0 20 40 60 80 100

Inhi

bito

ry ra

te (

)

Du145HepG2

HeLa

A549

PC12Vero







(a) (b)

(c) (d)







101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625017

28

186

11

(a)

101

102

103

104

100 101 102 103 104100

FL2

-HFL1-H

20140625019

558

576076

(b)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625015

132 712

513

(c)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625014

674

816300

(d)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625010

503

1268114

(e)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625009

144 1429

544

(f)


4 Discussion








0

50

100

Time (h)

mRN

A ex

pres

sion

( o

f0h

actio

n)

0 15 3 6 9 12

COX-25-LOX

FLAP12-LOX


0

100

200

300

Caspase-3Survivin

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(a)

0

100

200

BaxBcl-2

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(b)




H22





0

Com

poun

d 1

Com

poun

d 2

Com

poun

d 3

Com

poun

d 4

Com

poun

d 5

Com

poun

d 6

Docking result

COX-2LOX-5

LOX-12LOX-15

minus14

minus12

minus10

minus8

minus6

minus4

minus2




5 Conclusion


Abbreviations




doederleinii





Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)







101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625017

28

186

11

(a)

101

102

103

104

100 101 102 103 104100

FL2

-HFL1-H

20140625019

558

576076

(b)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625015

132 712

513

(c)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625014

674

816300

(d)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625010

503

1268114

(e)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625009

144 1429

544

(f)


4 Discussion








0

50

100

Time (h)

mRN

A ex

pres

sion

( o

f0h

actio

n)

0 15 3 6 9 12

COX-25-LOX

FLAP12-LOX


0

100

200

300

Caspase-3Survivin

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(a)

0

100

200

BaxBcl-2

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(b)




H22





0

Com

poun

d 1

Com

poun

d 2

Com

poun

d 3

Com

poun

d 4

Com

poun

d 5

Com

poun

d 6

Docking result

COX-2LOX-5

LOX-12LOX-15

minus14

minus12

minus10

minus8

minus6

minus4

minus2




5 Conclusion


Abbreviations




doederleinii





Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625017

28

186

11

(a)

101

102

103

104

100 101 102 103 104100

FL2

-HFL1-H

20140625019

558

576076

(b)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625015

132 712

513

(c)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625014

674

816300

(d)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625010

503

1268114

(e)

101

102

103

104

100 101 102 103 104100

FL2

-H

FL1-H

20140625009

144 1429

544

(f)


4 Discussion








0

50

100

Time (h)

mRN

A ex

pres

sion

( o

f0h

actio

n)

0 15 3 6 9 12

COX-25-LOX

FLAP12-LOX


0

100

200

300

Caspase-3Survivin

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(a)

0

100

200

BaxBcl-2

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(b)




H22





0

Com

poun

d 1

Com

poun

d 2

Com

poun

d 3

Com

poun

d 4

Com

poun

d 5

Com

poun

d 6

Docking result

COX-2LOX-5

LOX-12LOX-15

minus14

minus12

minus10

minus8

minus6

minus4

minus2




5 Conclusion


Abbreviations




doederleinii





Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

Time (h)

mRN

A ex

pres

sion

( o

f0h

actio

n)

0 15 3 6 9 12

COX-25-LOX

FLAP12-LOX


0

100

200

300

Caspase-3Survivin

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(a)

0

100

200

BaxBcl-2

Time (h)

mRN

A ex

pres

sion

( o

f0h

actin

)

0 15 3 6 9 12

(b)




H22





0

Com

poun

d 1

Com

poun

d 2

Com

poun

d 3

Com

poun

d 4

Com

poun

d 5

Com

poun

d 6

Docking result

COX-2LOX-5

LOX-12LOX-15

minus14

minus12

minus10

minus8

minus6

minus4

minus2




5 Conclusion


Abbreviations




doederleinii





Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

Com

poun

d 1

Com

poun

d 2

Com

poun

d 3

Com

poun

d 4

Com

poun

d 5

Com

poun

d 6

Docking result

COX-2LOX-5

LOX-12LOX-15

minus14

minus12

minus10

minus8

minus6

minus4

minus2




5 Conclusion


Abbreviations




doederleinii





Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















selaginella doederleinii hieron in vitro and in...

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