Evaluation of an Epitypified Ophiocordyceps formosana (Cordyceps ...

Research ArticleEvaluation of an Epitypified Ophiocordyceps formosana(Cordyceps sl) for Its Pharmacological Potential

Yen-Wen Wang1 Tzu-Wen Hong2 Yu-Ling Tai1 Ying-Jing Wang1 Sheng-Hong Tsai2

Pham Thi Kim Lien3 Tzu-Ho Chou1 Jui-Ya Lai2 Richard Chu2 Shih-Torng Ding45

Kenji Irie36 Tsai-Kun Li57 Shean-Shong Tzean1 and Tang-Long Shen15

1Department of Plant Pathology and Microbiology National Taiwan University Taipei 10617 Taiwan2Mucho Biotechnology Inc Taipei 10684 Taiwan3Graduate School of Comprehensive Human Sciences University of Tsukuba 1-1-1 Tennodai Tsukuba 305-8575 Japan4Department of Animal Science and Technology National Taiwan University Taipei 10672 Taiwan5Center for Biotechnology National Taiwan University Taipei 10617 Taiwan6Laboratory of Molecular Cell Biology Faculty of Medicine University of Tsukuba 1-1-1 Tennodai Tsukuba 305-8575 Japan7Graduate Institute of Microbiology College of Medicine National Taiwan University Taipei 10051 Taiwan

Correspondence should be addressed to Tsai-Kun Li tsaikunlintuedutw Shean-Shong Tzean sstntuedutw andTang-Long Shen shentlntuedutw

Received 10 March 2015 Revised 13 July 2015 Accepted 16 July 2015

Academic Editor Min Li

Copyright copy 2015 Yen-WenWang et alThis 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 substantial merit of Cordyceps sl spp in terms of medicinal benefits is largely appreciated Nevertheless only few studies havecharacterized and examined the clinical complications of the use of health tonics containing these species Here we epitypifiedC formosana isolates that were collected and characterized as Ophiocordyceps formosana based on morphological characteristicsmolecular phylogenetic analyses and metabolite profiling Thus we renamed and transferred C formosana to the new protologueOphiocordyceps formosana (Kobayasi amp Shimizu)Wang Tsai Tzean amp Shen comb novAdditionally the pharmacological potentialof O formosana was evaluated based on the hot-water extract from its mycelium The relative amounts of the known bioactiveingredients that are unique to Cordyceps sl species in O formosana were found to be similar to the amounts in O sinensis and Cmilitaris indicating the potential applicability ofO formosana for pharmacological uses Additionally we found that O formosanaexhibited antioxidation activities in vitro and in vivo that were similar to those of O sinensis and C militaris Furthermore Oformosana also displayed conspicuously effective antitumor activity compared with the tested Cordyceps sl species IntrinsicallyO formosana exhibited less toxicity than the otherCordyceps species Together our data suggest that themetabolites ofO formosanamay play active roles in complementary medicine

1 Introduction

The entomopathogenic fungus Cordyceps sl (sensu lato)species including Ophiocordyceps sinensis and Cordycepsmilitaris are among the prestigious traditional Chinesemedicines and have a long history of use as tonics andfolk medicines that can be applied to cancer and diabetestreatments and used as antioxidants among other uses [1]Rather than the safety of the use of Cordyceps sl (sensu lato)species much attention has been focused on their high valuedue to their pharmacological and therapeutic benefits in

the treatment of respiratory and cerebrovascular diseases [2]enhancement of immunoregulatory function [3] regulationof liver metabolism [4] and treatment of cancer [5] In thisregard in recent years much effort has been devoted todiscovering the bioactive ingredients pharmacological effi-cacies and mechanisms of action of the secondary metabo-lites that are derived from Cordyceps sl spp For instanceCordyceps sl spp have been well characterized as possessingabundant bioactive compounds such as cordycepin D-mannitol (cordycepic acid) adenosine polysaccharides vita-mins and enzymes Among these compounds cordycepin

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 189891 13 pageshttpdxdoiorg1011552015189891

2 Evidence-Based Complementary and Alternative Medicine

(3-deoxyadenosine) is uniquely produced in Cordyceps sppis a key active constituent of medicines and confers broad-spectrum biological activity due to the steroidogenic [6]and antitumor proliferation and antimetastasis activities [78] that are mediated by its antioxidation effects [9] Addi-tionally reactive oxygen species (ROS) have been found tocontribute to cellular necrosis and a variety of pathologicalconditions including cancer [10] degenerative diseases inneurons [11] hepatopathies [12] atherosclerosis [13] andeven aging [14] Therefore Cordyceps sl species have beenrepeatedly reported as antioxidants [15] due to their freeradical scavenging abilities

Cordyceps sl is a large paraphyletic group in Clavicipi-taceae sl that comprises species with a wide range of hoststhat span from insects and spiders to Ophiobolus and truffle-like fungi [16 17] Initially the classification was based onthe color and shape of the stromata the perithecia the sizesand shapes of asci and ascospores and the host speciesAccording to these characteristics Kobayasi classified thegenus into three subgenera Cordyceps Ophiocordyceps andNeocordyceps [17] Later two extra subgenera were intro-duced Racemella and Cryptocordyceps by Mains [18] Sincethen the definitions of several original subgenera have expe-rienced drastic changes Specifically for rapid and accurateidentification and phylogenetic studies molecular markerssuch as internal transcribed spacers (ITSs) RNA polymeraseII (RPB) and elongation factor (EF) have been widely used[19 20] In 2007 Sung et al revised the classification basedon phylogenetic analyses of multiple genes ConsequentlyClavicipitaceae sl was divided into three families Cordy-cipitaceae Clavicipitaceae ss and OphiocordycipitaceaeAmong these families Cordyceps sl was separated intoseveral genera that included Cordyceps ss ElaphocordycepsMetacordyceps and Ophiocordyceps Indeed the connectionsbetween themorphological characteristics and themolecularphylogeny provide invaluable information about Cordycepssl that can be used to identify species and genera based onmolecular evidence [21]

Ophiocordyceps formosana was first reported in 1981 byKobayasi [22] and the etymology referred to its originaldiscovery as an indigenous Cordyceps sl fungus in TaiwanHowever the characteristics of O formosana were poorlydocumented with respect to the morphological descriptionPhysiological and biochemical analyses and descriptions ofits pharmacological applications were completely absentAccordingly the growth habits and morphological featuresof the known herbal Cordyceps sl species attracted ourattention for their potential medicinal applications Never-theless until now only few and scattered findings regardingO formosana have been reported in Taiwan and China andsufficient specimens and samples for further characterizationand investigation are lacking

The safety and medicinal efficacy of Cordyceps sl andrelated products have been the focus of the developmentof complementary and traditional medicine However dueto its limited distribution high price overexploitation anddifficulty related to artificial culture O sinensis has becomean endangered species Here we further established anepitypified O formosana that is phylogenetically related to

O sinensis Additionally we evaluated its effective pharma-cological potentials in terms of antitumor and antioxidationbioactivities to elucidate the medicinal merit ofO formosanaas a substitute for O sinensis and C militaris

2 Materials and Methods

21 Sample Collection and Maintenance Ophiocordyceps for-mosana specimens were collected from mummified dark-ling beetle bodies residing in decayed wood in Lala ShanTaoyuan County Taiwan (latitude 24∘42101584012010158401015840N longitude121∘251015840492010158401015840E) in the summer of 2013The collected sampleswere immediately photographed and brought back to theApplied Mycology Laboratory of the National Taiwan Uni-versity in Taipei for spore and mycelium isolation accordingto a standard decontamination procedure Several isolatesincluding O formosana MUCHO 815 and O formosanaNTU00035 were successfully obtained and cultivated onPDA and S-DAY plates at 25∘C Every 3-4 weeks thecolonies were harvested The harvested colonies were firstdesiccated at 55∘C overnight and then stored in a desiccatorfor future use To maintain and preserve the O formosanasamples small cubic mycelia-containing agars were routinelytransferred to new PDA or S-DAY plates for maintenanceCordycepsmilitarismycelia and fruiting bodies were obtainedfromMucho Biotechnology Inc as described previously [23]Themycelia and fruiting bodies ofO sinensiswere purchasedfrom Tongrentang Co Ltd Beijing China

22 Micromorphological Characteristic Examination Micro-scopic examinations were performed via cryosectioning andexamination under a compound light microscope (Olym-pus Japan) The samples were fixed with 4 formaldehydein PBS for one day with one change of solution Afterthe samples were mounted in optimal cutting temperature(OCT) compound cryosectioning was performed with aLEICA CM3050-S cryostat (Leica Biosystems Germany) toobtain sections ranging in thickness from 5120583m to 10 120583mThe sections were examined and photographed under anOlympus BH2 light microscope (Olympus Japan) equippedwith a Canon-ds126 camera

23 Genomic DNA Extraction from Cordyceps spp ThegenomicDNA extraction fromCordyceps spp was performedaccording to a modified version of the protocol of Doyle[24] Briefly either the fungal samples were directly cut downfrom the stroma and stalks of Cordyceps or mycelia wereharvested from culture plates The collected samples werelyophilized and ground to powder with a pestle with 500 120583Lof hot CTAB buffer (2 CTAB 14M NaCl 20mM EDTA100mMTris at pH 80 and 2 PVP-40) in combination with3 120583L of fresh 120573-mercaptoethanol After incubation at 60∘Cfor 20 minutes 500120583L of chloroform isoamyl alcohol (CI24 1) was added and mixed gently for 10 minutes prior tocentrifugation at 13200 g for 2 minutes The supernatant wascarefully transferred to a new Eppendorf tube following theadministration of a 06x volume of isopropanol Next themixture was allowed to incubate at minus20∘C for 30 minutes to

Evidence-Based Complementary and Alternative Medicine 3

precipitate the DNA The DNA was harvested by centrifuga-tion at 13200 g for 30 minutes along with a single 75 coldethanol wash drying with a vacuum (LABCONCO USA)and resuspension in 50120583L of ddH

2O

24 DNA Amplification and Sequencing PCR was employedfor the amplification of specific DNA fragments as describedbelow In general the PCR reactionmixtures contained 25120583Lof 10x reaction buffer 1 120583L of 10 120583M of each specific primer(see Supporting Table 1 in Supplementary Material avail-able online at httpdxdoiorg1011552015189891) 05 120583L of10mM dNTPs 2 120583L of template DNA and 03 120583L of 1 U Taqpolymerase in a total volume of 25 120583L The RPB1 sequenceswere amplified using the forward primer cRPB1 and thereverse primer RPB1c-r [25] according to the following stepsdenaturation at 95∘C for 5 minutes 40 cycles of 95∘C for 1minute 55∘C for 1 minute and 72∘C for 1 minute and anadditional extension step at 72∘C for 10 minutes The RPB2sequences were amplified with the forward primer fRPB2-5f and the reverse primer fRPB2-7cr [26] according to thefollowing steps denaturation at 95∘C for 5 minutes 40 cyclesof 95∘C for 50 seconds 55∘C for 1 minute and 72∘C for90 seconds and an additional extension step at 72∘C for10 minutes The EF1-120572 sequences were amplified using theforward primer EF1-2218R and the reverse primer EF1-983F[27] according to the following steps denaturation at 94∘C for5minutes 40 cycles of 94∘C for 1minute 46∘C for 30 secondsand 72∘C for 2 minutes and an additional extension step at72∘C for 7 minutes The PCR products were electrophoresedand eluted for sequencing using an ABI 3730XL system(Applied Biosystems MA USA) The obtained sequenceswere then assembled and trimmed using a ContigExpress ofvector NTI

25 Phylogenetic Analysis The nucleotide sequences of threegenes that is the largest and second largest subunits ofRNA polymerase II (RPB1 RPB2) and translation elongationfactor 1120572 (EF1-120572) were either obtained from our sequencedPCR products of the O formosana isolates retrieved fromthe NCBI database following the criteria described by Sunget al [21] or acquired by whole genome ortholog BlastNs ofC militaris CM01 A total of 51 speciesisolates were includedin this study (Supporting Table 3) The nucleotide sequencesof these three genes were concatenated aligned and testedin models using MEGA 60 [28] The best model accordingto the AIC and BIC values was the GTR+I+G model Thealigned sequences were analyzed using CIPRES portal-basedRaxml blackbox (RAxML 727) [29] formaximum likelihood(ML) tree searching with the default settings an estimation ofthe gamma distribution invariable sites and Bayesian infer-ence of the phylogenywithMrBayes 323 with the parametersobtained from the model test The resulting phylogenetictrees were saved and plotted using Adobe Illustrator CS3

26 Cordyceps Extract Preparation and HPLC Analysis Allof the desiccated Cordyceps samples that were subjected toHPLC (HITACHI D-2000 HSM Japan) analyses for de novosecondarymetaboliteswere pulverizedwith a homogenizer at1300 rpm and subsequently stored in a humidity-controlled

desiccator at room temperature The preparation of theCordyceps extracts was performed according to the stan-dard operation procedure described below Briefly 1 g ofCordyceps powder was dissolved with deionized water at asolid-to-solvent ratio of 1 40 by vortexing for 30 secondsin a 50mL falcon tube (BD Biosciences USA) Next theextraction was performed with 50∘C hot water reflux for2 hours (including the first 30 minutes of ultrasonication)After 20min of centrifugation at 4000 rpm (approximately3200 g) the supernatant was collected and filtered with a022 120583m filter The resulting extracts were directly subjectedto HPLC analyses or stored at minus20∘C

High-performance liquid chromatography (HPLC) wasperformed on RP-18 column (150252 Purospher STAR RP-18 endcapped (5 120583m) LiChroCART 250-4 Merck) at a flowrate of 1mLmin The composition of the mobile phase was20 methanolH

2O Three of the indicated components

D-mannitol adenosine and cordycepin were detected at260 nm using a DAD detector and the corresponding reten-tion times of the individual standard compounds in the sameHPLC conditions Quantitation was preformed according tothe established standard curve of each compound

27 Cell Culture and MTT Assay The MTT assay was usedto measure the abilities of the Cordyceps extracts to inhibithuman cancer cell proliferation Approximately 5 times 103 cellswere seeded onto each well of a 96-well plate contain-ing 200120583L of culture medium (per well) 24 hours beforetreatment After exposure to various concentrations of theindicated extracts for 96 hours the cells were incubated with20120583L of MTT (Thiazolyl Blue Tetrazolium Bromide SigmaUSA) solution for 4 hours Cell proliferation was measuredby the absorption at 570 nm using a spectrophotometer(SpectraMax 190 UV-Vis Microplate Molecular DevicesUSA) The measurements were performed in triplicateThe percentages of viable cells were calculated as (sampleOD570

nmMock OD570

nm) times 100 () [30]

28 DPPH Antioxidation Assay The scavenging of DPPHradicals was estimated by vigorously mixing equal amountsof the Cordyceps extract and DPPH solution (01mM inmethanol) The mixture was then incubated at room tem-perature for 50 minutes before measurement at 517 nm usinga spectrophotometer (SpectraMax 190 UV-Vis MicroplateMolecular Devices USA) The scavenging activity was deter-mined by comparing the blank (100) that contained onlyDPPH and solvent The scavenging efficiency percentageswere calculated as (sampleOD

517nmMockOD

517nm)times 100

() [31]

29 Cell-Based ROS Scavenging Assay Approximately 5 times104 CHO-K1 cells were seeded in each well of a 12-well

plate and exposed to the indicated Cordyceps extracts for 1hour after 16 hours of serum starvation Here the 10mM N-acetylcysteine treatment served as a positive control AfterH2O2(1800 120583M) challenge for 5 minutes the cells were

incubated with fluorescent probes (CM-H2DCFDA SigmaUSA) for 30minutes in a 37∘C incubator (light was excluded)


Finally the cells were trypsinized and subjected to ROSquantification via the detection of the DCF levels whichcorresponded to the relative ROS amounts at 525 nm usinga flow cytometer (BD FACSCanto USA)

210 Tumor Xenografts An in vivo tumorigenesis assaywas conducted as previously described [32] Briefly 5 times106 MDA-MB-231 breast cancer cells were inoculated into

the left thighs of 10-week-old nude mice (BALBcAnNCg-Foxn1nuCrlNarl) via subcutaneous injections The oral gav-age treatments with O formosana extract began daily imme-diately after 10 days when the tumor growthwas palpableThemice were divided into three groups two groups were treatedwith O formosana water extracts at concentrations of 1x(25mgmL 100 120583Lday) and 5x (125mgmL 100120583Lday) anda control group was treated with PBS The tumor sizes weremeasured every four days and calculated with the formulatumor size = 04 times length times width2 according to previousreports [33] Statistical strategy was based on Kursquos study [34]After 26 days of O formosana water extract treatment thetumors in the mice were photographed

211 Statistical Analyses All data are expressed as the meansplusmn the SEMs based on at least three independent experimentsThe statistical analyses were performed using one-way anal-yses of variance (ANOVA) followed by Tukeyrsquos tests andsignificance was measured as indicated

3 Results

31 Collection and Morphological Features of Ophiocordycepsformosana We collected Cordyceps spp from varied poten-tial niches in Taiwan to assess their potentialmedicinal valuesand applications InTaoyuanCounty of Taiwan (GPS altitude1600m latitude 24∘42101584012010158401015840N longitude 121∘251015840492010158401015840Ecollectors Y-W Wang S-H Tsai T-W Hong J-Y LaiT-L Shen and others Figure 1(h)) two Ophiocordycepsformosana samples were found growing on darkling beetlelarvae (Tenebrionoidea) that resided on fallen decayed woodin August of 2013 Based on examination of the macroscopicand microscopic details of the newly collected O formosanathe morphological features were described and documentedas shown in Figure 1

Obviously the ascostromata arose from the heads orabdomens of the infected insect hosts (here darkling beetlelarva of the Tenebrionoidea superfamily) and the stalksexhibited long-cylindrical shapes of 10ndash30mm times 05ndash2mmin size that were orange (6A7) in color and bore short hairsbut lacked membranous sheaths The oblong ascostromataheads were 4ndash6mm times 1ndash4mm in size and largely consistedof pseudoparenchymatous epidermal tissues Moreover theperithecia were embedded within the stromata and werebrownish orange (7C8) and ovoid shaped (360ndash480 times 240ndash320 120583m) with short necks with ostioles of 60120583m in widthThe perithecial wall was 20120583m thick underneath withapproximately 40 120583m-thick epidermal tissue Furthermorethe asci displayed long-cylindrical shapes with attenuatedbases (65ndash79 120583m times 160ndash240 120583m) and thick dome-shaped

apices (39ndash53 times 32ndash46 120583m) along with narrow slits Theasci contained eight ascospores and hyaline were cylindricaland filamentous and were often fragmented into more than10ndash20 cylindrical and truncated partspores (26ndash30 times 65ndash73 120583m) at maturity Colonies grown on potato dextrose agar(PDA) were orange (5A7) to white in color and pulvinateto umbonate in shape and exhibited velutinous to floccosemycelia that were whitish light-orange to pink in color andexudation droplets The anamorphic state was similar toHir-sutella type and the conidiogenous cells were monophialidichyaline and elongated ampuliform-shaped (15ndash23 times 86ndash170 120583m) The conidia were hyaline and cylindrical (16ndash23 times36ndash69 120583m in size)

32 Molecular Identification of Ophiocordyceps formosana byRPB1 RPB2 and EF1-120572 In addition to the morphologicalfeatures we amplified the 790- 1062- and 1068-base pair(bp) nucleotide sequences of the RPB1 RPB2 and EF1-120572genes from the isolates of O formosana respectively bypolymerase chain reaction (PCR) with the correspondingspecific primers listed in Supporting Table 1 The obtainedand sequenced genes were subjected to BLAST in the NCBIdatabase (httpwwwncbinlmnihgov) The top hits ofthe 3 sequences were Cordyceps formosana TNM F13893Ophiocordyceps coenomyia NBRC 106964 and Cordycepsformosana TNM F13893 with identities of 100 (635635)90 (9561065) and 99 (847849) respectively Althoughthe top hit for RPB2 was not Cordyceps formosana thesequence shared 100 identity with the Cordyceps formosanaTNM F13893 with 47 coverage (Supporting Table 2) TheITS sequences were not utilized due to time and reagentlimitations However the multigene analysis results of thethree genes were sufficient to indicate that our collectedspecimens were identical to Cordyceps formosana TNMF13893 as verified by the molecular markers

Indeed the phylogenetic trees based on the maximumlikelihood method and Bayesian principles resulted in twoidentical trees with different support strengths A maximumlikelihood bootstrap tree is shown in Figure 2 and the pos-terior probability ML bootstrap support values are denotedafter the slashes The topologies of Cordyceps sl in the threenewly created families Ophiocordycipitaceae Cordycipi-taceae and Clavicipitaceae ss were largely congruent withthe results of Sung et al [21]O formosana resided within theOphiocordycipitaceae clade with a strong bootstrap supportvalue (BS = 10PP = 1) rather than in the other allied taxaof Cordycipitaceae and Clavicipitaceae ss PhylogeneticallyO formosana obviously exhibited a closer relatedness to Osinensis than to C militaris However in contrast C militariswas placed in the Cordycipitaceae clade which was distantlyrelated to O sinensis and O formosana Intriguingly the Oformosana collected in Taiwan from the varied geographicregions in this studywere in the clade ofO formosana isolatedby another group Based on the unambiguous morphologicaltraits and molecular phylogenetic study we recombined thepreviously erected Cordyceps formosana as Ophiocordycepsformosana and the epitypic specimen was designated as OformosanaMUCHO 815


(a) (b) (c)

(d) (e) (f)

(g)

Alishan Nantou

Guanwu Hsinchu

Lala Shan Taoyuan

(h)

Figure 1 Morphological and anatomical features of the Ophiocordyceps formosana collected from Taoyuan County Taiwan (a) The fungalascostromata arise from the host Tenebrionoidea insect corpses with cylindrical stalks Bar = 10mm (b) The orange stromatal head exhibitsan oblong shape Bar = 3mm (c) An ovoid perithecium embedded in a stroma is shown in cross section The asci reside within the ovoidperithecium and obliquely align with the perithecium Bar = 100120583m (d)The ostiole of a perithecium showing a narrow opening in the necksurrounded by a thick perithecial wall Bar = 25120583m (e) Cylindrical partspores derived from fragmented ascospores Bar = 10120583m (f) Thecolonies were velutinous or floccose with exudating droplets and orange to white in color on a PDA culture plate (g) AHirsutella anamorphof O formosana the branched conidiophore is curl-shaped and bears baciloid-shaped conidia Bar = 10120583m (h) The documented habitats ofO formosana specimens in Taiwan [22 35] (source Taiwan 23∘461015840509010158401015840N and 120∘571015840505410158401015840E from Google Earth)

33 Chemical Profiles of the Ophiocordyceps formosana WaterExtracts The chemical profiles of the extracts from themycelia and fruiting bodies of the various Cordyceps speciesincluding O sinensis and C militaris were compared withO formosana To perform the investigations the hot waterextracts of the tested Cordyceps samples were subjected toHPLC analyses The HPLC profile of O formosanaMUCHO815 (Figure 3(a)) was reminiscent of those of O sinensis

and C militaris which indicated that the biochemicalproperties of O formosana were similar to those of otherwell-known Cordyceps species Additionally we comparedthree predominatemetabolites that were unique inCordycepsspp cordycepin adenosine and D-mannitol As shown inFigure 3(b)O formosanaMUCHO815 possesses amounts ofthese threemajor bioactive compounds in terms of quantitiesand percentiles that were similar to other Cordyceps species


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Lecanicillium psalliotae CBS 53281Lecanicillium psalliotae CBS 101270

Lecanicillium attenuatum CBS 40278

Cordyceps ochraceostromata ARSEF 5691Cordyceps bifusispora EFCC 8260Cordyceps bifusispora EFCC 5690

Cordyceps takaomontana NHJ 12623

Cordyceps kyusyuensis EFCC 5886Cordyceps militaris CM01

Microhilum oncoperae AFSEF 4358

Cordyceps cardinalis OSC 93610

Torrubiella alba CBS 72673a

Torrubiella wallacei CBS 101237

Nectria sp CBS 47875

Pochonia chlamydosporia CBS 50466Nomuraea rileyi CBS 80671

Paecilomyces marquandii CBS 18227

Paecilomyces carneus CBS 23932Paecilomyces carneus CBS 39959

Cordyceps sp NHJ 12118Cordyceps sp OSC 110996

Pochonia bulbillosa CBS 14570

Pochonia rubescens CBS 46488Tolypocladium parasiticum ARSEF 3436

Aschersonia placenta BCC 7869Torrubiella luteorostrata NHJ 12516

Balansia epichloe AEG 96-15aClaviceps purpurea SA cp11Shimizuomyces paradoxus EFCC 6564Shimizuomyces paradoxus EFCC 6279

Ophiocordyceps entomorrhiza KEW 53484Elaphocordyceps subsessilis OSC 71235

Cordyceps variabilis ARSEF 5365Ophiocordyceps variabilis OSC 111003

Ophiocordyceps heteropoda EFCC 10125

Ophiocordyceps gracilis EFCC 8572Ophiocordyceps gracilis EFCC 3101

Ophiocordyceps formosana MUCHO 815Ophiocordyceps formosana TNM F13893

Ophiocordyceps formosana NTU 00035

Cordyceps ravenelii OSC 110995Ophiocordyceps nigrella EFCC 9247

Ophiocordyceps konnoana EFCC 7315

Ophiocordyceps sinensis EFCC 7287Cordyceps stylophora OSC 110999

Ophiocordyceps rhizoidea NHJ 12529Ophiocordyceps rhizoidea NHJ 12522

Ophiocordyceps irangiensis OSC 128579

Haptocillium zeosporum CBS 33580

Paecilomyces lilacinus CBS 28436Paecilomyces lilacinus CBS 43187

Roumegueriella rufula GJS 91-164

Ophiocordycipitaceae

Clavicipitaceae ssC

ordycipitaceae

Figure 2 Phylogenetic tree ofCordyceps slPhylogenetic cladogramofCordyceps sl showing the three newly created families Clavicipitaceaess Cordycipitaceae and Ophiocordycipitaceae as inferred by the maximum likelihood (ML) method and Bayesian inference of thephylogeny using three concatenated genes (RPB1 RPB2 and EF1-120572) The bootstrap support values are denoted on the branches in frontof the posterior probabilities The underlined Ophiocordyceps formosana NTU 00035 and MUCHO 815 samples were collected from Taiwanin this study

Collectively we epitypified O formosana based on detailedstudies of its morphological molecular and biochemicalfeatures

34 The Ophiocordyceps formosana Water Extract DisplayedAntioxidation Activity Ophiocordyceps formosana displayedbiochemical features that were analogous to those of othervaluable medicinal Cordyceps spp slWe furthermore exam-ined its biological and pharmacological properties Thenature of the antioxidant activity of the hot water extractfrom O formosana was evaluated based on DPPH assayand cell-based ROS scavenging bioactivity As shown in

Figures 4(a) and 4(b) the hot water extract of O formosanaMUCHO 815 apparently exhibited a strong antioxidant capa-bility compared with the extracts from the mycelia of Osinensis and C militaris (Figure 4(a)) that was even compa-rable with that of the extract derived from the O sinensisfruiting body (Figure 4(b)) Coincidently an in vivo cell-basedROS scavenging assay usingCHO-K1 cells revealed thatO formosana exhibited a nonstatistically significantly greaterantioxidant effect than the mycelial extracts (Figure 4(c))Notably the extract from the O formosana mycelia was alsocomparable to the extracts derived from the O sinensis andC militaris fruiting bodies (Figure 4(d)) Our data imply that


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D-Mannitol Adenosine CordycepinRetentiontime (min)Cordycepsspp

Concentration(mgmL)

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Concentration(mgmL)

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Concentration(mgmL)

permil(mgg)

10683 427 676 27 2314 09220245 810 353 14 4029 16137119 1484 282 11 3016 12616197 647 375 15 2616 10425740 102 667 26 1732 069

311ndash321 679ndash689 742ndash749

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CMFB

(b)

Figure 3 HPLC profiles and analyses of extracts from various Cordyceps species (a) The HPLC retention times are shown for D-mannitol(designated as ldquoDrdquo) adenosine (designated as ldquoArdquo) and cordycepin (designated as ldquoCrdquo) (A) The HPLC profiles extracted from the Oformosana mycelia (B OFMY) the O sinensis mycelis (C OSMY) and fruiting bodies (D OSFB) and the C militaris mycelia (E CMMY)and fruiting bodies (F CMFB) (b)The quantitative results for the indicated compounds were calculated according to the area under the peakthat corresponded to the standard calibration r2 gt 0999


OFMY OSMYCMMY

lowastlowast

125 25 5 100Concentration (mgmL)

0

10

20

30

40

Scav

engi

ng effi

cien

cy (

)

(a)

OFMY OSFBCMFB


0

20

40

60

80

Scav

engi

ng effi

cien

cy (

)

(b)

Blanktimes103

times10350 100 150 200 250

FSC-A

50

100

150

200

250

SSC-

A

H2O2 (1800 120583M)

0

25

50

75

100

Cou

nt

103 104 105102

FITC-A103 104 105102

FITC-A

OSFB

0

50

100

150

Cou

nt

103 104 105102

FITC-A

0

25

50

75

100

125

Cou

nt

CMFB

Blank

105104103102

FITC-A

0

50

100

150

Cou

nt

0

25

50

75

100

125

Cou

nt

103 104 105102

FITC-A

OFMY CMMY

0

25

50

75

100

125C

ount

103 104 105102

FITC-A103 104102 105

FITC-A

0

25

50

75

100

125

Cou

nt

OSMY

(c)

AB AB

CBC

A

CMMY OSMY CMFB OSFBOFMY

0

20

40

60

80

100

Scav

engi

ng effi

cien

cy (

)

(d)

Figure 4 The in vitro and in vivo antioxidation activities of the extracts from various Cordyceps species (a) The in vitro DPPH antioxidantactivities of the water extracts of O formosana C militaris and O sinensis mycelia (designated as OFMY CMMY and OSMY resp) (b) TheDPPH antioxidant activity of theO formosanamycelial water extract compared to those of the water extracts of theCmilitaris andO sinensisfruiting bodies (designated as CMFB and OSFB resp) (c d) The in vivo cell-based ROS scavenging effect as measured by flow cytometry (c)and the quantification results (d) ABCBar graphs without the same letter are significantly different from one another in each group (119901 lt 005)


O formosanamay serve as an alternative andor substitute fortonic and medicinal uses of O sinensis and C militaris

35 The Ophiocordyceps formosana Water Extract ExhibitsAnticancer Activity To determine the anticancer potentialof O formosana the hot water extracts from the myceliaand fruiting bodies of O formosana O sinensis and Cmilitaris were tested against various human cancer cell linesincluding A549 lung cancer MDA-MB-231 breast cancerHuh7 liver cancer and HL-60 leukemia cells at differentdosages As shown in Figure 5(a) the O formosana extractexerted anticancer activities IC

50s of 10mgmL for the A549

cells 053mgmL for the MDA-MB-231 cells 044mgmLfor the Huh7 cells and 019mgmL for the HL-60 cellsApparently theO formosana extract exhibitedmore effectivecancer cell suppression activities than the extracts from theOsinensis and C militaris mycelia that were even comparableto those of the extract derived from the O sinensis fruitingbodies in all tested cancer cell lines (Table 1) Strikingly theOformosana extract yielded the lowest toxicities (maintaining asurvival rate greater than 90) to the tested normal cell lineswhich included HCEC normal human corneal epithelial cellsandMCF10A normal human breast epithelial cells among allof the other Cordyceps extracts as shown in Figure 5(b)

36 In Vivo Anticancer Activity of the Ophiocordyceps for-mosana Water Extracts To further validate the antitumoractivities of the extracts from O formosana we conductedan in vivo antitumorigenesis assay using tumor xenograftmouse models Briefly nude mice were first implementedwith the human MDA-MB-231 breast cancer cells and thentreated with the water extracts of O formosana at low(25mgday) and high doses (125mgday) daily by oralgavages (Figure 6)The results showed that the treated groupsexhibited decreases in tumor size compared to the nontreatedgroup in a dose-dependent manner Our study highlights thepotential future applications of O formosana in tonics andmedicines

4 Discussion

Ophiocordyceps formosana was originally collected fromXitao Nantou County Taiwan and described and namedCordyceps formosana in the subgenus Eucordyceps byKobayasi [17 22] Nevertheless Ophiocordyceps formosanadid not receive much attention until a revisit based on a newcollection found in the Guanwu National Forest RecreationArea of Hsinchu County Taiwan [35] Consistent withour discovery the indigenous Ophiocordyceps formosanaseemed to prefer to inhabit Tenebrionoidea larva in areas oftemperate broadleaf forests at approximately 1500ndash1800m inaltitude (Figure 1(h)) More recently O formosana was alsofound in Huangshan Anhui Providence of China by Li etal which extended the distribution of O formosana beyondTaiwan [36]

Morphologically our collected samples were nearlyalmost identical to the holotype of Cordyceps formosanadescribed by Kobayasi [22] and Li et al [36] which thereforejustified the identification of the entomopathogen discovered

in this study as C formosana (Figure 1) However themorphology and the genotype of C formosana had notbeen clearly defined and linked To address this issuethe nucleotide sequences of the RPB1 RPB2 and EF1-120572 housekeeping genes that were obtained and sequencedfrom our fungal samples were aligned and compared withthe documented allied sequences of the reported C for-mosana TNM F13893 to further validate the identities ofboth fungal specimens The reason that we used RPB1RPB2 and EF1-120572 to infer the species phylogeny was basedon a hypothesis that essential genes are more likely tobe subjected to the same selection forces and convergentevolution in amino acid sequences is less likely to occurthis hypothesis has also been supported in several previousstudies of the phylogenies of the Cordyceps relatives [20 21]Other approaches involving the use of a neutral locus suchITS should be appropriate for inferring species phylogenyhowever ITS sequences change too fast and are thereforeunsuitable for inferring the phylogenies of distantly relatedspecies Indeed we failed to obtain the ITS sequence of Cformosana using the ldquoso-calledrdquo conserved primer sequencesTherefore in this study we were dealing with distantlyrelated species so these three genes were selected to infer thespecies phylogeny Interestingly the phylogenetic tree-basedmaximum likelihood and Bayesian inference phylogeneticmethods both revealed the same topology although due torestrictions in computer capacity we were unable to rule outthe possibility that the trees were trapped in local optimaHowever we replicated two runs of the BI analyses and theresults were identical which supports our hypothesis thatOphiocordyceps formosana resides in OphiocordycipitaceaeDue to the clear evidence from not only the morphologicalcharacteristics but also the molecular linkages we epitypifiedC formosana to O formosana Moreover the anamorphicstate ofHirsutella type in culture was also consistent with thedescription from Li et al [36] According to the definitionof Ophiocordyceps proposed by Kobayasi the anamorphs ofthis genus consist of four genera Hirsutella HymenostilbeParaisaria and Syngliocladium types [22] The results of thephylogenetic investigation further support our suggestionfor combining and transferring C formosana to the newprotologue Ophiocordyceps formosana (Kobayasi amp Shimizu)Wang Tsai Tzean amp Shen comb nov (see Supporting Text 1for details) although the comparisons of several phylogeneti-cally relatedOphiocordyceps spp indicated thatO formosanaapparently displays a brighter yellow color of its stromata thatdistinguishes it from other species in this clade (Figure 1)For example O ravenelii exhibits superficially distributedperithecia and dark brown-colored clavate stromata [37]The stromata of O nigrella are also dark brown and clavatebut the perithecia are embedded in mycelial tissues [38]whereas O konnoana exhibits yellow clavate stromata andsuperficial perithecia [39] However O heteropoda and Ogracilis exhibit embedded perithecia and capitate stromata[40 41] which indicates that these two species may becomparatively less related to O formosana Interestingly theclade that was phylogenetically allied to O formosana andincluded O ravenelii O nigrella and O konnoana appearsto exhibit a similar host preference that is Coleoptera


120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1

Concentration (mgmL)

A549

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

MAD-MB-231120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


0

Huh7120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

HL60120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(a)

HCEC120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

MCF10A120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(b)

Figure 5 Antitumor activities of the extracts from various Cordyceps species (a) Survival rates of various cancer cell lines including thehuman lung cancer A549 human breast cancer MDA-MB-231 human liver cancer Huh7 and human leukemia HL60 cell lines exposed tothe water extracts of O formosana (OFMY) C militaris (CMMY and CMFB) or O sinensis (OSMY and OSFB) (b) The survival rates of humannormal cell lines human corneal epithelial HCEC cells and human lung epithelial MCF10A cells that were exposed to the water extracts ofO formosana C militaris or O sinensis


PBS

4 8 12 16 200Days (of treatment)

1X OFMY

5X OFMY

0

50

100

150

Tum

or v

olum

e (m

m3)

(a)

PBS 1X OFMY 5X OFMY

(b)

Figure 6 In vivo antitumor activities of the extracts from O formosana (a) Mice were xenografted with human MDA-MB231 breast cancercells and then orally treated with the water extracts of O formosana (OFMY) with 1x (25mg 119899 = 3) 5x (125mg 119899 = 3) or PBS only dailyThe tumor sizes were recorded every four days and showed decreases in tumor volumes in accordance with the dosage of treated OFMY waterextracts Note that bar is shown as SEM (b) Photos of tumors in the mice with or without treatments on day 20

Table 1 The IC50

values for each of the extracts of the various Cordyceps spp with antitumor activities

Cell line (IC50

) OFMY CMFB CMMY OSFB OSMY

A549 100 plusmn 109 028 plusmn 006 170 plusmn 021 131 plusmn 062 054 plusmn 057MDA-MB-231 053 plusmn 015 020 plusmn 009 085 plusmn 008 275 plusmn 011 686 plusmn 006Huh7 044 plusmn 016 001 plusmn 001 044 plusmn 013 268 plusmn 011 116 plusmn 043HL-60 019 plusmn 013 007 plusmn 001 072 plusmn 017 032 plusmn 015 114 plusmn 015OFMY O formosanamycelium CMFB C militaris fruiting body CMMY C militarismycelium OSFB O sinensis fruiting body OSMY O sinensismycelium

larvae In contrast O heteropoda and O gracilis parasitizeAuchenorrhyncha (cicada) nymphs and Lepidopteran lar-vae respectively Indeed regarding phytopathogens linkagesbetween host ranges and evolution have been suggested[42] Although it is currently recognized as belonging toCordyceps sl the strict host specificities of some of theclades such as Elaphocordyceps may be due to recent hostjumping [21 43] that might have given rise to strong drivingforces for an evolutionarily genetic drift Moreover this cladealso shares morphological features of the stromata howeverthis seems to be only a symplesiomorphic character withinOphiocordyceps spp [43]

The adverse effects of oxidative stress on human healthhave attracted considerable interest Indeed the exposure oforganisms or cells to excessive amounts of reactive oxygenspecies (ROS) results in striking homeostatic imbalances[44] Eventually high levels of ROS are capable of inducingcellular and DNA damage triggering cell apoptosis andcontributing to deleterious complications such as aging highblood pressure inflammatory responses and even cancerTwo Cordyceps spp that is O sinensis and C militaris arecommonly used to ameliorate conditions associated withaging [45] and several malignant complications includinglung disorders [46] diabetes [47] kidney failure [48] andcancer [23] It is believed that the antioxidation activitiesderived from different preparations or fractions of Cordy-ceps are effective in therapeutics [49] Using the in vitro

DPPH assay and the cell-based anti-ROS scavenging assay(Figure 4) the extract from the mycelia of O formosanawas found to exhibit prominent antioxidative activities thatexceeded those of the mycelia of C militaris and O sinensisandwere even comparable to those of the fruiting bodies ofOsinensis Hence our evidence-based results suggest the valueof the complementary and alternativemedicineO formosanafor oxidation-related complications

In recent years increasing amounts of evidence haveindicated that Cordyceps spp are competent as alternativeantitumor medicines and adjuvants for chemo- and radio-therapy in the treatment of various cancers [50] Notablythe hot water extract from the mycelia of O formosanaexhibits superior antitumor activities against various cancercell lines via induction of cancer apoptosis (unpublisheddata) including breast lung and liver cancers and leukemiacompared to extracts from the mycelia of O sinensis and Cmilitaris (Figure 5(a)) Moreover the O formosana mycelialextract even exhibited an anticancer efficacy that was com-parable to that of the extract from the O sinensis fruitingbodies (Table 1) therefore benefits are available for manymarkets because a massive supply is achievable Intriguinglythe relatively lower toxicity of O formosana to normal cellsrenders O formosana as more competent as an alternativemedicine for the treatment of cancer (Figure 5(b)) In supportof this idea we have observed no apparent deleterious effectson any of the dissected tissuesorgans or in the in vivo


biochemistries of mice that have been orally treated with Oformosana for 14 successive days at concentrations of up to2 gkg (unpublished data)

5 Conclusions

In conclusion we established an evidence-based alterna-tive indigenous medicinal Cordyceps sl named Ophiocordy-ceps formosana that exhibits pharmacological potential asan anticancer and antioxidative stress-related complicationagent With the support of the HPLC analyses the chemicalprofile indicated that key bioactive ingredients includingD-mannitol adenosine and cordycepin were present inOphiocordyceps formosana at amounts that were comparableor even exceeded those in other known traditional medicinalCordyceps species In addition to its high level of pharma-cological efficacy O formosana also exhibited lower toxicitythan the documented valuable Cordyceps species O sinensisand C militaris Together these findings support the notionthat this epitypified Ophiocordyceps formosana (Cordycepssl) can serve as an alternative medicinal fungus

Conflict of Interests

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

Acknowledgments

The authors gratefully thank Drs Bo-Chi Chen Jun-YangLiou Lee-Yen Sheen and Shang-Ming Chou for their criticalreading and comments on this paper

References

[1] C-Q Ling X-Q Yue and C Ling ldquoThree advantages ofusing traditional Chinese medicine to prevent and treat tumorrdquoJournal of Integrative Medicine vol 12 no 4 pp 331ndash335 2014

[2] J Wang Y-M Liu W Cao K-W Yao Z-Q Liu and J-YGuo ldquoAnti-inflammation and antioxidant effect of cordymina peptide purified from the medicinal mushroom Cordycepssinensis in middle cerebral artery occlusion-induced focalcerebral ischemia in ratsrdquo Metabolic Brain Disease vol 27 no2 pp 159ndash165 2012

[3] B Shi Z Wang H Jin Y W Chen Q Wang and Y QianldquoImmunoregulatory Cordyceps sinensis increases regulatory Tcells to Th17 cell ratio and delays diabetes in NOD micerdquoInternational Immunopharmacology vol 9 no 5 pp 582ndash5862009

[4] N Manabe Y Azuma M Sugimoto et al ldquoEffects of themycelial extract of cultured Cordyceps sinensis on in vivohepatic energymetabolism and blood flow in dietary hypoferricanaemic micerdquo British Journal of Nutrition vol 83 no 2 pp197ndash204 2000

[5] T Jayakumar C-C Chiu S-H Wang D-S Chou Y-KHuang and J-R Sheu ldquoAnti-cancer effects of CME-1 a novelpolysaccharide purified from the mycelia of Cordyceps sinensisagainst B16-F10melanoma cellsrdquo Journal of Cancer Research andTherapeutics vol 10 no 1 pp 43ndash49 2014

[6] H-Y Pao B-S Pan S-F Leu and B-M Huang ldquoCordycepinstimulated steroidogenesis in MA-10 mouse Leydig tumor cellsthrough the protein kinase C pathwayrdquo Journal of Agriculturaland Food Chemistry vol 60 no 19 pp 4905ndash4913 2012

[7] A Sato N Yoshikawa E Kubo et al ldquoInhibitory effect of cordy-cepin on experimental hepatic metastasis of B16-F0 mousemelanoma cellsrdquo In Vivo vol 27 no 6 pp 729ndash732 2013

[8] J Y Wu Q X Zhang and P H Leung ldquoInhibitory effects ofethyl acetate extract of Cordyceps sinensismycelium on variouscancer cells in culture and B16 melanoma in C57BL6 micerdquoPhytomedicine vol 14 no 1 pp 43ndash49 2007

[9] T Ramesh S-K Yoo S-W Kim et al ldquoCordycepin (31015840-deoxyadenosine) attenuates age-related oxidative stress andameliorates antioxidant capacity in ratsrdquo Experimental Geron-tology vol 47 no 12 pp 979ndash987 2012

[10] F A Faisal D Sundi J L Cooper et al ldquoRacial disparitiesin oncologic outcomes after radical prostatectomy long-termfollow-uprdquo Urology vol 84 no 6 pp 1434ndash1441 2014

[11] I N Tiurenkov E V Volotova D V Kurkin D A BakulinI O Logvinov and T A Antipova ldquoNeuroprotective effectof neuroglutam under conditions of activated free radicaloxidationrdquo Eksperimentalrsquonaia i Klinicheskaia Farmakologiiavol 77 no 8 pp 16ndash19 2014

[12] K Matsui Y Kawaguchi T Ozaki et al ldquoEffect of active hexosecorrelated compound on the production of nitric oxide inhepatocytesrdquo Journal of Parenteral and Enteral Nutrition vol 31no 5 pp 373ndash380 2007

[13] E-S Park D-H Kang M-K Yang et al ldquoCordycepin 31015840-deoxyadenosine prevents rat hearts from ischemiareperfusioninjury via activation of AktGSK-3120573p70S6K signaling pathwayand HO-1 expressionrdquo Cardiovascular Toxicology vol 14 no 1pp 1ndash9 2014

[14] D-B Ji J Ye C-L Li Y-H Wang J Zhao and S-Q CaildquoAntiaging effect of Cordyceps sinensis extractrdquo PhytotherapyResearch vol 23 no 1 pp 116ndash122 2009

[15] S P Li K J Zhao Z N Ji et al ldquoA polysaccharide isolated fromCordyceps sinensis a traditional Chinese medicine protectsPC12 cells against hydrogen peroxide-induced injuryrdquo LifeSciences vol 73 no 19 pp 2503ndash2513 2003

[16] Y Kobayasi ldquoThe genusCordyceps and its alliesrdquo Science Reportsof the Tokyo Bunrika Daigaku Section B vol 5 no 84 pp 53ndash260 1941

[17] Y Kobayasi ldquoKeys to the taxa of the genera Cordyceps andTorrubiellardquo Transaction of the Mycological Society of Japan vol23 pp 329ndash364 1982

[18] E B Mains ldquoNorth American entomogenous species of Cordy-cepsrdquoMycologia vol 50 no 2 pp 169ndash222 1958

[19] C L Schoch K A Seifert S Huhndorf et al ldquoNuclear riboso-mal internal transcribed spacer (ITS) region as a universal DNAbarcode marker for fungirdquo Proceedings of the National Academyof Sciences of the United States of America vol 109 no 16 pp6241ndash6246 2012

[20] Y Tanabe M Saikawa M M Watanabe and J SugiyamaldquoMolecular phylogeny of Zygomycota based on EF-1120572 and RPB1sequences limitations and utility of alternative markers torDNArdquoMolecular Phylogenetics and Evolution vol 30 no 2 pp438ndash449 2004

[21] G-H Sung N L Hywel-Jones J-M Sung J J Luangsa-ardB Shrestha and J W Spatafora ldquoPhylogenetic classification ofCordyceps and the clavicipitaceous fungirdquo Studies in Mycologyvol 57 pp 5ndash59 2007


[22] Y Kobayasi ldquoThe genus Cordyceps and its allies from Taiwan(Formosa)rdquo Bulletin of the National Science Museum Series B(Tokyo) vol 7 no 4 pp 113ndash122 1981

[23] S-M ChouW-J Lai T-WHong et al ldquoSynergistic property ofcordycepin in cultivated Cordyceps militaris-mediated apopto-sis in human leukemia cellsrdquo Phytomedicine vol 21 no 12 pp1516ndash1524 2014

[24] J J Doyle ldquoIsolation of plant DNA from fresh tissuerdquo Focus vol12 pp 13ndash15 1990

[25] L A Castlebury A Y Rossman G-H Sung A S Hyten andJ W Spatafora ldquoMultigene phylogeny reveals new lineage forStachybotrys chartarum the indoor air fungusrdquo MycologicalResearch vol 108 no 8 pp 864ndash872 2004

[26] Y J Liu S Whelen and B D Hall ldquoPhylogenetic relationshipsamong ascomycetes evidence from an RNA polymerse IIsubunitrdquo Molecular Biology and Evolution vol 16 no 12 pp1799ndash1808 1999

[27] S A Rehner and E Buckley ldquoA Beauveria phylogeny inferredfrom nuclear ITS and EF1-120572 sequences evidence for crypticdiversification and links to Cordyceps teleomorphsrdquoMycologiavol 97 no 1 pp 84ndash98 2005

[28] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[29] A Stamatakis ldquoRAxML-VI-HPC maximum likelihood-basedphylogenetic analyses with thousands of taxa and mixed mod-elsrdquo Bioinformatics vol 22 no 21 pp 2688ndash2690 2006

[30] J Mondal K Bishayee A K Panigrahi and A R Khuda-Bukhsh ldquoLow doses of ethanolic extract of Boldo (Peumusboldus) can ameliorate toxicity generated by cisplatin in normalliver cells ofmice in vivo and inWRL-68 cells in vitro but not incancer cells in vivo or in vitrordquo Journal of Integrative Medicinevol 12 no 5 pp 425ndash438 2014

[31] S Saha F Hossain M Anisuzzman andM K Islam ldquoPharma-cological evaluation of Musa seminifera Lour fruitrdquo Journal ofIntegrative Medicine vol 11 no 4 pp 253ndash261 2013

[32] P-Y Chu T-K Li S-T Ding I-R Lai and T-L Shen ldquoEGF-induced Grb7 recruits and promotes ras activity essential forthe tumorigenicity of Sk-Br3 breast cancer cellsrdquoThe Journal ofBiological Chemistry vol 285 no 38 pp 29279ndash29285 2010

[33] K-H Lu Y-F Chang P-H Yin et al ldquoIn vitro and in vivoapoptosis-inducing antileukemic effects ofMucunamacrocarpastem extract on HL-60 human leukemia cellsrdquo IntegrativeCancer Therapies vol 9 no 3 pp 298ndash308 2010

[34] H H Ku ldquoNotes on the use of propagation of error formulasrdquoJournal of Research of the National Bureau of Standards vol 70no 4 pp 263ndash273 1966

[35] Y-Z Wang andW-N Chou Investigation of the Environmentaland Ecological IndicatorsmdashThe Research Report for GuanwuMushroom Shei-Pa National Park Construction and PlanningAgency Ministry of the Interior Taichung Taiwan 2004

[36] C-R Li C-R Xia Y-R LinM-Z Fan andZ-Z Li ldquoHirsutellahuangshanensis sp nov The anamorph of Cordyceps formosanaand its infection on Tenebrio molitorrdquoMycosystema vol 24 no3 pp 349ndash355 1985

[37] E B Mains ldquoCordyceps Stylophora and Cordyceps raveneliirdquoMycologia vol 33 no 6 pp 611ndash617 1941

[38] Y Kobayasi ldquoCordyceps species from Japan 6rdquo Bulletin of theNational Science Museum Series B (Tokyo) vol 9 no 1 pp 1ndash21 1983

[39] Y Kobayasi and D Shimizu ldquoCordyceps species from Japan 2rdquoBulletin of the National Science Museum Tokyo Series B vol 6no 3 pp 77ndash96 1980

[40] I A Gorbunova V Y Kryukov and E G Zibzeev ldquoFirstrecords of the entomopathogenic fungus Ophiocordyceps gra-cilis (Ascomycota Hypocreales) from Siberiardquo Euroasian Ento-mological Journal vol 10 no 1 pp 17ndash18 2011

[41] Y Kobayasi ldquoOn the genus Cordyceps and its allies on cicadaefrom Japanrdquo Bulletin of the Biogeographical Society of Japan vol9 pp 145ndash176 1939

[42] P Schulze-Lefert and R Panstruga ldquoA molecular evolutionaryconcept connecting nonhost resistance pathogen host rangeand pathogen speciationrdquo Trends in Plant Science vol 16 no3 pp 117ndash125 2011

[43] N Nikoh and T Fukatsu ldquoInterkingdom host jumping under-ground phylogenetic analysis of entomoparasitic fungi of thegenus Cordycepsrdquo Molecular Biology and Evolution vol 17 no4 pp 629ndash638 2000

[44] J M C Gutteridge and B Halliwell ldquoFree radicals and antiox-idants in the year 2000 A historical look to the futurerdquo Annalsof the New York Academy of Sciences vol 899 pp 136ndash147 2000

[45] X-T Li H-C Li C-B Li D-Q Dou and M-B GaoldquoProtective effects on mitochondria and anti-aging activity ofpolysaccharides from cultivated fruiting bodies of Cordycepsmilitarisrdquo American Journal of Chinese Medicine vol 38 no 6pp 1093ndash1106 2010

[46] M Chen F W K Cheung M H Chan et al ldquoProtective rolesof onCordyceps lung fibrosis in cellular and rat modelsrdquo Journalof Ethnopharmacology vol 143 no 2 pp 448ndash454 2012

[47] Y Dong T Jing Q Meng et al ldquoStudies on the antidiabeticactivities of cordyceps militaris extract in diet-streptozotocin-induced diabetic sprague-dawley ratsrdquo BioMed Research Inter-national vol 2014 Article ID 160980 11 pages 2014

[48] H-P Wang C-W Liu H-W Chang J-W Tsai Y-Z Sungand L-C Chang ldquoCordyceps sinensis protects against renalischemiareperfusion injury in ratsrdquoMolecular Biology Reportsvol 40 no 3 pp 2347ndash2355 2013

[49] B-J Wang S-J Won Z-R Yu and C-L Su ldquoFree radical scav-enging and apoptotic effects of Cordyceps sinensis fractionatedby supercritical carbon dioxiderdquo Food and Chemical Toxicologyvol 43 no 4 pp 543ndash552 2005

[50] W-D Wu Z-M Hu M-J Shang et al ldquoCordycepin down-regulates multiple drug resistant (MDR)HIF-1alpha throughregulating AMPKmTORC1 signaling in GBC-SD gallbladdercancer cellsrdquo International Journal of Molecular Sciences vol 15no 7 pp 12778ndash12790 2014

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


(3-deoxyadenosine) is uniquely produced in Cordyceps sppis a key active constituent of medicines and confers broad-spectrum biological activity due to the steroidogenic [6]and antitumor proliferation and antimetastasis activities [78] that are mediated by its antioxidation effects [9] Addi-tionally reactive oxygen species (ROS) have been found tocontribute to cellular necrosis and a variety of pathologicalconditions including cancer [10] degenerative diseases inneurons [11] hepatopathies [12] atherosclerosis [13] andeven aging [14] Therefore Cordyceps sl species have beenrepeatedly reported as antioxidants [15] due to their freeradical scavenging abilities

Cordyceps sl is a large paraphyletic group in Clavicipi-taceae sl that comprises species with a wide range of hoststhat span from insects and spiders to Ophiobolus and truffle-like fungi [16 17] Initially the classification was based onthe color and shape of the stromata the perithecia the sizesand shapes of asci and ascospores and the host speciesAccording to these characteristics Kobayasi classified thegenus into three subgenera Cordyceps Ophiocordyceps andNeocordyceps [17] Later two extra subgenera were intro-duced Racemella and Cryptocordyceps by Mains [18] Sincethen the definitions of several original subgenera have expe-rienced drastic changes Specifically for rapid and accurateidentification and phylogenetic studies molecular markerssuch as internal transcribed spacers (ITSs) RNA polymeraseII (RPB) and elongation factor (EF) have been widely used[19 20] In 2007 Sung et al revised the classification basedon phylogenetic analyses of multiple genes ConsequentlyClavicipitaceae sl was divided into three families Cordy-cipitaceae Clavicipitaceae ss and OphiocordycipitaceaeAmong these families Cordyceps sl was separated intoseveral genera that included Cordyceps ss ElaphocordycepsMetacordyceps and Ophiocordyceps Indeed the connectionsbetween themorphological characteristics and themolecularphylogeny provide invaluable information about Cordycepssl that can be used to identify species and genera based onmolecular evidence [21]

Ophiocordyceps formosana was first reported in 1981 byKobayasi [22] and the etymology referred to its originaldiscovery as an indigenous Cordyceps sl fungus in TaiwanHowever the characteristics of O formosana were poorlydocumented with respect to the morphological descriptionPhysiological and biochemical analyses and descriptions ofits pharmacological applications were completely absentAccordingly the growth habits and morphological featuresof the known herbal Cordyceps sl species attracted ourattention for their potential medicinal applications Never-theless until now only few and scattered findings regardingO formosana have been reported in Taiwan and China andsufficient specimens and samples for further characterizationand investigation are lacking

The safety and medicinal efficacy of Cordyceps sl andrelated products have been the focus of the developmentof complementary and traditional medicine However dueto its limited distribution high price overexploitation anddifficulty related to artificial culture O sinensis has becomean endangered species Here we further established anepitypified O formosana that is phylogenetically related to

O sinensis Additionally we evaluated its effective pharma-cological potentials in terms of antitumor and antioxidationbioactivities to elucidate the medicinal merit ofO formosanaas a substitute for O sinensis and C militaris

2 Materials and Methods

21 Sample Collection and Maintenance Ophiocordyceps for-mosana specimens were collected from mummified dark-ling beetle bodies residing in decayed wood in Lala ShanTaoyuan County Taiwan (latitude 24∘42101584012010158401015840N longitude121∘251015840492010158401015840E) in the summer of 2013The collected sampleswere immediately photographed and brought back to theApplied Mycology Laboratory of the National Taiwan Uni-versity in Taipei for spore and mycelium isolation accordingto a standard decontamination procedure Several isolatesincluding O formosana MUCHO 815 and O formosanaNTU00035 were successfully obtained and cultivated onPDA and S-DAY plates at 25∘C Every 3-4 weeks thecolonies were harvested The harvested colonies were firstdesiccated at 55∘C overnight and then stored in a desiccatorfor future use To maintain and preserve the O formosanasamples small cubic mycelia-containing agars were routinelytransferred to new PDA or S-DAY plates for maintenanceCordycepsmilitarismycelia and fruiting bodies were obtainedfromMucho Biotechnology Inc as described previously [23]Themycelia and fruiting bodies ofO sinensiswere purchasedfrom Tongrentang Co Ltd Beijing China

22 Micromorphological Characteristic Examination Micro-scopic examinations were performed via cryosectioning andexamination under a compound light microscope (Olym-pus Japan) The samples were fixed with 4 formaldehydein PBS for one day with one change of solution Afterthe samples were mounted in optimal cutting temperature(OCT) compound cryosectioning was performed with aLEICA CM3050-S cryostat (Leica Biosystems Germany) toobtain sections ranging in thickness from 5120583m to 10 120583mThe sections were examined and photographed under anOlympus BH2 light microscope (Olympus Japan) equippedwith a Canon-ds126 camera

23 Genomic DNA Extraction from Cordyceps spp ThegenomicDNA extraction fromCordyceps spp was performedaccording to a modified version of the protocol of Doyle[24] Briefly either the fungal samples were directly cut downfrom the stroma and stalks of Cordyceps or mycelia wereharvested from culture plates The collected samples werelyophilized and ground to powder with a pestle with 500 120583Lof hot CTAB buffer (2 CTAB 14M NaCl 20mM EDTA100mMTris at pH 80 and 2 PVP-40) in combination with3 120583L of fresh 120573-mercaptoethanol After incubation at 60∘Cfor 20 minutes 500120583L of chloroform isoamyl alcohol (CI24 1) was added and mixed gently for 10 minutes prior tocentrifugation at 13200 g for 2 minutes The supernatant wascarefully transferred to a new Eppendorf tube following theadministration of a 06x volume of isopropanol Next themixture was allowed to incubate at minus20∘C for 30 minutes to



2O









nmMock OD570

nm) times 100 () [30]


517nmMockOD

517nm)times 100

() [31]









3 Results







(a) (b) (c)

(d) (e) (f)

(g)

Alishan Nantou

Guanwu Hsinchu

Lala Shan Taoyuan

(h)





74099

74099

1001

57-

47098

55095

22077

681

54098

600931001

1001

9911001

1001

9911001

1001

10011001

9811001

1001

92-51064

29-1001

991

891

44094

60096

4707488099

1001

71093

100186098

1001

1001

921

1001

86098

981

9911001

1001

880981001

1001


































Clavicipitaceae ssC

ordycipitaceae






(A)

1713

2033

2907

3507

4340

4660

6653

7320

8227

0 2 4


D A C

6 8 10 12 14

00

02

04

06

08Ab

sorb

ance

(AU

)

D

A

C

249

32

960

320

73

407

399

34

260

458

75

120

618

7

670

0 728

0

930

0


000

005

010

015

020

Abso

rban

ce (A

U)

(B)

D

CA

252

02

980

316

73

493

401

34

180

459

34

747

540

7

680

0

746

7

882

7


00

01

02

03

04

05

Abso

rban

ce (A

U)

(C)

D

AC

255

32

813

297

33

167

349

34

013

417

34

573

476

05

053

539

3

616

76

773

744

7

816

0

882

0

000

005

010

015

020

025

030

Abso

rban

ce (A

U)


(D)

(E)

D

A

252

7

315

33

493

387

34

167

473

35

053

673

3

808

7


000

005

010

015

020

025

030

035

Abso

rban

ce (A

U)

(F)

D

A

C

252

02

960

340

7

398

74

300 6

173

732

0

822

7

928

0


00

01

02

03

04

05

Abso

rban

ce (A

U)

(a)


Concentration(mgmL)

permil(mgg)

Concentration(mgmL)

permil(mgg)

Concentration(mgmL)

permil(mgg)

10683 427 676 27 2314 09220245 810 353 14 4029 16137119 1484 282 11 3016 12616197 647 375 15 2616 10425740 102 667 26 1732 069


OFMYOSFBOSMY

CMMY

CMFB

(b)



OFMY OSMYCMMY

lowastlowast


0

10

20

30

40

Scav

engi

ng effi

cien

cy (

)

(a)

OFMY OSFBCMFB


0

20

40

60

80

Scav

engi

ng effi

cien

cy (

)

(b)

Blanktimes103

times10350 100 150 200 250

FSC-A

50

100

150

200

250

SSC-

A

H2O2 (1800 120583M)

0

25

50

75

100

Cou

nt

103 104 105102

FITC-A103 104 105102

FITC-A

OSFB

0

50

100

150

Cou

nt

103 104 105102

FITC-A

0

25

50

75

100

125

Cou

nt

CMFB

Blank

105104103102

FITC-A

0

50

100

150

Cou

nt

0

25

50

75

100

125

Cou

nt

103 104 105102

FITC-A

OFMY CMMY

0

25

50

75

100

125C

ount

103 104 105102

FITC-A103 104102 105

FITC-A

0

25

50

75

100

125

Cou

nt

OSMY

(c)

AB AB

CBC

A


0

20

40

60

80

100

Scav

engi

ng effi

cien

cy (

)

(d)








4 Discussion





120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


A549

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

MAD-MB-231120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


0

Huh7120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

HL60120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(a)

HCEC120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

MCF10A120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(b)



PBS


1X OFMY

5X OFMY

0

50

100

150

Tum

or v

olum

e (m

m3)

(a)

PBS 1X OFMY 5X OFMY

(b)


Table 1 The IC50


Cell line (IC50









5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2O









nmMock OD570

nm) times 100 () [30]


517nmMockOD

517nm)times 100

() [31]









3 Results







(a) (b) (c)

(d) (e) (f)

(g)

Alishan Nantou

Guanwu Hsinchu

Lala Shan Taoyuan

(h)





74099

74099

1001

57-

47098

55095

22077

681

54098

600931001

1001

9911001

1001

9911001

1001

10011001

9811001

1001

92-51064

29-1001

991

891

44094

60096

4707488099

1001

71093

100186098

1001

1001

921

1001

86098

981

9911001

1001

880981001

1001


































Clavicipitaceae ssC

ordycipitaceae






(A)

1713

2033

2907

3507

4340

4660

6653

7320

8227

0 2 4


D A C

6 8 10 12 14

00

02

04

06

08Ab

sorb

ance

(AU

)

D

A

C

249

32

960

320

73

407

399

34

260

458

75

120

618

7

670

0 728

0

930

0


000

005

010

015

020

Abso

rban

ce (A

U)

(B)

D

CA

252

02

980

316

73

493

401

34

180

459

34

747

540

7

680

0

746

7

882

7


00

01

02

03

04

05

Abso

rban

ce (A

U)

(C)

D

AC

255

32

813

297

33

167

349

34

013

417

34

573

476

05

053

539

3

616

76

773

744

7

816

0

882

0

000

005

010

015

020

025

030

Abso

rban

ce (A

U)


(D)

(E)

D

A

252

7

315

33

493

387

34

167

473

35

053

673

3

808

7


000

005

010

015

020

025

030

035

Abso

rban

ce (A

U)

(F)

D

A

C

252

02

960

340

7

398

74

300 6

173

732

0

822

7

928

0


00

01

02

03

04

05

Abso

rban

ce (A

U)

(a)


Concentration(mgmL)

permil(mgg)

Concentration(mgmL)

permil(mgg)

Concentration(mgmL)

permil(mgg)

10683 427 676 27 2314 09220245 810 353 14 4029 16137119 1484 282 11 3016 12616197 647 375 15 2616 10425740 102 667 26 1732 069


OFMYOSFBOSMY

CMMY

CMFB

(b)



OFMY OSMYCMMY

lowastlowast


0

10

20

30

40

Scav

engi

ng effi

cien

cy (

)

(a)

OFMY OSFBCMFB


0

20

40

60

80

Scav

engi

ng effi

cien

cy (

)

(b)

Blanktimes103

times10350 100 150 200 250

FSC-A

50

100

150

200

250

SSC-

A

H2O2 (1800 120583M)

0

25

50

75

100

Cou

nt

103 104 105102

FITC-A103 104 105102

FITC-A

OSFB

0

50

100

150

Cou

nt

103 104 105102

FITC-A

0

25

50

75

100

125

Cou

nt

CMFB

Blank

105104103102

FITC-A

0

50

100

150

Cou

nt

0

25

50

75

100

125

Cou

nt

103 104 105102

FITC-A

OFMY CMMY

0

25

50

75

100

125C

ount

103 104 105102

FITC-A103 104102 105

FITC-A

0

25

50

75

100

125

Cou

nt

OSMY

(c)

AB AB

CBC

A


0

20

40

60

80

100

Scav

engi

ng effi

cien

cy (

)

(d)








4 Discussion





120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


A549

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

MAD-MB-231120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


0

Huh7120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

HL60120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(a)

HCEC120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

MCF10A120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(b)



PBS


1X OFMY

5X OFMY

0

50

100

150

Tum

or v

olum

e (m

m3)

(a)

PBS 1X OFMY 5X OFMY

(b)


Table 1 The IC50


Cell line (IC50









5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results







(a) (b) (c)

(d) (e) (f)

(g)

Alishan Nantou

Guanwu Hsinchu

Lala Shan Taoyuan

(h)





74099

74099

1001

57-

47098

55095

22077

681

54098

600931001

1001

9911001

1001

9911001

1001

10011001

9811001

1001

92-51064

29-1001

991

891

44094

60096

4707488099

1001

71093

100186098

1001

1001

921

1001

86098

981

9911001

1001

880981001

1001


































Clavicipitaceae ssC

ordycipitaceae






(A)

1713

2033

2907

3507

4340

4660

6653

7320

8227

0 2 4


D A C

6 8 10 12 14

00

02

04

06

08Ab

sorb

ance

(AU

)

D

A

C

249

32

960

320

73

407

399

34

260

458

75

120

618

7

670

0 728

0

930

0


000

005

010

015

020

Abso

rban

ce (A

U)

(B)

D

CA

252

02

980

316

73

493

401

34

180

459

34

747

540

7

680

0

746

7

882

7


00

01

02

03

04

05

Abso

rban

ce (A

U)

(C)

D

AC

255

32

813

297

33

167

349

34

013

417

34

573

476

05

053

539

3

616

76

773

744

7

816

0

882

0

000

005

010

015

020

025

030

Abso

rban

ce (A

U)


(D)

(E)

D

A

252

7

315

33

493

387

34

167

473

35

053

673

3

808

7


000

005

010

015

020

025

030

035

Abso

rban

ce (A

U)

(F)

D

A

C

252

02

960

340

7

398

74

300 6

173

732

0

822

7

928

0


00

01

02

03

04

05

Abso

rban

ce (A

U)

(a)


Concentration(mgmL)

permil(mgg)

Concentration(mgmL)

permil(mgg)

Concentration(mgmL)

permil(mgg)

10683 427 676 27 2314 09220245 810 353 14 4029 16137119 1484 282 11 3016 12616197 647 375 15 2616 10425740 102 667 26 1732 069


OFMYOSFBOSMY

CMMY

CMFB

(b)



OFMY OSMYCMMY

lowastlowast


0

10

20

30

40

Scav

engi

ng effi

cien

cy (

)

(a)

OFMY OSFBCMFB


0

20

40

60

80

Scav

engi

ng effi

cien

cy (

)

(b)

Blanktimes103

times10350 100 150 200 250

FSC-A

50

100

150

200

250

SSC-

A

H2O2 (1800 120583M)

0

25

50

75

100

Cou

nt

103 104 105102

FITC-A103 104 105102

FITC-A

OSFB

0

50

100

150

Cou

nt

103 104 105102

FITC-A

0

25

50

75

100

125

Cou

nt

CMFB

Blank

105104103102

FITC-A

0

50

100

150

Cou

nt

0

25

50

75

100

125

Cou

nt

103 104 105102

FITC-A

OFMY CMMY

0

25

50

75

100

125C

ount

103 104 105102

FITC-A103 104102 105

FITC-A

0

25

50

75

100

125

Cou

nt

OSMY

(c)

AB AB

CBC

A


0

20

40

60

80

100

Scav

engi

ng effi

cien

cy (

)

(d)








4 Discussion





120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


A549

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

MAD-MB-231120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


0

Huh7120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

HL60120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(a)

HCEC120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

MCF10A120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(b)



PBS


1X OFMY

5X OFMY

0

50

100

150

Tum

or v

olum

e (m

m3)

(a)

PBS 1X OFMY 5X OFMY

(b)


Table 1 The IC50


Cell line (IC50









5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)

(d) (e) (f)

(g)

Alishan Nantou

Guanwu Hsinchu

Lala Shan Taoyuan

(h)





74099

74099

1001

57-

47098

55095

22077

681

54098

600931001

1001

9911001

1001

9911001

1001

10011001

9811001

1001

92-51064

29-1001

991

891

44094

60096

4707488099

1001

71093

100186098

1001

1001

921

1001

86098

981

9911001

1001

880981001

1001


































Clavicipitaceae ssC

ordycipitaceae






(A)

1713

2033

2907

3507

4340

4660

6653

7320

8227

0 2 4


D A C

6 8 10 12 14

00

02

04

06

08Ab

sorb

ance

(AU

)

D

A

C

249

32

960

320

73

407

399

34

260

458

75

120

618

7

670

0 728

0

930

0


000

005

010

015

020

Abso

rban

ce (A

U)

(B)

D

CA

252

02

980

316

73

493

401

34

180

459

34

747

540

7

680

0

746

7

882

7


00

01

02

03

04

05

Abso

rban

ce (A

U)

(C)

D

AC

255

32

813

297

33

167

349

34

013

417

34

573

476

05

053

539

3

616

76

773

744

7

816

0

882

0

000

005

010

015

020

025

030

Abso

rban

ce (A

U)


(D)

(E)

D

A

252

7

315

33

493

387

34

167

473

35

053

673

3

808

7


000

005

010

015

020

025

030

035

Abso

rban

ce (A

U)

(F)

D

A

C

252

02

960

340

7

398

74

300 6

173

732

0

822

7

928

0


00

01

02

03

04

05

Abso

rban

ce (A

U)

(a)


Concentration(mgmL)

permil(mgg)

Concentration(mgmL)

permil(mgg)

Concentration(mgmL)

permil(mgg)

10683 427 676 27 2314 09220245 810 353 14 4029 16137119 1484 282 11 3016 12616197 647 375 15 2616 10425740 102 667 26 1732 069


OFMYOSFBOSMY

CMMY

CMFB

(b)



OFMY OSMYCMMY

lowastlowast


0

10

20

30

40

Scav

engi

ng effi

cien

cy (

)

(a)

OFMY OSFBCMFB


0

20

40

60

80

Scav

engi

ng effi

cien

cy (

)

(b)

Blanktimes103

times10350 100 150 200 250

FSC-A

50

100

150

200

250

SSC-

A

H2O2 (1800 120583M)

0

25

50

75

100

Cou

nt

103 104 105102

FITC-A103 104 105102

FITC-A

OSFB

0

50

100

150

Cou

nt

103 104 105102

FITC-A

0

25

50

75

100

125

Cou

nt

CMFB

Blank

105104103102

FITC-A

0

50

100

150

Cou

nt

0

25

50

75

100

125

Cou

nt

103 104 105102

FITC-A

OFMY CMMY

0

25

50

75

100

125C

ount

103 104 105102

FITC-A103 104102 105

FITC-A

0

25

50

75

100

125

Cou

nt

OSMY

(c)

AB AB

CBC

A


0

20

40

60

80

100

Scav

engi

ng effi

cien

cy (

)

(d)








4 Discussion





120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


A549

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

MAD-MB-231120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


0

Huh7120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

HL60120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(a)

HCEC120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

MCF10A120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(b)



PBS


1X OFMY

5X OFMY

0

50

100

150

Tum

or v

olum

e (m

m3)

(a)

PBS 1X OFMY 5X OFMY

(b)


Table 1 The IC50


Cell line (IC50









5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















74099

74099

1001

57-

47098

55095

22077

681

54098

600931001

1001

9911001

1001

9911001

1001

10011001

9811001

1001

92-51064

29-1001

991

891

44094

60096

4707488099

1001

71093

100186098

1001

1001

921

1001

86098

981

9911001

1001

880981001

1001


































Clavicipitaceae ssC

ordycipitaceae






(A)

1713

2033

2907

3507

4340

4660

6653

7320

8227

0 2 4


D A C

6 8 10 12 14

00

02

04

06

08Ab

sorb

ance

(AU

)

D

A

C

249

32

960

320

73

407

399

34

260

458

75

120

618

7

670

0 728

0

930

0


000

005

010

015

020

Abso

rban

ce (A

U)

(B)

D

CA

252

02

980

316

73

493

401

34

180

459

34

747

540

7

680

0

746

7

882

7


00

01

02

03

04

05

Abso

rban

ce (A

U)

(C)

D

AC

255

32

813

297

33

167

349

34

013

417

34

573

476

05

053

539

3

616

76

773

744

7

816

0

882

0

000

005

010

015

020

025

030

Abso

rban

ce (A

U)


(D)

(E)

D

A

252

7

315

33

493

387

34

167

473

35

053

673

3

808

7


000

005

010

015

020

025

030

035

Abso

rban

ce (A

U)

(F)

D

A

C

252

02

960

340

7

398

74

300 6

173

732

0

822

7

928

0


00

01

02

03

04

05

Abso

rban

ce (A

U)

(a)


Concentration(mgmL)

permil(mgg)

Concentration(mgmL)

permil(mgg)

Concentration(mgmL)

permil(mgg)

10683 427 676 27 2314 09220245 810 353 14 4029 16137119 1484 282 11 3016 12616197 647 375 15 2616 10425740 102 667 26 1732 069


OFMYOSFBOSMY

CMMY

CMFB

(b)



OFMY OSMYCMMY

lowastlowast


0

10

20

30

40

Scav

engi

ng effi

cien

cy (

)

(a)

OFMY OSFBCMFB


0

20

40

60

80

Scav

engi

ng effi

cien

cy (

)

(b)

Blanktimes103

times10350 100 150 200 250

FSC-A

50

100

150

200

250

SSC-

A

H2O2 (1800 120583M)

0

25

50

75

100

Cou

nt

103 104 105102

FITC-A103 104 105102

FITC-A

OSFB

0

50

100

150

Cou

nt

103 104 105102

FITC-A

0

25

50

75

100

125

Cou

nt

CMFB

Blank

105104103102

FITC-A

0

50

100

150

Cou

nt

0

25

50

75

100

125

Cou

nt

103 104 105102

FITC-A

OFMY CMMY

0

25

50

75

100

125C

ount

103 104 105102

FITC-A103 104102 105

FITC-A

0

25

50

75

100

125

Cou

nt

OSMY

(c)

AB AB

CBC

A


0

20

40

60

80

100

Scav

engi

ng effi

cien

cy (

)

(d)








4 Discussion





120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


A549

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

MAD-MB-231120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


0

Huh7120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

HL60120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(a)

HCEC120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

MCF10A120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(b)



PBS


1X OFMY

5X OFMY

0

50

100

150

Tum

or v

olum

e (m

m3)

(a)

PBS 1X OFMY 5X OFMY

(b)


Table 1 The IC50


Cell line (IC50









5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(A)

1713

2033

2907

3507

4340

4660

6653

7320

8227

0 2 4


D A C

6 8 10 12 14

00

02

04

06

08Ab

sorb

ance

(AU

)

D

A

C

249

32

960

320

73

407

399

34

260

458

75

120

618

7

670

0 728

0

930

0


000

005

010

015

020

Abso

rban

ce (A

U)

(B)

D

CA

252

02

980

316

73

493

401

34

180

459

34

747

540

7

680

0

746

7

882

7


00

01

02

03

04

05

Abso

rban

ce (A

U)

(C)

D

AC

255

32

813

297

33

167

349

34

013

417

34

573

476

05

053

539

3

616

76

773

744

7

816

0

882

0

000

005

010

015

020

025

030

Abso

rban

ce (A

U)


(D)

(E)

D

A

252

7

315

33

493

387

34

167

473

35

053

673

3

808

7


000

005

010

015

020

025

030

035

Abso

rban

ce (A

U)

(F)

D

A

C

252

02

960

340

7

398

74

300 6

173

732

0

822

7

928

0


00

01

02

03

04

05

Abso

rban

ce (A

U)

(a)


Concentration(mgmL)

permil(mgg)

Concentration(mgmL)

permil(mgg)

Concentration(mgmL)

permil(mgg)

10683 427 676 27 2314 09220245 810 353 14 4029 16137119 1484 282 11 3016 12616197 647 375 15 2616 10425740 102 667 26 1732 069


OFMYOSFBOSMY

CMMY

CMFB

(b)



OFMY OSMYCMMY

lowastlowast


0

10

20

30

40

Scav

engi

ng effi

cien

cy (

)

(a)

OFMY OSFBCMFB


0

20

40

60

80

Scav

engi

ng effi

cien

cy (

)

(b)

Blanktimes103

times10350 100 150 200 250

FSC-A

50

100

150

200

250

SSC-

A

H2O2 (1800 120583M)

0

25

50

75

100

Cou

nt

103 104 105102

FITC-A103 104 105102

FITC-A

OSFB

0

50

100

150

Cou

nt

103 104 105102

FITC-A

0

25

50

75

100

125

Cou

nt

CMFB

Blank

105104103102

FITC-A

0

50

100

150

Cou

nt

0

25

50

75

100

125

Cou

nt

103 104 105102

FITC-A

OFMY CMMY

0

25

50

75

100

125C

ount

103 104 105102

FITC-A103 104102 105

FITC-A

0

25

50

75

100

125

Cou

nt

OSMY

(c)

AB AB

CBC

A


0

20

40

60

80

100

Scav

engi

ng effi

cien

cy (

)

(d)








4 Discussion





120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


A549

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

MAD-MB-231120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


0

Huh7120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

HL60120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(a)

HCEC120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

MCF10A120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(b)



PBS


1X OFMY

5X OFMY

0

50

100

150

Tum

or v

olum

e (m

m3)

(a)

PBS 1X OFMY 5X OFMY

(b)


Table 1 The IC50


Cell line (IC50









5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OFMY OSMYCMMY

lowastlowast


0

10

20

30

40

Scav

engi

ng effi

cien

cy (

)

(a)

OFMY OSFBCMFB


0

20

40

60

80

Scav

engi

ng effi

cien

cy (

)

(b)

Blanktimes103

times10350 100 150 200 250

FSC-A

50

100

150

200

250

SSC-

A

H2O2 (1800 120583M)

0

25

50

75

100

Cou

nt

103 104 105102

FITC-A103 104 105102

FITC-A

OSFB

0

50

100

150

Cou

nt

103 104 105102

FITC-A

0

25

50

75

100

125

Cou

nt

CMFB

Blank

105104103102

FITC-A

0

50

100

150

Cou

nt

0

25

50

75

100

125

Cou

nt

103 104 105102

FITC-A

OFMY CMMY

0

25

50

75

100

125C

ount

103 104 105102

FITC-A103 104102 105

FITC-A

0

25

50

75

100

125

Cou

nt

OSMY

(c)

AB AB

CBC

A


0

20

40

60

80

100

Scav

engi

ng effi

cien

cy (

)

(d)








4 Discussion





120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


A549

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

MAD-MB-231120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


0

Huh7120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

HL60120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(a)

HCEC120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

MCF10A120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(b)



PBS


1X OFMY

5X OFMY

0

50

100

150

Tum

or v

olum

e (m

m3)

(a)

PBS 1X OFMY 5X OFMY

(b)


Table 1 The IC50


Cell line (IC50









5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















4 Discussion





120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


A549

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

MAD-MB-231120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


0

Huh7120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

HL60120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(a)

HCEC120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

MCF10A120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(b)



PBS


1X OFMY

5X OFMY

0

50

100

150

Tum

or v

olum

e (m

m3)

(a)

PBS 1X OFMY 5X OFMY

(b)


Table 1 The IC50


Cell line (IC50









5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


A549

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

MAD-MB-231120

100

80

60

40

20

0

Surv

ival

rate

()

0 0125 025 05 1


0

Huh7120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

HL60120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(a)

HCEC120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

MCF10A120

100

80

60

40

20

Surv

ival

rate

()

0 0125 025 05 1


0

OFMY OSFBOSMY

CMMY

CMFB

OFMY OSFBOSMY

CMMY

CMFB

(b)



PBS


1X OFMY

5X OFMY

0

50

100

150

Tum

or v

olum

e (m

m3)

(a)

PBS 1X OFMY 5X OFMY

(b)


Table 1 The IC50


Cell line (IC50









5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PBS


1X OFMY

5X OFMY

0

50

100

150

Tum

or v

olum

e (m

m3)

(a)

PBS 1X OFMY 5X OFMY

(b)


Table 1 The IC50


Cell line (IC50









5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusions




Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Evaluation of an Epitypified Ophiocordyceps formosana (Cordyceps ...

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Transcript of Evaluation of an Epitypified Ophiocordyceps formosana (Cordyceps ...