Isolation and Characterization of Biologically ActiveSecondary Metabolites from Endophytic Fungi of

lndigenous Species of Tsxus Plant

By

NIGHAT FATIMA

Department of Biotechnology

Quaid-i-Azam University

Islamabad, Pakistan

2013

Isolation and Characterization of Biologically ActiveSecondary Metabolites from Endophytic Fungi of

lndigenous Species of TuvusPlant

A thesis submitted to Depa ment of Biotechnology,

Quaid-i-Azam University, Islamabad, Pakistan in the partial fullillment oftherequirements for the degree of

Doctor of Philosophy

in

Biotechnology

By

NIGHAT FATIMA

Department of Biotechnology

Quaid-i-Azam University

lslamabad, Pakistan

2013

()

IN THE NAME OF ALLAH, THE

MOST GRACIOUS, THE MERCIFUL

CERTIFICATE

This thesis, submitted by Nighat Fatima, is accepted in its present form by

the Depatment of Biotechnology, Faculty of Biological Sciences, Quaid-i-

Azam University, Islamabad as satisSing the thesis requirements for the

degree of

Doctor of Philosophy (Ph.D.)

Supervisor:

Chailperson Department of MicrobiologyQuaid-i-Az-am University, Islamabad

Extemal Examiner:Dr. Azra YasminAssociate Professor & ChairpersonDepartment of Environmental SciencesFatima Jinnah Women UniversityRa*alpindi

External Examiner:

Associate Prof. Department of BiochemistryPMAS-University of Arid Ag cultureRawalpindi

Chairman:Prof. Dr. Zabta Khan Shinwa

Professor Drl Safia Ahmed

Dr GhaTala Koukrb

Dated: Julv 9. 2013

DEDICATED TO

My Futher & all of My Fumily

DECLARATION

The material and inlormation contained in this thesis is my original work.

I have not previously presented any pafi of this work elsewhere lbr any

other degree. Material presented in the thesis has not been copied from

anv other source.

NIGHAT FATIA,LA

Chaptcr Il.t1.2

Chapter 2

2.1

2.2

l l.l1.t.22.i.12.1

2.1.t2.1.2

2.4.2.1

1.1.2.2

2.1.2.i

2.1.2.4

2.4.2.5

2.13l.,l.l.l2.5

Chapter 3

l. r

3.2

3.2.1

3 2.2

i.:.1l.l.l I

3.2..r

t.2.,1.1

1.2.:l.l.l1.2.1. L2i.:..+.1 .i3.2.4.2

L2.4.2 I

3.2.1.2.2

Conlenls

List oftables iI i"t ol figure' iiiList ofabbrer iations r i

List ot appendice, r iriAcl,no\a ledBemenr. i,(Abslracl \iI[troduction ]

Introduction 2

Aims and objectives 6

Lilerature Review '/

Microbial metabolites and drug discovery 8

Endophytes as emerging source ofDovel metabolifes I I

Role ofendophytes in drug discovery 14

Antimicrobial nletaboliles 14

CaDcer chemopreventive and cytoioxic metaboliles l6Anti-diabetic and immunosuppressivc metaboliles 23

Bioassays and drug discovery 26

Antimicrobial assays 26

Caicer chemoprevenlive assays 26

lnhibition ofTNF-o activated nuclear f'actorkappa B assay 2'7

Aromatase inhibition assay 28

Inhibition of nitric oxide (NO) producrion in lipopolysaccharide (LPS)- 3laclivated murine macrophage RAW 264.7 cells assay

DPPH free radical scar,enging assay

Quinone reductase I (QRl) induction assay

Cytotoxicit) assay

Sulforhodamine B (SRB) assay

Techniques used for purification and characterization ofnatural

3)32

33

3ll5

lsolation, Biological Screening and Solcction ol Endophytic Fungi 38

Ilrtroduction

Material and methods

Collection of plant sample

Isolation ofendophytes from different plant pafts

Cuhivalion of fungi for production of metabolites

Solid state fermentatioD and extraction ofmctabolites

BiologicalscreeningAntimicrobial screening

AntibacterialassayAntifungalassayHyphae formatioD inhibilion assay

Cancer chemopreveDtive screening

Inhibition ofTNF-u activated nuclear factor-kappa B (N!'KB) assay

Aronutase inhibition assay

39

40

40

10

4l,11

14

44

14

14

45

47

11

48

3.2.4.2.3

3.2.4.2.1

3.2.4.2.5

3.2.4.3

3.2.4.3.t3.2.5

3.3

3.3.1

i.i.23.3.3

3.3.3.1

3.3.3.1.1

3.3.3.2

i.1.3.2.i

3.3.3.2.5

3.3.3.3

3.3.4

3.4

Chapte t

4.1

4.2

4.2.t

4.2.3

1.2.3.1

4.2.3.1 .I4.2.3.1.2

4.2.1.2

4.2.3.2.1

1.2.3.2.2

4.2.3.2.3

4.2.4

4.2.4.t

Conlehls

Inhibition of nitric oxide (NO) production in lipopolysaccharide (LpS)- 48activaled murire macrophage RAW 264.7 cells assay

Quinone reduclase I (QRl) induction assay 49

DPPH free radical scavenging assay 49

Cytotoxicity assay 50

SulforhodamiDe B (SRB) assay 50

Molecular identification offungal isolates 5lResuhs 52

Isolation ofendophytic fungi 52

Fermentation and extraction 52

Biological screeDiDg 51

Antimicrobial screening 57

Results ot'antibacterial assay 57

Results of aDt ifun gal assay 57

Results hyphac formation inhibition (HFI) assay 6lCaDcer chemopreventive assays 63

Results ofnuclear factor-kappa B (NF(B) inhibition assay 63

Results ofaromatase inhibition assay 63

Results ol inhibifion of lipopolysaccharide (LPS)-aclivatcd nitric oxide 67

(NO) production in murine macrophage RAW 264.7 cells (iNOS)

Results ofquinone reductase I (QRt) induction assay 6/Results ofDPPH free radical scavenging assay 1lResults ofcytotoricity assay (Sulfbrhodamine B Assay) 1lMolecular ideDtification ofthe active endophytic fungi 75

Conclusion 85

Isolation and Biological Evaluation of Cancer Chemopreventive and 86

Cytotoxic Compounds lrom Selected Endophytes

Introduction 87

Materialand merhods 88

Bioassays 88

Cuhivation aod extractioD of secondary metabolites ftom Epicocc m 88

"igrurl, NFWI and Psricill1 fi sp. NFW9

Fractionation and purification of crude extract of Epicocc m nigrum 90

Solvenlsolvent extractior ofcrude extract 90

Normal phase column chromatography ofNFWSH fraction 90

Norma! phase column chromatography ofNFwlE fraclion 90

Purification ofcompounds from selectcd fractions NFWlHl3, NI'W3E9 95

and NFW3EI I

lsolation and purification ofNFW3H ll-l-Fatima compound 95

Isolarion and purification ofNFWSEgE-Fatima compound 97

Isolation and purificatioi ofNFW3El lC compound 97

Fractionation and purilication ofcrude extract ofP.rl.illirm sp. NFW9 100

Normal phase column chromatography ofcrude extract ofNFwg l0t)

1.2.4.1.l

4.2.1.2

4.2.4.2.1

4.2.4.2.2

4.2.4.2.3

1.2.4.2.1

1.2.5

4.3,1.t.1.

4.3.1.l4.3.t.24.3.2

4.3.2.1

1.3.3

4.4

Chapter 5

5.1

5.2

5.2.\5.2.2

5.3

5.3.1

5.1.1 . l

5.3.1.2

5.1.2

5.3.2.1

5.3.2.2

5.3.2.3

5.3.2.4

5.1.2.5

5.4

Chapter 6

6.1

Cohteds

Normal phase column chromatography ofselected fraction NFW9C l0lPurification ofcompounds ltom selected fiacrions NFWgC-15, NFW9C- 106

17. NFWgC-25 and NFW9C-IlIsolation and purificalioD of NFW9C,6, NFW9C-]l and N[WqC-]5 I06compounds

Isolation and purification ofNFWgC- I 7 compound 106

lsolation and purification ofNFWgC-25 compound I l0lsolation and puriflcation ofNFW9C-33 compound I l0Sample preparation olpure compounds isolaled fiom Epi.oc.?rn nigrun 110NFW3 and Penrilllrm sp. NFW9 for bioassays

Results ll4Results of cancer chemoprevenfive assays for lractions of Epicoccum 114

,igr&m NFWS strain

Assay results ofNFwlll fraclions 114

A,ssay results ofNFWlE fractions I 17

Biological acrivities ol tiactions ot' NFW9 sample obtained by normal ll9phase column chromalography

Assay results ofNFWgC fractioDs 119

Assay results ofpure compounds 122

Conclusion 124

Characterization of lsolated Compounds frolm Epioccutt nigrum 125

NFw3 and P?rlril/iffi sp. NFW9lnlroduction

Materials aDd methods

Sample preparafion for NMR speclrometry

Sample preparation for mass spectrometry

Results

Structure elucidalion of compounds fto|m Epico(unstrain

Structure elucidatioD ol NFW3E9E-I -Fatima

Structure elucidation olNFWlEl lCStructural elucidation ol compounds from isolated from

NFW9 straiD

Structure elucidation of NFW9C-1 I

Structure elucidation of NFw9C-l 5

Structure elucidation of NFW9C-1 7

Structure elucidation of NFW9C-25

Structure elucidation of NFw9C-33Conclusions

DiscussionDiscussion

Conclusionhuture prospects

t26t26t26t26t21

,?igrrm NFW3 129

129

t36Penicillium sp. 143

t43152

160

169

179

188

189

190

200

20r

Conte s

References

Appendices

Publications

202

230

210

Table No.

List of Tables

Title P#No.

2.1 DiafereDr endophyric fuDgi and rheir host plants I32.2 Biological activifies and bioactive compounds isolated from I8

endophytic fungi3.1 Composition ofmodified raxol nedium (TM) 123.2 Conrposition oflSP4 nedium 163.1 Plant parts and nrorphologl ofendophytic f'ungal isolates of laus 55

3 .4 Weight of crude ethy I acetate extract after so lid state fermentation 5 6i.5 Antibacterial activites of crude ethyl acelate exkacts ofendophytic 59

fungal isolates ol Tuxus Juana measured as zone of inhibition in(mm)

3.6 ADtifungal activites of crude ethyl acetate extract of endophltic 60fungal isolates of Tarus fuana measured as zone of iDhibition in(mm)

3.7 Hyphae formdtion inhibition (HFI) assay of crude ethyl acerare 62extracts ofcndophytic fungi isolated from \rood and leaves of ldirrr

3.8 Results of TNF-o activated NFXB inhibilion assay of crude extracts 65ofendophytic fungal isolates of T&xus rt1ana

3.9 Results ofaromatase inhibition assay ofcrude exlracts ofendophytic 66fungal isolates of Taras /r.rana

3.10 Results of iNOS assay of crude extracls of endophytic fuDgal 69isolates ol Taxus Juana

3.11 Results of quinone reductase I (QRl) iDduction assay of crude 70exlracls ofendophytic fungal isolales of Tair.s/udrd

l.l2 Results of DPPH assay of cnide extracts of endophytic fungal /3isolates of la.rr.t lrdnd

3.ll Results ofcytotoxicity (SRB) assay olcrude extracfs ofendophytic 14fungal isolates of fajrr.r l dnd

3.14 Percent homology aDd accession numbers of the l8S rRNA 76neucleotide scquences olactive endophytic fuDgal isolates

4.1 Combinafion scheme of NFWIH fraclions prepared by normal phase 93column chromatography on the basis oI fLC analysis

4.2. Combinalion scheme ofNFW3E lractions prepared by Dormal phase 94

column chromatography on the basis ofTLC analysis4.3 Combiiation scheme ofNFw9 fractions prepared by norrnal phase I02

column chromatography on ihe basis ofTLC analysis

4.1 CoDbinalion scheme oiNFwqC fractions prepared by normal phase 105

column chromatography on the basis ofTLC analysis4.5 Sample preparation of pure compounds isolated of NFW3 and 113

NFW9 fbr bioassays4.6 Res u hs of cancer chenoprevent ive and cytotoxic ity assays of NFW3 I I 5

lrection5 aner sol\ enl-.olv(nt exlrdclion4.7 Results ofcancer chemopreventive assays ofNFW3H fractions after 116

normal phase column chromatography4.8 Results of cancer cheDroprcvenlive assays of NFWIE fractions 118

prepared by normal phase columD chromalography4.9 Results ofcancer chemopreventive and cytoioxicily assays ofNFw9 120

fractions prepared bt normal phase column chromatography,1.10 Results of cancer chemoprevenlive and cytoioxicity assays of 121

NFWgC fractions prepared by normal phase column

4.ll

5.1

5.2

5.3

5.4

5.5

5.6

5.7

5.8

List of Tobles

chromatographySummarized results of cancer chemopreventive and cytotoxicityassays of purified compounds from selected strains Penicillium sp.NFW9 and Epicoccum niglrm NFW3Nanre of the compounds isolated fiofi Penicillium sp. NFWS andEpicoccum nigrum NFW3, solvents used and quaDtity of thecoinlounds for NMR \pecrroscopyH and FC NMR dara {400 \4Hl. rn MeOH-J, ol Nt WIFqE-l-

Fatima, d- in ppmrH and rrc NMR dara (400 MHz, in MeOH,r4) of NFW3E] lC(]0),d in ppm

'U an,l ''c Nua data (,100 MH4 in CDCI:) of NFWgc-I 1, a- inppmrH and !' \M R dala i 400 M Il,/. in D\.4 SU-z/ , ol'Nl WqC- t), /J inppmlH and rrc NMR dara (400 MH4 in DMSO-d6) ol NFW9C,I7-pa-

rH and rrc NMR Spectroscopic Dara (400 MH4 in DMSO-d) ofNFWgC-25-novqth, d iD ppm.

'H and LrC NMR dara (400 MHa in MeOH-./r) of NFW9C-33, zi inppm.

123

t28

134

l4l

150

158

167

r1'/

186

ii

Lisl ol Figures

fig. No. Titte p#No.

2.1 AD overview of ceDtral mctabolic pathways of fungi (Adapted from l0Khan 2007)

2.2 Outline of host-endophyte relationship showing response of host planr t2and endophytes against phytopafhogens by producing bioactivecompounds aDd their potential applications (retrieved from Zhao et dl.,2010)

2.3 Mechanism of cancer prevenlion b) natural compounds (adapted from 20Tsuda er dl., 2004)

2.4 Chemical structures of different anlicancer aDd cancer chemopreveDtive 25

compounds.

2.5 Schematic diagram of NFKB activation pathways and its role in 29

carcinogenesis (adapted fiom lchikawa e1dl., 2006).

2.6 Steroido8enesis showing that aromalase inhibition will not affect dre l0production of othcr useful sleroids

2.7 Attacking pattern of hydroxyl free radical (OH) on the C8 position of 34

guaniDe (Valko el a1.,2004)

3.1 Schematic representation of cultivation and exlrartion of metabolites 43

after solid state fermentation

3.2 Morphologicalcharacterization ofendophytic fungal isolates from leaves 53

ol Ttxus fi&na3.3 Morphological characferization olendophytic fingal isolates from wood 54

patts of l axus litana3.4 Phylogenetic tree showing the evolutionary relationship ofNFWl isolate 77

with 1,1 taxa

1.5 Phylogenetic tree showing the evolutionary relationship ofNFW3 isolate 78

with 16 trx,3.6 Phylogenetic tree shovring the evolutionary relationship ofNFW5 isolate 79

with I8 taxa

3.1 Phylogenetic lree showing the evolurionary relationship ofNFw6 isolate 80

\|ith l4 closely related taxa

3.8 Phylogenetic Iree showingthe evolutionary relatioDship ofNFwT isolate 8lwith 2l closely related ta-\a

3.9 Phylogenetic trce showing the evolutioDary relalionship ofNFW8 isolate 82

with I7 closely relaled raxa

3.10 Phylogenetic tree showing the evo,utionary relationships of NFw9 83

isolate with l2 closely relaled taxa

3.1 I Phylogenetic tree showing the evolutionary rclationships ofNFL2 isolate 8,1

with l7 closely related taxa

4.1 Schematic representation of preparation of crude exfracts of Epico..rm 89

nrgran NFW3 and Perl.i/l,rm sp. NFW94-2 Schematic representation of fracrionation ofcrJ?e extracr of Epicoccum 92

,?lgi,rr? N FWI

1.4

1.6

1.5

4.8

.1.j

1.9

4.10

4.ll

5.1

5.2

5.3

5.4

5.5

5.6

5.1

5.8

5.9

5. l0

5.1 I

5.12

5.13

5. l45.1 5

5.16

5.1 7

5.18

5.19

5205.21

5.22

5.23

List of Figures

Schematic representation of isolation and purification of compound 96NFW3HI3-1-FatimaSchematic representation of isolation and purification of compound 9gNFW3EqE-l -FatimaSchematic representation ot' isolalion and purification of compound 99NFW3EI lCSchenre for preparalion of fractions of Penicilliun sp. NFW9 by usingnormal phase column chromatography

Schematic representation of tiactiooation of NFW9C by using normalphase column chromatography

Schematic representation of isolation and purification of corrpoundsNFW9C-6, NFW9C,l 1, NFWgC-15ScheDratic representation of isolatioD and purification of compoundNFWgC.] 7

Schemafic representation of isolation and purificalion of compoundNFW9C-25

Schematic representalion of isolation and puritication of conrpound

NFW9C-33rH NMR spectra (400 MIIz, in MeOH-d4) ofNFW3E9E-l -Farimar3C NMR spectra (400 MHz, in MeOH-d4) of NFW3 EgE- I -FatimaHSQC specira (400 MHz, in MeOH-d,{) ofNFW3EgE- I -Fatinra

HMBC spectra (400 MHz, in CDCII) ofNFW3E9E-l,FatimaProposed structure of oew compound (NFW3E9E-I-F) identified as

epicoccamidc analoguerH NMR speclra (400 MHz, in MeOH-dr) of NFWIEI I C(]0) 137rrc NMR spectra (400 Mllz, in McOLI-./r) of NFW3EI lC(10) ll8HSQC spectra (400 MHz, iD MeOH-dr) ofNFW3El lC(I0) 139

HMBC speclra (,{00 MHz, in MeOH-d, ofNFW3El lC(10) 140

Proposed slructure ofNFW3El lC(10) idenlified as epicoccamide 142

analoguerH NMR spectra (400 MHa in CDCL) ofNFWgC-I1 145rrc NMR spectra (400 MHz, in CDCI]) of NFWgC-l I 146

I ISQC spectra (400 MHz, in CDCL) of NFwqC- l I 141

HMBC spectra (400 MHz, in CDCIr) olNFW9C-l I 148

COSY speclra (400 MH4 in CDCIr) ofNFWgC-l I 149

Proposed structure ofNFWgC-I1 identificd as wortmin (an azaphilone l5lderivative)rH NMR spectra (400 MIIz, in DMSO-d,) of NFW9C- 15rrC NMR spe$ra (,100 MHz, in DMSOd6) of NFWgC-15

HSQC spectra (400 MHz. in DMSO-d,r) of NFWgC-15

HMBC spectra (,100 MHz. in DMSO-d6) of NFW9C-i 5

COSY spectra (400 Mllz, in DMSO-d,,) of NFWgC-15

Proposed saructure ofNFW9C-15 compound identified as anthraquinonerH NMR speclra (400 MHz, in DMSO-d) of NFW9C-17

t08

t0l

10.1

109

l

112

1t0111

132

lll115

153

15,1

155

r56157

159

162

5.24

5.25

5.26

5.27

5.28

5.29

5.10

5.31

5.32

5.ll5.34

5.15

5.3 6

5.3 7

5.18

5.39

5.,{0

5.41

l8l182

r83

1 8,1

r85

r87

List of Figarcs

irC NMR spectra (,+00 MHz, in DMSO-r/) of NFWSC-17HSQC spectra (400 MHz. in DM5(]-r/6) of NFW9C-t7HMBC spectra (400 Mllz, in DMSOd, of NFW9C- l7COSY spectra (400 MHz, in DMSO-dr) ofNFW9C-l7Structure and key HMBC correlation and Structurescompound identified as wortmanninrH NMR spe$ra (400 MHz. in DMSO-d6) of NFWSC-25LrC NMR spe$ra (400 MHz, in DMSO-d, of NFWgC-25HSQC spectra (400 MHz. in DMSO-d, ofNFW9C-25HMBC spectra (400 MHz, in DMSO-d, ofNFW9C-25COSY spectra (400 MHz, in DMSO-./, of NFW9C 25

NOESY spectra (400 MHz, in DMSO-dd) olNFW9C-25Proposed slruclure of compound NI.W9C-25

'tl NMR spect'a i+00 l,t Hz, in MeOH,{) of NFWSC-33rrC NMR specrra (400 MFIZ, in MeOH-dr) of NFW9C-33HSQC spectra (400 MIIz, ir MeOH-d]) ofNFW9C-33HMBC spectra (400 MH4 in MeOH-dr) of NFW9C,33NOESY speclra (,100 MHz, in MeOH-.1r) of NFW9C-33

l6lt64165

r66of NFW9C-17 168

171

112

173

174

t15t76

identified as I78

Proposed structure of NFW9C-ll identified as I I -desacety lwortmann in

or I l-deacetylwortmannin

List ofAbbrcuialions

Abbreviations Definitions'OH Hydroxyt fiee radicalrrc NMR rrCarbon nuclear magnetic resonancelD-\\,lR One dimensroril nuLlear mduneric re,onanccH NVR Proron nuclear magneric -esonance

2D-NMR Two dimensional nuclear magneric resonanceA. llatus AspetgillusflawsA flnigar s Aspetgillusfrnigatt$A. niser /tspetgillus nigerA. terrec Asperyi us teffeutAC Absorbance ofcontrolAS Absorbance oftesl sampleATar {Teucdn l)pe.ulJre collecrionBLAST Basic local alignment search loolC albicdns Cdndida albican}CD Concentration required to double the enzyme activilyCOSY Correlalion speciroscopyCTAB Cetyl trimerhylammonium bromideDEPT Distodionless enhsncemenl by polarization transferDMEM Dulbecco s modified eagle mediumDMSO DimethylsultbxideDMSO- d6 Dimethyl sulfoxide with six deuterium alomsDPPH 2'2-diphenyl- 1 -picrylhydrazylE. cali E cheichia coliESI-MS ElecrrosprayionizalionmassspectrometryFBS Felal bovine serumFCBP First fungalculture bank ofPakistanGC Gass chromalographyHEK cells Human embryonic kidney cellsHepa lclc 7 Murine hepaloma cellsHFI Hyphae tbmation inhibilionHL-60 Human peripheral blood leukemia cell lineHMBC Heleronuclear mulliple bond correlalionH\1C-(Oc H)dro\) meth) lBlurarll-r"AHPLC High performance liquid chromalographyhn HonrsHSQC Heteronuclear sin8le quantum coherencelC o aoncenrrdlion dr 50" . inh.birioriNOS Inducible nitric oxide synthaseIPP lsopentenyl pyrophosphatIR lnduclion ratioISP4 Inrcrnational streplomyces projecl inorganic salls starch agarIU International unitK pnelnaniae Klebsie a pneunoniaeKB Human epidermis carcinoma in mouth cellsLC Liquid column chromatographyLC Liquid chromatosraphyL-NMMA Na-L monomethyl arsinineLPS LipopolysaccharideM. hnetr Micrococcus luteu:rnlz Mass to charge ratioMALDI-TOF-MS Matrix'assisted laser desorplion/ionisation-time offlight mass spectrometryMCI'-7 Hormon€ responsive human brcasr cancer cellsNfDA-MB-231 Eslrogen receplor negative huma. breast cancer cellsMED MycoepoxydieneMEGA Molecular evolutionary genetic analysis

List of Abbrcviatio,ts

MEM-0 Minimum essential mediumMeOH MethanolMeOH-da Melhanol with four deulerium atomsMPLC Medium pressure liquid chromatogmphyMS Mass speclrometryMTI 3-(4,5dimethyhhiazo-2-yl)-2,5-diphenyltetrazoliumbromideN.A. Not applicableNADP Nicolinamide adenine diphosphateNADPII NicotimmideadeninediphosphaleNaOCI Sodium hypochloriteNCBI Nalional center for biotechnology informaiionNCI Narional cancer institule (USA)NFXB Nuclear trcto. kappa BNJ NeighborjoiningNO Nitric oxideNOESY Nuclear overhauser effecl spectroscopyNPCC Normal phase liquid column chromalogaphyNPCC Normal phase column chromarographyNT Nor testedOFR Oxygen free radicalsP oetusinosd Pseu.la onas deruginosaPBS Phosphate saline bufferPCI Human prosrare.an.er .ell inePCR Polymerase chain rcactionPDA Potalo dexlrose agarPDT Podophylloloxinp\,1 Pico moleppm Parts per millionQRI Quinone reductase I

Apperulices

Title P#No.Fig. No.

AppendixA]

AppendixA2

Table A1

'I able A2

Morphological features of eDdophytic fungal strains isolated from leaves 231of Taxus fuana

Morphological features of endophytic fungal strains isolated from wood 233parls of Taxur Juana

Cherricals supplies and apparatus used for cancer cheDopreventive assays. 237

Chemicals supplies and apparatus used for chromatographic techniques. 239

Ackno )lelgeuents

I am grateful lo Almightv Allah, the Omnipotenf and ihe most Merciful and beneficent. who is

the Creator of universe, who blessed the man with the ability to dream and with the courage Io

lry. His blessings enabled me to achieve my goals. fremulous venerations are for His Holy

Prophet Muhammad (PBUH), who is everlasting torch of guidance and knowledge for

humanity.

I am lacking with the words, to express my sincere and deepest heartfelt gratitude to professor

Dr. Safia Ahm€d, Chairperson, Deparhnenl of Microbiology, Quaid-i-Azam University

Islamabad, Pakistan for her scholastic guidance, continuous encouragemelrt and sincere criricism

lhroughout the study and presentation of this manuscript. I am extremely grafeful to faculty

members of DeparlmeDt of Microbiology for their consistenl suppor.

I am obliged 1() Dr. Zrbta Khan Shinwari, Professor and Chairman, Department of

Biotechnology, Quaid-i-Azam Universi\, lslamabad for his suppon during my PhD. I am

thankful to Professor Dr. Bushra Mirza. Chairperson Department of Biochemistry, Quaid-i,

Azam University Islamabad, Pakistan for her suppon. I cxpress my gratitude for Dr. Ihsan-ul-

Haq lecturer at Department of Pharmacy Quaid-i-Azam University Islamabad, Pakistan for his

consistcnt technical and moral support.

lam thankful to

tillowship and IR scholarship. I believe that HEC funding adds to my strength a 191-bm also

ancer I-nstitute usA additional suppon under program

prq ,l8l12, CoP, UHH US atural inhibitor of carcinoelenesis" to complete this

I am extremely grateful to Dr. Leng C. Chang, Assistant Professor and Dr. John M. Pezzuto,

Professor and Dcan, College ot' Pharrnacy (CoP), University of Hawaii at Hilo (UHII), USA for

providing research facililies lo carry oul rny research project iD their laboratories and also for

usef'ul suggestioDs and guidancc. I am also thankful 1() Dr. Philip Williams, Assisfant professor.

DepartmeDt of Chemistry, University of Hawaii al Manoa. for his kind cooperation to provide dre

facility fbr mass spectrometry. I am also lhankful to Dr. Espcrunza J. Carcache de Blarco

associate professor College of Pharmacyr The Ohio State University USA for lrer support to

complete mechanism based chemopreventive sfudy of pure compounds. I lvould like lo extend

Higher Education Commission (HEC) Pakistan for providing me indigenous

my deepest appreciation 1() those people, who helped me iD one way or another during my sray at

Department of Microbilogy, QAU lslarrabad, pakistan and Cop, UHH, USA. DuriDg my phD

research, I worked with a great number ofpeople; I wish to convey my gratitude to all ofthem.

i would like 1() pay special thaDks to my fellows especially Muneer Ahmed eazi, MunibaJadoon, lbrar Khan, Dr. Aun M and Mlrssrat S for their consistent, inseparable suppo( and

prayers. I am also thanklul to Dr.Farah M, Sadia M, Nida K. Rabia L, Irum p, Aysha S. Dr.

Naima A. Syed Zceshan H. and Mohsin A for their support and prayers. h is my pleasure to

nentioD Dr. Tamara P. Kondratyuk, AssisGlll Specialist and Laboratory Manager and Dr. Chai

XY. Dr. Lee S, Dr. Youn UJ, Dr. Park LJ. Marler LE ior rheir great cooperation and support

during my stay at CoP, UHH, USA. I believe that withour their help my PhD project may nor

have been furnished so quickly and nicely.

I would like to pay very special tribute to my family, who helped me and guided me in e\er)

aspecl of life. I owe non payable debit to my loving pareDts whose wishes motivate me to strive

for higher education. I owe my loving thanks to my husband Nazar Hussain for his understanding

and continuous support as well lny loving son Ali Abuzar because wi(hout his prayers it would

have been irnpossible for me to finish this work. My special gratitude is for my parents, my

brother Shoukat AIi, Mrs. Hina Shoukat and my nieces Maryam and Amna for their loving

suppofl.

Finally, I would like to express my apology to those who ever had a soft corner for me but I

missed to mention them persoDally.

Abstract

Endophltic microorganisms symbolize one of the promising and rather overlooked

sources of unique and potent biochemical entities. fhis study reports the isolation of l5endophltic frmgal strains from wood and leaf parts of faxas ,4lata of Himalayan region

of Pakistan. These stlains \r'ere screened for their antimicrobial and cancer

chemopreventive potential. tnitially isolates \\'ere cultured via solid state fermentation on

two dilferent agar media and crude extract was obtained using organic solvent ethyl 'acetate. The extract exhibited significant antimicrobial and c;mcer chemopre\entilc

potential.

Different assays used were antimicrobial, hyphae formation inhibitiol, canccr

chemopreventive such as inhibition of TNF-o induced NF(B, inhibition of nit c oxide

production in lipopolysaccharide -activated RAW 264.7 cell, aromatase inhibition, free

radical scavenging and induclion of quinine reductase and cytotoxicity assays. NFKB

activity plays a critical role in cancer development, progrcssion and therapy and six ofthe

f'ungal samples showed more than 50 % inhibition of TNF-o activated NFKB. The fungal

sijiains Epicoccum rrgrzrn NFW3 and Penicillium sp. NFW9 showed signiticant

inhibition of more than 70 oZ in aromatase assay. Of the tested fungal samples lour

exhibited more than 65 % inhibition ofnitric oxide synthase. Oxygen free radicals are the

products of rormal cellular metabolism and can produce endogenous DNA lesions

leading to carcinogenesis. DPPH free radical scavenger has the potential to scavenge free

oxygen radicals and four tested samples shoued more than 80 o% scavenging activity in

antioxidant assay. Quinone reductase (QR) is an enzyme involved in melabolic

detoxification of chemical carcinogens and protects the cell against redox cycling,

oxidant stess and carcinogenesis. Three ofthe tested samples showed potent induction in

cell based assay with CD (concenlntion required to double the enzyme activity) < 5

pg/ml. A total of four samples showed cytotoxicity in sulphorhodamine B assay against

MCF-7 cell line and ICr6 values were < 20 pg/ml. 1'he overall results ofinitial sr:reening

showed that wood isolates NFWl, N!-w3, N!-W6, NFW7. NFWS. NFW9 and one leaf

isolate NFLI showed promising activit"v in multiple bioassays used. Some ofihc posili\e

endophyic fungi uere identified at thc molecular level by nnalyzing the 18S ribosomal

DNA (rRNA). The results of molecular identification showed that genus Ep icoccum was

dominant among $,ood isolates while other isolates belong to Mucot,, Trichoderma,

Penicillium and (;hdetomium sp.

After initial screening, two strains Epicoccum ,?lgl&rt NFW3 and perlcil1lam sp_ NI-W9

were selected for isolation and purification of pharmacologically relevant oompouflds.

For this purpose, ,plcoccum nigrumNFW3 a]ird Penicilliutn sp. NFWg were cultured on

solid media lbr 21 days and extraction was done with an organic solvent the elhyl acetate.

Further, crude ethyl acetate extracts \tere fractionated and subjected 10 biological

evaluatio[ using chemopreventive and c]totoxicity assays to identily the active fractions.

Active liactions were lurther proccssed through sequential fractionation utilizing r arious

chromatographic techniques, which led to isolation of three biologically active pure

compounds from rpicoccum nigrumNFW3 ar.rd 6 from Penicilliam sp. NFW9. Structura.l

elucidation ofpule compounds was carried out by utilizing mass spectometry (MS), one

dimensional nuclear magnetic resonance (lD-NMR) and two dimensional nuclear

mag[etic resonance (2D-NMR) techniques. It was found that two compounds isolated

from NFW3 belonged to an unusual class of biologically active fungal metabolites

epicoccamide, consisting of three distinct subunits including one new analogue.

Compounds isolated from NFW9 belonged to azaphilone, anthraquinone and worlmannin

classes of biologically active metabolites. On the basis of existing reports and

spectroscopic analysis. it is concluded that out of these 7 compoulds; I is new and 6 are

known. 'l'his study is a contribution towards the exploratioD of distinct classes of fungal

secondary mctabolites as well as their potential use in cancer chemopreventive and

anticancer drug discovery.

Chapter 1

Introduction

1

Chaptel 1

I.1 INTRODUCTION

Human beings through ages; have relied on natural resources for their basic necessities

such as food. sheltcr and mcdicine. Naturc has been a reservoir of several accustomed

products with diverse chemical naturc, derived from plants, animals, insects and

microorganisms. As chemicals, natural products conprise ofdifferent chemical classes of

compounds such as carbohydrates, proteins, peptides, terpenoids, polyketidcs, amino

acids, lipids, nucleic acid bases, ribonucleic acid (RNA), deoxyribonucleic acid (DNA),

etc. Since they :Lre highly diverse ard often provide highly specific biological activity,

natural products have been playing primary role in human diseases treatment being a

major source ofnew drugs (Chin et a/., 2006).

Although unique natural and synthctic remedies exist, but continuous emergence of

resistance and different life threatening diseases especially cancer, has called for the

exploration of novel natuml sources to discover new chemical entities with improved

biological activities. Among the ailments faced by the man today, cancet appears zts one

of thc most deadly disease. It is the second leading cause of the deaths in developed

countries and its incidence in the developing countries is also at rise. It has bccn

estimated that 13 % (7.5 million) of the deaths around the world are caused by cancer

each year (Bray et al., 2012). Abont 72 yo of these deaths occured in low- and middle-

income countrics where basic medical facilities are not adequate. In Pakistan, two-thirds

of the cancer cases are caused by environmental problems and the remaining, by

nutritional and reproductive probiems. Around 320,000 (per annum) new cancer cases are

rcported in Pakislan, yet there arc only 20 cancet hospitals in the country ttearing over

400,000 cancer patients (Achakzai, 2008).

Canccl is the resull of the abnormal ccll groMh involving the initiation, promotion and

progression stages of tumor in specific pafis of the body. Carcinogenesis can be viewcd

as a multistage process culminating in tumorigenesis. Initiation involves a change in the

genetic makeup of a cell, possibly due to carcinogens or damage to a DNA repair

mechanism. Dudng promotion, the mutated cell is stimulated to grow and divide,

becoming a population of highly proliferative cclls. These cells can progress to expand

Chapter l

further as tumor cells, eventually outnumbering their normal cell counterpads (Reddy e,

a/., 2003; Kinghom et a1.,2.004). Cancer is mainly caused by several factors includingenvironmental factors, viral infections and genetic reasons and can be attributed tosmoking, improper diet, drinking, chronic inflammation of any part of thc body. The

onset of cancer can be prevented by the improved defense mechanisms of the person,

protection Aom the overexposure of the carcinogcns, modifications in life styles and

chemoprevention (Cancer'lrcnds Progress Report - 201 l/2012 Update).

Despitc cunent treatment strategies, lherc is recurrent cancer incidence and damaging

side eflccts of therapeutic agents. 'lhis advocatcs a continuous need to design a strategy

that can offcl site specific remedy at initial stages as well as halt the metastasis. Cancer

chemoprevention is a strategy for rcducing cancer mortality and i[volves thc use ofnatural, dictary, or pharmaceutical agents to delay, inhibit, or reverse thc development ofcancer before malignancy occlrrs. This urgcs the need for discovery of novel compounds

u'ith enhanced chemopreventive potcntial particularly though natural sources (park and

Pezzuto, 2002; Kinghom et aL, 2004; Balunas and Kinghom, 2005).

Now a day's major challenge for the ph.maccutical industries is to accelerate the drug

discovery by improving their research zmd developme[t. This will only be successfu]ly

achieved if the methods for identification of lead compounds and elucidation of drug

targets are optimized. Howcver, varieties of stucfurally diverse natural products can be

created only with the implementation ofnew ideas to identily pertinent lead compounds.

Natural products have established their worth as valuable tools in molccular, cellular

biology and biological chemistry because of their therapeutic role and contribution to

Lrnderstand various biochemical pathways.

Among natural sourccs, microorganisms have been a prolific domain for lead compounds

and new pharmaceuticals. Sevcral secondary metabolites from microbes have potential

anti-cancer, anti-fungal, antimala al, anti'mycobacterial, and anti-viral therapeutic

indications. Ihe compounds isolatcd from microbial sources also show great divemity of

chemical stuctures and biological aclivities. Bndophltic microorganisms have gained

immense importance during rcccnt ycars due to ptoduction of compounds with

Chapter 1

impressive biological activitics (Phongpaichit et al.,2OO7). Endophytes constitute a

remarkably multifarious group of microorganisms ubiquitous in plants and maintain an

imperceptible association with their hosts for at least a part of thcir life cycle. Their

enormous biological diversiq, coupled with their capability to biosynthesize bioactive

secondary metabolites has provided the impetus for a number of investigations on

endophyes (Kusari et rr/.,2012).'l'hese endophytes arc though associated with all higher

plants however; endophltic microflora of medicinal plants like Tnrzs species holds a

distinguished position in tcms of their biological activities. Studies show an estimated

number of onc million endophltic species of medicinal imporlance (\u et al., 2010).

Howevcr, to date, only few of the plants have been explored for their endophyic

diversity and bioactive potential ofthose endophytes (Khan, 2007).

Thc role of endophytes in novel drug discovery emcrged after the disoovery of billion

dollar anticancer drug tzxol from endophyic fungi Tatxotfiyces andrednee isolaled from

pacrlic yew Teuus bretifolia (Strobel et al., 1993). l'axol is a very potent anticancer agent

and was first isolated ftom the bark oI Taxus brevilblia commonly knom as Pacific

Yew. This drug acts through unique mechanism by stabilization of microtubules leading

to cell death or apoptosis (Wani el a/., l97l; Stein, 1999). lt is prcsent in varling

concentration in the cell wall ofthe Ta{us plant and is difficult to synthesize chemically

(Russin €/ d1., 1995). Therefore attention was diverted towards other possible souces.

lhc qucst tumed out to be successful with ta\ol production ftom endophltic fungi

Peslaloliop[is microspora, Peslaloliopsis guepini all,d Tubercularia isolated fiom Taxus

'|allichidna, Wollemi pine ar,d T.*us mailie respectively (Stobel et a/., 20041Wang et

a1., 2000). The addition ofthis clinically useful drug from endophytic fungi has prompted

great interest for the discovery of other drugs in this domain especially anticancer and

chemopreventive agent. lt is considered that there exists a great potential for the

discovery of novel fungal species, with new bioactivc compounds among endoph)tes

(Hawksworth,200l). The need of novel metabolites and discovery of taxol and other

cancer chemopreventive agents from Taxus associated endophltic fungi have further

patronized these studies. For discovery of novcl cancd chcmopreventives, a battery of

mechanism-based ir-rit,,o assays is uscd for screening as well as purification ofpotential

drug leads. Different assays which are employed include, inhibition of TNFo- induced

4

Chaptet 1

NF(B, aromataseJ and of nitric oxide (NO) production in lipopolysaccharide (LpS)-

activated RAV/ 264.7 cells, induction of quinine rcductase 1 (QRl) and c).totoxicity

against MCF-7 and MDA-MB-231 cancer cell lines. These assays can be utilized for

evaluation of compounds to monitor potential inhibition of multiplc stages ofcarcinogcnesis (Kinghotn et al., 2004).

Owing to this fact, Tdrus species have particularly gained prime importance for

potentially aclive endophyic fungi (Lin et al., 2009). Thesc plants are found throughout

the world. Noflhem areas of Paldstan also inhabit laxrs plants. However, reports on

bioactive potential of endoph),tes associatcd with them aie scant. Therefore this study

was proposed to explore the endophyic fungi associated \trth TcL:xlts fuana fonnd in

Pakistan, for the production, isolation and purilication ofanticancer and chemoprcventive

drugs with enhanced therapeutic potential.

5

1.2 AIM AND OBJECTIVES

The main aim ofthis study was isolation and characterization of canqer chemopteventive

compounds from endophyic fitngi of Taxus.fuana, an indigenous medicinal plant ofPakistan.

lhe objectir cs of the present stud) were:

. Isolation and molecular identification of cndophytic fu,ngi of Taxzrsfala.

. Evaluation of biological activities of crude organic extract of endophytic fungi to

find their antimicrobial, cancer chemopreventive and cfotoxic potential.

. Isolation and pudficatio[ ofbioactive compounds from selected strains.

. Characterization of pure compounds by using modem structue elucidation

techniques.

. Comparative analysis ofthe biological activities ofthe pure compounds.

6

Chaptet 2

Chapter 2

Review of Literuture

1

Chapter 2

2.1 MICROBIAL METABOLITES AND DRUG DISCOVERY

Long before the era of high-thoughput screening and genomics, drug discovery relied

heavily on the inspiration from nature. Medicalions available today contair acrive

ingredients originating ftom natural products. Among the myriad of natltal sources,

microorganisms have been a prolific source for lead compounds and new

pharmaceuticals. Several natural products ftom microbes have potential therapeutic

indications. Metabolites derived from microorganism are being used as

immunosuppressive agents (rapamycin), aDtineoplastic agents (mitomycin),

hypocholesterolemic agents (pravastatin), antiparasitic agents (salinomycin), Exzymc

inhibitors (desferal), herbicides (bialaphos), antimigraine agents (ergor alkaloids), and

bioiDsecticides (tetranactin) (Demain. 1998). In addition to promising pharmaceutical

potential, these compounds also showed the great diversity ofchemical stuctures.

The metabolic processes form the basis of all sofis of life cycles since they provide the

key elements allowing the growth, reproduction and maintenance of cells in their

environment. Microbial metabolism is broadly classified as primary and secondary

metabolism. Primary metabolism involves the processes required for cell maintenance

and proliferation \\hile secondary metabolism refers to the production of metabolites nol

required for cell growth (Kliebenstein, 2004). Secondary metabolites are usually

produced in late stages of gro\rth cycle especially at the end ofexponential stage or later

because of limitation ofessential nuhients. These melabolites include peptide, antibiotics,

polyketides and many other groups ofnovel biologically active compoulds. The products

of secondary microbial metabolism havc gaincd immense importance owing to unique

biological functions assigned to them by nature. within microbial domain l'ungi hare

proved to be a remarkable sourcc of diverse natural products. The large biodiversity of

higher fungi provides a huge resource for extending the chemo diversity of natural

substances and for the discovery of new lead structures. There are three recognised

pathways ol secondary rnetabolism in fungi, which contribute significartly to diversity oI

chemical structuesi an over view of central metabolic palhway ol t'ungi is shown in Fig.

2.t.

8

Chapter 2

In mevalonic acid pathway Acetyl-CoA is starting point (Ganaway and Evans. 1984).

Two molecules of acetyl-CoA condense to lbrm acetoacetyl CoA. Acetoactyle-CoA

reacts with another molecule of acetyl-CoA to form hydroxymethylglutaryl-CoA (HMG-

CoA) and finally mevalonic acid. Mevalonic acid then undergoes phosphorylation

followed by decarboxylation to form isopentenyl pyrophosphate (lPP). Isopentenyl

pyrophosphate is the lilst most important molecule coDtaining isoprene carbon skeleton

in this pathway. It serves as chain initiating unit for synthesis of large terpenoids.

Mevalorate is the key intemediate in terpene biosynthesis which leads to va ety of

secondary metabolites tkough cyclization reactions. These metabolites could be

diterpenes, triteryenes, steroidal lanosterol and other sterol derivatives. The 20 carbon

containing diteryene can serve as the precursor tbr a number of biologically impotant

compounds (Khar1, 2007).

Polyketides are an important class of fungal secondary metabolites. Difl'erent types of

metabolites are produced by this pathway. They are formed by the condensation of one

molecule of acetyl-CoA with three malotyl-CoA molecules. In this pathway aldol

condensation is involved which leads to a variety of aromatic compounds like orsellinic

acid, dihydroxydimethyl benzoic acid,6-methyl salicylic acid and acetylphloroglucinol.

These aromatic compounds can generate variety ofcompounds by moditications through

reduction, hydroxylation, oxidation. decarboxylation and methylation. Metabolites

generated through this pathway intemct with those liom other metabolic pathways

leading to generation ofunique chemical entities (Khan, 2007).

Shikimic acid pathway is responsible for the synthesis of a wide variety of aromatic

compounds and is common in plants, fungi and bacteria (Garaway and Evans, 1984).

The pathway is initiated *'ith condensation ol two glycolytic intermediates

phosphoenolp).ruvate and erythdose-4-phosphate, to fonn dehydroquinic acid which is

converted into shikimic acid and finally to chorismic acid. Chorismic acid leads to

synthesis of aromatic amino acids phenylalanine, tyrosine, tryptophane, etc. These amino

acids serve as precuNor lor synthesis of more complex compounds. The imponMt

products of this pathway including amino acids are cinamic acid de vatives such as

coumarin and antibiotics (penicillin. ccphalosporin) and alkaloids (Khan, 2007).

llonos,.cheider ----------.-.--+ srroctuhlklncose, eic)

- potl.3a.charjd€s

)';-^' I ---r...-)";1" I I \-,*,-*.

I I D,. n. hr- i".

i ',:'. ;i:.:::," *.rroorrn,ni.o,.id5lI

s.(obdr^ .,.,*"^!"..*0,llerrbol'r.! rptt, qi dphtdc por. d.h,drosh(dooe po4i

,/ I /,,,.*,,,".*",.( I -.-r' P\ rI\ dre .-.------------ > \Jioted)I,l/t/ t '"lt:::i.'^Sr.Dtt

F2iF rleroEi,co.r

- A(€lll coenqme-{ ---------.> \lernloo"t" + r..obdan.I | ..-,--^,,l

i^ I I 'li'i'""i*

-.>

orgdi. a.ids-----.--

,.,oo n.ro,

Fig. 2.1: An overview ofcentral metabolic pathways of fungi (adapted from Khan. 2007).

Energl

10

Chapter 2

2.2 ENDOPIIYTES AS EMERGING SOURCE OF NOVEL METABOLITES

Endoph),tes are microorganism (bacte a, fungi and actinomycetes) which complete part

or whole oftheir life cycle inside a plant by colonizing infer- and/or intra-cellularly (Tan

and Zou, 2001). Among the endophytes, endoph)1ic fungi are a polyphyletic group of

highly diverse, primarily ascomycetous fungi defined functionally by their occurence

\\ithin asymptomatic tissues of plants. Almost all classes of vascular plants studied to

date have been reported to host endophltic microorganisms (Zhang et al , 2006).

Endoph)-tes can be transmitled vefiically as well as horizontally- Vertical transmission

occu$ through seeds and vegetative propagation of the host and horizontal transmission

occws through spores, external to host tissues (Canoll, 1988).

Till 1970's, endophytes were considered to be neutral as they were believed to cause

neither any harm nor the benefit to the plant. However; later studies revealed that

endophytes play an important role in host protection against predators and pathogens

(Azevedo e, al., 2OOOI They provide protection 10 their host plant from adverse

environmental conditions, insccts, pests and herbivores by secreting a plethora of

bioactive secondary metabolites (Azevedo et a\.,2000, Strobel and Daisy,2003; Strobel

et at..2004; Pimentel er a/., 2010; KIan et a1.,2010) and in retum receive nutrition,

protection and propagation opponunilies liom their host (Clay and Schardl,2002: Khan

et al-, 2O1O). This mutalistic association results in reprogramming of metabolic

machinery of the host and the endophltes leading to accumulation of unique classes of

secondary metabolites (Fig. 2.2). As a rcsult, a new era of pharmaceutical invesligation is

generated making these plants attractive for not only cultivation but also isolation of

associated endophytes. The rationale for the host plant selection is crucial to increase the

chances of isolating novel microorganisms and new bioaclive compounds Selection of

the plants is usually based on their unique environmental setting, ethnobotanical history,

endemism, umrsual longevity, and large areas of biodiveNity (Strobel aDd Daisy, 2003)

Many scientists have isolated interesting genera of fungi iiom different host plants

mainly Taxus sp. such as Taxomyces, Periconia, Trichoderma, Penicillium and

Perenniporia sp. (Strobel el 41., 1993a Zhang et al.,2007; Raghunath ?l 41.. 2012; Wu e,

.r1., 2013) shown in Table. 2.1.

11

Chdpter 2

Host plauts

Co-evolution

relations

PLrytopathogedc

Bioactive

Plart Geletic

ellgueerll1gbioteclurology

Chemical

process

\,ficLobial

l'emierrlatior

Fig. 2.2: Outline of host-endophyte relationship showing response of host plant and

endophytes against phytopathogens by producing bioaclive compor:nds and their

potential applications (retrieved from Zhao et aI.,2010)

12

e3 _ E-d=G

=-=lE -r;.:'-=-i 3 t i -lE n R 5 = ; . E-:: x:- ^

d '-i 6 : - - - - J 5 X -'= 9p! - i!\ ! ; = i != t t ! s a a t:e;;-Exi. : : i 'i : : : i ;: i - .- :--E EE n -,; -,- : : >: !

= _i ! J s c oB;*3;i 6

{ 3 i k i =:! z 4 air .i d : 6\-qr=: t:=

': : a .- € : 5 : E - r la ._li d aa = i. > : !. J > < > 6 J A '/. < j<4":; O,

s't$65

Edl.rfE i ..! - + I\ < :t;s i ! 6t I i f . $ i.- - . , i .6. f =.:.. E::i:!:i i i " :i ! !:!i ii;*ss:ni!: E:GiiGs 3 + i $s: S i I i : iSE:! : l:6 -! i a : N { .ii .: : .€ i f,'a y.S 6 i ,a E

-1'F. 5<r- J Q-l : R A a:s

B]

!

s!:io\q::Bo-58"i\*.sbn-: -s + :i ]:.ii 9o;-t-/la:rEs:s ':E s ! : + ;IS",d:*:S:NF..i=c<Sea-:oi:!9:r>ns.->! : ! * s I t a.e { * -s : FF -.o i ! : 3 s i A r I - :{ i i

i -i?i *ia€ i 3i I - i.4l i.3i 1-!E: :.i1 E r l: S - S?", * . i:': c.!. k Ji = I < !? \ "ii t li i :.-, s s F S.S=.: .l Bi \ i i: -r s+ , : ^: =n : i-:! ! !"\.! : > ; :; ^- :.:i.; n : !-! : +:i I ii -," : - i i r i *nia I { &

-a-i.i2-

Ch.tpter 2

2.3 ROLE OF ENDOPHYTES IN DRUG DISCOVERY

Endophytes are capable of synthesizing hundreds of natural products \\'ith unique

shuctures such as alkaloids, terpenoids, llavonoids, steroids and quinones with interesting

biological activities (Tan and Zou, 2001; Pimentel el al., 2010; Joseph and Priya, 201l)'

Crag and Newman, (2007) presented a list of all approved agents from l98l to 2006,

which indicatcs that signilicant number of natural drugs are produced either by

endoph),tes or other microorganisms. A single endophyte may be able to produce several

bioactive metabolites due to which thc role of endophytes in the production of novel

structures for exploitation in medicine is receiving increased allention (Wang el a/', 2000:

Gunatilaka, 2006).

2.3.1 Antimicrobial metabolites

Antimicrobial potential of the endophytes isolated from various plants in different

regions of the world has been widely explored Endophytes are believed to catry out a

resistance mechanism to overcome pathogen invasion by the production of secondary

metabolites (Tan and Zou, 2001). These secondary metabolites are potential candidates

lbr antimicrobial compounds (Table 2 2). The discovery of novel antimicrobial

metabolites is an impofiant altemative to overcome the drug resistance being acquired by

human and plant pathogens. Antimicrobial metabolites isolated i'rom endophyes belong

to diverse classes such as alkaloids, peptides, steroids' terpenoids. phenols, quinines and

flavonoids (Yu e1.r/., 2010; Joseph and Priya, 2011).

Qin et al., (2009) reported the chamcterizaiion of alkaloidal compounds chaetoglobosins

A and chaetoglobosins C being produced by endophytic Chaetomium globosum isolated

fiom leaves ot Ginkgo biloba. These alkaloides exhibit broad spectrum antifimgal

activity. Another alkaloid phomoenamide has been reported fiom an endophyic fungus

Phonopsis sp. exhibiting inhibitory concentration 6.25 pg/ml against Mycohacterium

tuberculosis (krkachaisi kul et al., 2008; Yu et al, 2010) Studies reported the isolation

and characterization of peptidal compounds such as leusnostatin A, cryptocandin'

echnicocandins (A, B, C, D, H) produced by endophltes snch as Acremoniun sp '

14

Chapter 2

Cryptosporiopsis sp. and Pezicula sp. (Strobel et al., 1999i Yu el a/., 2010) which also

exhibit antilungal ,ind antimal ial acti\ iry.

Dai et al., (2006) repofied isolation of antimicrobial llavonoid derivatives from culture

extract of endoph).tic fllt'tgrs Nodulisporlrn sp., isolated from .luniperus cedre otGomera Island. Similarly, there are repofis about the production of urdque antimicrobial.

fungal metabolitc, containing l3-membcred lactone ring from endophtlLc Penicillium sp.,

isolated lrom rcot of Punax ginscng (Yu et a1.,2010). Han et al., (2008) reported the

production of two new altibiotics by endophtlic Penicillium sp.. tsolated from mangrove

plant Cerberu ma/tghats. Thesc compounds were active against methicillin-resistant

St ap hy I oco c cu s aur e us.

Methylalatemin and altersolanol A are the quinines synthesized by endophytic

Ampelomyces sp. displaying antimicrobial activity with MIC value of 12.5-25 pg/ml,

against gram positive pathogells such as S. aale r, S- epidermidis and Enlerococcctls

.t'aecalis (Aly et dl., 20OB). Antimicrobjal terpenoid compounds have also been purified

fuom Phomospsis sp. isolated fiom Plumeria ucutfulia platt (Nithya and Muthumary,

2010). Methylcoumarin isolated ftom Xylaria sp. an endophyte of Ginkgo biloba

showed antimicrobial activity (Liu et d1..2008). Guanancastepene A, guanacastepene,

periconicin A and perieoniein B were four novel diterpenoid antibiotics isolated from

endophyles (Brady e, a1.. 2001; Yu el dl., 2010). Totg et al., (2011) investigated

antimicrobial activities of methanolic and ethanolic extract obtained from fermentalion

broth of 72 endophytic fungi isolated liom flowers of medicinal hetb Orthosporium

stamineus. Al-tined et a/., (201 I ) reported three new antimicrobial metabolites along with

six other known compounds from the endophytic for.,g:us Phomopsis sp. chenically two

were new chromones (phomockomone A and B) and one new natural cyclopentenone

derivative (phomotenone). Zhang et al., (2012) reported a Mrcol .tp. SPS-I1 isolated

fiom Artemisia an u.t Linn, with antimicrobial potential against Rhizoctonia ceredlis, E.

coliandS aureus. Li et ul.. (2012) also reported antimicrobial potential of39 fungal

metabolites including two ne* alkaloids isolated from the fermentation broth of

Aspergillus fumigatus LN-4. an endophytic fungus isolated from the stem bark of Melia

azedarach. Shan eI al., (2012) reported thal two out 48 endophltic fungi isolated from

Chapter 2

foots of Macleayd corduta sho'Ned potent antibacterial potential. Griesofulvin; another

antifungal compound \\as isolated from Nr'grosporur sp. an endophlte of Moringa oleifera

(Zhao et aI.,2012).

So far, many antimicrobial compounds have been isolated from diflerent endoph),tes:

however, the species isolated and screened occupy only a small portion of total

endophytic population. This suggests a great opportunity, within endoph),tic communiry,

to find reliable and novel antimicrobial compounds with the future use as clinically

effective antibiotics (yn et a1.,2010).

2.3.2 Cancer chemoprevetrtive and cytotoxic metabolites

Cancer is a group of diseases characterized by uncontrolled proliferation of abnormal

cells which ultimately lcads to death (Pimentel e/ a/.. 2010). Curent treatment strategies

i[volve chemotherapy. surgery, radiation therapy, immunotherapy and monoclonal

antibody therapy. These strategies have many side efl'ects particularly non-specilic

c).totoxicity of anticancer drugs (Gangadevi and Muthumary, 2008). Despite availability

of multiple ffeatment choices. there is continuous rise in the magnitude as well as

mortality rate of cancer. This indicates ineffectuality of existing therapies suggesting a

need to re-evaluate different options to attain more intensive and cuEtive approaches to

treat cancer.

Chemoprevention is a promising approach to reduce the prevailing chances of cancer

growth by controlling the deregulation of path\\'ays of cell division. The thought of

cancer prevention is gaining escalating consideration because it is a more beneficial

alternative to cancer treatment (Amin et.r1.,2009). Cancer chemoprevention approaches

target each of the steps in carcinogenic process (Fig.2.3) and include anti-initiation and

anti-promotior/anti-progression shategies (Greenwald, 2002; Tsao et al-, 2004; Balunas

and Kinghom,2005). Cancer chemopreventive agents are basically to act belbte onset of

cancer and should be either non-toxic or less toxic. However; in case of agents Llsed to

prevent cancer recurence, slightly higher toxicity is acceptable. Regardless ofsignificant

diversity and complexity of molccular events in cancer, natural products from plants and

microorganisms have conferred signiiicant inhibition without any rccognizable adverse

76

Chaptet 2

effects (Wang and Jiang, 2012a). Natural products from endoph)tes and their articancer

potential were evaluated by many scientists (Table 2.2). Structures ofsome commercially

important anticancer compounds are given in Fig. 2.4.

1'l

- - E ;- - ; - .E60=u

E E / A E A i ':i =

'= 6 P 6 d E o

a

:

t: ! - -l

" : i; i ! *.s Si I i t i i *.i a : .! r i i E ::rtti: Ytis!ititi!SriSt*i:!t*;Sit:rl. I€ N S =l € o i 'j'5 S d S =o e S ,": S

*ils : s !si g t i ; s rt .i i u i ; s i i iii:ssssi s: :siSsiSi

E

.2

E

,9

o.j!!-

(,

Lllqb

a_o=

a9U.--o7aa.aio-.=x;!- O i5 &c 5 : '.o f j

,=:*rlS

n.s d E i ti s st -r i : 3 rt i -c 3! n ; ;: ; <S s ! !E S : ! -- tF t\ s 3 E .r sI Y ! rI i ! r, J -s \ q !

z

jtc0

:-:!co+siui::le!$- i * r s s ! +"-is(\r!i'7i8ii*Sr:9!:Eo::S'i:S.i'ir.:==in->:s!i.ii:.'i'iiq\;iii:-€lis!.Y8.!t:s:--l

-A.a:aa-r\\a-7.

Chaptet 2

Antioxidant

Anti-T

oncoeene t ]

inflammatory -..> ._ Cell proliferation

hnnrune +. Cell differentiationenhancilg

Anti- hormone ...'>

ModificatioI1 of Phase

l/II enzyme

lL'<- Apoprosis I

Anti-zulgiogenesis

Fig. 2.3: Mechanism of cancer prevention by natural compounds (adapted from Tsuda e'

al.,2004\.

(lnc.r{-hr ropru\ cntile

20

Chdpter 2

Anticancer potential of several secondary metabolites produced by endoph)-tes has becn

invcstigated and lead to the discovery ofnovel diteryenoid taxol also known as paclitaxel

(Cheewarat, 2006; PimeDtel et al.,2010 Jospeh and priya,20l1). Since irs discovery,

this compound has received considerable attention than any other anticancer drug due to

its unique mode of action as compared to other agents (Firakova et al_, 2OOj; plmenLel et

a/., 2010). It was first isolated from the bark of tree Taxus brefifulia but since the trees

are rare and slow growing and also their use impose environmental degradation concems

therefbre the market p ce of the drug is quier high (G:uo et al., 2008). 'l he isolation oftaxol producing endophyte Taxamyces undrearae has paved the path for such altematives

since they can be exploited to obtain cheaper product via microbial fermentation (Stierle

et al., 1993, Pimentel e/ al., 2010). Deng. (2009) reported isolarion of endoph)-tic

Fusarium solani from Taxas crirer?l.ii grown in China with the potential application of

taxol production. Gangadevi and Muthumary, (2008) were successful in the isolation of

novel taxol producing endophytic lungus Chaetomella raphigera frofi medicinal plant

Terminalia atjuna in lndia (Table 2.2). Taxol has also been found in several other genera

of fungal endophytes which may or may not be associated with zrxas species like

Taxodium distichium, Wollenia nobilis, l'hyllosticta spin.trum, Ba aliniu robillardoide,

Pestalotiopsis termindliae and Botryodiltlodia thcobromae (Kunlar3,n e, dl, 2008; Pandi

el.rl, 2010; Pimentel et Ltl.,2010).

Pentacyclic quinoline alkaloidal compound camptothecin (C:oHroN:O,r): a potent

anti[eoplastic agent was first isolated from the wood of Camptotheca acuminate ('Vlall et

.r1., 1996). Campotothecin and l0-hydroxycamptothecin are two important precursoE for

the synthesis of clinically useful drugs; topotecan and i notecan. Pu e/ a/., (2005) first

reported an endoph].tic fungus Entrcphospora inliequens obtained lrom Nolhttpodyles

foetida rhathad, a,bt]i.ty to produce camptothecin (Table 2.2). Later; RehmaD sl al., (2008)

reported camptothecin producing endophyic fungi Neurospora sp- and Fusarium solani

isolated from No/iapodytes lbelida and Camptotheca acuminata.

Podophyllotoxin (PDI) is a well-known aryltetralin Iignan with potent antrcancer,

antiviral, immunostimulation. antibacterial, antioxidant, and anti-rheumatic propefiies. lt

is used clinically to treat testicular cancer, small-cell lung carcinoma, lymphomas and

27

Chupter 2

other cancers (Nobili e, al., 2009). podophyllotoxin is mainly obrained from

Sinopodophyllum plants, however Yar,g ct ul., (2003) first time reported thar

podophyllotoxin is produced by endoph).tic fungi isolated fiom Sirutpodophyllum

hexandru , Diphylleia tinensis d17d Dysosma veitchii plants. puri e/ a/., (2006) and Kour

e/ a1., (2008) reported podophyllotoxin producing endophytic fungj Trametes hirsute a\d

Fusarium oxysporun isolated from Sinopodophyllum hexandrum ard Sabika recuna

respectively.

Kakadumycin Ai a potent anticancer molecule exhibiting IC56 of 4.5 ng/ml against

human breast cancer cell line BT20; was produced from endophylic Streptomyces sp.

NRRL 30566 of Grevillea pteridifoli.t (Castillo e/ al , 2003). Another imponant

anlicancer compound produced by endophlte is ergollavin. Ergoflavin (C3oH26O14) is

dimeric xanthene which belo[gs to the compourd class ergochromes. This novel

compound has been isolated from endophyic fungi of lndian medicinal plant Mr,r?rropj

e/ergi (Deshmukh et d1.,2009). Similarly toneyanic acid is an unusual dimeric quinone

isolated from endophytic f:]ngus Pestalotiopsis microrporu of T.rus taxifolia and. has

shown selective cytotoxicity in cell lines sensitive to protein kinase C agonists (Lee et.r1.,

1996; Pimentel e1a1., 2010).

Screening of endophytic fungi isolated from pharmaceutical plants in China showed that

13.4 0/o endophytes were cytotoxic to HL-60 cells while 6.4 % to KB cells (Huang el a/.,

2001). Zhang et al., (2010) reported anticancer activity of 14 anthracenedione deri\clives

separated from secondary metabolites of endophytic fungi Halorosellinia sp. (No. 1403)

Guignardia sp. (l.Jo. 4382). These endophytes were isolated from Mangrove plant and

\!ere potent enough to inhibit the growth of KB and KBv200 cells (Zhang et al., 2010).

Radu and Kqueen, (2002) reported antiiumor activity in isopropanol extract of

endophytic ftmgi isolated from medicinal plants in Malaysia.

Mycoepoxydiene (MED) polyketide isolated from endophytic flungns Diaporthe sp.

suppressed NFKB to disrupt cell proliferation (Wang er a/., 2012b). Chaetoglobosin and

hispidin derivatives isolated from endophltlc fungi Chctelomium globo,sum and Phellinus

,,rrmii respectively; showed potent NFKB inhibitory activity (Dolu et 41.,2011, Wn et al..

22

Chapter 2

2011). Panepoxydone is also a class offungal metabolites which showed NF(B inhibition(Zaidmian et u|.,20051Erkel et aI..2007).

Depsidone metabolites isolated liom endophytic fungi Aspergillus unguis antlCorynespora cassiicola L36 showed aromatase inhibition potential (Chomcheon e/ a/.,

2009; Sureram et al.,2012). Aromatase inhibitors can block the production of estrogen

which inturn reduced the emergence of breast cancer. Moderate aromatase inhibitory

activity of monomeric xanthones and a benzophenone isolated from marine fungal strain

Monodictys putredikis was also obser,,ed (Ktick et al.,2OOj)_

Antioxidants are important to overcome degenemtive conditions since they possess anti-

inllammatory, antitumor, antimutagenic, anticarcinogenic. antibacte al or antiviml

activities in higher or lower level (Owen et al., 2000; Cozma, 2004; Sala et al_, 2002;

Joseph and P ya,20l1). Pestacin and isopestacin l-3, dihydro isobenzofurans are the

compounds obtained from cndophytic fungrs Pcstaloliopsis micrcspora isolated from

Terninaliu norobensis (Hatpet et a1.,2003; Strobel e1 a1., 2002). Pestacin is believed to

have antioxidant activity 1l times greater than trolox; a vitamin E derivative; p ma ly

via cleavage of reactive bonds (Harper et dl., 2003). Isopestacin cxhibit antioxidanr

activity by scavenging both superoxide and hydroxyl free radicals and is stmctually

similar to flavonoids (Stobel €l a1., 2002). Tianpanich et al., (2011) also repoded five

kno*n isocoumarins and a trew phthalide isolated from the endophytic fungus

t ollir,.'trichun,p- u ith antioriJant artir it1.

2.3.3 Anti-diabetic and immunosuppressive metabolites

Zhang et al., (1999) repofted the isolation ol non-peptidal fungal metabolite (L-783,281)

fiom endophyic fungus Pseudomassa,'la sp. collected from African rainlorest in the

Democratic Republic of Congo. This compound acts like insulin mimetic and can be

give[ orally since it is not destroyed in the digestive tract (Zhang et al-, 1999).

Endophltic fungi havc been found to produce metabolites with immunosuppressive

prope ies (Joseph and Priya, 201 l). Endoph)tic ful.g\ts F'usdtiun subglutinans has beer.

repofied to produce immunosuppressive but non cytotoxic diterpene pyrone subglutinol

A and B (Lee er.rl, 1995). Hiseh el a/., (2009) evaluated extracts of 12 endoph)-tes from

13 Taiwanese plants for cytotoxicity, anti-platelet aggregation and anti_inflammatory

activity. Extracts exhibited inhibitory eff'ects on collagen induced platelet aggregalion

with IC50 values of42.80-61.54 pg/ml-.

24

Chapter 2

Taxol

Fig. 2.4: Chemical structures

compounds.

Rl

Camptothecin Rl, R2, R3 = H

Topotecan Rr= 0H, R2 = CH2N(CH)r.HCl

R2 R3

9nIrinotecan R = -0-C-N FN )

Rr=H \-/ \JRr = CH7-CH]

of differcnt anticancer and cancer chemoprcventive

25

Chaptet 2

2.4 BIOASSAYS AND DRUG DISCOVERY

Bioassay can be defined as the determination of the relative strength ofa substance (as a

drug) by comparing its effect on a test organism with that of a standard prepamtion. The

role of bioassays is obvious to determine the biological activity ofheterogeneous natural

products. The selection of appropriate, sensitive, predictive. reproducible and reliable

bioassay plays a key role in drug discovery (Dey and Harborne, l99l).

2,,1.1 Antimicrobialassays

Antibiotics were deemed "miracle drugs" in the 1940s to treat infectious conditions.

Various classes ofantibiotics are currently in use to treat infections. Diffbrent techniques

are used to ascefiain the role of natural products for infectious conditions. Most

commonly used assays are antimicrobial assays (antibacterial and antifungal). In addition.

other assays are also there like aniiviral, antimalarial assays etc. However; the approach

used lor the discovery of penicillin is still in use today. This primitive method of agar

diffusion laid a foundation for antimicrobial study (Sing and Barrett,2006). This method

used as indicative assay for bioassay guided fractionation leading the isolation of active

natural products. Nevertheless; it is imperative that antimicrobial assays are playing

remarkable role ill the discovery and dcvelopment of antimicrobial agents. Therefore;

understanding assays for antimicrobial activity is mandatory to evaluate samples having

polential lbr new antimicrobials.

2.4.2 Cancer chemopreventive assays

To detemine cancer chemopreventive potency of natural products more sophisticated

bioassays are used as compared to antimicrobial assays.,l, ui*o bioassays used to

measue aDticancer and chemopreventivc potential can be divided into two groups:

subcellular or molecular assays and cellular assays. In case of subcellular assays isolated

systems such as receptoN, enzymes and DNA etc. are used while intact cells are used in

cellular assays. There are several types of assays that are used to find the cancer

chemopreventive potential of natural sources. Some important assays are. inhibition of

TNF-d activated nuclear factor kappa B (NFKB). aromatase inhibition. inhibition ofnitric

26

Chapter 2

oxide s)nthase (iNOS), jnducrion of quinone reductase I (eRl). 2,2_diphenyl_ l _picryl

hydrazyl (DPPH) free mdicai scavenging and inhibition of MCF-7(ATCC number H.IB_22) cancer cell line.

2.4,2.1 Lthibition of TNF-L qctieotel nucleqr factor-kappa B dssq)

All lypes ofcancers involved abnormal or uncontrolled cell growth because ofdisruption

of normal signalling pathways of cell cycle regulation. TNF-0 is one of the activatoN ofnuclear f'actor-kappa B (NFKB) and NFr<B is an inducible transcription faclor that plays

an important role in the regulation of apoptosis. cell differentiation and cell migration

(Fig. 2.5). Its activation may promote cell proliferation and further prevent programmed

cell death through transcriptional activation of genes that suppress apoptosis (Baldwin,

2001; Karin, 2006). As an important regularor in cell fate decisions and being critical in

tumo genesis, inhibition of NFKB signalling has its potential application for cancer

cortrol (Aggarwal ?t a/., 2004; Schupp e, a/., 2009). Because ofcritical role ofNFrB incarcinogenesis and regulation ofcell fate decisions, its inltibition has potential role for the

treatnent or prevention of cancer (Aggarwal et a1..2004 Schupp e/ a/., 2009). Many

natural products have been investigated for their NFKB inhibition potential. It is evident

that NFKB inhibition played a central role for anticancer properties of natural products

(Kondrat)'uk and Peza.rto, 2004). Rether et al., (200'1) repofted suppression of TNF-0

synthesis by inhibiting the activation of extracellular signal regulated kinase from fungal

piperazile netabolite gliovirin. Gliovirin could be evaluated fufther because of its NFKB

inhibition as an antitumor lead compound. Wu ea a1., (2011) also reported NFKB

inhibition activity of phenolic compounds isolated from fungus Phellinus baufiii. Haq el

al., (2012) repoded cancer chemopreventive study of six medicinal plants by using

difl'erent assays including NFr<B inhibition assay. Mycoepoxydiene a fungal polyketide

was evaluated for molecular mechanism of apoptosis on the basis of NF(B assay as an

antitumor drug lead (Wang et a1..2012b). Antiprolil'erative mechanjsm of cr).topleu ne

an alkaloid isolated from Boehmeria pannosa plant by using NFI<B assay was also

reported by Jin et a1., (2012).

21

2.4.2,2 Atotfiatase inhibition assay

The second major cause of cancer related deaths is the breast cancer. Breast cancer is

most liequent cancer among the women. Majority of post-menopausal women have

hormone receptor positive tumors because estrogen hormones are involved indevelopment of breast cancer (Maitl et al., 2007). Aromatase is a cytochome p450

enzyme complex required for the conversion of androgen to estrogen (Jongen e/ a/.,

2005). Aromatase inhibitors can block the production ofestrogen, which in turn can trimdown the groMh of estrogen receptor positive breast cancer cells. Because estrogen

production by aromatase is the last step in synthetic pathway of steroid production (Fig.

2.6). selective inllibition of aromatase will not affect the production of other useful

steroids such as adrenal corticoids (Maiti et a1.,2007). Iherelbre; aromatase inhibitors

have become attractive therapeutic agents in the treatment of estrogen dependent breast

cancer. A large number of small molecule natural product secondary metabolites (plant

and n'ricroorganisms), of various chemical classes have been tested for their aromatase

inlibitory potential (Balunas and Kinghorn. 2005; Balunas s/ a/., 2008; Chomcheon et

a/.. 2009; Sureram e, a/., 2012).

Chapter 2

cscDOXIL-1 F TN F

U ,E-4lt(6""b -:**E

( |RAK-4)

(.IR,i1K-rj--) I

._ar*--;;) @ITAB2I- \ ]

-(sr, I

m-6' -q59;

Antia popto sis

l

tFa'plTtsr-r/a;l

trAPTlnvasionl - I lcaM.r II t___-l

l

l!

Proliferation

Fig.2.5r Schematic diagram ofNFrB actilation pathways and its role in carcinogenesis

(adapted from Ichikawa et a|.,2006).

TAA 1 .IRAF9TAA2 -TAKt

l(Ba degradatlon

p5o-p65 DHA bIndlng

NF-rB activataon

2-o

p65 phosphorylallon

Chaptet 2

Caffar localonol ellzFnes

Flt"ct'""d.1.

S"."ah "'n"pr-'1.trri.ulum

Fig. 2.6: St€roidogqresis showing that aromatase inhibition will not affect the production

of other useful steroids.

htto://upload.wikimedia.ors/wikioedia/commons/8/8flSteroidoeenesis.eif (Accessed on

December 13.2012)

30

Mhenloco.tcoiG ^ o

Chaptet 2

2,4.2.3 Inhibitiott o/ ittic oxi.le (NO) prodactioh in lipopolysaccharide (LPS)-

aclfuated m rihe macrophage RAll264.7 cells assay

lnflammation is a serious element of tumor progression. Many cancers begin from sites

ofchronic ilritation, inlection and inflammation. There are several reporls suggesting that

the tumor microenvironment, which is largely surrounded by inflammatory cells. is a

crucial member in the neoplastic process, promoting proliferation, cell survival, and

migration (Fearon arld Vogelstein, 1990; Hanahan and Weinberg, 2000; Pan e/ al, 2008).

Macrophages are involved in chronic inflarnmation by producing various inflammatory

mediators including cytokines. interleron, colony-stimulating faclors. chemokines.

lysozymes, proteases, growth factors, eicosanoids, and nitric oxide (NO). Among these,

N0 is excessively synthesized endogenously from L-arginine by one of the pro-

inflammatory enzymes iNOS, and consequently results in diverse diseases including

arthritis, asthma, multiple sclerosis, psoriasis. colitis, neurodegcnerative disorders, tumor

development and transplant rejection of septic shock (Cheenpracha et al., 2010a). A

consistent relationship between up-regulation of iNOS and cancers of the prostate,

bladder, ovary, oral cavity, and esophagus has been observed. Moreover, deregulation

appears to occur during ezrly tumor devclopment in these organs, suggesting that use of

iNOS inhibitors may be a possible chemopreventive strategy (Cro$,ell et al., 2003;

Nomelini el al., 2008\. Therefore, it is ol interest to find new inhibitors of NO

prcduction.

lnhibitor,v effects of radicicol a l'ungal antibiotic on iNOS suggest that this porenl

antifungal agent may represent a useful anti-inflammatory agent (Jeon €, a/.,2000).

Three novel iNOS i ibitors namely, sporogen, Sl,l-95 and S-curvularin were isolated

from different Penicillium species (Yao et al.,2003). Anti-inflammatory effects of

Asperlin isolated from ma ne derived fungus Aspergillus sp. SF-5044 are mediated

though iNOS inlibition (Lee et al.. 20ll). Fermentation products of the fungus

Morascrs are also repofied as a potential source of iNOS inhibitols (Hsu et a1., 2012).

31

Chapter 2

2.4.2.4 DPPH free radical scavenging assay

It has been estimated that one human cell is exposed to approximately 10,000 oxidative

hits per day from OH (free radical) and other such species (Halliwell and Aruoma, 1991:

Dreher and Junod, 1996; Jaruga and Dizdarogluo, 1996; Wang et al, 1998; Marnett,

2000; Dizdarogluo el til.,2002). These agents cause permanent modification of genetic

material by oxidative damage (Fig. 2.7), which represents the first step towards

mutagenesis involved in carcilogenesis and aging (Halliwell and Gutteridge, 1989;

Valko e/ a/., 2004). There are two different mechanisms thought to be involved in

oxidative damage leading to lhe dcvelopment of carcinogenesis. The first mechanism by

rvhich oxidative damage can affect carcinogenesis is through the modulation of gene

expression. The second mechanism involves the radical induce genetic alterations, such

as mutations and chromosomal rearangcments, which in tum can play a role in the

initiation of carcinogenesis (Guyton and Kensler, 1993; Cerda and Weitzman, 1997i

Valko et a/., 2004). The initiation potential of oxidants may be contribution to

carcinogenesis due to their ability to induce DNA base changes in certain oncogenes and

tumor suppressor genes (Jackson, 1994). To protect the cells against lhe ftee radicals

attack, the search for new free radicals scavengcr from the natural sources is exceedingly

required. A large number of plant and microbial species have been studicd for their

antioxidant potential by using DPPH free radical scavenging assay (Zheng et a/, 2008;

Srinivasane/a/.,2OlO:Artatltietal.,2011lHaqetal.,2Ol2.RavindBnelal.,20l2).

2.4.2.5 Quinone rcductase I (QRI) ihtluction assay

Carcinogenesis is a multifaceted and protracted process; however it can be initiated by a

single event in which a cellular macro molecule is damaged by an endogenous or

exogenous agent (Cuendet ca ol., 2.006) Approaches for protecting cells from these

initiating events includc decreasing metabolic enzymes (Phase 1 cnzymes) responsible

for generating reactive species and/or increase phase II enzyme that can deectivate

radicals and electrophiles known to intervene in nomal cell plocesses. An imponant

detoxification pathway involves reduction of electrophile quinones by QRI to

hyroquinones and reduces oxidative cycling. Therefore the compounds that induce phase

32

Chapter 2

II enzyme selectively are more attractive candidate for cancer chemoprevention (Cuendet

et a1.,2006).

Induction of quinone reductase I (QRl) $ith cultured Hepa lclcT (murine hepatoma)

cells is one of i,? ,i/ro bioassay. Quinone reductase I rise ir? viro and i,? yivo systems has

been shown to be associated with induction of other protective phase ll enzymes and

offers a logical biomarker for the promising chemopreventive effect of test samples

againsl cancer initiation (Su et a1.,2004 Cuendet el .r1., 2006). Therefore induction of

QR, at the tumor initiation stage, is suggestive Ior cancer prevention potential but it has

also been established that inlibition of carcinogenesis at later stages is also possible.

Quinone reductase induction potential of metabolites of medicinal plants and

microorganisms oan also favour to identify leads for cancer chemopreventive drug

development (Gusman et a|.,2001.5D et al.- 200,1; Balunas and Kinghorn, 2005; Pontius

et aI.,2008; Haq et a|.,2012).

2.4.3 Cytotoxicityassay

2.4,3,1 Su$orhodamine B (SRB) essay

Sulforhodamine B assay; a rapid, sensitive and inexpensive method, developed by

Skehan and his colleagues and reported in 1990; remained one ofthe most widely used

methods for l, yir,.o c),totoxicity screening (Skehan et al., 1990). The assay is based on

the capability of SRB to bind to protein components of cell that have been fixed to

bottom ofccll tissue culture plate by trichloroacetic acid (TCA). Sullbrhodamine B is a

bright pink aminoxanthine dye with two sulfonic groups that u[der mild acidic conditions

bind to basic amino acid residues and detach under basic conditions. The binding of SRB

is directly propoftional to the cell mass and strong intensity of SRB staining permits the

assay to be carried out in 96-well plates. Fufthemrore according to Skehan e/ a/., (1990),

sensitivity of this assay is comparable to that of fluorescent dye staining methods and

even much superior to other protein staining methods using conventional visible dye.

33

ll

i-Y') "" l"*i^'' ---i:Y'X'.a"."n- -"A.i"'

R"! __/ r-8 nq d tu\.r-d.!,l or

i r' """'"'gt *r"&*'* ..].---*i1* .)l-.-.-\"r-, ,fL-X A,I->",? -hyd ro- 8,hydroxygx a D ine 8-h y droxy gun ine

\, /;-\. r;-L -,",-.,"

1-"-[-*.2,6-diaminc4-hydroxy-5-fomamidoplmmdine

Fig. 2.7: Attacking pattem of hydroxyl free radical (OH) on the C8 position of guanine

(Valko er.r/., 2004).

Chapter 2

This assay can detect densities as low as 1000-2000 cells per well and with signal tonoise ratio 4.83 at a density of 5000 cells per well. Results from SRB assay show a lineardynamic range over densities of7500 180,000 cells per well. This assay has been

extensively used lbr drug toxicity testilg against various types of cancerous and non-cancerous cell lines (Vichai and Kirtikara, 2006). Cfotoxicity assays played imponanrrole in development of various natural products as anticancer agents such as taxol,camptothecin. podophyllotoxin, vincristine. torreyanic acid, kakadumycin A and

ergoflavin (Castillo e t al., 2OO3; Kour el a1., 2008; Kusari er a/., 200g; Deshmukh e, a/..

2009; Wang and Tang, 201 1).

2.5 TECHNIQUES USED FOR PURIFICATION AND CHARACTERIZATION

OF NATURAL PRODUCTS

The area of natural product isolation has gained rattling intercst in the last lew decades.

Multiple strategies have been developed to isolate the natural products. First step

involved in the isolation of natural products is the sepamtion of medicinally active

components from inert or inactive moieties by usjng different extraction techniques such

as maceration, infusion, percolation. hot continuous extraction (Soxhlet), digestion.

decoction, counter-curelt extBction, aqueous alcohoiic extraction by fermentation,

ultrasound exlmction and supercritical lluid extraction. Different organic solvents like n-

Hexane, Ethyl acetate, Butanol. Ethanol and Melhanol arc used for extraction ofsecondary metabolites from plants as well as microorgatljsms (Yao et al.,2007, Wtlght et

a1..2003; Haq et aI.,2012).

Active crude organic extracts are used for fractionation to separate difl'erent components

on the basis oftheir physicochemical properties by using two immiscible organic solvents

in separating funnel. Individual components ofpartially purified mixture can be separated

by using difl'erent chromatographic techniques such as column chromatography (nomal

phase and reverse phase column chromatography), thin layer chomatography, size

exclusion chromatography, medium pressure column chromatography(MPlC) and highly

sophisticated and reliable technique high pertbrmance liquid chromatography (HPLC).

Slructural elucidation of pure compounds is based on different techniques. Over the past

fifty years nuclear magnetic resonance specftoscopy (NMR), has become the preeminent

35

Chapter 2

technique for detennining the structue of organic compourds (Rabi el a/., 1938). Of all

the spectroscopic methods involved in structural elucidation, it is the only powerful, non-

selective, analltical tool that erMbles to asceftain molecular structure including relative

configuration, relative and absolute concentmtions. and even intermolecular interaclions

without the destruction of the anal),te. Small and simpler organic molecules can be

analysed with l-dimensional proton or carbon-llC NMR while complex molecules

require 2-dimensional NMR techniques (Marlin and Zekter, 1988). Various complex

organic molecules are completely characterized structually by using 2D NMR methods

such as Heteronuclear Single Quantum Coherence (HSQC), Heteronuclear Multiple Bond

Corelation (HMBC), Conelation Spectroscopy (COSY) and Nuclear Overhauser effect

Spectroscopy (NOESY) (Wright et al.. 2003; Y ao et al., 2007; Chet et a1..201 1 ). Mass

spectrometry is another technique used to accurately determine the mass ofthe molecular

ion in structure elucidation to identili or confirm the molecular formula for a compound

(Sparkman, 2000). It is a supportive technique usually used individually or coupled with

LIPLC, LC, GC, MALDI-TOF etc. These tcchniques of purification and characterization

played an impofiant role in modern drug discovery.

The MeOH extract obtained from the cultivation of Pestalotiopsis guepini, aftet

chromatographic separation yielded tkee anthraquino[es derivatives (Oliveira e/ a/..

2011). Bioassay guided l'ractionation of ethyl acetate extracl of Chaetomium globos

fungus lead to isolatiofl and purification of lbur azaphilone compounds including one

novel chlorinated derivative (Qin et a1.,2009). Sequenrial iactionation of exhact ofPenicillium commune by using normal phase column chromatography, sephadex LH20

and finally HPLC lead to isolation and purification of six new azaphilone de vatives

(Gao e/ a1..201l). Proton and carbon NMR data ofa new compound epicoccamide gave

evidences that it is composed ofthrce distinct subunits. glycosidic, an alipharic chain and

tetramic acid moiety. The structure was further confirmed with 2DNMR and ESI-MS

experiments (Wright e/ dl., 2003). The structure of two new diketopiperazlnes

(Epicoccins E and F) possessing thc characleristic internal sull'ur bridges was determined

by Cuo ei a/., (2009). The structure oftwo novel benzoquinone metabolites isolared from

fungus -Y_r1.rla sp. was also repoded by Tanswan er 4l., (2007). New azaphilones were

also repofied from Chuebmiwn and Penicillium fungi by Borges et al., (2011) and

36

Chapter 2

Htlr,ng et al.. (201 1) respectively. Oliveira ei a/., (201 I ) and Chen er a/., (201 l ) reporled

structures of new anthraquinone derivatiles from fungi. Structural characterization ofunusual epicolactone and new sesquiterpenoid isolated fuom Epicoccufi nigrum and

Perenniporia tephropora lungi respectively was reported by Araujo e1al., (2012) and Wuet al-, (2013). Different bioassays, purification arld structural elucidation techniques

enabled the researchers to obtain drug leads from natural soulces.

Chapter 3

Isolution, Biologicul Screening and

Selection of Endophytic Fangi

38

Chapten

Chapter 3

3.1 INTRODUCTION

Bioactivity screening has been an integral parl of the modern drug discovery process

fbr decades. The demand to discover natural products fbr required pharmacological

potential has i[creased the need to screen more targets with minimum cost. highest

reliability and reproducibility. Due to recent advances in assay technology,

instrumentation and automation. the use of mammalian cell based assays has

expanded to all stages of the lead generation process, including primary screening.

Historically, the mostly exploited natural source has been the plant kingdom and

many compounds. medicinal plant extracts and herbs have been studied for calscr

prevention which can help to sustain human health without identifiable side elfects

(Kinghorn et a/.. 2004; Wang and Jiang, 2012a; Haq et d1.,2012). The second most

successlul natural source has been fungi, from which breakthroughs like antibiorics;

immunosuppressants and anticancer drugs have been discovered (Tulip and Bohlin,

200,1). Among the iulgi endoph).tes represent an enormous diversity and their

specialized habituations make them a stimulating field of study (Ower and Hundley,

2004). The role of endophl,tes for a[timicrobial and cancer chemoprevemtive

potential is needed to explore as an emerging less studied domain.

The aim of this study was the investigation of bioactive metabolites of endophltic

fungi derived from laxus plant of Pakistarl. In order to sclect the most promising

fungal srrains, the isolated fungi *ere grown on a small scale by using two different

production media and secondary metabolites were extracted \\'ith organic solvent

ethyl acetate. Then crude ethyl acetate extracts were tested for antimicrobial, cancer

chemopreventive and c),totoxic potential. Antimicrobial screening was done by using

antibacterial and antifungal assays. Different bioassays used for chemopreventive and

cltotoxic potential evaluation were, inhibition of tumor necrosis factor (TNF-0)

activated nuclear factor kappa-B (NFXB), aromatase inhibition, inhibition of

lipopolysaccharide (LPS)-activated nitric oxide (NO) production in macrophage cells

(iNOS assay), induction ofquinone reductase I (QRl), and inhibition of proliferarion

of hormone .esponsive human breast cancer MCF-7(ATCC HTB-22), human

prostrate PC-3 (CRL-1435), human peripheral blood leukemia l-1L60 (CCL-2,10) and

estrogen receptor negative human breast MDA-MB-231 (ATCC llT8-26) cancer cell

lines.

39

Chdpter 3

MATERIAL AND METHODS

3.2.1 Collection ofplant samplc

Healthy and mature plants (showing no symptom of disease) ol T.$us fuonct were

selected for sample collection in April 2008 liom Nathia gali (a mountain resod town

or hill station in Hazara, Klryber Pakhtunliwa, pakistan). Leaves and woody pads

(stem. bark) were collected randomly lbr study. The plant material was brought to

laboratory in slerile bags and undcr cold condition for fufiher processing. The plant

was identilied at Dcpartment of Plant Sciences euaid-i-Azam University by prof. Dr.

Mir Ajab KIar after comparing with Lryrs y,allichiana preserved specimen ofvoucher no. 2978-ZB and accession no. of 52035 in the Herbarium of pakistan (lSL).

Later it was taxonomically identified as Taxus fuaha natiye to eastern Himalaya and

southwestem China (Shah et al.. 2008). The plant material was processed immediately

to avoid contamination.

3.2.2 Isolation of endophytes from dilferent plant pafts

Isolation o1'l'ungi from plant parts was done by using surface sterilization method

described by Petrini. (1986), with slight modification. Sodium hypochlorite (NaOCl)

and 75 % ethanol were used foa surface sterilization. The concentration and trealment

time of tissues *,ith sodium hypochlorite was changed according to type of tissues.

The concentration of sodium hypochlorite varied between l-13 oZ concentration and

time of sterilization was varied bet$een 3-10 min. Plant pa s leaves and wood were

cut into small pieces of length 0.5-l cm. Each plant part was treated with 75 7o

ethanol for I min. lbllowed by treatment \\,ith sodium hypochlorite and again with 75

0/o ethaDol for 30 second. Lastly the segments were rinsed three times with steriie

distilled water and 3-6 plant segments \{ere placed on water agar media as well as

potato dextrose agar medium supplemented with antibiotics penicillin G 100 unit and

streptomycin 100 [g/ml-.

These petri dishes were sealed with paralilm and incubated at 25 .C for 2-4 weeks by

using 16:8 lights and dark cycle. After rwo weeks of incubation fungal hyphae

emcrged fiom diflerenl plant parts wcre transl'ered carefully to fresh PDA plates to

obtain pure cultures.

40

Chapter 3

3.2.3 Cultivation offungi for production of metabolites

For production of metabolites liom ftutgal isolates, growth was carried out on potato

dextrose agar (PDA Oxoid) and modificd taxol medium (TM) (Xt et at..2006\.

3,2.3.1 Solid stdte ferrfientation ahd extructiot of uetabolites

Solid state fementation $,as carried out in PDA and TM agar. The composition ofTM agar is given in Tables 3.1. Almost 3 liters ofeach media prepared in petri plates.

A mycelial disc of 8 mm diameter from fieshly grown colony of fungal isolates was

transferred in the center ofpiates and incubated at 25 .C for 2l days. After 21 days ofincubation mycelia aJong with agar was blended Iith equal volume oforganic solvenl

ethyl acetate and left ovemighl on shaker. Then organic solvent was decanted and

filtered this process was repeated three times aIId organic filtrate was pooled and

concenhated by using rotary evaporator to obtain crude ethyl acetate extract for initial

biological screeningasshoun in fig. J.l.

41

Chaptet 3

Table 3.lr Composilion ofmodified taxol medium (TM).

Name ofchemical Ouantity (g/L)

Sucrose

Phenylalanine

Peptone

Yeast extract(NHr,SO{

MgSO{.7HrO

KII,POlNaCl

Sodium acetate

Sodium benzoate

40

0.01

0.5

0.83.0

0.5

2.00.6

0.5

0.r

t5

42

Chaptet 3

Iucul,ated for 21di)-s at 25'C

I

FiltrrtioE of tLe extrrcted sok.at

I

Crude eth)I acetate e ract

Fig. 3.1: Schematic representation of cultivation and extraction of metabolites alter

solid state fermcntation.

Filtrate

I

43

Chapter 3

3.2.4 Biologicalscreening

Fifteen fungal strains were used for biological screening. All these strains cultLrred in

TM ard PDA agar media and crude ethyl acetate extracts obtained (Fig. 3.1) were

tested by using battery ofantimicrobial and chemopreventive assays.

3.2.4.1 Attlihtictobial sctuehing

3. 2.1. l. l Antibacterial as.tay

Both gram positive and gram negative bacterial strain ]fitclndlng Staphylococcus

aweus (ATCC 6538), Micrococcus luteus (ATCC 10210), Escherichia coli (ATCC

25922). Pseudomonas aerugi osa (ATCC 9721) ard Klebsiella pneurnniue (ATCC

13883) uere used to evaluate antibacterial potential of the samples. Agar u'ell

diffusion method was employed as repofied by Nair et a/., (2005).

Nutrient agar (Oxoid) was used as medium for bacte al growth. Test bacterial

cultures were refreshed by streaking on nut ent agar plates and 24 hrs old bacterial

cultues were used to fotm bacte al suspension. Turbidity of the culture was

maintained by comparing with 0.5 % McFarland solution. Lawn of the test organism

was made using nutrient agar plate with sterile swabs. Plates were kept for some dme

and then u,ells u,ere made using sterile met. lic borer of 8 mm diameter. Then 100 pL

ofeach sample (4 mg/ml in DMSO) was added to the respective well. Tetacycline

was used as positive control (30 pg/well) and DMSO \\'as used as negative control.

Plates were labeled carefully and incubated at 37 "C lbr 24 hrs during which activity

was evidenced by the fomation ofzone of inhibition suffounding the well. The assay

was performed in triplicate. Antibacterial activity was expressed as diameter of the

zone ofinhibition and measured in millimetre (mm) by using microscale.

3.2.1.1 2,lntifunqal assav

Antii'ungal assay was perfomed by agar well diffusion method as rcpofted by Kanan

and A1- Najar (2008). Fungal test cultures used in this assay include Aspergillus Jlaws(ICBP 0064), Asperyillus fumigatus (FCBP 66), Aspergillus riger (FCBP 0198),

Aspergillus teteus (FJ654131) ard Candida albicans (Cl.l4043). Potato dextrose

agar (PDA, Oxoid) was used as gro\\th media lor fungal test cultures. Aliquot of 100

44

Chapter 3

pl spores suspension (1xl08 spores/ml) of each tesl isolate was spread evenly on the

surlace of PDA plates by using sterile glass rod. Wells were made at appropriatc

distance using sterilized cork borer of 8 mm diameter and 100 pL of crude ethyl

acetate extract at conccntration of 10 mg/ml in DMSO was dispensed carefully in the

respective wells. Nystatin (100 pg/\r'ell) antifungal drug was used as positive control

and solvent DMSO as negative control. Plates were incubated at 27 oC and results

were [oled after 24 hrs 1n case of Candida species and after 48 hrs in case of other

l'ungi. The assay \las performed in hiplicate. Antifungal activity was expressed as

diameter ofzone ofinhibition and measured in millimetre (mm) by using microscale.

3.2.1.1.3 Htohde formution inhibition assaN

An llFl assay in Streptomyces 85E was performed by a method previously repofied

(Waters el ul.,2002). An aliquot of 5 prl of Sheptomyces 85E spore stock was

inoculated into tryptic soy broth (TSB) and grown overnight in a shaking incubator at

30'C. After 24 hrs the fermentation broth was dilutcd 10 times with TSB and 50 pL

of diluted broth containing mycelia fragments of Streplomyces 85E uere spread on

minimal medium (lntemational Streptomyces Project Inorganic Salts Starch Agar)

ISP4 (Table 3.2) agar plates to produce the bacterial lawn. The impregnated paper

disks (with a dose of 80 pg/disk of sample) were applied directly on the suface of

agar plates seeded with Streptomyces 85E. The zone of inhibition was recorded after

30 hrs of incubation at 30 'C. T*'o types of zones \ere observed: a clear zone of

inhibition and/or a bald (not clear) around the disk. Staurosporine, a protein kinase C

inhibitor (20 gg/disk) nas used as positive control and solvent I)MSO was used as a

negative conhol- An inhibition zone of greater than 8 mm of sample \\,as considered

as an active sample. The assay was performed in triplicate.

45

Chapter 3

lable 3.2: Composition oflSP4 medium.

Name ofchemicAl Quantity (g/L)

Agar

Soluble slarch

CaCOr

(NFIr)?SOJ

MgSO,1.THrO

KTHPO4

NaCI

FeSOr.THrO

MnClz.THrO

ZnSOr.TH:O

20

IO

2

2

1

l

i

0.001

0.001

0.001

16

Chaptet 3

3.2.4.2 Cqncer cltemoprcve live screenifig

3.2.1.2.I lnhibition oI TNF-t actituted nuclear factor-kaopa B (NFt B) assav

ln this assay 293,4.JFkB-Luc HtrK cells \r'ere used. Dulbecco's Modified Eagles

Medium (DMEM) containing l0 %o Felal Bovine Serum (FBS), penicillin G sodium

100 lU/mL and streptomycin sulphate 100 pg/ml u,as used to maintain cells. Cells

were added in white walled 96 $ells plate ar 20 x l0r cells per 200 pL and incubated

for 48 hrs at 37 "C and 5 % CO2. After 48 hrs medium was replaced with fresh

medium and test samples at concentration of 20 prg/ml were added. The plate was

again incubated for 6 hrs after the addition of TNF-o to make a final corcentation l0

ng/ml. Phosphate Saline Buffer (PBS) was uscd to wash cells and cells were

subjected to freeze /thaw cycle (-80/37'C) after adding 50 pL lx reporter lysis buffer.

Phosphate Saiine BulGr (PBS) was used to wash the cells and cells were subjected to

freeze /thaw cycle (-80/37 "C) after adding 50 pL lx reporter lysis buf'fer. Luciferase

assay system was used and inhibition of TNF-0 activated NFkB was measured in a

luminometcl (BMG Labtechnologies, Durham, NC) to calculate % inhibition. The

samples which were considered to be active (*ith more thal,'l} yo inhibition) at

concentration ol 20 pg/ml were selected to determine IC;o (Haq e, al_. 2012).

Cytotoxicity, sulphorhodamine B (SRB) assay was used parallei to avoid false

positive results ofsamples due to toxicity.

47

Chaortr J

3.2.4.2.2 Atomatase inhihilion assav

The method repo ed by Haq e/ d/.. (2012) was used to determine aromatase inhibition

potential of samples. Dibenzylfluorescine was used as a substnte lbr aromatase and

fluorescent intensity of hydrolltic product fluorescine is considered a measure ofinhibition potential. A black coloured 384 well plate was used for this assay.

Nicotinamide adenine diphosphate (NADPH) 30 trrL regenerating system (2.6 mM

NADP+,0.8 ll/mL glucose 6 phosphate dehyclrogenase, 7.6 mM glucose 6-phosphare,

13.9 mM MgCl2 and I mg/ mL albumin in 50 mM potassium phosphate, pII 7.4) was

incubated for 10 min at 37 "C along with test sample (3.5 gL). Then 33 pL ofaromatase enzyme along with substrate mixture (80 pL/mL aromatase enzyme, 0.4

pM dibenzylfluorescein, and 4 lrg/ml albumin in 50 mM potassium phosphate, p]l7.4) was added and incubated for 30 min at 37 oC to complete enzyme reaction. The

reaction was stopped by adding 25 trrL of 2 N NaOH and kepr on shaker for 5 minrc.

The plates were again incubated for 2 hrs at 37 oC and then fluorescent was measured

at 530 nm (emission) and 485 nm (excirarion) by rising synergy II fluorescent plate

reader with Gen5 software. The ICso values and dose response curves were

determined for active samples (\\,hich showed more than 70 % inhibition at 20

pg/ml) and Naringenin (lCso = 0.23 pM) was used as a posirive control.

3.2.1.2.3 lnhibition ol itric oxide (NO) production in lipopolysaccharide (LPS)-

aclivated murine macrophaye R4W 261.7 cells Ltssay

Inhibition of nitric oxide (NO) production in lipopolysaccharide (LPs)-activated

murine macrophage RAW 26,1.7 cells was determined using test samples according to

the method described by Haq et Ltl., (2012).

Cells at concentration of200 prl of50xl0a cell/ml per well were incubated for 24 hrs

in humidified atlnosphere in DMEM medium contairing l0 % l.-BS, 100 IU/mL

penicillin G sodium, 100 pg/ml streptomycine sulphate and 0.25 pg/ml

amphotericin B. Affer 24 hrs of incubation media was replaced with 190 [L of tiesh

media containing 10 % FBS and lacks phenol red. Test samples (10 pL) prepared in

10 %o DMSO were used io ireat cells lbr l5 min folloued by addition of I pg/ml o1

LPS for 24 hls at 37 "C in humjdified atmosphere. Alter 24 h 100 pL of thc mcdia

was shifted 1() ne\r' 96-well plales and Griess reagent [90 pl of N-(1-naphthyl)

ethylene diamine in 2.5 % HrPOq and 90 LrL of I % sulfanilamidel was added to

48

( hapter 3

measure absorbance at 5,10 nm. Sulpharhodamine B (SRB) assay \\,as run

simultaneously to determine cytotoxic effects of samples. Samples showed more than

70 % inhibirion at 20 Llg/ml concentration were lufther proceeded to calculate IC5o.

In this assay Hepa lclc7 (murine hepatoma) cells were used.200 pL of 0.5 x lOa

cells/ml per well were added in 96 well plate containing MEM- o (minimum

essential medium) supplemented u,ith antibioric/antimycotic agents, l0 % FBS but itdidn't contain ribonucleosides or deoxyribonucleoside. After 24 hrs ofincubation in a

CO2 incubator at 37 "C, previous media was replaced with fresh media and test

sample (10 pL). Plates were incubated again lbr 48 hrs. Enzyme activiry was

detemined by the reduction of MTT (3-(4, 5-dimethylthiazo-2-yl)-2, 5-

diphenyltetrazolium bromide to blue lbrmazan and digitonin was used to pemeabilize

cell membmnes. Absorption was measured at 595 nm (Song er a/., 1999) to quantify

production of formazan. To test cytotoxic effects of test samples a total protein assay

using crystal violet stain was perlbrmed simultaneously (Su er a/.,2004).4,-Bromoflavone (CD = 0.01 pM) was used as a positive control.'lhe samples which

showed induction ratio >2 at 20 pg/ml u,ere tested at three fold serial dilution to find

cD.

3 2 I ) 5 DPPH fla LlJ dl scovcntinp a:fiv

The free radical scavenging assay was perfolmed according to method reported by

Lee et al., (1998) in which 2, 2-diphcnyl- I -picryl-hydrazyl (DPPH) was used. Test

samples were dissolvcd in 100 % DMSO and 95 UL DPPH solution (316 pM in

methanol) and 5 pL of test solution were added in each well of 96-well plate. The

plate was incubated at 37'C for I h after thorough mixing. Absorbance was measured

at 515 run using micro plate reader with highest final concentration ofthe tested

sample 200 pglml. Ihe samples which showed more than 70 o% scavenging activit)

\\,ere further processed to determine ICs6. Ascorbic acid (lcso:35.6114 [M) and

pure DMSO were used as positive and negative conhol. The test was performed in

ftiplicate. The following formula was used to calculate percentage scavenging a(ti!it)

and lC50 value was calculated by using table cuNe soii\\are.

49

Scatengmg eIf ecL (o/o.) = ll - +l \ 100AC'

Where "Ac" is Absorbance ofcontrol and ,,As,'is Absorbance ofthe test sample.

3.2.4.3 Cytotoxiciqassay

J 2,t 3.1 .\ullbrhoddminc B rSRBt o'$v

Sulphorhodamine B colorimetic assay described previously by H aq et al., (2012) was

used to determine c),totoxic potential oftest samples against MCF-7, pC-3 and HL-60

cancer cells. Dulbecco's Modified Eagles Mediurn (DMEM) supplemented wirh 100

IU/mL pcnicillin G sodium, 0.25 pg/ml amphotericine B, streptomycine sulphare 100

pg/ml and 10 % FBS u,as used to culture cell lines. Cells were incubated in 96 well

plate in humidified atmosphere at 37'C and 5 % CO2 for 72 hrs to get required

confluence. Alier changing old media with fresh cells \\,ere again incubated for 24

hrs.'Ihese cells were trypsonised and diluted to get 5xl0a cells/ml-. Cells (190 pl)

were transferred to new 96 well plates and 10pL of test samples (in l0 % DMSO in

PBS) were added and incubated at 37 'C in a CO2 incubaror for 72 hrs. 50 pL cold 20

70 trichloroacetic acid was used to fix the cells. TCA was removed from cells and

cells *ere washed wi& tap water 4 times. After drying 50 prl of 0.4 % SRB in I %acetic acid was used to stain cells for 30 min at room temperature. I o/o acetic acid was

used to $ash wells and plates were dried ovemight. Then plates were placed on

gyratory shaker for l0 min after adding 200 LrL l0 mM Tris base, pH 10 to solubilize

the bound dye and optical density was measured at 515 nm by using micro plate

reader (Bio-tek). In each case, a zcro-day control was performed by adding an

equivalent number of cells to sixteen wells, incubaling at 37 "C for 30 min, and

pocessing as described above. Percent of cell survival was calculated using the

formula:

O. D. cells + tested samples - O. D. day 0

O. D. cells + 10%DMSo - O. D. day 0x 100

50

Chaptefi

3.2.5 Molecular identification of fungal isolates

Molecular identification of selected endophytic 1irngi was conducted by analyzing the

18S ribosomal DNA (rRNA) sequence using polymerase chain reaction. DNA was

extracted ftom f'ungal mycelia by CTAB method (pa]r,l et a1..2007). A pair ofuniversal primers ITS1 (5'TCC GTA GGT CAA CCT GCG G 3,) (Fermentas) and

lTS4 (5'TCC TCC GCT TAT TGA TAT GC 3') (Fermentas) were used to amplify

the target genes encoding 18s rDNA. PCR was caried out in a programmable

themrocycler (MJ Mini Biorad). The rcaction mixture (25 pL) contained 5 pL ofDNA template, 3 pL of 25 mM MgCl2 100 pM of each dNTp, 25 pM ol each primer

and I U ofTaq DNA polymerase (Fermentas). The amplification conditions involved

preheating at 95 'C for 5 min lbllowed by 35 cycles with a denaturation step at 95 .C

for 30s, annealing step at 55 "C lbr I min and an extension step at 72 "C lbr 1 min,

followed by linal extension at 72 "C lbr 6 min. The purified DNA was sequenced by

Macrogen inc. Korea. The sequences obtained were compared with NCBI database by

using Basic Local Alignment Search Tool (BLAST) of the GenBank

(httpr//\r'ww.ncbi.nlm.nih.sov).

The l8S rRNA gene sequences of fungal isolates were submitted to NCBI ge[e bank

database using Banklt program and the accession numbem were obtained.

Phylogenetic analysis was conducted and phylogenetic trees were constructed using

MEGA4 software (Tamura e/ a/., 2007). The evolutionary history was inlbrred using

the Neighbor-Joining method (Saitou and Nei, 1987). The percentages of replicate

trees in which the associated taxa clustered together in the bootsttap 1est. The tree was

drawn to scale, with branch lengths (next to the branches) in the same units as those

of the evolutionary distances used to infer the phylogenetic trees. The evolutionar)

distances were computed using the maximum compositc likelihood method (Tamura

et a/., 2004) and are in the units ofnumber ofbase substitutions per site. All positions

containing gaps and missing data *ere eliminated from the dataset.

51

(lhapter 3

RESULTS

3.3.1 Isolation of endophytic fungi

A total of fifteen fungal isolates were obtained from dillerent parts (woody parts and

leaves) of ftrras fata. These filieen fungal isolates were selected to work on the

basis of their morphology and in vitro culturing pattern shown in Table 3.3. Among

them l0 were isolated from wood (NFWI, NFW3, NFW4, NFW5, NFW6, NFW7.

NFW8, NFW9, NFWI() and NFW12) and five uere isolated from leaves (NFLI.

NFL2, NFL3. NFL5 and NFL6) [(Fig. 3.2 and 3.3) (enlarged view ol all plate irnages

are given in appendix Aland A2)1. Some of wood isolates showed production ofcoloured metabolites on PDA agar plares. The strains NFWI, NFW3, NFW7, NFWS

and NFW9 produced pink, orange yello$. yellowish brown, dark brown. purplish

pink and greenish yellow pigments respectively. Among leaf isolates, growth was

mostly manifested as whitish colony excepr in NFL2. This isolate initially showed

gro*th as whitish colony but later gave rise to dark green spores. All these srains

were maintained as pure culture on PDA plates for fufther processing (Table 3.3).

3.3.2 Fermentation and extraction

All fifteen strains were cultured initially by using PDA and TM medium. The quantity

of crudc organic extract obtained after solid state fermentation was considered good

enough for l'unher processing (Table 3.4). The weights of crude extract ol strains in

TM and PDA medium range between (1.73-3.76 g). NFW3 showed highest weighr of

J.76 g crude metabolites in TM medium while NFW9 showed highest production of

metabolites 2.89 g PDA medium.

52

o

,li

'&oEBz{

f!

6

H.

d

;E

E

E

o

'E

's

sz

s6

\

Chapter 3

Table 3.3:Plant pafis and morphology ofendophytic fingal isolates of Taxus;t'uana.

S. No Isolation Code Plant/Pafi Morphological fcatures

1. NFWI Wood Pinkish white colony

2. NFWS Wood Peach white colony with dark pink, yellowishmargins later on turns orange yellow

3. NFW4 Wood Otlwhite colony with light pink shades

,1- NFw5 Wood Off white flat colony

5. NFW6 Wood Light brown mat like colony with thread likestructures

6. NFWT wood Peach pink colony later on lurn yellowish brown

7. NFWs Wood Dark purplish pink colony,

8. NFWg Wood Yellow green colony with exudates

9. NFwlo Wood Green colony

10. NFwl2 Wood Colony with \rhite marginal area and brownishcentet

11. NFLI Leaf White colony with mesh like mat

12. NFL2 Leaf Dark green mat like colony

13. NFL3 Leaf White mat like colony

14. NFL5 Leaf Brown colony with thread like structures

15. NFL6 Leaf Flat white colony

55

Chdpler 3

Table 3.4: Weight ofcrude ethyl acetate extract after solid state fermentation.

Weight (g) ofcrude ethyl acetatc extract obtained from 3L mediumStrain code

TM medium PDA medium

NFWI

NFW3

NFW4

NFW5

NFW6

NFWT

NFW8

NFW9

NFW]O

NFW] I

NFW] 2

NFL]

NFL2

NFL]

NFL5

NFL6

2.t0

3.7 6

t.92

2.1 I

2.00

2.50

2.18

2.11

1.85

2.23

1.73

1.87

2.1I

1.98

2.7t

1.91

2.13

2.56

2.07

1.88

2.45

2.64

2.32

2.89

2.63

2.52

2.66

2.21

2.1t

1.93

2.30

55

Chapter 3

3.3.3 Biologicalscreening

Cmde ethyl acetate extacts of fifteen fungal isolates after culturing in both TM and

PDA media were used for initial screening by using a battery of bioassays Thc results

ofinitial scieening are described below.

3.3.3,1 A limicrcbial screening

3-3-3.1.1 Resuhs ol anlibacterial assav

Crude ethyl acetate extracts of all endophytic fungal isolates were tested against 5

bacterial stains. Most of wood isolates showed significant activily against test strains

with zone of inhibition ranging from 9.2-23.2 mm The summa zed results are giren

in Table 3.5. A wood isolate NFW1 showed promising effect against L coli, K

pnu.ono)1i.k, M. luleus and S arreas with zone of inhibition of 16'2' 141' 18'2 and

23.2 mm respectively, \\'hen grown in TM medium This isolate also showed good

activity with PDA sample against these test bacterial strains NFW3 also showed

signiticant activity in TM sample against-E. co1i, K pneumoniac, M luteus and S

.rrrer.r with zone ofinhibition of 15.8'13.9. 15.9and 18.5 mm respectively Sample of

NFW6 (in TM) sho$ed maximum zone of inlibition of 17 8 mm agajl-st M luteus'

NFWS showed more than 16 mm of zone of inhibition against '

coli and S' auteus'

PDA sample of NFW9 showed good activity against E' coli' P' deruginosd, M l teus

and S. aureus with zone of inhibition 13.8, 15.6, 133 and 223 mm respectively'

lsolate NFWI2 and NFL2 were active against M' luteus and S alreas and zone of

inhibition were 12.6, 14.4. 12.1 and 16.4 mm respectively' NFL6 was found to be

active against E- coli and K. pnetutoniue Rest of the isolates expressed no

antibacterial activity in either ofthe tested media Tetracycline used as positive control

showed zone of inhibition ranging ltom 21-24 mm lhese results indicated that wood

isolates showed promising activity and could be explored further for purification of

antibacterial compounds.

3 3 3.1.2 Resuks ofantifunqal assav

Crude ethyl acetate extacts oI endophyes werc lested against 5 fungal stains Wood

isolates showed significant antifungal activity against test fungal stlains The

summarized results are given in Table 3.6. Maximum antifungal activily was expressed

57

Chapter 3

by NFW1 which inhibited the grouth ofall test fungal strains by fbrming clear zone of

i ribition ranging from 9.2-19.7 mm. lt showed 19.7, 16.9, 11.5,9.2 and 14.3 mm of

zone of inJribition against C. albicans, A. Jumigatus, A- Jlarus, A- fiiger and A- terreus

respectively. NFW3 also showed zone ofinhibition of 15.1, 14.2, 9.8, 9.6 and 16.1 mm

against all fungal strains respectively. NFWT also showed zone of inhibition of 14.2

mm against a. a/bic.rrr. NFW9 showed signilicant rcsults when cultured in PDA and

zones of inhibition uere I 5.2, I 3.2 and 16.5 mm against al albicans, A. fitmigatus and

,4. /e/rer.r respeotively. Three ieaves isolates NFLI, NFL2 and NFL6 showed zone of

inhibition of l2.6, 15.9 and 13.1 mm agair,st A. ./umigatus, C. .tlhica s and A. terreus

respectively. Rest ofthe endophytic fungi did not show afltifungal activity against test

stmins. Nystatin was used as positive control showed zone of inhibition ranging 21-27

mm. The overall results of this assay showed that used endophytic fungi had potential

to biosynthesis potent antifungal metabolites especially when cultured in TM medium

except NFW9 which showed better rcsults in PDA medium.

58

Chapler 3

Table 3.5: Antibacterial activites of crude ethyl acetate extracts of endophytic fungal

iso\ates of Taxus fuanq measured as zone of inhibition in mm.

Zone ofinhibition (mm)

Isolatc P.Mlen budettul nruitt!

E. coli

NFWItMPDA

16_2+ 0_2

r3.5 + 0.3

14.1+ 0.4

9.0 = 0.6

+ 18.2:0-5

t3.t + 0.3

23.2-O.2

17.3 + 0.8

NIWSTM

PDA

15.8 + 0.3

13.2+ 0.1

13.9 + 1.0

12.1:r 0.2

15.9+ 0.2

+

18.5 + 0.2

l5.l + 0.6

NFW4TM

PDA

Nt-w5TM

PDA

NFW6TM

PDA

I l.l= 0.5

9.9+t3I1.3 + 0.7

9.5 r 0.6

17.8 r 0.8

15.3 + 0.6

t2.3 + l.0ll0.l + 0.6

NITWTTM

PDA I4.5 + 1.6

12.9 t0.2115 = 0.7

14.3 + 0.4 t6.2+0.214.6 * 1.0

NTW8TM

PDA

I6.l+ 0.2

lt.4 + 0.2

10.,1+ 0.0 16.4 + 0.2

l4.l j 0.4

NFW9TM

PDA

13.2 + 0.4

13.8+ 0.8

1,1.3 + 1.0

15.6 + 0.2

9.9 + 0.2

I t.3 + 0.3

16.l + 0.2

22_3 + 0_2

Nfwr01M

PDA

NI--Wt2TM

PDA

12.6+ 1.0

10.6 + 0.6 t2_8+0.2

NFLlTM

PDA

12.4 - 0.6

8.9 r 0.2

NI]L2TM

PDA

l2.l + 1.0

10.3 - 1.3

t6-4!O-2

t2_3 +O_2

NFL3TM

PDA

NFL5TM

PDA

NFL6TM

PDA

1i.1- 0.1

12-2+ l-]L

t).2 + 0.2

10.I = 0.2

Tc 21.,1 + 0.6 21.6 = 0.8 15.6+ 0.3 22.8 = 0.8 24.1+0.16

All datashowed above is mean ofduplicate or tr;plicate test r standard eror

+ = zone of inhibition less than 8.5, Tc= Tetracycline used as posilive and DMSO as negative control

P. M production medium,

59

Chapter 3

Table3.6:Antifungalactivitesofcrudeethylacetateextractofendophlticfungal

isolates of Taxus Juana measured as zone ofinhibition in mm'

All data showed above is mean ofduplicate or triplicate test + = standard error

+ =zone ofinhibition less than 8.5, Nyslatin used as positive control and DMSo as negalive control'

P. M = produciion medium. -: no zone ofinhibition

Zone ofinhibition (mm)

Testfung strai s

---A. funisttu' I A. llawi I A. niget I A. tcfieusC.

lsolate P.M

NFW]TMPDA

19.? + 0.5

14.8 + 0.,+

16.9 +0.2+

11.5 + 0.5+

9.2 + t.0+

t4.3 + 0.4t2.3 +0.2

NI.'W]TMPDA

l5.l + 0.613.2 + 0.2

14.2 + 1.6

12.3 + L09.8 a 0.3

+

+8.9 + 0.2

I6.1+ 0.2l5.l + 0.6

N l'w4TMPDA

NFW5TMPDA

NIiW6TMPDA

13.4 r 0.2

19.3 + 1.0

11.2 + 0.7

9.6 + 0.6

10.2 + 1.0

+

NFWTTMPDA

14.2 + 0.1

12.6 + 0.210.2 + 0.0 I l.t + 0.2

9.8 + 0.2 +

+

10.2 r 1.0

NFW8TMPDA

NI]W9TMPDA

10.9 + 0.415.2 t 0.2

10.3 + 1.0

t 3.2 + 0.1

+I 1.2 + 1.0

+10.7 + 1.0

13.8 + 0.3

16.5 + 0.2

NFWIOTMPDA

NFWI2TMPDA

10.6 + 0.6+

11.2 + 0.29.2 + 1.0

12.3 + 0.2

t0.l + 1.0

NFLITt\,1

PDA.4 + 0.2

10.2 + 0.7

12.6 + 0.2

11.9 + 0.6I0.1 + 0.7

+

NFL2IM

PDA

10.4 + 1.0+

15.9 + 1.0

12.1+0.2

NFI,JTMPDA

NFL5TMPDA

NFL6TMPDA

I1.1 + 0.2

11.9 r 1.0

Nystalin 22.8 + 0.1 21.2,r 0.8 27 .3 + 1.0 24.8 + 0.2 21.6 + 0.8

60

Chapter j

3 3.3.1.3 Results of hvphae folmotion inhibitiot't IHI I) ossav

A prokaryotic whole cell assay. hyphae formation inhibition (llFl) in Streptunyces

85E. was ulilized as a rapid screen for general serine/threonine kinase inlibitors

(Waters e, al., 2002) Tuo phenotypes were observed liom the HFI assay A clear

zoneofinhibitionindicatedthatthctestedsamplesha\,ecytotoxicelTectu,hilebald

zone showed the presence of protein kinase inhibitors Out of fifteen isolates six

showed significant rcsults in this assay' The summarized results are described in

fable 3.7.'fhe isolates NFWI. NFW3. NFW6 and NFL6 showed clear zone of

inhibition when cullLlred in TM media u'ith values of 25, 16, 28'1 and 19 mm

respectively. However NFW1, NFW3, NFW6, NFWT and NFW9 also showed good

activity after culturing in PDA media with values of l5'2, 16' 20, 14 and 15 mm of

clear zone of inhibition respectively. But NFW9 showed clear as well as bald zone of

15 mm. These results indicated that these isolates could be the promising source of

c),totoxic and anticancer compounds The behavior ofNFWg indicated that thjs strain

may contain cltotoxic as well as protein kinase inhibitors. Staurosporine, a protein

kinase C inlibitor. exhibited a 12.8 mm bald phenotype (20 pg/disk) was used as

positive contlol. ln sum these results indicated that wood isolates could be an

excellent souce ofcytotoxic compounds and protein kinase inhibitors'

61

Cfu)pter 3

Table 3.7: Results of hyphae formation inhibition (HFl) assay of crude ethyl acetate

extracts ofendoph)-tic fungi isolated from wood and leaves of latus '/aaaa'

[solate nameP.M

Zone ofinhibition(mm)

NFW]TMPDA

25 + 0.8

15.2 + 0.2

C

C

NFW3TMPDA

l6 + 0.6

22.5 + 1 .0

C

C

NFW4TMPDA

NFW 5TMPDA

NFW6TMPDA

28.1 + 0.2

20 + 0.6

C

C

\]FW7TMPDA

12.3 + 0.2

l4 i 0.3

C

C

NFW8TMPDA 8r 1.0 C+B

NF\\ 9't-N,l

PDA 15 + 0.6

C+BC+B

NFWIOTMPDA

NFWI2TMPDA

ll+0.7 C+B

NFLITMPDA

N[12TMPDA

NITL3TMPDA

q.2 + 0.2 B

NFL5fM

PDA

NFL6TMPDA

t9 + 0.5 C

Stauosporine 12.8 + 0.2

All data showed above is mean ofduplicale or triplicale test + = standard error

P. M=production m€dium, C= clear zone ofinhibition, B= bald zone ofinhibition

-: no zone ofinhibition

62

3.3.3.2 Cancer Chernoprcventire Assq)s

All crude extracts of endoph)'tic lungi and later on fractions were subjected to a battery

of cancer chemopreventive and cytotoxicity assays- Results of different bioassays are

described below.

3.3.3.2.I Results o/ nucleo factorkapD.t B INFKB) inhibitbn assLl'l

Nuclear factor-kappa B (Nl-KB), an induciblc transcription lbctor plays an important role

in the regulation of apoptosis, cell differentialion, and cell migration is activated by an

activatolTNF.q,TheactivationofNF(Bpromotescellproliferationandsuppresses

apoptosis (Baldwin, 2001; Karin. 2006). Therelbre inhibition of NF(B signaling has

potential role fbr the prevention and treatment of cancer (Aggarwal sl l]l ' 2004; Schupp

et al.,2}Og). Crude eth"vl acetate extracls of fifteen endoph)tes were tested for theil

NFKB inhibition potential. The isolate NFWI showed maximum inhibitory activity in

both samples of l'M and PDA media with 70 inhibition oI99 1 and 99'60 respectively'

While the lc5o value was calculated fbr TM media sample only which was 0 18 pLg/ml-'

The results are summarized in Table 3.8 Total of six samples showed more than 50 70

inhibition at 20 pg/ml concentration. Out of these six samples five isolates NFW3'

NFW7, NFW9, N!'Ll including NFWI showed significant inhibition (more than 55 7o at

20 pg/rnl conc.) with ICio values betwecn 0.18-5.78 pglnl NFW3 showed 66.90 oZ

inhibition in fM medium and ICso value was 0.27 prg/ml. NFW8 showed 63'8 %

inhibition in PDA medium at ICio value of 17.05pg/ml- while NFWT showed 52 50 %

inhibition. Among leaf isolates only one strain NFLl showed excellent inhibition of 72'0

% in TM medium with lcro value of 5.78 pg/ml. Overall result of this assay indicated

that five wood and one leaf isolate could be the potential candidates of NFKB inhibitors

(Table 3.8).

3. 3. 3. 2. 2 Re s uhs ol aromat use inhibi t ion ai sav

Aromatase is a c)'lochrome P450 enzyme complex responsible for the conversion of

androgens to eshogens (Jongen et al., 2005) which can play an important role in many

physiological processes in human body. Esterogens and related hormones also have

Chaprer 3

criticalrolesincertaindiseaseconditions,pafiiculallyinmammaryandendometrial

cancers (Maiti et at., 2OO7). Aromatase inhibitors can block the synthesis of estogens'

$hich in turn can be helpl'ul to reduce the growth of estrogen receptor posilive breast

cancer cells.

Crude ethyl aceiate exhacts of fifteen fungal isolates were used to determine aromatase

inhibitory potential only two isolates showed morc than 50 7o inhibition and are

considered to be active at 20 [g/ml conc. The two isolates NFW3 and NFWq showed

'13.3 Yo and 76.4 % inhibition with ICso values of 12 18 and 10'5 Pg/mL respectivel,v'

Other isolates exhibit 1ow Ieveled aromatase inhibition ranging from 9 to 40 %' These

results sho\\'ed that NFw3 and NFW9 isolate could be potential source of aromatase

inlibitors and needed to explole furthel. The summarized results are given in Table 3'9'

64

C ?,2

3E }E

9: q -^.

e: i 5;A9.t2,=

l4?!>

t. sra,.r: e;:

_2

e

e

I

p

EO

z

ZF

{

.lq-o

trtrll;llllllll q;:;I

U-,rrllllll^+-llll

=oa

.i.:qon6!n -o,.1 --ru--i-^i =-; ",: + I - == - I r- |

J^cI@:6_7zcr.^qo;.'"r:ci Io<; ^:-F^65 as -

=:-^- ^.l?r7ft73aa=====227227222272277Q.9

I

Is

l-

F

9

eu t

OE

;;;al;E 3

N A€ E

s

E

E

a

F

e

I

:rttt-tttttlt

E3

:l-ltttttttttttl

!

4 L - F , n - t-, - d

i iR;x.l /;v/. z s'o3g:9odi3 n6d+--

Eir

09 n n o.:-o9s: ^]rva-l'r'r r- a?3::i 33r =:u ez tK9a;.ieo<i^i -rs+: .-;

-.i

_:s6€1.rq9:_^_-.33j3a.aa2.. z7 / / 27 227 z7 z z

rotluctitn in murine macrophasLfuU-2!7-JJ!ll!14!).

Chdptet 3

3.3.3.2.3 Resltlts ol inhibition ol lipopotvsaccharide &P9'ctcti\)ated nitric oxide (NO)

Nitric oxide (NO), reactive specie is synthesized endogenously by an inducihle nitric

oxide synthase (iNOS) enzyme and is most consistently associated with lumor production

(Nomelini et a1., 2008). A consistent relationship between up-rcgulation of iNOS and

cancers of the prostate, bladder' ovary, oral cavity and oesophagus has been observed'

Thercfbre use of iNOS inhibitors may be a possible chemopreventive strategy (Crowell e/

a/., 2003; Nomelini et dl.,2OOg). Crude ethyl acetate extracts of fifteen fungal isolates

\rere tested for iNOS inhibilion potential. It was obserr"ed that a total of 5 isolates NFwl -

NFW3. NFw6, NFWT and NFLI showed iNOS inhibition greater than 50 vo at 20

prg/ml conc. (Table 3.10) The samples $hich showed 170 % inhibition and had 170 %

cell survival at 20 pg/ml concentration were selected for dose dependence to detemine

IC5o values. L-NMMA was used as positive control in this assay (lC:o : 19 7 pM)'

Among positive strains NFWI, NFW3 and NFLI showed highest activity with %

inhibition of 99.6, 69.7. 84.4 and ICso values of 0.32, 7 .'16 arLd 4 42 pg/m[' respectively'

NFWT also showed 79.73 % inhibition in TM medium. Other strains did not exhibit

positive etfects in this assay. These results indicated thal wood isolates NFWl and NFW3

and one leaf isolate NFLI are promising source of iNOS inhibitors The summarized

results are given in'l'able 3.10.

3 3 3-2-1 Results ofouinone reductase I (ORl) induclion assav

lnduction of quinone reductase I (QRl) enzyme is an example of elevation of phase Il

enzyme levels. In this assay Hepa lclcT (murine hepatoma) cells were used Quinone

reductase I is an enzyme widely distributed in mammalian tissues and can be used as an

indicative enzyme to measure. showjng a larger inducer response (Su ct 4i.. 2004) The

induction of QR1 at the tumor initiation slage for detoxification of chemical carcinogens

is suggestive for cancer prevention potcntial.

In the presenl stLtdy. crude ethyl acetate extacts offi11een endophltic fungal isolates was

tested for QRl induction potential at 20 pg/ml. The summarized results are gilen in

Table 1.11. The rcsuhs showed that a total of 3 samples NFW3' NFWT and NFL1 were

61

Chapter 3

found to be active and lR (induction ratio) values were more than 2 (2 6' 2'5 and 6 9

respectively). These isolates showed 7o survival of 68 1' 7068 and 55 6 and CD

(concentration requircd to double activity) values of 5 54' 049 and 0'21 gg/ml

respectively iD TM medium. Thcse results indicated that these strains have the capability

to induce quinone reductase enzyme and could be explored funher'

6a

9 eP

-UN;

Y c 26

i;Poi+.au;iZv

* i es;

:E;\,

I ; d5 =L=i>

e : er

a : a u-u < aiz

it:

j

I

Cz

O

4F

(,

I I I I I I I I I

!.t+tttl11r11":lllsx d o!

o, .:

a

119.rr1..lvl ".::::- P -l^^_o^e.z/c^4466

r33:33';j S=EEE

:F

".: qo.-^:i -."9:'9: 1.:r. . l | {'^^:- -1??Csii=?=2=='17=3Ra5rE5;5=-= s-

=:----.>>rt7?>1=5-====7772772222,/7zzz

E

il^

cl-

4a*.

=;q:HI de

,!,=, >

ooco9ct9:EaZEi

!.i-6:>{i

.->5EAF{r'nnaz;ian -. e_t E - tI goj==i9-=

&

o

F

rlrllllllllllll.,]

U r rl:rlllllllllfi r+ N; Rc> a

^,a6d-cicic:-o, " i r , - - =+

I

'"=6d 6i635.; a?^- --.ioo-- eo

3>>>>>>->5=---i.772272272722'/77

Ohapter 3

3.3 3.2.5 Results ol DPPH free radical scavensins assav

Various endogenous and exogenous stimuli can cause a seties of cellular and molecular

changes which contribute in cancer development A product of normal cellular

metabolismoxygenfreeradicals(oFR)alsocalledasreactiveoxygenspecies(RoS)can

cause endogenous damage. Antioxidants can either directly scavenge or prevent

genemtion of OFR-/ROS. To determine the free radical scavenging potential of the crude

ethyl acetatc extracts of fif1een endophytic fungal isolates, DPPH assay was used in this

study.

lnitial screening \\'as car ed out at ooncentration of 200 pg/ml and the samples which

showed 2 70 o% scavenging were tested to three fold serial dilution in order to find thejr

lcjo values. Ethyl acetate extracts of 15 fungal isolates cultured in two production media

were used for free radical scavenging activity, among them four extracts NFW3, NFW6,

NFWT and NFwg exhibiled : 70 scavcnging at 200 [g/ml concentration All these

samples NFW3, NFW6, NFWT and NFWS showed 88.8.90.21, 86.69 and 84'38 %

activity rcspectively with IC5o values of (117, 53.92, 30.84 and 50 1). NFw3 was

considered to be most potent free radical scaveDger with IC'0 value of 1 1.7 pglml' These

results suggest that NFW3 could be a source of polent antioxidant compounds Other

strains showed low levels ofactivity ranging 3-34 % (Table 3.12)

3.3,3.3 Results of cytoloxicity ass.E (Sanothodamine B Assa!)

Sulforhodamine B assay, is a rapid, sensitive and inexpensive method the most widely

used methods for ir virro cytotoxicity screening (Skehan el a/., 1990). Al1 fungal samples

were evaluated for their c)'lotoxicity potential by using SRB assay. In SRB assay, fbur

samples showed cytotoxicity with MCF-7 cell culture (1'able 3.13). NFW1 showed %

survival of 0.20 and 16.60 in TM and PDA media samples with IC5i values of 0.56 and

6.82 pg/ml respectively. NFWS and NFW9 showed o/o survival of 30.2 and 45.7 at ICso

values of 12.4 and 17.5 pg/ml respectively in PDA media sample. A leaf isolate NFLI

shor,,,ed o/o survival of 45.7 iD TM media sample with I 1.3 pg/ml IC50 value. Overall

results of cytotoxic activity showed that samples of NFWI, NFWS, NFW9 and NFLI

11

Chapter 3

could be a good source of cytotoxic compounds Other strains showed high 7n suNival

and low levels oftoxicity.

J2

P

6>

-o:-os6 -

:,:

gii g

C= A

q

ttlrrrl:llllll-

U

r: r r:; I I I I I I

c-.?o o=.=.s lo=o:l:l:;:r':-:;r:;erl:;=!izz7'.i9.3;Jg;-:a\; :o loo

r,]

B

--:r.o!.9=a-. --o,3?>-=-ari=:-==zz,/ 7z/ zzzT z'z'z/ zoi.:

F

p

AE;

ET

9u

-rrrrt:-lllllllo9

^*

(,

FO

,.]

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'ttrrttlll-llll

"lE

9!----->>-a>>->r5i==--ZZ7 7 Z 2 Z 2 22 z '/ 7 7 7

E

;;

Chapter j

3.3.4 Molecular identification of the active endophytic fungi

The molecular phylogenetic analyses with closely related taxa revealed the identity of

endophyic fungi. Phylogenetic trees $'ere constructed using MEGA4 software (Tamura

et al., 2001) (F ig. 3.4 - 3.ll).

NFWland NFW3 shains were identilied as Epicoccum nigrdm with Acccssion No'

JX402049.1 and JX838792.1 showing 89 % and 98 % homology with 'picoccum

nigrom

strains PC4-3 and EpNil respectively. Another \\'ood isolate NFWT \\ith Accession No'

JX838793.1 sho\ied 98 oZ homologJ with Epicoccum sp. The isolate NFW5 identified as

Tritarichium sp. (Accession No. JX845570.1) showed 94 7o homology with 'l'ritdrichi m

sp. (Accession No. JXl56380.1). Blast rcsults of NFWS and NFW9 showed 97 and 99 Yo

homology with Penicillium sp. (Accession No. JN226993.1) and Ctukto iun sp

ATT039 (Accession: HQ607810.1) and sequcnces are submitted in genebank to obtain

accession no. The leaf isolate NFL2 was identified as Ttichoderma asperellum

(Accession No. JX838791.1 showed 96 % homology with TrichodermQ asperellum sllain

T77 (Table 3.14).

l5

Chdptet 3

Table 3.14: Percent homology and accession

se{luences of active endophytic fungal isolates'

numbers of the 18S iRNA nucleotide

Closest related sPeciesGenBank accession numberStraitr Codc

98%

94%

94%

98%

99%

91%

96%

Epicoccum nigramPC4-3

[Accession: JX914480.1]

Epicoccum nigram EpNilIAccession: JQ387578. 1]

DFFSCS0I2 [Accession:JX 156380.I l

Mucor hiemalisMHl[Accession: JF]03856. ll

Epicoccun sp. XAE 132

[Accession: JN03] 0l4.llChaetamiun sp. ATT039

lAccession: HQ6078l0.il

PeniciLliun sp. PX201 lalAccession: JN226993. l]

Ttichoderma asperelL m

T77 [Accession:GU176454.r

JX,{020,19.1

JX838792.1

JX845570.1

JX84557t.1

JX838793.l

JX838791.1

NFW]

NFW5

NFW6

N F'W7

NFW8

NFW9

NFL2

16

Epicoccun nignlm isolo? PC+3 11X914...

Epicaccun nigrun stlin NRRL 54519 1H...

Fungal sp. B1B W-2A11 1HM439531.11

Eptct)ccun nigrun isolate tom63 |HM061...

Epicaccun nigrun ttain CLjjlj 11n41...

Epicaccun ni!run tsalate Ny7255a lHM9...

Fungal sp. 0cwWB21 1 5 llQ070 47 B.1 l

Epicoccunsp. C 'B lKCl39499 1I

Epicaccun sp. ttxain HS'1 11F694747.11

Ep i ca c cwn sp. A U CMB n a 12 i B I H Q9 1 4 87 B. 1 I

Unculturcd fungus 1A8520273.11 dane:...

Uncultured endaphyfrc fungus clane lE..

Phosparllet sp italatt |KAT JHFts2AB,.

Epi t)cc un sp N lW7 llXB3B793.1 I

Wcoccum sp, NFWI M02M9,11 )

Fig. 3.4: Phylogenetic tree showing the evolutionary relationship of NFWI isolate with

14 taxa.

71

Chapter 3

Epieoccun nigrun NFw3 gbllX\3\7g2l fEpkaccrn sp. ASR'79 gblcu973662 1I

Epicaccun nigrun tsak? EpNi2 gbllQi...

Dakideamlcetes sp lena\pe 478 isol..

Epicaccun nigtum ttlin E-a00535641 !..

Epicoccun nilnln slrain DAAM 185469 !..

Epica ccun sp. yXN14 gblKc13947 8.1 I

Epic\ccum sp. CHTAM6 gblJF773637 U

Epica ccurn sp. 22MSAI gb llx27 A 580.1 I

Epicoccunnigtu stain cMT2 9b11Q754...

Epica ccun nigrun 9bllN862q0 5.1 I

Epicoccum nigrun isalote EpNil \bllQ3.

Epicaceun nig.li,n st[in I DR1aa1 02 81 Z 7...

Epicoccun tp. CBMAI 1A28 gblCW7A377.1l

Epicaccun nigtun tsalate 2481 !b|8U82...

[picaccun sp. CBMAI 1a29 gbl1Qi7a37q 1l

Epicoccum sp. itollk 5A20 gblFR66795 .

Fig. 3.5: Phylogenetic tree showing the evolutionary relationship of NFW3 isolate with

16 taxa.

18

Chapter 3

Ti ti nchiun sp. j9 gb 18U497945.1 I

Beaatenl alb| 84U18961 9b1U18961.1 I

Ttidmchiun sp. lAM 14522 9b14810976...

Ttitimchium sp. D[FSCSA34 gbl]X15638..

Ckdaspo nu n sp. K N UC255 gb ll N 084020.1 I

Ti ti t0 chi u n sp. isall E LF 562 g b I F R8...

Engyadanaun 1lbum isollte DFFS[5022 . .

Peni ci lli um sp. MTP09 3 gb lH Q8277 86.1 I

Enlladanttunsp. tsahY ATU 058 endo...

F u ng o I tp. BM P29A6 lb lfi Q83297 1.1 I

Ascanfcete sp. 5/97-57 gblAl279474 1l

Tti b tochi u n s p. C2 -Z gb llQT 1 7 341. 1 I

Tntiachiun sp. )UCMBI 101AAB gbvq,

Tntrdchiunsp. 3 gblEU497949.1I

T ritit or/tium sp NFw 5 gUIxA 557 A \ fSardananycezs sp. M60 XS-2012 gbllx..

Engladantiun albun shlin NRRL 2312 9..

Beautena sp. ATAC sbllQ781827.1l

Fig. 3.6: Phylogenetic tree showing the evolutionary relationship of NFW5 isolate with

18 taxa.

/9

Chuptet 3

Mucot sp. TM5.2011 vauchet MSjp 50'5 ...

Mucar hiemalis sttain MHZ gbllfia3g,7 1l

Muc ot racenasus lblAll7 6659.2 I

Mucot hienlts stlin FSU6519 gbl|Q22,.

Mucar hiemolis strain MH4 gbllfiA3872.1l

Mucot hiendlis isallte UASWSA442 ghlq...

Mucar hienlils iokte Mj2I gbllQ6832.'

Mucat hiernlhs stdin MH3 gbllFiA3B|g.ll

Mucot hiemals isalo? XSLI 98 gblEu3z,.

Mucor sp. 1523 A pb\ 861 41 3.1 1

Mucat hienlhs siIin KACC 46A74 gbll..

Mucot sp. 11MA02 !UN7A$7.11

Mucar hienllis f. coticala sttain KC. .

Mucor hienllis NFw6 gbllx845571,1l )Mucot hienalis stioin MH1 gblll303q56.1l

Fig. 3.7: Phylogenetic tlee showing the evolutionary relationship ofNFW6 isolate with

14 closelv related taxa.

80

Chupter 3

Epicaccun nignnn isalate A28N gbllQ71. .

tpicaecun sp. PV Wi 57e gblEU74A401 1l

Ep icacc un sp. 0UCMB11 a 12 40 gbl H Q9 1 487...

Epico ccun nlg rrn gb 1fi4a219 2.1 I

[pica ccun ni g run ob ]1/,40219 1.1 I

Epi ca ccun sp I gb I F17881 33.1 I

Epicaccun nigrum isolaL HLJ9 gbllN]..

Epicoccun nigrum isoll? A30C gbllQ7g. .

Epicoccum sp. C0nS.45 gbll F817322.1 I

Epic1ccun nigrun )salItc Hbs K04 tblq...

Epicaccunnigrunstrain P163 D2 36 9b...

Epieaecum nigtun isallte A12C ghllQ78..

Epicaccum ni!run isolate UASWSA679 9b...

tpicaccum cf . nigru n T MBW a2 gb I lQ67 6..

Unculturcd Epicaccun clone SW 2d Ca9..

tpicaccum cf. niqtun BXln2 gbllQ6762...

Epicocumsp isol e NM7 ghllx838793.1l )Epic occum sp. XAE 1 3 2 0b I I N 03 1 0 14.1 I

F un!01 enda ph! u sp APl 53 gb I F M20 0 45...

Epicoceun nigrun isalo? 2AA2 !b11Q76...

lungal endaphtte sp. AP485 !b1FM20067,.

Ep tcocc um nlgrum isa l0 te A H7 lbl H M 59 5..

Fig. 3.8: Phylogenetic tree showing the evolutionary relationship of NFWT isolate with

2l closely related taxa.

81

Chapter 3

Poec ilo myces sp. A1'10j7 g b lH Q607 808. 1 I

Pdecik nyces sp. TMS-2011 gblHQ631041.1l

So rdl riales sp. A P-2 A 12 stn i n ThN MAq...

Chaeto ni u n sp. EF 12 g b lA QI 7 62 7 9. 1 I

Chaetnniun sp. R01 gblCU2A7839.1l

ChIetoniun sp EF7 gblGQ17627A.1l

Cho eto mi un sp. CAL| gb ll F 681 94 5. 1 I

Choeto ni u n sp. ATT10 6 g b I H Q607 837. 1 I

Chaetnmi u m sp. Anljg g b I HQ607 8a9. 1 I

Chaetanium strunoiun stuin 11:22 gb1. .

Chaetaniun luEun gbllN 5640A4.11

Achaeta niun stufiIiL1n stroin SC lAtr9...

Ac hle b ni u n stuna i u n stro i n SG LAll 9...

Achoett ni u m sttunori u n g b I Ay681 2 04 1 I

Chaeto mi un s p. tN B I 2 -2 60 emb l A l 62 A...

Cha eto ni un sp. Anfig gb I H Q607 81 0. 1 |

a NFwBrrs4 )

sho*ing the evolutionary relationship of NFWS isolate withFig. 3.9r Phylogenetic tree

17 closely related taxa.

a2

Chapter 3

Penic illiun ca nnune llx1i97 A3. 1l sto.,.

Penici iun sp.WZLAAl1 IfiA29066.11 t...

Pen i cillj um sp. PX.Z A11a ll N22 6993. 1 1...

Pen I c i llium crustosun I I N22697 i. 1 I it..

Penx i lli u tn c tustasun ll N2 269 35.11 is...

Penicilliun cannune llN9B67 56U strl...

Penicilliun sp, Nfl4l9llsl1BJ rrrv, ra.. ]Eu pe n t c i lli un crust\c eun lH F !4639 1.1 I .

Pe n i cilli un chry sogenum ll N851002.1 1...

Penicilkum c hrysagenun ll N22 69 64,U...

Pen t c ill um ch rysal en un llN9867 60.1 1...

Pent c i lli u n ch rysa gen un lH F546389. 1 ls...

Penl c illiun sp. PX-2011 b ll N226982.1 1...

Fig. 3.10: Phylogenetic tree showing the evolutionary relationships ofNFW9 isolate with

l2 closely related taxa.

83

Chaptet 3

Tnchodemo asperc umstainn9 gbl...

Tichaderna aspetellLn saain 7128H 9...

fichadenno ospere un isalltt NBAII...

Tnchodenno aspercllun stain T5 gblc ..

Tichodem1 aspercllun isllate lARl M..

hi cha derma aspercllu n i salaz Th-CAR...

Tnchaderno ospere um strlin UTP.16 ..

H),pacrca koningii stain 1i6L gblAU17..

Tnchademo Isperellun strain yS-27 9...

thchodemo sp. TRS060186 gblJ a4974.1I

hi cho de mo sp TRH 0 61 61 I gbll F 3A4971.1 I

Fungol sp. enrichnentcularc clone 2...

Tnchadermo sinensis tiain n gblGUl. .

Ttichadenna osperelun stoin n7 gbl. .

Trichoderma asryellun isolateNFL2 g,,. )Tichaderna atperellun siain f164 9b...

Trichodemo ospere un strain na !b1...

Tnchodemo vinde stoin Ts1 gblCU17...

Fig. 3.I 1: Phylogenetic hee showing the evolutionary relationships of NFL2 isolate with

17 closely related taxa.

84

3.1 CONCLUSION

This study provides promising scientilic information aboul newly isolated endophlticfungi from Taxus .fuana medicinal plant of pakistan. Detemination of chemopreventive

potential of endophltes of Tarrs plant from pakistan is done for the first time. This part

of initial screening gives preliminary information about biological potential of secondary

metabolites of fungi cultured in two different media.

Comparison of all tested samples for their biological activities has led to the conclusion

that most ofthe isolates showed better results u'hen cultured in TM media like NFWI.NFW3, NfW6 and NFLI. These strains were active in multiple assays. Only few strains

showed better results iD PDA media.

On the basis of these results four fungal isolates (NFWl, NFW3, NFWS and NFLI) were

considered promising candidates lbr further work to isolate bioactive compounds. NFW3

exhact of TM media showed 11.7, 7 .76, 12.18 and 0.27 pglml- IC56 values in DppH,

iNOS. aromatase and NFrB assay respectively but showing IR value of2.6 in eRl assay.

NFW9 extract of PDA medium showcd 5.37, 10.5. 50.1 and 17.5 pgmL IC56 values in

N!'r<B, aromatase, DPPH and SRB assay respectively. Therefore in present study

initially two sijains Epicoccum,ig,. ,, NFW3 and Penicillium sp. NFW9 were selected

to isolates c)-totoxic and cancer chemopreventive compounds.

85

ChaPler 4

Chapter 4

Isolution and Biological Evuluation of

Cuncer Chemopreventive and Cytotoxic

Compo ands from Selected Endophytes

85

Chdpter I

,t.l INTRODUCTION

Natural products have been recognized as one of the promising source for antitumor

compounds. A substantial amount ofresearch into cltotoxic natural products has been

caried out in the last 50 years, and significant advances in cancer treatment have been

made (Rocha er a1., 2001).

The role of endophltes in anticancer, chemopreventive drug discovery is

tremendously increased, after the discovery of taxol producing endophytic fungus

Taxomyces arulreanae (Strobel et at-, l9()3) isolated from Taxus brevifolia- Laler on

many scientists reported cytotoxic and anticancer compounds from endophltes ol

z,rr"t plants (Wu el a/., 2013; Wang and l ang 201 1; Zhou ea ai ' 2009)'

Molecular identification of fungal isolate indicated that three isolates (NFWl, NFW]

and NFWT) belonging to genus Eprcoccam showed promising activities in bioassays.

The genus Epicoccum have been studied lbr their natunl products such as

epicorazines A and B, epirodin and triornicin indicating membcrs of the genus

Epicoccumha're ahighly developcd and diverse secondary metabolism (Wright er a/',

2003). Other strains b elongto Pe icillium, Mucor. Chaelonium ardTrichoderma sp'

On the basis ofconclusion drawn liom previous chapter two fullgal slrains Epicoccum

nigraz NFW3 alrd I'}enicittium sp.NFWS were selected for cultivation on large scale'

purilication of cytotoxic and cancer chemoprcventive compounds. Crude ethyl acetate

extract of NFW3 and NFW9 affcr cultuing in modified taxol medium (TM) and

potato dextrose agar (PDA) medium respectively \\'as used. Va ous separation

methods, chromatographic techniques as well as bioassays were applied to isolate and

purily required chemical entity from crude mixture ofmetabolites. It was intended 1()

perform bioassay-guided isolation, using the same cytotoxicity-guided fractionation

lbr the screening but r;mdom isolation was done from aclive fractions, rather than

st ctly following bioassay-guided isolation.

87

Chapter I

,I.2 MATERIAL AND METHODS

,1.2.1 Bioassays

In the present study, we employed the approach of bioassay-guided fractionation and

random pu fication from biologically active fractions. Fractions and pure isolates

(compounds) were evalualed fbr their biological activities in cancer chemoprevention

and c)totoxicity assays. Following bioassays were used (NFKB) and (iNOS) assay

lHaq et a1.,2012). DPPH assays (Lee et al.. 1998) and SRB assay (Haq et ol.,2012)

and complete proccdures were described in chapter 3.

4.2.2 Cultivation and extraction of secondary metabolites from Epicoccum

,rigrtrz NFW3 and PerricrTlizrn sp, NFW9

Cultivation of Epicoccum rigrrn NFW3 and Penicillium sp. NFW9 was carricd out

using 50 L of modified taxol (TM) and 40 L of PDA media rcspecrively to obtain

cmde extract. After 21 days of incubation at 25 'C fungal growth along with agar was

blended with ethyl acetate ( I000 mL solvent /1000 mL medium) and left ovemight on

shaker in each case. Al'ter 24 hrs organic layer was removed and filtered. This

extraction process was done three times with equal volumes of ethyl acetate. Filtrate

was dried in rotary evaporator. Crude extract 70 and 40 g was obtained in case ofNFW3 and NF'W9 respectively and stored at -20 oC for further processing

respectively (Fig. 4. l).

88

Chapter 4

,'/-".-.Gt

,2--<'-Nfll!

+ L.rrdr&.!rd4F <-.r25.C

Itr@l..&rn-.dd.tndtEr. t.H dlt&.6.

Oeo t rdri : I00 rL x3..rEr. r!&i4

I016ltl ntrdtg OE tuB.*rdr t dtErc rh.n

xF&9aq,tq{r.ab

IIW9 (40 OlT\v3 O0 t)

Fig. 4.1: Schematic represe[tation of prepaxatio[ of crude extact of Epicoccum

hi grum l{FW 3 alnd P eni ci I li um sp. l{FW 9.

89

Chaptet I

.1.2.3 Fractionation and purilication of crude extract ol Epicoccum nigrum

NFW3

4,2.3.1 Solvett-solte l extraction ofct de extruct

Cnlde extract was partitioned initially by using immiscible organic solvents described

in Fig.4.2. Briefly, 70 g ofNFWl crude extract was suspended in 1000 mL ol 10 %

methanol. Then the suspension was extracted dlree times with n-hexane (1000 mL x

3) using a separatory funnel. The n-hcxane layer was dried in rotary evaporator at 35

"C and 12.5 g of nhexane fraction (NFW3H) was obtained. Methanolic layer was

again extmcted with ethyl acetate threc times similarly to obtain ethyl acelate ftaction

40 g (NFW3E). The remaining methanolic lraclion was also dried in rotary evapomtor

at 40 'C and 14.5 g ofmethanolic fraction (NFWIM) \l'as oblained. All sub fractions

of NFW3 (NFW3H, NFW3E, NFWM) uere dissolved in DMSO at concentmtion of 4

mg/ml and were subjected to bioassays. On the basis of bioassay results, NFW3H

and \fwlE \\ere selecteJ lor lunher proce.sing.

1.2. 3. l. I Normal ohase column chromutffi

NFW3I'I fraction (12.5 g) was dissolved in a solvent system of nHex: CHCI3/1:l and

was adsorbed on silica gel 60 (70-230 mesh. Merck. Germany) in the ratio of 1 g

sample on 1.5 g silica. Sample was loaded on the top ofglass column tilled *ith silica

gel 60 (230-400 mesh, Merck, Getmany) along with protective layer ofsilica gel 1.5

cm. The column loaded with NFW3H was eluted with gradient change in mobile

phasc; starting with 100 7o nhexane to 100 % CHCIr and then CHCI3: MeOH /l:1. A

total of 90 fractions (50 mL each) were collected. Schematic representation of

fractionation ofhexane and ethyl acetate portions is given in Fig. 4.2. All fractions of

NFW3H were subjected to normal phase J]LC analysis. Total 18 liactions were

obtained after combining them on the basis of TLC analysis (those having same types

of spots and same Rf values were combined) liom NHW3HI to NFw3H18 an.l used

ibr bioassays (Table 4.1).

1.2.3. L2 Normul phase column chromatoqrdDhy ol NFW3E fraclion

The sub ftaction NFW3E (40 g) *as dissolved in a solvent system of EA: MeOH /1:1

and was adsorbed on silica gel 60 (70-230 mesh. Merck, Cermany) in the mtio of I g

90

sample on 1.5 g silica. Then this prepared sample was loaded on the top of glass

column fil1ed with 600 g of silica gel 60 (230-400 mesh, Merck, Cermary). The

sample was fractionated using a stepwise elution with EA: MeOH/100:1_l:1. Total 85

fractions (150 mL each) were collected and dried in rotary evaporator at j5 oC.

Schematic represemation is given in Fig.4.2. All fractions ofNFW3E were subjected

to normal phase TLC analysis and finally I5 fractions alier combination were used for

bioassays. Table ,1.2 showed combination offtactions.

91

Chaptel 4

c-\'Elr3E)12.5 s

oiFNsfDl:,.5 g

E6}l rcdn t c-tio!6Tr15D

zog

::E::::- _ _ __- - - r - --- -- - - - -- -- -l-- --,I rrossrvs I i

---I- ---- -----v--------------+----'EieU!'.dn e Eigl'b r.rnc

f.'-*,:.ffi*-"-lI ctrco.t"gnptr II siuo r.160 I

I Oo-230 6.'i) |

TI .-*--.r";-lI t"ecttors I

rllv ___________+____,

:::::":::"_1'::"1::"_"_"_::::':::"-1i::'l

Fig. 4.2: Sohsmatic rqresentation of fractionatiotr of clude eatract of Epicoccam

zr'gnra NFW3.

)iod.l ph.r. .olud

3ilio tel60(70-230 Derh)

92

Chdpler I

Table 4.1: Combination scheme ol NFW3H fractions prepared by normal phase

column chromatography on the basis ofTLC analysis

Combination scheme of NFW3H fractions

Fractions combiued

NI-Wt H I

NFW3H2

NFW3H]

NFWJH4

NfwlH5

NFW3H6

NI]W3 H7

NFW]H8

NFW]H9

NFW]H1()

NFW]HI ]

NFW3H12

N FW3I I1 ]NFW]H I,+

NFW]III5

NFW3Hi 6

NFW]HI7

NFW3H] 8

1.1 0

0.30

0.42

0.31

0.58

0.12

0.45

0.81

0.3 5

0.25

0.42.

0.85

2.O2

0.42

0.50

0.70

0.80

t.l0

Fraction (l-5)

Fraction (6- 12)

Fraction (13 17)

Fraction (l8-19)

lraction (20-22)

Fraction (23-26)

Fraction (27-33)

Fraction (34-36)

Fraction (37-40)

Fraction (,11)

Fraction (42-44)

traction (45-48)

Fraction (49-55)

Fraclion (56-61)

Fraction (62-64)

Fraction (65-72)

Fraction (73-83)

Fraction (84-90)

I

2

l4

5

6

7

8

9

l0

t2

I3

l4

15

16

17

18

Chaplet I

Table ,1.2: Combination scheme of NFW3E liactions

column chromatography on the basis ofTLC analysis'

prepared by normai Phase

Combination scheme of NFW3E frnctions

Name of fractionWeisht (g)Fractions combincd

NFW]E1

NFW3E2

NFW3E]

NF'WJE4

NFW]E5

NFW3E6

NFW3E7

NFW3E8

NFW3E9

NFW3ElO

NFW3E I ]

NFW3E] 2

NFW]EI ]NFW]E],I

NFW]EI5

1.32

1.20

1.93

0.90

l.l01.50

0.90

1.50

3.10

6.50

3.12

4.02

i.0l1.45

Fraction (l-5)

Fraction ( 6-12)

Fraction ( I l-17)

Fraction ( 18-i9)

FractioD ( 20-23)

Fraction ( 24-26)

Fraction ( 27-30)

Fraction ( ll-33)

Fraction (3,+-40)

Fraction ( 41-47)

Fractions ( 48-55)

Fractions ( 56-59)

Fractions ( 60-67)

Fractions ( 68-75)

Fractions ( 76-85)

I

2

5

6

1

8

9

10

llt2

t3

I4

l5

94

Chdptet I

4.2.3.2 Purirtcation of conpouxds Jrcn' selected fisctians NFW3H13' NF\Y3E9

antl NFll3El l

Chromatographic processing of NFW3 crude extract followed by bioassays resrrlted in

few significant fractions which can be used further for isolation ol bioactive

compounds. Therefore above mentioned thrce fractions which showed biological

activity were selected curently to isolate pure compounds'

1.2.3.2.I Isoldtion .tnd Dutilication of NFW3H 13-] -Fdtina comDoul1d

One of the selectcd fractions NFW3Hll was subjected to normal phase column

chromatogmphy (silica gel 60,230-400 mesh) using mobile phase nHex: CHCh/10:l

- 0:1. A tolal of 26 tiactions (100 ml, each) were collected and combined again to get

fractions A, B. C and D. From these fractions, fraction C was fullher fractionated by

using normal phase silica gel 60 (230-'100 mesh) eluted with nHex: Cl-lclr/3:1-0: I )'

A total of 12 fractions (150 mL each) were collected. Fractions 6-7 were comhined

and fractionated by using medium prcssure liquid chromatography (MPLC, silica gel

60, 18-25 pm) eluted with nHex: CI-ICIr/3:1-0:1. A total of 18 fractions were

obtained. Crystals were fomed in vials of fractions 12 and 13 These crystals were

washed and labeled as NFW3H 13- l-Fatima. A schematic rcpresentation is given in

Fig.4.3.

95

Fig. 4.3: Schematic rcpresentation of isolation and purification of compoutrd

NFW3Hl3-1-Fatima.

96

M',lv3El3Cot@ choorlogr.PltY

Si[.. grl 60 (23G{00 nes!)

[E r : CHCL r: l0l -0126 (A,3,c,D) hclim

Siica gel $ 010-.ltl0 mcsh)

nHer : CHCIj :: 3l -0112 Facd)os

NFW3II13-lhtimrcoopo!!d

\Ieshin! ofd\slalsFnclioo 12. 13

Cry$,niz.d

Chdpter I

1.2.3. 2. 2 Isolation ttntl rturification r'tf N FW3 E9E-Futima comoound

oneoflhesrrbfractionsofNFw3E'NFW3E9(5.50g)wassubjectedtonormalphase

column chromatography (silica gel 60,230-400 mesh) using mobile phase EA:

MeOLI/IO:l-0:1. Total 36 l'ractions (100 mL each) were obtained and combined after

TLC analysis to get fractions A, B, C and D From these ftactions' ftaction C was

further processed by using normal phase silica gel 60 eluted with EA: MeOH/5:1-0:1

and a total of32 fractions ( 100 mL each) were collected and combined again to get A'

B, C, D, E and F fractions. Fraction E was subjected to gel illtration column

chromatography (Sephadex LH20) using 100 % MeOH as mobile phase' and a total of

18 fractions of 10 mL each were collected Out of these fractions, fractions 12 and 13

were combined and again 'fhese combined fractions were again subjected to column

chromatography by using Sephadex LI120 using 100 % MeOH and a total 01 20

fiactions (2 mL each) were collected. Fractions 9 and 10 wele again combined and

eluted with 100 % MeoH by using Sephadex LH20 column and total l0 fractions

obtained. Fraction I was found to have a single dragendorffs positive spot after TLC

analysis and labeled as NFW3E9E- l-Fatima. Schematic reprcsentation is giren in

Fig.4.4.

1.2.3.2.3 Isolatio and purifictttion ol NFW3El1C compg!!)!!

One of the sub fractions. obtained liom NFW3E normal phase column

cfuomatography, NFW3El1 (65 g) nas again subjected to normal phase column

chromatography (silica gel 60, 230-400 mesh) using mobile phase EA: MeOH/)0:l-

0:1. Total 46 fractions (150 mL each) were obtained and combined again to get

fractions A, B, C, D, E. F- and G. Fraotion C and G contained dragendorffs positive

spots (indicating presence ol alkaloids) were selected for compound isolation'

Fraction C was further fractionated by using normal phase silica gel 60 (230-400

mesh ) eluted with EA: MeOH/ 5:l-0:1 and total 12 liactions (100 mL each) were

collected and t'ractions 6-7 \!ere combined and subjected to gel filtration column

chromatography (Sephadex LH20 ) using 100 % MeOH as mobile phase and a total of

40 fractions of2 mL each $ere collected. Out ofthese l'ractions, vials of fraction l0

and I I were found to have a similar and single dragendorlls positive spot. Both vials

were then mixed and labeled as NFw3EllC. Schematic representation is gi\en in

Fig.4.5.

9t

NFW3E9Column cbronlliognphy

slhr 8el 60 (230-|00 o.sh)EA: M€oH :: lol -0:l

( A, B. c. D) fraclio$_

Fnctbr 12. 13

CC s.phr&i LH20MeOH

Fig. 4.4: Schematic represetrtation ofNFW3E9E-l-Fatima.

isolation and purifioation of comPound

Frrclbo 9, r0cc s.phlder Lg20

M.Olt!0 iictbtrs

98

CC S€ph.dcx LH20M€OH

sho$ed sbgle spolaff€r

NFW3E9E-l-frtlDrco@ourd

Chapter 4

Fig. 4.5: Scheruatic representation of isolation and pulification of compound

NFW3E1lC.

99

Nrw3E11coluon ckooalog4try

Siha gel 60 (21G100 mesb)

EA:MeoE:: l0l - 0:l

frrctio! C

\TV3E1ICCompound

Fractiotr 11,12

containiu siule slot

ftrcdor G7CC Sepha&i tfi20

MeoH

Purity checkcd by

lLC

Chapter I

4.2.;l Fractionation and purification ofcrude extract ofPenicil/lrz sp. NFWS

4.2.4.1 Nofttal phase column chruhatography of crude extldct ofNFW9

'l'he crLrde ethyl acctate extract ol Penicilliunt sp. NFWg \las funhcr processed by

using normalphase column chromatography using silica gel 60 (70-230 mesh, Merck.

Germany). The loaded column with NFW9 sample was eluted with gradient change in

mobile phase; starting liom n-Hex: EA/l00:1-1:100 then with EA: MeOH/I00:1-l:1.

A total 01 92 fractions (150 mL each) were collected and d ed in rotary evaporate at

35 'C. Schematic representation is given in Fig.4.6. These ftactions were subjected to

normal phase TLC for analysis and combined into 7 fractions (A-G) were then used

for bioassa-vs as shown in Table 4.3. Out of all eight fractions only ofle liaclion

NFW9C showed moderate activity as well as contains similar types of spots with

different ,l?l values when observed by after TLC analysis stained with 10 % sulphuric

acid.

100

Chapter 4

Cmde ertr.clliIW9 ({0 g)

,t

/<il;;;\I r___,*__--ZI N"-"t rt"- I| .ohDr I I Eluted rith tr.EerlDe-fttll rl

| .t r.t"u.*l l'--'+ | o.n ritr Etrlt.cehter| ----, I

i EroAssAvs i

Fig. 4.6: Scheme for prepamtion of fractions of Penicillium sp. NFW9 by usingnomal phase columa cbromatography.

M,C ['OR. COMtsINAI]ION OF LIKE TR.ACTTONS

Eluted ritl &EeDDe-Ett]t rcettte (100:l to l:100) lndtt.I ri& Ettlt.cehte-UeoE (100:l to l:1)

Totd 92 fnctiors (150 Dl e.c[) collected rld &ied irrot:r!' errpontor .t 35 oC

101

Chapter 4

Table 4.3: Combination scheme of NFW9 fractions preparcd by normal phase column

chromatography on the basis ofTLC analysis.

S. No.Combination scheme of NFW9 fractions

Fractions combined weishl (g) Fraction ramc

I

2

4

5

6

,7

NFW9(1 I9.)

NFW9(20 32)

NFW9(33-45)

NFW9(46-s9)

NFW9(60-7s)

NFW9(76-82)

NFW9(83-92)

7.00

,1. t0

9.80

3.5 0

3.60

5.50

5.10

NFW9A

NFW9B

NFW9C

NFW9D

NFW9E

NFW9F

NFW9G

fi2

Chdpter I

1.2.1.l.I Normal phase column chromatosraphv ofselected ltaction NFWgC

NFWgC was subjected to normal phase column chromatography. Sample (9.8 g) was

Ioaded on silica gel 60 (70-230 mcsh, Merck. Germany). Glass column filled with

silica gel 60 (230-400 mesh, Merk, Germany) and dried sample along with protective

layer was loaded on the top of filled column. The sample was eluted with nHex:

EA/5rl-0:1 and then EA: MeOH/lr0-1:1. Each fiaction of 100 mL was collected and

dried in rotary evaporator at i5 'C. Total 33 fractions were collected. Schematic

representation is given in Fig.4.7. Fractions were then analyzed by using normal

phase TLC and after combination total seven fractions NFW9C-5 to NFWgC-3j were

obtained and subjected to bioassays as shown in Table 4.,1. Some moderately active

fractions NFW9C-l5, 17, (19+25) and 33 were used for isolation and purification ofcompounds.

103

Nonml phase

coholchoE.rogr.pl].

o-Pcc)

f,hred rith !-EeI..ne-EtS.cet re (5rI to 0:t) .!drl.i rirb Eo$ r..t tc-\t.ox (t:0 to t:l)

Told 33 fndiols (100 sl e&L) coll€cted rrd dded inmt r.r eripor.tor.t 35 oC

\n]9C,l5

Combiratiol after TLC atrrhsis/Bioassa].s

\n\9C,lr- \n\ 9C-25 sn\'9c-33

tLI IItt

i- __---__--_ _ _ -_-__ __

i SuD-frrctio[s frnIer subiect€d to comporld pormcrfor i

Fig.4.7: Schematic rcpresentation of fractionation ofNFWgC by using normal phase

colua:n chromato gr4phy.

704

Table 4.4: Combination scheme of NFW9C fractions prepared by normal phase

column chromatography on the basis ofTLC a-nalysis.

S. No.Combinalior schem( of NF\\ 9C tractions

Fractions combined Weight (g) Name ollractionI

2

3

4

5

6

7

Fraction ( l-5)

Fraction ( 6-15)

Fraction ( l6-17)

Fraction ( l8-19)

Fraction ( 20-25)

Fraction 26

Fraction ( 27-31)

0.30

2.10

t.i0

1.40

L50

0.50

2.10

NFW9C.5

NFWgC-r 5

NFWgC-t7

NFWgC-l9

NFW9C-25

NFW9C-26

NFW9C-33

10s

Chapter l

4.2.4.2 Putirtcation of compounds from selecledfractiors NFW9C-15, NFWqC-17,

N F ll9C-2 5 an rl N Fl/9 C-3 3

After TLC analysis. combination and bioassays four moderalely active fractions were

selected for compound purillcation.

121.2.1 Isolation ond lurifi,dtion of NFW9C-6. NFll/gC-11 dnd NFWqC-15

utmpounds

NFW9C-15 fraction was subjected to normal phase column chromatography (silica

gel 60,230-400) using mobile phase nHex: EA/3:1-0:1. Total 19 fractions (150 mL

each) $ere obtained. From these fractions (8-19) and (1-7) were mixed for further

purification. Fractions 8-19 were fractionated by using gel filtration column

chromatography (Sephadex LH20 ) using 100 % MeOH as mobile phase and total 16

fractions (10 mL each) were obtained. Fractions (7-15) were mixed and eluted by

using silica gel 60 (5-40 pm) \'ith gradient solvenls nHex: EA/l0:1-0:1 and total 20

fractions collected. Fraction 15 was precipitated and then these precipitates were

washed with ethyl acetate and checked for purity and labeled as compound NFWgC-

15. While fractions 8-1 1 were processed by using normal phase HPLC with CHCL:

Meol.i/ 9:l to collect different peaks (at 65 and 90 minutes of retention time) after

detection at shon UV wave length and one peak was checked by TLC and was found

as single spot and labeled NFW9C-11 compound. Initial (l-7) fractions were also

processed separately by using sephadex LH20 with 100 % MeOH and 10 fractions

(20 mL each) were collected. Fractions 5 and 6 were crystallized washed and checked

for purity by TLC and labeled as NFW9C-6. Schematic representation is given in

Fig.4.8.

1.2.1.2.2 Isolation and purification ol NFW9C-17 compound

Another sub liaction ofNFWgC, NFWgC-17 fraction was subjected to normal phase

column chromatogiaphy (silica gel 60,230-,100 mesh) using mobile phase nHexl

EA/3:1- 0:lto EA: MCOH/5:l.Total 20 tiactions (150 ml each) were obtained. From

fiese fractions (8-16) were mixed for further purification by using nomal phase silica

gel 60 (18-25 um) using mobile phase nHex: EA,/l:l- 0:lto EA: MeOH/ 3:1 total 20

fractions (100 mL each) \\,ere collected. Fractions 15-18 were f'ractionated again by

usirg gel filtration column chromatography (Sephadex LH20) \a'ith 100 % MeOH as

106

Chaptet I

mobile phase and total 20 f'racrions (10 mL each) were obtained. Fractions 15. l6 and

17 were crystallized and crystais were washed with MeOH and checked for purity and

labeled as NI-WgC-17 compound. Schematic representation is given iI1 Fig.4.9.

107

Fig. 4.8: Schernatic representation ofisolation and purification ofcompound NFW9C-6, NFW9C-11, NFWgC-15.

108

C.b &@dr+iy (CC)

Siir sd 60 (230100 6cd)

iHslA :: 31 - 0l

rtrenlion llN deteted

MW9C-11CoDpoo!d

Chapter 4

NF1V9C-r7

coDpolrd

\\'asnhg ofo]siat!$itl MeoH rhrte tin s

Fig. 4.9: Schernatic representation ofisolation and pudfication ofcompoutrd NFWgC-t1.

109

Nrw9c-r7C& &ldrdy (CC)

saq d 60exrft..i)ltlalA : ll -01i0 tl-\t!OH (t:l

Fmction 8-16

CC Silica gel 60 (18'2J l]e),rHexfA :r ll- 0lro EA:VeOH (31)

:0 fractio!5

Fnclio[ 15,16,17

crysrrh b iills

Fractioas l5-18CC S€phader tlro

M.OII20 i!.iioos

ChaPter I

1.2.1-2-3 lsotation an l Durilicatiofi ol NFI4/9C-25 compound

Other two sub fractions mixed (NFW9C-19.25) were subjected to normal phase

column chromatography (silica gcl 60.230-400 mesh) using mobi)e phase nHex: EA'i

2:1-0:1 to EA: MeOH/3:1. Total 15 fractions (100 mL each) were obtained. From

these fractions (9-13) were mixed for fufther purificalion by using normal phase silica

gel 60 (5-40 pm) using mobile phase nHex: EA/0.5:1-0:l to EA: MeOH/1:l total 10

Iractions (100 mL each) were collected. Fractions 5-8 were subjected to gel filtration

column chromatography (Sephadex I-H20) using 100 % MeOH as mobile phase and

total 27 fraclions (10 mL each) were obtained. Fractions 25 and 26 were precipitated

and precipitates were washed with ethyl acetate and checked for purity and labeled as

NFW9C-25 compound (Fig.'1. 10).

1.2.1.2.1 ^olation

and putilicatio ofNFIYqC-33 compound

Total l9 fractions (100 mL each) were obtained after normal phase column

chromatography (silica gel 60, 230-400) of NFW9C-33 fraction by using mohile

phase nHex: EA/0.5:1-0:l to EA: MeoH/l: 1. From these fractions (9-16) were mixed

for further purilication by using gel filtration column chromatography (Sephadex

LH20) using 100 % MeOH as mobile phase and 12 fractions were collected.

Fractions 6-7 were lractionated by using RP-MPLC (C18) using MeOH: HzO/30:70.

Total 18 fractions were collected and fractions 12-15 were mixed for further

purification by using gel filtration column chromatography (Sephadex LH20) using

100 % MeOH as mobile phase and total 50 fractions of 2 mL each were collected.

Fractions 33 and 34 were combined and appeared as single spoi on TLC. Schematic

reprcsentation is given in Fig.4.l l.

4.2.5 Sample preparation of pure compounds isolated from Epicoccam fiigtunNFW3 and Peaicil/iaa sp. NFW9 for bioassays

Pue compounds were dissolved in DMSO and Chloroform at conce[tration of 4

mg/ml and sent for biological study again as shown in the Table 4.5.

110

ChaPtet 4

Fig. 4.10: Schenatic rcpresentation of isolation and purification of compound

NFW9C-25.

111

Nrw9c-25CdD droios.p!, (CC)

s&a sd 60Ci0 $nci)!H6tA :: lrl -Olto IAM.OH {3:l)

Fnctio! 9'13

NF!V9C-2s

compoundCC S ica gel60 (5.{0llm)

nH.x:EA ::0.5:l- 0:l

IoEAMeOH (l:l)l0 fmctiors

PrEcipihLd wm cteledfor puity by IlCfncfio! 25 rld 26

Ercipihled

FnctiossCC Sepha&xlH2n

27 tactiou

Chapter 4

NtrW9C33Cotm0 ct omatoF4hy (Cq

SIica g.l m e3G.l6{nash):: 0.51 -qlio E*MeoH

19 Frrclhtrs

RP-MPLC (C l8)McOIlrl:O]30:r0

l8 t?crioDs

Fig. 4.11: Schematic representation of isolation and purification of compoundNFW9C-33.

LL2

Frsctlo! 9-16

CC Sephader LH.lo

MeOH

ll ftactioDs

Fndlors 33,3{coi$in ded.lect d

byrLC

frscdols 12-15

CC SQhrdeilH2oMeOH

50 i'aclicms

Nnv9c,33

compould

Chdpter 4

Table4.5: Sample preparation of pure compounds isolated of NFW3 and NFW9 lbt

bioassays.

S. No Compound code Conc./mL Solvent

I

2

3

1

5

6

7

8

9

NFW3H13

NFW3E9E

NFW3E1lC

NFW9C.6

NFW9C I ]

NFW9C.] 5

NFW9C-17

NFW9C-25

NFW9C-33

4mg

4mg

4mg

4mg

4 'ng

,1 lnc

4mg

4mg

4mg

DMSO

DMSO

DMSO

Chloroform

DMSO

DMSO

DMSO

DMSO

DMSO

113

4.3 RESULTS

4.3.1 Results of cancer chemoprcventive assays of fractiors of Epkoccum

zigram NFW3 strain

Crude elhyl acetate extract of Epicoccum aigla"r NFW3 was first liactionated by

using separating funnel to obtain NFW3H' NFW3E and NFW3M fractions which

were sent for bioassays. The results ofbioassays showed, fraction NFW3H was highly

active in iNOS and cytotoxicity assay with % iniibition and % survival of 99 61 and

0.00 respectively. lhe other two l'ractions NFW3E and NFW3M were also active'

NFwlts showed 78.80 and 8i.75 % inhibitior in NF(B and DPPH assay respectively

as well as 0.00 o% survival in cytotoxicity assay. NFW3M showed 7o inhibition of

66.60 and 76.32 in NF(B and DPPH respectively NFW3M was also active in SRB

assay with 7o survival of 6.30 Results showed that all three lractions are workable to

isolate polent bioactive compounds (Tablc 4.6) lnitially two fractions NFW3H and

NFW3E were processed for compound isolation.

4.3.1.1 Assay re$ults of NFW3Hfrqclions

Total 18 fractions obtained alier column chromatogiaphy of NFW3H were subjected

to bioassays. Results indicated that 16 fractions showed more than 50 % inhibjtion in

NFrB while 4 fractions showed more than 80 % inhibition in iNOS assay. Fracrion

NFW3Hl3 showed signilicant % inhibition of 78.30 and 94.50 in NFrB and jNOS

assays respeclively (Table 4.7). Another fraction NFW3H14 showed 7o inhibjtion of

75.50 and 90.80 in NkB and iNOS assays rcspectively. Fractions NFW3EHI l and

12 showed % inhibition of89.20 and 90 10 in iNOS assays respectively. Fractions

NFW3H3-9 shoued 7o inhibilion of 65.50, 71 20, 72.60, '15 60,76.20, '12 20 afi62.90 in NF(B assay respectively. Three fractions N!'W3H16-18 also showed more

than 65 % inhibition in NFrB assay (Table 4 7). overall results of these assays

indicated that fractions NFW3tl13 and NFW3HI4 could be the first choice for

compound isolation. lnitially fiaction NFW3H13 was used lbr further processing.

1-14

q

=

adal ,

E,=

Z

i Q1;Z

?, c7-o <

",t;r=z.af(r4.*

E E! o reyroaa'i z a

,:E!!>.v.a:-

2r:"tz

t v, !i

": u i 6

<z<;z

,.

E

e

Z

,2

6

Z

.

*

+++l

22,Is

+t ++

U is

++nrl

x

+l

a++

z

t ++lr

+++l

Itr-zzz

I

!

:

= (-)

3:

t2

-z

.a -, E

; (""

a;9

;<=

,-E.

EI

E

,i

'F

z

E12

(

F

i?:i;:?:?;l=!1i:iE;EEEEEEE;;::=!.-E;<

*

..t.t \ !q - 1 ! -.l - d - q I 09 n - q

"-"Tlnr-1,-t'"E5555=oac.3;Cqeqqc.C= 999 :! i = 9 = 9r: y: 33 E 3 5-3:

=: -:: --

= =@ @

-

-t9..9\q 99 ql .-: ---; +.i.j -: -.i -l .+ r;o -.1 -i.rd d'+ + + +l + + + + + +l + + +l ll + + + +

;;;- -i -; o.i .6o o a!rd;eo qj

-;.i, i r r

Chapter 4

4.3.1.2 Assa! rcsults ofNFW3E fiactions

Total l5 ftactions (NFW3E1-NFW3E15) obtained after column chromatography of

NFW3E were subjecled to bioassays. Bioassay results showed that only two fracrions

NFW3E11 and NFW3E9 showed 94.70 and 60.30 % inhibition in NF(B assav

respectively and were selected for compound isolation. Overall results ofall ftactions

are shown in Table 4.8. Rest ofthe ftactions showed % inhibition below 50 and were

considererl inactive. Rest of the fraclions showed % inhibition below 50 and were

considered inactive.

711

:a ,\E}

=i

9

f-

,E€

zi

r9"ja;9

=<a

9€;

P E-5

<zz

e

E

3

I!

z

g

E

=a

F

z

i

*09...1q ci ; - -i -i - ri -r _jr.io-r rlr r-l; .t 09

erir<i5rc-^i^-

z

r6.1 o9 "l ,o ecj.r -.i-i .l _r.i.i.r- -ilonii=oov.r,aa:ac+r;=i+r::=oo

;;;-;-a=-a;;j 311;, s i> r, u

z

z

Chuptet I

4.3.2 Biological activities of fractions ol NFWg sample obtained by normal phase

column chromatograPhY

NFWS fungal strain was cultured in PDA media and crude ethyl acetate extract was

fractionated by using normal phase column chromatography All 8 (A 10 G) fractions

were tested lor biological activity. The results showed that only two fractions NFW9C

and NFWSD were active in PC-3 cell line assay with o; surival of 36 60 and 44 50

respectively. These t\\'o fiactions were also active in DPPH assay 1lith % inhibition of

72.40,82.20 respectively as shown in Tablc 4.9. These two fractions wele considered

lor further processing.

4,3,2.1 Assa! resuhs of NFllgCfructiotts

NFWSC fraction on the basis of previous rcsults was fractionated by using normal

phase column chromatography and combined seven fractions were used lbl bioassays

again. Four indicative assays SRB, DPPH, NFKB and iNOS were perfonned with

these fractions again. The rcsults showed that four liactions NFW9C-15, 17' 19, 25

were active in c)lotoxicity assay in which PC-3 cell line was used and showing 7o

survival 30.90. I1.80. 27.30 and 31.60 respectively These liactions were also actire

in iNOS assay with 71.30, 84.00,78.30 and 48.50 % inlibition respectively' Another

fraction 33 was active in DPPH assav with % inhibition of 85'10 These fractions

\\'ere selected for compound isolation and processed further' The surnmarized results

are given in Table'1.10.

119

.h

.= 6',

-,2.9=

i

-zIF

!.q

9=:-d ?;4

;. ) a S:;

^.26 u .zJ

=a>t9

'r;=-=a c 6 -E

v i a -rE - Fi.

= 7,!33< t- z<z

E

E

,9

z

e

c

F

a

z

qvrq'l:ci o+++++l+:838333=*';ndi@rX;Ao6=

l+ + ! + + + +l; s i s 3 i ?lo ri ^ \ +l.+

A

q.1 cq+++l +

| "1 1q+++

z

a!

Uq

ts

+++rl+++l

<cnuoLi.r!-oai6d'

L:rlJ-rLlLrzzzzzzz

z

,9

€

;

9,:.;a

a' a 2J

u ali

a 9-4O;9'3 5 q=

; z<z

,9

+

4

E

E

E

E

()

Z

I

E

do

F

90-nY.d.j-i^i-n--,; j3a;; E 3 io:-r,-i^;959R;$d:=

"1:?ZZC3;'I;5X?a

J. I o d - o

=+oFi€r5.:d-^id

'a _=

c1=.

aqdia6-F

aeY_;_

-t11:,192.-V?;Y;+--L.c.F.

O

-e=23F9E318'.t 3 y5>B=-.4zzzzzzz

Chapter I

4.3.3 Assay Results ofPure Compounds

The compounds isolated from active fractions of Epicoccum '?igr''' NFW3 fungal

StrainsshowedinhibiloryactivityinN!-(Bassay.outthrcecompoLlndstwo

NFWSE9E-F and NFW3EI lC showed 77 and 80 % NFnB inhibition respectivelv

The ICso value of NFW3EllC compound was 3 5 pg/ml However NFW3HlS-1-F

compould showed least activity. Cltotoxicity potential of these compounds against

diflerent cell lin(s is not delennined )et

Compounds isolated ftom Penicillium sp NFWS also showed potent biological

activity. NFW9C-I1 and NFWSC-15 showed 74 and 68 9 % NFrB inhibition and

ICio values *ere 2.84 and 2.2 ug/ml respectively' Both of these compounds showed

slight cylotoxic activity against HeLa Cells and HT-29 cell lines Two out of other

thee (NFW9C-17, 25, 33) pure compounds of this strain which are apparently

analogr:es showed potent NFKB inhibition. NFWgC-17 and NFW9C-25 showed ?3 73

afi85.12% inhibition and ICl \'alues were 0.2 and 0.72 pg/ml respectively These

two compounds showed potent cytotoxic effects against breast cancer cell line MDA-

MB-231 with similar [Cso value of 0.00372 PM. In case of HeLa cells 61 an 52 To

inhibition was observed with NFW9C-17 and NFW9C-25 compound respectively'

The third analogue NI"W9C-33 showed less than 50 % inhibition in NFrB assay'

Paclitaxel was used as positive control fbr c)'totoxicity assays showed ICio values of

0.0029 and 0.0016 pM against HeLa cells and HT-29 cell lines respectively Overall

results of pure compounds advocated that these compounds are good candidates for

anticancer and chemopreventive drug development (Table 4 1 1).

122

Z

5!

.G

\,=6z

zF

a i=

dE

2.1 a'

-dF -d

i= 3

<2;

E

E

ES

Ei

-i -o

l-Z

+

,-Z

Fz

FAnAA

q.l

,l

U

aaael??s9d59g=ZL=9q

i,-noao!,)arat!'?999 '):>iri-':6-

IU

.l+

FFi;ZZ

F.-t-z

'!

U

*rrl=9;8-i ^od ;i

-d-;o9.1-.nod;;

U

q:E:.i:ze;>?zta;z

dd,idqa33EhITHzzzz

Chapter I

4.1 CONCLUSION

Twostrainswereselectedinitiallylbrstudytoisolatechemopreventiveandc}totoxic

compoulds. In case of NFW3 bioactivity guided isolation was performed to some extent

andfourcompoundswereisolatedbyusingnormalphasecolumnchromatography'gel

filtration column chromatography and HPI'C The compounds isolated from NFW3 strain

uere dragendorffs positive showing that they could be alkaloid in nature chemically'

Ho$evel,sixcompoundswereisolatedfromNFwgrandomlyalongwithlittleindication from biological study by using same isolation methods but the staining

solution which was preferred in this case was 107o sulphuric acid Out of these

compounds NFWSC-I l, 17, 25 and 33 were found to be analogue on the basis of TLC

slaining panern with dil'[ercnt R, values.

All these compounds were sent for biological evaluation as well as structual elucidation

tkough NMR and Mass spectroscopy analysis So l'ar some compounds are evaluated

biologically and showing good activity in cancer chemopreventive and c)totoxicity

assays while some compounds are in process

124

Chapter 5

Chapter 5

C h ur ucterizutio n of I s o late d C o mp o an ds

from Epicoccam nigrum NFW3 and

Penicillium sp. NFW9

725

Chapter 5

5.1 INTRODUCTION

Different chromatographic tcchniques (normal phase column chromatography,

reversed phasc column chromatography. gel filtation column chromatography as well

as HPLC) were used to isolate and purify bioactive compounds flom selected active

fractions. l'hese techniques and methods of isolation enabled us to purify 9

conpounds; 3 from active fractions of Epicoccum nlgraa NFW3 and 6 from

Penicillium sp. NFWg after solid statc fermentation. Bioassays used to evaluate the

cancer chemopleventive and c).totoxic potcntial of thc isolated compounds were

inhibition TNF-0 activated nuclear factor kappa B (NF(B) and cltotoxicity against

HL60. PC-3 and MCI-7 cells proliferation.

Structural elucidation of isolated pure compounds was carried out by using a series of

lD, 2D Nuclear magnetic resonance (NMR) and mass spectroscopy (MS). NMR is

considered to be the most powerlul technique and iirst choice of natural product

chemist for structural elucidation.

5.2 MATERIALS AND METHODS

Bruker AVANCE 400 MHz NMR spectrometer was used under kind supervision of

Dr. Leng Chee Chang, Assistant Professor at the Department of Pharmaceutical

Sciences, College of Pharmacy, University of Hawaii at Hilo, Hilo. USA. Mass

spectroscopy was done *ith kind cooperation of Dr. Philip William at Departmenl of

Chemistry at Manoa campus, University of Hawaii USA. lnteryretation of data was

done in Dr Leng Chee Chang's lab.

5.2,1 Sample preparation for NMR spcctrometry

NMR solvents (DMSO-d6 CDCI3 and MeOH-d) uere used to prepare samples fbr

analysis accordingly. Detail of the quantity of each sample and solvent used for NMR

is given in Table (5.1). lsolation methodology of each compound was previously

described in Chapter 4.

5.2.2 Sample preparation for mass spectrometry

Mass specta were obtail1ed on a Varian LCQ ion-trap mass spectrometer using the

ESI source in the positive and negatjve ion mode. The sample was dissolved in

126

Chapter 5

methanol ancl introduccd into the source by inlusion with a sy nge pump at rate of 5

pllmin. Accurate and high resolution mass spectra for compound uas obtained on a

BioTOF ll fitted with an Analytical Electrospray Source (ESl) mass spectrometer.

The sample was dissolved in mcthanol and introduced in to the source by direct

infusion with a syringe pump at a rate of60 pl-/min.

5.3 RESULTS

All pure compounds were analyzed lbr structure detemination using ID and 2D

experiments including rH, 'tC, uSqC, HMBC, COSY and NOESY. Molccular

weight of the pure compounds was determined by MS spectrum, and high resolution

mass spectra were recorded for a new compound. NMR spectra along with thei'

description are given below according to respective compound.

727

Chapter 5

Table 5.1: Name of the

Epicoccum zigran NFW3,

specffoscopy.

compounds isolated from Pe icilliun sp. NFWg and

solvcnts used and quantity of the compounds 1br NMR

S. No. Compound name Solvent Quanlily

NFW9C-6

NFW9C-l I

NFW9C-15

NFW9C-t 7

NFW9C-25

NFW9C-33

NFW3H13-l -F

NFW3E9E-I-F

NFW3E] ]C

DMSO-d6

CDCI]

DMSO-d6

DMSO-d,

DMSO-dd

MeOD-dr

Pyridine

MeOD-r/r

MeOD-dr

6.0 mg

5.0 mg

8.0 mg

10.0 mg

10.0 mg

7.0 mg

4.2 mg

6.0 mg

10.0 mg

128

1.

2.

3.

4.

5.

6.

7.

8.

9.

Chu7rer 5

5,3.1 Structure elucidation of compounds isolated from Epicoccufi igrum

NFW3 strain

Three compounds isolated from NFW3 strain by using different isolation techniques'

lhe structure eluci<lation of one compound (NFW3H 13-1-Fatima) is still in process

Two compourds NFW3EgE-l-Fatima and NFW3EI lC(10) were characterized lbr

structure and details are given below.

5.3.1.1 Sttuclurc eluci(lation of NFW3EgE-l-Fatima

Compound NFw3EgE \\'as isolated as a light brown solid' lt showed a [M+ll+Na]+

ion at rt/z 471.2987 (C::H+oNNaO, in the llR-ESl-MS spectrum, implying 2

unsaturation in this molecule. NFW3EgE also sho*ed positive reaction with

dmgendrofls reagent such as NFW3El1C(10). All above data suggesting that this

compound contained nitrogen. The rrc NMR spectral data of NFW3E9E (Table 5 2)

displayed 23 carbons including one carbonyl (6C 18 1 .6) From fie 'H Nun datu lFig'

5.1 and Table 5.2) showed also D-marmose sugar unit including at 6H 4 49 (br s, H-

l'). 3.86 GD, H-6b), 3.84 (m, H-2').3.7s (dd' J:6 0, t2.4Hz.H-6a).3 56 (m, H-4')'

3.44 (dd, J = 3.6, '7.6 Hz, H-3') and 3.20 (m, H-5') were similsr to those of

NFW3E1lC(10). The rrc NMR (Fig.5.2) also indicated one anomeric (6C102'5; C-

1'), four oxymethine (6C 79.0r C-5', '/61. C3'.73.4: C-2'and 69'4; C-4') and one

oxymethylere carbons (3C 63.61 C-6'). -fhe

signals of aliphatic chain also observed at

6H 1.24-1.37, located position C-'1 to C-13 (6C 31.4-31 6)' On the basic of HMBC

spectrun (Fig.5.4). sugar unit a1ld aliphatic chain ate linked to each other at 5H 3 93

(m, H-16a) and 3.51 (m, H-l6b) to anomeric 6C 102 5 (C-1). All 'H andrrc NMR

spectrun ofNFW3EgE were very closely related to that ofNFW3El lC(10)' excepted

for the disappearance ofthe signal of tetramic acid moiety to amide moiety because of

2J and 3J HMBC correlation between Me-17 [6H 1.13 (d, J: 8'1 Hz)] and H-2 [6H

2.35 (dd,8.4, 14.8)l with 181.6(C-l)(Fig 5.l-54) Thus, compound NFW3E9E was

ac{igned ,r5 d nen compoLlnd (fig.5 5 )

r29

.!

rI]!!

z

oz

dz

"i

;

dr{$i{

:ILIIIafr

{41

*l

zil

fil[il

i

E

:3

glE1

>zeo

II

zzo

\*tr

('ll

llti

mB-

*INl -

$J

IuddlIJ

f i i.i.i r 6 f F F E i

&.

::

::

1.]:

.. Elpl

-z

:::>

0' ;

-+. I. :="dk

{sio

,

I ,t+afd :{,*f;

iri I

i)o:E

i

e:3;9 s3 P B 3 E: B i ! E E B !

+.st'*#s*

IrL'ddlrj

!

O C!,tt

&i&t

,

o

,<!.

.+0

0

,

@ oo

,-

a

8"

I

s

."'

+ii5,nT;];:i!

q.l

Lll

z

UoU

.-)

=n

Chupler 5

Table 5.2i rH and rrC NMR data (400 MHz. in MeoH-dr) of NFW3EgE-1-Fatima, d

in ppm.

Position 6C 6H (multi., J, Hz) HMBC

I

2

15

16

1',

2',

3',

1',

t1

4-r3

l1

5',

6',^

6',b

181.6 q

4t.5 CH

3 5.8 CH:

3l.4-31.6 CHI

28.0 CH:

3r.6 CHz

71.4 CH1

18.,1 CHr

102.5 cH

73.4 CH

76.1 CH

69.4 CH

79.0 CH

63 .6 CH1

58 (m), 1.29 (m)

L24-) .3 /

1.40 (m)

1.61 (m)

3.93 (m),3.51 (m)

l.l3 (d, J = 8.4 Hz)

4.49 (br s)

3.84 (m)

3 .44 (dd, I = 3 .6. 1 .6 Hz)

3.56 (m)

3.20 (m)

1.75 (dd, J = 6.0, 12.4 Hz),

3.86 (m)

2.3s (dd, J:8.4. 1,1.8 Hz) r 8r.6 (c-1), 3s.8 (C-3), r 8.4(c,17)

r8 r.6 (c-r ), 4l.s (c-2), 31.4(c-1), 18.4 (C-17)

31.6 (C-r l), 71.4 (C-16),31.6 (C,ls)

28.0 (C-r4), 7r.4 (C-16)

28.0 (C-r4), 3 r.6 (C-15),102.5 (c-r )l8 r.6 (c-l ), 11.5 (C-2), 35(c-3)

7 1.4 (C-t 6), 73.4 (C-2'), 7 6. I(c-3),7e.0 (c-s)

r 02.5 (c-r'), 76.1 (c-3),6q.4 G-4'.\

73 .4 (C-2',), 69 .4 (C4',), 19 .0(c-5')

79.0 (C-5',), 63.6 (C-6',)

102.5 (c-l), 76.1 (C-3',),

69.4 (C-4), 63.6 C-6',

69.4 (C,4), 79.0 (C-s)

134

Chapter 5

OH6'1 0H

Ho-N QHoi:^li

Fig.5.5: Proposed structure

epicoccamide analogue.

compound (N-FW3E9E-1-F) identilied asof new

135

Chapter 5

5.3.1.2 Struclurc elucidation of NFW3El lC

Compound NFW3EI lC(10) was obtained as brown coloured viscous oil' and showed

a pseudomolecular ion peak at nJz 580 0 ([M+Na]t' calcd 580 3) in the LR-ESI-MS'

coresponding to an elemental fomula of C:qHsrNOe The llc NMR spectral data of

NFW3E1lC(10) displayed 29 carbons including thee carbonyl (enol form) at dc

176.9 (C-l), 198.0 (C-3) and 202 2 (C-7)' and 'H NMR exhibited '\'-Me at dH 2 91 (H-

6), suggesting this compound contained tetramic acid moiety ' From the rH NMR data

(Fig. 5.6 and Table 5.3) showed a hexose sugar unit at ds 4'49 (br s' H-l')' 3 87 (m'

H-6b), 3.s5 (br d, -r = 2.8 Hz, l1-2'),3.71(dd' I = 6.8, 10 8 Hz. H'6a),3 51 (r' I: 9'6

Hz, H-4'),3.45 (dd, ./ = 2.8, 9 6 Hz, H-3') and 3 20 (m, H-5') The ''CNMRulto

indicated one anomeric (5c1027: C-l')' lbur oxymethine (5c 790; C-5" 762; Cl''

73.4: C-2'and 69.4; C-4') and one oxymethylene carbons (66 63 6; C-6') ThelH and

llc Nw iFig. 5.6 and 5.7) chemical shifts were very similar to those of D-mannose

(Wright el 41., 2003). The signals o1'aliphatic chain also observed at drr 1 26-1 38'

located position C-l0 to C-19 (dc 31.4-31 7). ln the HMBC spectrum of

NFW3EI IC(10), three units are linked to each other at dH 3 52 (m. H-22a), 3 89 (m,

H-22b), 3.85 (br d, "r: 2.8 Llz,H-2).3.20 0n. H-5') to dc 102.7 (C-1), and dn 3 8'l

(m, H-8) and drr 3.5,1(m, H-4) to dc 102.6 (C-2) (Fig 5.9). Furthermore, the lH NMR

spectrum exhibited two methyl groups at d|I 1.2'] (d, J : 8.0 Hz,l.l-5) and 1 02 (m, H-

23). ]-herelbre, the structure of NFW3EI1C(I0) was identified to be a known

compound, epicoccamide (wright el al . 2003), which is composed of three

biosynthetically clistinct subunits; glycosidic, Iatty acid and tetmmic acid (amino acid)

Fig.5.10.

136

r,68

tlit!-,r3rm.:tc35!m.a2a't',g,ts't .

E!59'! IA'9.L Jd.{a'

I

:

))

)

t

I

I

fl

{

t'

U

E]

zo

oz

&z

I

IIE'ClrSl'E0tlIrzttfa

!!lt t5Z!Cf9tvflit9ttSstt

!$5

-O

t!Fz

\ioz

Ie

dzz(-)

a:

J

noF]

z

\o

z

Oda1E

t

E

a

I

sJ

00l' l

&

tI]

z

oz

z

Ua

a

o

it

J

a'hapt€r :

Table 5.3: rH and rrc NMR data (400 MHz, in MeOH-d) of NFW3E11C(10), d in

ppm.

Position 6c 6H (multi., "/, Hz) HMBC

I

2

3

4

6

7

8

9

l0- l9

21

22

23

1',

2',

3',

1',

5',

6',a

6',b

116.9

102.6

198.0

62.9

r 6.5

27.0

4t.0

I5.5

31.4-31.7

27.5

31.4

71.5

I8.5

t02.'/

73.1

16.2

69.4

79.0

63.6

3.54 (m)

1.27 (d,J= 8.0 Hz)

2.91 (br s)

3.8,1(m)

1.26 (m), 1.70 (m)

1.26- 1.t8

1.40 (m)

1.64 (m)

3.52 (m),3.89 (n)

1.02 0n)

4.49 (br s)

1.85 (brd,J= 2.8 Hz)

3 .45 (dd. J = 2.8. 9 .6 Hz)

3.s7 (t, J = 9.6 Hz)

3.20 (m)

3.71 (dd,./ = 6.8, r0.8 Hz),

3.87 (m)

c-1, c-2, c-3, c-5, c-6

c-3, c,4, c-6

c-1, c-4

c-2

c-10, c-1 I

c-r8, c-r9, c-21

c-19,C-20,C-22

c-20, c-21, c- l'

c-7, c-8, c-9

c-1" c-3" c-4'

c-5,, c-6'

c-1" c-3" c-4" c-6'

c-4" c-5'

147

6'1 0H0

3'1

Fig: 5.10: Proposed

epicoccamide.

structure of NFW3E11C(10) compound identified as

142

a'/1r11€r S

5.3.2 Structural clucidation of compounds isolated from Pezicl/iaz sp. NFW9

strain

5.3.2.1 Sttucturc elucidation of NFWgC-l1

Compound NF'W9C-I I was obtained as a yeliow-brownish amoryhous powder. The

high-resolution ESIMS spectrum displayed ion peaks at n'/z ,145.1926 [M+tl] *.

coresponding to the molecular formula C:,rH:sOg.

The LH NMR spectroscopic data (Table 5.4) displayed two meta-coupled aromatic

proton signals at EH 6.29 (br s, H-3') and 6.26 (br s, H-5'), together with two ole{inic

proton signals at 5u 5.53 (dd, J: 17.2, 6.8 Hz, H-10), and 5.80 (m. H-11).

Additionally, resonances lbr one methoxy group at 6H 3.7'7 (4'-OCH1), two methyl

singlets at EH 1.56 (s, H-9) and ilH 2.49 (H-7') and one methyl doublet at 611 1.73 (d, J

: 6.8 Hz, H-12) were also obsened. The rrc NMR and DEPT spectra (Table 5.4)

revealed the presence of one acetyl, four nethyls (with one methoxy), three

methylenes, six methines (\\'ith two oxygenated, two olefinic and two aromatic) and

l0 quaternary (with one oxygenated, two olefinic, four aromatic and firee keto)

carbon atoms inNFWSC-I1. The general features of its'Hand'lC NMR data (Fig.

5.1 1 and Fig. 5.12) suggested the presence 01- azaphilone and methoxylated orsellinic

acid moieties in NFW9C-l1, which closely resembled those of wofimin, an

azaphilone derivative repo(ed by Merlini et ol., (1913).

The correlatioD peaks in the HMBC spectrum (Fig. 5.14) ofNFW9C-I l are listed in

Table 5.4. The tertiary methyl protons at 3H 1.56 (s. H-9) were correlated with the

conjugated carbonyl carbon at 6q 192.0 (C-8), oxygen-bearing carbon at 66 83.7 (C-7)

and the carbon bearing the hydroxyl group at 6c 70.0 (C-6). Co(elation peaks of the

olefinic proton at 611 5.53 (dd, I : 17.2,6.8. H-10), which coupled \r'ith the proton at

6u 5.80 (m, H-l1) were observed with the carbons at 6c 130.5 (C-10), 18.3 (C-12),

and 73.8 (C-3). The oxygenated methine proton at EH 6.18 (dd, J: 10.4,6.4, H-6)

were corelated $,ith the acetyl oarbon at 5c 170.2, oxygen-bearing carbon at 6c 83.7

(C-7). rertiary methyl at 6c 17.5 (C-9), and the methylene carbon at 6c 35.1 (C-5). The

placement of a methoxy group at C-,1' ot' the orsellinic acid moiety was confirmed by

the observed rJ-HMBC corelation from the methoxy protons to C-.1'. On the basis of

thc above evidence, the structure of NFW9C-I 1 was determined $'as 2"-hydroxy-4"-

143

ehnplet :

methoxy-6"-methylbenzoic acid with 6-acetoxy-3,4'5,6,7,8-hexahydro-7-methyl-8-

oxo-3-( 1-propenyl)- I H-2-benzolelpyran-7-yl-ester (Fig. 5.16). It is a known

compound.

144

g

.iO

z

OIJO

I

zz

Ifiln*,t1.{:rkn}a.1

t{rtr'..I-'.:

ltrtI..r

f...r

l--{

ts(

lnia1{'#1t".4rH

.?i;

I

O

z

(,oU

zU:.i

I

s

I

(ls-

tl!!-

7.U-

.g

-i

Q

z

O

U

Ni

:r

.-)c.,

r;

ec I i ? 3:',] 98:!1 xY:(+5. + ? i"ai

:I

TY:l)2E{

I

II

-1*-1

I

_lI

tI

OE

a

:U

z

UoU

i

lr!ddllj

r ? e ? ?.?.t-? ?-l-

a{

a;.rra,

-. !.- ..b " .t

I'a a

a.a

vT

ii

il.,*!*T.

t,O

d0

t.81'10

la.l:8

0 ..i

o

z

OIJ(-)

o(Jiiri

s8ts

]FF<r

-lI

II

* 'sg0?

[TrI

t

€ha7ta :

Table 5.4: rlI and rrc NMR data (400 MHz, in cDCls) of NFw9c-11, d in ppm.

\o. d. dn ("/ in Hz) HMBC

2

3

6

1

8

8a

9

I

5

l0

1l

12

1.,

t;".J'

4',

I :: "','l;

,*

ocoI o.osr,

4a

61.8 CIl,

36.7

3 5.1 CHI

CH

CH:

70.0 cH

83.7 q

r92.0 q

I10.5 q

17.5 CHI

130.5 cH

I29.1 CH

18.3 CH1

105.6 q

166.0 q

99,I CI'I

t64.4 q

5 5.7 q

111.6 CH

143.3 q

24.6 CHI

170.1 q

170.2 q

21.3 CHr

4.32 d (t1 .6)

1.s8 d (17.6)

4.08 br S

2.22 br d (19.2\;2..3'7

dd (19.2, r 0.8)

149.5

2.57 br d (11.6);2.70

dd (16.8,5.6)

6.18 dd (10.1,6.4)

1.56 s

5.53 dd ( r7.2, 6.8)

5.80 m

1.73 d (6.8)

ll.0s6.29 br s

1.77 s

6.26 bt s

2.49 s

2.0/ s

130.s (c-10), 63.8 (c-l)

73.8 (C-3), r49.5 (C-4a)

130.5 (C-r0)

110.5 (C-8a),

73.8 (C-3), 149.5 (C-4a), 130.5 (C-8a)

36.7 (C-4),70.0 (C-6). IJ3.7 (C'7). 14e.5

(Ll-4a), l3 0.5 (C-84)

l5. r (c,s), 83.7 (c-7), r 70.3. r 7.s (c-9)

7o.o (c-6),83.7 (C-7), r92.0 (C-8)

r30.5 (c-10), 129.3 (C-rr), t8.l (c-12),

73.8 (C-3)

130.5 (c-10), r29.3 (C'll)

10s.6 (c-r), 166.0 (c-2),99.1 (C-3',)

24.6 (CH.-6',), 105.6 (C-r),99.1 (C-3',)

164.4 (C,4',)

143.3 (C-6), r 6 (C-s',), 105.6 (C-1',)

150

-hqtcr -;

Fig.5.16: Proposed structure of NFW9C-11 compound idenlified as wortmin (an

azaphilone derivative).

151

€h,tpto !

5,3.2.2 Structure elucfulation of NFWqC-|5

Compound NFW9C-15 was obtained as a red powder. Its molecular formula.

Cr0Hr8Or0, was deduced by LR-ESI-MS (mlz 537.1[M H]., calcd for 537. 0822).

Analysis ofthe 'H and 'lC NMR spectroscopic ciata (Table 5.5) as well as LR-ESI-

MS indicated that compound NFW9C-15 had thc charactedstic signals of the

antluaquinone compounds, and the structure is symmet cal. The 'H and l3C NMR

(Fig. 5.17 and 5.18) spectrum oI NFW9C-15 indicated signals for two phenolic

hydroxyl groups [6] 12.0 (5-OH, s); 6q 161.8 (C-5)] and [6u 12.76 (l-OH, s); 6c

165.3 (C-1)1, three aromatic proton signals at [6s 6.74 (H-4, s); 6c 108.0 (C-4)].7.14

(H-6, d, J =1.5 Hz);6(. 124.0 (C-6) and 7.27 (H-8. d, J = 1.5 Hz); 6c 121.8 (C-8)l and

one methyl at [6H 2.33 (1l-CHr, ,; 5( 22.0 (C-l l)]. The 'rC NMR spectrum (Table

5.5) exhibited 15 carbon signals. consisting ofone methyl carbon at 5c 22.0 (C-ll),

lhree methine carbons at 6c 108.0 (C-4), 124.0 (C-6), 121.8 (C-8), three oxygenated

quatemary carbons at 6c 165.3 (C-l), 165.3 (C-3), 161.8 (C-5), six quaternary carbons

at 6c 123.9 (C-2), 132.3 (C-4a), 109.8 (C-9a), 134.3 (C-8a), 114.0 (C-10a). 149.3 (C-

7) and two carbonyl carbons at 6c 182.8 (C-9), 190.8 (C-10). The mass specha data

suppo{ing it is an anthraquinone dimer (CroHrsOro). HMBC (Fi9.5.20) correlations

liom H-4 (6H 6.74) to 109.8 (C-9a), 123.9 (C-2), 190.8 (C-10), 165.3 (C-3), from H-6

(6H 7.1,1) to 22.0 (CH3-l1), 114.0 (C-l0a), 121.8 (C-8), 161.8 (C-5), liom H-8 (6s

7.27) to 22.0 (CH3-t t), 114.0 (C-l0a), 124.0 (C-6), 182.8 (C-9) and 149.3 (C-7) and

from H-l1 (5H2.33) ro 124.0 (C-6), 149.3 (C-7) and 121.8 (C-8) u'ere observed

(Table 5.5). Therefore, the structure of NFW9C-15 was identified to be a known

compound anthraquinone (Chen et a/., 2011) and structure is given in Fig. 5.22.

\52

O

z

\'oaa

e

z

ri

9lrr9

00rftEItrT

I!.LI oz

fltt I

;

a

z!-t0l0t -flzt'501,tt{0rlI

!!rE'lilts8ftat

I iero-zi

_i

O

\'

lrC

si

o'

4

i-:

:{Jdq r: (!

o.,t!.

II

I

I

I

I

I

J)

I

I

Ij

I

I

I

I

I

-iQ

z

\_o

o

@

=5c.l

tt!l

sti

l.i(.)

,z

o

A

zI

IoOJ..!

5

8

it

t

B

t

h

sr

$

-i

No. ,c .rH (./in Hz) HMBC

I

2

3

4

4^

5

6

7

8

8a

9

9^

10

l0a

1t

r-oH

4-OH

5-OH

7-OH

I65.3 q

123.9 q

I65.3 q

108.0 cH

132.3 q

161.8 q

124.0 cH

149.3 q

l2t.8 cH

134.3 9

182.8 q

109.8 q

190.8 q

114.0 q

22.0 CHI

6.7.+ (1H, s)

7.11(lH,d,J=1.5)

7.27 (l H, d. J =1.0)

2.32 (3H, s)

12.76 ( IH, s)

12.02 (l H, s)

109.8 (C'9a), 121.9 (C-2), 190.8 (C,

l0), 165.3 (c-3)

22.0 (CH1-11), 114.0 (C-10a), 121.8

(c-8), r 61.8 (c-5)

22.0 (CHr,11), 114.0 (C-t0a), 124.0

(c-6), 182.8 (C-9), 119.3 (C-7)

149.3 (C-7), 121.0 (C-6), 121.8 (C-

8)

a'hErct i

rable 5.5: rH and rrc NMR data (400 MHz, in DMSO-d) of NFWSC-15, d in pprrr.

158

€krTtcr S

oo

Fig.5.22: Proposcd structure ofNFW9C-15 compound identified as anthfaquinone.

159

€haptr :'

5.3.2,3 Slruclare elucitlntion of NFll9C-17 compound

The NMR spectra of compounds NFW9C-17 were acquired on a Bruker AVANCE

(400 MHz) NMR spectometer. The following experiments were conducted: lH. l3C,

DEPT, HSQC, HMBC, and COSY. The rH NMR chemical shifts (obtained in

DMSO) and the rrC NMR chemical shifts are lisred in Table 5.6.

Compound NFWgC-I7 was obtained as a white solid with a molecular ion at rrlz

429.1,145 [M + H] + in the HR-ESIMS, corresponding to a molecular formula of

CrH21O8. The rH Nltn 1nig. 5.23) spectrum ofNFWSC-I7 displayed characte stic

signals for two methyl groups at 6H 0.84 (3H, s, CH3-18), and 1.62 (3H. s, CHr-19),

an olefinic proton at 6H 8.91 (1H, s, H-21), an oxygenated methylene at 3H 3.45 (dd, J

: l,9, 11.28 Hz,ll-2oa), 3.10 (m, H-20b), two oxygenated methines at 5rr 4.87 (1H, d,

J =1 1.0 Hz, H-1). 5.94 (1H, t, J : 9.2 Hz. H- I 1), a methoxyl at 3g 3.09 (3H, s, OCH]-

22), and an acetoxyl at 3g 2.09 (3H, s, COCH3).

The 'rC NMR (Fig. 5.24) spectrum of NFWSC-17 revealed 23 carbons: a conjugared

carbonyl (6c 173.3, C-7), a ketone (56 217.3, C-I7), a lactone (6c. 158.7, C-3), seven

quatemary carbons (6c 114.5, C-,1; 6c 143.5, C-5; 3c 145.0, C-6i 6c 141.0, C-8;5c

149.8, C-9; 5c 40.0, C-10: 6c 49.5. C-l3), an oleflnic carbon (5c. 152.9, C-21), four

methylenes (5c 36.4, C-12;6c 23.6, C-15; 5c 36.4, C-l6;6c 73.1, C-20), two teniary

methyls (56 26.5, C-l9; 6c 15.3, C-l8), two oxygenated methines (5c 71.0, C-l1; 66

88.6, C-l), a methoxyl (6c 59.5, C-22), an acetyl group (6c 170.3: 6c 21.7), and a

methine (66 43.6, C-l,l) (Table 5.6). These proton and carbon NMR data were closely

related to those of the knorvn $,ofimannin, suggested the presence of steroidal

furanoid skeleton in NFW9C-l7. The structure of NFWgC-17 was established on the

basis of heteronuclear single quantum coherence (HSQC) and heteronuclear multiple

bond connectivity (HMBC) expe nents (Fig. 5.25 and 5.26). The HSQC spectra

revealed two tertiary methyl groups at [6H 0.84 s)/66 15.3 (CH1-]8) and 5rr 1.62 (s)/6c

26.5 (CHr-19)1, which showed correlation peaks with C-l7lC-l4lC-12 and C-9/C-

l0/C-5/C-l in the HMBC spectum. respectively. In addition, an oxygenated methine

proton at 5H 5.9,1 t (J:9.2) uas observed in the rH NMR spectrum. which showed

t\\,o- and three-bond HMBC correlation peaks with carbons (C-8, C-9, and C-12),

\.hich suggested that the oxygenated methine group was connected to C-11 ofthe

150

€. haVa' S

molecule. Fuihermore, a second oxygenated methine proton at ds 4.87 d (J = 1 l )

was observed in the IH NMR spectrum, which showed two- and three-bond HMBC

correlation peaks with carbons (C-3, C-5, C-10, and C-20), which suggested that the

oxygenated methine group was connected to C-l ofthe molecule. An olefinic singlet

at 5H 8.91, which showed two- and three-bond HMBC conelation peaks with carbons

(C-4 and C-5 the relative stereochemistry ofH-11 and H-l *,as deduced from the

NOESY spectrum ofNFW9C-17, in which corelations berween protons H-1/CHr_lg

indicated that the H-l is at o-configuration). Finally its structue was futherconfirmed bylH,rrc NMR, HSeC and HMBC analyses (Fig. 5.23-5.26) as

wortmamin, and compared with the literatures (Brain e/ a/., 1957).

161

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Table 5.6: rH and

rrc NMR data (400 MH4 in DMSo-r'i6) of NFWSC-I7-pa-71h oct' d

in ppm.

aH (J in Hz)

40.0 (C-10), 1,13.5 (C-5), 1s8.7 (C-l)

141.0 (c-8), 149.8 (C-9),36.4 (C-12),

r70.3 ( oqocH.)

r5.3 (C-18), 4e.5 (C-1i). 71.0 (c-l I )

43.6 (C-14),71.0 (C-11), 149.8 (C-9)

.41.6 (C-r4), 14 r.0 (c-8).217.3 (C- 17)

211 .3 (C-t',I)

36.4 (C-12), 43.6 (C-r4), 49.5 (C-13),

2t1.3 (C-17)

40.0 (c-10), 88.6 (c-l), 143.5 (c-s),

149.8 (C,S)

59.5 (OCHr),88.6 (C-r)

r43 s (c-5), 114.5 (C-4)

73.1 (C-20)

t'70.3

4.87 d (r l)

5.94 t (9 -2)

1.6 m,

2.-4 m

3.0 m

2.05 m,2.85 m

2.2 m,2.53 m

1.62 s

3.4s dd (r.9. r 1.8).3.r

8.91 s

3.09 s

88.6 CH

158.7 q

I14.5 q

143.5 q

145.0 q

173.1q

141.0 q

149.8 q

40.0 q

71.0 cH

]6,4 CH:

49.5 q

43.6 CH

23,6 CH,

36.4 CHr

2 t7.i q

t5 I CHI

26.5 CHr

73,1 CH,

t52.9CH

59.5 CH3

170.3 q

21.7 q

l9

20

2l

ocHl

ogocHtr

ocogHl

767

No. I

I

3

I5

6

e hdl,ln S

Fig- 5.28: Structure and key HMBC corelation and structures of NFW9C-17

compound identified as wofimannin.

168

\<Ior.

a'hqt'r i

5.3.2.4 Structurc elucidation of NFW9C-25

Compound NFW9C-25 gave a molecular ion peak at ,r/z 341.1365 [M + H] .

(calcd

for C:oH:rOs, 34I .1305) (1 1 degrees of unsaturation) in the HRISIMS, corresponding

to an elemental formula of CzoH:oO:. The rH NMR (Fig. 5.29) spectrum of NFWSC-

25 (Table 5.7) displayed the presence of two methyl groups at 5p 0.83 (3H, s, CH3-

l8), and 1.65 (3H, s, CHr.l9), an olefinic proton at 611 8.05 (1H, s, H-20), an

oxymethine proton at 5.19 (1H, t, J:2.88 Hz, H-3), and the epoxide protons at 5H

3.74 (lH, d, J =3.8 Hz, H-1), and 3.4,1(lH, dd, J = 3.8,2.88 Hz, H-2) Table 5.7.

The lrc NMR (Fig. 5.30) spectrum ol NFwgC-25 revealed 20 carbons: a conjugated

carbonyl [66 173.9 (C-7)), a ketone [3(.217.1 (C-17)]. seven quaternary carbons [6c

121.1 (C,4), 111.6 (C-s), r45.0 (C-6), r33.9 (C-8), 160.2 (C-9),41.r (C-10),47.7 (C-

13)1, an olefinic carbon [56 1,16.3 (C-20)], four methylenes [6c 24.4 (C-1]), 27.7 (C-

12),23.1 (C-15), 36.7 (C-16)1, t\&o tertiary methyls [3c 28.5 (C-19) and 13.9 (C-18)],

one oxymelhine [5c 59.4 (C-3)], an epoxide group [3c 54.6 (C-l) and 53.6 (C-2)], and

a methine [56 43.6 (C- 14)]. Comparison of the rH and rrC NMR data of NFwgC-25

and NFW9C-17 (Tables 5.7 and 5.6) indicated that they were closely related

analogues, except for the presence of an additional epoxy group at 6H 3.74 d/3c 54.6

and at 611 3.44 dd/6c 53.6, an oxygenated methine at 3H 5.19 t/8c 59.4, and a

methylene at 3H 2.8 m/6(. 24.,1 in NFW9C-25, instead of two oxygenated carbons at

6c 88.6 and 71.0. and a lactone (6. 158.7) found in NFW9C-17. The structure of

NFW9C-25 was established on the basis of heteronuclear single quantum coherence

(HSQC) and heteronuclear multiple bond connectivity (HMBC) experiments (Fig.

5.31 and 5.32). For example, the HSQC spectra revealed two tertiary methyl groups at

[3H 0.83 s)/Ec 13.9 (CHr-l8) and 611 1.65 (s)/66 28.5 (CH]-19)1, which showed

conelation peaks with C-l'IlC-141C-12 and C-glC- l0/C-5/C- I in the HMBC

spectrum, respectively. In the HMBC spectrum of NFW9C-25. olefinic proton at 3H

8.05 G, H-20) exhibited correlation peaks rith carbons at 5c121.1 (C-4),3c141.6 (C-

5), and 5c145.0 (C-6). In addilion, an ox).rnethine proton at 5H 5.19 (t, H-3) exhibited

corelation peaks with carbons at 5c53.6 (C-2), 5c121.1 (C-4), 56J41.6 (C-5). and

5c146.3 (C-20). FLrrthermore. a methylene at EH 2.8 (m. H-l1) showed two- and three-

bond HMBC correlation peaks with carbons 5c133.9 (C-8) and 6c160.2 (C-9), which

169

€hqlo i

suggested that the methylene group was connected to C_11 of the molecule. The

epoxy group at 5H 3.7,1 (d, H-l) showed HMBC conelation peaks with carbons at

6c53.6 (C-2),6c141.6 (C-5),6c41.1 (C-10), and 6q28.5 (C-19), \.hich suggested that

the epoxy group was cornected to C-l/C-2 ofthe molecule. Ihese assignments were

confirmed by extensive HMBC analysis. The configuration of the C_l and C-2

protors \\,as deduced from a NOESY spectrum (Fig. 5.34). The NOESY corelations

between CHr-18 and CHr-19, suggested these two methyls are at Eoriented. The H-lwas p-o ented. as conf,rmed by NOISY correlations of II-1 \rith CL[-19. and I{-

1lp. The COSY (Fig. 5.33) correlarion berween H-2p and H-3, suggest that the H-3

uas located at a-configuration. Thus, rhe structue of NFWSC-25 (Fig. 5.35) u,as

determined as a known conpound, wortmaflnolone (Blight and Grove, 1986).

fia

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No. ,c a-rr (J in ltz) HMBC

I

2

3

3

,l

5

6

7

8

9

10

11

t2

l3

l,t

l5

l6

t1

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l9

20

54.6 CH

53.6 CII

59.4 CH

OH

l2l.l q

1,11.6 q

1,+5.0 q

I73.9 q

133.9 q

160.2 q

4l. r q

21.4 CLlz

27 .1 CH1

47.7 q

13.6 CH

23.I CHI

36.7 CHl

2.t7.t q

r3.9 CH:

28.5 CHr

I46.3 CI.I.

1.74 d (3.8)

3.44 dd (2.88, 3.8)

5.19 t (2.88)

6.0r d (4)

2.8 m

1.58 m, 1.82 m

2.69 m

1.95 m,2.90 m

2.21 m.2.54 m

0.8i s

1.65 s

8.05 s

41.1 (C-10), s3.6 (C-2), 141.6 (C-5), 14s.0(c-6), r60.2 (c,9)

1r.l (c- r 0), 5e.4 (c-l), r21.1 (c-4)

53.6 (C-2), r2r.r (C-4), r41.6(C-5), r46.3(c-20)

59.4 (C-3),53.6 (C 2), 121.1 (C 4)

r33.9 (C-8), 160.2 (C-9)

,17.7 (C-13),43.6 (C"r4), 13.9 (C-18)

23.r(C-15)

217.1 (c-11)

23.1 (C-ls). 217.1 (C-17)

2',7 .1 (C-t2),43.6 (C-14), 47.7 (C- 13),2t7.1 (c-],7\

4l. r (c-10), s4.6 (c-1), 141.6 (C,5), 160.2(c-e)

121.1 (C-4), r,{1.6 (C-5), 145.0 (C-6)

177

€haf/o S

ll 13

9tl

Fi9.5.35:Proposed structure ofcompound NI'W9C-25 identified as wofimannolone.

1la

(|h,Eta -i

5.3.2.5 Structure elucidation of NFll9C-33

Compound NFW9C-33 was isolated as a yello\i.bro$n solid, and the HRESIMS

spectrum showed the molecular lbrmula as C2rH22O7 with ion peak at m,tz 409.1316

[M + Na]+ (11 degrees of unsaturation). Ihe'H NMR (Iig. 5.36) spectum of

NFW9C-33 (Table 5.8) showed signals representing two methyl groups at 6H 1.00

(3H,s,CH3-18),andl.81(3H,s,CHr-19),anolefinicprotonatSl 8.23 (1H, s, H-21),

an oxygenated methylene at 6H 3.00 (m. H-20a), 3.30 (m, H-20b), two oxymethine

protons at 5H 4.8,1 (1H, m, H-1), 5.18 (1H, m, H-11), and a methoxyl at 6H 2.99 (3H,

s. OCHr-22).

The rrc NMR and HSQC (Fig. 5.37 and 5.38) spectra displayed 21 carbon signals:

one conjugated carbonyl at 5c 177.8 (C-7); a ketone at 6c 217.0 (C-17); a lactone at

Ec 158.7 (C-3); seven quatemary carbons at 6c 127.0 (C-4), 5c ( 140.1) (C-5), 6c 150.5

(C-6). 5c 130.1 (C-8), 6c 163.3 (C-9). 5c 47.2 (C-10), 5c 50.5 (C-13); an olefinic

carbon at 3c 152.8 (C-21); fbur meth,vlenes at 6c 35.8 (C-12),23.6, (C-15),36.9 (C-

16), 75.7 (C-20); two tediary methyls at 6c 26.3 (C-19) and 14.9 (C-18); two

oxymethane carbons at 6c 75.4 (C-11) and 86.6 (C-1), and a methoxyl at 6c 59.6 (C-

22), and, a methine ar 6c 46.6 (C-1,1). The rH and 'rC NMR data of NFWSC-13

exhibited characteristic signals for a steroidal furanoid skeleton. I he NMR and HSQC

spectra revealed two tertiary methyl groups at [6H 1.0r/dc 14.9 (CHr-18) and 3H 1.81

(s)/6c 26.3 (CHr-19)1, which showed correlation pcaks with C-l7lc-l4lc-l3lc-12

and C-9/C-10/C-5/C-1 in fie HMBC (Fig. 5.39) spectrum, respeclively. In addition,

an oxymethinc proton at 6H 5.18 m nas observed in the rH NMR speclrum. which

showed lwo- and three-bond HMBC conelation peaks with carbons (C-8, C-9, and C-

l2), which suggested thdt the oxymethine proton was connected to C-11 of the

molecule. Futhcmore, a second oxymethiDe proton at 6H 4.8,1 d (J = 1l) was

observed in the tH NMR spectrum, which showed two- and three-bond HMBC

correlation peaks with carbons (C-5, C-9, C-10, C-19, and C-20), which suggested

that the oxymethine group u,as connected to C-l of the molecule. An olelinic singlet

at 6 8.23, \,hich sho$,ed t\\o- and three-bord HMBC corelation peaks with carbons

(C-4, C-5, and C-6). lherrC NMR dara for NFW9C-33 and NFW9C-17 (Tables 5.8

and 5.6) \rere almost identical, with the lollowing exceptionst signals for an acetyl

179

€hcpra i

group (6c 170.3; 6c 21.7) in NFWgC-3i were absent; instead the OH group u'as

replaced with the acetoxyl gtoup for C-11 in NFWSC-33 The configuration ofthe C-

1l OH group was deduced from a NOESY (Fig 5.40) spectrum The OH-l1 was a-

oriented, as confimed by NOESY correlations of H-l 1 with CH3-I8, CH:-19 and H-

12g (6 2.42 dd), rcspectively. Its structure was fu her confirmed by rH' 'rC NMR,

HSQC and HMBC analyses as I I -deacetylwortmannin, and compared with the

literature. The structure is given in Fig 5.4l.

180

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No. dc drr ("Iin Hz) HMBC

I

3

1

6

7

8

9

10

l1

t2

13

l4

l5

16

l7

l8

l9

20

21

oeH.

158.7 q

127.0 q

1,10.1 q

150.5 q

t'/'1.8 I130.1 q

163.3 q

41 .2 9

7 5.4 CH

3 5.8 CH,

50.5 q

46.6 CH

23.6 CH)

36,9 CH,

217.0 q

r,1.9 CHr

26.1 CHI

'75.',7 CH1

r52.8 CH

59.6 CH:r

86.6 CH

5.18 m

l.4l t (11.1)

2..12 dd (6 .9,

ll.l)

2.78 m

2.1 m

2.9 m

2.25 n,2.66 dd

1.0 s

1.81 s

3.0 m,3.3

8.23 s

2.99 s

1.84 d (l r)47.2 (C-10), r 40.r (C-5).

26.3 (C- r9)

163.3 (C-9), 75.7 (C-20),

3s.8 (C,12), r30.r (C-8), 163.3 (C-9)

46.6 (C-14), 7s.4 (C-11), 50.5 (C-13), I4.9 (C-18)

46.6 (C-14), 75.4 (C-1 1), s0.5 (C- r3), r4.9 (C-18),

r63.3 (C-e)

130.r (c-8), 163.3 (C-9), 50.s (C-13),36.9 (C-r6),

r4.9 (C-18)

217 .o(c-t-/),46.6 (C-14), 130.1 (C-8)

217.0 (c-17), 46.6 (C-r 4), 23.6 (C- r5)

ls.8(c-12),46.6(C,14), 50.s (C-r3),2r7.0(C-

17)

47.2 (C-10), 86.6 (C-l), 110.1 (C-s), 163.3 (C-e)

59.6 (OCHr), 86.6 (C-r )

140.1 (c-s), 127.0 (c-4), 150.s (c-6)

'7 5.7 (C-20)

185

€hapter 5

Iig. 5.41: Proposed structure of NFW9C-33 compound identified as 11-

desacell lr,,onmannin or I I -deacetl lr.lonmannin.

187

Io\zo

:

€l4ta -i

5.4 CONCLUSIONS

OLrt of three pure compourds isolated from NFW3 two N}'W3E9E-1-F and NFW3El1C

(10) compounds have been fully characte zed while one is in process These compounds

are epicoccamides most unusual glycosylated tetramic acid derivatives' composed of

three biosynthetically distinct subunits: glycosidic, fatty acid and tetramic acid (amino

acid). Tetramic acid derived natural products are interesting due to their pronounced

biological activilies. The compound NFW3EgE-1-F is a ne\\'compound'

Compounds isolated ftom NFWS displayed charactedstic NMR spectrum of steroidal

furanoid type skelelon called wortmamin fungal metabolite. Five compounds have been

characterized successfully One compound NFW9C-33 is reported first time ftom natffe

already repolted as synthetic compound. NFWSC-I7 and NFW9C-25 are already

rcported as \\,ell knoun protein kinase inhibitors isolated from Penicillium sp All these

three compounds NFW9C-17, 25 and 33 belong to wortmannin (furanosteroid class of

fungal metabolites) and all are analogues While other two compounds NFW9C-I1 and

NFW9C-15 belong to wortmin (an azaphilone) and anthraquinone class respectively'

All pure compounds have been tested for biological activities desc bed in chapter 4,

which reveals that these compounds can be a very good candidate for the drug

developmcnt 1br cancer chemoprevention. The compounds isolated from these

eldophytes revealed that Ta.ra.t plalt is a good reservoir ofimportant microbial flora'

188

L-h,|Pt€r b

Chapter 6

Discussion

189

€hEta t

6.1 Discussion

Modem therapeutics; mailly bascd on natura] products; are considered cornerstones for

treatment of many diseases either used directly or after synthetic modification (Chin e/

al., 2006). Nature provides an immense diversity of organic molecules with impressile

biological activities. These compounds ate used directly or after synthetic modification.

Among natural products plants played an invaluable role irom beginning of human life

and considered as direct ancestor to modem medicine (Fabricant and Franswo(h,2001).

However in recent times, along with plants, microorganisms are considered to be an

emerging source ofdrug development against cerlain diseases especially cancer and other

immunosuppression ailments. Among microorganisms endophytes as an important

component of plant micro-ecosystems are considered suitable source for efficiently

producing the scarce and valuable bioaqtive compounds (Gunatilaka, 20061Zhon et al.,

2009).

In the present study, filieen endophyic fungi isolated from medicinally important plant

Taxus fuana were investigated first time 1br biological activities. These samples were

tested for their antimicrobial activities initially followed by their cancer chemopreventive

and c),totoxic evaluation enabled us to isolate medicinally important pure compounds.

Fifteen endophytic fungi 10 from wood and 5 from leaf parts were isolated from lar,s

Juana. After initial screening six wood and one leaf isolates was identilled at molecular

level. Three isolates liom wood NFWI, NFW3 and NFWT belonged to genera

Epicoccun. These hndings coffespond to repofis suggesting the frequent isolation oI

Aureohasidium pullulans and Epicoccum n lgra, as endophytes of various crops (F6varo

et a1.,2012: Stuart e/ d1., 2010; Martini et e1..2009| Stain NFW5 was idenliiied as

Ttitirachium fungus. Li e,41., (2012) also reported Trilirachium as an endophyte from

Quercus pannosa alnd Rhododendron sp.lsolate NFW6 was idenlifled as Mucor hiemalis.

Mrcor sp. as an endophyte has also been isolated ftom Taxus chinen"^is (Zhot et al.,

2009; Miao ct al., 2009t Tayung and Jha, 2010). The isolate NFW9 belonged to

Penicillium sp. Thcse findings corresponds \\'ith reports of stierle €/ al , (1997) and

Raghunath el dl., (2012) that Penicillium sp. reside in inner balk and u'ood pans of

Tdxus breNilolid and Taxus 6accdla respectively. Among leaf isolates NFL2 was

190

identified as Trichoderma sp. which was previously reported previously as an endophlte

fiom laxas plants (Zh ang et al..200't ,Lir et al ' 2OO9;Zhao et ol ''2010)'

Antimicrobial potcntial of isolated fungal endophltes was evaluated by using

antibacterial and antif'nngal assays. It was observed that crude organic extracts ofmost of

the fungal endophytes used in the present study showed antimicrobial activities agaimt

tested microbial strains. Five isolates Nl-Wl. NFW3' NFW6, NFWT and NFW9 showed

promjsing effects with zone of inhibition in the range of 9.5-23 2 and 8 9-19'7 mm

against bacterial anr:l fungal strains respectively (Table 3 5 and 3 '6) Three orrt of five

active isolates belonged to Epicoccum geneta Epicoccum genra is able to produce

diverse classes of biologically aclive metabolites such as epicoccin' epicorazies and

epicoccamide having antimicrobial potential (Favaro e/ 4/., 2012; Wang e/ a1', 2010; Guo

€r.r1.,2009; Musetli et al..2OO'7: Wtight et at.,2003). Penicillium sp'NFW9 and Mzrcor

sp. NFW6 isolates also showed good antimicrobial effects which corespond to the

tindings of Zhang el al., (2012) that Mucor sp. SPS-Il isolated from Arlemitia annua

showed antimicrobial potential dgainst Rhizoctonia cerealis, E. coli and S aureus'

Anlimicrobial potential of extracts of Mucor and, Penicilli ,, endoh)'tes of taxus plant is

also reported by (Tayung and Jha,2010) By now, at leasl 19 genera of endophytic fungi

mainly isolated from larr.t plant have ability lo produce taxol which also have antifungal

activity (Zhao e1 d/., 2010).

The hyphae formation inhibition (HFl) assay is an excellent primary assay for screening

of crude extacts for protein kinase inhibitors Protein kinases, play a key rcle in cancer

and this has led to extensive efforts to develop kinase inhibitors for the treatment of a

wide array of cancers (Fabbro et Ltl..2OO2: Dancey and Sausville, 2003). During this

study, crude extracts of isolated endophyles were evaluated for protein kinase inhibition

potential which revealed promising activities of extracts (Table 3.7). Out of fifteen strains

six showed zone of inhibition of 16-28 mm (Table 3.7). Ihese findings are corelated

with the reports about HFI potential of secondary metabolites such as crilinin, glycosides,

alkaloids and aminocouma nes from marine coral, ma ne sponge' steptomyces and

l-ungi (Ankudey et al., 2008, \ao et al .2003; Cheenparacha et dl.. 2010b. Yao et al.,

2011).

191

a'hdPlo 6

Because of critical role oI NFXB in carcinogenesis and regulation of cell fate decisions'

its inhibition has potential role in the treatment or plevention of cancer (Aggarwal. el d/.'

2004; Schupp e, dl., 2009). In the prcsent study crude ethyl acetate extracts of 15 fungal

endophyes were used to evaluate their NF-KB inhibitory potential (Table 3 8) Six

isolatcs showed more than 50 % inhibition Three isolates NFWI' 3 and 7 were

Epicocum species which exhibited poient activity while other two strains Chaeloniun sp'

NI-WS and Penicittium sp. NFWS also showed significant inhibition ofNFxB Different

compounds from endophytic fungi shou'ing NFrB inhibitory activity have been repofied

includjng t chodion (Erkel,2000), tencyclic acid (Wijeratne el a/,2003)' panepoxydone

(Etkcl et al.,2007), hispidin derivatives (Wu e1 'r1

, 2011), chaetoglobosin (Dou e' a/ '

2011) and mycoepoxydiene (Wang e, a/ , 2012b)'

Aromatase inhibitors can be the best entities to trim down the growth of estrogens

receptor posiiive breast cancer through blocking thc production of estrogens Although

aromatase inhibitors are alrcady in clinical use as chemopreventive agents (Lubet el 4/,

1994, Gubson et al., lgg5) but the slrategies adopted pose side effects therefore search

for more efficient aromatase inhibitors continues (Balunas and Kinghom, 2010)' In

present study aromatase inhibition potential of crude extmct of 15 endophyes was

evaluated (Table 3.9). Two samples NFW3 and NFW9 showed '73 3 and '76 4 Yo

inhibition with lc5o values of 12.18 and 10.5 Pg/mL respectively. Fungal metabolites

\lith azol ng have been reported for their capability to inhibit different P450 enzymes

including aromatase (Andersen e1 aI ,2002; Zhatg et a1.,2002) Various other fungal

metabolites showed aromatase inhibitory activity such as monomeric xanthones,

benzophenone, depsidones and diaryl etheN (Krick el d1.,2007; Chomcheon e/ a/ ' 2009:

Sureram er a/., 2012).

lnducible nitric oxide synthase (iNOS) is most consistently associated $ith chronic

inllammation and tumor production Q'lomelini e1 a/., 2008). The up-regulation of iNOS

has beel observed in many cancers though over expression appears to occur during early

tumor devclopment, suggesting the role oF iNOS inhibitors as cancer chemopre\'enti\'e

(Crowell et a/., 2003; Nomelini et dl ,2OO8). ln present study the inhibition of NO

production in LPs-activated mu ne macrophage RAW 264.7 cell was used as an indirect

192

c4lupto t:

marker to monitor iNOS activity of crude exracts of endophytic isolates and five of the

isolates showed significant inhibition of more than 50 % (Table 3.10). Among them

three isolates NFWI, NFW3 and NFLI showed highest activity with % inhibition of

99.6. 69.7.84.4 and IC50 values of 0.32,7 .16 afi 4.12 pg/ml, respectively (Table 3.10).

Three Fungal metaboliles radicicol, Sporogen, Sl4-95, S-Curvularin and fifteen

Maphilones showed iNOS inhibiton (Jeon et al.,2000; \ao et a1.,2003; Quang et a/,

2006). Two 1 lactone derivatives (1, 2) isolated from ethyl acetate extract of fermented

broth of Neosartorya sp. ofTaiwan showed iNOS inhibition $ith ICro values of 12.2 and

11.4 pM respectively (yatg et u1..2010). Another fungal metabolite isolated from

Aspergillus sp. SF-504,1 sho\ied anti-inflammatory ellects through iNOS inhibition (Lee

et a|..2011).

Strategies for protecting cells from qancer initiation events also include increasing phase

Il enzymes. lnduction of quinone rcductase I (QRl) with cultured Hepa 1clc7 (murine

hepatoma) cells is representative of the overall elevation of phase II enzyme levels.

Therefore, induction of QRl at the tunor initiation stage is an indicator of cancer

prevention (Cucndet ?1 a/.,2006). Quinone rcductase potential of 15 endophytic fungal

extracts was determined with reference to induction values. It was observed that three

isolates NFW3, NFWT and NFL1 showed signiflcant results wilh IR and CD values of

(2.6, 2.5 and 6.9) and (5.54, 0.49 and 0.21 pg/ml) respectively (Table 3 11). These

results are comparable to quinone reductase (QR) induction potential of two xanthone

de vatives isolated liom marine algicolous fingus Monodictys prrledlr?is showing CD

values of22.1 and 24.8 4M, respectively (Pontius e, ai.. 2008).

The discovery of novel and safer antioxidants from natural products to combat and/or

prevent diseases is a continuous process- To detcrmine the free radical scavenging

potential of 15 fungal extracts, DPPH free radical scavenging assay \\'as utilized in this

study. Four fungal extracts NFW3, 6, 7 and 9 showed 88.8. 90.21, 86 69 and 84.38 %

scavenging activity respectively (Table 3.12). Metabolites from NFW3 wcre considered

to be the most potent free radical scavengers and IC5o value of 11.7 Lrg/ml was obseNed.

Antioxidant potential ol organic extracts of various endophylic i'ungal species [ere

repofied such as Fusarium, Aspergillus. Penicillium, Mucor- Phomopsis- Xylaria and

193

a'hqw 6'

Colletotrichum (Lfu et a/., 2007; Tianpanich et dl.,2011t Attanti et a/., 201 1; Ravindran

e t al .. 2012, Mlltthy et aI ., 201 1 ; N u et aI .. 2013).

C)'totoxicity studies of synthetic as well as natual materials against various cancer cell

lines are considered mandatory for anticancer drug development. MCF-7 breast cancer

cell line, MDA-MB 231 cstrogen receptor negative breast cancer cell line, PC-3, HL-60

and HeLa cervical cells were used to evaluate cytotoxic potential ofextracts of l5 f'ungal

endophltes ( l'able 3.13). Four fungal isolates NFWl, NFW8, NFW9 ard NFL1 showed

cytotoxic activity against MCFT cell line with % survival of0.20, 30.2, 46.2 and 45.7 and

lcio values were 0.56, 12.4, 17.5 and 11.3 [g/ml respectively. These extracts showcd

activity that was within the cutoff point set by the National Cancer Institute for

cltotoxicity (ICi6 < 20 pg/rnl) (Lee and Houghton 2005). Differert fungal metabolites

exhibited cytotoxic activity $hen tested against cancerous cell lines such as taxol,

norsesquitelpene peroxides, cyclohcptapeptides, Iactones and chlorinated anthraquinones

(Wang and Tang, 20I l; Li er a/., 201 l; Chen et a|.,2012. Song et a|.,2012; Hrang et ul.,

20t2).

After initial screening it was found that seven out of fifteen endophyes showed

biological activities. Two isolates were identified as Penicilliufi sp. NIW9 and

Epicoccum nigrum NFW3, and wcrc sclected with reference to two different media for

fermentation, extraction and compound isolation. During fractionation it was observed

that flash column chromatography as well as gel filhation were suitable techniques for

isolation and relatively less polar or medium poiar mobile phases were preferable.

The crude extract fror., Penicilliutn sp. NFW9 obtained after fermentation by using PDA

medium showed modemte activity irl NF-kB assay along with SRB and aromatase assays.

Furthermore fractions oI NFW9 strain prepared using nomal phase column

chromatography showed moderate c)totoxic effect against PC-3 human prostate cancer

cell line. However after first column chromatography, fraction NFW9C showed better

cylotoxicity as compared to other fractions was selected for further analysis. Its TLC

analysis showed the presence of relevant compounds (similar behavior after sraining with

10 % HrSOa) with different R/values.

194

C'hEln L;

Therelbre a targeled approach based on various chromatographic tcchniques was adopted

for isolation ofpure compounds. Thus a sequential change ofmobile phase and stationary

phases led to isolation of four structurally very closely related compounds with

significant biological activities. In case of Epicoccum nigrum NFW3 same strategy vv.as

adopted for isolation ofbiologically active pure compotulds_ Fractions fiom crude exhact

of NFW3 strain also shou,ed chemopreventive and cltotoxic activities when tested with

NFkB, iNOS and cytotoxicity assays. lllitially three active fractions were selected for

compound isolation leading to three pure compounds including one new compound

NFWSE9E-1-Fatima.

Both of NFW3E9E and NFW3EllC(10) compounds showed positive reacrion \\,ith

dragendoffs reagent. The rrc NMR spectral data (Table 5.2) ofNFW3E9E displayed 23

carbons including one carbonyl one anomeric, four oxymethine, one oxymethylene

carbons and signals of aliphatic chain. Thc lH NMR data showed also D-mannosc sugar

unit. All IH and IrC NMR spectrum of NFW3ESE were very closely related to that ofNFWIEI lCl(10) a known epicoccamide derivative . except for the disappearance of the

signal of tetramic acid moiet), to amjde moiety. Thus, compound NFW3E9E was

assigned as a new compound.

While 'rC NMR spectral data ofNFW3E1lC(10) (Table 5.3) displayed 29 carbons

including three carbonyl; rH NMR exhibited N-Me at .rH 2.91 (H-6), suggesting this

compound contained tetramic acid moiety. The rH NMR data showed a hexose sugar unit

at i\t 4.49. The rrc NMR also indicated one anomeric, four oxymethine and one

oxymelhylene carbons. The lH and llc NMR chemical shifts were very similar to those

of D-mannose (Wright et a/., 2003). The signals of aliphatic chain also observed at du

1.26-1.38, located position C-10 to C-l9 ()c 31.4-31.7). On the basis ofNMR data the

structure of NFW3EIIC(IO) was identified (Fig.5.10) ro be a known conpound,

epicoccamide previously isolated ilom Epicoccum purpzrrascers endophyic fungus ofjellylsh Aurelia aurira (W ght et u1..2003). Epicoccamide is quire unusual secondary

metabolite of fungi since it is composed of three biosynthetically distinct subunits;

glycosidic, fatty acid and tetmmic acid (amino acid). Both compounds from NFW3,

NFW3E9E-l-F and NFW3EIlC showed significant 80 and 77 % NFKB inhibirion

respectively. NFW3E9E-l-F exhibited ICro value of 3.5 pg/ml showed its potential as

195

a'hdplo 6

chemo preventive entity (Tabie 4.11). Gerus Epicoccum have a highly developed and

diversc secondary metabolism as natural products such as epicorazines A and B. epirodin

and triornicin all being produced by E. nigum (Wright e, al.,2003). Wangun e/ a1.,

(2007) reponed three new epicoccamides B, C and D isolated from Epicoccum

endophltic fungts of Pholiota squatosa. The long chain derivative Epicocamide D

exhibited modemte cytotoxicity to HeLa cell lines (CC56 17.0 pM) and antiproliferative

effects toward mouse flbroblast (L-929) and human leukemia cell lines (K-562) wirh

growth inhibition (GI5n) of50.5 and 33.3 prM, respectively (WangulL et a|.,2007).

Out ol tohl 6 compounds isolated lrom NFW9 strain, NFW9C-6 is identified as known

ergosterol (proton and carbon NMR specta) and was not processed lbr structural

elucidation. Other compounds were struclurally characterized and described in chapter 4.

They were lested for biological activities by using NFKB and c)'toroxicity assays with

dill'erent cell lines.

The lH NMR spectrum ofNFWgC-ll a pure compound from NFW9 showed signals oftwo olefinic protons. one methoxy, two methyl singlets and one methyldoublets. The lrc

NMR spectrum showed presence of acetoxyl, methyls, methoxy, thiee methylenes, six

methieens and 1 I quatemary carbon atoms (Table 5.4). The presence of azaphi]one and

methoxylated osellinic acid moieties in structure suggested its resemblance with

wortmin: an azaphilone derivative reported by Merlini e/ .r1., (1973). Azaphilones can be

defined as a structurally diverse class of fungal secondary metabolites (polykctide

derivates), mainly pigments with pyrone-quinone structures containing a highly

oxygenated bicyclic core and a chiral quatemary centre (Sturdikova et al.,2OOO. Zh! et

a1.,2005; Dorg et al., 2006). Musso et a1., (2010) repofled isolation of azaphilones from

perfect and imperfect stages of ascomycetes, such as ,,{rpergll/rj, Penicillium,

Hypoxylon, and Mona:^cus sp. Recently, seyeral Penicillium species isolated as

endophytes have been rcported to produce metabolites with antimicrobial and c).totoxic

activity (Mapa e, a/..2010. Li, et aI.,2010. Hnang et al.,2011). These metabolites have

been repofted to exhibit a u,ide range of biological activities, including monoamrne

oxidase inhibition, tumor promotjon inhibition, clrotoxicity and sphingosine kinase

inhibition (Musso et d1.,2010). The compound NFWgC-11 sho$,ed moderate c).rotoxjc

196

a'hq\"t 6

activity and NFKB inhibition (Table 4.11) which coresponds to the findings of Gao el

al.- (2011) about azaphilones ftom Penicillium commuke. Cylotoxjc activity and

pre)iminary structure activity relationship (SAR) results of these Maphilones indicated

that the double bond at C-10 and lhe location ofthe oNellinic acid unit at C-6 in these

azaphilones are important for the antibacte al activity and cltotoxicity, respectively. An

azaphilone aspergilone A isolated lrom fungus Aspergillus sp. showed c).totoxicity

towards HL-60 human promyelocyic leukemja. MCF-7 human breast adenocaruinoma

and A-5,19 human lung carcinoma cell lines with lC5e values of 3.2, 25.0 and 37.0 pg/mlrespectively (Shao e, a/..2011). Pyran ring is also considered to be impo(ant for

antiproliferative activity of these metabolites (Hsu e/ a1., 2010). New azaphilone

derivatives from fungus Mona.rcus purpureus with cytotoxic activity were also reported

(Cheng e/ a/., 201 l; Hso et dl.,2012). Recently ne$ azaphilones have also been reported

from endophltic fnngt, Dothideomycete sp.. ald Fusarium sp., (Sendeera et a1., 2012;

Yang et al., 2012). ID another study Li et al. (2013) also reported isolation ofthree novel

cytotoxic azaphilones from endophytic fungus of Ginkgo bilobu.

Compourd NFW9C-I5 was identified as an antlraquinone de varive (Table 5.5). The

NMR spectrum of NFW9C-15 showed characteristic signals of anthraquinone derivative.

The 'H NMR spectrum showed two phenolic hydroxyi groups, three aromatic proton

signals and one methyl signal. TherlC-NMR spectrum not only showed tu,o ketone

carbonyl groups but also shou'ed signals for 15 carbons including three melhines. one

methyl group and 11 quaternary carbon atoms. This data showed that compound is

similar with anthraquinone compound2 reported from seed of Rhodom))rtus tementosa

(Cher et a1.,2011). NFW9C-I5 compound showed 68.91 % NFKB inhibirion with ICjo

value of2.2 Lrg/ml and was also found slightly cytotoxic against HT-29 and HeLa cancer

cell lines (Table 4.ll). These findings were in accordance wirh repoft by Chen er a/.,

(2011) that two anthraquinone compounds I and 2 showed cylotoxic activity against KB

with IC5o values of 17.1 and 18.1 pg/mt, respectively. Other bioactive antkaquinones

from Penicillium sp. were also reported (Zha\ et al-.2004; Marinho er a/., 2005). A new

anthmquinone de vative from the marine endophytic l].t'tg]os Fusarium sp. was also

reported by Shao el a/., (2010). Huang et al..2011 reported three new bianthraquinone

derivatives, alterporriol K, L and M, along with six known compounds obtained Irom

191

C'hqt b

extracts of the endoph)1ic fnngus Alternarid sp. ZJ9-6B, isolated from the miurgrove.

Alterponiol K and L exhibited modemte clroroxic activiry towards MDA-MB-435 and

MCF-7 cells with ICso values ranging liom l3.l to 29.1 pM. Zhl et d/., (2012) also

reported a marine anthraquinone SZ-685C overrides adriamycin_resistance in breast

cancer cells through suppressing Akt signaling. Hawas ef a/., 2012 also reponed

antfuaquinones from Aspergillus endophlte ftom red algea with antimicrobial and

anticancer activities.

The rH NMR spectrum ofNFW9C-17 (Table 5.6) displayed characteristic signals for rwo

methyl groups at 6H 0.84 and 1.62,an olelinic proton at AH 9.91, an oxygenated

methylere at 6H 3.45, two oxygenated methines ar 511 4.87 and 5.94, a methoxyl at 3H

3.09 and an acetoxyl at 6H 2.09. Therrc NMR specrrum of NFWSC-I7 revealed 23

carbons: a conjugated carbonyl (C-7), a ketone (C-17). a lactone (C-3), seven quatemary

carbons, an olefinic carbon (C-21), four methylenes, two tertiary methyls, two

oxygenated methines, a methoxyl, an acctyl group and a methane group. These proton

and carbon NMR data were closely related to those ofthe know[ $,onmannin repofied by

Brain et al., (1957) suggested rhe presence ofsteroidal furanoid skeleroD in NFW9C-t7.

The proton and carbo[ NMR of NFW9C-25 compould also showed signa]s related to

wo mannin and comparison of the rH an,l r3C NMR ,lata of NFWSC-25 an<i N!.W9C-17

(Tables 5. 7 and 5.6) indicated that they were closely related analogues, except for the

presence of an additional epoxy group at 6p 3.74 d/56 54.6 and at 6H 3.44 dd/6c 53.6, an

oxygenated methine at 5rr 5.19 t/6c 59.4. and a methylene at 6H 2.8 m/6c 24.4 in

NFW9C-25. instead of two oxygenated carbons at 5c 88.6 and 71.0 and a lactone (Ac

158.7) fbund in NFW9C-17. The compotu.rd NFW9C-25 was also a known analogue ofwortmannin derivative which is wortmannolone (Blight ard Grove, 1986).

Therrc NMR <.lata for NF-W9C-33 and NFWSC-I7 (Tables 5.8 and 5.6) were almost

identical, with the following exceptions. The acetyl group at position C-l I in NFW9C-17

was replaced with the hydroxyl group at C-l I position in NFW9C-33 and signals 1br an

acctyl group in NFW9C-33 were absent. Thc configuration of the C-l1 O[l group was

deduced from a NOESY spectrum. lhe structlLre ofNFW9C-33 was fuflher confirmed by

'H,'tC NMR, HSQC and HMBC analyses (Table 5.7) as I I -deacetylwortmannin. and

198

C'h,tl 6

compared with the literature. I 1-deacetylwortmannin is a potent and specific

phosphatidylinositol 3-kinase (Pl3-K) intribitor wirh an IC,\o of 1.6 ng/Ml.

The lH NMR and rlc NMR data revealed that these compounds are wortmannin

analogues, a well-known furanosteroid metabolite of the fungus belonging to the viridin

group. Various fungal species inc|odirrg Penicillium Juniculosum, Talaromyces

Penicillium wofimannii produce this class ofmetabolites (Kim ea a/., 2012). Wortmannin

is a potent and specific phosphatid),lirositol 3-kinase (PI3-K) inhibitor, with an ICro of2-4 nM. Wortmannin binds to the pl10 catal),tic subunit of Pl3K, noncompetirively and

irreversibly inhibiting the enzyme (Powis et dl., 1994). Wortmannin acts by opening ofthe electrophilic furan ring at the C-20 position, to a iysine within the ATP binding region

ofPI3-K. Electrophilicity offumn ring in the struclure is the cenrral to inhibitory activity

Two out of other tltee pure compounds of Pehicillium sp. NlWg strain which were

analogues showed potent Nr'KB inlibition. NFW9C-17 and NFWSC-25 showed 73.73

and 85.12 % inldbition and IC5x values were 0.2 arLd 0.'12 pg/ml respectively. These two

compounds showed potent cytoloxic effects against breast cancer cell line MDA-MB-231

u'ith similar IC56 value 0f0.00372 pM. In case ofHeLa cells 6l and 52 % inhibition was

observed with NFWgC-17 and NFWgC-25 compound respectively. The third analogue

NFW9C-33 showed less than 50 % inhibition in NFKB assay. pacliraxel which was used

as positive control for cytotoxicity assays showed IC56 values of 0.0029 and 0.0016 FMagainst HeLa cells and HT-29 cell lines respectively. Overall results ol pure compounds

advocated that these compounds are good candidates for anticancer and chemopreventive

drug development (Tablc 4.11). Our findings are correlated with repofis ofpowis er 4/.,

(1994) and Manna and AggaNal. (2000) about potent NFrB inhibitory porerrial ofwoftmannin. Wortmannin and its analogues are also reported for anti-proliferative effects

against human MCF-7 breast and colo[ cancer cell lines (Akter et al., 2012t Shan et al..

2013). These bioactive compounds also exhibit other phamacological activities such as

neuroprotective effects in epileptic rats as u,ell as protective efTects in case ofmyocardial

inlarction (Li et al., 2010: Wiedemann et al., 2013).

199

alhaptn b

Conclusions

r Seven endophytic isolates NFWI, 3, 6, 7, 8 and 9 (wood) and NFL1 (lea| showed

potent eff'ects irl antimicrobial as well as in chemopreventive assays.

o Molecular identification ofactive isolates revealed endophytic fungal diversity ofTaxus .fauna of Himylian region ofPakistan.

. These endoph),tes can be a potential source of phamacologically active natural

products.

. Cancer chemopreventive compounds from Epicoccum nigrum NFW3 an<l

Penicillium sp. NFW9 were obtained by using bioacrivity guided isolation

strategy.

. Solvent-solvent extaction and combination of different chromatographic

techniques proved to be an ef'ficient strategy to obtain diverse chemical entities.

. Total 9 compounds (6 from NFW9 and 3 from NFW3) belonging to azaphilone,

anthraquinone, $,ortmannin and epicoccamide classes of fungal bioactive

metabolites are reported first time in the present study from endophltes ofHimalay an T LL\ u s fu dn d.

o The neu,compound NFW3E9E-1-F obtained from ,picoccum nigrum belongs to

epicoccanide class showed pronisirg activity in NF(B chemopreventive assay.

. Two out ofthree $ortmarmin analogues isolated from penicilliura sp. NFWSC_I7

and NFW9C-25 have potential for cancer chemopreventive drug development.

. The compounds obtained from thesc endoph)1es revealed that Znras plant is a

good resenoir ofuseful microbial flora.

2AO

Future strategies may includes

These purified compounds can be a promising source for drug development tocombat cancer. Isolated compounds can be used as lead compounds to synthesizemore potent derivatives.

Studies for preclinical trail could be carried out to evaluate the lz ,iyo activity andtoxicity ofthe isolated compounds to establish their safety and efficacy.

Several new bioactive compounds with cancer chemopreventive and cytotoxicactivities can be isolated from these endophltes with additional pharmacological

activities.

Isolation of more wortmannin analogues from penicilli m sp. can be done forestablishment of a broader SAR to find out the most active compound fbllowedby development ofsynthetic approach for most active analogues.

Computational and. i silico drug designing of newly isolated and structurallycharacterized compounds against cancerous and infectious potential targets can be

done.

All these endophytes can be explorcd for their taxol production potential. These

isolates can also be explored for presence of genes involved in tarol biosynthetic

pathway.

Optimization of operational parameters for enhanced production of porential

bioactive compounds.

20L

Relerences

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Zheng CJ, Li L. Zou JP, Han T, Qin LP (2012) Identification of a

quinazoline alkaloid proiluced by Penicilliun ikdceum, a\ endophytic fungus

fron, Crocus sativus.Pharm Biol 50(2): 129-33

Zheng LP, Gao LW, Zhou JQ, Sima YH, Wang JW (2008) Antioxidant

activity of aqueous exhact of a Tollpocladium sp' fungus isolated from wild

Cordyceps sinensis. Afr J Biotechnol 7(17): 300'1-3010

7.hou L. Zhao J. Xu L, Huang Y. Ma Z, Wang J' Jiang W (2009)

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producing endophyic fungus EFY-36. Afr J Biotechnol 8(l l): 2623-2625

Zhu X. He Z, Wu J, Yuan J, Wen W' Hu Y. Jiang Y, Lin C, Zhang Q' tin

M, Zhang H, Yang W, Chen H. Zhong L' She Z, Chen S, Lin Y' Li M

(2012) A marine anthmquinone SZ-685C ovenides adriamycin-resistance in

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229

APPENDICES

230

Appendices

Morphologic&l features ofendophltic fimg&l streins isolated from leaves of fax,l,,luanqi

Fungus: UnidentifiedCoder NFLIFeatures: White coloDy v/ith mesh likemat

GenBank Accession No: ln prccess

F rntgtts I Ti c ho d e rma a.sp el e llunlCode: NFL2Featuresi Dark greeD eat like colony

GenB&Dk Accession No: JX838791.1

Futrgus: UnidefltifiedCode: NFL3Features: White mat like colony

G€nBank Accession No: In process

231

NFL2

\

t\

NFL3

Appendices

Fungus: UnidentifiedIsolate Code: NFL5Featuies: Brow colotry with th€ad like

GenBaDk Accession No: In process

Fungus: UddentifiedIsolate Code: NFL6Fertures: Flat white colotry

G€DBank Accessiolr No: In process

232

l

Appendices

Aooendix A2:

Morphologic&l features ofendophltic fungal Btr.ins isolated from wood palts ofTaxus fuanal

Ftnguaz Epicoccun nigramIsoLte Code: NFWIFeatuies: Pinkish white colony

GenBank Accession No: JX402049.1

Fnnignsr Epicoccum nigramIsolrte Code: NFW3Features: Peach White colony with dalk pink,yetlov,,ish margins later oI1 tums orange yellow

GenBank Accession No: JX838792.1

Fulgus: UnidentifiedIsolate Code: NFW4Features: Offwhite colotry with light pinkshades

GerBalrk Accersion No: In process

233

7A:

F tn,trglrs i Tlitaichium sp.Isolate Code: NFWsFeaturcs: Offwhite flat colony

G€nBank Accession No: IX845570.1

B urtgttst Mucor hiemalisIsolate Code: NFW6Features: Light brown mat like colony withthrcad lile structures

G€nB&nk Acce$sion No: JX845571.1

Frngtrs: Epicoccum sp.I$olate Code: NFWTFeatu!'es: Peach pink colony later on tumyellowish bro*n

GenBank Accessiotr No: JX838793.1

234

NFW6

\

N

Fn,ignsi Chaetomhan sp.Isolate Code: NFW8Features: Dart purplish piDk colotry

GelBaDk Accessiotr No: ID ptocess

trungus: UoidetrtifiedIsol&tiotr Code: NFW9Featuresi Yellow greetr colooy wi(hexudates

GenBank Accession Noi Io prccess

Fungus: UnideltifiedIsolation Code: NFWI0Features: Green colony

GenBrnk Accession No: In process

235

/ppend,ces

Appendices

Fungus: Uaidentified

Isolot'on Code: NFW12Fe&turcs: Colotry $.ith white marginal areaand brownish ceder

GenBank Accession No: In process

236

NFW12

Appendices

Table Al I Chemicals supplies and apparatus used for cancer chemopreventive assays.

Inhibition of TNF-0 activated nuclear factor-kappa B (NFkB) assay

293,NFrB-Luc llEK cells (Panomics caralog nLunber RC00l4), LUI!,1lsrar Galaxy

Luminomete. (BMG Labtechnologies, Durham, NC), letal bovine serum (FBS, ATCC

catalog number 30-2020), dulbecco's modified eagles medium (DMEM, penicillin G

sodium and sheptomycine sulfate, Forma series II water jacketed C02 incubator, Gibco

catalog number 12800-058). 20 o% l chloroacetic acid (TCA). 0.4 % sulforhodamine B

(SRB) in 1% acetic acid, phosphate saline buffer (PBS), lX reporrer lysis buffer

(Promega catalog nunber 83971), rest samples (0.4 mg/mL in 10 o/o DMSO), tumor

necrosis lactor - o (TNF-(I, Calbiochem catalog number 654205), refrigerator -80.C.

incubator 37"C, Srerile white-walled 96 well plates, sterile transparent 96 \\'ell plates,

gyratory shaker , micro plate reader (Biotek).

Aromatase inhibitioo assay

Nicotinamide adenine diphosphate (NADP*), sodium dydroxide (NaOH), Naringenin,

glucose 6-phosphate, potassium phosphate, glucose 6 phosphate dehydrogenase,

aromatase enzyme (CYP19, BD Biosciences, Magnesium chloride (MgCl2), albumin,

San Jose. CA), dibenzylfluorescein, synergy II lluorescent plate reader, gyratoty shaker

and i84 well black micro tite. plate, i[cubator.

Inhibition of nitric oxide (NO) production in lipopolysaccharide (LpS)-activated

murine macrophagc RAW 26d.7 cells assay

Murine rnacrophagc RAW 264.7 cells. dulbecco's modil-red eagles medium (DMEM,

Gibco catalog number 12800-058), penicillin G sodium, streptomycine sulfate,

amphotericin B, fetal bovine scrum (FBS, A'I'CC caralog number 30-2020),

iipopolysaccharide, c ess reagent, 0.4 % sulforhodamine B (SRB) in I % acetic acid,

acetic acid, test samples (0..1 mg/ml in l0 % DMSO), l0 yo rrichloroacetic acid (TCA),

l0 mM l'ris base pH 10. Micro plate readcr (Biorek). Sterile rrarNparent 96 well plates,

gyratory shaker and lbrma series Il waterjacketed CO2 incubalor.

Quinone reductase 1 (QR1) induction assa)-

llepa 1clc7 (murinc hepatoma) cells. MEM-(, (minimum essenrial mcdium) \r'ifiout

Appendices

ribonucleoside. or.l rat UoGi

serum (FBS, Gibco), amphotericirl B peniciliin G sodium 3_(4,5-dimethylthiazo-2_yl)_2,5- diphenylterrazolium bromide- 4,,Rromoflavone_ Forma series II \\ater jacketed CO2incubator, gyratory shaker, stedre transparent 96 \l'eir plates, micro plate reader (Biotek).

DPPH free radical scavenging assay

Methanol, dimethyt sulfoxide (DMSO). 2,2_diphenyl_ t -picrythydrazyt (DppH), ascorbicacid test sampres. Micro prate reader (Biotek), transparent 96-we prates. incubator.

Cytotoxicity assay, Sulforhodamine B (SRB) assay

Human lung carcinoma cells LU_l (established from depaftment of Surgical UncologyUniversity of lllinois. College of Medicine at Chicago), Hormone responsive breastcancer cell line MC|-7(ATCC number HTB_22), phosphate saline buffer , estrogenreceptor negative breast cancer call line MDA_MB_231 (ATCC number H.l.B_26).DMSO, FBS, DMEM, streptomycine sulfate, penicillin C sodium, amphorericin B, l0mM Tris base pH 10, 20 o/o rrichloroaceric acid (TCA), 0.4 % sulforhodamine B (SRB) inI yo acetic acid, acetic acid. Mjcro plate reader (Biotek), sterile transparent 96_u,.ell

plates, Forma se es II waterjacketed CO2 incubator, gyratory shake.

238

l'able A2: Chemicals supplies and appa.atus used lbr chromatographic techniques.

Chemicals supplies ana apparatus

Organic solvents (nHexane. chlorofornr, ethyl acetate, ethanol and methanol), Silica gel,gel 60 (70-230, 230-400. 18-25 and S-,10 mesh.merk,Germany) for normal phasecolumn chromatography and Sephadex LH20 lbr gel f,iltration column chromatography.Glass columns ofdiflerent diameters. Thin layer chromatography plates (TLC), MPLC(Medium pressure liquid chromatography) and HPLC (High performance liquidchromatography).

Tlc visualing reagenti

Dragendorff Reagent (preparation: Solution A: r.7 g basic bismuth nitrate in 100 mLu,ater/acetic acid (4:l).solution B: 40 g potassium iodide in 100 mL of .,,ater. Mixreagents together as fbllows: 5 mL A + 5 mL B + 20 mL acetic acid + 70 mL water.Spray plates, orange spots deverop). Sulphuric acid reagent ( 1 5 % sulphu c acid in disrilwater. Spray and heat to develop TLC plates).

239

Publications

PABLICATIONS

240

Certificare #Obtained

from an endophyticfungus ofa Taxusplant

'lech.lD.20t3-0t6Applied for ,

20t2

University ofllawaii, The Ohio

USA, Quaid-i-

Islamabad

Provisionalpatent

Papers published / Submitted

Patent Applied:

Fatima N, Zai M, Rehman R, Rizvi ZF, Ahmed F, Mirza B, Chaudhary MF(2009) Biological activities of nrnex dentalus L: Evaluation of melhanol andhexane extracts. Afr J Biotech 8(24)j 6945_6951

Aimed N, Fazal H, Abbasi BH, Rashid M, Mahmood.t., Fatima N (2010)

Efficient regeneration and antioxidant potential in regenerated tissues of pDe,"

igtum L. Plant Cell Tissue Organ Culr 102(t):129_134. doi: 10.1007/s11240_

010-9712-x

Young UJ, Fatima N, Chen QC, Chae S. Hung 1.M, Min B-S (2012) Apoptosis_

inducing and antitumor activity of neolignans isolated from Magnolia o./Iicinalis

in Hela cancer cells. Phltother Res. doi: 10. I 002/ptr.4g93.

Masood F, Chen P, Yasin T, Fatima N, Hassan F, Hameed Abdul (2013)

Encapsulation of ellipticine in poly-(3-hydroxyburyrate-co-3-hydroxyvalerate)

based nanoparlicles and irs in vitro application. Mater Sci Eng C 33(3): 1054-

1060 hltprr,'dx.doi.ou.l1 0. I016/i.msec.20 i 2.1 1.025

Fatima N, Tamara P. Kondratyuk TP., park E-J., Marler LE., Muniba Jadoon M..

Qazi MA , Khan L. Atiq N.. Chang LC., pezzuto JM and Ahmed S. Cancer

chemopreventive and antimicrobial activity of endophyic llngi isolated fromTaxus fuana (baccata); Submitted ir planta Medica March 201j.

241

Publicotions

Abstracts published

. Acuta UM, Fatima N, Ahmed S, Chang LC, Carcache de Blanco EJ (2012)

Potent NF-(B inhibitors from endophytic l'ungus of a Taxus plant Planta Med

78PL24. doi: 10. 10554-0032-1321 358

Oral presentatioIls

Nighat Fatima, tbrar Khan, lhsan-ul-Haq, Muneer Ahmed Qazi, Abdul Hameed

and Safia Ahmed, "Biological Evaluation and Preliminary Screening of

Endophytes of Tctxus fuana (baccata) lbr Paclitaxel Production." ln: gth

lntemational Biennial Conference of Pakistan Society for Microbiology, January

28-l1, 2013, Karachi, Pakistan, pp. 27.

Muneer Ahmed Qazi, Saira Azecm, Zulfiqar Ali Malik, Nighat Fatima, Abdul

Hameed and Safia Ahmed. "Antimicrobial Potential of the Biosudactant

Produced by Pseudomonas putida SOL-10," In: gth Intemational Biennial

Conference of Pakistan Society for Microbiology, January 28-31,2013, Kamchi,

Pakistan, pp.26.

Adam Sher Khan. Nighat Fatima, Safia Ahmed, Rabaab Zahra, "Evaluation of

Antimicrobial Activity of Natural Fungal Products against Resistant

Microorganisms," In: 9th International Biennial Conference of Pakistan Societv

for Microbiology, January 28-31,2013, Karachi, Pakistan, pp.29.

Poster presented

Fatima N, Ahmed S, Kondratluk TP, Park E-J. Marler LE, Youn UJ, Qazi MA,

Pezzuto JM, Chang LC, "Cancer Chemoprevenlive Potential of Eudophytic Fungi

Isolated from Taxus;fuana," Poster session ptesented at: 52nd Annual Meeting of

Amcrican Society ofPharmacognosy (July 30-August 3. 2011). The University of

Califomia, San Diego. USA

Nighat Fatima, Safia Ahmed. 'famara P. Kondratyuk. Eun-Jung Park, Laura E.

Marler. Ui Joung Youn, Muneer Ahmed Qazi, John M. Pezzuto and Leng Chee

Chang, "Evaluation of Secondary Metabolites of Fungi of laxru.rar?a as Protein

242

Publications

Kinase lnhibitors," In: HI-ASM Spring Meeting (Hawaii Branch ' American

society ofMicrobilogy) USA , Apil 23' 2011'

Nighat Fatima, Usman Mukhtar, Muneer Ahmed Qazi' Muniba Jadoon' Naima

Atiq, Abdul Hameed, Safia Ahmed, "lsolation and Antimicrobial Screening of

Endophytic Fungi oI -/rs/acia adhatoda," ln: lst National Symposium on "New

Horizons ofMicrobiology" on 7-8 November' 2012' at FUUAST Karachi

Muniba Jadoon, Nighat Fatima, Sjdra Murtaza' Leng Chee Chang' Safia Ahmed

and Naeem Ali, "lsolatiol and Biological Evaluation of Proteinaceous Extract of

Endoph.vtic Fungal sffain NFWI"' Inl 9th International Biennial Conference of

Pakistan Society for Microbiology, January 28-31' 201i' Karachi' Pakislan' pp'

6'1.

Muneer Ahmed Qazi, Mishal Subhan, ZLrlfiqar Ali Malik' Nighat Fatima' Safia

Ahmed,Abdr.rlHameedandMuhammadtshtiaqAli'(RoleofBiosurfactant

Produced by Fusarium sp. BS-8 in Enhanced Oil Recovery (EoR) Through Sand

Pack Column," ln; 9th Intemational Biennial Conlercnce of Pakistan Society for

Microbiology, January 28-l1.2013, Kamchi' Pakistan' pp 77'

Salma Gul Shah, Farwa Rubab, Nighat Fatima, Sidra Hafeez' Naeem AIi' Abdul

Hameed and Safia Alnned. "Evaluation of FLrngal Isolate NFW8 as a Source of

Natuml Coloured Secondary Metabolites," In: 9th lnternational Biennial

Conference of Pakistan Society for Microbiology' January 28-31' 2013' Karachi'

Pakistan, pp.63.

243