Interactions of Desmethoxyyangonin, a Secondary Metabolite...

Research ArticleInteractions of Desmethoxyyangonin a Secondary Metabolitefrom Renealmia alpinia with Human Monoamine Oxidase-Aand Oxidase-B

Narayan D Chaurasiya1 Francisco Leoacuten2 Yuanqing Ding1 Isabel Goacutemez-Betancur3

Dora Benjumea3 Larry A Walker12 Stephen J Cutler14 and Babu L Tekwani12

1National Center for Natural Products Research School of Pharmacy The University of Mississippi University MS 38677 USA2Department of Biomolecular Sciences School of Pharmacy The University of Mississippi University MS 38677 USA3Programa de OfidismoEscorpionismo Sede de Investigacion Universitaria Facultad de Ciencias Farmaceuticas y AlimentariasUniversidad de Antioquia Torre 2 Laboratorio 631 Medellın Colombia4College of Pharmacy University of South Carolina Columbia SC 29208 USA

Correspondence should be addressed to Babu L Tekwani btekwaniolemissedu

Received 23 April 2017 Revised 24 June 2017 Accepted 17 July 2017 Published 24 August 2017

Academic Editor Cheorl-Ho Kim

Copyright copy 2017 Narayan D Chaurasiya et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Renealmia alpinia (Zingiberaceae) a medicinal plant of tropical rainforests is used to treat snakebites and other injuries and alsoas a febrifuge analgesic antiemetic antiulcer and anticonvulsant The dichloromethane extract of R alpinia leaves showed potentinhibition of human monoamine oxidases- (MAOs-) A and B Phytochemical studies yielded six known compounds includingpinostrobin 1 41015840-methyl ether sakuranetin 2 sakuranetin 3 pinostrobin chalcone 4 yashabushidiol A 5 and desmethoxyyangonin6 Compound 6 displayed about 30-fold higher affinity for MAO-B than MAO-A with Ki values of 31 and 922 nM respectivelyKinetic analysis of inhibition and equilibrium-dialysis dissociation assay of the enzyme-inhibitor complex showed reversiblebinding of desmethoxyyangonin 6 with MAO-A and MAO-B The binding interactions of compound 6 in the active site ofthe MAO-A and MAO-B isoenzymes investigated through molecular modeling algorithms confirmed preferential binding ofdesmethoxyyangonin 6 with MAO-B compared to MAO-A Selective reversible inhibitors of MAO-B like desmethoxyyangonin6 may have important therapeutic significance for the treatment of neurodegenerative disorders such as Parkinsonrsquos disease andAlzheimerrsquos disease

1 Introduction

The Renealmia genus (Zingiberaceae) belongs to the rareclass of amphi-Atlantic plants well represented in tropicalAfrica and Americas [1 2] Several Renealmia species havebeen used for folk medicine and food on both sides of theAtlantic Decoctions of fruits from R congoensis are usedto treat stomachache in Cameroon children [3] Fruits ofR cincinnata are commonly used as a spice and utilized bytraditional healers to treat infectious diseases in NorthwestCameroon [4] In the Americas several species of Renealmiahave been reported to treat different diseases [5]R thyrsoideais used to treat skin infections associated with leishmaniasisand to reduce fever [6] The crushed stem or infusion of R

alpinia is used to treat headaches stomachaches and bodyfatigue by the Amazon-Yanomami Indians [7] Edible fruitsof R alpinia are valuable for taste in the Sierra Norte dePuebla (Mexico) and Ecuador [8] In Trinidad R alpiniacrushed fruitsmixedwith the juice ofCostus scaber have beenfound effective for treatment of snakebites [9] Otero et al[10] reported extensive investigations on the nativeNorthwestColombian medicinal plants used by indigenous Embera-Katios tribes They highlighted the use of R alpinia rhizomesorand leaves as aqueous extracts decoctions or poultice forthe treatment of snakebites Moreover the aqueous extractsof R alpinia showed neutralizing effect against Bothropsasper venom through inhibition of proteinases present in thesnake toxin [11] Pinostrobin the main bioactive constituent

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 4018724 10 pageshttpsdoiorg10115520174018724

2 Evidence-Based Complementary and Alternative Medicine

O

O

O

O

OH

OH O

O

O

O

O

O

1 2

4 6

3

5

（3

（3

（3 （3

（3

OHOH

OH

OH

OHOH

Figure 1 Chemical structures of Renealmia alpinia constituents Pinostrobin 1 41015840-methyl ether sakuranetin 2 sakuranetin 3 pinostrobinchalcone 4 yashabushidiol A 5 and desmethoxyyangonin 6

showed inhibitory effects on the enzymatic anticoagu-lant myotoxic and edema inducing activities of phospho-lipase A2 (PLA2) isolated from Crotalus durissus venom[12]

Additionally the extracts of R alpinia showed inhibitiontowards indirect hemolytic coagulant effects and proteolyticactivity produced by B asper venom Pinostrobin was foundto be the bioactive component in the extract responsiblefor this effect [13] Recently we explored the antinociceptiveeffects of methanol and aqueous extracts of R alpinia inin vivo models Comprehensive phytochemical analysis ofR alpinia yielded pinostrobin 1 along with two flavonoids(naringenin 741015840-dimethyl ether 2 and naringenin 7-methylether 3) one chalcone (2101584061015840-dihydroxy-41015840-methoxychalcone4) one diarylheptanoid (35-heptanediol-17-diphenyl 5) andone kavalactone (desmethoxyyangonin 6) [14] (Figure 1)This was the first report on the isolation of compound 6 froma Renealmia species [15]

Increasing efforts have been made to identify dual mo-noamine oxidases (MAOs) inhibitory and anti-inflamma-tory agents which enhance cognitive functions and de-layprevent progression of neurodegenerative diseases [16ndash18] Dichloromethane extract of R alpinia and individualconstituents isolated from this extract were evaluated invitro against the recombinant human MAO-A and MAO-B Binding interactions of desmethoxyyangonin 6 the mostprominentMAO inhibitory constituent in R alpinia extractsin the active site of the MAO-A and MAO-B isoenzymeswere investigated through enzyme-kinetics assays enzyme-inhibitor complex binding equilibrium-dialysis dissociationanalyses and computationalmolecularmodeling algorithmsThese studies may have implications for future research andscientifically validate traditional use of R alpinia as a poten-tial therapeutic agent for treatment of neurodegenerativedisorders as well as the use ofR alpinia as a functional dietarybenefit for the local populations

2 Materials and Methods

21 Reagents and Chemicals Pure recombinant humanmonoamine oxidases (MAO-A and MAO-B) enzymes over-expressed in baculovirus (Autographa californica) infectedinsect cells (BTI-TN-5B1-4) were purchased from BD Bio-sciences (Bedford MA USA) Kynuramine bromide 4-hydroxyquinoline clorgyline R (minus) deprenyl and DMSOwere obtained from Sigma Chemicals Company (St LouisMO USA) R alpinia extract and six evaluated compounds1ndash6 were obtained from the isolation procedures describedpreviously [14] These compounds have been kept at minus20∘Cuntil evaluation Previous to assay to verify the stabilityof compounds 1ndash6 the spectrometric and spectroscopicanalysis was done No degradation products were detected

22 Determination of MAOs Inhibition Activity of the Com-pounds In vitro assays were performed to measure theinhibitory effects of R alpinia dichloromethane extractand its purified compounds 1ndash6 on human recombinantMAO-A and MAO-B activity The dichloromethane extract(0001 to 100 120583gmL) purified compounds (10minus9 to 10minus2M)and standard MAO inhibitors (phenelzine clorgyline anddeprenyl) (10minus12 to 10minus5M) were tested on human MAO-A and MAO-B enzymes [19] Stock solutions of the testcompoundsextracts were prepared in DMSO and dilutedfurther in 01M potassium phosphate buffer (pH 74) toobtain the desired concentrations MAO-A and MAO-B activities were determined by fluorometric kynuraminedeamination assay set up in 384-well solid white flat-bottomplates [20] MAO-A and MAO-B inhibition activities (IC50values) were determined using fixed concentration of thesubstrate kynuramine and varying concentrations of thetest compounds or extracts The enzyme reactions werecarried out in 01M potassium phosphate buffer (pH-74)Reactionmixture (total volume 75120583L in each well) containedpotassium phosphate buffer (01M pH 74) kynuramine(80 120583M for MAO-A assay and 50120583M for MAO-B assay) the

Evidence-Based Complementary and Alternative Medicine 3

test compound or extract (to the desired concentration) andthe enzyme (375 ng for MAO-A or 9375 ng for MAO-B)The reaction mixtures with buffersubstrateinhibitor werepreincubated for 10 minutes at 37∘C followed by addition ofMAO-A and MAO-B to initiate the reaction The solid whitemicroplates were incubated for 20 minutes at 37∘C and theenzymatic reaction was stopped by addition of 28 120583L of 2NNaOH to each well The deaminated product of kynuraminewhich spontaneously cyclizes to 4-hydroxyquinoline wasmeasured fluorometrically at 320 nm excitation and 380 nmemission wavelengths with a plate reader (SpectraMax M5Molecular Devices Sunnyvale CA USA) The IC50 valueswere computed by XL-Fit from the dose-response inhibitioncurves

23 Enzyme Kinetics and Mechanism of Inhibition of MAOsThe potential inhibitor desmethoxyyangonin 6 was selectedfor inhibition kinetic studies with MAO-A and MAO-BFor the enzyme-kinetics analysis assays were performed atvarying concentrations of kynuramine (190120583M to 500 120583M)for determination of the enzyme inhibition constants (Ki) forinhibition ofMAO-AandMAO-Bwith compound6 In addi-tion to controls without inhibitors two concentrations (onebelow and one above IC50 values) of the inhibitors [forMAO-A phenelzine (0450 120583M and 0900 120583M) desmethoxyyango-nin 6 (0125 120583M and 0250 120583M) and for MAO-B phenelzine(0050120583Mand 0100 120583M) desmethoxyyangonin 6 (0045 120583Mand 0090 120583M)] were tested Results are presented as doublereciprocal Lineweaver-Burk plots The kinetic data namely119870M 119881max and Ki values were calculated by SigmaPlot123 with enzyme-kinetics module using Michaelis-Mentenequation The results were also analyzed for the type ofinhibition

24 Equilibrium Dialysis Assay for Analysis of Binding ofDesmethoxyyangonin 6 with MAOs Binding and inhibitionof MAO-A and MAO-B with desmethoxyyangonin 6 werefurther examined by incubating the enzyme with high con-centrations of the inhibitor followed by extensive dialysisof the enzyme-inhibitor complex and recovery of enzymeactivities MAO-A (005mgmL protein) was incubated withdesmethoxyyangonin 6 (20120583M and 1000 120583M) and MAO-B(005mgmL protein) was incubated with desmethoxyyan-gonin 6 (150 120583M and 200120583M) in an enzyme incubationmixture of 1mL containing 100mM potassium phosphatebuffer (pH 74) Formation of enzyme-inhibitor complex wasallowed by incubation of the reaction mixtures for 20min at37∘C The enzyme-inhibitor mixtures after incubation werechilled transferred to the dialysis bags and dialyzed against25mM potassium phosphate buffer (pH 74) for 14ndash16 hoursat 4∘C (changing the dialysis buffer three times) The enzymecatalytic activities were measured before and after dialysis

25 Time-Dependent Enzyme Inhibition Assay MAO-Aand MAO-B enzymes were preincubated for different timeperiods (0ndash15min) with the inhibitor Concentrations ofthe inhibitor tested for time-dependent inhibition with20120583gmL MAO-A were 750120583M (desmethoxyyangonin 6)and 0600120583M (phenelzine) and with 50 120583gmL MAO-B

were 040 120583M (desmethoxyyangonin 6) and 0100 120583M(phenelzine) Controls without inhibitors were also runsimultaneously The enzyme activities were determined asdescribed above

26 Computational Methods and Software Packages Thecrystal structures of MAO-A and MAO-B were downloadedfrom the Protein Data Bank with PDB IDs 2Z5X for MAO-A [21] and 1OJ9 for MAO-B [22] Water molecules wereremoved and protein structures were preprocessed reviewedmodified and refined [23] using the Protein PreparationWizard [24] A ldquostandardrdquo mode of the ProtAssign algorithmwas run to optimize the hydrogen-bonding (H-bond) net-work at neutral pH The Impref module of Impact [25] andthe OPLS 2005 force field [26ndash28] were employed to relaxthe entire structure in the recommended protein preparationprotocol The docking calculations were carried out usingthe Glide software [29] The centroid of ligands harmine(HRM) in 2Z5X and 14-diphenyl-2-butene (1PB) in 1OJ9was set as the center of active site The dock ligand lengthwas set as 25 A and the XP module in Glide was used torank the obtained binding poses Prior to docking the liganddesmethoxyyangonin 6 was prepared using the LigPrep[30] Molecular mechanics combined with the generalizedBorn surface area continuum solvationmethod (MMGBSA)were employed to calculate the protein-ligand binding freeenergies of the docking conformations [31] using Prime[32] of the Schrodinger software suite [24] All residuesthat have atoms inside 5 A from the ligand were treatedflexibly

3 Results

31 MAOs Inhibitory Properties of R alpinia Extract andCompounds 1ndash6 R alpinia dichloromethane extract showedsignificant inhibitory effect with IC50 values of 375 and170 120583gmL for the MAO-A and MAO-B respectively Theextract was subjected to purification using a silica gelcolumn chromatography yielding six purified compounds1ndash6 (Figure 1) The structures of these constituents wereelucidated on the basis of spectroscopic data (EIMS 1HNMRand 13CNMR HSQC and HMBC) by comparison withthose reported in the literature [14] Compounds 1ndash6 wereevaluated against MAO-A and MAO-B inhibition assaysCompounds 1ndash5 showedmoderate inhibition effect howevercompound 6 exhibited potent MAO inhibition (Table 1)Noticeably desmethoxyyangonin 6 showed 15-fold morepreferential inhibition ofMAO-B thanMAO-A evidenced bythe IC50 values of 1850 and 0123 120583M forMAO-A andMAO-B respectively (Table 1 Figure 2)

32 Enzyme Kinetics and Mechanism of Inhibition of MAOIsoenzymes with Desmethoxyyangonin 6 We further eval-uated the kinetics and mechanism of inhibition of humanMAO-A and MAO-B isoenzymes by desmethoxyyangonin6 (Figures 3 and 4) To comprehend the type of inhibitionwe examined 6 against both MAO-A and MAO-B at vary-ing concentrations of kynuramine a nonselective substrateDesmethoxyyangonin 6 was tested at two concentrations


Table 1 Inhibition (IC50 values) of recombinant human MAO-A and MAO-B by R alpinia extract and isolated compounds 1ndash6lowast

Samples Monoamine oxidase-AIC50 (120583M)

Monoamine oxidase-BIC50 (120583M) Index MAO-AB

Renealmia alpiniadichloromethane extract 3750 plusmn 0283a 1700 plusmn 02121a 2201

Pinostrobin 1 23895 plusmn 1346 45547 plusmn 4314 052441015840-Methyl ethersakuranetin 2 31400 plusmn 4577 gt100 mdash

Sakuranetin 3 45482 plusmn 5715 36505 plusmn 6626 1246Pinostrobin chalcone 4 6326 plusmn 0206 10036 plusmn 3237 0610Yashabushidiol A 5 gt100 35384 plusmn 0121 mdashDesmethoxyyangonin 6 1850 plusmn 0086 01233 plusmn 00095 15040Phenelzine 0235 plusmn 00218 0150 plusmn 00095 1566Clorgyline 00046 plusmn 0003 mdash mdashDeprenyl mdash 0032 plusmn 0012 mdashlowastThe IC50 values computed from the dose response inhibition curves are Mean plusmn SD of triplicate observations a120583gmL

01 1 10

100

80

60

40

20

Concentration (－)

Activ

ity (

)

100

80

60

40

20

0

001 01 1

Concentration (－)

Activ

ity (

)

MAO-A phenelzine

MAO-B phenelzine

(a)

100

80

60

40

20

0

001 01 1

Concentration (－)

Activ

ity (

)

1 10

100

80

60

40

20

0

Concentration (－)

Activ

ity (

)

MAO-A desmethoxyyangonin

MAO-B desmethoxyyangonin

(b)

Figure 2 Concentration dependent analysis of inhibition of recombinant human monoamine oxidase-A and monoamine oxidase-B bydesmethoxyyangonin 6 and phenelzine


1V

1[S]

07

06

05

04

03

02

01

00

minus01minus005 000 005 010 015 020 025 030

000 － phenelzine0250 － phenelzine0550 － phenelzine

(a)

1V

1[S]

07

06

05

04

03

02

01

00

minus01minus005 000 005 010 015 020 025 030

160 － desmethox000 － desmethox

320 － desmethox

(b)

Figure 3 Kinetics analysis of inhibition of recombinant human MAO-A with (a) desmethoxyyangonin 6 and (b) phenelzine 119881 =nmolesminmg protein and S = substrate (kynuramine) concentration (120583M)

1V

1[S]

06

05

04

03

02

01

00

minus01

minus005 000 005 010 015 020 025 030


(a)

1V

1[S]minus005 000 005 010 015 020 025 030

12

10

08

06

04

02

00

000 － desmethox0125 － desmethox0250 － desmethox

(b)

Figure 4 Kinetics analysis of inhibition of recombinant human MAO-B with (a) desmethoxyyangonin 6 and (b) phenelzine 119881 =nmolesminmg protein and S = substrate (kynuramine) concentration (120583M)

one above and the other below the IC50 value For eachexperiment three sets of assays were done at variableconcentrations of the substrate two concentrations of theinhibitorcompound and one control without inhibitors Theresults are presented as double reciprocal Lineweaver-Burkplots and the kinetic data namely 119870119872 119881max and 119870119894 valueswere computed by SigmaPlot 123 with enzyme-kineticsmodule using Michaelis-Menten equation (Table 2) Theresults suggest that desmethoxyyangonin 6 binds as a mixedinhibitor with the human MAO-A (Figure 3) HoweverMAO-B inhibition by 6 was competitive (Figure 4) Thebinding affinities of compound 6 with MAO-A and MAO-Bwere compared with reference MAO inhibitors

33 Analysis of Time-Dependent Enzyme Inhibition and Bind-ing of Compound 6 with MAOs In order to examine thetime-dependent binding inhibition of MAO-A and MAO-B the enzymes were preincubated with inhibitor for theindicated time (0ndash15min) at concentrations that causednearly 60ndash70 inhibition (Figure 5) The control enzymeswithout inhibitor were also run concurrently The resultsshow that inhibition of MAO-A and MAO-B (Figure 5) bydesmethoxyyangonin 6 was not time-dependent The bind-ing characteristics of desmethoxyyangonin 6 with MAO-A and MAO-B were examined by equilibrium dialysis tomeasure dissociation of the enzyme-inhibitor complex (Fig-ure 6) MAO-A and MAO-B were incubated with high


Table 2 Inhibitionbinding affinity constants (119870119894) for inhibition of recombinant human MAO-A and MAO-B by desmethoxyyangonin 6and phenelzinelowast

Compounds Monoamine oxidase-A Monoamine oxidase-B119870119894 (nM) Type of Inhibition 119870119894 (nM) Type of Inhibition

Desmethoxyyangonin 6 9229 plusmn 0025 Mixedreversible 310 plusmn 0003 CompetitivereversiblePhenelzine 1460 plusmn 0009 Mixedirreversible 1100 plusmn 0005 MixedirreversiblelowastValues are mean plusmn SD of triplicate experiments

ac

tivity

Time (min)

ControlPhenelzine (060 －)

Desmethoxyyangonin (750 －)

150

125

100

75

50

25

0

0 5 10 15

MAO-A time-dependent

(a)

ac

tivity

150

125

100

75

50

25

0

Time (min)0 5 10 15

Phenelzine (010 －)


Control

MAO-B time-dependent

(b)

Figure 5 (a) Time-dependent inhibition of recombinant human MAO-A by phenelzine (0600 120583M) and desmethoxyyangonin 6 (750 120583M)Each point representsmeanplusmn SDof triplicate values (b) Time-dependent inhibition of recombinant humanMAO-Bby phenelzine (0100 120583M)and desmethoxyyangonin 6 (0400120583M) Each point represents mean plusmn SD of triplicate values

concentration of desmethoxyyangonin 6 for 20 minutes at37∘C to allow binding of inhibitor with the enzyme andformation of enzyme-inhibitor complex The mixtures ofenzyme-inhibitor complex were dialyzed overnight at 4∘Cagainst 25mMKHPO4 (pH-74) bufferThe enzyme activitieswere examined before and after dialysis Through overnightdialysis the recombinant human MAO-A enzyme lost about10ndash15 of activity Incubation of MAO-A with 200 and100 120583M concentrations of desmethoxyyangonin 6 inhibitedmore than 65 of the enzyme activity (Figure 6) Afterthe dialysis more than 90 catalytic activity of MAO-Awas recovered from enzyme-desmethoxyyangonin 6 incu-bation mixtures Similarly MAO-B lost about 10ndash15 ofthe enzyme activity during overnight dialysis Incubationof desmethoxyyangonin 6 (150 and 200 120583M) with MAO-B caused more than 90 inhibition of enzyme activitywhich was fully recovered after dialysis (Figure 6) Theseobservations suggest that the inhibition of MAO-A andMAO-B by desmethoxyyangonin 6 was reversible due todissociable nature of their enzyme-inhibitor complexes

34 Molecular Modeling-Based Analysis of Interaction ofDesmethoxyyangonin 6 with MAO-A and MAO-B Selectiveinhibition of MAO-B compared to MAO-A by desmethox-yyangonin 6 led us to the investigations on interactions of

Table 3 Docking scores and MMGBSA binding energies ofpreferred binding poses of desmethoxyyangonin 6 in MAO-A andMAO-B

Methods MAO-Aa MAO-Bb

DSXPc minus389 minus798MMGBSAd minus3510 minus6295aHuman monoamine oxidase-A (pdb id 2Z5X) bHuman monoamineoxidase-B (pdb id 1OJ9) cDocking scores obtained by using extra precisionmodule (Kcalmol) dBinding free energy that does not include contributionsfrom receptor or ligand strain (Kcalmol)

compound 6 with the human MAO-A and MAO-B employ-ing computational molecular modeling algorithms The pre-ferred binding poses of desmethoxyyangonin 6 in MAO-A and MAO-B are shown in Figure 7 and their dockingscores and MMGBSA binding energies are listed in Table 3The docking scores for the favored complexes with MAO-A and MAO-B are minus389 and minus798 kcalmol respectivelyThese scores confirm better binding of desmethoxyyangonin6 to MAO-B than to MAO-A and support the experimentalobservations on selective inhibition of MAO-B compared toMAO-A with desmethoxyyangonin 6 MMGBSA bindingenergies were computed as minus3510 and minus6295 Kcalmol forthe MAO-A and MAO-B complexes respectively These


0

20

40

60

80

100

120

activ

ity

Phen

elzi

ne (2

50)

Phen

elzi

ne (2

50)

Con

trol

DM

A (1

000

)

DM

A (2

00)

Concentration (－)

Before dialysisAfter dialysis

(a)

0

20

40

60

80

100

120

ac

tivity

Phen

elzin

e (030

)

Phen

elzin

e (10

0)

Con

trol

DM

A (1

50

)

DM

A (2

00)


Concentration (－)

(b)

Figure 6 (a) Analysis of binding of phenelzine and desmethoxyyangonin 6with recombinant humanMAO-A Recovery of catalytic activityof the enzyme after equilibrium dialysis of the enzyme-inhibitor complex Each bar shows mean plusmn SD of triplicate values (b) Analysis ofbinding of phenelzine and desmethoxyyangonin 6 with recombinant human MAO-B Recovery of catalytic activity of the enzyme afterequilibrium dialysis of the enzyme-inhibitor complex Each bar shows mean plusmn SD of triplicate values DMA = desmethoxyyangonin

observations are consistent with their docking scores andobserved experimental IC50 and 119870119894 values Further analysisof these interactions indicates that the pyranone moietyof compound 6 120587-120587 stacks with the phenol moiety ofresidue Y326 in MAO-B complex (Figure 7(b)) which wascorrespondingly replaced by residue I335 inMAO-A complex(blue-circled in Figures 7(c) and 7(d) resp) To facilitatethis 120587-120587 stacking interaction compound 6 in MAO-B staysfarther away from FAD than that in MAO-A (Figures 7(e)and 7(f)) The sulfhydryl hydrogen of residue C172 in MAO-B complex also interacts with pyran and ketone oxygens of6 which was correspondingly replaced by residue N181 incomplex MAO-A (white-circled in Figures 7(c) and 7(d)resp) In spite of these differences residues interactingwith the pyranone moiety are very similar in both MAO-A and MAO-B complexes Specifically the pyranone moietyinteracts with I207 Y444 Y69 Y407 F352 Q215 and L337 inMAO-A complex and correspondingly with I198 Y435 Y60Y398 F343Q206 and L328 inMAO-B complex respectivelyThe residues which interact with phenyl vinyl moiety ofdesmethoxyyangonin 6 are quite different in MAO-A andMAO-B complexes Specifically inMAO-B complex residuesP104 W119 and F168 contact with phenyl vinyl moiety ofdesmethoxyyangonin 6 whereas only one aromatic residueF208 was found to be involved in interaction with phenylvinyl moiety in MAO-A complex (yellow-circled in Figures7(c) and 7(d)) In addition phenyl vinyl moiety was found tohave contact alsowith residues I180 I325 L97 A111 C323 andV210 in MAO-A complex but with residues I199 I316 L164P102 L167 and L171 in MAO-B complex (green-circled in

Figures 7(c) and 7(d))These differences in interaction mightcause rotation of phenyl vinyl moiety of approximately 180∘above the (pyranone) C-C(vinyl) bond from its position inMAO-A to that inMAO-B (Figures 7(c) and 7(f)) and furtherimply better binding of desmethoxyyangonin 6 to MAO-Bthan to MAO-A

4 Discussion

Many herbs remedies contain MAO inhibitors without theunpleasant side effects Recently Carradori et al [33] havelisted common natural sources and the chemical featuresresponsible for inhibition of MAO-B justifying the potentialuse of folk herbs and natural products for treatment ofneurodegenerative diseases Desmethoxyyangonin 6 is oneof the main kava-pyrone derivatives known as kavalactonesKavalactones are bioactive principles of the traditional bev-erage kava-kava made from Piper methysticum used to treatanxiety [34] The neurobiological activities of kavalactonesprimarily include modulation of gamma-aminobutyric acidtype A receptors (GABA) [35] Isolated kavalactones haveshown other neurological activities potential for the treat-ment of neurodegenerative diseases In fact some potentialtherapeutic actions namely anxiety tension and restless-ness of standardized extracts of kava-kava roots have beenattributed to kava pyrones desmethoxyyangonin 6 and(+minus)-methysticin through inhibition of platelet MAO-B[36] Similarly kavapyrones have also been reported to showseveral psychotropic and neuropharmacological propertiesnamely relaxation euphoria sleepiness skeletal muscle


(a) (b)

(c) (d)

(e) (f)

Figure 7 (a c) The most preferred binding poses of desmethoxyyangonin 6 in MAO-A and (b d) for MAO-B (e f) The superposition ofthe complexes

relaxation anticonvulsant properties neuroprotection andanalgesia Treatment of rats with kava extract caused changesin their behavior and increased the levels of dopamine innucleus accumbens [37] Methysticin kavain and yangoninhave been reported to induce ERK12 phosphorylation whiledihydro-56-dehydrokavain and desmethoxyyangonin havebeen shown to inhibit peroxide-induced P38 phosphory-lation [38] Desmethoxyyangonin 6 has been reported toprevent inflammation and hepatitis in mice through attenu-ation of lipopolysaccharide- (LPS-) stimulated inflammation

in murine macrophages and LPSD-galactosamine- (LPSD-GalN-) induced fulminant hepatitis in mice [15] Moreoverkavalactones such as desmethoxyyangonin 6 have not beenlisted under PAINS (pan assay interference) and 6 does notcontain any PAIN-like motif [39 40]

The presence of pinostrobin 1 and desmethoxyyango-nin 6 in R alpinia further explains the potential bene-fits of this plant for human health and nutrition Anti-inflammatory properties and selective reversible inhibition ofhuman MAO-B by desmethoxyyangonin 6 suggest potential


therapeutic use of this kavalactone for treatment of neurode-generative diseases like Parkinsonrsquos disease

5 Conclusion

In this study phytochemical analysis of dichloromethaneextract of R alpinia after fractionation and purificationproduced pure desmethoxyyangonin 6 which was furtherevaluated for the inhibitory effects against recombinanthuman MAO-A and MAO-B enzymes Desmethoxyyango-nin 6 was identified as a prominent MAO-A and MAO-B inhibitory constituent from R alpinia The computa-tional docking and thermodynamic analysis of MAO-A andMAO-B complexed with desmethoxyyangonin 6 support theexperimental results regarding 29-fold selective inhibition ofMAO-B compared to MAO-A The presence of prominentMAO-B constituent supports the R alpinia folkloric use anddesmethoxyyangonin 6 can be used as a lead compound forrational design of anti-Parkinsonrsquos disease agents

Disclosure

The content is solely the responsibility of the authors anddoes not necessarily represent the official views of theNIGMS or the NIH A portion of the results reported in thispaper was presented at the 15th Annual Oxford InternationalConference on the Science of Botanicals (ICSB) Oxford MSUSA and the abstract has been published in a supplementissue of Planta Medica (Planta Med 2015 81 ndashPB19) (httpsthieme-connectcomproductsejournalsabstract101055s-0035-1545174)

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This study has been supported in part by the NationalInstitute of General Medical Sciences (NIGMS) (Grant noP20GM104932) a component of the National Institutes ofHealth (NIH) and the Sustainability Strategy 2015-2016of the OphidismScorpionism Program and Serpentariumof University of Antioquia Furthermore this investigationwas conducted in a facility constructed with support fromresearch facilities improvement program C06RR14503 fromthe NIH National Center for Research Resources (NCRR)The authors are thankful to the National Center for NaturalProducts Research for the facilities to support this work

References

[1] T E Sarkinen M F Newman P J M Maas et al ldquoRecentoceanic long-distance dispersal and divergence in the amphi-Atlantic rain forest genus Renealmia Lf (Zingiberaceae)rdquoMolecular Phylogenetics and Evolution vol 44 no 3 pp 968ndash980 2007

[2] A Antonelli and I Sanmartın ldquoWhy are there so many plantspecies in the Neotropicsrdquo Taxon vol 60 no 2 pp 403ndash4142011

[3] D A Focho W T Ndam and B A Fonge ldquoMedicinal plantsof Aguambu-Bamumbu in the Lebialem highlands SouthwestProvince of Cameroonrdquo African Journal of Pharmacy andPharmacology vol 3 no 1 pp 1ndash13 2009

[4] M H K Tchuendem J A Mbah A Tsopmo et al ldquoAnti-plasmodial sesquiterpenoids from the African Renealmiacincinnatardquo Phytochemistry vol 52 no 6 pp 1095ndash1099 1999

[5] R R B Negrelle ldquoRenealmia lf Botanical pharmacologicaland agronomical aspectsrdquo Revista Brasileira de Plantas Medici-nais vol 17 no 2 pp 274ndash290 2015

[6] B J Cabanillas A-C Le Lamer D Olagnier et al ldquoLeishmani-cidal compounds and potent PPAR120574 activators from Renealmiathyrsoidea (Ruiz amp Pav) Poepp amp Endlrdquo Journal of Ethnophar-macology vol 157 pp 149ndash155 2014

[7] W Milliken and B Albert ldquoThe use of medicinal plants by theYanomami Indians of Brazilrdquo Economic Botany vol 50 no 1pp 10ndash25 1996

[8] M J Macıa ldquoRenealmia alpinia (Rottb) Maas (Zingiberaceae)an edible plant from Sierra Norte de Puebla (Mexico)rdquo Analesdel Jardın Botanico de Madrid vol 60 no 1 pp 183ndash187 2002

[9] C Lans T Harper K Georges and E Bridgewater ldquoMedicinaland ethnoveterinary remedies of hunters in Trinidadrdquo BMCComplementary and Alternative Medicine vol 1 article 10 17pages 2001

[10] R Otero R Fonnegra S L Jimenez et al ldquoSnakebites andethnobotany in the northwest region of Colombia Part Itraditional use of plantsrdquo Journal of Ethnopharmacology vol 71no 3 pp 493ndash504 2000

[11] A Camilo Patino D Marıa Benjumea and J Andres PereanezldquoInhibition of venom serine proteinase and metalloproteinaseactivities by Renealmia alpinia (Zingiberaceae) extracts Com-parison of wild and in vitro propagated plantsrdquo Journal ofEthnopharmacology vol 149 no 2 pp 590ndash596 2013

[12] I Gomez-Betancur J A Pereanez A C Patino and DBenjumea ldquoInhibitory effect of pinostrobin from Renealmiaalpinia on the enzymatic and biological activities of a PLA2rdquoInternational Journal of Biological Macromolecules vol 89 pp35ndash42 2016

[13] I Gomez-Betancur D Benjumea A Patino N Jimenez and EOsorio ldquoInhibition of the toxic effects of Bothrops asper venomby pinostrobin a flavanone isolated from Renealmia alpinia(Rottb) MAASrdquo Journal of Ethnopharmacology vol 155 no 3pp 1609ndash1615 2014

[14] I Gomez-Betancur N Cortes D Benjumea E Osorio FLeon and S J Cutler ldquoAntinociceptive activity of extracts andsecondarymetabolites fromwild growing andmicropropagatedplants of Renealmia alpiniardquo Journal of Ethnopharmacology vol165 pp 191ndash197 2015

[15] T-W Chou J-H Feng C-C Huang et al ldquoA plant kavalactonedesmethoxyyangonin prevents inflammation and fulminanthepatitis in micerdquo PLoS ONE vol 8 no 10 Article ID e776262013

[16] N Gokhan-Kelekci S Yabanoglu E Kupeli et al ldquoA new ther-apeutic approach in Alzheimer disease Some novel pyrazolederivatives as dual MAO-B inhibitors and antiinflammatoryanalgesicsrdquo Bioorganic and Medicinal Chemistry vol 15 no 17pp 5775ndash5786 2007

[17] J G Villarinho K D V Pinheiro F D V Pinheiro et alldquoThe antinociceptive effect of reversible monoamine oxidase-A inhibitors in a mouse neuropathic pain modelrdquo Progress inNeuro-Psychopharmacology and Biological Psychiatry vol 44pp 136ndash142 2013


[18] W Liu A Rabinovich Y Nash et al ldquoAnti-inflammatoryand protective effects of MT-031 a novel multitarget MAO-Aand AChEBuChE inhibitor in scopolamine mouse model andinflammatory cellsrdquo Neuropharmacology vol 113 pp 445ndash4562017

[19] N D Chaurasiya V Gogineni K M Elokely et al ldquoIsolationof acacetin from Calea urticifolia with inhibitory propertiesagainst human monoamine oxidase-A and -Brdquo Journal ofNatural Products vol 79 no 10 pp 2538ndash2544 2016

[20] S Parikh S Hanscom P Gagne C Crespi and C PattenldquoA Fluorescent-based high-throughput assay for detectinginhibitors of humanrdquo Tech Rep S02T081R2 BD BiosciencesDiscovery Labware Woburn Mass USA 2002

[21] S-Y Son J Ma Y Kondou M Yoshimura E Yamashitaand T Tsukihara ldquoStructure of human monoamine oxidaseA at 22-A resolution The control of opening the entry forsubstratesinhibitorsrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 105 no 15 pp 5739ndash5744 2008

[22] C Binda M Li F Hubalek N Restelli D E Edmondson andA Mattevi ldquoInsights into the mode of inhibition of humanmitochondrial monoamine oxidase B from high-resolutioncrystal structuresrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 100 no 17 pp 9750ndash9755 2003

[23] G Madhavi Sastry M Adzhigirey T Day R Annabhimojuand W Sherman ldquoProtein and ligand preparation parametersprotocols and influence on virtual screening enrichmentsrdquoJournal of Computer-Aided Molecular Design vol 27 no 3 pp221ndash234 2013

[24] ldquoSchrodinger Suite 2015-3 Protein Preparation Wizard Epikversion 33 Schrodingerrdquo Impact version 68 Schrodinger LLCNew York NY USA 2015 Prime version 41 Schrodinger LLCNew York NY USA 2015

[25] J L Banks H S Beard Y Cao et al ldquoIntegrated ModelingProgram Applied Chemical Theory (IMPACT)rdquo Journal ofComputational Chemistry vol 26 no 16 pp 1752ndash1780 2005

[26] W L Jorgensen and J Tirado-Rives ldquoThe OPLS [optimizedpotentials for liquid simulations] potential functions for pro-teins energy minimizations for crystals of cyclic peptides andcrambinrdquo Journal of the American Chemical Society vol 110 no6 pp 1657ndash1666 1988

[27] G A Kaminski R A Friesner J Tirado-Rives and W LJorgensen ldquoEvaluation and reparametrization of the OPLS-AA force field for proteins via comparison with accuratequantum chemical calculations on peptidesrdquo Journal of PhysicalChemistry B vol 105 no 28 pp 6474ndash6487 2001

[28] D Shivakumar J Williams Y WuW Damm J Shelley andWSherman ldquoPrediction of absolute solvation free energies usingmolecular dynamics free energy perturbation and the opls forcefieldrdquo Journal of Chemical Theory and Computation vol 6 no5 pp 1509ndash1519 2010

[29] Glide version 68 Schrodinger LLC New York NY USA 2015[30] LigPrep version 35 Schrodinger LLC New York NY USA

2015[31] C Mulakala and V N Viswanadhan ldquoCould MM-GBSA be

accurate enough for calculation of absolute proteinligand bind-ing free energiesrdquo Journal of Molecular Graphics andModellingvol 46 pp 41ndash51 2013

[32] Prime Schrodinger LLC New York NY USA 2014

[33] S Carradori M DrsquoAscenzio P Chimenti D Secci and ABolasco ldquoSelective MAO-B inhibitors A lesson from naturalproductsrdquoMolecular Diversity vol 18 no 1 pp 219ndash243 2014

[34] A R Bilia S Gallori and F F Vincieri ldquoKava-kava and anxietyGrowing knowledge about the efficacy and safetyrdquo Life Sciencesvol 70 no 22 pp 2581ndash2597 2002

[35] H C Chua E T H Christensen K Hoestgaard-Jensen etal ldquoKavain the major constituent of the anxiolytic kavaextract potentiates gabaa receptors Functional characteristicsand molecular mechanismrdquo PLoS ONE vol 11 no 6 Article IDe0157700 2016

[36] R Uebelhack L Franke and H-J Schewe ldquoInhibition ofplatelet MAO-B by kava pyrone-enriched extract from Pipermethysticum forster (kava-kava)rdquo Pharmacopsychiatry vol 31no 5 pp 187ndash192 1998

[37] S Sallstrom Baum R Hill and H Rommelspacher ldquoEffect ofkava extract and individual kavapyrones on neurotransmitterlevels in the nucleus accumbens of ratsrdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 22 no 7 pp1105ndash1120 1998

[38] Y-M Tzeng and M-J Lee ldquoNeuroprotective properties ofkavalactonesrdquo Neural Regeneration Research vol 10 no 6 pp875ndash877 2015

[39] J Baell and M A Walters ldquoChemistry chemical con artists foildrug discoveryrdquo Nature vol 513 no 7519 pp 481ndash483 2014

[40] J B Baell ldquoFeeling naturersquos pains natural products natu-ral product drugs and pan assay interference compounds(PAINS)rdquo Journal of Natural Products vol 79 no 3 pp 616ndash628 2016

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


O

O

O

O

OH

OH O

O

O

O

O

O

1 2

4 6

3

5

（3

（3

（3 （3

（3

OHOH

OH

OH

OHOH

Figure 1 Chemical structures of Renealmia alpinia constituents Pinostrobin 1 41015840-methyl ether sakuranetin 2 sakuranetin 3 pinostrobinchalcone 4 yashabushidiol A 5 and desmethoxyyangonin 6

showed inhibitory effects on the enzymatic anticoagu-lant myotoxic and edema inducing activities of phospho-lipase A2 (PLA2) isolated from Crotalus durissus venom[12]

Additionally the extracts of R alpinia showed inhibitiontowards indirect hemolytic coagulant effects and proteolyticactivity produced by B asper venom Pinostrobin was foundto be the bioactive component in the extract responsiblefor this effect [13] Recently we explored the antinociceptiveeffects of methanol and aqueous extracts of R alpinia inin vivo models Comprehensive phytochemical analysis ofR alpinia yielded pinostrobin 1 along with two flavonoids(naringenin 741015840-dimethyl ether 2 and naringenin 7-methylether 3) one chalcone (2101584061015840-dihydroxy-41015840-methoxychalcone4) one diarylheptanoid (35-heptanediol-17-diphenyl 5) andone kavalactone (desmethoxyyangonin 6) [14] (Figure 1)This was the first report on the isolation of compound 6 froma Renealmia species [15]

Increasing efforts have been made to identify dual mo-noamine oxidases (MAOs) inhibitory and anti-inflamma-tory agents which enhance cognitive functions and de-layprevent progression of neurodegenerative diseases [16ndash18] Dichloromethane extract of R alpinia and individualconstituents isolated from this extract were evaluated invitro against the recombinant human MAO-A and MAO-B Binding interactions of desmethoxyyangonin 6 the mostprominentMAO inhibitory constituent in R alpinia extractsin the active site of the MAO-A and MAO-B isoenzymeswere investigated through enzyme-kinetics assays enzyme-inhibitor complex binding equilibrium-dialysis dissociationanalyses and computationalmolecularmodeling algorithmsThese studies may have implications for future research andscientifically validate traditional use of R alpinia as a poten-tial therapeutic agent for treatment of neurodegenerativedisorders as well as the use ofR alpinia as a functional dietarybenefit for the local populations

2 Materials and Methods

21 Reagents and Chemicals Pure recombinant humanmonoamine oxidases (MAO-A and MAO-B) enzymes over-expressed in baculovirus (Autographa californica) infectedinsect cells (BTI-TN-5B1-4) were purchased from BD Bio-sciences (Bedford MA USA) Kynuramine bromide 4-hydroxyquinoline clorgyline R (minus) deprenyl and DMSOwere obtained from Sigma Chemicals Company (St LouisMO USA) R alpinia extract and six evaluated compounds1ndash6 were obtained from the isolation procedures describedpreviously [14] These compounds have been kept at minus20∘Cuntil evaluation Previous to assay to verify the stabilityof compounds 1ndash6 the spectrometric and spectroscopicanalysis was done No degradation products were detected

22 Determination of MAOs Inhibition Activity of the Com-pounds In vitro assays were performed to measure theinhibitory effects of R alpinia dichloromethane extractand its purified compounds 1ndash6 on human recombinantMAO-A and MAO-B activity The dichloromethane extract(0001 to 100 120583gmL) purified compounds (10minus9 to 10minus2M)and standard MAO inhibitors (phenelzine clorgyline anddeprenyl) (10minus12 to 10minus5M) were tested on human MAO-A and MAO-B enzymes [19] Stock solutions of the testcompoundsextracts were prepared in DMSO and dilutedfurther in 01M potassium phosphate buffer (pH 74) toobtain the desired concentrations MAO-A and MAO-B activities were determined by fluorometric kynuraminedeamination assay set up in 384-well solid white flat-bottomplates [20] MAO-A and MAO-B inhibition activities (IC50values) were determined using fixed concentration of thesubstrate kynuramine and varying concentrations of thetest compounds or extracts The enzyme reactions werecarried out in 01M potassium phosphate buffer (pH-74)Reactionmixture (total volume 75120583L in each well) containedpotassium phosphate buffer (01M pH 74) kynuramine(80 120583M for MAO-A assay and 50120583M for MAO-B assay) the








3 Results










01 1 10

100

80

60

40

20

Concentration (－)

Activ

ity (

)

100

80

60

40

20

0

001 01 1

Concentration (－)

Activ

ity (

)

MAO-A phenelzine

MAO-B phenelzine

(a)

100

80

60

40

20

0

001 01 1

Concentration (－)

Activ

ity (

)

1 10

100

80

60

40

20

0

Concentration (－)

Activ

ity (

)



(b)



1V

1[S]

07

06

05

04

03

02

01

00

minus01minus005 000 005 010 015 020 025 030


(a)

1V

1[S]

07

06

05

04

03

02

01

00

minus01minus005 000 005 010 015 020 025 030


320 － desmethox

(b)


1V

1[S]

06

05

04

03

02

01

00

minus01

minus005 000 005 010 015 020 025 030


(a)

1V

1[S]minus005 000 005 010 015 020 025 030

12

10

08

06

04

02

00


(b)








ac

tivity

Time (min)



150

125

100

75

50

25

0

0 5 10 15


(a)

ac

tivity

150

125

100

75

50

25

0

Time (min)0 5 10 15



Control


(b)









0

20

40

60

80

100

120

activ

ity

Phen

elzi

ne (2

50)

Phen

elzi

ne (2

50)

Con

trol

DM

A (1

000

)

DM

A (2

00)

Concentration (－)


(a)

0

20

40

60

80

100

120

ac

tivity

Phen

elzin

e (030

)

Phen

elzin

e (10

0)

Con

trol

DM

A (1

50

)

DM

A (2

00)


Concentration (－)

(b)




4 Discussion



(a) (b)

(c) (d)

(e) (f)







5 Conclusion


Disclosure




Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























3 Results










01 1 10

100

80

60

40

20

Concentration (－)

Activ

ity (

)

100

80

60

40

20

0

001 01 1

Concentration (－)

Activ

ity (

)

MAO-A phenelzine

MAO-B phenelzine

(a)

100

80

60

40

20

0

001 01 1

Concentration (－)

Activ

ity (

)

1 10

100

80

60

40

20

0

Concentration (－)

Activ

ity (

)



(b)



1V

1[S]

07

06

05

04

03

02

01

00

minus01minus005 000 005 010 015 020 025 030


(a)

1V

1[S]

07

06

05

04

03

02

01

00

minus01minus005 000 005 010 015 020 025 030


320 － desmethox

(b)


1V

1[S]

06

05

04

03

02

01

00

minus01

minus005 000 005 010 015 020 025 030


(a)

1V

1[S]minus005 000 005 010 015 020 025 030

12

10

08

06

04

02

00


(b)








ac

tivity

Time (min)



150

125

100

75

50

25

0

0 5 10 15


(a)

ac

tivity

150

125

100

75

50

25

0

Time (min)0 5 10 15



Control


(b)









0

20

40

60

80

100

120

activ

ity

Phen

elzi

ne (2

50)

Phen

elzi

ne (2

50)

Con

trol

DM

A (1

000

)

DM

A (2

00)

Concentration (－)


(a)

0

20

40

60

80

100

120

ac

tivity

Phen

elzin

e (030

)

Phen

elzin

e (10

0)

Con

trol

DM

A (1

50

)

DM

A (2

00)


Concentration (－)

(b)




4 Discussion



(a) (b)

(c) (d)

(e) (f)







5 Conclusion


Disclosure




Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























01 1 10

100

80

60

40

20

Concentration (－)

Activ

ity (

)

100

80

60

40

20

0

001 01 1

Concentration (－)

Activ

ity (

)

MAO-A phenelzine

MAO-B phenelzine

(a)

100

80

60

40

20

0

001 01 1

Concentration (－)

Activ

ity (

)

1 10

100

80

60

40

20

0

Concentration (－)

Activ

ity (

)



(b)



1V

1[S]

07

06

05

04

03

02

01

00

minus01minus005 000 005 010 015 020 025 030


(a)

1V

1[S]

07

06

05

04

03

02

01

00

minus01minus005 000 005 010 015 020 025 030


320 － desmethox

(b)


1V

1[S]

06

05

04

03

02

01

00

minus01

minus005 000 005 010 015 020 025 030


(a)

1V

1[S]minus005 000 005 010 015 020 025 030

12

10

08

06

04

02

00


(b)








ac

tivity

Time (min)



150

125

100

75

50

25

0

0 5 10 15


(a)

ac

tivity

150

125

100

75

50

25

0

Time (min)0 5 10 15



Control


(b)









0

20

40

60

80

100

120

activ

ity

Phen

elzi

ne (2

50)

Phen

elzi

ne (2

50)

Con

trol

DM

A (1

000

)

DM

A (2

00)

Concentration (－)


(a)

0

20

40

60

80

100

120

ac

tivity

Phen

elzin

e (030

)

Phen

elzin

e (10

0)

Con

trol

DM

A (1

50

)

DM

A (2

00)


Concentration (－)

(b)




4 Discussion



(a) (b)

(c) (d)

(e) (f)







5 Conclusion


Disclosure




Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1V

1[S]

07

06

05

04

03

02

01

00

minus01minus005 000 005 010 015 020 025 030


(a)

1V

1[S]

07

06

05

04

03

02

01

00

minus01minus005 000 005 010 015 020 025 030


320 － desmethox

(b)


1V

1[S]

06

05

04

03

02

01

00

minus01

minus005 000 005 010 015 020 025 030


(a)

1V

1[S]minus005 000 005 010 015 020 025 030

12

10

08

06

04

02

00


(b)








ac

tivity

Time (min)



150

125

100

75

50

25

0

0 5 10 15


(a)

ac

tivity

150

125

100

75

50

25

0

Time (min)0 5 10 15



Control


(b)









0

20

40

60

80

100

120

activ

ity

Phen

elzi

ne (2

50)

Phen

elzi

ne (2

50)

Con

trol

DM

A (1

000

)

DM

A (2

00)

Concentration (－)


(a)

0

20

40

60

80

100

120

ac

tivity

Phen

elzin

e (030

)

Phen

elzin

e (10

0)

Con

trol

DM

A (1

50

)

DM

A (2

00)


Concentration (－)

(b)




4 Discussion



(a) (b)

(c) (d)

(e) (f)







5 Conclusion


Disclosure




Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















ac

tivity

Time (min)



150

125

100

75

50

25

0

0 5 10 15


(a)

ac

tivity

150

125

100

75

50

25

0

Time (min)0 5 10 15



Control


(b)









0

20

40

60

80

100

120

activ

ity

Phen

elzi

ne (2

50)

Phen

elzi

ne (2

50)

Con

trol

DM

A (1

000

)

DM

A (2

00)

Concentration (－)


(a)

0

20

40

60

80

100

120

ac

tivity

Phen

elzin

e (030

)

Phen

elzin

e (10

0)

Con

trol

DM

A (1

50

)

DM

A (2

00)


Concentration (－)

(b)




4 Discussion



(a) (b)

(c) (d)

(e) (f)







5 Conclusion


Disclosure




Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

activ

ity

Phen

elzi

ne (2

50)

Phen

elzi

ne (2

50)

Con

trol

DM

A (1

000

)

DM

A (2

00)

Concentration (－)


(a)

0

20

40

60

80

100

120

ac

tivity

Phen

elzin

e (030

)

Phen

elzin

e (10

0)

Con

trol

DM

A (1

50

)

DM

A (2

00)


Concentration (－)

(b)




4 Discussion



(a) (b)

(c) (d)

(e) (f)







5 Conclusion


Disclosure




Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e) (f)







5 Conclusion


Disclosure




Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion


Disclosure




Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Interactions of Desmethoxyyangonin, a Secondary Metabolite...

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Transcript of Interactions of Desmethoxyyangonin, a Secondary Metabolite...