Research Article Bioassay Directed Isolation and ...downloads.hindawi.com/journals/bmri/2014/204340.pdf · root acetone extract. e isolated compounds then checked for its antioxidant

Research ArticleBioassay Directed Isolation and Biological Evaluation ofCompounds Isolated from Rubus fairholmianus Gard

Blassan Plackal George1 Parimelazhagan Thangaraj1 Cheruthazhakkatt Sulaiman2

Shanmughavel Piramanayagam3 and Sathish Kumar Ramaswamy3

1 Bioprospecting Laboratory Department of Botany School of Life Sciences Bharathiar UniversityCoimbatore Tamil Nadu 641 046 India

2 Phytochemistry Division Centre for Medicinal Plants Research Arya Vaidya Sala Kottakkal 676 503 India3 Department of Bioinformatics Bharathiar University Coimbatore Tamil Nadu 641 046 India

Correspondence should be addressed to ParimelazhaganThangaraj drparimelgmailcom

Received 13 February 2014 Revised 27 March 2014 Accepted 8 April 2014 Published 1 September 2014

Academic Editor Kuo-Chen Chou

Copyright copy 2014 Blassan Plackal George 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

The in vitro and in silico analysis of Rubus fairholmianus acetone extract for antioxidant antiproliferative and anti-inflammatoryactivity led to the isolation of six compounds Amongst all the six isolated compounds tested 1-(2-hydroxyphenyl)-4-methylpentan-1-one (compound 1) and 2-[(3-methylbutoxy) carbonyl] benzoic acid (compound 2) were found to be more active in inhibitingBRCA and COX target proteins which also showed the better results for DPPH andABTS radical scavenging assaysThe promisingresults of this investigation emphasize the importance of using R fairholmianus in the treatment of radical generated disordersmainly cancer and other inflammatory diseases

1 Introduction

The genus Rubus is very diverse includes over 750 species in12 subgenera and is found on all continents except Antarctica[1] Due to useful ethnomedicinal and pharmacological prop-erties Rubus species has been used in folk medicine [2] Theleaf extract of R fairholmianus (R moluccanus L) collectedin the early morning has been used by Koch-Rajbongshi andRangia tribes to reduce headache by [3]The leaves have beenreported to possess insecticidal properties fruits are edibleand stimulant [4]The total phenolic content and antioxidantand analgesic potential of R fairholmianus extracts havebeen evaluated by George et al [5] R fairholmianus rootacetone extract showed significant anti-inflammatory andwound healing properties which may be due to the presenceof analogues of quercetin and other related polyphenoliccompounds [6]

The literature showed that the berries (Rubus sp) ofRosaceae family have been reported for their strong antiox-idant and pharmacological properties [5ndash9] and various

bioactive free radical scavenging compounds were isolated[10ndash14] Berry fruits are characterized by a high content andwide diversity of bioactive compounds such as phenolic com-pounds organic acids tannins anthocyanins and flavonoids[15] Oxidative stress is caused by an imbalance betweenantioxidant systems and the production of oxidants whichis associated with many diseases like cancers cardiovasculardiseases and inflammation related disorders [16] A largenumber of naturally occurring antioxidant compounds havebeen identified from different plant sources as free radicalor active oxygen scavengers [17 18] Antioxidants protectorganisms against free radicals and they are vital to neutralizethe destruction caused by the radicals with a sufficient supplyof antioxidants [19]

Molecular docking is an approach to help researchersto screen a large set of small molecules by orienting andscoring them in the binding site of target proteins Top rankedcompounds have been tested in vitro and further they maybecome lead compounds for drug development The Glide

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 204340 15 pageshttpdxdoiorg1011552014204340

2 BioMed Research International

score number of H-bonds distance of bonds interacted pro-tein residues and ligand atom were observed from dockingstudies This docking study helps us in understanding thebinding mode of the isolated compounds with the targetproteins

In the present scenario the antioxidant researchers aremainly focusing on the identification and isolation of newnatural antioxidant compounds from different plants speciessince it can protect the human body from various free radicalgenerated disorders A survey of literature revealed that thephytochemical aspects of this plant have not been evaluatedThis study aimed to investigate the antioxidant activitiesof different extracts of R fairholmianus This study focuseson the isolation and identification of bioactive antioxidantcompounds from R fairholmianus through chromatographictechniques based on the activity guided fractionation of theroot acetone extract The isolated compounds then checkedfor its antioxidant potential Further we adopted dockingstudies to identify inhibiting activity of the compoundsagainst BRACA and COX proteins

2 Materials and Methods

21 Plant Collection and Extraction The fresh plant partsof R fairholmianus were collected from Marayoor Sholaforest Kerala India during the month of September 2010The collected plant material was identified and authenticatedby (Voucher specimen number BSISRC5232010-11Tech1657) Botanical Survey of India SouthernCircle CoimbatoreTamil Nadu The roots were extracted successively usingacetone in a soxhlet apparatus for 72 hours The extract wasconcentrated to dryness under reduced pressure in a rotaryevaporator

22 Determination of In Vitro Antioxidant Activities Thedifferent fractions and isolated compounds from root acetoneextracts of R fairholmianus were analyzed for their antioxi-dant activity using DPPH assay and ABTS assay

221 DPPH Radical Scavenging Activity The DPPH assaywas done as per the method described by Blois [20] Negativecontrol was prepared by adding 100 120583L of methanol in 5mLof 01mM methanolic solution of DPPH∙ The tubes wereallowed to stand for 20 minutes at 27∘C Radical scavengingactivity of the samples was expressed as IC

50

which is theconcentration of the sample required to inhibit 50 ofDPPH∙ concentration

222 Trolox Equivalent Antioxidant Capacity (TEAC) AssayThe total antioxidant activity was measured by ABTS radicalcation decolourization assay according to Re et al [21]Triplicate determinations were made at each dilution ofthe standard and the percentage inhibition was calculatedagainst the blank (ethanol) with an absorbance at 734 nmand then was plotted as a function of Trolox concentrationThe unit of total antioxidant activity (TAA) is defined asthe concentration of Trolox having equivalent antioxidantactivity expressed as 120583Mg sample extracts

23 Chromatographic Separation of the Root Acetone ExtractBased on the in vitro antioxidant studies RFRA (R fairholmi-anus root acetone) showed maximum antioxidant activityand it was selected for the further isolation of phytocon-stituents The preliminary screening was done using thinlayer chromatography by toluene ethyl acetate acetic acid(6 3 05) as mobile phase The extract (50 g) was adsorbedon activated silica (230ndash400 mesh) The column (90 times 5 cm)was packed with activated silica gel (230ndash400 mesh) intoluene and it was eluted with 400mL of each of differentsolvents such toluene (100) toluene ethyl acetate (9 1 7 36 4 5 5 4 6 2 8) ethyl acetate chloroform (9 1 7 35 5 3 7 1 9) chloroform methanol (8 2 6 4 5 5 4 6)diethyl ether (100) ethanol (100)methanol (100) aceticacid (2) and acetone (100) A total of 183 fractions werecollected and the fractionswith similar chemical profileswerepooled based on TLC analysis

Based on the TLC the fractions with similar bandingpattern were clubbed and 16 fractions were obtained Thevarious fractions such as F1 (34ndash37) F2 (38ndash54) F3 (55ndash58)F4 (59ndash72) F5 (76ndash80) F6 (84ndash88) F7 (89ndash110) F8 (111ndash116)F9 (121ndash126) F10 (132ndash136) F11 (137ndash141) F12 (142) F13 (143ndash147) F14 (148ndash151) F15 (159ndash162) and F16 (163ndash183) weresubjected to antioxidant studies The most active fractionssuch as F5 F7 F9 and F16 were selected for further isolationprocess

The 4 active fractions further yielded 6 subfractionsmethanol and ethyl acetate subfractions of F5 (F5M andF5EA) chloroform subfractions of F7 and F9 (F7C and F9C)and methanol and diethyl ether subfractions of 16 (F16M andF16DE) All these fractionswere analysed by TLCHPLC andso forth

24 Purification of Isolated Compounds The isolated com-pounds were purified by semipreparative TLC and prepar-ative HPLC The bands visualized under UV at 254 nmand 366 nm were scrapped out and dissolved in respectivesolvents The time based collection was performed usingpreparative fraction collector on Agilent 1200 high pressureliquid chromatographic system equipped with prep pump arheodyne injector and diode array detector in combinationwith Chem32 Chemstation software C18-scalar (5120583m 46times 150mm) Agilent column was used for separation with abinary gradient elution Mobile phase 01 acetic acid inacetonitrile (A) methanol water (60 40) (B) 0ndash2 70A2ndash5 60A 5ndash10 50A 10ndash20 70A The flow rate wasmaintained as 15mLminute

25 Characterization of Isolated Compounds

251 UV Spectroscopy The UV-visible spectrum of the iso-lated compound inHPLCgrademethanolwas recordedusinga Shimadzu 160AUV-visible spectrophotometer at a range of280ndash400 nm

252 Fourier Transform Infrared Spectrometry (FTIR) TheFTIR spectrum was recorded using a Nicolet 5700 (Thermoelectron Madison WI USA) spectrometer at room temper-ature The bioactive compound was dissolved in dimethyl

BioMed Research International 3

sulfoxide (DMSO) and scanned in the range of 4000ndash400 cmminus1

253 Nuclear Magnetic Resonance Spectroscopy (NMR)NMR spectra were recorded on a Bruker DRX 500 NMRinstrument operating at 500MHz for 1H and 125MHz for13C at room temperature A region from 0 to 12 ppm for 1Hand 0 to 200 ppm for 13Cwas employed Signals were referredto as the internal standard tetramethylsilane (TMS) About10mg of the sample dissolved in 05mL of CDCl

3

was usedfor recording the spectra

254 Mass Spectrometry LC-ESI-MS analysis was con-ducted on Agilent 6520 accurate mass Q-TOF LCMS cou-pled with Agilent LC 1200 equippedwith Extend-C18 columnof 18 120583m 21 times 50mm Gradient elution was performed withmethanol (solvent A 70) and 01 formic acid (solventB 30) at a constant flow rate of 03mLmin Columntemperature was maintained at 30∘C The MS analysis wasperformed using ESI in the negativemodeThe conditions formass spectrometrywere as follows drying gas (nitrogen) flow5 Lmin nebulizer pressure 50 psig drying gas temperature325∘C capillary voltage + 3000V fragmentor volt 250V OctRf Vpp 750V

26 Molecular Docking of Bioactive Compounds againstBRCA and COX Proteins Molecular docking has becomean increasingly important tool for drug discovery It is auseful vehicle for investigating the interaction of a proteinreceptor with its ligand and revealing their binding mecha-nism as demonstrated by a series of studies [22ndash27] We haveemployed flexible docking strategies in this study to identifythe most suitable ligand binging sites in the BRCA andCOX target proteins Flexible molecular docking operationsrequire no prior knowledge of the ligand conformation andsuch an approach becomes necessary if there is no usefulinformation on the conformation of a particular ligand

261 Protein Retrieval and Preparation Three-dimensionalstructures of BRCA1 BRCA2 COX-1 and COX-2 proteinswere obtained from PDB database (PDB id 1T15 3EU71EQH 3LN1) (PDB httpwwwrcsborg)The retrieved pro-tein has been prepared for docking using Protein PreparationWizard [28] Finally the grid was generated for the preparedproteins

262 Inhibitory Molecules Retrieval and Preparation Inhibi-tory molecules were retrieved from PubChem database in3D SDF format The compounds such as cyclophospha-mide letrazole doxorubicin paclitaxel and tamoxifen (Pub-Chem id CID 2907 CID 3902 CID 31703 CID 36314CID 2733526) are the commercially available standarddrugs for the treatment of cancer diclofenac indomethacinparacetamol aspirin and morphine (PubChem id CID3033 CID 3715 CID 1983 CID 2244 CID 5288826) arethe NSAIDs used commonly for the treatment of inflam-mation related diseases About 6 compounds such as

3(imino methyl)-24-dimethyl phenol isopentyl benzoateor 3-methylpentyl benzoate 4-methylpentyl benzoate 2-(isopentyloxycarbonyl) benzoic acid 2-(5-methylhexyl) ben-zoic acid and 1-(2-hydroxyphenyl)-4-methyl pentan-1-oneisolated from R fairholmianus root acetone extract werescreened for their inhibitory activity against BRCA and COXproteins Retrieved molecules were prepared for dockingusing LigPrep module [29] Finally the prepared compoundswere used for docking with BRCA and COX proteins

263 Molecular Docking of Target Proteins with InhibitoryMolecules The docking was performed using Glide module[30ndash32] through blind docking approach in which com-mercially available drugs and newly isolated compoundswere docked against BRCA and COX proteins Dockedcomplex was examined with an emphasis on visual ratherthan numerical appraisal so we used XP visualizer

27 Statistical Analysis All the values are expressed asmeanplusmnSEMThe values are analyzed using one-wayANOVAand thesignificance of the difference betweenmeans was determinedby Duncanrsquos Tukey Kramer multiple comparisons usingSPSS

3 Results and Discussion

31 In Vitro Antioxidant Activity Figure 1 schematicallyrepresents the isolation procedure of bioactive compoundsall the compounds reported in this study were isolated forthe first time from R fairholmianus The antioxidant activityof different fractions and isolated compounds was evaluatedusing DPPH and ABTS radical scavenging assaysThe resultsare given in Table 1 Among the different fractions F9 showedhighest scavenging activity against DPPH (IC

50

value 361)The IC

50

values of the isolated compounds ranged between1235 and 323 120583gmL Compound 1 (1-(2-hydroxyphenyl)-4-methylpentan-1-one) showed maximum DPPH radicalscavenging activity with IC

50

323 120583gmL The ABTS rad-ical scavenging activities of the fractions ranged between114074 and 497472 120583MTEg The isolated compounds alsoshowed commendable ABTS radical scavenging activitieswith highest value for 2-[(3-methylbutoxy) carbonyl] benzoicacid (compound 2) (923320 120583MTEg)

No previous reports are available on DPPH radicalscavenging activity of this species The phenolic compoundsin berries have been well characterized as natural antiox-idants which are believed to play a major role in certainhealth benefits The naturally occurring phenolic antioxi-dants encompass a diverse range of chemical classes thatprotect against the damage caused by ROS toDNA andmem-brane and cellular components [33] The strong antioxidantactivities of R fairholmianus may be due to the phenoliccompounds Previously Vadivelan et al [34] reported theantioxidant properties of R ellipticus rootThe root methanolextracts showed strongest radical scavenging activity (IC

50

122 120583gmL) against DPPH∙ and (IC

50

25 120583gmL) againstABTS radical Comparing these results the DPPH and ABTSradical scavenging activities of the isolated column fractions


Petroleum ether Chloroform Acetone Methanol

Silica gel columnchromatography I

(227 fractions eluted)

Out of 20 pooled fractionsF5 F7 F9 and F1

found to be more active

eluted fractions

89ndash110) ethyl acetate

eluted fractions


eluted fractions

163ndash183) diethyl

methanol (5 5 4 6)eluted fractions

Compound 5

Compound 6

Compound Compound 4

Methanol dissolved F16 fraction

Silica gel columnchromatography II(33 fractions eluted)

Compounds 2 and 3

F5(76ndash80) ethyl acetatechloroform (9 1) chloroform (9 1)chloroform (7 3 5 5 3 7) ether (100) chloroform

Diethyl ether (100) dissolved fractions

13ndash32) chloroform isopropyl alcohol (7 3)chloroform dimethyl ether methanol (6 3 1) and

chloroform tetrahydroxy furan (7 3) eluted fractions

F7( F9( F16(

F(

Rubus fairholmianus root powder

1

6 are

Figure 1 Schematic representation showing the isolation procedure of bioactive compounds from R fairholmianus

and compounds ofR fairholmianus are highly significant andare almost equivalent and more than that of the standardsThe DPPH radical scavenging activity of an allied species Rsanctus has also been reported which was found to scavengethe DPPH radical by 8327 compared to vitamin C andBHT (9715 and 9647) [35] Comparing the literatures theisolated compounds and fractions of R fairholmianus rootacetone extract have a commendable antioxidant activity inthe tested assays

32 Identification of the Isolated Bioactive Compounds Thestructure and molecular formula of the isolated compoundsare shown in Table 2

Compound 1 The fraction F5 yielded yellowish white amor-phous solid (102mg) after recrystallization onmethanol UV(methanol) 120582max nm 280 IR (KBR) cmminus1 343853 172941161415 127943 1H NMR (CDCl

3

) 120575 773 773 754 753 4843 155 207 098 099 13C NMR 120575 16943 16001 1341613266 13061 12211 7356 4356 3146 2949 2091MS (mz)[M-H] 19198 The compound was identified as 1-(2-hydroxyphenyl)-4-methyl pentan-1-one

Compound 2 Fraction F7 presented brownish crystallinesolid (846mg) on evaporation UV (methanol) 120582max nm290 IR (KBR) cmminus1 343090 292630 172588 127861 1HNMR (CDCl

3

) 120575 803 792 789 771 754 751 430 429 205204 173 170 098 13C NMR 120575 16771 12569 12884 6655The molecular formula of this compound was establishedbased on the mass spectrum MS (mz) [M-H] 23512The structure was confirmed as cis-2-(isopentyloxycarbonyl)benzoic acid

Compound 3 [2-(5-Methylhexyl) benzoic acid] 679mgwhite amorphous solid recrystallised on evaporation of chlo-roform from fraction 7 (F7) UV (methanol) 120582max nm 290IR (KBR) cmminus1 342720 292685 172835 162605 128120 and112782 1HNMR (CDCl

3

) 120575 771 770 769 753 752 751 750424 423 422 420 419 418 13C NMR 120575 16773 1423913249 13236 13088 13086 12880 6619 3876 3668 35503250 2880 MS (mz) [M-H] 21919

Compound 4 [4-Methylpentyl benzoate] 618mg whiteamorphous crystals from the diethyl ether fraction of F9 UV(methanol) 120582max nm 280 IR (KBR) cmminus1 295800 172747146212 128011 and 112588 1H NMR (CDCl

3

) 120575 771 770


Table 1 DPPH and ABTS radical scavenging activities of fractions and compounds of R fairholmianus

Fractions and compoundsDPPH radical scavenging

activity(IC50 120583gmL)

ABTS radical cation scavengingactivity

(120583MTrolox equivalentsg extract)1 1473 224099 plusmn 32552 1795 136349 plusmn 23383 1870 374960 plusmn 5854 2031 307461 plusmn 35565 716 349648 plusmn 51966 1801 447860 plusmn 35567 955 337498 plusmn 23388 2556 233549 plusmn 40929 361 465072 plusmn 154710 1536 437735 plusmn 116911 2197 223424 plusmn 58512 1761 212286 plusmn 325513 2779 114074 plusmn 309314 2463 476547 plusmn 478515 1639 497472 plusmn 456616 1341 386098 plusmn 4215Compound 1 323 534445 plusmn 2494Compound 2 509 923320 plusmn 5432a

Compound 3 1076 642123 plusmn 8446d

Compound 4 857 875478 plusmn 4229b

Compound 5 1235 565632 plusmn 3853Compound 6 943 745554 plusmn 6298c

Butylated hydroxy toluene (BHT) 1318 mdashButylated hydroxy anisole (BHA) 488 mdashRutin 581 mdashQuercetin 412 mdashValues are mean of triplicate determination (119899 = 3) plusmn standard deviation Statistically significant at 119875 lt 005 where a gt b gt c gt d

769 753 751 426 423 421 170 168 167 140 099 098094 092 13C NMR 120575 16776 13396 13248 12881 68183876 3028 1404 MS (mz) [M-H] 20523

Compound 5 [3-(Iminomethyl)-24-dimethylphenol] whitecrystals (1058mg) obtained from F16 UV (methanol) 120582maxnm 272 IR (KBR) cmminus1 345140 292673 157691 143696121830 and 107745 1HNMR (CDCl

3

) 120575 841 680 657 547427 428 438 438 246 232 13C NMR 120575 11848 115134507 2080 14949 13597 13419 12646 1824MS (mz) [M-H] 14808

Compound 6 [3-Methyl benzoate] (1208mg) was obtainedas yellow amorphous crystals from methanolic fraction ofF16 UV (methanol) 120582max nm 275 IR (KBR) cmminus1 352578295846 172790 128265 and 112762 1H NMR (CDCl

3

) 120575803 751 792 429 432 206 207 168 173 094 099 13CNMR120575 16771 12569 12884 13090 13599 3058 2774 19181915 MS (mz) [M-H] 19110

There are many reports available on the phytochemicalcharacterization and activity guided studies of the related

species of Rubus Most of the compounds isolated from theRubus species belong to cyanidin type compounds antho-cyanins and tannins Ruiz et al [36] reported thepresence of total anthocyanins (034ndash092 120583molg) mono-meric anthocyanins (031ndash116 120583molg) and cyanidin-3-sam-bubioside in the fruits of R geoides Kubota et al [37] isolatedand purified the anthocyanins cyanidin-3-O-glucoside andpelargonidin-3-O-glucoside from R croceacanthus fruitsand pelargonidin-3-O-glucoside and pelargonidin-3-Oruti-noside from R sieboldii fruits Porter and coworkers [38]reported the minor flavonoid components in R idaeusEstupinan et al [39] Vasco et al [40] Garzon et al [41]Mertz et al [11] Kim et al [42] Ku and Mun [43] Bae et al[44] Deighton et al [45] Wyzgoski et al [46] Dossett et al[47] Ling et al [48] Dossett et al [49] Tulio et al [50] andTian et al [51] reported the presence of cyanidin-3-glucosideand cyanidin-3-rutinoside cyanidin-3-sambubioside cyani-din-3-xylosylrutinoside pelargonidin-3-glucoside and pel-argonidin-3-rutinoside cyanidin glycoside lambertianin Cand sanguiin H-6 and 2 ellagitannins in various Rubusspecies Bowen-Forbes et al [52] reported the presence of


Table 2 Structure of the isolated compounds

Sl No Name Structure Molecularformula

Molecularweight

1 1-(2-Hydroxyphenyl)-4-methylpentan-1-one

CH3

CH3

HO

O

C12H16O2 19225

2 2-[(3-Methylbutoxy)carbonyl]benzoic acid

CH3

CH3

HO

OOO

C13H16O4 47253

3 2-(5-Methylhexyl) benzoic acid

CH3

CH3

HO

O

C14H20O2 22031

4 4-Methylpentyl benzoate

CH3

CH3

OO

C13H18O2 20628

5 3-(Iminomethyl)-24-dimethylphenol

CH3

CH3

OH

NH C9H11NO 14919

6 3-Methylbutyl benzoate CH3

CH3OO

C12H16O2 19225

eight hydroxyursane type compounds euscaphic acid 1-b-hydroxyeuscaphic acid hyptatic acid B 19a-hydroxyasiaticacid trachelosperogenin 4-epi-nigaichigoside F1 nigai-chigoside F1 and trachelosperoside B-1 in the ethyl acetateextract of R rosifolius fruits

33 Molecular Docking of Isolated Compounds against BRCAand COX Proteins Isolated compounds and standard drugsfor cancer treatment were docked against BRCA1 Thedocking score and hydrogen bond interactions of the com-pounds are showed in Table 3 1-(2-Hydroxyphenyl)-4-methylpentan-1-one (119866 score minus47) has high binding with

active site of BRCA1 protein followed by 2-[(3-methylbutoxy)carbonyl] benzoic acid (119866 score minus386) The standard drugssuch as doxorubicin and letrazole also showed strong bindingaffinity (119866 score minus482 and minus418 resp) The docking resultsof BRCA1 revealed that the mode of molecular interac-tions of the isolated compounds and standard drugs wasalmost similar (Figure 2) Meanwhile 1-(2-hydroxyphenyl)-4-methylpentan-1-one and 4-methylpentyl benzoate alsoshowed fairly good binding affinities towards the targetprotein BRCA2 with a 119866 score of minus45 and minus38 respectively2-[(3-Methylbutoxy) carbonyl] benzoic acid also dockedwith a moderate 119866 score (minus31) the binding affinities were


Table 3 Docking score and H-bond interaction of ligands with BRCA1 protein (1T15)

Ligand 119866 score Number ofH-bonds

Distance(A) Protein atom Ligand

atom

Doxorubicin minus482 5

24492418247022032013

SER 6 H (HG)LEU1657 (O) OASN1678 (O) OPRO9 (O) O

LYS1702 (H) HZ2

OHHHO

Letrazole minus418 2 21092056

GLY 1656 (H) HSER 6 H (HG)

NN

Cyclophosphamide minus338 3217219441753


LYS1702 (H) HZ2

OOO

Tamoxifen minus2 1 2004 SER 6 H (HG) N

1-(2-Hydroxyphenyl)-4-methylpentan-1-one minus47 3222320982012

LYS1702 (H) HZ2GLY 1656 (H) HSER 6 H (HG)

OOO

2-[(3-Methylbutoxy)carbonyl] benzoic acid minus386 5

22572142207019161709

SER 6 H (HG)GLY 1656 (H) HGLY 1656 (H) HSER 6 H (HG)

LYS1702 (H) HZ2

OOOOO

4-Methylpentyl benzoate minus383 3203217761677


LYS1702 (H) HZ2

OOO

2-(5-Methylhexyl) benzoic acid minus37 1 2066 LEU1701 (H) H O

3-(Iminomethyl)-24-dimethylphenol minus36 3247720091999

LYS1702 (H) HZ2PRO9 (O) O

LEU1701 (H) H

NHO

3-Methylbutyl benzoate minus34 1 1947 LYS1702 (H) H OGlide score number of H-bond interactions distance of H-bond interaction and involved protein atom and ligand atom in interaction of isolated and standarddrugs with BRCA1 protein obtained from docking studies

found to be more than that of the standards letrazole andpaclitaxel (119866 score minus30 and minus29) The standard drugdoxorubicin showed a 119866 score of minus111 followed by tamoxifen(119866 score minus59) Among the 6 isolated compounds only threecompounds were found to have good docking with targetprotein The molecular interaction and docking score of thecompounds are shown in Table 4 and Figure 3Themoleculardocking results showed that the isolated compound 1-(2-hydroxyphenyl)-4-methylpentan-1-one is highly significantin binding (119866 score minus47) and formed three hydrogen bondsagainst SER 6 H (HG) GLY 1656 (H) H and LYS 1702 (H)HZ2 residues of BRCA1 The residues involved in bindingwere more or less similar with that of standard drugs Thedocking score of 2-[(3-methylbutoxy) carbonyl] benzoic acid4-methylpentyl benzoate 2-(5-methylhexyl) benzoic acid 3-(iminomethyl)-24-dimethylphenol and 3-methylbutyl ben-zoate compounds ranged between minus34 and minus383 and thedocking score of the standard drugs falls between minus20and minus482 Docking score and H-bond interaction of all 6isolated compounds were closely related to standard drugsThe docking results of BRCA2 with the isolated com-pounds 1-(2-hydroxyphenyl)-4-methylpentan-1-one and 4-methylpentyl benzoate are significant 1-(2-Hydroxyphenyl)-4-methylpentan-1-one formed three hydrogen bonds with

the ALA 874 (O) O ALA874 (H) H and GLY1166 (H)H residues of BRCA2 protein and 4-methylpentyl benzoateformed 2 hydrogen bonds with the ALA 1063 (H) Hand LYS 1062 (H) HZ2 residues of BRCA2 Some of thebinding residues were common in both standard drugsand the isolated compounds The 119866 score of isolated com-pounds ranged between minus45 and minus31 whereas the stan-dard drugs were in between minus111 and minus32 BRCA1 andBRCA2 predispose individuals to breast ovarian and othercancers These proteins are required for the maintenance ofgenetic stability and have function in DNA damage [53]Previously Raja et al [54] studied the docking analysisof mangrove-derived compounds and revealed that amongsix compounds triterpenoid stigma sterol and pyrethrinwere efficient in destroying BRCA1 Saravanakumar andcoworkers [55] proposed the docking of fungal metabolitesagainst BRCA1 The docking score of phthalic acid waslowest (minus1371 kcalmol) followed by 23-dihydro-1H-inden-2-yl acetate and 3-methylcyclopentan-1-ol having the valueof minus1111 and minus908 respectively However there is no reporton the docking of isolated compounds from R fairholmianusagainst BRCA proteins So the information gained from thisstudy will be useful for further development of novel breastcancer inhibitors


Table 4 Docking score and H-bond interaction of ligands with BRCA2 protein (3EU7)



atom


20431994192119261843

LYS1062 (H) HZ3ASP1122 (O) OD1ASP1125 (H) H

ASP1125 (O) OD2ASP1122 (O) OD1

OHOHH

Tamoxifen minus59 2 21252022


HH


VAL969 (H) HHID1061 (H) HD1

NN

Cyclophosphamide minus32 2 22111923

LYS1062 (H) HZ3GLU1018 (H) H

OO

Paclitaxel minus29 4

2429193318191408

LYS1062 (H) HZ3LYS1062 (H) HZ3GLY971 (O) OHID1061 (O) O

OOHH


GLY1166 (H) HALA874 (H) HALA874 (O) O

OOH

4-Methylpentyl benzoate minus38 2 21492215

ALA1063 (H) HLYS1062 (H) HZ2

OO

2-[(3-Methylbutoxy)carbonyl] benzoic acid minus31 mdash mdash mdash mdashGlide score number of H-bond interactions distance of H-bond interaction and involved protein atom and ligand atom in interaction of isolated and standarddrugs with BRCA2 protein obtained from docking studies

(a) (b)

(c) (d)

Figure 2 Close view of BRCA1 docked with (a) doxorubicin (b) letrazole (c) 1-(2-hydroxyphenyl)-4-methylpentan-1-one and (d) 2-[(3-methylbutoxy)carbonyl]benzoic acid Isolated compounds (green color ball stick mode) docked with target protein BRCA1 (stick mode)through hydrogen bond interaction (pink color dot)


(a) (b)

(c) (d)

Figure 3 Close view of BRCA2 docked with (a) doxorubicin (b) tamoxifen (c) 1-(2-hydroxyphenyl)-4-methylpentan-1-one and (d) 4-methylpentyl benzoate Isolated compounds (green color ball stick mode) docked with target protein BRCA2 (stick mode) through hydrogenbond interaction (pink color dot)

The binding pocket of BRCA1 contains two chainswith two fragments (BCRT1 and BCRT2) BRCA1 is inter-acting with protein c-terminal helicase 1The highly conserv-ed C-terminal BRCT repeats that function as a phospho-serinephosphothreonine-binding module Clapperton et al[56] reported the X-ray crystal structure at a resolution of185 A of the BRCA1 tandem BRCT domains in complex witha phosphorylated peptide representing the minimal interact-ing region of the DEAH-box helicase BACH1 The structurerevealed the determinants of this novel class of BRCA1 bind-ing events Transcriptionantitumor protein BRCA2 containstwo chains with a fragment c-terminalWD40 domain havingresidues 835ndash1186 The synonym is PALB2 Fanconi anemiagroup n protein 19-meric peptides have been identified fromBRCA2 protein The second fragment interacts with PALB2having residues 21ndash39 The early onset protein (BRCA2)is central to the repair of DNA damage BRCA2 recruitsrecombinase RAD51 to sites of damage regulates its assemblyinto nucleoprotein filaments and thereby promotes homolo-gous recombination Localization of BRCA2 to nuclear focirequires its association with the partner and localizer ofBRCA2 (PALB2) mutations which are associated with cancerpredisposition as well as subtype n of Fanconi anaemiaOliver et al [57] have determined the structure of the PALB2carboxy-terminal beta-propeller domain in complex with aBRCA2 peptide

The molecular docking insights provide isolated com-pounds and standard drugs were docked against the COX-1and COX-2 proteins The results of COX-1 docking showedamong 6 compounds only 4 compounds were found to bedocked firmly with the target protein The docking score

and the interaction of the compounds seem to be highlysignificant 1-(2-Hydroxyphenyl)-4-methylpentan-1-one and2-[(3-methylbutoxy) carbonyl] benzoic acid showed strongbinding affinities with 119866 score of minus58 and minus44 respectivelyThe 119866 score of the compounds were found to be higherthan the majority of the standard drugs used Howeverindomethacin also showed good results (119866 score minus667) The119866 score of the isolated compounds 2-(5-methylhexyl) benzoicacid and 3-(iminomethyl)-24-dimethylphenol were minus343and minus323 respectively whereas the 119866 scores of the standarddrugs such as aspirin diclofenac paracetamol andmorphinewereminus565minus377minus341 andminus336 respectively (Table 5)Thetopmost interacted compounds against target proteins areshowed in Figure 4 The binding of isolated compounds andstandard drugs to the active sites of COX-2 protein is shownin Figure 5 The docking score and the interaction of thecompounds were tabulated (Table 6)The119866 score of the com-pounds 2-[(3-methylbutoxy) carbonyl] benzoic acid and 1-(2-hydroxyphenyl)-4-methylpentan-1-one was minus29 and minus24The 119866 score of the standard drug diclofenac is minus34

The results of the COX-1 docking with the isolatedcompounds are found to be significant The 119866 score ofthe three isolated compounds (minus323 to minus528) was higherthan standard drugs such as diclofenac paracetamol andmorphine (minus377 minus341 and minus336) The hydrogen bondinteraction of 2-[(3-methylbutoxy) carbonyl] benzoic acidand diclofenac seems to be similar 1-(2-Hydroxyphenyl)-4-methylpentan-1-one formed two hydrogen bonds with theASN 375 (H) H and ASN 375 (O) O residues of COX-1 and 2-[(3-methylbutoxy) carbonyl] benzoic acid formedthree hydrogen bonds with the ARG 374 (H) HH21 ARG


Table 5 Docking score and H-bond interaction of ligands with COX-1 protein (1EQH)



atom

Indomethacin minus667 3223022711986

ARG 374 (H) HH22ARG 374 (H) HH21ARG 374 (H) HE

OOO

Aspirin minus565 5

20662043219918571781

ARG 374 (H) HH22ASN 375 (H) HARG 374 (H) HE

ARG 374 (H) HH12ARG 374 (H) HH21

OOOOO

Diclofenac minus377 3223820171824

ARG 374 (H) HH22ASN 375 (H) H

ARG 374 (H) HH22

OOO

Paracetamol minus341 2 21391936

ARG 374 (H) HH21GLY225 (O) O

OH

Morphine minus336 1 1984 ARG 374 (H) HH22 O

1-(2-Hydroxyphenyl)-4-methylpentan-1-one minus528 2 19641734

ASN 375 (H) HASN 375 (O) O

OH


ARG 374 (H) HH21ARG 374 (H) HH12ASN 375 (H) H

OOO

2-(5-Methylhexyl) benzoic acid minus343 1 2044 ASN 375 (H) H O

3-(Iminomethyl)-24-dimethylphenol minus323 2 22261700

ASN 375 (H) HASN 375 (O) O

OO

Glide score number of H-bond interactions distance of H-bond interaction and involved protein atom and ligand atom in interaction of isolated and standarddrugs with COX-1 protein obtained from docking studies

(a) (b)

(c) (d)

Figure 4 Close view of COX-1 docked with (a) indomethacin (b) aspirin (c) 1-(2-hydroxyphenyl)-4-methylpentan-1-one and (d) 2-[(3-methylbutoxy)carbonyl]benzoic acid Isolated compounds (green color ball stick mode) docked with target protein COX-1 (stick mode)through hydrogen bond interaction (pink color dot)


(a) (b)

(c) (d)

Figure 5 Close view of COX-2 docked with (a) diclofenac (b) indomethacin (c) 2-[(3-methylbutoxy) carbonyl] benzoic acid and (d) 1-(2-hydroxyphenyl)-4-methylpentan-1-one Isolated compounds (green color ball stick mode) docked with target protein COX-2 (stick mode)through hydrogen bond interaction (pink color dot)

374 (H) HH12 and ASN 375 (H) H residues of COX-2The docking results of isolated compounds with COX-2 aresignificant when compared with the standard drugs The 119866score of the compounds ranged between minus29 and minus04 2-[(3-methylbutoxy) carbonyl] benzoic acid had the highest119866 score in the isolated compounds bound with HID 228(H) HD1 LYS 543 (H) HZ1 and LYS 543 (H) HZ1 residuesof the COX-2 protein The binding residues (LYS 543 (H)HZ3 LYS 543 (H) HZ2 and THR 547 (H) HG1) of thediclofenacwere also similar to 2-[(3-methylbutoxy) carbonyl]benzoic acid Recent studies have shown that stellatin hasCOX-1 and COX-2 inhibitory activities which was isolatedfromDysophylla stellata Docking study revealed the bindingorientations of stellatin and its derivatives into COX-1 andCOX-2 and thereby helps to design potent inhibitors [58]Olgen et al [59] reported the COX inhibitory activities ofindomethacin derivatives (N-substituted indole-2-carboxylicacid esters) All the derivatives were shown to be dockedat the site where intact flurbiprofen was embedded forCOX-1 and s-58 (1-phenylsulphonamide-3-trifluoromethyl-5-para-bromophenylpyrazole) for COX-2 Three series ofspiro derivatives have been synthesized and docked to COX-2 by Amin et al [60] The results showed nearest value ofindomethacin Abdel-Aziz et al [61] designed a group ofcyclic imides (1ndash13) and evaluated its selective COX-2 inhi-bition The molecular docking study showed that the CH

3

Osubstituents of 5b were inserted firmly to COX-2 where theO-atoms of such group underwent a H-bonding interactionwith HIS 90 (243 283 A) ARG 513 (289 A) and TYR

355 (334 A) This revealed a similar binding mode to SC-558 a selective COX-2 inhibitor Basile and coworkers [62]have synthesised 14 sulfonilamidothiopyrimidone deriva-tives Compounds 2ndash5 were able to fit into the active site ofCOX-2 with highest scores and interaction energies Further-more compound 2 which showed an inhibition of around50onPGE2 production was best scored El-Sayed et al [63]designed and synthesized new arylhydrazone derivatives anda series of 15-diphenyl pyrazoles from 1-(4-chlorophenyl)-444-trifuorobutane-13-dione 1 The designed compoundsdocked into the COX-2 Docking study of the synthesizedcompounds 2f 6a and 6d into COX-2 revealed a similarbinding mode to SC-558 a selective COX-2 inhibitor

Synonym of prostaglandin H2 synthase-1 is COX-1 Non-steroidal anti-inflammatory drugs block prostanoid biosyn-thesis by inhibiting prostaglandin H (2) synthase Selinsky etal [64] reported the crystal structure of COX-1 complex withthe inhibitors ibuprofen methyl flurbiprofen flurbiprofenand alclofenac at resolutions ranging from26 to 275AThesestructures allow direct comparison of enzyme complexeswith reversible competitive inhibitors (ibuprofen and methylflurbiprofen) and slow tight-binding inhibitors (alclofenacand flurbiprofen) All the inhibitors bind to the same siteand adopt similar conformations In all four complexesthe enzyme structure is essentially unchanged exhibitingonly minimal differences in the inhibitor binding site Thedifferent apparent modes of NSAID binding may result fromdifferences in the speed and efficiency with which inhibitorscan disturb the hydrogen bonding network around Arg-120


Table 6 Docking score and H-bond interaction of ligands with COX-2 protein (3LN1)



atom


LYS543 (H) HZ3LYS543 (H) HZ2THR547 (H) HG1

(O) O3O

(O) O3

Indomethacin minus32 2 20411767

ARG297 (H) HLYS543 (H) HZ2

O(O) O4

Aspirin minus28 3211221402049

LYS 543 (H) HZ3THR547 (H) HG1ARG297 (H) H21

(O) O20(O) O20(O) O3

Paracetamol minus24 3238322642014

GLU539 (O) OE2TRP531 (H) HE1GLU539 (O) OE1

HOH

Morphine minus10 5

24712249202519891974

ARG297 (H) HEASN556 (O) OD1ARG297 (H) HH21ARG297 (H) HH21ARG297 (H) HH22

(O) O2(H) H38(O) O21(O) O2(O) O3


HID228 (H) HD1LYS 543 (H) HZ1LYS 543 (H) HZ1

(O) O8(O) OS2(O) O13


TRP531 (H) HE1ASP348 (O) OD1

(O) O7H

3-Methylbutyl benzoate minus21 1 1898 THR547 (H) HG1 O


LYS543 (H) HZ2THR547 (H) HG1

(O) O35O

2-(5-Methylhexyl) benzoic acid minus13 1 2156 TRP531 (H) HE1 O3-(Iminomethyl)-24-dimethylphenol minus04 1 2171 ASN546 (H) HD21 OGlide score number of H-bond interactions distance of H-bond interaction and involved protein atom and ligand atom in interaction of isolated and standarddrugs with COX-2 protein obtained from docking studies

and Tyr-355 Chou [65] studied the various methods adoptedto understand the molecular mechanism of proteins andconduct structure based drug design by timely using theupdated information of newly found sequences He hasalso reported three main strategies developed in structuralbioinformatics that is pure energetic approach heuristicapproach and homology modeling approach as well astheir underlying principles in a review These approacheshelp to understand the action mechanisms of proteins andstimulating the course of drug discovery In the literature thebinding pocket of a protein receptor to a ligand is usuallydefined by those residues that have at least one heavy atom(ie an atom other than hydrogen) with a distancelt 5 A froma heavy atom of the ligand Such a criterion was originallyused to define the binding pocket of ATP in the Cdk5-Nck5alowastcomplex [66] and has later proved quite useful in identifyingfunctional domains and stimulating the relevant truncationexperiments The similar approach has also been used todefine the binding pockets of many other receptor-ligandinteractions important for drug design

Computational molecular docking is a useful tool forinvestigating the interaction of a target protein receptorwith its ligand and revealing their binding mechanism asdemonstrated by various studies [22ndash27] In this study wellknown docking program Glide module is used to dock the

isolated compounds of R fairholmianus root acetone and thestandard drugs to the active pockets of BRCA andCOX targetproteins to study the mode of interaction and inhibitoryproperties Many marvelous biological functions in proteinsand DNA and their profound dynamic mechanisms such asswitch between active and inactive states [67] cooperativeeffects [68] and allosteric transition [69] can be revealedby studying their internal motions [70] Likewise to reallyunderstand the actionmechanismbetween a drug compoundand its target protein we should consider not only thestatic binding structure but also the dynamical informationobtained by simulating their internal motions or dynamicprocess We will consider this in our future studies

4 Conclusions

The present study describes the bioactivity guided isolationstructure elucidation and antioxidant activities and molecu-lar docking of 6 compounds 1-(2-hydroxy phenyl)-4-methylpentan-1-one (1) cis-2-(isopentyloxycarbonyl) benzoic acid(2) 2-(5-methylhexyl) benzoic acid (3) 4-methylpentylbenzoate (4) 3-(iminomethyl)-24-dimethylphenol (5) andisopentyl benzoate or 3-methyl benzoate (6) from the rootacetone extract of R fairholmianus No previous reports areavailable on the compound isolation and other bioactivity


studies of this plant The investigation describes the iso-lated compounds from acetone extract of R fairholmianusroot docked to the active pockets of BRCA and COXproteins to predict analogous binding mode of the BRCAand COX inhibitors The results revealed that the com-pounds were docked more firmly and inhibited the breastcancer related BRCA1 and BRCA2 oncoproteins and COX-2 and COX-1 inflammatory proteins Among the 6 com-pounds 1-(2-hydroxyphenyl)-4-methylpentan-1-one and 2-[(3-methylbutoxy) carbonyl] benzoic acid were found tobe more active in inhibiting BRCA and COX proteinswhich also revealed the better results for antioxidant assaysTherefore these compounds may be considered as the leadcompounds for further development of therapeutic drugs

Conflict of Interests

The authors declare that there is no conflict of interests

References

[1] C E Finn ldquoRubus spp blackberryrdquo in The Encyclopedia ofFruits and Nuts J Janick and R E Paull Eds pp 348ndash351CABI Cambridge Mass USA 2008

[2] A V Patel J Rojas-Vera and C G Dacke ldquoTherapeuticconstituents and actions of Rubus speciesrdquo Current MedicinalChemistry vol 11 no 11 pp 1501ndash1512 2004

[3] N J Das S P Saikia S Sarkar and K Devi ldquoMedicinal plantsof north kamrup district of Assam used in primary health caresystemrdquo International Journal of Traditional Knowledge vol 5no 22 pp 489ndash493 2006

[4] J Barukial and JN Sarmah ldquoEthnomedicinal plants used by thepeople of Golaghat district Assam Indiardquo International Journalof Medicinal and Aromatic Plant vol 1 no 3 pp 203ndash211 2011

[5] B P George T Parimelazhagan and R Chandran ldquoEvaluationof total phenolic content antioxidant and analgesic potential ofRubus fairholmianus gardrdquo International Journal of Pharmacyand Pharmaceutical Sciences vol 5 no 3 pp 484ndash488 2013

[6] B P George T Parimelazhagan and R Chandran ldquoAnti-inflammatory and wound healing properties of Rubusfairholmianus Gard rootmdashan in vivo studyrdquo Industrial Cropsand Products vol 54 pp 216ndash225 2014

[7] B P George T Parimelazhagan and S Saravanan ldquoAnti-inflammatory analgesic and antipyretic activities of Rubusellipticus smith leaf methanol extractrdquo International Journal ofPharmacy and Pharmaceutical Sciences vol 5 no 2 pp 220ndash224 2013

[8] B PGeorge T Parimelazhagan S Saravanan andRChandranldquoAnti-inflammatory analgesic and antipyretic properties ofRubus niveusThunb root acetone extractrdquo Pharmacologia vol4 no 3 pp 228ndash235 2013

[9] B P George T Parimelazhagan T K Yamini and T SajeeshldquoAntitumor and wound healing properties of Rubus ellipticusSmithrdquo Journal of Acupuncture and Meridian Studies 2013

[10] K R Maatta-Riihinen A Kamal-Eldin and A R TorronenldquoIdentification and quantification of phenolic compounds inberries of Fragaria andRubus species (family rosaceae)rdquo Journalof Agricultural and Food Chemistry vol 52 no 20 pp 6178ndash6187 2004

[11] C Mertz V Cheynier Z Gunata and P Brat ldquoAnalysisof phenolic compounds in two blackberry species (Rubus

glaucus and Rubus adenotrichus) by high-performance liquidchromatography with diode array detection and electrosprayion trap mass spectrometryrdquo Journal of Agricultural and FoodChemistry vol 55 no 21 pp 8616ndash8624 2007

[12] G E Pantelidis M Vasilakakis G A Manganaris and GDiamantidis ldquoAntioxidant capacity phenol anthocyanin andascorbic acid contents in raspberries blackberries red currantsgooseberries and Cornelian cherriesrdquo Food Chemistry vol 102no 3 pp 777ndash783 2007

[13] J Reyes-Carmona G G Yousef R A Martınez-Peniche andM A Lila ldquoAntioxidant capacity of fruit extracts of blackberry(Rubus sp) produced in different climatic regionsrdquo Journal ofFood Science vol 70 no 7 pp S497ndashS503 2005

[14] S Y Wang and H Lin ldquoAntioxidant activity in fruits and leavesof blackberry raspberry and strawberry varies with cultivarand developmental stagerdquo Journal of Agricultural and FoodChemistry vol 48 no 2 pp 140ndash146 2000

[15] A Szajdek and E J Borowska ldquoBioactive compounds andhealth-promoting properties of berry fruits a reviewrdquo PlantFoods for Human Nutrition vol 63 no 4 pp 147ndash156 2008

[16] M Laguerre J Lecomte and P Villeneuve ldquoEvaluation of theability of antioxidants to counteract lipid oxidation existingmethods new trends and challengesrdquoProgress in Lipid Researchvol 46 no 5 pp 244ndash282 2007

[17] P Duh ldquoAntioxidant activity of burdock (Arctium lappa linne)Its scavenging effect on free-radical and active oxygenrdquo Journalof the American Oil Chemistsrsquo Society vol 75 no 4 pp 455ndash4611998

[18] W Zheng and S Y Wang ldquoAntioxidant activity and phenoliccompounds in selected herbsrdquo Journal of Agricultural and FoodChemistry vol 49 no 11 pp 5165ndash5170 2001

[19] V Sharma H V Kumar and L J M Rao ldquoInfluence of milkand sugar on antioxidant potential of black teardquo Food ResearchInternational vol 41 no 2 pp 124ndash129 2008

[20] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958

[21] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[22] K C Chou D Q Wei and W Z Zhong ldquoBinding mechanismof coronavirus main proteinase with ligands and its implicationto drug design against SARSrdquo Biochemical and BiophysicalResearch Communications vol 308 no 1 pp 148ndash151 2003

[23] QDu SWangDWei S Sirois andKChou ldquoMolecularmod-eling and chemical modification for finding peptide inhibitoragainst severe acute respiratory syndrome coronavirus mainproteinaserdquoAnalytical Biochemistry vol 337 no 2 pp 262ndash2702005

[24] L Cai Y Wang J Wang and K Chou ldquoIdentification ofproteins interacting with human SP110 during the process ofviral infectionsrdquo Medicinal Chemistry vol 7 no 2 pp 121ndash1262011

[25] Q H Liao Q Z Gao J Wei and K C Chou ldquoDockingand molecular dynamics study on the inhibitory activity ofnovel inhibitors on epidermal growth factor receptor (EGFR)rdquoMedicinal Chemistry vol 7 no 1 pp 24ndash31 2011

[26] Y Ma S Q Wang W R Xu R L Wang and K C ChouldquoDesign novel dual agonists for treating type-2 diabetes bytargeting peroxisome proliferator-activated receptors with corehopping approachrdquo PLoS ONE vol 7 no 6 Article ID e385462012


[27] J Wang and K Chou ldquoInsights into the mutation-inducedHHH syndrome from modeling human mitochondrialornithine transporter-1rdquo PLoS ONE vol 7 no 1 Article IDe31048 2012

[28] 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

[29] Lig Prep Schrodinger Release 2013-1 LigPrep version 26Schrodinger LLC New York NY USA 2013

[30] R A Friesner R B Murphy M P Repasky et al ldquoExtraprecision glide Docking and scoring incorporating a model ofhydrophobic enclosure for protein-ligand complexesrdquo Journalof Medicinal Chemistry vol 49 no 21 pp 6177ndash6196 2006

[31] T A Halgren R B Murphy R A Friesner et al ldquoGlidea new approach for rapid accurate docking and scoring 2Enrichment factors in database screeningrdquo Journal of MedicinalChemistry vol 47 no 7 pp 1750ndash1759 2004

[32] R A Friesner J L Banks R B Murphy et al ldquoGlide a newapproach for rapid accurate docking and scoring 1 Methodand assessment of docking accuracyrdquo Journal of MedicinalChemistry vol 47 no 7 pp 1739ndash1749 2004

[33] F Shahidi and M Naczk Phenolics in Food and NutraceuticalsCRC Press Boca Raton Fla USA 2nd edition 2003

[34] P Vadivelan R R Kumar S Bhadra A Shanish K Elango andB Suresh ldquoEvaluation of antioxidant activity of root extracts ofRubus ellipticus Smithrdquo Hygeia vol 9 no 1 pp 74ndash78 2009

[35] S M Motamed and F Naghibi ldquoAntioxidant activity of someedible plants of the Turkmen Sahra region in northern IranrdquoFood Chemistry vol 119 no 4 pp 1637ndash1642 2010

[36] A Ruiz I Hermosın-Gutierrez C Vergara et al ldquoAnthocyaninprofiles in south Patagonian wild berries by HPLC-DAD-ESI-MSMSrdquo Food Research International vol 51 no 2 pp 706ndash7132013

[37] M Kubota C Ishikawa Y Sugiyama S Fukumoto T Miyagiand S Kumazawa ldquoAnthocyanins from the fruits of Rubuscroceacanthus and Rubus sieboldii wild berry plants fromOkinawa Japanrdquo Journal of Food Composition and Analysis vol28 no 2 pp 179ndash182 2012

[38] E A Porter A A van Den Bos G C Kite N C Veitchand M S J Simmonds ldquoFlavonol glycosides acylated with3-hydroxy-3-methylglutaric acid as systematic characters inRosardquo Phytochemistry vol 81 pp 90ndash96 2012

[39] D C Estupinan S J Schwartz and G A Garzon ldquoAntioxidantactivity total phenolics content anthocyanin and colour sta-bility of isotonic model beverages coloured with Andes berry(Rubus glaucus Benth) anthocyanin powderrdquo Journal of FoodScience vol 76 no 1 pp S26ndashS34 2011

[40] C Vasco K R Riihinen J Ruales and A Kamal-EldinldquoPhenolic compounds in rosaceae fruits from Ecuadorrdquo Journalof Agricultural and Food Chemistry vol 57 no 4 pp 1204ndash12122009

[41] G A Garzon K M Riedl and S J Schwartz ldquoDeterminationof anthocyanins total phenolic content and antioxidant activityin Andes berry (Rubus glaucus Benth)rdquo Journal of Food Sciencevol 74 no 3 pp 227ndash232 2009

[42] H Kim S J Park S Hyun et al ldquoBiochemical monitoring ofblack raspberry (Rubus coreanus Miquel) fruits according tomaturation stage by 1H NMR using multiple solvent systemsrdquoFood Research International vol 44 no 7 pp 1977ndash1987 2011

[43] C S Ku and S PMun ldquoOptimization of the extraction of antho-cyanin from Bokbunja (Rubus coreanus Miq) marc producedduring traditional wine processing and characterization of theextractsrdquo Bioresource Technology vol 99 no 17 pp 8325ndash83302008

[44] J Bae S S Lim J Cho and Y Kang ldquoProtective actions ofRubus coreanus ethanol extract on collagenous extracellularmatrix in ultraviolet-B irradiation induced human dermalfibroblastsrdquo Nutrition Research and Practice vol 1 no 4 pp279ndash284 2007

[45] N Deighton R Brennan C Finn and H V Davies ldquoAntiox-idant properties of domesticated and wild Rubus speciesrdquoJournal of Agricultural and Food Chemistry vol 80 no 9 pp1307ndash1313 2000

[46] F J Wyzgoski L Paudel P L Rinaldi et al ldquoModeling rela-tionships among active components in black raspberry (Rubusoccidentalis L) fruit extracts using high-resolution1H nuclearmagnetic resonance (NMR) spectroscopy and multivariatestatistical analysisrdquo Journal of Agricultural and Food Chemistryvol 58 no 6 pp 3407ndash3414 2010

[47] M Dossett J Lee and C E Finn ldquoVariation in anthocyaninsand total phenolics of black raspberry populationsrdquo Journal ofFunctional Foods vol 2 no 4 pp 292ndash297 2010

[48] Y Ling C Ren S R Mallery et al ldquoA rapid and sensitive LC-MSMS method for quantification of four anthocyanins and itsapplication in a clinical pharmacology study of a bioadhesiveblack raspberry gelrdquo Journal of Chromatography B vol 877 no31 pp 4027ndash4034 2009

[49] M Dossett J Lee and C E Finn ldquoInheritance of phenologicalvegetative and fruit chemistry traits in black raspberryrdquo Journalof the American Society for Horticultural Science vol 133 no 3pp 408ndash417 2008

[50] A Z Tulio Jr R N Reese F J Wyzgoski et al ldquoCyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside as primary pheno-lic antioxidants in black raspberryrdquo Journal of Agricultural andFood Chemistry vol 56 no 6 pp 1880ndash1888 2008

[51] Q Tian M M Giusti G D Stoner and S J SchwartzldquoCharacterization of a new anthocyanin in black raspberries(Rubus occidentalis) by liquid chromatography electrosprayionization tandemmass spectrometryrdquo Food Chemistry vol 94no 3 pp 465ndash468 2006

[52] C S Bowen-ForbesVMulabagal Y Liu andMGNair ldquoUrso-lic acid analogues non-phenolic functional food components inJamaican raspberry fruitsrdquo Food Chemistry vol 116 no 3 pp633ndash637 2009

[53] A R Venkitaraman ldquoFunctions of BRCA1 and BRCA2 in thebiological response toDNAdamagerdquo Journal of Cell Science vol114 no 20 pp 3591ndash3598 2001

[54] S P Raja K Kathiresan and S Sahu ldquoIn silico docking analysisof mangrove-derived compounds against breast cancer protein(BRCA1)rdquo International Multidisciplinary Research Journal vol1 no 1 pp 9ndash12 2011

[55] K Saravanakumar S K Sahu and K Kathiresan ldquoIn-silicostudies on fungal metabolites against breast cancer protein(BRCA1)rdquoAsian Pacific Journal of Tropical Biomedicine pp 1ndash32012

[56] J A Clapperton I A Manke D M Lowery et al ldquoStruc-ture and mechanism of BRCA1 BRCT domain recognition ofphosphorylated BACH1 with implications for cancerrdquo NatureStructural and Molecular Biology vol 11 no 6 pp 512ndash5182004


[57] A W Oliver S Swift C J Lord A Ashworth and L H PearlldquoStructural basis for recruitment of BRCA2 by PALB2rdquo EMBOReports vol 10 no 9 pp 990ndash996 2009

[58] R Gautam S M Jachak V Kumar and C G MohanldquoSynthesis biological evaluation and molecular docking stud-ies of stellatin derivatives as cyclooxygenase (COX-1 COX-2) inhibitors and anti-inflammatory agentsrdquo Bioorganic andMedicinal Chemistry Letters vol 21 no 6 pp 1612ndash1616 2011

[59] S Olgen E Akaho and D Nebioglu ldquoSynthesis and receptordocking studies ofN-substituted indole-2-carboxylic acid estersas a search for COX-2 selective enzyme inhibitorsrdquo EuropeanJournal of Medicinal Chemistry vol 36 no 9 pp 747ndash770 2001

[60] K M Amin M M Kamel M M Anwar M Khedr andY M Syam ldquoSynthesis biological evaluation and moleculardocking of novel series of spiro [(2H3H) quinazoline-211015840-cyclohexan]-4(1H)- one derivatives as anti-inflammatory andanalgesic agentsrdquo European Journal of Medicinal Chemistry vol45 no 6 pp 2117ndash2131 2010

[61] A A- Abdel-Aziz K E H Eltahir and Y A Asiri ldquoSynthesisanti-inflammatory activity and COX-1COX-2 inhibition ofnovel substituted cyclic imides Part 1 Molecular dockingstudyrdquo European Journal of Medicinal Chemistry vol 46 no 5pp 1648ndash1655 2011

[62] L Basile S Alvarez A Blanco et al ldquoSulfonilamidothiopy-rimidone and thiopyrimidone derivatives as selective COX-2inhibitors synthesis biological evaluation and docking stud-iesrdquo European Journal of Medicinal Chemistry vol 57 pp 149ndash161 2012

[63] M A-A El-Sayed N I Abdel-Aziz A A-M Abdel-Aziz AS El-Azab Y A Asiri and K E H Eltahir ldquoDesign synthesisand biological evaluation of substituted hydrazone and pyrazolederivatives as selective COX-2 inhibitors molecular dockingstudyrdquo Bioorganic amp Medicinal Chemistry vol 19 no 11 pp3416ndash3424 2011

[64] B S Selinsky K Gupta C T Sharkey and P J Loll ldquoStructuralanalysis of NSAID binding by prostaglandinH2 synthase time-dependent and time-independent inhibitors elicit identicalenzyme conformationsrdquo Biochemistry vol 40 no 17 pp 5172ndash5180 2001

[65] K Chou ldquoStructural bioinformatics and its impact to biomed-ical sciencerdquo Current Medicinal Chemistry vol 11 no 16 pp2105ndash2134 2004

[66] K Chou K D Watenpaugh and R L Heinrikson ldquoA modelof the complex between cyclin-dependent kinase 5 and theactivation domain of neuronal Cdk5 activatorrdquo Biochemical andBiophysical Research Communications vol 259 no 2 pp 420ndash428 1999

[67] J F Wang and K C Chou ldquoInsight into the molecular switchmechanism of human Rab5a frommolecular dynamics simula-tionsrdquo Biochemical and Biophysical Research Communicationsvol 390 no 3 pp 608ndash612 2009

[68] K C Chou ldquoLow-frequency resonance and cooperativity ofhemoglobinrdquo Trends in Biochemical Sciences vol 14 no 6 pp212ndash213 1989

[69] J R Schnell and J J Chou ldquoStructure andmechanism of theM2proton channel of influenza A virusrdquo Nature vol 451 no 7178pp 591ndash595 2008

[70] K Chou ldquoLow-frequency collective motion in biomacro-molecules and its biological functionsrdquo Biophysical Chemistryvol 30 no 1 pp 3ndash48 1988

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MEDIATORSINFLAMMATION

of


score number of H-bonds distance of bonds interacted pro-tein residues and ligand atom were observed from dockingstudies This docking study helps us in understanding thebinding mode of the isolated compounds with the targetproteins

In the present scenario the antioxidant researchers aremainly focusing on the identification and isolation of newnatural antioxidant compounds from different plants speciessince it can protect the human body from various free radicalgenerated disorders A survey of literature revealed that thephytochemical aspects of this plant have not been evaluatedThis study aimed to investigate the antioxidant activitiesof different extracts of R fairholmianus This study focuseson the isolation and identification of bioactive antioxidantcompounds from R fairholmianus through chromatographictechniques based on the activity guided fractionation of theroot acetone extract The isolated compounds then checkedfor its antioxidant potential Further we adopted dockingstudies to identify inhibiting activity of the compoundsagainst BRACA and COX proteins

2 Materials and Methods

21 Plant Collection and Extraction The fresh plant partsof R fairholmianus were collected from Marayoor Sholaforest Kerala India during the month of September 2010The collected plant material was identified and authenticatedby (Voucher specimen number BSISRC5232010-11Tech1657) Botanical Survey of India SouthernCircle CoimbatoreTamil Nadu The roots were extracted successively usingacetone in a soxhlet apparatus for 72 hours The extract wasconcentrated to dryness under reduced pressure in a rotaryevaporator

22 Determination of In Vitro Antioxidant Activities Thedifferent fractions and isolated compounds from root acetoneextracts of R fairholmianus were analyzed for their antioxi-dant activity using DPPH assay and ABTS assay

221 DPPH Radical Scavenging Activity The DPPH assaywas done as per the method described by Blois [20] Negativecontrol was prepared by adding 100 120583L of methanol in 5mLof 01mM methanolic solution of DPPH∙ The tubes wereallowed to stand for 20 minutes at 27∘C Radical scavengingactivity of the samples was expressed as IC

50

which is theconcentration of the sample required to inhibit 50 ofDPPH∙ concentration

222 Trolox Equivalent Antioxidant Capacity (TEAC) AssayThe total antioxidant activity was measured by ABTS radicalcation decolourization assay according to Re et al [21]Triplicate determinations were made at each dilution ofthe standard and the percentage inhibition was calculatedagainst the blank (ethanol) with an absorbance at 734 nmand then was plotted as a function of Trolox concentrationThe unit of total antioxidant activity (TAA) is defined asthe concentration of Trolox having equivalent antioxidantactivity expressed as 120583Mg sample extracts

23 Chromatographic Separation of the Root Acetone ExtractBased on the in vitro antioxidant studies RFRA (R fairholmi-anus root acetone) showed maximum antioxidant activityand it was selected for the further isolation of phytocon-stituents The preliminary screening was done using thinlayer chromatography by toluene ethyl acetate acetic acid(6 3 05) as mobile phase The extract (50 g) was adsorbedon activated silica (230ndash400 mesh) The column (90 times 5 cm)was packed with activated silica gel (230ndash400 mesh) intoluene and it was eluted with 400mL of each of differentsolvents such toluene (100) toluene ethyl acetate (9 1 7 36 4 5 5 4 6 2 8) ethyl acetate chloroform (9 1 7 35 5 3 7 1 9) chloroform methanol (8 2 6 4 5 5 4 6)diethyl ether (100) ethanol (100)methanol (100) aceticacid (2) and acetone (100) A total of 183 fractions werecollected and the fractionswith similar chemical profileswerepooled based on TLC analysis

Based on the TLC the fractions with similar bandingpattern were clubbed and 16 fractions were obtained Thevarious fractions such as F1 (34ndash37) F2 (38ndash54) F3 (55ndash58)F4 (59ndash72) F5 (76ndash80) F6 (84ndash88) F7 (89ndash110) F8 (111ndash116)F9 (121ndash126) F10 (132ndash136) F11 (137ndash141) F12 (142) F13 (143ndash147) F14 (148ndash151) F15 (159ndash162) and F16 (163ndash183) weresubjected to antioxidant studies The most active fractionssuch as F5 F7 F9 and F16 were selected for further isolationprocess

The 4 active fractions further yielded 6 subfractionsmethanol and ethyl acetate subfractions of F5 (F5M andF5EA) chloroform subfractions of F7 and F9 (F7C and F9C)and methanol and diethyl ether subfractions of 16 (F16M andF16DE) All these fractionswere analysed by TLCHPLC andso forth

24 Purification of Isolated Compounds The isolated com-pounds were purified by semipreparative TLC and prepar-ative HPLC The bands visualized under UV at 254 nmand 366 nm were scrapped out and dissolved in respectivesolvents The time based collection was performed usingpreparative fraction collector on Agilent 1200 high pressureliquid chromatographic system equipped with prep pump arheodyne injector and diode array detector in combinationwith Chem32 Chemstation software C18-scalar (5120583m 46times 150mm) Agilent column was used for separation with abinary gradient elution Mobile phase 01 acetic acid inacetonitrile (A) methanol water (60 40) (B) 0ndash2 70A2ndash5 60A 5ndash10 50A 10ndash20 70A The flow rate wasmaintained as 15mLminute

25 Characterization of Isolated Compounds

251 UV Spectroscopy The UV-visible spectrum of the iso-lated compound inHPLCgrademethanolwas recordedusinga Shimadzu 160AUV-visible spectrophotometer at a range of280ndash400 nm

252 Fourier Transform Infrared Spectrometry (FTIR) TheFTIR spectrum was recorded using a Nicolet 5700 (Thermoelectron Madison WI USA) spectrometer at room temper-ature The bioactive compound was dissolved in dimethyl




3











50

value 361)The IC

50


50



50


50








eluted fractions


eluted fractions


eluted fractions



Compound 5

Compound 6

Compound Compound 4



Compounds 2 and 3





F7( F9( F16(

F(


1

6 are





3



3



3

) 120575 771 770 769 753 752 751 750424 423 422 420 419 418 13C NMR 120575 16773 1423913249 13236 13088 13086 12880 6619 3876 3668 35503250 2880 MS (mz) [M-H] 21919


3

) 120575 771 770











769 753 751 426 423 421 170 168 167 140 099 098094 092 13C NMR 120575 16776 13396 13248 12881 68183876 3028 1404 MS (mz) [M-H] 20523


3

) 120575 841 680 657 547427 428 438 438 246 232 13C NMR 120575 11848 115134507 2080 14949 13597 13419 12646 1824MS (mz) [M-H] 14808


3

) 120575803 751 792 429 432 206 207 168 173 094 099 13CNMR120575 16771 12569 12884 13090 13599 3058 2774 19181915 MS (mz) [M-H] 19110






Molecularweight


CH3

CH3

HO

O

C12H16O2 19225


CH3

CH3

HO

OOO

C13H16O4 47253


CH3

CH3

HO

O

C14H20O2 22031


CH3

CH3

OO

C13H18O2 20628


CH3

CH3

OH

NH C9H11NO 14919


CH3OO

C12H16O2 19225








atom


24492418247022032013


LYS1702 (H) HZ2

OHHHO



NN



LYS1702 (H) HZ2

OOO




OOO


22572142207019161709


LYS1702 (H) HZ2

OOOOO



LYS1702 (H) HZ2

OOO




LEU1701 (H) H

NHO








atom


20431994192119261843



OHOHH



HH



NN


LYS1062 (H) HZ3GLU1018 (H) H

OO


2429193318191408


OOHH



OOH


ALA1063 (H) HLYS1062 (H) HZ2

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)










atom



OOO

Aspirin minus565 5

20662043219918571781


ARG 374 (H) HH12ARG 374 (H) HH21

OOOOO


ARG 374 (H) HH22ASN 375 (H) H

ARG 374 (H) HH22

OOO


ARG 374 (H) HH21GLY225 (O) O

OH



ASN 375 (H) HASN 375 (O) O

OH



OOO



ASN 375 (H) HASN 375 (O) O

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)



3








atom



(O) O3O

(O) O3



O(O) O4



(O) O20(O) O20(O) O3



HOH

Morphine minus10 5

24712249202519891974


(O) O2(H) H38(O) O21(O) O2(O) O3



(O) O8(O) OS2(O) O13



(O) O7H




(O) O35O





4 Conclusions






References














































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




3











50

value 361)The IC

50


50



50


50








eluted fractions


eluted fractions


eluted fractions



Compound 5

Compound 6

Compound Compound 4



Compounds 2 and 3





F7( F9( F16(

F(


1

6 are





3



3



3

) 120575 771 770 769 753 752 751 750424 423 422 420 419 418 13C NMR 120575 16773 1423913249 13236 13088 13086 12880 6619 3876 3668 35503250 2880 MS (mz) [M-H] 21919


3

) 120575 771 770











769 753 751 426 423 421 170 168 167 140 099 098094 092 13C NMR 120575 16776 13396 13248 12881 68183876 3028 1404 MS (mz) [M-H] 20523


3

) 120575 841 680 657 547427 428 438 438 246 232 13C NMR 120575 11848 115134507 2080 14949 13597 13419 12646 1824MS (mz) [M-H] 14808


3

) 120575803 751 792 429 432 206 207 168 173 094 099 13CNMR120575 16771 12569 12884 13090 13599 3058 2774 19181915 MS (mz) [M-H] 19110






Molecularweight


CH3

CH3

HO

O

C12H16O2 19225


CH3

CH3

HO

OOO

C13H16O4 47253


CH3

CH3

HO

O

C14H20O2 22031


CH3

CH3

OO

C13H18O2 20628


CH3

CH3

OH

NH C9H11NO 14919


CH3OO

C12H16O2 19225








atom


24492418247022032013


LYS1702 (H) HZ2

OHHHO



NN



LYS1702 (H) HZ2

OOO




OOO


22572142207019161709


LYS1702 (H) HZ2

OOOOO



LYS1702 (H) HZ2

OOO




LEU1701 (H) H

NHO








atom


20431994192119261843



OHOHH



HH



NN


LYS1062 (H) HZ3GLU1018 (H) H

OO


2429193318191408


OOHH



OOH


ALA1063 (H) HLYS1062 (H) HZ2

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)










atom



OOO

Aspirin minus565 5

20662043219918571781


ARG 374 (H) HH12ARG 374 (H) HH21

OOOOO


ARG 374 (H) HH22ASN 375 (H) H

ARG 374 (H) HH22

OOO


ARG 374 (H) HH21GLY225 (O) O

OH



ASN 375 (H) HASN 375 (O) O

OH



OOO



ASN 375 (H) HASN 375 (O) O

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)



3








atom



(O) O3O

(O) O3



O(O) O4



(O) O20(O) O20(O) O3



HOH

Morphine minus10 5

24712249202519891974


(O) O2(H) H38(O) O21(O) O2(O) O3



(O) O8(O) OS2(O) O13



(O) O7H




(O) O35O





4 Conclusions






References














































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







eluted fractions


eluted fractions


eluted fractions



Compound 5

Compound 6

Compound Compound 4



Compounds 2 and 3





F7( F9( F16(

F(


1

6 are





3



3



3

) 120575 771 770 769 753 752 751 750424 423 422 420 419 418 13C NMR 120575 16773 1423913249 13236 13088 13086 12880 6619 3876 3668 35503250 2880 MS (mz) [M-H] 21919


3

) 120575 771 770











769 753 751 426 423 421 170 168 167 140 099 098094 092 13C NMR 120575 16776 13396 13248 12881 68183876 3028 1404 MS (mz) [M-H] 20523


3

) 120575 841 680 657 547427 428 438 438 246 232 13C NMR 120575 11848 115134507 2080 14949 13597 13419 12646 1824MS (mz) [M-H] 14808


3

) 120575803 751 792 429 432 206 207 168 173 094 099 13CNMR120575 16771 12569 12884 13090 13599 3058 2774 19181915 MS (mz) [M-H] 19110






Molecularweight


CH3

CH3

HO

O

C12H16O2 19225


CH3

CH3

HO

OOO

C13H16O4 47253


CH3

CH3

HO

O

C14H20O2 22031


CH3

CH3

OO

C13H18O2 20628


CH3

CH3

OH

NH C9H11NO 14919


CH3OO

C12H16O2 19225








atom


24492418247022032013


LYS1702 (H) HZ2

OHHHO



NN



LYS1702 (H) HZ2

OOO




OOO


22572142207019161709


LYS1702 (H) HZ2

OOOOO



LYS1702 (H) HZ2

OOO




LEU1701 (H) H

NHO








atom


20431994192119261843



OHOHH



HH



NN


LYS1062 (H) HZ3GLU1018 (H) H

OO


2429193318191408


OOHH



OOH


ALA1063 (H) HLYS1062 (H) HZ2

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)










atom



OOO

Aspirin minus565 5

20662043219918571781


ARG 374 (H) HH12ARG 374 (H) HH21

OOOOO


ARG 374 (H) HH22ASN 375 (H) H

ARG 374 (H) HH22

OOO


ARG 374 (H) HH21GLY225 (O) O

OH



ASN 375 (H) HASN 375 (O) O

OH



OOO



ASN 375 (H) HASN 375 (O) O

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)



3








atom



(O) O3O

(O) O3



O(O) O4



(O) O20(O) O20(O) O3



HOH

Morphine minus10 5

24712249202519891974


(O) O2(H) H38(O) O21(O) O2(O) O3



(O) O8(O) OS2(O) O13



(O) O7H




(O) O35O





4 Conclusions






References














































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of











769 753 751 426 423 421 170 168 167 140 099 098094 092 13C NMR 120575 16776 13396 13248 12881 68183876 3028 1404 MS (mz) [M-H] 20523


3

) 120575 841 680 657 547427 428 438 438 246 232 13C NMR 120575 11848 115134507 2080 14949 13597 13419 12646 1824MS (mz) [M-H] 14808


3

) 120575803 751 792 429 432 206 207 168 173 094 099 13CNMR120575 16771 12569 12884 13090 13599 3058 2774 19181915 MS (mz) [M-H] 19110






Molecularweight


CH3

CH3

HO

O

C12H16O2 19225


CH3

CH3

HO

OOO

C13H16O4 47253


CH3

CH3

HO

O

C14H20O2 22031


CH3

CH3

OO

C13H18O2 20628


CH3

CH3

OH

NH C9H11NO 14919


CH3OO

C12H16O2 19225








atom


24492418247022032013


LYS1702 (H) HZ2

OHHHO



NN



LYS1702 (H) HZ2

OOO




OOO


22572142207019161709


LYS1702 (H) HZ2

OOOOO



LYS1702 (H) HZ2

OOO




LEU1701 (H) H

NHO








atom


20431994192119261843



OHOHH



HH



NN


LYS1062 (H) HZ3GLU1018 (H) H

OO


2429193318191408


OOHH



OOH


ALA1063 (H) HLYS1062 (H) HZ2

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)










atom



OOO

Aspirin minus565 5

20662043219918571781


ARG 374 (H) HH12ARG 374 (H) HH21

OOOOO


ARG 374 (H) HH22ASN 375 (H) H

ARG 374 (H) HH22

OOO


ARG 374 (H) HH21GLY225 (O) O

OH



ASN 375 (H) HASN 375 (O) O

OH



OOO



ASN 375 (H) HASN 375 (O) O

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)



3








atom



(O) O3O

(O) O3



O(O) O4



(O) O20(O) O20(O) O3



HOH

Morphine minus10 5

24712249202519891974


(O) O2(H) H38(O) O21(O) O2(O) O3



(O) O8(O) OS2(O) O13



(O) O7H




(O) O35O





4 Conclusions






References














































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




Molecularweight


CH3

CH3

HO

O

C12H16O2 19225


CH3

CH3

HO

OOO

C13H16O4 47253


CH3

CH3

HO

O

C14H20O2 22031


CH3

CH3

OO

C13H18O2 20628


CH3

CH3

OH

NH C9H11NO 14919


CH3OO

C12H16O2 19225








atom


24492418247022032013


LYS1702 (H) HZ2

OHHHO



NN



LYS1702 (H) HZ2

OOO




OOO


22572142207019161709


LYS1702 (H) HZ2

OOOOO



LYS1702 (H) HZ2

OOO




LEU1701 (H) H

NHO








atom


20431994192119261843



OHOHH



HH



NN


LYS1062 (H) HZ3GLU1018 (H) H

OO


2429193318191408


OOHH



OOH


ALA1063 (H) HLYS1062 (H) HZ2

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)










atom



OOO

Aspirin minus565 5

20662043219918571781


ARG 374 (H) HH12ARG 374 (H) HH21

OOOOO


ARG 374 (H) HH22ASN 375 (H) H

ARG 374 (H) HH22

OOO


ARG 374 (H) HH21GLY225 (O) O

OH



ASN 375 (H) HASN 375 (O) O

OH



OOO



ASN 375 (H) HASN 375 (O) O

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)



3








atom



(O) O3O

(O) O3



O(O) O4



(O) O20(O) O20(O) O3



HOH

Morphine minus10 5

24712249202519891974


(O) O2(H) H38(O) O21(O) O2(O) O3



(O) O8(O) OS2(O) O13



(O) O7H




(O) O35O





4 Conclusions






References














































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





atom


24492418247022032013


LYS1702 (H) HZ2

OHHHO



NN



LYS1702 (H) HZ2

OOO




OOO


22572142207019161709


LYS1702 (H) HZ2

OOOOO



LYS1702 (H) HZ2

OOO




LEU1701 (H) H

NHO








atom


20431994192119261843



OHOHH



HH



NN


LYS1062 (H) HZ3GLU1018 (H) H

OO


2429193318191408


OOHH



OOH


ALA1063 (H) HLYS1062 (H) HZ2

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)










atom



OOO

Aspirin minus565 5

20662043219918571781


ARG 374 (H) HH12ARG 374 (H) HH21

OOOOO


ARG 374 (H) HH22ASN 375 (H) H

ARG 374 (H) HH22

OOO


ARG 374 (H) HH21GLY225 (O) O

OH



ASN 375 (H) HASN 375 (O) O

OH



OOO



ASN 375 (H) HASN 375 (O) O

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)



3








atom



(O) O3O

(O) O3



O(O) O4



(O) O20(O) O20(O) O3



HOH

Morphine minus10 5

24712249202519891974


(O) O2(H) H38(O) O21(O) O2(O) O3



(O) O8(O) OS2(O) O13



(O) O7H




(O) O35O





4 Conclusions






References














































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





atom


20431994192119261843



OHOHH



HH



NN


LYS1062 (H) HZ3GLU1018 (H) H

OO


2429193318191408


OOHH



OOH


ALA1063 (H) HLYS1062 (H) HZ2

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)










atom



OOO

Aspirin minus565 5

20662043219918571781


ARG 374 (H) HH12ARG 374 (H) HH21

OOOOO


ARG 374 (H) HH22ASN 375 (H) H

ARG 374 (H) HH22

OOO


ARG 374 (H) HH21GLY225 (O) O

OH



ASN 375 (H) HASN 375 (O) O

OH



OOO



ASN 375 (H) HASN 375 (O) O

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)



3








atom



(O) O3O

(O) O3



O(O) O4



(O) O20(O) O20(O) O3



HOH

Morphine minus10 5

24712249202519891974


(O) O2(H) H38(O) O21(O) O2(O) O3



(O) O8(O) OS2(O) O13



(O) O7H




(O) O35O





4 Conclusions






References














































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(a) (b)

(c) (d)










atom



OOO

Aspirin minus565 5

20662043219918571781


ARG 374 (H) HH12ARG 374 (H) HH21

OOOOO


ARG 374 (H) HH22ASN 375 (H) H

ARG 374 (H) HH22

OOO


ARG 374 (H) HH21GLY225 (O) O

OH



ASN 375 (H) HASN 375 (O) O

OH



OOO



ASN 375 (H) HASN 375 (O) O

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)



3








atom



(O) O3O

(O) O3



O(O) O4



(O) O20(O) O20(O) O3



HOH

Morphine minus10 5

24712249202519891974


(O) O2(H) H38(O) O21(O) O2(O) O3



(O) O8(O) OS2(O) O13



(O) O7H




(O) O35O





4 Conclusions






References














































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





atom



OOO

Aspirin minus565 5

20662043219918571781


ARG 374 (H) HH12ARG 374 (H) HH21

OOOOO


ARG 374 (H) HH22ASN 375 (H) H

ARG 374 (H) HH22

OOO


ARG 374 (H) HH21GLY225 (O) O

OH



ASN 375 (H) HASN 375 (O) O

OH



OOO



ASN 375 (H) HASN 375 (O) O

OO


(a) (b)

(c) (d)



(a) (b)

(c) (d)



3








atom



(O) O3O

(O) O3



O(O) O4



(O) O20(O) O20(O) O3



HOH

Morphine minus10 5

24712249202519891974


(O) O2(H) H38(O) O21(O) O2(O) O3



(O) O8(O) OS2(O) O13



(O) O7H




(O) O35O





4 Conclusions






References














































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(a) (b)

(c) (d)



3








atom



(O) O3O

(O) O3



O(O) O4



(O) O20(O) O20(O) O3



HOH

Morphine minus10 5

24712249202519891974


(O) O2(H) H38(O) O21(O) O2(O) O3



(O) O8(O) OS2(O) O13



(O) O7H




(O) O35O





4 Conclusions






References














































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





atom



(O) O3O

(O) O3



O(O) O4



(O) O20(O) O20(O) O3



HOH

Morphine minus10 5

24712249202519891974


(O) O2(H) H38(O) O21(O) O2(O) O3



(O) O8(O) OS2(O) O13



(O) O7H




(O) O35O





4 Conclusions






References














































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





References














































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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