Research Article Bioactivities of Compounds from...
8
Research Article Bioactivities of Compounds from Elephantopus scaber, an Ethnomedicinal Plant from Southwest China Jianjun Wang, 1 Ping Li, 1 Baosai Li, 2 Zhiyong Guo, 1 Edward J. Kennelly, 1,3 and Chunlin Long 1,4 1 College of Life and Environmental Sciences, Minzu University of China, 27 Zhong-Guan-Cun South Avenue, Haidian District, Beijing 100081, China 2 School of Chinese Materia Medica, Beijing University of Chinese Medicine, 11 ird Ring Road, Chaoyang District, Beijing 100029, China 3 Department of Biological Science, Lehman College, and Graduate Center, City University of New York, 250 Bedford Park Boulevard West, Bronx, NY 10468, USA 4 Kunming Institute of Botany, Chinese Academy of Sciences, 132 Lanhei Road, Kunming 650201, China Correspondence should be addressed to Chunlin Long; [email protected] Received 2 April 2014; Accepted 30 April 2014; Published 19 May 2014 Academic Editor: Shi-Biao Wu Copyright © 2014 Jianjun Wang et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Elephantopus scaber is an ethnomedicinal plant used by the Zhuang people in Southwest China to treat headaches, colds, diarrhea, hepatitis, and bronchitis. A new -truxinate derivative, ethyl, methyl 3,4,3 ,4 -tetrahydroxy--truxinate (1), was isolated from the ethyl acetate extract of the entire plant, along with 4 known compounds. e antioxidant activity of these 5 compounds was determined by ABTS radical scavenging assay. Compound 1 was also tested for its cytotoxicity effect against HepG2 by MTT assay (IC 50 = 60M), and its potential anti-inflammatory, antibiotic, and antitumor bioactivities were predicted using target fishing method soſtware. 1. Introduction Elephantopus is a genus comprised of about 30 species worldwide, mainly distributed in South America, with only 2 species E. scaber and E. tomentosus found in Southwest China [1]. From 2008 to 2012, our ethnobotanical investigation in the traditional medicinal market, held during the Dragon- Boat Festival in the fiſth month of the Chinese lunar calendar with a history of over 700 years, found that Elephantopus scaber L. (Asteraceae) is a common medicinal plant used by the Zhuang people in Jingxi County of Southwest China. e local Zhuang people use E. scaber commonly as a traditional herbal medicine to treat many ailments including headaches, colds, diarrhea, hepatitis, and bronchitis. To date, 30 compounds have been reported from E. scaber, including 4 sesquiterpene lactones, 9 triterpenes, and 5 flavones. Previous bioactivity studies on E. scaber demon- strated that the extracts or compounds from this species have antibiosis, antivirus, and cytotoxicity activities [2]. e sesquiterpene lactones in particular have been explored for their anti-inflammatory and hepatoprotective activities [3], which partially proved the traditional knowledge of E. scaber. In this paper, the isolation and structure elucidation of a new ethyl, methyl 3,4,3 ,4 -tetrahydroxy--truxinate (1, Figure 1) is reported, together with 4 known compounds, 5- O-caffeoylquinic acid (2)[4], chlorogenic acid methyl ester (3)[5], deoxyelephantopin (4), and isoscarbertopin (5)[6]. e radical scavenging activity of these 5 compounds was conducted using the ABTS method. e cytotoxicity effect against HpeG2 cell line of the new compound was determined by MTT assay, and the IC 50 value (24.0 g/mL) was obtained. In addition, the potential activity of 1, calculated with target fishing, which used 3D structures of compounds to identify their interacting proteins by virtual screening [7], is also presented. 2. Materials and Method 2.1. Plant Material. e whole plant of E. scaber was collected from the traditional medicinal market during the Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2014, Article ID 569594, 7 pages http://dx.doi.org/10.1155/2014/569594
Transcript of Research Article Bioactivities of Compounds from...
Research ArticleBioactivities of Compounds from Elephantopus scaberan Ethnomedicinal Plant from Southwest China
Jianjun Wang1 Ping Li1 Baosai Li2 Zhiyong Guo1
Edward J Kennelly13 and Chunlin Long14
1 College of Life and Environmental Sciences Minzu University of China 27 Zhong-Guan-Cun South AvenueHaidian District Beijing 100081 China
2 School of Chinese Materia Medica Beijing University of Chinese Medicine 11 Third Ring RoadChaoyang District Beijing 100029 China
3Department of Biological Science Lehman College and Graduate Center City University of New York250 Bedford Park Boulevard West Bronx NY 10468 USA
4Kunming Institute of Botany Chinese Academy of Sciences 132 Lanhei Road Kunming 650201 China
Correspondence should be addressed to Chunlin Long longmailkibaccn
Received 2 April 2014 Accepted 30 April 2014 Published 19 May 2014
Academic Editor Shi-Biao Wu
Copyright copy 2014 Jianjun Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Elephantopus scaber is an ethnomedicinal plant used by the Zhuang people in Southwest China to treat headaches colds diarrheahepatitis and bronchitis A new 120575-truxinate derivative ethyl methyl 343101584041015840-tetrahydroxy-120575-truxinate (1) was isolated from theethyl acetate extract of the entire plant along with 4 known compounds The antioxidant activity of these 5 compounds wasdetermined by ABTS radical scavenging assay Compound 1 was also tested for its cytotoxicity effect against HepG2 by MTT assay(IC50= 60 120583M) and its potential anti-inflammatory antibiotic and antitumor bioactivities were predicted using target fishing
method software
1 Introduction
Elephantopus is a genus comprised of about 30 speciesworldwide mainly distributed in South America with only 2species E scaber and E tomentosus found in Southwest China[1] From 2008 to 2012 our ethnobotanical investigation inthe traditional medicinal market held during the Dragon-Boat Festival in the fifthmonth of the Chinese lunar calendarwith a history of over 700 years found that Elephantopusscaber L (Asteraceae) is a common medicinal plant used bythe Zhuang people in Jingxi County of Southwest ChinaThelocal Zhuang people use E scaber commonly as a traditionalherbal medicine to treat many ailments including headachescolds diarrhea hepatitis and bronchitis
To date 30 compounds have been reported from Escaber including 4 sesquiterpene lactones 9 triterpenes and5 flavones Previous bioactivity studies on E scaber demon-strated that the extracts or compounds from this specieshave antibiosis antivirus and cytotoxicity activities [2] Thesesquiterpene lactones in particular have been explored for
their anti-inflammatory and hepatoprotective activities [3]which partially proved the traditional knowledge of E scaber
In this paper the isolation and structure elucidationof a new ethyl methyl 343101584041015840-tetrahydroxy-120575-truxinate (1Figure 1) is reported together with 4 known compounds 5-O-caffeoylquinic acid (2) [4] chlorogenic acid methyl ester(3) [5] deoxyelephantopin (4) and isoscarbertopin (5) [6]The radical scavenging activity of these 5 compounds wasconducted using the ABTS method The cytotoxicity effectagainstHpeG2 cell line of the new compoundwas determinedbyMTT assay and the IC
50value (240 120583gmL) was obtained
In addition the potential activity of 1 calculated with targetfishing which used 3D structures of compounds to identifytheir interacting proteins by virtual screening [7] is alsopresented
2 Materials and Method
21 Plant Material The whole plant of E scaber wascollected from the traditional medicinal market during the
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 569594 7 pageshttpdxdoiorg1011552014569594
2 Evidence-Based Complementary and Alternative Medicine
OH
OHOH
OH
OHOH
OH
OH
OHOH
OH OH
O
O
OOO
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OO
HOHO HO
CH3
179
19984007998400999840011998400
5
3
3998400
5998400
1 2 3
4 5
Figure 1 The chemical structures of compounds obtained from Elephantopus scaber
Dragon-Boat Festival of Jingxi County (Guangxi) SouthwestChina and identified by Professor Chunlin Long A voucherspecimen was deposited in the Herbarium of Minzu Univer-sity of China numbered 201006023
22 Extraction and Isolation Theair-dried and groundwholeplant of E scaber (40 kg) was extracted with EtOH H
2O
(90 10) at reflux for 3times 3 hThe solvent was evaporated underreduced pressure to yield dark brown material (3724 g) Thelatter was suspended in H
2O (3 L) and individually parti-
tioned with petroleum ether (3 times 3 L) Chloroform (2 times 3 L)EtOAc (3 times 3 L) and n-BuOH (3 times 3 L) to obtain petroleumether (1694 g) Chloroform (338 g) EtOAc (469 g) and n-BuOH (1223 g) phase
The EtOAc phase was separated by silica gel columnchromatography (CC) eluted with CHCl
3 CH3OH in order
of increasing polarity to give seven fractions on the basisof TLC Fraction 3 was subjected to MCI CC eluted withCH3OH H
2O to seven fractions A
1ndashA7 Fraction A
1was
isolated by Sephadex LH-20 ODS CC (CH3OH H
2O =
44 56) and Si gel CC (CHCl3 CH3OH = 14 1) successively
to afford compound 1 (240mg) Fraction A2was purified
with Sephadex LH-20 to give six subfractions Subfraction2 was subjected to ODS CC (CH
3OH H
2O = 30 70) and
silica gel CC (CHCl3 CH3OH = 12 1) successively to give
compound 3 (170mg) Subfraction 3 was subjected to ODSCC (CH
3OH H
2O = 48 52) and Sephadex LH-20 to afford
compound 2 (270mg)The petroleum ether phase was separated by silica gel
CC eluted with petroleum ether EtOAc (100 1ndash0 100) togive ten fractions Fraction 8 was purified by MCI CC usingCH3OH H
2O (60 100ndash100 0) to afford four fractions B
1ndash
B4 Fraction B
2was subjected to Sephadex LH-20 and ODS
CC (CH3OH H
2O = 83 17) to give compound 4 (90mg)
FractionB3was isolated byODSCC (CH
3OH H
2O=80 20)
and Sephadex LH-20 to give compound 5 (70mg)
23 Antioxidant Assay The antioxidant activity of com-pounds 1ndash5 was evaluated with ABTS radical scavengingassay as described previously [8] The IC
50was expressed as
millimoles per liter (mM)
24 Cytotoxicity Assay Compound 1 was tested for cytotoxi-city using a slightly modifiedMTTmethod [9] Briefly 150120583L(10 120583M 20120583M 30 120583M and 40 120583M) of samples was added to96-well plate containing a confluent HepG2 cell monolayerin sextuplicate 10 120583gmL of norcantharidin (NCTD) andblank medium were used as the positive and control grouprespectively After a 72 h incubation at 37∘C 100 120583L MTTsolution (5mgmL phosphate buffered saline) was addedto each well which was further incubated for 4 h for theformation of the formazan product After removing themedium 150 120583L DMSO was added to dissolve the formazancrystals The optical density (OD) was measured at 550 nmwith amicroplate readerThe rate of inhibitionwas calculatedby the following formula rate of inhibition = (1 minus sampleOD)control OD The concentration causing inhibition ofviable cells by 50 (IC
50) was determined from a dose-
response curve which was based on triplicate measurements
25 Virtual Screening The potential activity of compound1 was predicted by the ldquoTarget Fishingrdquo functional modelsoftware (Discovery Studio) The target fishing process wasconducted as follows The DockScore energy function wasutilized tominimize the energy of compound 1 conformationSetting full minimization as minimization gave the smartconformation of compound 1 Then pharmacophore search
Evidence-Based Complementary and Alternative Medicine 3
was set to be screened and profiled Screen and profile wasset to be ligand profiler PharmaDB pharmacophores wereset to be all Conformation generation was set to be the bestMaximum conformation was set to be 200 Energy thresholdwas set to be 10 Saved conformations were set to be true andother parameters were set to be default
Top 14 candidate receptors were ranked according tothe fit value (as shown in Table 2) which is based on forcefield approximation and specifically examined the compoundinternal energy and the compound-receptor interactionenergy which is taken as the sum of van der Waal force andelectrostatic energy [10]
26 Ethyl Methyl 343101584041015840-tetrahydroxy-120575-truxinate Lightyellow oil [120572]
119863minus20∘ (c 0018 MeOH) UV (in MeOH) 120582max
284 and 228 nm IR ]max ATR (cmminus1) 3436 2924 2854 1736and 1600ndash1450HRESIMS (mz) 4031286 [M+H]+ 1HNMR(300MHz CD
3OD) 120575H 673 (4H d-like) 662 (1H t J = 60
30Hz) 659 (1H t J = 60 30Hz) 419 (2H q J = 71Hz)373 (3H s) 343 (1H d-like J = 29Hz) 340 (1H d-like J= 25Hz) 330 (1H d-like J = 31Hz) 327 (1H d-like J =35Hz) and 126 (3H t J = 142 71 Hz) 13C NMR (75MHzCD3OD) 120575C 1735 1730 1450 1441 1328 1176 1150 1135
607 512 502 500 462 458 and 132
3 Results
Compound 1 (280mg) was separated from the ethyl acetateextract of E scaber whole plant as a light yellow oil Themolecular formula C
21H22O8was determined by themolecu-
lar ion observed atmz 4031359 [M+H]+ in the LC-TOF-MS(positive mode) which requires 11 degrees of unsaturationThe IR spectrum presented bands in the 1600ndash1450 cmminus11736 cmminus1 2854 cmminus1 2924 cmminus1 and 3436 cmminus1 regionwhich corresponded to aromatic ester methyl or methyleneand phenolic hydroxyl groups respectively The structure ofcompound 1 was further elucidated by examination of its 1D13C (75MHz) DEPT (90∘ and 135∘) and 1H (300MHz)NMRspectra and HMQC HMBC COSY and NOESY spectra inMeOH-d
4 Only 13 carbon resonance signals and the other
two carbon signals overlapped by the solvent carbon signalswere found in 13C NMR and DEPT spectra respectivelywhich suggested that there are many identical parts in thismolecule Further analysis of the 13C NMR spectrum ofcompound 1 suggested each signal of 120575C 1450 1441 13281176 1150 and 1135 is comprised of two overlapping carbonsignals [120575C 1450 (C-3 and C-31015840) 1441 (C-4 and C-41015840) 1328(C-1 and C-11015840) 1176 (C-6 and C-61015840) 1150 (C-5 and C-51015840)and 1135 (C-2 and C-21015840)] the other signals were assignedto two carbonyl carbon atoms [120575 1735 (C-9) and 1730 (C-91015840)] one methoxy group (120575C 607 C-10) one submethoxy(120575C 512 C-101015840) methyl (120575C 132 C-111015840) and four methinecarbons [120575C 502 (C-71015840) 500 (C-7) 462 (C-81015840) and 458 (C-8)] From the number of unsaturations and carbons thesefour methane carbons were deduced to be cyclobutane ringFrom the analysis of 1H NMR spectrum of compound 1 twophenylpropanoid units were presented at 120575H 673 (4H d-likeH-2 H-5 H-21015840 H-51015840) 120575H 662 (1H t J = 60 30Hz H-6H-61015840) and 659 (1H t J = 60 30Hz H-61015840H-6) Four
Table 1 The radical scavenging activity of 5 compounds from Ele-phantopus scaber
Compound Name IC50 (mM)a
1 Ethyl methyl 3 4 3101584041015840-tetrahydroxy-120575-truxinate 044 plusmn 0039
2 5-O-caffeoylquinic acid 096 plusmn 0096
3 Chlorogenic acid methyl ester 089 plusmn 0140
4 Deoxyelephantopin NR5 Isoscarbertopin NR6b Trolox 133 plusmn 0187
aThe inhibition was recorded at 10min of reaction (ABTS method) andIC50 value was measured using PROBIT model PROBIT (119901) = intercept +119861119883 (covariates 119883 are transformed using the base 10000 logarithm) Eachvalue corresponds to the mean and standard deviation of duplicates at fiveconcentrationsbPositive control groupNR No reaction at the conditions discribed
OH
OH
OH
OH
O
O
O
O
5
1
3
79
1998400
3998400
5998400
7998400999840011998400
Figure 2 Selected HMBC (proton to carbon) correlations ofcompound 1
methane carbons of the cyclobutane ring were observed at120575H 343 (1H d-like J = 29Hz H-7) 340 (1H d-like J =25Hz H-71015840) 330 (1H d-like J = 31Hz H-81015840) and 327(1H d-like J = 35Hz H-8) and the relative configurationof the cyclobutane ring was determined by comparing thechemical shift of compound 1 with reported 1HNMR data ofother 120575-truxinate derivatives [11] Other signals of 1H NMRspectra were assigned to submethoxy [120575H 419 (2H q J =71Hz H-101015840)] methoxy [120575H 373 (3H s H-10)] and methyl[120575H 126 (3H t J = 142 71 HzH-111015840)]Meanwhile theHMBCspectrum of compound 1 presented the correlations from H-10 toC-9H-8 toC-9 andC-81015840 H-7 toC-2 andC-6H-111015840 toC-101015840 fromC-101015840 toH-91015840 andH-111015840 fromC-81015840 to C-91015840 and fromC-71015840 to H-21015840 and H-61015840 respectively (Figure 2) Consequentlythe structure of compound 1 was deduced to be ethylmethyl 343101584041015840-tetrahydroxy-120575-truxinate which was furtherconfirmed by HMQC COSY and NOESY spectra Thispaper reports a new 120575-truxinate derivative in Elephantopusgenus for the first time Compounds 2ndash5 were identifiedrespectively as 5-O-caffeoylquinic acid (2) chlorogenic acidmethyl ester (3) deoxyelephantopin (4) and isoscarbertopin(5) by comparing their NMR and MS data with reportedliterature values
The antioxidant activity of 5 compounds isolated fromE scaber was evaluated by the ABTS radical scavengingassay and the results are presented as IC
50in Table 1
4 Evidence-Based Complementary and Alternative Medicine
Table 2 The potential bioactivity screening results of compound 1
Pharmacophore Name of pharmacophore Type Fit value Biological function(s) Reference2zb8-01-s Prostaglandin reductase 2 Protein 405271 Inflammation [14]
3kjs-01 Dihydrofolate reductase-thymidylatesynthase Protein 39615 Malarial parasites anticancer and
inflammation [15ndash17]
2uue-01 Cell division protein kinase 2 Protein 360758 Cell division [18 19]2w4i-01-s Glutamate racemase Protein 355547 Antibiotics [20ndash24]3k6l-01 Peptide deformylase Protein 351102 Antibiotic [25 26]3md7-01 beta-lactamase-like Protein 341887 Antibiotic [27 28]
2ovy-01 Phosphodiesterase 10A Protein 341834 Schizophrenia andnervous system [29ndash31]
3ac8-01 Protooncogene tyrosine-protein kinase LCK Protein 339142 Antitumor [32 33]3f7z-01 G17 glycogen synthase kinase-3-beta Protein 330076 Antitumor and neurodegenerative disease [34]3cgy-01 Virulence sensor histidine kinase phoQ Protein 328695 Antibiotic [35 36]2qe5-01 RNA-directed RNA polymerase Protein 328198 Antivirus [37 38]1dvx-01 Transthyretin Protein 327421 Antitumor and obesity [39 40]
2brc-01 ATP-dependent molecular chaperoneHSP90 Protein 326648 Antitumor and antivirus [41 42]
1c1b-01-s HIV-1 reverse transcriptase (A-chain) Protein 325549 Anti-HIV [43ndash45]
The most active radical scavengers were the new compoundethyl methyl 343101584041015840-tetrahydroxy-120575-truxinate (IC
50=
044 plusmn 0039mM) The other 2 quinic acid derivatives 5-O-caffeoylquinic acid and chlorogenic acid methyl ester alsoshowed radical scavenging potential (IC
50= 096 plusmn 0096 and
089 plusmn 0140mM resp) while the antioxidant activity of theother 2 sesquiterpene lactone compounds deoxyelephantopinand isoscabertopin was not detected Comparing thestructures of these 5 compounds the different antioxidantactivities were attributed to the existence of phenolichydroxyl groups in compounds which were supported bythe previous reports [12]
Compound 1 was also tested for in vitro cytotoxicityagainst HepG2 cell line with norcantharidin (NCTD 60 120583M)as positive control at 72 h incubation (Figure 3) Compound 1exhibited a dose-response inhibition curve from 27 growthinhibition at 10120583gmL to 81 at 40 120583gmL demonstratingthat it has significant and dose-dependent inhibition on thegrowth of HepG2 (IC
50= 60 120583M) Further work will be
conducted on the mechanism by which compound 1 inducesapoptosis
With the rapid development of computer-aided drugdesign (CADD) virtual screening technique has been usedmore and more widely in drug design and bioactivityscreening of compounds [13] The potential bioactivities ofcompound 1 have been predicted by the target fishingmethodwhich was based on the Discovery Studio software andProtein Date Bank (PDB) including over twelve thousand 3Dmacromolecular structure data determined experimentallyby X-ray crystallography and NMR The top 14 biologicalmolecular targets ranked as the fit value (FV) are reported(Table 2)
Theoretically FVgt 3means this target should be exploredexperimentally The strongest activity of compound 1 waspredicted to be anti-inflammatory (FV = 405271) and anti-AIDS (FV = 325549) respectively (Table 2) Further exper-iments on the biological functions of 1 should be directed
10
08
06
04
02
00
Gro
wth
inhi
bitio
n (
)lowast
lowastlowast
lowast
lowast
25120583M 50120583M 75120583M 100120583M NCTD (60120583M)
Figure 3 Growth inhibition of compound 1 on HepG2 cell lineswhere each value represents mean plusmn standard deviation of 6 repli-cates (119899 = 6) as compared to the positive control norcantharidin(60120583M) ( lowast119875 lt 0001 for comparison of control cell with cells treatedwith compound 1 andNCTD) 72 h for the incubation period of cellsafter being treated with compound 1 and NCTD
towards its potential anti-inflammatory antibiotic antivirusand anticancer activities
4 Discussion and Conclusion
With more and more present modern drugs discovered fromtraditional medical knowledge the traditional knowledge isgetting more extensive attention which also led to the devel-opment of important drugs such as reserpine (a treatmentfor hypertension) podophyllotoxin (the base of an importantanticancer drug) and vinblastine (used in the treatment ofcertain cancers) [46]
Previous studies showed that pulmonary oxidant stresscan cause some disease conditions such as acute lung injury
Evidence-Based Complementary and Alternative Medicine 5
radiation injury COPD (chronic obstructive pulmonary dis-ease) and inflammation [47] Meanwhile previous clinicaland experimental studies described that antioxidant supple-mentation including flavonoids and vitamins may inversethe oxidant-mediated cough depression by modulating theinflammatory process in lung disease [48 49] Interestinglyour work using ABTS assay demonstrated that compounds1ndash3 showed strong antioxidant activity especially compound1 (IC50
= 044 plusmn 0039mM) Moreover E scaber was alsoreported as the source of a number of sesquiterpene lactonessuch as compounds 4 and 5 which have shown significantcontribution to the anti-inflammatory activity of plants [50]Some of the sesquiterpenes from the genus Elephantopushave demonstrated significant anti-inflammatory as well ashepatoprotective activities and are being considered as druglead compounds [3] Based on the above analysis we hypoth-esize that Zhuang people use this plant to treat headachesbronchitis and hepatitisdue to its anti-inflammatory andantioxidant effects
According to the in vitro cytotoxicity assay with NCTD(60 120583M) as control group and activity virtual screeningcompound 1 exhibited good (IC
50= 60120583M) and dose-
response inhibition on HepG2 cell line and potential anti-inflammatory antibiotic antivirus and anticancer activitieswhich indicated that the further research of E scaber couldbe focused on anticancer and anti-inflammatory activityThepresent work further developed the usage of this traditionalmedicine plant
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors thank Vanya Petrova (City University of NewYork) for assistance with the antioxidant assay and WeiguoLiu (Minzu University of China) for assistance with usingthe Discovery Studio software This work was supported inpart by the National Natural Science Foundation of China(31161140345 and 31070288) the Ministry of Science amp Tech-nology of China (2012FY110300) the Ministry of Educationof China through its 111 and 985 Projects (B08044 MUC985-9 and MUC98506-01000101) and JSPS Asian Core program(JSPSAP109080)
References
[1] Z Y Wu and P Raven Flora of China vol 20-21 of AsteraceaeScience Press Beijing China Missouri Botanical Garden StLouis Mo USA 2007
[2] J JWang Y J Liu J X Xu AWang Y LWang andC L LongldquoStudies on medicinal plants of Elephantopus (Compositae)rdquoNatural Products Research and Development vol 25 pp 401ndash409 2013
[3] C C Huang K J Lin Y W Cheng C A Hsu S S Yang andL F Shyur ldquoHepatoprotective effect and mechanistic insightsof deoxyelephantopin a phyto-sesquiterpene lactone against
fulminant hepatitisrdquo Journal of Nutritional Biochemistry vol 24no 3 pp 516ndash530 2013
[4] K Schutz D Kammerer R Carle and A Schieber ldquoIdentifi-cation and quantification of caffeoylquinic acids and flavonoidsfrom artichoke (Cynara scolymus L) heads juice and pomaceby HPLC-DAD-ESIMSnrdquo Journal of Agricultural and FoodChemistry vol 52 no 13 pp 4090ndash4096 2004
[5] X Zhu X Dong Y Wang P Ju and S Luo ldquoPhenolic com-pounds from Viburnum cylindricumrdquo Helvetica Chimica Actavol 88 no 2 pp 339ndash342 2005
[6] Q L Liang and Z D Min ldquoSesquiterpene lactones fromElephantopus scaberrdquoChinese Chemical Letters vol 13 no 4 pp343ndash344 2002
[7] L R Wang and X Q Xie ldquoComputational target fishing whatshould chemogenomics researchers expect for the future of insilico drug design and discoveryrdquo Future Medicinal Chemistryvol 6 no 3 pp 247ndash249 2014
[8] S B Wu K Dastmalchhi C L Long and E J KennellyldquoMetabolite profiling of jaboticaba (Myrciaria cauliflora) andother dark-colored fruit juicesrdquo Journal of Agriculture and FoodChemistry vol 60 no 30 pp 7513ndash7525 2012
[9] A L Liu H D Wang S M Lee Y T Wang and G H DuldquoStructure-activity relationship of flavonoids as influenza virusneuraminidase inhibitors and their in vitro anti-viral activitiesrdquoBioorganic and Medicinal Chemistry vol 16 no 15 pp 7141ndash7147 2008
[10] C Y C Chen ldquoVirtual screening and drug design for PDE-5receptor from traditional Chinese medicine databaserdquo Journalof Biomolecular Structure and Dynamics vol 27 no 5 pp 627ndash640 2010
[11] YDeng YWChinH B Chai et al ldquoPhytochemical and bioac-tivity studies on constituents of the leaves of Vitex quinatardquoPhytochemistry Letters vol 4 no 3 pp 213ndash217 2011
[12] D Huang O U Boxin and R L Prior ldquoThe chemistry behindantioxidant capacity assaysrdquo Journal of Agricultural and FoodChemistry vol 53 no 6 pp 1841ndash1856 2005
[13] J H Nettles J L Jenkins A Bender Z Deng J W Daviesand M Glick ldquoBridging chemical and biological space ldquotargetfishingrdquo using 2D and 3D molecular descriptorsrdquo Journal ofMedicinal Chemistry vol 49 no 23 pp 6802ndash6810 2006
[14] I Inoue S I Goto K Mizotani et al ldquoLipophilic HMG-CoA reductase inhibitor has an anti-inflammatory effectreduction of MRNA levels for interleukin-1120573 interleukin-6cyclooxygenase-2 and p22phox by regulation of peroxisomeproliferator-activated receptor 120572 (PPAR120572) in primary endothe-lial cellsrdquo Life Sciences vol 67 no 8 pp 863ndash876 2000
[15] B I Schweitzer A P Dicker and J R Bertino ldquoDihydrofolatereductase as a therapeutic targetrdquoThe FASEB Journal vol 4 no8 pp 2441ndash2452 1990
[16] L Jiang P C Lee J White and P K Rathod ldquoPotent andselective activity of a combination of thymidine and 1843U89a folate-based thymidylate synthase inhibitor against Plasmod-ium falciparumrdquo Antimicrobial Agents and Chemotherapy vol44 no 4 pp 1047ndash1050 2000
[17] N Sienkiewicz S Jarosławski S Wyllie and A H FairlambldquoChemical and genetic validation of dihydrofolate reductase-thymidylate synthase as a drug target in African trypanosomesrdquoMolecular Microbiology vol 69 no 2 pp 520ndash533 2008
[18] L A Pinna and F Meggio ldquoProtein kinase CK2 (ldquocaseinkinase-2rdquo) and its implication in cell division and proliferationrdquoProgress in Cell Cycle Research vol 3 pp 77ndash97 1997
6 Evidence-Based Complementary and Alternative Medicine
[19] S Ortega I Prieto J Odajima et al ldquoCyclin-dependent kinase 2is essential for meiosis but not for mitotic cell division in micerdquoNature Genetics vol 35 no 1 pp 25ndash31 2003
[20] M Ashiuchi K Tani K Soda and H Misono ldquoProperties ofglutamate racemase from Bacillus subtilis IFO 3336 producingpoly-120574-glutamaterdquo Journal of Biochemistry vol 123 no 6 pp1156ndash1163 1998
[21] K Y Hwang C S Cho S S Kim H C Sung Y G Yu andY Cho ldquoStructure and mechanism of glutamate racemase fromAquifex pyrophilusrdquo Nature Structural Biology vol 6 no 5 pp422ndash426 1999
[22] A de Dios L Prieto J A Martın et al ldquo4-substituted D-glutamic acid analogues the first potent inhibitors of glutamateracemase (MurI) enzyme with antibacterial activityrdquo Journal ofMedicinal Chemistry vol 45 no 20 pp 4559ndash4570 2002
[23] G S Basarab P J Hill A Rastagar and P J H WebbornldquoDesign of Helicobacter pylori glutamate racemase inhibitorsas selective antibacterial agents a novel pro-drug approach toincrease exposurerdquo Bioorganic andMedicinal Chemistry Lettersvol 18 no 16 pp 4716ndash4722 2008
[24] B Geng G Basarab J Comita-Prevoir et al ldquoPotent andselective inhibitors of Helicobacter pylori glutamate racemase(MurI) pyridodiazepine aminesrdquo Bioorganic and MedicinalChemistry Letters vol 19 no 3 pp 930ndash936 2009
[25] C M Apfel H Locher S Evers et al ldquoPeptide deformylaseas an antibacterial drug target target validation and resistancedevelopmentrdquo Antimicrobial Agents and Chemotherapy vol 45no 4 pp 1058ndash1064 2001
[26] J A Leeds and C R Dean ldquoPeptide deformylase as an antibac-terial target a critical assessmentrdquo Current Opinion in Pharma-cology vol 6 no 5 pp 445ndash452 2006
[27] S Kumar U Narain S Tripathi and K Misra ldquoSynthesesof curcumin bioconjugates and study of their antibacterialactivities against120573-lactamase-producingmicroorganismsrdquoBio-conjugate Chemistry vol 12 no 4 pp 464ndash469 2001
[28] Q Li J Y Lee R Castillo et al ldquoNB2001 a novel antibacterialagent with broad-spectrum activity and enhanced potencyagainst 120573-lactamase-producing strainsrdquo Antimicrobial Agentsand Chemotherapy vol 46 no 5 pp 1262ndash1268 2002
[29] T M Coskran D Morton F S Menniti et al ldquoImmunohis-tochemical localization of phosphodiesterase 10A in multiplemammalian speciesrdquo Journal of Histochemistry and Cytochem-istry vol 54 no 11 pp 1205ndash1213 2006
[30] F S Menniti T A Chappie J M Humphrey and C J SchmidtldquoPhosphodiesterase 10A inhibitors a novel approach to thetreatment of the symptoms of schizophreniardquo Current Opinionin Investigational Drugs vol 8 no 1 pp 54ndash59 2007
[31] C J Schmidt D S Chapin J Cianfrogna et al ldquoPreclinicalcharacterization of selective phosphodiesterase 10A inhibitorsa new therapeutic approach to the treatment of schizophreniardquoThe Journal of Pharmacology and Experimental Therapeuticsvol 325 no 2 pp 681ndash690 2008
[32] J B Bolen R B Rowley C Spana and A Y Tsygankov ldquoTheSrc family of tyrosine protein kinases in hemopoietic signaltransductionrdquoThe FASEB Journal vol 6 no 15 pp 3403ndash34091992
[33] E Buchdunger J Zimmermann H Mett et al ldquoInhibition ofthe Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivativerdquo Cancer Research vol 56pp 100ndash104 1996
[34] P Mishra S Senthivinayagam A Rana and B Rana ldquoGlyco-gen synthase kinase-3120573 regulates snail and 120573-catenin during
gastrin-induced migration of gastric cancer cellsrdquo Journal ofMolecular Signaling vol 5 article 9 2010
[35] D Beier andRGross ldquoRegulation of bacterial virulence by two-component systemsrdquo Current Opinion in Microbiology vol 9no 2 pp 143ndash152 2006
[36] W J Gooderham S L Gellatly F Sanschagrin et al ldquoThe sensorkinase PhoQ mediates virulence in Pseudomonas aeruginosardquoMicrobiology vol 155 no 3 pp 699ndash711 2009
[37] T Dalmay A Hamilton S Rudd S Angell and D CBaulcombe ldquoAn RNA-dependent RNA polymerase gene inArabidopsis is required for posttranscriptional gene silencingmediated by a transgene but not by a virusrdquo Cell vol 101 no5 pp 543ndash553 2000
[38] F Simmer M Tijsterman S Parrish et al ldquoLoss of the putativeRNA-directed RNA polymerase RRF-3 makes C elegans hyper-sensitive to RNAirdquo Current Biology vol 12 no 15 pp 1317ndash13192002
[39] L HWu and C TWoodbridge ldquoRegulation of transthyretin totreat obesityrdquo Patent Application publication 0160394 A1 2002
[40] S K Frey J Spranger A Henze A F H Pfeiffer F JSchweigert and J Raila ldquoFactors that influence retinol-bindingprotein 4-transthyretin interaction are not altered in overweightsubjects and overweight subjects with type 2 diabetes mellitusrdquoMetabolism Clinical and Experimental vol 58 no 10 pp 1386ndash1392 2009
[41] J Hu and C Seeger ldquoHsp90 is required for the activity of a hep-atitis B virus reverse transcriptaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no3 pp 1060ndash1064 1996
[42] J R Sydor E Normant C S Pien et al ldquoDevelopment of17-allylamino-17-demethoxygeldanamycin hydroquinonehydrochloride (IPI-504) an anti-cancer agent directed againstHsp90rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 103 no 46 pp 17408ndash17413 2006
[43] C Tantillo J Ding A Jacobo-Molina et al ldquoLocations ofanti-AIDS drug binding sites and resistance mutations in thethree-dimensional structure of HIV-1 reverse transcriptaseImplications for mechanisms of drug inhibition and resistancerdquoJournal of Molecular Biology vol 243 no 3 pp 369ndash387 1994
[44] S G Sarafianos B Marchand K Das et al ldquoStructure andfunction of HIV-1 reverse transcriptase molecular mechanismsof polymerization and inhibitionrdquo Journal of Molecular Biologyvol 385 no 3 pp 693ndash713 2009
[45] K M Frey M Bollini A C Mislak et al ldquoCrystal structures ofHIV-1 reverse transcriptasewith picomolar inhibitors reveal keyinteractions for drug designrdquo Journal of the American ChemicalSociety vol 134 no 48 pp 19501ndash19503 2012
[46] M Idu ldquoCurrent trends in ethnobotanyrdquo Tropical Journal ofPharmaceutical Research vol 8 no 4 pp 295ndash296 2009
[47] M Christofidou-Solomidou and V R Muzykantov ldquoAntioxi-dant strategies in respiratory medicinerdquo Treatments in Respira-tory Medicine vol 5 no 1 pp 47ndash78 2006
[48] M Brozmanova J PlevkovaV Bartos L PlankM Javorka andM Tatar ldquoThe interaction of dietary antioxidant vitamins andoxidative stress on cough reflex in guinea-pigs after long termoxygen therapyrdquo Journal of Physiology and Pharmacology vol57 supplement 4 pp 45ndash54 2006
[49] M Brozmanova V Bartos L Plank J Plevkova and M TatarldquoDietary intake of flavonoids and hyperoxia-induced oxidativestress related cough in guinea pigsrdquo Bratislava Medical Journalvol 108 no 12 pp 489ndash494 2007
Evidence-Based Complementary and Alternative Medicine 7
[50] M Chadwick H Trewin F Gawthrop and C Wagstaff ldquoSes-quiterpenoids lactones benefits to plants and peoplerdquo Interna-tional Journal of Molecular Sciences vol 14 no 6 pp 12780ndash12805 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Evidence-Based Complementary and Alternative Medicine
OH
OHOH
OH
OHOH
OH
OH
OHOH
OH OH
O
O
OOO
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OO
HOHO HO
CH3
179
19984007998400999840011998400
5
3
3998400
5998400
1 2 3
4 5
Figure 1 The chemical structures of compounds obtained from Elephantopus scaber
Dragon-Boat Festival of Jingxi County (Guangxi) SouthwestChina and identified by Professor Chunlin Long A voucherspecimen was deposited in the Herbarium of Minzu Univer-sity of China numbered 201006023
22 Extraction and Isolation Theair-dried and groundwholeplant of E scaber (40 kg) was extracted with EtOH H
2O
(90 10) at reflux for 3times 3 hThe solvent was evaporated underreduced pressure to yield dark brown material (3724 g) Thelatter was suspended in H
2O (3 L) and individually parti-
tioned with petroleum ether (3 times 3 L) Chloroform (2 times 3 L)EtOAc (3 times 3 L) and n-BuOH (3 times 3 L) to obtain petroleumether (1694 g) Chloroform (338 g) EtOAc (469 g) and n-BuOH (1223 g) phase
The EtOAc phase was separated by silica gel columnchromatography (CC) eluted with CHCl
3 CH3OH in order
of increasing polarity to give seven fractions on the basisof TLC Fraction 3 was subjected to MCI CC eluted withCH3OH H
2O to seven fractions A
1ndashA7 Fraction A
1was
isolated by Sephadex LH-20 ODS CC (CH3OH H
2O =
44 56) and Si gel CC (CHCl3 CH3OH = 14 1) successively
to afford compound 1 (240mg) Fraction A2was purified
with Sephadex LH-20 to give six subfractions Subfraction2 was subjected to ODS CC (CH
3OH H
2O = 30 70) and
silica gel CC (CHCl3 CH3OH = 12 1) successively to give
compound 3 (170mg) Subfraction 3 was subjected to ODSCC (CH
3OH H
2O = 48 52) and Sephadex LH-20 to afford
compound 2 (270mg)The petroleum ether phase was separated by silica gel
CC eluted with petroleum ether EtOAc (100 1ndash0 100) togive ten fractions Fraction 8 was purified by MCI CC usingCH3OH H
2O (60 100ndash100 0) to afford four fractions B
1ndash
B4 Fraction B
2was subjected to Sephadex LH-20 and ODS
CC (CH3OH H
2O = 83 17) to give compound 4 (90mg)
FractionB3was isolated byODSCC (CH
3OH H
2O=80 20)
and Sephadex LH-20 to give compound 5 (70mg)
23 Antioxidant Assay The antioxidant activity of com-pounds 1ndash5 was evaluated with ABTS radical scavengingassay as described previously [8] The IC
50was expressed as
millimoles per liter (mM)
24 Cytotoxicity Assay Compound 1 was tested for cytotoxi-city using a slightly modifiedMTTmethod [9] Briefly 150120583L(10 120583M 20120583M 30 120583M and 40 120583M) of samples was added to96-well plate containing a confluent HepG2 cell monolayerin sextuplicate 10 120583gmL of norcantharidin (NCTD) andblank medium were used as the positive and control grouprespectively After a 72 h incubation at 37∘C 100 120583L MTTsolution (5mgmL phosphate buffered saline) was addedto each well which was further incubated for 4 h for theformation of the formazan product After removing themedium 150 120583L DMSO was added to dissolve the formazancrystals The optical density (OD) was measured at 550 nmwith amicroplate readerThe rate of inhibitionwas calculatedby the following formula rate of inhibition = (1 minus sampleOD)control OD The concentration causing inhibition ofviable cells by 50 (IC
50) was determined from a dose-
response curve which was based on triplicate measurements
25 Virtual Screening The potential activity of compound1 was predicted by the ldquoTarget Fishingrdquo functional modelsoftware (Discovery Studio) The target fishing process wasconducted as follows The DockScore energy function wasutilized tominimize the energy of compound 1 conformationSetting full minimization as minimization gave the smartconformation of compound 1 Then pharmacophore search
Evidence-Based Complementary and Alternative Medicine 3
was set to be screened and profiled Screen and profile wasset to be ligand profiler PharmaDB pharmacophores wereset to be all Conformation generation was set to be the bestMaximum conformation was set to be 200 Energy thresholdwas set to be 10 Saved conformations were set to be true andother parameters were set to be default
Top 14 candidate receptors were ranked according tothe fit value (as shown in Table 2) which is based on forcefield approximation and specifically examined the compoundinternal energy and the compound-receptor interactionenergy which is taken as the sum of van der Waal force andelectrostatic energy [10]
26 Ethyl Methyl 343101584041015840-tetrahydroxy-120575-truxinate Lightyellow oil [120572]
119863minus20∘ (c 0018 MeOH) UV (in MeOH) 120582max
284 and 228 nm IR ]max ATR (cmminus1) 3436 2924 2854 1736and 1600ndash1450HRESIMS (mz) 4031286 [M+H]+ 1HNMR(300MHz CD
3OD) 120575H 673 (4H d-like) 662 (1H t J = 60
30Hz) 659 (1H t J = 60 30Hz) 419 (2H q J = 71Hz)373 (3H s) 343 (1H d-like J = 29Hz) 340 (1H d-like J= 25Hz) 330 (1H d-like J = 31Hz) 327 (1H d-like J =35Hz) and 126 (3H t J = 142 71 Hz) 13C NMR (75MHzCD3OD) 120575C 1735 1730 1450 1441 1328 1176 1150 1135
607 512 502 500 462 458 and 132
3 Results
Compound 1 (280mg) was separated from the ethyl acetateextract of E scaber whole plant as a light yellow oil Themolecular formula C
21H22O8was determined by themolecu-
lar ion observed atmz 4031359 [M+H]+ in the LC-TOF-MS(positive mode) which requires 11 degrees of unsaturationThe IR spectrum presented bands in the 1600ndash1450 cmminus11736 cmminus1 2854 cmminus1 2924 cmminus1 and 3436 cmminus1 regionwhich corresponded to aromatic ester methyl or methyleneand phenolic hydroxyl groups respectively The structure ofcompound 1 was further elucidated by examination of its 1D13C (75MHz) DEPT (90∘ and 135∘) and 1H (300MHz)NMRspectra and HMQC HMBC COSY and NOESY spectra inMeOH-d
4 Only 13 carbon resonance signals and the other
two carbon signals overlapped by the solvent carbon signalswere found in 13C NMR and DEPT spectra respectivelywhich suggested that there are many identical parts in thismolecule Further analysis of the 13C NMR spectrum ofcompound 1 suggested each signal of 120575C 1450 1441 13281176 1150 and 1135 is comprised of two overlapping carbonsignals [120575C 1450 (C-3 and C-31015840) 1441 (C-4 and C-41015840) 1328(C-1 and C-11015840) 1176 (C-6 and C-61015840) 1150 (C-5 and C-51015840)and 1135 (C-2 and C-21015840)] the other signals were assignedto two carbonyl carbon atoms [120575 1735 (C-9) and 1730 (C-91015840)] one methoxy group (120575C 607 C-10) one submethoxy(120575C 512 C-101015840) methyl (120575C 132 C-111015840) and four methinecarbons [120575C 502 (C-71015840) 500 (C-7) 462 (C-81015840) and 458 (C-8)] From the number of unsaturations and carbons thesefour methane carbons were deduced to be cyclobutane ringFrom the analysis of 1H NMR spectrum of compound 1 twophenylpropanoid units were presented at 120575H 673 (4H d-likeH-2 H-5 H-21015840 H-51015840) 120575H 662 (1H t J = 60 30Hz H-6H-61015840) and 659 (1H t J = 60 30Hz H-61015840H-6) Four
Table 1 The radical scavenging activity of 5 compounds from Ele-phantopus scaber
Compound Name IC50 (mM)a
1 Ethyl methyl 3 4 3101584041015840-tetrahydroxy-120575-truxinate 044 plusmn 0039
2 5-O-caffeoylquinic acid 096 plusmn 0096
3 Chlorogenic acid methyl ester 089 plusmn 0140
4 Deoxyelephantopin NR5 Isoscarbertopin NR6b Trolox 133 plusmn 0187
aThe inhibition was recorded at 10min of reaction (ABTS method) andIC50 value was measured using PROBIT model PROBIT (119901) = intercept +119861119883 (covariates 119883 are transformed using the base 10000 logarithm) Eachvalue corresponds to the mean and standard deviation of duplicates at fiveconcentrationsbPositive control groupNR No reaction at the conditions discribed
OH
OH
OH
OH
O
O
O
O
5
1
3
79
1998400
3998400
5998400
7998400999840011998400
Figure 2 Selected HMBC (proton to carbon) correlations ofcompound 1
methane carbons of the cyclobutane ring were observed at120575H 343 (1H d-like J = 29Hz H-7) 340 (1H d-like J =25Hz H-71015840) 330 (1H d-like J = 31Hz H-81015840) and 327(1H d-like J = 35Hz H-8) and the relative configurationof the cyclobutane ring was determined by comparing thechemical shift of compound 1 with reported 1HNMR data ofother 120575-truxinate derivatives [11] Other signals of 1H NMRspectra were assigned to submethoxy [120575H 419 (2H q J =71Hz H-101015840)] methoxy [120575H 373 (3H s H-10)] and methyl[120575H 126 (3H t J = 142 71 HzH-111015840)]Meanwhile theHMBCspectrum of compound 1 presented the correlations from H-10 toC-9H-8 toC-9 andC-81015840 H-7 toC-2 andC-6H-111015840 toC-101015840 fromC-101015840 toH-91015840 andH-111015840 fromC-81015840 to C-91015840 and fromC-71015840 to H-21015840 and H-61015840 respectively (Figure 2) Consequentlythe structure of compound 1 was deduced to be ethylmethyl 343101584041015840-tetrahydroxy-120575-truxinate which was furtherconfirmed by HMQC COSY and NOESY spectra Thispaper reports a new 120575-truxinate derivative in Elephantopusgenus for the first time Compounds 2ndash5 were identifiedrespectively as 5-O-caffeoylquinic acid (2) chlorogenic acidmethyl ester (3) deoxyelephantopin (4) and isoscarbertopin(5) by comparing their NMR and MS data with reportedliterature values
The antioxidant activity of 5 compounds isolated fromE scaber was evaluated by the ABTS radical scavengingassay and the results are presented as IC
50in Table 1
4 Evidence-Based Complementary and Alternative Medicine
Table 2 The potential bioactivity screening results of compound 1
Pharmacophore Name of pharmacophore Type Fit value Biological function(s) Reference2zb8-01-s Prostaglandin reductase 2 Protein 405271 Inflammation [14]
3kjs-01 Dihydrofolate reductase-thymidylatesynthase Protein 39615 Malarial parasites anticancer and
inflammation [15ndash17]
2uue-01 Cell division protein kinase 2 Protein 360758 Cell division [18 19]2w4i-01-s Glutamate racemase Protein 355547 Antibiotics [20ndash24]3k6l-01 Peptide deformylase Protein 351102 Antibiotic [25 26]3md7-01 beta-lactamase-like Protein 341887 Antibiotic [27 28]
2ovy-01 Phosphodiesterase 10A Protein 341834 Schizophrenia andnervous system [29ndash31]
3ac8-01 Protooncogene tyrosine-protein kinase LCK Protein 339142 Antitumor [32 33]3f7z-01 G17 glycogen synthase kinase-3-beta Protein 330076 Antitumor and neurodegenerative disease [34]3cgy-01 Virulence sensor histidine kinase phoQ Protein 328695 Antibiotic [35 36]2qe5-01 RNA-directed RNA polymerase Protein 328198 Antivirus [37 38]1dvx-01 Transthyretin Protein 327421 Antitumor and obesity [39 40]
2brc-01 ATP-dependent molecular chaperoneHSP90 Protein 326648 Antitumor and antivirus [41 42]
1c1b-01-s HIV-1 reverse transcriptase (A-chain) Protein 325549 Anti-HIV [43ndash45]
The most active radical scavengers were the new compoundethyl methyl 343101584041015840-tetrahydroxy-120575-truxinate (IC
50=
044 plusmn 0039mM) The other 2 quinic acid derivatives 5-O-caffeoylquinic acid and chlorogenic acid methyl ester alsoshowed radical scavenging potential (IC
50= 096 plusmn 0096 and
089 plusmn 0140mM resp) while the antioxidant activity of theother 2 sesquiterpene lactone compounds deoxyelephantopinand isoscabertopin was not detected Comparing thestructures of these 5 compounds the different antioxidantactivities were attributed to the existence of phenolichydroxyl groups in compounds which were supported bythe previous reports [12]
Compound 1 was also tested for in vitro cytotoxicityagainst HepG2 cell line with norcantharidin (NCTD 60 120583M)as positive control at 72 h incubation (Figure 3) Compound 1exhibited a dose-response inhibition curve from 27 growthinhibition at 10120583gmL to 81 at 40 120583gmL demonstratingthat it has significant and dose-dependent inhibition on thegrowth of HepG2 (IC
50= 60 120583M) Further work will be
conducted on the mechanism by which compound 1 inducesapoptosis
With the rapid development of computer-aided drugdesign (CADD) virtual screening technique has been usedmore and more widely in drug design and bioactivityscreening of compounds [13] The potential bioactivities ofcompound 1 have been predicted by the target fishingmethodwhich was based on the Discovery Studio software andProtein Date Bank (PDB) including over twelve thousand 3Dmacromolecular structure data determined experimentallyby X-ray crystallography and NMR The top 14 biologicalmolecular targets ranked as the fit value (FV) are reported(Table 2)
Theoretically FVgt 3means this target should be exploredexperimentally The strongest activity of compound 1 waspredicted to be anti-inflammatory (FV = 405271) and anti-AIDS (FV = 325549) respectively (Table 2) Further exper-iments on the biological functions of 1 should be directed
10
08
06
04
02
00
Gro
wth
inhi
bitio
n (
)lowast
lowastlowast
lowast
lowast
25120583M 50120583M 75120583M 100120583M NCTD (60120583M)
Figure 3 Growth inhibition of compound 1 on HepG2 cell lineswhere each value represents mean plusmn standard deviation of 6 repli-cates (119899 = 6) as compared to the positive control norcantharidin(60120583M) ( lowast119875 lt 0001 for comparison of control cell with cells treatedwith compound 1 andNCTD) 72 h for the incubation period of cellsafter being treated with compound 1 and NCTD
towards its potential anti-inflammatory antibiotic antivirusand anticancer activities
4 Discussion and Conclusion
With more and more present modern drugs discovered fromtraditional medical knowledge the traditional knowledge isgetting more extensive attention which also led to the devel-opment of important drugs such as reserpine (a treatmentfor hypertension) podophyllotoxin (the base of an importantanticancer drug) and vinblastine (used in the treatment ofcertain cancers) [46]
Previous studies showed that pulmonary oxidant stresscan cause some disease conditions such as acute lung injury
Evidence-Based Complementary and Alternative Medicine 5
radiation injury COPD (chronic obstructive pulmonary dis-ease) and inflammation [47] Meanwhile previous clinicaland experimental studies described that antioxidant supple-mentation including flavonoids and vitamins may inversethe oxidant-mediated cough depression by modulating theinflammatory process in lung disease [48 49] Interestinglyour work using ABTS assay demonstrated that compounds1ndash3 showed strong antioxidant activity especially compound1 (IC50
= 044 plusmn 0039mM) Moreover E scaber was alsoreported as the source of a number of sesquiterpene lactonessuch as compounds 4 and 5 which have shown significantcontribution to the anti-inflammatory activity of plants [50]Some of the sesquiterpenes from the genus Elephantopushave demonstrated significant anti-inflammatory as well ashepatoprotective activities and are being considered as druglead compounds [3] Based on the above analysis we hypoth-esize that Zhuang people use this plant to treat headachesbronchitis and hepatitisdue to its anti-inflammatory andantioxidant effects
According to the in vitro cytotoxicity assay with NCTD(60 120583M) as control group and activity virtual screeningcompound 1 exhibited good (IC
50= 60120583M) and dose-
response inhibition on HepG2 cell line and potential anti-inflammatory antibiotic antivirus and anticancer activitieswhich indicated that the further research of E scaber couldbe focused on anticancer and anti-inflammatory activityThepresent work further developed the usage of this traditionalmedicine plant
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors thank Vanya Petrova (City University of NewYork) for assistance with the antioxidant assay and WeiguoLiu (Minzu University of China) for assistance with usingthe Discovery Studio software This work was supported inpart by the National Natural Science Foundation of China(31161140345 and 31070288) the Ministry of Science amp Tech-nology of China (2012FY110300) the Ministry of Educationof China through its 111 and 985 Projects (B08044 MUC985-9 and MUC98506-01000101) and JSPS Asian Core program(JSPSAP109080)
References
[1] Z Y Wu and P Raven Flora of China vol 20-21 of AsteraceaeScience Press Beijing China Missouri Botanical Garden StLouis Mo USA 2007
[2] J JWang Y J Liu J X Xu AWang Y LWang andC L LongldquoStudies on medicinal plants of Elephantopus (Compositae)rdquoNatural Products Research and Development vol 25 pp 401ndash409 2013
[3] C C Huang K J Lin Y W Cheng C A Hsu S S Yang andL F Shyur ldquoHepatoprotective effect and mechanistic insightsof deoxyelephantopin a phyto-sesquiterpene lactone against
fulminant hepatitisrdquo Journal of Nutritional Biochemistry vol 24no 3 pp 516ndash530 2013
[4] K Schutz D Kammerer R Carle and A Schieber ldquoIdentifi-cation and quantification of caffeoylquinic acids and flavonoidsfrom artichoke (Cynara scolymus L) heads juice and pomaceby HPLC-DAD-ESIMSnrdquo Journal of Agricultural and FoodChemistry vol 52 no 13 pp 4090ndash4096 2004
[5] X Zhu X Dong Y Wang P Ju and S Luo ldquoPhenolic com-pounds from Viburnum cylindricumrdquo Helvetica Chimica Actavol 88 no 2 pp 339ndash342 2005
[6] Q L Liang and Z D Min ldquoSesquiterpene lactones fromElephantopus scaberrdquoChinese Chemical Letters vol 13 no 4 pp343ndash344 2002
[7] L R Wang and X Q Xie ldquoComputational target fishing whatshould chemogenomics researchers expect for the future of insilico drug design and discoveryrdquo Future Medicinal Chemistryvol 6 no 3 pp 247ndash249 2014
[8] S B Wu K Dastmalchhi C L Long and E J KennellyldquoMetabolite profiling of jaboticaba (Myrciaria cauliflora) andother dark-colored fruit juicesrdquo Journal of Agriculture and FoodChemistry vol 60 no 30 pp 7513ndash7525 2012
[9] A L Liu H D Wang S M Lee Y T Wang and G H DuldquoStructure-activity relationship of flavonoids as influenza virusneuraminidase inhibitors and their in vitro anti-viral activitiesrdquoBioorganic and Medicinal Chemistry vol 16 no 15 pp 7141ndash7147 2008
[10] C Y C Chen ldquoVirtual screening and drug design for PDE-5receptor from traditional Chinese medicine databaserdquo Journalof Biomolecular Structure and Dynamics vol 27 no 5 pp 627ndash640 2010
[11] YDeng YWChinH B Chai et al ldquoPhytochemical and bioac-tivity studies on constituents of the leaves of Vitex quinatardquoPhytochemistry Letters vol 4 no 3 pp 213ndash217 2011
[12] D Huang O U Boxin and R L Prior ldquoThe chemistry behindantioxidant capacity assaysrdquo Journal of Agricultural and FoodChemistry vol 53 no 6 pp 1841ndash1856 2005
[13] J H Nettles J L Jenkins A Bender Z Deng J W Daviesand M Glick ldquoBridging chemical and biological space ldquotargetfishingrdquo using 2D and 3D molecular descriptorsrdquo Journal ofMedicinal Chemistry vol 49 no 23 pp 6802ndash6810 2006
[14] I Inoue S I Goto K Mizotani et al ldquoLipophilic HMG-CoA reductase inhibitor has an anti-inflammatory effectreduction of MRNA levels for interleukin-1120573 interleukin-6cyclooxygenase-2 and p22phox by regulation of peroxisomeproliferator-activated receptor 120572 (PPAR120572) in primary endothe-lial cellsrdquo Life Sciences vol 67 no 8 pp 863ndash876 2000
[15] B I Schweitzer A P Dicker and J R Bertino ldquoDihydrofolatereductase as a therapeutic targetrdquoThe FASEB Journal vol 4 no8 pp 2441ndash2452 1990
[16] L Jiang P C Lee J White and P K Rathod ldquoPotent andselective activity of a combination of thymidine and 1843U89a folate-based thymidylate synthase inhibitor against Plasmod-ium falciparumrdquo Antimicrobial Agents and Chemotherapy vol44 no 4 pp 1047ndash1050 2000
[17] N Sienkiewicz S Jarosławski S Wyllie and A H FairlambldquoChemical and genetic validation of dihydrofolate reductase-thymidylate synthase as a drug target in African trypanosomesrdquoMolecular Microbiology vol 69 no 2 pp 520ndash533 2008
[18] L A Pinna and F Meggio ldquoProtein kinase CK2 (ldquocaseinkinase-2rdquo) and its implication in cell division and proliferationrdquoProgress in Cell Cycle Research vol 3 pp 77ndash97 1997
6 Evidence-Based Complementary and Alternative Medicine
[19] S Ortega I Prieto J Odajima et al ldquoCyclin-dependent kinase 2is essential for meiosis but not for mitotic cell division in micerdquoNature Genetics vol 35 no 1 pp 25ndash31 2003
[20] M Ashiuchi K Tani K Soda and H Misono ldquoProperties ofglutamate racemase from Bacillus subtilis IFO 3336 producingpoly-120574-glutamaterdquo Journal of Biochemistry vol 123 no 6 pp1156ndash1163 1998
[21] K Y Hwang C S Cho S S Kim H C Sung Y G Yu andY Cho ldquoStructure and mechanism of glutamate racemase fromAquifex pyrophilusrdquo Nature Structural Biology vol 6 no 5 pp422ndash426 1999
[22] A de Dios L Prieto J A Martın et al ldquo4-substituted D-glutamic acid analogues the first potent inhibitors of glutamateracemase (MurI) enzyme with antibacterial activityrdquo Journal ofMedicinal Chemistry vol 45 no 20 pp 4559ndash4570 2002
[23] G S Basarab P J Hill A Rastagar and P J H WebbornldquoDesign of Helicobacter pylori glutamate racemase inhibitorsas selective antibacterial agents a novel pro-drug approach toincrease exposurerdquo Bioorganic andMedicinal Chemistry Lettersvol 18 no 16 pp 4716ndash4722 2008
[24] B Geng G Basarab J Comita-Prevoir et al ldquoPotent andselective inhibitors of Helicobacter pylori glutamate racemase(MurI) pyridodiazepine aminesrdquo Bioorganic and MedicinalChemistry Letters vol 19 no 3 pp 930ndash936 2009
[25] C M Apfel H Locher S Evers et al ldquoPeptide deformylaseas an antibacterial drug target target validation and resistancedevelopmentrdquo Antimicrobial Agents and Chemotherapy vol 45no 4 pp 1058ndash1064 2001
[26] J A Leeds and C R Dean ldquoPeptide deformylase as an antibac-terial target a critical assessmentrdquo Current Opinion in Pharma-cology vol 6 no 5 pp 445ndash452 2006
[27] S Kumar U Narain S Tripathi and K Misra ldquoSynthesesof curcumin bioconjugates and study of their antibacterialactivities against120573-lactamase-producingmicroorganismsrdquoBio-conjugate Chemistry vol 12 no 4 pp 464ndash469 2001
[28] Q Li J Y Lee R Castillo et al ldquoNB2001 a novel antibacterialagent with broad-spectrum activity and enhanced potencyagainst 120573-lactamase-producing strainsrdquo Antimicrobial Agentsand Chemotherapy vol 46 no 5 pp 1262ndash1268 2002
[29] T M Coskran D Morton F S Menniti et al ldquoImmunohis-tochemical localization of phosphodiesterase 10A in multiplemammalian speciesrdquo Journal of Histochemistry and Cytochem-istry vol 54 no 11 pp 1205ndash1213 2006
[30] F S Menniti T A Chappie J M Humphrey and C J SchmidtldquoPhosphodiesterase 10A inhibitors a novel approach to thetreatment of the symptoms of schizophreniardquo Current Opinionin Investigational Drugs vol 8 no 1 pp 54ndash59 2007
[31] C J Schmidt D S Chapin J Cianfrogna et al ldquoPreclinicalcharacterization of selective phosphodiesterase 10A inhibitorsa new therapeutic approach to the treatment of schizophreniardquoThe Journal of Pharmacology and Experimental Therapeuticsvol 325 no 2 pp 681ndash690 2008
[32] J B Bolen R B Rowley C Spana and A Y Tsygankov ldquoTheSrc family of tyrosine protein kinases in hemopoietic signaltransductionrdquoThe FASEB Journal vol 6 no 15 pp 3403ndash34091992
[33] E Buchdunger J Zimmermann H Mett et al ldquoInhibition ofthe Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivativerdquo Cancer Research vol 56pp 100ndash104 1996
[34] P Mishra S Senthivinayagam A Rana and B Rana ldquoGlyco-gen synthase kinase-3120573 regulates snail and 120573-catenin during
gastrin-induced migration of gastric cancer cellsrdquo Journal ofMolecular Signaling vol 5 article 9 2010
[35] D Beier andRGross ldquoRegulation of bacterial virulence by two-component systemsrdquo Current Opinion in Microbiology vol 9no 2 pp 143ndash152 2006
[36] W J Gooderham S L Gellatly F Sanschagrin et al ldquoThe sensorkinase PhoQ mediates virulence in Pseudomonas aeruginosardquoMicrobiology vol 155 no 3 pp 699ndash711 2009
[37] T Dalmay A Hamilton S Rudd S Angell and D CBaulcombe ldquoAn RNA-dependent RNA polymerase gene inArabidopsis is required for posttranscriptional gene silencingmediated by a transgene but not by a virusrdquo Cell vol 101 no5 pp 543ndash553 2000
[38] F Simmer M Tijsterman S Parrish et al ldquoLoss of the putativeRNA-directed RNA polymerase RRF-3 makes C elegans hyper-sensitive to RNAirdquo Current Biology vol 12 no 15 pp 1317ndash13192002
[39] L HWu and C TWoodbridge ldquoRegulation of transthyretin totreat obesityrdquo Patent Application publication 0160394 A1 2002
[40] S K Frey J Spranger A Henze A F H Pfeiffer F JSchweigert and J Raila ldquoFactors that influence retinol-bindingprotein 4-transthyretin interaction are not altered in overweightsubjects and overweight subjects with type 2 diabetes mellitusrdquoMetabolism Clinical and Experimental vol 58 no 10 pp 1386ndash1392 2009
[41] J Hu and C Seeger ldquoHsp90 is required for the activity of a hep-atitis B virus reverse transcriptaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no3 pp 1060ndash1064 1996
[42] J R Sydor E Normant C S Pien et al ldquoDevelopment of17-allylamino-17-demethoxygeldanamycin hydroquinonehydrochloride (IPI-504) an anti-cancer agent directed againstHsp90rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 103 no 46 pp 17408ndash17413 2006
[43] C Tantillo J Ding A Jacobo-Molina et al ldquoLocations ofanti-AIDS drug binding sites and resistance mutations in thethree-dimensional structure of HIV-1 reverse transcriptaseImplications for mechanisms of drug inhibition and resistancerdquoJournal of Molecular Biology vol 243 no 3 pp 369ndash387 1994
[44] S G Sarafianos B Marchand K Das et al ldquoStructure andfunction of HIV-1 reverse transcriptase molecular mechanismsof polymerization and inhibitionrdquo Journal of Molecular Biologyvol 385 no 3 pp 693ndash713 2009
[45] K M Frey M Bollini A C Mislak et al ldquoCrystal structures ofHIV-1 reverse transcriptasewith picomolar inhibitors reveal keyinteractions for drug designrdquo Journal of the American ChemicalSociety vol 134 no 48 pp 19501ndash19503 2012
[46] M Idu ldquoCurrent trends in ethnobotanyrdquo Tropical Journal ofPharmaceutical Research vol 8 no 4 pp 295ndash296 2009
[47] M Christofidou-Solomidou and V R Muzykantov ldquoAntioxi-dant strategies in respiratory medicinerdquo Treatments in Respira-tory Medicine vol 5 no 1 pp 47ndash78 2006
[48] M Brozmanova J PlevkovaV Bartos L PlankM Javorka andM Tatar ldquoThe interaction of dietary antioxidant vitamins andoxidative stress on cough reflex in guinea-pigs after long termoxygen therapyrdquo Journal of Physiology and Pharmacology vol57 supplement 4 pp 45ndash54 2006
[49] M Brozmanova V Bartos L Plank J Plevkova and M TatarldquoDietary intake of flavonoids and hyperoxia-induced oxidativestress related cough in guinea pigsrdquo Bratislava Medical Journalvol 108 no 12 pp 489ndash494 2007
Evidence-Based Complementary and Alternative Medicine 7
[50] M Chadwick H Trewin F Gawthrop and C Wagstaff ldquoSes-quiterpenoids lactones benefits to plants and peoplerdquo Interna-tional Journal of Molecular Sciences vol 14 no 6 pp 12780ndash12805 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 3
was set to be screened and profiled Screen and profile wasset to be ligand profiler PharmaDB pharmacophores wereset to be all Conformation generation was set to be the bestMaximum conformation was set to be 200 Energy thresholdwas set to be 10 Saved conformations were set to be true andother parameters were set to be default
Top 14 candidate receptors were ranked according tothe fit value (as shown in Table 2) which is based on forcefield approximation and specifically examined the compoundinternal energy and the compound-receptor interactionenergy which is taken as the sum of van der Waal force andelectrostatic energy [10]
26 Ethyl Methyl 343101584041015840-tetrahydroxy-120575-truxinate Lightyellow oil [120572]
119863minus20∘ (c 0018 MeOH) UV (in MeOH) 120582max
284 and 228 nm IR ]max ATR (cmminus1) 3436 2924 2854 1736and 1600ndash1450HRESIMS (mz) 4031286 [M+H]+ 1HNMR(300MHz CD
3OD) 120575H 673 (4H d-like) 662 (1H t J = 60
30Hz) 659 (1H t J = 60 30Hz) 419 (2H q J = 71Hz)373 (3H s) 343 (1H d-like J = 29Hz) 340 (1H d-like J= 25Hz) 330 (1H d-like J = 31Hz) 327 (1H d-like J =35Hz) and 126 (3H t J = 142 71 Hz) 13C NMR (75MHzCD3OD) 120575C 1735 1730 1450 1441 1328 1176 1150 1135
607 512 502 500 462 458 and 132
3 Results
Compound 1 (280mg) was separated from the ethyl acetateextract of E scaber whole plant as a light yellow oil Themolecular formula C
21H22O8was determined by themolecu-
lar ion observed atmz 4031359 [M+H]+ in the LC-TOF-MS(positive mode) which requires 11 degrees of unsaturationThe IR spectrum presented bands in the 1600ndash1450 cmminus11736 cmminus1 2854 cmminus1 2924 cmminus1 and 3436 cmminus1 regionwhich corresponded to aromatic ester methyl or methyleneand phenolic hydroxyl groups respectively The structure ofcompound 1 was further elucidated by examination of its 1D13C (75MHz) DEPT (90∘ and 135∘) and 1H (300MHz)NMRspectra and HMQC HMBC COSY and NOESY spectra inMeOH-d
4 Only 13 carbon resonance signals and the other
two carbon signals overlapped by the solvent carbon signalswere found in 13C NMR and DEPT spectra respectivelywhich suggested that there are many identical parts in thismolecule Further analysis of the 13C NMR spectrum ofcompound 1 suggested each signal of 120575C 1450 1441 13281176 1150 and 1135 is comprised of two overlapping carbonsignals [120575C 1450 (C-3 and C-31015840) 1441 (C-4 and C-41015840) 1328(C-1 and C-11015840) 1176 (C-6 and C-61015840) 1150 (C-5 and C-51015840)and 1135 (C-2 and C-21015840)] the other signals were assignedto two carbonyl carbon atoms [120575 1735 (C-9) and 1730 (C-91015840)] one methoxy group (120575C 607 C-10) one submethoxy(120575C 512 C-101015840) methyl (120575C 132 C-111015840) and four methinecarbons [120575C 502 (C-71015840) 500 (C-7) 462 (C-81015840) and 458 (C-8)] From the number of unsaturations and carbons thesefour methane carbons were deduced to be cyclobutane ringFrom the analysis of 1H NMR spectrum of compound 1 twophenylpropanoid units were presented at 120575H 673 (4H d-likeH-2 H-5 H-21015840 H-51015840) 120575H 662 (1H t J = 60 30Hz H-6H-61015840) and 659 (1H t J = 60 30Hz H-61015840H-6) Four
Table 1 The radical scavenging activity of 5 compounds from Ele-phantopus scaber
Compound Name IC50 (mM)a
1 Ethyl methyl 3 4 3101584041015840-tetrahydroxy-120575-truxinate 044 plusmn 0039
2 5-O-caffeoylquinic acid 096 plusmn 0096
3 Chlorogenic acid methyl ester 089 plusmn 0140
4 Deoxyelephantopin NR5 Isoscarbertopin NR6b Trolox 133 plusmn 0187
aThe inhibition was recorded at 10min of reaction (ABTS method) andIC50 value was measured using PROBIT model PROBIT (119901) = intercept +119861119883 (covariates 119883 are transformed using the base 10000 logarithm) Eachvalue corresponds to the mean and standard deviation of duplicates at fiveconcentrationsbPositive control groupNR No reaction at the conditions discribed
OH
OH
OH
OH
O
O
O
O
5
1
3
79
1998400
3998400
5998400
7998400999840011998400
Figure 2 Selected HMBC (proton to carbon) correlations ofcompound 1
methane carbons of the cyclobutane ring were observed at120575H 343 (1H d-like J = 29Hz H-7) 340 (1H d-like J =25Hz H-71015840) 330 (1H d-like J = 31Hz H-81015840) and 327(1H d-like J = 35Hz H-8) and the relative configurationof the cyclobutane ring was determined by comparing thechemical shift of compound 1 with reported 1HNMR data ofother 120575-truxinate derivatives [11] Other signals of 1H NMRspectra were assigned to submethoxy [120575H 419 (2H q J =71Hz H-101015840)] methoxy [120575H 373 (3H s H-10)] and methyl[120575H 126 (3H t J = 142 71 HzH-111015840)]Meanwhile theHMBCspectrum of compound 1 presented the correlations from H-10 toC-9H-8 toC-9 andC-81015840 H-7 toC-2 andC-6H-111015840 toC-101015840 fromC-101015840 toH-91015840 andH-111015840 fromC-81015840 to C-91015840 and fromC-71015840 to H-21015840 and H-61015840 respectively (Figure 2) Consequentlythe structure of compound 1 was deduced to be ethylmethyl 343101584041015840-tetrahydroxy-120575-truxinate which was furtherconfirmed by HMQC COSY and NOESY spectra Thispaper reports a new 120575-truxinate derivative in Elephantopusgenus for the first time Compounds 2ndash5 were identifiedrespectively as 5-O-caffeoylquinic acid (2) chlorogenic acidmethyl ester (3) deoxyelephantopin (4) and isoscarbertopin(5) by comparing their NMR and MS data with reportedliterature values
The antioxidant activity of 5 compounds isolated fromE scaber was evaluated by the ABTS radical scavengingassay and the results are presented as IC
50in Table 1
4 Evidence-Based Complementary and Alternative Medicine
Table 2 The potential bioactivity screening results of compound 1
Pharmacophore Name of pharmacophore Type Fit value Biological function(s) Reference2zb8-01-s Prostaglandin reductase 2 Protein 405271 Inflammation [14]
3kjs-01 Dihydrofolate reductase-thymidylatesynthase Protein 39615 Malarial parasites anticancer and
inflammation [15ndash17]
2uue-01 Cell division protein kinase 2 Protein 360758 Cell division [18 19]2w4i-01-s Glutamate racemase Protein 355547 Antibiotics [20ndash24]3k6l-01 Peptide deformylase Protein 351102 Antibiotic [25 26]3md7-01 beta-lactamase-like Protein 341887 Antibiotic [27 28]
2ovy-01 Phosphodiesterase 10A Protein 341834 Schizophrenia andnervous system [29ndash31]
3ac8-01 Protooncogene tyrosine-protein kinase LCK Protein 339142 Antitumor [32 33]3f7z-01 G17 glycogen synthase kinase-3-beta Protein 330076 Antitumor and neurodegenerative disease [34]3cgy-01 Virulence sensor histidine kinase phoQ Protein 328695 Antibiotic [35 36]2qe5-01 RNA-directed RNA polymerase Protein 328198 Antivirus [37 38]1dvx-01 Transthyretin Protein 327421 Antitumor and obesity [39 40]
2brc-01 ATP-dependent molecular chaperoneHSP90 Protein 326648 Antitumor and antivirus [41 42]
1c1b-01-s HIV-1 reverse transcriptase (A-chain) Protein 325549 Anti-HIV [43ndash45]
The most active radical scavengers were the new compoundethyl methyl 343101584041015840-tetrahydroxy-120575-truxinate (IC
50=
044 plusmn 0039mM) The other 2 quinic acid derivatives 5-O-caffeoylquinic acid and chlorogenic acid methyl ester alsoshowed radical scavenging potential (IC
50= 096 plusmn 0096 and
089 plusmn 0140mM resp) while the antioxidant activity of theother 2 sesquiterpene lactone compounds deoxyelephantopinand isoscabertopin was not detected Comparing thestructures of these 5 compounds the different antioxidantactivities were attributed to the existence of phenolichydroxyl groups in compounds which were supported bythe previous reports [12]
Compound 1 was also tested for in vitro cytotoxicityagainst HepG2 cell line with norcantharidin (NCTD 60 120583M)as positive control at 72 h incubation (Figure 3) Compound 1exhibited a dose-response inhibition curve from 27 growthinhibition at 10120583gmL to 81 at 40 120583gmL demonstratingthat it has significant and dose-dependent inhibition on thegrowth of HepG2 (IC
50= 60 120583M) Further work will be
conducted on the mechanism by which compound 1 inducesapoptosis
With the rapid development of computer-aided drugdesign (CADD) virtual screening technique has been usedmore and more widely in drug design and bioactivityscreening of compounds [13] The potential bioactivities ofcompound 1 have been predicted by the target fishingmethodwhich was based on the Discovery Studio software andProtein Date Bank (PDB) including over twelve thousand 3Dmacromolecular structure data determined experimentallyby X-ray crystallography and NMR The top 14 biologicalmolecular targets ranked as the fit value (FV) are reported(Table 2)
Theoretically FVgt 3means this target should be exploredexperimentally The strongest activity of compound 1 waspredicted to be anti-inflammatory (FV = 405271) and anti-AIDS (FV = 325549) respectively (Table 2) Further exper-iments on the biological functions of 1 should be directed
10
08
06
04
02
00
Gro
wth
inhi
bitio
n (
)lowast
lowastlowast
lowast
lowast
25120583M 50120583M 75120583M 100120583M NCTD (60120583M)
Figure 3 Growth inhibition of compound 1 on HepG2 cell lineswhere each value represents mean plusmn standard deviation of 6 repli-cates (119899 = 6) as compared to the positive control norcantharidin(60120583M) ( lowast119875 lt 0001 for comparison of control cell with cells treatedwith compound 1 andNCTD) 72 h for the incubation period of cellsafter being treated with compound 1 and NCTD
towards its potential anti-inflammatory antibiotic antivirusand anticancer activities
4 Discussion and Conclusion
With more and more present modern drugs discovered fromtraditional medical knowledge the traditional knowledge isgetting more extensive attention which also led to the devel-opment of important drugs such as reserpine (a treatmentfor hypertension) podophyllotoxin (the base of an importantanticancer drug) and vinblastine (used in the treatment ofcertain cancers) [46]
Previous studies showed that pulmonary oxidant stresscan cause some disease conditions such as acute lung injury
Evidence-Based Complementary and Alternative Medicine 5
radiation injury COPD (chronic obstructive pulmonary dis-ease) and inflammation [47] Meanwhile previous clinicaland experimental studies described that antioxidant supple-mentation including flavonoids and vitamins may inversethe oxidant-mediated cough depression by modulating theinflammatory process in lung disease [48 49] Interestinglyour work using ABTS assay demonstrated that compounds1ndash3 showed strong antioxidant activity especially compound1 (IC50
= 044 plusmn 0039mM) Moreover E scaber was alsoreported as the source of a number of sesquiterpene lactonessuch as compounds 4 and 5 which have shown significantcontribution to the anti-inflammatory activity of plants [50]Some of the sesquiterpenes from the genus Elephantopushave demonstrated significant anti-inflammatory as well ashepatoprotective activities and are being considered as druglead compounds [3] Based on the above analysis we hypoth-esize that Zhuang people use this plant to treat headachesbronchitis and hepatitisdue to its anti-inflammatory andantioxidant effects
According to the in vitro cytotoxicity assay with NCTD(60 120583M) as control group and activity virtual screeningcompound 1 exhibited good (IC
50= 60120583M) and dose-
response inhibition on HepG2 cell line and potential anti-inflammatory antibiotic antivirus and anticancer activitieswhich indicated that the further research of E scaber couldbe focused on anticancer and anti-inflammatory activityThepresent work further developed the usage of this traditionalmedicine plant
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors thank Vanya Petrova (City University of NewYork) for assistance with the antioxidant assay and WeiguoLiu (Minzu University of China) for assistance with usingthe Discovery Studio software This work was supported inpart by the National Natural Science Foundation of China(31161140345 and 31070288) the Ministry of Science amp Tech-nology of China (2012FY110300) the Ministry of Educationof China through its 111 and 985 Projects (B08044 MUC985-9 and MUC98506-01000101) and JSPS Asian Core program(JSPSAP109080)
References
[1] Z Y Wu and P Raven Flora of China vol 20-21 of AsteraceaeScience Press Beijing China Missouri Botanical Garden StLouis Mo USA 2007
[2] J JWang Y J Liu J X Xu AWang Y LWang andC L LongldquoStudies on medicinal plants of Elephantopus (Compositae)rdquoNatural Products Research and Development vol 25 pp 401ndash409 2013
[3] C C Huang K J Lin Y W Cheng C A Hsu S S Yang andL F Shyur ldquoHepatoprotective effect and mechanistic insightsof deoxyelephantopin a phyto-sesquiterpene lactone against
fulminant hepatitisrdquo Journal of Nutritional Biochemistry vol 24no 3 pp 516ndash530 2013
[4] K Schutz D Kammerer R Carle and A Schieber ldquoIdentifi-cation and quantification of caffeoylquinic acids and flavonoidsfrom artichoke (Cynara scolymus L) heads juice and pomaceby HPLC-DAD-ESIMSnrdquo Journal of Agricultural and FoodChemistry vol 52 no 13 pp 4090ndash4096 2004
[5] X Zhu X Dong Y Wang P Ju and S Luo ldquoPhenolic com-pounds from Viburnum cylindricumrdquo Helvetica Chimica Actavol 88 no 2 pp 339ndash342 2005
[6] Q L Liang and Z D Min ldquoSesquiterpene lactones fromElephantopus scaberrdquoChinese Chemical Letters vol 13 no 4 pp343ndash344 2002
[7] L R Wang and X Q Xie ldquoComputational target fishing whatshould chemogenomics researchers expect for the future of insilico drug design and discoveryrdquo Future Medicinal Chemistryvol 6 no 3 pp 247ndash249 2014
[8] S B Wu K Dastmalchhi C L Long and E J KennellyldquoMetabolite profiling of jaboticaba (Myrciaria cauliflora) andother dark-colored fruit juicesrdquo Journal of Agriculture and FoodChemistry vol 60 no 30 pp 7513ndash7525 2012
[9] A L Liu H D Wang S M Lee Y T Wang and G H DuldquoStructure-activity relationship of flavonoids as influenza virusneuraminidase inhibitors and their in vitro anti-viral activitiesrdquoBioorganic and Medicinal Chemistry vol 16 no 15 pp 7141ndash7147 2008
[10] C Y C Chen ldquoVirtual screening and drug design for PDE-5receptor from traditional Chinese medicine databaserdquo Journalof Biomolecular Structure and Dynamics vol 27 no 5 pp 627ndash640 2010
[11] YDeng YWChinH B Chai et al ldquoPhytochemical and bioac-tivity studies on constituents of the leaves of Vitex quinatardquoPhytochemistry Letters vol 4 no 3 pp 213ndash217 2011
[12] D Huang O U Boxin and R L Prior ldquoThe chemistry behindantioxidant capacity assaysrdquo Journal of Agricultural and FoodChemistry vol 53 no 6 pp 1841ndash1856 2005
[13] J H Nettles J L Jenkins A Bender Z Deng J W Daviesand M Glick ldquoBridging chemical and biological space ldquotargetfishingrdquo using 2D and 3D molecular descriptorsrdquo Journal ofMedicinal Chemistry vol 49 no 23 pp 6802ndash6810 2006
[14] I Inoue S I Goto K Mizotani et al ldquoLipophilic HMG-CoA reductase inhibitor has an anti-inflammatory effectreduction of MRNA levels for interleukin-1120573 interleukin-6cyclooxygenase-2 and p22phox by regulation of peroxisomeproliferator-activated receptor 120572 (PPAR120572) in primary endothe-lial cellsrdquo Life Sciences vol 67 no 8 pp 863ndash876 2000
[15] B I Schweitzer A P Dicker and J R Bertino ldquoDihydrofolatereductase as a therapeutic targetrdquoThe FASEB Journal vol 4 no8 pp 2441ndash2452 1990
[16] L Jiang P C Lee J White and P K Rathod ldquoPotent andselective activity of a combination of thymidine and 1843U89a folate-based thymidylate synthase inhibitor against Plasmod-ium falciparumrdquo Antimicrobial Agents and Chemotherapy vol44 no 4 pp 1047ndash1050 2000
[17] N Sienkiewicz S Jarosławski S Wyllie and A H FairlambldquoChemical and genetic validation of dihydrofolate reductase-thymidylate synthase as a drug target in African trypanosomesrdquoMolecular Microbiology vol 69 no 2 pp 520ndash533 2008
[18] L A Pinna and F Meggio ldquoProtein kinase CK2 (ldquocaseinkinase-2rdquo) and its implication in cell division and proliferationrdquoProgress in Cell Cycle Research vol 3 pp 77ndash97 1997
6 Evidence-Based Complementary and Alternative Medicine
[19] S Ortega I Prieto J Odajima et al ldquoCyclin-dependent kinase 2is essential for meiosis but not for mitotic cell division in micerdquoNature Genetics vol 35 no 1 pp 25ndash31 2003
[20] M Ashiuchi K Tani K Soda and H Misono ldquoProperties ofglutamate racemase from Bacillus subtilis IFO 3336 producingpoly-120574-glutamaterdquo Journal of Biochemistry vol 123 no 6 pp1156ndash1163 1998
[21] K Y Hwang C S Cho S S Kim H C Sung Y G Yu andY Cho ldquoStructure and mechanism of glutamate racemase fromAquifex pyrophilusrdquo Nature Structural Biology vol 6 no 5 pp422ndash426 1999
[22] A de Dios L Prieto J A Martın et al ldquo4-substituted D-glutamic acid analogues the first potent inhibitors of glutamateracemase (MurI) enzyme with antibacterial activityrdquo Journal ofMedicinal Chemistry vol 45 no 20 pp 4559ndash4570 2002
[23] G S Basarab P J Hill A Rastagar and P J H WebbornldquoDesign of Helicobacter pylori glutamate racemase inhibitorsas selective antibacterial agents a novel pro-drug approach toincrease exposurerdquo Bioorganic andMedicinal Chemistry Lettersvol 18 no 16 pp 4716ndash4722 2008
[24] B Geng G Basarab J Comita-Prevoir et al ldquoPotent andselective inhibitors of Helicobacter pylori glutamate racemase(MurI) pyridodiazepine aminesrdquo Bioorganic and MedicinalChemistry Letters vol 19 no 3 pp 930ndash936 2009
[25] C M Apfel H Locher S Evers et al ldquoPeptide deformylaseas an antibacterial drug target target validation and resistancedevelopmentrdquo Antimicrobial Agents and Chemotherapy vol 45no 4 pp 1058ndash1064 2001
[26] J A Leeds and C R Dean ldquoPeptide deformylase as an antibac-terial target a critical assessmentrdquo Current Opinion in Pharma-cology vol 6 no 5 pp 445ndash452 2006
[27] S Kumar U Narain S Tripathi and K Misra ldquoSynthesesof curcumin bioconjugates and study of their antibacterialactivities against120573-lactamase-producingmicroorganismsrdquoBio-conjugate Chemistry vol 12 no 4 pp 464ndash469 2001
[28] Q Li J Y Lee R Castillo et al ldquoNB2001 a novel antibacterialagent with broad-spectrum activity and enhanced potencyagainst 120573-lactamase-producing strainsrdquo Antimicrobial Agentsand Chemotherapy vol 46 no 5 pp 1262ndash1268 2002
[29] T M Coskran D Morton F S Menniti et al ldquoImmunohis-tochemical localization of phosphodiesterase 10A in multiplemammalian speciesrdquo Journal of Histochemistry and Cytochem-istry vol 54 no 11 pp 1205ndash1213 2006
[30] F S Menniti T A Chappie J M Humphrey and C J SchmidtldquoPhosphodiesterase 10A inhibitors a novel approach to thetreatment of the symptoms of schizophreniardquo Current Opinionin Investigational Drugs vol 8 no 1 pp 54ndash59 2007
[31] C J Schmidt D S Chapin J Cianfrogna et al ldquoPreclinicalcharacterization of selective phosphodiesterase 10A inhibitorsa new therapeutic approach to the treatment of schizophreniardquoThe Journal of Pharmacology and Experimental Therapeuticsvol 325 no 2 pp 681ndash690 2008
[32] J B Bolen R B Rowley C Spana and A Y Tsygankov ldquoTheSrc family of tyrosine protein kinases in hemopoietic signaltransductionrdquoThe FASEB Journal vol 6 no 15 pp 3403ndash34091992
[33] E Buchdunger J Zimmermann H Mett et al ldquoInhibition ofthe Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivativerdquo Cancer Research vol 56pp 100ndash104 1996
[34] P Mishra S Senthivinayagam A Rana and B Rana ldquoGlyco-gen synthase kinase-3120573 regulates snail and 120573-catenin during
gastrin-induced migration of gastric cancer cellsrdquo Journal ofMolecular Signaling vol 5 article 9 2010
[35] D Beier andRGross ldquoRegulation of bacterial virulence by two-component systemsrdquo Current Opinion in Microbiology vol 9no 2 pp 143ndash152 2006
[36] W J Gooderham S L Gellatly F Sanschagrin et al ldquoThe sensorkinase PhoQ mediates virulence in Pseudomonas aeruginosardquoMicrobiology vol 155 no 3 pp 699ndash711 2009
[37] T Dalmay A Hamilton S Rudd S Angell and D CBaulcombe ldquoAn RNA-dependent RNA polymerase gene inArabidopsis is required for posttranscriptional gene silencingmediated by a transgene but not by a virusrdquo Cell vol 101 no5 pp 543ndash553 2000
[38] F Simmer M Tijsterman S Parrish et al ldquoLoss of the putativeRNA-directed RNA polymerase RRF-3 makes C elegans hyper-sensitive to RNAirdquo Current Biology vol 12 no 15 pp 1317ndash13192002
[39] L HWu and C TWoodbridge ldquoRegulation of transthyretin totreat obesityrdquo Patent Application publication 0160394 A1 2002
[40] S K Frey J Spranger A Henze A F H Pfeiffer F JSchweigert and J Raila ldquoFactors that influence retinol-bindingprotein 4-transthyretin interaction are not altered in overweightsubjects and overweight subjects with type 2 diabetes mellitusrdquoMetabolism Clinical and Experimental vol 58 no 10 pp 1386ndash1392 2009
[41] J Hu and C Seeger ldquoHsp90 is required for the activity of a hep-atitis B virus reverse transcriptaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no3 pp 1060ndash1064 1996
[42] J R Sydor E Normant C S Pien et al ldquoDevelopment of17-allylamino-17-demethoxygeldanamycin hydroquinonehydrochloride (IPI-504) an anti-cancer agent directed againstHsp90rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 103 no 46 pp 17408ndash17413 2006
[43] C Tantillo J Ding A Jacobo-Molina et al ldquoLocations ofanti-AIDS drug binding sites and resistance mutations in thethree-dimensional structure of HIV-1 reverse transcriptaseImplications for mechanisms of drug inhibition and resistancerdquoJournal of Molecular Biology vol 243 no 3 pp 369ndash387 1994
[44] S G Sarafianos B Marchand K Das et al ldquoStructure andfunction of HIV-1 reverse transcriptase molecular mechanismsof polymerization and inhibitionrdquo Journal of Molecular Biologyvol 385 no 3 pp 693ndash713 2009
[45] K M Frey M Bollini A C Mislak et al ldquoCrystal structures ofHIV-1 reverse transcriptasewith picomolar inhibitors reveal keyinteractions for drug designrdquo Journal of the American ChemicalSociety vol 134 no 48 pp 19501ndash19503 2012
[46] M Idu ldquoCurrent trends in ethnobotanyrdquo Tropical Journal ofPharmaceutical Research vol 8 no 4 pp 295ndash296 2009
[47] M Christofidou-Solomidou and V R Muzykantov ldquoAntioxi-dant strategies in respiratory medicinerdquo Treatments in Respira-tory Medicine vol 5 no 1 pp 47ndash78 2006
[48] M Brozmanova J PlevkovaV Bartos L PlankM Javorka andM Tatar ldquoThe interaction of dietary antioxidant vitamins andoxidative stress on cough reflex in guinea-pigs after long termoxygen therapyrdquo Journal of Physiology and Pharmacology vol57 supplement 4 pp 45ndash54 2006
[49] M Brozmanova V Bartos L Plank J Plevkova and M TatarldquoDietary intake of flavonoids and hyperoxia-induced oxidativestress related cough in guinea pigsrdquo Bratislava Medical Journalvol 108 no 12 pp 489ndash494 2007
Evidence-Based Complementary and Alternative Medicine 7
[50] M Chadwick H Trewin F Gawthrop and C Wagstaff ldquoSes-quiterpenoids lactones benefits to plants and peoplerdquo Interna-tional Journal of Molecular Sciences vol 14 no 6 pp 12780ndash12805 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Evidence-Based Complementary and Alternative Medicine
Table 2 The potential bioactivity screening results of compound 1
Pharmacophore Name of pharmacophore Type Fit value Biological function(s) Reference2zb8-01-s Prostaglandin reductase 2 Protein 405271 Inflammation [14]
3kjs-01 Dihydrofolate reductase-thymidylatesynthase Protein 39615 Malarial parasites anticancer and
inflammation [15ndash17]
2uue-01 Cell division protein kinase 2 Protein 360758 Cell division [18 19]2w4i-01-s Glutamate racemase Protein 355547 Antibiotics [20ndash24]3k6l-01 Peptide deformylase Protein 351102 Antibiotic [25 26]3md7-01 beta-lactamase-like Protein 341887 Antibiotic [27 28]
2ovy-01 Phosphodiesterase 10A Protein 341834 Schizophrenia andnervous system [29ndash31]
3ac8-01 Protooncogene tyrosine-protein kinase LCK Protein 339142 Antitumor [32 33]3f7z-01 G17 glycogen synthase kinase-3-beta Protein 330076 Antitumor and neurodegenerative disease [34]3cgy-01 Virulence sensor histidine kinase phoQ Protein 328695 Antibiotic [35 36]2qe5-01 RNA-directed RNA polymerase Protein 328198 Antivirus [37 38]1dvx-01 Transthyretin Protein 327421 Antitumor and obesity [39 40]
2brc-01 ATP-dependent molecular chaperoneHSP90 Protein 326648 Antitumor and antivirus [41 42]
1c1b-01-s HIV-1 reverse transcriptase (A-chain) Protein 325549 Anti-HIV [43ndash45]
The most active radical scavengers were the new compoundethyl methyl 343101584041015840-tetrahydroxy-120575-truxinate (IC
50=
044 plusmn 0039mM) The other 2 quinic acid derivatives 5-O-caffeoylquinic acid and chlorogenic acid methyl ester alsoshowed radical scavenging potential (IC
50= 096 plusmn 0096 and
089 plusmn 0140mM resp) while the antioxidant activity of theother 2 sesquiterpene lactone compounds deoxyelephantopinand isoscabertopin was not detected Comparing thestructures of these 5 compounds the different antioxidantactivities were attributed to the existence of phenolichydroxyl groups in compounds which were supported bythe previous reports [12]
Compound 1 was also tested for in vitro cytotoxicityagainst HepG2 cell line with norcantharidin (NCTD 60 120583M)as positive control at 72 h incubation (Figure 3) Compound 1exhibited a dose-response inhibition curve from 27 growthinhibition at 10120583gmL to 81 at 40 120583gmL demonstratingthat it has significant and dose-dependent inhibition on thegrowth of HepG2 (IC
50= 60 120583M) Further work will be
conducted on the mechanism by which compound 1 inducesapoptosis
With the rapid development of computer-aided drugdesign (CADD) virtual screening technique has been usedmore and more widely in drug design and bioactivityscreening of compounds [13] The potential bioactivities ofcompound 1 have been predicted by the target fishingmethodwhich was based on the Discovery Studio software andProtein Date Bank (PDB) including over twelve thousand 3Dmacromolecular structure data determined experimentallyby X-ray crystallography and NMR The top 14 biologicalmolecular targets ranked as the fit value (FV) are reported(Table 2)
Theoretically FVgt 3means this target should be exploredexperimentally The strongest activity of compound 1 waspredicted to be anti-inflammatory (FV = 405271) and anti-AIDS (FV = 325549) respectively (Table 2) Further exper-iments on the biological functions of 1 should be directed
10
08
06
04
02
00
Gro
wth
inhi
bitio
n (
)lowast
lowastlowast
lowast
lowast
25120583M 50120583M 75120583M 100120583M NCTD (60120583M)
Figure 3 Growth inhibition of compound 1 on HepG2 cell lineswhere each value represents mean plusmn standard deviation of 6 repli-cates (119899 = 6) as compared to the positive control norcantharidin(60120583M) ( lowast119875 lt 0001 for comparison of control cell with cells treatedwith compound 1 andNCTD) 72 h for the incubation period of cellsafter being treated with compound 1 and NCTD
towards its potential anti-inflammatory antibiotic antivirusand anticancer activities
4 Discussion and Conclusion
With more and more present modern drugs discovered fromtraditional medical knowledge the traditional knowledge isgetting more extensive attention which also led to the devel-opment of important drugs such as reserpine (a treatmentfor hypertension) podophyllotoxin (the base of an importantanticancer drug) and vinblastine (used in the treatment ofcertain cancers) [46]
Previous studies showed that pulmonary oxidant stresscan cause some disease conditions such as acute lung injury
Evidence-Based Complementary and Alternative Medicine 5
radiation injury COPD (chronic obstructive pulmonary dis-ease) and inflammation [47] Meanwhile previous clinicaland experimental studies described that antioxidant supple-mentation including flavonoids and vitamins may inversethe oxidant-mediated cough depression by modulating theinflammatory process in lung disease [48 49] Interestinglyour work using ABTS assay demonstrated that compounds1ndash3 showed strong antioxidant activity especially compound1 (IC50
= 044 plusmn 0039mM) Moreover E scaber was alsoreported as the source of a number of sesquiterpene lactonessuch as compounds 4 and 5 which have shown significantcontribution to the anti-inflammatory activity of plants [50]Some of the sesquiterpenes from the genus Elephantopushave demonstrated significant anti-inflammatory as well ashepatoprotective activities and are being considered as druglead compounds [3] Based on the above analysis we hypoth-esize that Zhuang people use this plant to treat headachesbronchitis and hepatitisdue to its anti-inflammatory andantioxidant effects
According to the in vitro cytotoxicity assay with NCTD(60 120583M) as control group and activity virtual screeningcompound 1 exhibited good (IC
50= 60120583M) and dose-
response inhibition on HepG2 cell line and potential anti-inflammatory antibiotic antivirus and anticancer activitieswhich indicated that the further research of E scaber couldbe focused on anticancer and anti-inflammatory activityThepresent work further developed the usage of this traditionalmedicine plant
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors thank Vanya Petrova (City University of NewYork) for assistance with the antioxidant assay and WeiguoLiu (Minzu University of China) for assistance with usingthe Discovery Studio software This work was supported inpart by the National Natural Science Foundation of China(31161140345 and 31070288) the Ministry of Science amp Tech-nology of China (2012FY110300) the Ministry of Educationof China through its 111 and 985 Projects (B08044 MUC985-9 and MUC98506-01000101) and JSPS Asian Core program(JSPSAP109080)
References
[1] Z Y Wu and P Raven Flora of China vol 20-21 of AsteraceaeScience Press Beijing China Missouri Botanical Garden StLouis Mo USA 2007
[2] J JWang Y J Liu J X Xu AWang Y LWang andC L LongldquoStudies on medicinal plants of Elephantopus (Compositae)rdquoNatural Products Research and Development vol 25 pp 401ndash409 2013
[3] C C Huang K J Lin Y W Cheng C A Hsu S S Yang andL F Shyur ldquoHepatoprotective effect and mechanistic insightsof deoxyelephantopin a phyto-sesquiterpene lactone against
fulminant hepatitisrdquo Journal of Nutritional Biochemistry vol 24no 3 pp 516ndash530 2013
[4] K Schutz D Kammerer R Carle and A Schieber ldquoIdentifi-cation and quantification of caffeoylquinic acids and flavonoidsfrom artichoke (Cynara scolymus L) heads juice and pomaceby HPLC-DAD-ESIMSnrdquo Journal of Agricultural and FoodChemistry vol 52 no 13 pp 4090ndash4096 2004
[5] X Zhu X Dong Y Wang P Ju and S Luo ldquoPhenolic com-pounds from Viburnum cylindricumrdquo Helvetica Chimica Actavol 88 no 2 pp 339ndash342 2005
[6] Q L Liang and Z D Min ldquoSesquiterpene lactones fromElephantopus scaberrdquoChinese Chemical Letters vol 13 no 4 pp343ndash344 2002
[7] L R Wang and X Q Xie ldquoComputational target fishing whatshould chemogenomics researchers expect for the future of insilico drug design and discoveryrdquo Future Medicinal Chemistryvol 6 no 3 pp 247ndash249 2014
[8] S B Wu K Dastmalchhi C L Long and E J KennellyldquoMetabolite profiling of jaboticaba (Myrciaria cauliflora) andother dark-colored fruit juicesrdquo Journal of Agriculture and FoodChemistry vol 60 no 30 pp 7513ndash7525 2012
[9] A L Liu H D Wang S M Lee Y T Wang and G H DuldquoStructure-activity relationship of flavonoids as influenza virusneuraminidase inhibitors and their in vitro anti-viral activitiesrdquoBioorganic and Medicinal Chemistry vol 16 no 15 pp 7141ndash7147 2008
[10] C Y C Chen ldquoVirtual screening and drug design for PDE-5receptor from traditional Chinese medicine databaserdquo Journalof Biomolecular Structure and Dynamics vol 27 no 5 pp 627ndash640 2010
[11] YDeng YWChinH B Chai et al ldquoPhytochemical and bioac-tivity studies on constituents of the leaves of Vitex quinatardquoPhytochemistry Letters vol 4 no 3 pp 213ndash217 2011
[12] D Huang O U Boxin and R L Prior ldquoThe chemistry behindantioxidant capacity assaysrdquo Journal of Agricultural and FoodChemistry vol 53 no 6 pp 1841ndash1856 2005
[13] J H Nettles J L Jenkins A Bender Z Deng J W Daviesand M Glick ldquoBridging chemical and biological space ldquotargetfishingrdquo using 2D and 3D molecular descriptorsrdquo Journal ofMedicinal Chemistry vol 49 no 23 pp 6802ndash6810 2006
[14] I Inoue S I Goto K Mizotani et al ldquoLipophilic HMG-CoA reductase inhibitor has an anti-inflammatory effectreduction of MRNA levels for interleukin-1120573 interleukin-6cyclooxygenase-2 and p22phox by regulation of peroxisomeproliferator-activated receptor 120572 (PPAR120572) in primary endothe-lial cellsrdquo Life Sciences vol 67 no 8 pp 863ndash876 2000
[15] B I Schweitzer A P Dicker and J R Bertino ldquoDihydrofolatereductase as a therapeutic targetrdquoThe FASEB Journal vol 4 no8 pp 2441ndash2452 1990
[16] L Jiang P C Lee J White and P K Rathod ldquoPotent andselective activity of a combination of thymidine and 1843U89a folate-based thymidylate synthase inhibitor against Plasmod-ium falciparumrdquo Antimicrobial Agents and Chemotherapy vol44 no 4 pp 1047ndash1050 2000
[17] N Sienkiewicz S Jarosławski S Wyllie and A H FairlambldquoChemical and genetic validation of dihydrofolate reductase-thymidylate synthase as a drug target in African trypanosomesrdquoMolecular Microbiology vol 69 no 2 pp 520ndash533 2008
[18] L A Pinna and F Meggio ldquoProtein kinase CK2 (ldquocaseinkinase-2rdquo) and its implication in cell division and proliferationrdquoProgress in Cell Cycle Research vol 3 pp 77ndash97 1997
6 Evidence-Based Complementary and Alternative Medicine
[19] S Ortega I Prieto J Odajima et al ldquoCyclin-dependent kinase 2is essential for meiosis but not for mitotic cell division in micerdquoNature Genetics vol 35 no 1 pp 25ndash31 2003
[20] M Ashiuchi K Tani K Soda and H Misono ldquoProperties ofglutamate racemase from Bacillus subtilis IFO 3336 producingpoly-120574-glutamaterdquo Journal of Biochemistry vol 123 no 6 pp1156ndash1163 1998
[21] K Y Hwang C S Cho S S Kim H C Sung Y G Yu andY Cho ldquoStructure and mechanism of glutamate racemase fromAquifex pyrophilusrdquo Nature Structural Biology vol 6 no 5 pp422ndash426 1999
[22] A de Dios L Prieto J A Martın et al ldquo4-substituted D-glutamic acid analogues the first potent inhibitors of glutamateracemase (MurI) enzyme with antibacterial activityrdquo Journal ofMedicinal Chemistry vol 45 no 20 pp 4559ndash4570 2002
[23] G S Basarab P J Hill A Rastagar and P J H WebbornldquoDesign of Helicobacter pylori glutamate racemase inhibitorsas selective antibacterial agents a novel pro-drug approach toincrease exposurerdquo Bioorganic andMedicinal Chemistry Lettersvol 18 no 16 pp 4716ndash4722 2008
[24] B Geng G Basarab J Comita-Prevoir et al ldquoPotent andselective inhibitors of Helicobacter pylori glutamate racemase(MurI) pyridodiazepine aminesrdquo Bioorganic and MedicinalChemistry Letters vol 19 no 3 pp 930ndash936 2009
[25] C M Apfel H Locher S Evers et al ldquoPeptide deformylaseas an antibacterial drug target target validation and resistancedevelopmentrdquo Antimicrobial Agents and Chemotherapy vol 45no 4 pp 1058ndash1064 2001
[26] J A Leeds and C R Dean ldquoPeptide deformylase as an antibac-terial target a critical assessmentrdquo Current Opinion in Pharma-cology vol 6 no 5 pp 445ndash452 2006
[27] S Kumar U Narain S Tripathi and K Misra ldquoSynthesesof curcumin bioconjugates and study of their antibacterialactivities against120573-lactamase-producingmicroorganismsrdquoBio-conjugate Chemistry vol 12 no 4 pp 464ndash469 2001
[28] Q Li J Y Lee R Castillo et al ldquoNB2001 a novel antibacterialagent with broad-spectrum activity and enhanced potencyagainst 120573-lactamase-producing strainsrdquo Antimicrobial Agentsand Chemotherapy vol 46 no 5 pp 1262ndash1268 2002
[29] T M Coskran D Morton F S Menniti et al ldquoImmunohis-tochemical localization of phosphodiesterase 10A in multiplemammalian speciesrdquo Journal of Histochemistry and Cytochem-istry vol 54 no 11 pp 1205ndash1213 2006
[30] F S Menniti T A Chappie J M Humphrey and C J SchmidtldquoPhosphodiesterase 10A inhibitors a novel approach to thetreatment of the symptoms of schizophreniardquo Current Opinionin Investigational Drugs vol 8 no 1 pp 54ndash59 2007
[31] C J Schmidt D S Chapin J Cianfrogna et al ldquoPreclinicalcharacterization of selective phosphodiesterase 10A inhibitorsa new therapeutic approach to the treatment of schizophreniardquoThe Journal of Pharmacology and Experimental Therapeuticsvol 325 no 2 pp 681ndash690 2008
[32] J B Bolen R B Rowley C Spana and A Y Tsygankov ldquoTheSrc family of tyrosine protein kinases in hemopoietic signaltransductionrdquoThe FASEB Journal vol 6 no 15 pp 3403ndash34091992
[33] E Buchdunger J Zimmermann H Mett et al ldquoInhibition ofthe Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivativerdquo Cancer Research vol 56pp 100ndash104 1996
[34] P Mishra S Senthivinayagam A Rana and B Rana ldquoGlyco-gen synthase kinase-3120573 regulates snail and 120573-catenin during
gastrin-induced migration of gastric cancer cellsrdquo Journal ofMolecular Signaling vol 5 article 9 2010
[35] D Beier andRGross ldquoRegulation of bacterial virulence by two-component systemsrdquo Current Opinion in Microbiology vol 9no 2 pp 143ndash152 2006
[36] W J Gooderham S L Gellatly F Sanschagrin et al ldquoThe sensorkinase PhoQ mediates virulence in Pseudomonas aeruginosardquoMicrobiology vol 155 no 3 pp 699ndash711 2009
[37] T Dalmay A Hamilton S Rudd S Angell and D CBaulcombe ldquoAn RNA-dependent RNA polymerase gene inArabidopsis is required for posttranscriptional gene silencingmediated by a transgene but not by a virusrdquo Cell vol 101 no5 pp 543ndash553 2000
[38] F Simmer M Tijsterman S Parrish et al ldquoLoss of the putativeRNA-directed RNA polymerase RRF-3 makes C elegans hyper-sensitive to RNAirdquo Current Biology vol 12 no 15 pp 1317ndash13192002
[39] L HWu and C TWoodbridge ldquoRegulation of transthyretin totreat obesityrdquo Patent Application publication 0160394 A1 2002
[40] S K Frey J Spranger A Henze A F H Pfeiffer F JSchweigert and J Raila ldquoFactors that influence retinol-bindingprotein 4-transthyretin interaction are not altered in overweightsubjects and overweight subjects with type 2 diabetes mellitusrdquoMetabolism Clinical and Experimental vol 58 no 10 pp 1386ndash1392 2009
[41] J Hu and C Seeger ldquoHsp90 is required for the activity of a hep-atitis B virus reverse transcriptaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no3 pp 1060ndash1064 1996
[42] J R Sydor E Normant C S Pien et al ldquoDevelopment of17-allylamino-17-demethoxygeldanamycin hydroquinonehydrochloride (IPI-504) an anti-cancer agent directed againstHsp90rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 103 no 46 pp 17408ndash17413 2006
[43] C Tantillo J Ding A Jacobo-Molina et al ldquoLocations ofanti-AIDS drug binding sites and resistance mutations in thethree-dimensional structure of HIV-1 reverse transcriptaseImplications for mechanisms of drug inhibition and resistancerdquoJournal of Molecular Biology vol 243 no 3 pp 369ndash387 1994
[44] S G Sarafianos B Marchand K Das et al ldquoStructure andfunction of HIV-1 reverse transcriptase molecular mechanismsof polymerization and inhibitionrdquo Journal of Molecular Biologyvol 385 no 3 pp 693ndash713 2009
[45] K M Frey M Bollini A C Mislak et al ldquoCrystal structures ofHIV-1 reverse transcriptasewith picomolar inhibitors reveal keyinteractions for drug designrdquo Journal of the American ChemicalSociety vol 134 no 48 pp 19501ndash19503 2012
[46] M Idu ldquoCurrent trends in ethnobotanyrdquo Tropical Journal ofPharmaceutical Research vol 8 no 4 pp 295ndash296 2009
[47] M Christofidou-Solomidou and V R Muzykantov ldquoAntioxi-dant strategies in respiratory medicinerdquo Treatments in Respira-tory Medicine vol 5 no 1 pp 47ndash78 2006
[48] M Brozmanova J PlevkovaV Bartos L PlankM Javorka andM Tatar ldquoThe interaction of dietary antioxidant vitamins andoxidative stress on cough reflex in guinea-pigs after long termoxygen therapyrdquo Journal of Physiology and Pharmacology vol57 supplement 4 pp 45ndash54 2006
[49] M Brozmanova V Bartos L Plank J Plevkova and M TatarldquoDietary intake of flavonoids and hyperoxia-induced oxidativestress related cough in guinea pigsrdquo Bratislava Medical Journalvol 108 no 12 pp 489ndash494 2007
Evidence-Based Complementary and Alternative Medicine 7
[50] M Chadwick H Trewin F Gawthrop and C Wagstaff ldquoSes-quiterpenoids lactones benefits to plants and peoplerdquo Interna-tional Journal of Molecular Sciences vol 14 no 6 pp 12780ndash12805 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 5
radiation injury COPD (chronic obstructive pulmonary dis-ease) and inflammation [47] Meanwhile previous clinicaland experimental studies described that antioxidant supple-mentation including flavonoids and vitamins may inversethe oxidant-mediated cough depression by modulating theinflammatory process in lung disease [48 49] Interestinglyour work using ABTS assay demonstrated that compounds1ndash3 showed strong antioxidant activity especially compound1 (IC50
= 044 plusmn 0039mM) Moreover E scaber was alsoreported as the source of a number of sesquiterpene lactonessuch as compounds 4 and 5 which have shown significantcontribution to the anti-inflammatory activity of plants [50]Some of the sesquiterpenes from the genus Elephantopushave demonstrated significant anti-inflammatory as well ashepatoprotective activities and are being considered as druglead compounds [3] Based on the above analysis we hypoth-esize that Zhuang people use this plant to treat headachesbronchitis and hepatitisdue to its anti-inflammatory andantioxidant effects
According to the in vitro cytotoxicity assay with NCTD(60 120583M) as control group and activity virtual screeningcompound 1 exhibited good (IC
50= 60120583M) and dose-
response inhibition on HepG2 cell line and potential anti-inflammatory antibiotic antivirus and anticancer activitieswhich indicated that the further research of E scaber couldbe focused on anticancer and anti-inflammatory activityThepresent work further developed the usage of this traditionalmedicine plant
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors thank Vanya Petrova (City University of NewYork) for assistance with the antioxidant assay and WeiguoLiu (Minzu University of China) for assistance with usingthe Discovery Studio software This work was supported inpart by the National Natural Science Foundation of China(31161140345 and 31070288) the Ministry of Science amp Tech-nology of China (2012FY110300) the Ministry of Educationof China through its 111 and 985 Projects (B08044 MUC985-9 and MUC98506-01000101) and JSPS Asian Core program(JSPSAP109080)
References
[1] Z Y Wu and P Raven Flora of China vol 20-21 of AsteraceaeScience Press Beijing China Missouri Botanical Garden StLouis Mo USA 2007
[2] J JWang Y J Liu J X Xu AWang Y LWang andC L LongldquoStudies on medicinal plants of Elephantopus (Compositae)rdquoNatural Products Research and Development vol 25 pp 401ndash409 2013
[3] C C Huang K J Lin Y W Cheng C A Hsu S S Yang andL F Shyur ldquoHepatoprotective effect and mechanistic insightsof deoxyelephantopin a phyto-sesquiterpene lactone against
fulminant hepatitisrdquo Journal of Nutritional Biochemistry vol 24no 3 pp 516ndash530 2013
[4] K Schutz D Kammerer R Carle and A Schieber ldquoIdentifi-cation and quantification of caffeoylquinic acids and flavonoidsfrom artichoke (Cynara scolymus L) heads juice and pomaceby HPLC-DAD-ESIMSnrdquo Journal of Agricultural and FoodChemistry vol 52 no 13 pp 4090ndash4096 2004
[5] X Zhu X Dong Y Wang P Ju and S Luo ldquoPhenolic com-pounds from Viburnum cylindricumrdquo Helvetica Chimica Actavol 88 no 2 pp 339ndash342 2005
[6] Q L Liang and Z D Min ldquoSesquiterpene lactones fromElephantopus scaberrdquoChinese Chemical Letters vol 13 no 4 pp343ndash344 2002
[7] L R Wang and X Q Xie ldquoComputational target fishing whatshould chemogenomics researchers expect for the future of insilico drug design and discoveryrdquo Future Medicinal Chemistryvol 6 no 3 pp 247ndash249 2014
[8] S B Wu K Dastmalchhi C L Long and E J KennellyldquoMetabolite profiling of jaboticaba (Myrciaria cauliflora) andother dark-colored fruit juicesrdquo Journal of Agriculture and FoodChemistry vol 60 no 30 pp 7513ndash7525 2012
[9] A L Liu H D Wang S M Lee Y T Wang and G H DuldquoStructure-activity relationship of flavonoids as influenza virusneuraminidase inhibitors and their in vitro anti-viral activitiesrdquoBioorganic and Medicinal Chemistry vol 16 no 15 pp 7141ndash7147 2008
[10] C Y C Chen ldquoVirtual screening and drug design for PDE-5receptor from traditional Chinese medicine databaserdquo Journalof Biomolecular Structure and Dynamics vol 27 no 5 pp 627ndash640 2010
[11] YDeng YWChinH B Chai et al ldquoPhytochemical and bioac-tivity studies on constituents of the leaves of Vitex quinatardquoPhytochemistry Letters vol 4 no 3 pp 213ndash217 2011
[12] D Huang O U Boxin and R L Prior ldquoThe chemistry behindantioxidant capacity assaysrdquo Journal of Agricultural and FoodChemistry vol 53 no 6 pp 1841ndash1856 2005
[13] J H Nettles J L Jenkins A Bender Z Deng J W Daviesand M Glick ldquoBridging chemical and biological space ldquotargetfishingrdquo using 2D and 3D molecular descriptorsrdquo Journal ofMedicinal Chemistry vol 49 no 23 pp 6802ndash6810 2006
[14] I Inoue S I Goto K Mizotani et al ldquoLipophilic HMG-CoA reductase inhibitor has an anti-inflammatory effectreduction of MRNA levels for interleukin-1120573 interleukin-6cyclooxygenase-2 and p22phox by regulation of peroxisomeproliferator-activated receptor 120572 (PPAR120572) in primary endothe-lial cellsrdquo Life Sciences vol 67 no 8 pp 863ndash876 2000
[15] B I Schweitzer A P Dicker and J R Bertino ldquoDihydrofolatereductase as a therapeutic targetrdquoThe FASEB Journal vol 4 no8 pp 2441ndash2452 1990
[16] L Jiang P C Lee J White and P K Rathod ldquoPotent andselective activity of a combination of thymidine and 1843U89a folate-based thymidylate synthase inhibitor against Plasmod-ium falciparumrdquo Antimicrobial Agents and Chemotherapy vol44 no 4 pp 1047ndash1050 2000
[17] N Sienkiewicz S Jarosławski S Wyllie and A H FairlambldquoChemical and genetic validation of dihydrofolate reductase-thymidylate synthase as a drug target in African trypanosomesrdquoMolecular Microbiology vol 69 no 2 pp 520ndash533 2008
[18] L A Pinna and F Meggio ldquoProtein kinase CK2 (ldquocaseinkinase-2rdquo) and its implication in cell division and proliferationrdquoProgress in Cell Cycle Research vol 3 pp 77ndash97 1997
6 Evidence-Based Complementary and Alternative Medicine
[19] S Ortega I Prieto J Odajima et al ldquoCyclin-dependent kinase 2is essential for meiosis but not for mitotic cell division in micerdquoNature Genetics vol 35 no 1 pp 25ndash31 2003
[20] M Ashiuchi K Tani K Soda and H Misono ldquoProperties ofglutamate racemase from Bacillus subtilis IFO 3336 producingpoly-120574-glutamaterdquo Journal of Biochemistry vol 123 no 6 pp1156ndash1163 1998
[21] K Y Hwang C S Cho S S Kim H C Sung Y G Yu andY Cho ldquoStructure and mechanism of glutamate racemase fromAquifex pyrophilusrdquo Nature Structural Biology vol 6 no 5 pp422ndash426 1999
[22] A de Dios L Prieto J A Martın et al ldquo4-substituted D-glutamic acid analogues the first potent inhibitors of glutamateracemase (MurI) enzyme with antibacterial activityrdquo Journal ofMedicinal Chemistry vol 45 no 20 pp 4559ndash4570 2002
[23] G S Basarab P J Hill A Rastagar and P J H WebbornldquoDesign of Helicobacter pylori glutamate racemase inhibitorsas selective antibacterial agents a novel pro-drug approach toincrease exposurerdquo Bioorganic andMedicinal Chemistry Lettersvol 18 no 16 pp 4716ndash4722 2008
[24] B Geng G Basarab J Comita-Prevoir et al ldquoPotent andselective inhibitors of Helicobacter pylori glutamate racemase(MurI) pyridodiazepine aminesrdquo Bioorganic and MedicinalChemistry Letters vol 19 no 3 pp 930ndash936 2009
[25] C M Apfel H Locher S Evers et al ldquoPeptide deformylaseas an antibacterial drug target target validation and resistancedevelopmentrdquo Antimicrobial Agents and Chemotherapy vol 45no 4 pp 1058ndash1064 2001
[26] J A Leeds and C R Dean ldquoPeptide deformylase as an antibac-terial target a critical assessmentrdquo Current Opinion in Pharma-cology vol 6 no 5 pp 445ndash452 2006
[27] S Kumar U Narain S Tripathi and K Misra ldquoSynthesesof curcumin bioconjugates and study of their antibacterialactivities against120573-lactamase-producingmicroorganismsrdquoBio-conjugate Chemistry vol 12 no 4 pp 464ndash469 2001
[28] Q Li J Y Lee R Castillo et al ldquoNB2001 a novel antibacterialagent with broad-spectrum activity and enhanced potencyagainst 120573-lactamase-producing strainsrdquo Antimicrobial Agentsand Chemotherapy vol 46 no 5 pp 1262ndash1268 2002
[29] T M Coskran D Morton F S Menniti et al ldquoImmunohis-tochemical localization of phosphodiesterase 10A in multiplemammalian speciesrdquo Journal of Histochemistry and Cytochem-istry vol 54 no 11 pp 1205ndash1213 2006
[30] F S Menniti T A Chappie J M Humphrey and C J SchmidtldquoPhosphodiesterase 10A inhibitors a novel approach to thetreatment of the symptoms of schizophreniardquo Current Opinionin Investigational Drugs vol 8 no 1 pp 54ndash59 2007
[31] C J Schmidt D S Chapin J Cianfrogna et al ldquoPreclinicalcharacterization of selective phosphodiesterase 10A inhibitorsa new therapeutic approach to the treatment of schizophreniardquoThe Journal of Pharmacology and Experimental Therapeuticsvol 325 no 2 pp 681ndash690 2008
[32] J B Bolen R B Rowley C Spana and A Y Tsygankov ldquoTheSrc family of tyrosine protein kinases in hemopoietic signaltransductionrdquoThe FASEB Journal vol 6 no 15 pp 3403ndash34091992
[33] E Buchdunger J Zimmermann H Mett et al ldquoInhibition ofthe Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivativerdquo Cancer Research vol 56pp 100ndash104 1996
[34] P Mishra S Senthivinayagam A Rana and B Rana ldquoGlyco-gen synthase kinase-3120573 regulates snail and 120573-catenin during
gastrin-induced migration of gastric cancer cellsrdquo Journal ofMolecular Signaling vol 5 article 9 2010
[35] D Beier andRGross ldquoRegulation of bacterial virulence by two-component systemsrdquo Current Opinion in Microbiology vol 9no 2 pp 143ndash152 2006
[36] W J Gooderham S L Gellatly F Sanschagrin et al ldquoThe sensorkinase PhoQ mediates virulence in Pseudomonas aeruginosardquoMicrobiology vol 155 no 3 pp 699ndash711 2009
[37] T Dalmay A Hamilton S Rudd S Angell and D CBaulcombe ldquoAn RNA-dependent RNA polymerase gene inArabidopsis is required for posttranscriptional gene silencingmediated by a transgene but not by a virusrdquo Cell vol 101 no5 pp 543ndash553 2000
[38] F Simmer M Tijsterman S Parrish et al ldquoLoss of the putativeRNA-directed RNA polymerase RRF-3 makes C elegans hyper-sensitive to RNAirdquo Current Biology vol 12 no 15 pp 1317ndash13192002
[39] L HWu and C TWoodbridge ldquoRegulation of transthyretin totreat obesityrdquo Patent Application publication 0160394 A1 2002
[40] S K Frey J Spranger A Henze A F H Pfeiffer F JSchweigert and J Raila ldquoFactors that influence retinol-bindingprotein 4-transthyretin interaction are not altered in overweightsubjects and overweight subjects with type 2 diabetes mellitusrdquoMetabolism Clinical and Experimental vol 58 no 10 pp 1386ndash1392 2009
[41] J Hu and C Seeger ldquoHsp90 is required for the activity of a hep-atitis B virus reverse transcriptaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no3 pp 1060ndash1064 1996
[42] J R Sydor E Normant C S Pien et al ldquoDevelopment of17-allylamino-17-demethoxygeldanamycin hydroquinonehydrochloride (IPI-504) an anti-cancer agent directed againstHsp90rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 103 no 46 pp 17408ndash17413 2006
[43] C Tantillo J Ding A Jacobo-Molina et al ldquoLocations ofanti-AIDS drug binding sites and resistance mutations in thethree-dimensional structure of HIV-1 reverse transcriptaseImplications for mechanisms of drug inhibition and resistancerdquoJournal of Molecular Biology vol 243 no 3 pp 369ndash387 1994
[44] S G Sarafianos B Marchand K Das et al ldquoStructure andfunction of HIV-1 reverse transcriptase molecular mechanismsof polymerization and inhibitionrdquo Journal of Molecular Biologyvol 385 no 3 pp 693ndash713 2009
[45] K M Frey M Bollini A C Mislak et al ldquoCrystal structures ofHIV-1 reverse transcriptasewith picomolar inhibitors reveal keyinteractions for drug designrdquo Journal of the American ChemicalSociety vol 134 no 48 pp 19501ndash19503 2012
[46] M Idu ldquoCurrent trends in ethnobotanyrdquo Tropical Journal ofPharmaceutical Research vol 8 no 4 pp 295ndash296 2009
[47] M Christofidou-Solomidou and V R Muzykantov ldquoAntioxi-dant strategies in respiratory medicinerdquo Treatments in Respira-tory Medicine vol 5 no 1 pp 47ndash78 2006
[48] M Brozmanova J PlevkovaV Bartos L PlankM Javorka andM Tatar ldquoThe interaction of dietary antioxidant vitamins andoxidative stress on cough reflex in guinea-pigs after long termoxygen therapyrdquo Journal of Physiology and Pharmacology vol57 supplement 4 pp 45ndash54 2006
[49] M Brozmanova V Bartos L Plank J Plevkova and M TatarldquoDietary intake of flavonoids and hyperoxia-induced oxidativestress related cough in guinea pigsrdquo Bratislava Medical Journalvol 108 no 12 pp 489ndash494 2007
Evidence-Based Complementary and Alternative Medicine 7
[50] M Chadwick H Trewin F Gawthrop and C Wagstaff ldquoSes-quiterpenoids lactones benefits to plants and peoplerdquo Interna-tional Journal of Molecular Sciences vol 14 no 6 pp 12780ndash12805 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Evidence-Based Complementary and Alternative Medicine
[19] S Ortega I Prieto J Odajima et al ldquoCyclin-dependent kinase 2is essential for meiosis but not for mitotic cell division in micerdquoNature Genetics vol 35 no 1 pp 25ndash31 2003
[20] M Ashiuchi K Tani K Soda and H Misono ldquoProperties ofglutamate racemase from Bacillus subtilis IFO 3336 producingpoly-120574-glutamaterdquo Journal of Biochemistry vol 123 no 6 pp1156ndash1163 1998
[21] K Y Hwang C S Cho S S Kim H C Sung Y G Yu andY Cho ldquoStructure and mechanism of glutamate racemase fromAquifex pyrophilusrdquo Nature Structural Biology vol 6 no 5 pp422ndash426 1999
[22] A de Dios L Prieto J A Martın et al ldquo4-substituted D-glutamic acid analogues the first potent inhibitors of glutamateracemase (MurI) enzyme with antibacterial activityrdquo Journal ofMedicinal Chemistry vol 45 no 20 pp 4559ndash4570 2002
[23] G S Basarab P J Hill A Rastagar and P J H WebbornldquoDesign of Helicobacter pylori glutamate racemase inhibitorsas selective antibacterial agents a novel pro-drug approach toincrease exposurerdquo Bioorganic andMedicinal Chemistry Lettersvol 18 no 16 pp 4716ndash4722 2008
[24] B Geng G Basarab J Comita-Prevoir et al ldquoPotent andselective inhibitors of Helicobacter pylori glutamate racemase(MurI) pyridodiazepine aminesrdquo Bioorganic and MedicinalChemistry Letters vol 19 no 3 pp 930ndash936 2009
[25] C M Apfel H Locher S Evers et al ldquoPeptide deformylaseas an antibacterial drug target target validation and resistancedevelopmentrdquo Antimicrobial Agents and Chemotherapy vol 45no 4 pp 1058ndash1064 2001
[26] J A Leeds and C R Dean ldquoPeptide deformylase as an antibac-terial target a critical assessmentrdquo Current Opinion in Pharma-cology vol 6 no 5 pp 445ndash452 2006
[27] S Kumar U Narain S Tripathi and K Misra ldquoSynthesesof curcumin bioconjugates and study of their antibacterialactivities against120573-lactamase-producingmicroorganismsrdquoBio-conjugate Chemistry vol 12 no 4 pp 464ndash469 2001
[28] Q Li J Y Lee R Castillo et al ldquoNB2001 a novel antibacterialagent with broad-spectrum activity and enhanced potencyagainst 120573-lactamase-producing strainsrdquo Antimicrobial Agentsand Chemotherapy vol 46 no 5 pp 1262ndash1268 2002
[29] T M Coskran D Morton F S Menniti et al ldquoImmunohis-tochemical localization of phosphodiesterase 10A in multiplemammalian speciesrdquo Journal of Histochemistry and Cytochem-istry vol 54 no 11 pp 1205ndash1213 2006
[30] F S Menniti T A Chappie J M Humphrey and C J SchmidtldquoPhosphodiesterase 10A inhibitors a novel approach to thetreatment of the symptoms of schizophreniardquo Current Opinionin Investigational Drugs vol 8 no 1 pp 54ndash59 2007
[31] C J Schmidt D S Chapin J Cianfrogna et al ldquoPreclinicalcharacterization of selective phosphodiesterase 10A inhibitorsa new therapeutic approach to the treatment of schizophreniardquoThe Journal of Pharmacology and Experimental Therapeuticsvol 325 no 2 pp 681ndash690 2008
[32] J B Bolen R B Rowley C Spana and A Y Tsygankov ldquoTheSrc family of tyrosine protein kinases in hemopoietic signaltransductionrdquoThe FASEB Journal vol 6 no 15 pp 3403ndash34091992
[33] E Buchdunger J Zimmermann H Mett et al ldquoInhibition ofthe Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivativerdquo Cancer Research vol 56pp 100ndash104 1996
[34] P Mishra S Senthivinayagam A Rana and B Rana ldquoGlyco-gen synthase kinase-3120573 regulates snail and 120573-catenin during
gastrin-induced migration of gastric cancer cellsrdquo Journal ofMolecular Signaling vol 5 article 9 2010
[35] D Beier andRGross ldquoRegulation of bacterial virulence by two-component systemsrdquo Current Opinion in Microbiology vol 9no 2 pp 143ndash152 2006
[36] W J Gooderham S L Gellatly F Sanschagrin et al ldquoThe sensorkinase PhoQ mediates virulence in Pseudomonas aeruginosardquoMicrobiology vol 155 no 3 pp 699ndash711 2009
[37] T Dalmay A Hamilton S Rudd S Angell and D CBaulcombe ldquoAn RNA-dependent RNA polymerase gene inArabidopsis is required for posttranscriptional gene silencingmediated by a transgene but not by a virusrdquo Cell vol 101 no5 pp 543ndash553 2000
[38] F Simmer M Tijsterman S Parrish et al ldquoLoss of the putativeRNA-directed RNA polymerase RRF-3 makes C elegans hyper-sensitive to RNAirdquo Current Biology vol 12 no 15 pp 1317ndash13192002
[39] L HWu and C TWoodbridge ldquoRegulation of transthyretin totreat obesityrdquo Patent Application publication 0160394 A1 2002
[40] S K Frey J Spranger A Henze A F H Pfeiffer F JSchweigert and J Raila ldquoFactors that influence retinol-bindingprotein 4-transthyretin interaction are not altered in overweightsubjects and overweight subjects with type 2 diabetes mellitusrdquoMetabolism Clinical and Experimental vol 58 no 10 pp 1386ndash1392 2009
[41] J Hu and C Seeger ldquoHsp90 is required for the activity of a hep-atitis B virus reverse transcriptaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no3 pp 1060ndash1064 1996
[42] J R Sydor E Normant C S Pien et al ldquoDevelopment of17-allylamino-17-demethoxygeldanamycin hydroquinonehydrochloride (IPI-504) an anti-cancer agent directed againstHsp90rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 103 no 46 pp 17408ndash17413 2006
[43] C Tantillo J Ding A Jacobo-Molina et al ldquoLocations ofanti-AIDS drug binding sites and resistance mutations in thethree-dimensional structure of HIV-1 reverse transcriptaseImplications for mechanisms of drug inhibition and resistancerdquoJournal of Molecular Biology vol 243 no 3 pp 369ndash387 1994
[44] S G Sarafianos B Marchand K Das et al ldquoStructure andfunction of HIV-1 reverse transcriptase molecular mechanismsof polymerization and inhibitionrdquo Journal of Molecular Biologyvol 385 no 3 pp 693ndash713 2009
[45] K M Frey M Bollini A C Mislak et al ldquoCrystal structures ofHIV-1 reverse transcriptasewith picomolar inhibitors reveal keyinteractions for drug designrdquo Journal of the American ChemicalSociety vol 134 no 48 pp 19501ndash19503 2012
[46] M Idu ldquoCurrent trends in ethnobotanyrdquo Tropical Journal ofPharmaceutical Research vol 8 no 4 pp 295ndash296 2009
[47] M Christofidou-Solomidou and V R Muzykantov ldquoAntioxi-dant strategies in respiratory medicinerdquo Treatments in Respira-tory Medicine vol 5 no 1 pp 47ndash78 2006
[48] M Brozmanova J PlevkovaV Bartos L PlankM Javorka andM Tatar ldquoThe interaction of dietary antioxidant vitamins andoxidative stress on cough reflex in guinea-pigs after long termoxygen therapyrdquo Journal of Physiology and Pharmacology vol57 supplement 4 pp 45ndash54 2006
[49] M Brozmanova V Bartos L Plank J Plevkova and M TatarldquoDietary intake of flavonoids and hyperoxia-induced oxidativestress related cough in guinea pigsrdquo Bratislava Medical Journalvol 108 no 12 pp 489ndash494 2007
Evidence-Based Complementary and Alternative Medicine 7
[50] M Chadwick H Trewin F Gawthrop and C Wagstaff ldquoSes-quiterpenoids lactones benefits to plants and peoplerdquo Interna-tional Journal of Molecular Sciences vol 14 no 6 pp 12780ndash12805 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 7
[50] M Chadwick H Trewin F Gawthrop and C Wagstaff ldquoSes-quiterpenoids lactones benefits to plants and peoplerdquo Interna-tional Journal of Molecular Sciences vol 14 no 6 pp 12780ndash12805 2013
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
![Research Article Long-Term Treatment with Aqueous Garlic ...downloads.hindawi.com/journals/ecam/2014/381649.pdf · steroidal glycosides and lectins [ , ]. e sulfur compounds are responsible](https://static.fdocuments.in/doc/165x107/60194779d9786235b46ccdd6/research-article-long-term-treatment-with-aqueous-garlic-steroidal-glycosides.jpg)
