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Research ArticleAsparagus cochinchinensis Extract Alleviates MetalIon-Induced Gut Injury in Drosophila An In Silico Analysis ofPotential Active Constituents

Weiyu Zhang and Li Hua Jin

College of Life Science Northeast Forestry University Harbin 150040 China

Correspondence should be addressed to Weiyu Zhang zhangweiyu04nefueducn

Received 15 December 2015 Revised 24 February 2016 Accepted 13 March 2016

Academic Editor Ghee T Tan

Copyright copy 2016 W Zhang and L H Jin This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Metal ions and sulfate are components of atmospheric pollutants that have diverse ways of entering the human body We usedDrosophila as amodel to investigate the effect ofAsparagus cochinchinensis (A cochinchinensis) extracts on the gut and characterizedgut homeostasis following the ingestion of metal ions (copper zinc and aluminum) In this study we found that the aqueousA cochinchinensis extract increased the survival rate decreased epithelial cell death and attenuated metal ion-induced gutmorphological changes in flies following chronic exposure to metal ions In addition we screened out by network pharmacologysix natural products (NPs) that could serve as putative active components ofA cochinchinensis that prevented gut injury Altogetherthe results of our study provide evidence that A cochinchinensis might be an effective phytomedicine for the treatment of metalion-induced gut injury

1 Introduction

In recent years atmospheric pollution has become a rapidlygrowing international trend Atmospheric pollution not onlyis harmful to the Earthrsquos climate agriculture and industrybut also does immeasurable damage to humans Atmosphericpollution can cause respiratory system damage physiologi-cal abnormalities neurological abnormalities and digestivedisorders [1 2] Metal ions and sulfate are components ofatmospheric pollution stemming primarily from fuel com-bustion and large-scale industrial and mining enterprisesas well as from other man-made pollutants such as exhaustgas Metal ions enter the human body in diverse waysincluding inhalation swallowing and skin contact Due totheir nondegradation characteristics metal ions accumulatein the body where they are converted into more toxic metalcompounds by combining with organic matter This triggersa series of damaging effects that result in physiologicaldysfunctions [3] Previous studies have demonstrated thata dose of cadmium induces intestinal epithelial cell injuryin the Drosophila midgut [4] The intestinal epithelial isan important protective barrier between the internal and

external environment Mechanisms of immunity and tissueregeneration must be tightly regulated in order to maintainintestinal homeostasis [5 6] Dysregulation of inflammatoryresponses and tissue regeneration can lead to inflamma-tory bowel diseases and colorectal cancer in mammals [7]In recent years increasingly more people are plagued byintestinal inflammation and prolonged inflammation andtissue damage can lead to intestinal carcinogenesis and tumorformation

Asparagus cochinchinensis (A cochinchinensis) referredto as Tiandong in China is the root of A cochinchinensis(Lour) Merr (Liliaceae) that is distributed among manyprovinces of China A cochinchinensis (AC) has been usedin traditional Chinese medicine (TCM) for over 2000 yearsIts flavor is sweet bitter and cold The channel tropism isthe lungs kidney stomach and large intestine meridian Acochinchinensis has often been used for the treatment of fevercough throat pain swelling constipation and diabetes Thedried root has antibacterial antipyretic diuretic expectorantstomachic nervous stimulant and tonic properties [8] Mod-ern research has also demonstrated that A cochinchinensishas antitumor activity especially in lung cancer [9 10]

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 7603746 9 pageshttpdxdoiorg10115520167603746

2 Evidence-Based Complementary and Alternative Medicine

However the protective effect on intestinal injury and theactive components of A cochinchinensis affecting gut immu-nity remain poorly understood

Drosophila is a well studied and highly tractable geneticmodel organism Many basic biological and physiologicalproperties are conserved between Drosophila and mammalsand nearly 75 of human disease-related genes have a func-tional homolog in the fly [11] Thus the Drosophila is widelyused in basic and applied researches on a broad spectrumof human diseases including infectious diseases [12] cancers[13] neurodegenerative diseases [14] and metabolic diseases[15] Drosophila and human intestine have similar anatomyand physiological function [16 17] and they have alsosimilarities in cell and composition and underlying signalingpathways that maintain intestinal homeostasis [18]

Chinese medicinal herbs exert their therapeutic actionsthrough the synergistic effects of multiple compounds tar-gets and channels However it has been difficult to isolatethe effective components of these products and to identifyspecific therapeutic targets for treating disease Networkpharmacology has been used as an integrated approachto systematically investigate and explain the underlyingmolecular mechanisms of Chinese medicinal herbs Usingthe Computerized Virtual Screening Technique to explorepotential targetsmay help facilitate these investigations whilereducing manpower and material resources

Drosophila has emerged as a potential whole animalmodel for drug screening [19 20]Through a large number ofsurvival rate assays we identifiedA cochinchinensis as havinggood bioactivity against chemical reagents-induced stress inDrosophila (data was not shown) In this study we revealedthat the aqueous extract of A cochinchinensis exerts a protec-tive effect on gut injury in Drosophila induced by the chronicexposure to metal ions In addition we computationallyidentified the putative active ingredients ofA cochinchinensisusing network pharmacology Network analysis revealedsix constituents of A cochinchinensis that could potentiallymediate its protective effects on gut injury These results pro-vide new insight into the pharmacological basis of the antigutinjury activity of A cochinchinensis and will provide impetusfor preclinical drug discovery based on this medicinalplant

2 Materials and Methods

21 Drosophila Fly Stocks w1118 flies were obtained fromthe Bloomington Drosophila Stock Center Fly stocks weremaintained on a 12 h light12 h dark cycle at 25∘C and 60humidity

22 A cochinchinensis Extraction A cochinchinensis wasidentified by Professor XiuhuaWang at the Herbarium of theCollege of Life Sciences Northeast Forestry University andpurchased from Shiyitang Pharmacy of Harbin China Themethod of aqueous extraction of A cochinchinensis has beenpreviously described [21] Total aqueous-derived extract wasconsolidated and concentrated to a final concentration of 20(wv)

23 Drosophila Food Standard cornmeal-yeastmediumusedfor the control group consisted of 56 gL agar 168 gL yeast716 gL polenta 98 gL soybean flour and 60 gL sucroseStandard mediumwith 10 (wv) aqueousA cochinchinensisextract was used for the experimental group

24 Survival Experiments Procedures for the survival andfeeding experiments were performed as previously described[21] with the difference that adult flies which aged 3ndash5days (15 males and 15 females) were starved for 2 h withoutfood before being transferred to a vial containing chemicalcompounds in 5 (wv) sucrose solution serving as thecontrol group for all experiments The chemical compoundsincluded cupric sulfate (7mM Sigma) zinc sulfate (7mMSigma) and aluminum potassium disulfate dodecahydrate(20mM Sigma) The experimental group consists of flies fedwith sucrose solutions incorporating the chemical compoundwith added AC extract at 10 (ww) Fresh filter papersand solutions were provided every day and dead flies wereenumerated and evaluated daily

25 7-Amino-actinomycinD Staining and Imaging 7-Amino-actinomycin D (7-AAD) staining and imaging were per-formed as previously described [21 22]

26 Intestinal Morphological Analysis Female flies were usedfor intestinal morphological studies due to their larger size

Due to the bigger size of female flies we used them forintestinal morphological analysis experiment The guts of 3ndash5-day-old female flies that had orally ingested metal ions in5 (wv) sucrose with or without AC extract for 96 h weredissected at room temperature in phosphate buffered saline(PBS) and immediately observed under an Axioskop 2 plusmicroscope (Zeiss)

27 Targets Predictions of Natural Products (NPs) and Net-work Construction The information pertaining to naturalproducts (NPs) was obtained from the Universal NaturalProducts Database (UNPD) according to the scientific namesof the herbs [23] The information for approved drugs anddrug targets was obtained from DrugBank [24] We used thePharmMapper server to predict potential drug targets [25]The network was constructed by Cytoscape 321 [26]

28 Statistics For all experiments data are representative ofthree independent experiments Statistical analyses includ-ing Studentrsquos t-test the one-way ANOVA and log-rank(Mantel-Cox) test were performed using GraphPad Prism50 software (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 ns nosignificant difference)

3 Results

31 A cochinchinensis Increased Survival following the Inges-tion of Metal Ions As water decoction is the traditional for-mulation used in Chinese clinical medicine we used aqueousextraction in this study To assess the effect of A cochinchi-nensis (AC) extract on the development of Drosophila larvae

Evidence-Based Complementary and Alternative Medicine 3

Surv

ival

()

100

80

60

40

20

076543210

Day

ConAC

CuSO4

lowastlowastlowast

(a)

Surv

ival

()

100

80

60

40

20

0

76543210

Day

ConAC

ZnSO4

lowastlowastlowast

(b)

Surv

ival

()

100

80

60

40

20

076543210

Day

ConAC

lowastlowastlowast

KAl(SO4)2 middot 12H2O

(c)

Figure 1 A cochinchinensis improves the survival rate of flies that have ingested metal ions (a) The survival rates of AC extract and controlflies fed with 7mM CuSO

4

(b) The survival rates of AC extract and control flies fed with 7mM ZnSO4

(c) The survival rates of AC extractand control flies fed with 20mMKAl(SO

4

)2

sdot12H2

OThree replicates were used for the determination of survival rates 119875 value was calculatedby the log-rank test lowastlowastlowast119875 lt 0001 was considered statistically significant

we orally administered different concentrations of AC extractto flies from egg until the adult stage We found that ACextract demonstrated no cytotoxicity at doses of 10 (wv)(data not shown) therefore we used this concentration forthe experiments described in this paper

In order to investigate the effect of AC extract on theDrosophila gut following metal ion ingestion we first exam-ined whether AC extract affected survival We observed thatthe survival rate of flies fed with AC extract was significantlygreater compared with the control group following six daysof metal ion ingestion The survival rates associated with Cuingestion were 744 for the AC extract group and 25 forthe control group (Figure 1(a)) Six days following ingestionof Zn and Al the survival rates were 833 and 778 in theAC extract group and 52 and 24 in the control grouprespectively (Figures 1(b) and 1(c))These results indicate thatAC extract can increase survival in flies that have ingested

metal ions Based on this observation we hypothesized thatAC extract might have a protective effect on metal ion-induced gut injury in Drosophila

32 A cochinchinensis Protects the Gut from Metal Ion-Induced Epithelial Cell Death in Drosophila We furtherexamined our hypothesis that AC has protective effects onmetal ion-induced gut epithelial cell injury by evaluatinggut epithelial cell death After 4 days of ingesting variousmetal ions with or without AC extract we found that Cuand Al feeding was associated with significantly more celldeath compared with Zn feeding (Figure 2) Furthermoreonly very few dead cells were detected in the AC extractgroup comparedwith the control group (Figure 2 red signal)These results demonstrate that AC extract can maintain hosthomeostasis by protecting gut epithelial cells frommetal ion-induced damage


(a)

(b)

(c)

(d)

(e)

(f)

AC

Con

7-AAD

7-AAD

7-AAD

7-AAD

DAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

DAPI

DAPI

DAPI

7-AAD

7-AAD

DAPI

DAPI

AC

AC

Con

Con

CuSO

4Zn

SO4

lowastlowastlowast

lowastlowastlowast

lowastlowast

Con AC

200

150

100

50

0

Nec

rotic

cells

Nec

rotic

cells

Nec

rotic

cells

120

100

80

60

40

20

0

200

150

100

50

0

Con AC

Con AC

KAl(S

O4) 2middot12

H2O

Figure 2 A cochinchinensis protects gut epithelial cells from metal ion-induced cell death (a) The effect of AC extract on Cu-inducedepithelial cell death (b)The effect of AC extract on Zn-induced epithelial cell death (c)The effect of AC extract on Al-induced epithelial celldeath ((d)ndash(f))The comparison of gut necrotic cell quantity between control andAC groupThe number of necrotic cells was quantified usingImageJ software (10ndash15 guts were examined to quantify necrotic cells for each group) 119875 value was calculated by Studentrsquos 119905-test lowastlowastlowast119875 lt 0001lowastlowast

119875 lt 001 Con control flies fed with metal ions without AC for 96 h AC flies fed with metal ions with A cochinchinensis (AC) for 96 h7-AAD 7-amino-actinomycin D dead cells (red signal) DAPI 410158406-diamidino-2-phenylindole nucleus (blue signal) 7-AADDAPI (red andblue signal) Scale bar 100120583m


Table 1 The putative components of AC that mediate intestinal injury protection

UNPD ID Chemical name CAS number CIDUNPD133185 Coniferyl alcohol 32811-40-8|458-35-5 1549095UNPD43533 Nyasol 230292-85-0 6438674UNPD77220 Asparenydiol 166762-98-7 10084256UNPD135865 31015840-Hydroxy-41015840-methoxy-41015840-dehydroxynyasol NA 21575014UNPD68648 310158401015840-Methoxyasparenydiol NA NAUNPD96599 310158401015840-Methoxynyasol NA 25218067

ns

ns

Cu

Con AC Con AC Con AC

Zn Al

lowastlowastlowast

lowastlowastlowast

2500

2000

1500

1000

Gut

leng

th (120583

m)

5

sucr

ose

5

sucr

ose +

AC

Figure 3 A cochinchinensis prevents metal ion-induced gut atro-phy Con control flies fed with metal ion without AC for 96 h ACA cochinchinensis flies fed with metal ion with AC for 96 h Allexperiments were independently performed three times lowastlowastlowast119875 lt0001 ns no significant difference

33 A cochinchinensis Protects the Gut from Metal Ions-Induced Morphological Changes in Drosophila A large num-ber of reactive oxygen species (ROS) were rapidly producedafter feeding some toxic compounds [21 22] Howeverexcessive ROS can damage the host intestinal epithelial cellsStudy has shown that cadmium could change membranepermeability through inhibition of superoxide dismutaseactivity and result in necrotic organelles [27] A previousstudy has shown that an increase in gut epithelial cell deathassociated with morphological changes in Drosophila [28]therefore we examined the gut morphology of flies thathad been fed with metal ions Four days after induction weobserved that the length of the adult gut from the controlgroups was significantly shorter than that of the group fedwith 5 (wv) sucrose (Figure 3) In contrast the length ofACgroups was alleviated compared with control groups whichwere fed with Cu and Al However there was no significantdifference between the control group and the AC groupfollowing Zn feeding (Figure 3) These observations suggestthat AC extract prevents metal ion-induced morphologicalchanges in the adult fly gut and altogether the results fromour study demonstrate that AC extract has a protective effecton metal ion-induced gut injury in Drosophila

34 The Prediction of Potential Targets of A cochinchinensisNatural Products Based on our observations we hypoth-esized that AC has potential implications for the discov-ery of new intestinal anti-inflammatory drugs To test thishypothesis we obtained 29 natural products (NPs) derivedfrom AC from the UNPD Next we screened out 19 ther-apeutic proteins targeted by FDA-approved intestinal anti-inflammatory agents from DrugBank Finally using Phar-mMapper server we found that 19 of the 29 NPs werepredicted to bind to 3 of the 19 proteins targeted by FDA-approved intestinal anti-inflammatory agents (Supplemen-tary Table S1 in Supplementary Material available onlineat httpdxdoiorg10115520167603746) These three tar-gets were corticosteroid 11-beta-dehydrogenase isozyme 1(11-DH) glucocorticoid receptor (GR) and peroxisomeproliferator-activated receptor gamma (PPAR120574) PharmMap-per server is a web server for potential drug target identifica-tion using pharmacophore mapping approach [25 29 30]

Cytoscape software is a popular bioinformatics packagefor biological network visualization and data integration [2631] According to the docking results we constructed a NPand target network using theCytoscape 321 network analysissoftwareThe target protein was expressed by a node and theedges represented the relationship between NPs and targets(Figure 4) Using this network we found that 18 NPs targeted11-DH 16 NPs targeted GR and 7 NPs targeted PPAR120574 Thisresult indicates that ACmight serve as a useful newmedicinefor treating intestinal injury

35The Putative Components of A cochinchinensisThatMedi-ate Protection from Intestinal Injury Oral administrationa simple low-cost option that does not directly damagemucous membranes has become the most commonly usedmode of drug administration Our results demonstrated that7 A cochinchinensis NPs are predicted to bind three pro-teins targeted by FDA-approved intestinal anti-inflammatoryagents simultaneously (Figure 4) We narrowed down the listof 7 candidates to 6 NPs by applying Lipinskirsquos rule of five(Table 1) and considered these to be the putative componentsthat mediate the protective effect of AC on intestinal injury

36 The Drug-Likeness of the 6 NPs Molecular descriptorshave been extensively used in cheminformatics research andthe pharmaceutical industry for molecular clustering [32] Inorder to explore the drug-likeness of the 6 NPs screened outfrom A cochinchinensis (AC-NPs) we gathered informationon 11 FDA-approved intestinal anti-inflammatory agents


UNPD174302

UNPD206348

UNPD143968

UNPD206349

UNPD136991

GR 11-DH

UNPD135865

UNPD77220 UNPD96599

UNPD60722

UNPD96816

UNPD27847

UNPD43533

UNPD36432

UNPD68648

UNPD14125

UNPD130293

UNPD126821

UNPD133185

UNPD11158

PPAR120574

Figure 4 The NP-target network The target protein was expressed by a node and edges represented the relationship between NPs and thetarget protein 11-DH corticosteroid 11-beta-dehydrogenase isozyme 1 GR glucocorticoid receptor PPAR-gamma peroxisome proliferator-activated receptor gamma UNPD ID Universal Natural Products Database identification

from DrugBank (Supplementary Table S2) and calculatedthe following six descriptors molecular weight ALog119875number of hydrogen bond receptors (H Acceptors) numberof hydrogen bond donors (H Donors) number of rotatablebonds (Rotatable Bonds) and number of rings (Rings)(Supplementary Tables S2 and S3) Using this informationwe drew scatter plots to facilitate analysis of drug-likenessAs shown in Figure 5 the mean values of the descriptors ofAC-NPs tended to be smaller than those of FDA-approveddrugs Nevertheless ALog119875 of AC-NPs was larger than thatof approved drugs The mean value of ALog119875 was 36 plusmn 11for AC-NPs and 23 plusmn 08 for the FDA-approved drugs Thenumbers of H Acceptors and H Donors of approved drugswere primarily 5 and 3 but they were 3 and 2 for AC-NPs The mean number of rotatable bonds for AC-NPs wasslightly larger than those of the approved drugs and thisamounted to a significant difference Most of the approveddrugs had approximately 2 rotatable bonds while the AC-NPs had approximately 5 These results indicate that the 6AC-NPs have good drug-likeness and could potentially beused as intestinal anti-inflammatory drugs

4 Discussion

A cochinchinensis is primarily used in obstetrics and gynecol-ogy otolaryngology and ophthalmology Its use in the clinicalsetting has revealed its remarkable antiaging antitumor andantiproliferative effects [33ndash35] Previous studies have shownthat AC ethanol extract might have therapeutic potential inimmune-related cutaneous diseases [36] However the effectof AC aqueous extract on gut injury has not been previouslycharacterized In this study AC significantly amelioratedepithelial cell damage in adult flies following chronic metalion feeding (Figure 1) Due to the high level of conservationbetween Drosophila and mammalian intestinal properties[18] our results provide a theoretical basis for exploring thepotential use of AC for the clinical treatment of inflammatorybowel disease

Reverse docking of a small molecule compound (naturalproducts lead compounds and chemicals) to a probe is anapproachused to predict potential drug targets It is an impor-tant tool for drug research and is indispensable for drivingthe modernization of new drug discovery In this studyusing the PharmMapper server we observed AC-NP dockingwith targets that participate in several signaling pathwaysincluding those associated with cancer lipid metabolismneurodegenerative diseases the primary immunodeficiencypathway and nitrogen metabolism Moreover some NPsdemonstrated docking with intestinal anti-inflammatory tar-gets These observations support the hypothesis that ACexerts protective effects on metal ion-induced gut injury inDrosophila

PPAR120574 belongs to a subfamily of the nuclear hor-mone receptor superfamily of ligand-inducible transcrip-tion factors [37] PPAR120574 regulates genes related to lipidmetabolism as well as genes associated with immunityand inflammation [38ndash41] In our study we observedUNPD126821 UNPD133185 UNPD135865 UNPD43533UNPD68648 UNPD77220 and UNPD96599 docking withPPAR120574 (Figure 4) This result further supported the hypoth-esis that AC has a protective effect on metal ion-inducedgut injury in Drosophila Moreover this result provided apotential mechanism by which AC mediates its protectiveeffects on gut injury Altogether our results suggest that ACextracts may employ similar pharmacological mechanisms aswestern medicines to prevent gut injury

Nyasol was isolated from A cochinchinensis many yearsago but there are few reports on its pharmacological proper-ties [42] Nyasol has demonstrated anti-inflammatory effectsin LPS-activated BV-2 microglial cells [43] In our study wefound that UNPD43533 (Nyasol) targeted 11-DH GR andPPAR120574 (Figure 4) This result demonstrates that AC has thepotential to be used as an intestinal anti-inflammatory drugThe molecular descriptors of Nyasol were consistent withLipinskirsquos rules of five (Supplementary Table S3) indicatingthat Nyasol has good drug-likeness and is not likely to be


Approved drugsMolecular weight

AC-NPs

200

300

400

500

Mol

ecul

ar w

eigh

t

(a)

Approved drugs AC-NPs0

1

2

3

4

5

Num

ber

ALog P

(b)

Approved drugsH Acceptors

AC-NPs0

2

4

6

8

10

12

Num

ber

(c)

Approved drugsH Donors

AC-NPs0

1

2

3

4

5

Num

ber

(d)

Approved drugsRotatable Bonds

AC-NPs0

2

4

6

8

10

Num

ber

(e)

Approved drugsRings

AC-NPs0

1

2

3

4

5

6

Num

ber

(f)

Figure 5 Distribution of the six molecular descriptors of AC-NPs and FDA-approved drugs H Acceptors number of hydrogen bondreceptors H Donors number of hydrogen bond donors Rotatable Bonds number of rotatable bonds Rings number of rings Approveddrugs FDA-approved drugs AC-NPs A cochinchinensis natural products

associated with absorption problems Therefore Nyasol haspotential implications in intestinal anti-inflammatory drugdiscovery In addition we found that Nyasol docked withestradiol 17-beta-dehydrogenase 1 (HSD17B1) the insulinreceptor and other molecules HSD17B1 is known to be

involved in lipid transport and metabolism and the insulinreceptor is a key regulator of the insulin signaling pathwayTherefore we predict that Nyasol may also have potentialas an antidiabetic agent however this hypothesis requiresfurther experimental verification


5 Conclusion

Our results demonstrate that A cochinchinensis aqueousextract exerts a protective effect on metal ion-induced gutinjury in Drosophila In addition we screened out six con-stituents of A cochinchinensis that could potentially mediatethis effect In addition these NPs are associated with gooddrug-likeness Further studies will be required to delineatethe pharmacological properties associated with each of theputative active components ofA cochinchinensis and to deter-mine the mechanism by which A cochinchinensis mediatesits protective effects in metal ion-induced gut injury Takentogether our findings provide a basis to support the poten-tial use of A cochinchinensis for intestinal inflammationMoreover our studies provide helpful information and newinsights into support of the application of TCM-derivednatural products to drug discovery and development

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Fundamental ResearchFunds for the Central Universities (DL13EA08-01)

References

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[2] K Matus K-M Nam N E Selin L N Lamsal J M Reilly andS Paltsev ldquoHealth damages from air pollution in ChinardquoGlobalEnvironmental Change vol 22 no 1 pp 55ndash66 2012

[3] K T Palmer R McNeill-Love J R Poole et al ldquoInflammatoryresponses to the occupational inhalation of metal fumerdquo Euro-pean Respiratory Journal vol 27 no 2 pp 366ndash373 2006

[4] L Zhefeng C Qiongjie F Yuqi et al ldquoResearch on effects ofcadmium induced intestinal epithelial cell injury and regulationon intestinal stem cells regeneration and differentiation inDrosophilamid-gutrdquo Chinese Journal of Cell Biology vol 35 no5 pp 602ndash608 2013

[5] N Buchon N A Broderick S Chakrabarti and B LemaitreldquoInvasive and indigenousmicrobiota impact intestinal stem cellactivity through multiple pathways in Drosophilardquo Genes andDevelopment vol 23 no 19 pp 2333ndash2344 2009

[6] J Royet ldquoEpithelial homeostasis and the underlying molec-ular mechanisms in the gut of the insect model Drosophilamelanogasterrdquo Cellular and Molecular Life Sciences vol 68 no22 pp 3651ndash3660 2011

[7] W S Garrett J I Gordon and L H Glimcher ldquoHomeostasisand inflammation in the intestinerdquo Cell vol 140 no 6 pp 859ndash870 2010

[8] N B Samad T Debnath A Hasnat et al ldquoPhenolic con-tents antioxidant and anti-inflammatory activities ofAsparaguscochinchinensis (Loureiro) Merrillrdquo Journal of Food Biochem-istry vol 38 no 1 pp 83ndash91 2014

[9] M Park M S Cheon S H Kim et al ldquoAnticancer activity ofAsparagus cochinchinensis extract and fractions inHepG2 cellsrdquo

Journal of Applied Biological Chemistry vol 54 no 2 pp 188ndash193 2011

[10] J M Chun M S Cheon B C Moon A Y Lee B KChoo and H K Kim ldquoAnti-tumor activity of the ethyl acetatefraction fromAsparagus cochinchinensis in HepG2-xenograftednude micerdquo Journal of the Korean Society for Applied BiologicalChemistry vol 54 no 4 pp 538ndash543 2011

[11] T E Lloyd and J P Taylor ldquoFlightless flies Drosophila modelsof neuromuscular diseaserdquo Annals of the New York Academy ofSciences vol 1184 pp E1ndashE20 2010

[12] M S Dionne andD S Schneider ldquoModels of infectious diseasesin the fruit fly Drosophila melanogasterrdquo Disease Models andMechanisms vol 1 no 1 pp 43ndash49 2008

[13] M Vidal and R L Cagan ldquoDrosophila models for cancerresearchrdquoCurrent Opinion in Genetics and Development vol 16no 1 pp 10ndash16 2006

[14] M E Fortini andNM Bonini ldquoModeling human neurodegen-erative diseases in Drosophila on a wing and a prayerrdquo Trendsin Genetics vol 16 no 4 pp 161ndash167 2000

[15] P Leopold andN Perrimon ldquoDrosophila and the genetics of theinternal milieurdquo Nature vol 450 no 7167 pp 186ndash188 2007

[16] C Pitsouli Y Apidianakis and N Perrimon ldquoHomeostasis ininfected epithelia stem cells take the leadrdquo Cell Host ampMicrobevol 6 no 4 pp 301ndash307 2009

[17] D C Rubin ldquoIntestinal morphogenesisrdquo Current Opinion inGastroenterology vol 23 no 2 pp 111ndash114 2007

[18] Y Apidianakis and L G Rahme ldquoDrosophila melanogaster asa model for human intestinal infection and pathologyrdquo DiseaseModels and Mechanisms vol 4 no 1 pp 21ndash30 2011

[19] M Gladstone and T T Su ldquoChemical genetics and drugscreening inDrosophila cancer modelsrdquo Journal of Genetics andGenomics vol 38 no 10 pp 497ndash504 2011

[20] C Gonzalez ldquoDrosophila melanogaster a model and a tool toinvestigate malignancy and identify new therapeuticsrdquo NatureReviews Cancer vol 13 no 3 pp 172ndash183 2013

[21] W Li Q Luo and L H Jin ldquoAcanthopanax senticosus extractshave a protective effect on Drosophila gut immunityrdquo Journal ofEthnopharmacology vol 146 no 1 pp 257ndash263 2013

[22] C Zhu F Guan C Wang and L H Jin ldquoThe protective effectsof Rhodiola crenulata extracts on Drosophila melanogaster gutimmunity induced by bacteria and SDS toxicityrdquo PhytotherapyResearch vol 28 no 12 pp 1861ndash1866 2014

[23] J Gu Y Gui L Chen G Yuan H-Z Lu and X Xu ldquoUse ofnatural products as chemical library for drug discovery andnetwork pharmacologyrdquo PLoS ONE vol 8 no 4 Article IDe62839 2013

[24] C Knox V Law T Jewison et al ldquoDrugBank 30 a compre-hensive resource for ldquoOmicsrdquo research on drugsrdquo Nucleic AcidsResearch vol 39 no 1 pp D1035ndashD1041 2011

[25] X Liu S Ouyang B Yu et al ldquoPharmMapper server a webserver for potential drug target identification using pharma-cophore mapping approachrdquoNucleic Acids Research vol 38 no2 pp W609ndashW614 2010

[26] P Shannon A Markiel O Ozier et al ldquoCytoscape a softwareEnvironment for integratedmodels of biomolecular interactionnetworksrdquo Genome Research vol 13 no 11 pp 2498ndash25042003

[27] N Buchon N A Broderick M Poidevin S Pradervand and BLemaitre ldquoDrosophila intestinal response to bacterial infectionactivation of host defense and stem cell proliferationrdquo Cell Hostamp Microbe vol 5 no 2 pp 200ndash211 2009


[28] S Gupta M Athar J R Behari and R C SrivastavaldquoCadmium-mediated induction of cellular defence mechanisma novel example for the development of adaptive responseagainst a toxicantrdquo Industrial Health vol 29 no 1 pp 1ndash9 1991

[29] X Li X Xu J Wang et al ldquoA system-level investigation intothe mechanisms of chinese traditional medicine compounddanshen formula for cardiovascular disease treatmentrdquo PLoSONE vol 7 no 9 Article ID e43918 2012

[30] T Liu D Lu H Zhang et al ldquoApplying high-performance com-puting in drug discovery and molecular simulationrdquo NationalScience Review vol 3 no 1 pp 49ndash63 2016

[31] X Liu Z Hu B Zhou X Li and R Tao ldquoChinese herbal prepa-ration xuebijing potently inhibits inflammasome activation inhepatocytes and ameliorates mouse liver ischemia-reperfusioninjuryrdquo PLoS ONE vol 10 no 7 Article ID e0131436 2015

[32] R Todeschini andVConsonniHandbook ofMolecularDescrip-tors John Wiley amp Sons New York NY USA 2008

[33] J Li X Liu M Guo Y Liu S Liu and S Yao ldquoElectrochemicalstudy of breast cancer cells MCF-7 and its application inevaluating the effect of diosgeninrdquo Analytical Sciences vol 21no 5 pp 561ndash564 2005

[34] C Corbiere B Liagre A Bianchi et al ldquoDifferent contributionof apoptosis to the antiproliferative effects of diosgenin andother plant steroids hecogenin and tigogenin on human 1547osteosarcoma cellsrdquo International Journal of Oncology vol 22no 4 pp 899ndash905 2003

[35] B Liagre J Bertrand D Y Leger and J-L Beneytout ldquoDios-genin a plant steroid induces apoptosis in COX-2 deficientK562 cells with activation of the p38MAP kinase signalling andinhibition ofNF-120581B bindingrdquo International Journal ofMolecularMedicine vol 16 no 6 pp 1095ndash1101 2005

[36] D Y Lee B K Choo T Yoon et al ldquoAnti-inflammatoryeffects ofAsparagus cochinchinensis extract in acute and chroniccutaneous inflammationrdquo Journal of Ethnopharmacology vol121 no 1 pp 28ndash34 2009

[37] L Wang B Waltenberger E-M Pferschy-Wenzig et al ldquoNatu-ral product agonists of peroxisome proliferator-activated recep-tor gamma (PPAR120574) a reviewrdquo Biochemical Pharmacology vol92 no 1 pp 73ndash89 2014

[38] I Szatmari E Rajnavolgyi and L Nagy ldquoPPAR120574 a lipid-activated transcription factor as a regulator of dendritic cellfunctionrdquoAnnals of the NewYork Academy of Sciences vol 1088pp 207ndash218 2006

[39] L Szeles D Torocsik and L Nagy ldquoPPAR120574 in immunity andinflammation cell types and diseasesrdquo Biochimica et BiophysicaActa-Molecular and Cell Biology of Lipids vol 1771 no 8 pp1014ndash1030 2007

[40] WHuang andCKGlass ldquoNuclear receptors and inflammationcontrol molecular mechanisms and pathophysiological rele-vancerdquo Arteriosclerosis Thrombosis and Vascular Biology vol30 no 8 pp 1542ndash1549 2010

[41] C K Glass and K Saijo ldquoNuclear receptor transrepressionpathways that regulate inflammation in macrophages and Tcellsrdquo Nature Reviews Immunology vol 10 no 5 pp 365ndash3762010

[42] W-Y Tsui and G D Brown ldquo(+)-Nyasol from Asparaguscochinchinensisrdquo Phytochemistry vol 43 no 6 pp 1413ndash14151996

[43] H J Lee H Li H R Chang H Jung D Y Lee and J-H Ryu ldquo(minus)-Nyasol isolated from Anemarrhena asphodeloidessuppresses neuroinflammatory response through the inhibition

of I-120581B120572 degradation in LPS-stimulated BV-2 microglial cellsrdquoJournal of Enzyme Inhibition and Medicinal Chemistry vol 28no 5 pp 954ndash959 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


However the protective effect on intestinal injury and theactive components of A cochinchinensis affecting gut immu-nity remain poorly understood

Drosophila is a well studied and highly tractable geneticmodel organism Many basic biological and physiologicalproperties are conserved between Drosophila and mammalsand nearly 75 of human disease-related genes have a func-tional homolog in the fly [11] Thus the Drosophila is widelyused in basic and applied researches on a broad spectrumof human diseases including infectious diseases [12] cancers[13] neurodegenerative diseases [14] and metabolic diseases[15] Drosophila and human intestine have similar anatomyand physiological function [16 17] and they have alsosimilarities in cell and composition and underlying signalingpathways that maintain intestinal homeostasis [18]

Chinese medicinal herbs exert their therapeutic actionsthrough the synergistic effects of multiple compounds tar-gets and channels However it has been difficult to isolatethe effective components of these products and to identifyspecific therapeutic targets for treating disease Networkpharmacology has been used as an integrated approachto systematically investigate and explain the underlyingmolecular mechanisms of Chinese medicinal herbs Usingthe Computerized Virtual Screening Technique to explorepotential targetsmay help facilitate these investigations whilereducing manpower and material resources

Drosophila has emerged as a potential whole animalmodel for drug screening [19 20]Through a large number ofsurvival rate assays we identifiedA cochinchinensis as havinggood bioactivity against chemical reagents-induced stress inDrosophila (data was not shown) In this study we revealedthat the aqueous extract of A cochinchinensis exerts a protec-tive effect on gut injury in Drosophila induced by the chronicexposure to metal ions In addition we computationallyidentified the putative active ingredients ofA cochinchinensisusing network pharmacology Network analysis revealedsix constituents of A cochinchinensis that could potentiallymediate its protective effects on gut injury These results pro-vide new insight into the pharmacological basis of the antigutinjury activity of A cochinchinensis and will provide impetusfor preclinical drug discovery based on this medicinalplant

2 Materials and Methods

21 Drosophila Fly Stocks w1118 flies were obtained fromthe Bloomington Drosophila Stock Center Fly stocks weremaintained on a 12 h light12 h dark cycle at 25∘C and 60humidity

22 A cochinchinensis Extraction A cochinchinensis wasidentified by Professor XiuhuaWang at the Herbarium of theCollege of Life Sciences Northeast Forestry University andpurchased from Shiyitang Pharmacy of Harbin China Themethod of aqueous extraction of A cochinchinensis has beenpreviously described [21] Total aqueous-derived extract wasconsolidated and concentrated to a final concentration of 20(wv)

23 Drosophila Food Standard cornmeal-yeastmediumusedfor the control group consisted of 56 gL agar 168 gL yeast716 gL polenta 98 gL soybean flour and 60 gL sucroseStandard mediumwith 10 (wv) aqueousA cochinchinensisextract was used for the experimental group

24 Survival Experiments Procedures for the survival andfeeding experiments were performed as previously described[21] with the difference that adult flies which aged 3ndash5days (15 males and 15 females) were starved for 2 h withoutfood before being transferred to a vial containing chemicalcompounds in 5 (wv) sucrose solution serving as thecontrol group for all experiments The chemical compoundsincluded cupric sulfate (7mM Sigma) zinc sulfate (7mMSigma) and aluminum potassium disulfate dodecahydrate(20mM Sigma) The experimental group consists of flies fedwith sucrose solutions incorporating the chemical compoundwith added AC extract at 10 (ww) Fresh filter papersand solutions were provided every day and dead flies wereenumerated and evaluated daily

25 7-Amino-actinomycinD Staining and Imaging 7-Amino-actinomycin D (7-AAD) staining and imaging were per-formed as previously described [21 22]

26 Intestinal Morphological Analysis Female flies were usedfor intestinal morphological studies due to their larger size

Due to the bigger size of female flies we used them forintestinal morphological analysis experiment The guts of 3ndash5-day-old female flies that had orally ingested metal ions in5 (wv) sucrose with or without AC extract for 96 h weredissected at room temperature in phosphate buffered saline(PBS) and immediately observed under an Axioskop 2 plusmicroscope (Zeiss)

27 Targets Predictions of Natural Products (NPs) and Net-work Construction The information pertaining to naturalproducts (NPs) was obtained from the Universal NaturalProducts Database (UNPD) according to the scientific namesof the herbs [23] The information for approved drugs anddrug targets was obtained from DrugBank [24] We used thePharmMapper server to predict potential drug targets [25]The network was constructed by Cytoscape 321 [26]

28 Statistics For all experiments data are representative ofthree independent experiments Statistical analyses includ-ing Studentrsquos t-test the one-way ANOVA and log-rank(Mantel-Cox) test were performed using GraphPad Prism50 software (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 ns nosignificant difference)

3 Results

31 A cochinchinensis Increased Survival following the Inges-tion of Metal Ions As water decoction is the traditional for-mulation used in Chinese clinical medicine we used aqueousextraction in this study To assess the effect of A cochinchi-nensis (AC) extract on the development of Drosophila larvae


Surv

ival

()

100

80

60

40

20

076543210

Day

ConAC

CuSO4

lowastlowastlowast

(a)

Surv

ival

()

100

80

60

40

20

0

76543210

Day

ConAC

ZnSO4

lowastlowastlowast

(b)

Surv

ival

()

100

80

60

40

20

076543210

Day

ConAC

lowastlowastlowast


(c)


4



4

)2

sdot12H2







(a)

(b)

(c)

(d)

(e)

(f)

AC

Con

7-AAD

7-AAD

7-AAD

7-AAD

DAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

DAPI

DAPI

DAPI

7-AAD

7-AAD

DAPI

DAPI

AC

AC

Con

Con

CuSO

4Zn

SO4

lowastlowastlowast

lowastlowastlowast

lowastlowast

Con AC

200

150

100

50

0

Nec

rotic

cells

Nec

rotic

cells

Nec

rotic

cells

120

100

80

60

40

20

0

200

150

100

50

0

Con AC

Con AC

KAl(S

O4) 2middot12

H2O






ns

ns

Cu


Zn Al

lowastlowastlowast

lowastlowastlowast

2500

2000

1500

1000

Gut

leng

th (120583

m)

5

sucr

ose

5

sucr

ose +

AC








UNPD174302

UNPD206348

UNPD143968

UNPD206349

UNPD136991

GR 11-DH

UNPD135865

UNPD77220 UNPD96599

UNPD60722

UNPD96816

UNPD27847

UNPD43533

UNPD36432

UNPD68648

UNPD14125

UNPD130293

UNPD126821

UNPD133185

UNPD11158

PPAR120574



4 Discussion







AC-NPs

200

300

400

500

Mol

ecul

ar w

eigh

t

(a)


1

2

3

4

5

Num

ber

ALog P

(b)


AC-NPs0

2

4

6

8

10

12

Num

ber

(c)


AC-NPs0

1

2

3

4

5

Num

ber

(d)


AC-NPs0

2

4

6

8

10

Num

ber

(e)

Approved drugsRings

AC-NPs0

1

2

3

4

5

6

Num

ber

(f)





5 Conclusion


Competing Interests


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Surv

ival

()

100

80

60

40

20

076543210

Day

ConAC

CuSO4

lowastlowastlowast

(a)

Surv

ival

()

100

80

60

40

20

0

76543210

Day

ConAC

ZnSO4

lowastlowastlowast

(b)

Surv

ival

()

100

80

60

40

20

076543210

Day

ConAC

lowastlowastlowast


(c)


4



4

)2

sdot12H2







(a)

(b)

(c)

(d)

(e)

(f)

AC

Con

7-AAD

7-AAD

7-AAD

7-AAD

DAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

DAPI

DAPI

DAPI

7-AAD

7-AAD

DAPI

DAPI

AC

AC

Con

Con

CuSO

4Zn

SO4

lowastlowastlowast

lowastlowastlowast

lowastlowast

Con AC

200

150

100

50

0

Nec

rotic

cells

Nec

rotic

cells

Nec

rotic

cells

120

100

80

60

40

20

0

200

150

100

50

0

Con AC

Con AC

KAl(S

O4) 2middot12

H2O






ns

ns

Cu


Zn Al

lowastlowastlowast

lowastlowastlowast

2500

2000

1500

1000

Gut

leng

th (120583

m)

5

sucr

ose

5

sucr

ose +

AC








UNPD174302

UNPD206348

UNPD143968

UNPD206349

UNPD136991

GR 11-DH

UNPD135865

UNPD77220 UNPD96599

UNPD60722

UNPD96816

UNPD27847

UNPD43533

UNPD36432

UNPD68648

UNPD14125

UNPD130293

UNPD126821

UNPD133185

UNPD11158

PPAR120574



4 Discussion







AC-NPs

200

300

400

500

Mol

ecul

ar w

eigh

t

(a)


1

2

3

4

5

Num

ber

ALog P

(b)


AC-NPs0

2

4

6

8

10

12

Num

ber

(c)


AC-NPs0

1

2

3

4

5

Num

ber

(d)


AC-NPs0

2

4

6

8

10

Num

ber

(e)

Approved drugsRings

AC-NPs0

1

2

3

4

5

6

Num

ber

(f)





5 Conclusion


Competing Interests


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

(b)

(c)

(d)

(e)

(f)

AC

Con

7-AAD

7-AAD

7-AAD

7-AAD

DAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

7-AADDAPI

DAPI

DAPI

DAPI

7-AAD

7-AAD

DAPI

DAPI

AC

AC

Con

Con

CuSO

4Zn

SO4

lowastlowastlowast

lowastlowastlowast

lowastlowast

Con AC

200

150

100

50

0

Nec

rotic

cells

Nec

rotic

cells

Nec

rotic

cells

120

100

80

60

40

20

0

200

150

100

50

0

Con AC

Con AC

KAl(S

O4) 2middot12

H2O






ns

ns

Cu


Zn Al

lowastlowastlowast

lowastlowastlowast

2500

2000

1500

1000

Gut

leng

th (120583

m)

5

sucr

ose

5

sucr

ose +

AC








UNPD174302

UNPD206348

UNPD143968

UNPD206349

UNPD136991

GR 11-DH

UNPD135865

UNPD77220 UNPD96599

UNPD60722

UNPD96816

UNPD27847

UNPD43533

UNPD36432

UNPD68648

UNPD14125

UNPD130293

UNPD126821

UNPD133185

UNPD11158

PPAR120574



4 Discussion







AC-NPs

200

300

400

500

Mol

ecul

ar w

eigh

t

(a)


1

2

3

4

5

Num

ber

ALog P

(b)


AC-NPs0

2

4

6

8

10

12

Num

ber

(c)


AC-NPs0

1

2

3

4

5

Num

ber

(d)


AC-NPs0

2

4

6

8

10

Num

ber

(e)

Approved drugsRings

AC-NPs0

1

2

3

4

5

6

Num

ber

(f)





5 Conclusion


Competing Interests


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















ns

ns

Cu


Zn Al

lowastlowastlowast

lowastlowastlowast

2500

2000

1500

1000

Gut

leng

th (120583

m)

5

sucr

ose

5

sucr

ose +

AC








UNPD174302

UNPD206348

UNPD143968

UNPD206349

UNPD136991

GR 11-DH

UNPD135865

UNPD77220 UNPD96599

UNPD60722

UNPD96816

UNPD27847

UNPD43533

UNPD36432

UNPD68648

UNPD14125

UNPD130293

UNPD126821

UNPD133185

UNPD11158

PPAR120574



4 Discussion







AC-NPs

200

300

400

500

Mol

ecul

ar w

eigh

t

(a)


1

2

3

4

5

Num

ber

ALog P

(b)


AC-NPs0

2

4

6

8

10

12

Num

ber

(c)


AC-NPs0

1

2

3

4

5

Num

ber

(d)


AC-NPs0

2

4

6

8

10

Num

ber

(e)

Approved drugsRings

AC-NPs0

1

2

3

4

5

6

Num

ber

(f)





5 Conclusion


Competing Interests


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















UNPD174302

UNPD206348

UNPD143968

UNPD206349

UNPD136991

GR 11-DH

UNPD135865

UNPD77220 UNPD96599

UNPD60722

UNPD96816

UNPD27847

UNPD43533

UNPD36432

UNPD68648

UNPD14125

UNPD130293

UNPD126821

UNPD133185

UNPD11158

PPAR120574



4 Discussion







AC-NPs

200

300

400

500

Mol

ecul

ar w

eigh

t

(a)


1

2

3

4

5

Num

ber

ALog P

(b)


AC-NPs0

2

4

6

8

10

12

Num

ber

(c)


AC-NPs0

1

2

3

4

5

Num

ber

(d)


AC-NPs0

2

4

6

8

10

Num

ber

(e)

Approved drugsRings

AC-NPs0

1

2

3

4

5

6

Num

ber

(f)





5 Conclusion


Competing Interests


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















AC-NPs

200

300

400

500

Mol

ecul

ar w

eigh

t

(a)


1

2

3

4

5

Num

ber

ALog P

(b)


AC-NPs0

2

4

6

8

10

12

Num

ber

(c)


AC-NPs0

1

2

3

4

5

Num

ber

(d)


AC-NPs0

2

4

6

8

10

Num

ber

(e)

Approved drugsRings

AC-NPs0

1

2

3

4

5

6

Num

ber

(f)





5 Conclusion


Competing Interests


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















5 Conclusion


Competing Interests


Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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