Research ArticleEffects of Total Ginsenosides on the Feeding Behavior and TwoEnzymes Activities of Mythimna separata (Walker) Larvae

Ai-Hua Zhang1 Shi-Qiang Tan12 Yan Zhao1 Feng-Jie Lei1 and Lian-Xue Zhang1

1College of Chinese Medicinal Materials Jilin Agricultural University Changchun 130118 China2Tianjin University of Traditional Chinese Medicine Tianjin 300193 China

Correspondence should be addressed to Ai-Hua Zhang fengjie lei163com and Lian-Xue Zhang zlx863163com

Received 30 May 2014 Revised 26 August 2014 Accepted 24 September 2014

Academic Editor Shilin Chen

Copyright copy 2015 Ai-Hua Zhang 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

Ginsenosides the main effective components of Panax ginseng CA Meyer and Panax quinquefolius L are importantallelochemicals of ginseng Although many studies have targeted the pharmacological chemical and clinical properties ofginsenosides little is known about their ecological role in ginseng population adaptation and evolution Pests rarely feed onginseng and it is not known why This study investigated the effects of total ginsenosides on feeding behavior and activities ofacetylcholinesterase (AChE) and glutathione s-transferase (GST) in Mythimna separata (Walker) larvae The results showed thatthe total ginsenosides had significant antifeeding activity against M separata larvae determined by nonselective and selectiveantifeeding bioassays In addition the total ginsenosides had inhibitory effects on the activities of GST and AChEThe antifeedingratio was the highest at 8 h then decreased and was the lowest at 16 h Both GST and AChE activities decreased from 0 h to 48 hin all total ginsenosides treatments but increased at 72 h Total ginsenosides had antifeeding activity againstM separata larvae andinhibitory effects on the activities of GST and AChE

1 Introduction

In the long process of adaptation to the environment plantshave developed a chemical response to stress called allelopa-thy the release of allelochemicals [1 2] Terpenoids are a typeof allelochemical and play an important role in regulatingplant populations and ecological systems [3 4]

Ligularia virgaurea secretes terpenoids that inhibit seedgermination of other species [5 6] Duranta repens extractshave been shown to inhibit oviposition feeding and develop-ment of Plutella xylostella [7 8] Terpenoids from Meliaceaeplants have shown insecticidal action against Pieris occiden-talis and Leucania separata [9ndash12]

Ginseng (Panax ginseng CA Meyer) is a highly popularand valuable traditional Chinese medicine and tonic Themajor ginseng producers of theworld areChina Korea Japanand Russia [13 14] Ginseng can grow for more than onehundred years but it requires strict ecological conditions thatare limited Wild ginseng populations maintain a specific dis-tance internally between plants [15] Ginseng has triterpene

saponins and few insects eat its stems or leaves Ginsengtriterpene saponins change the soil microbial populationstructure and inhibit ginseng seed and seed germination byother species [13ndash20]

Ginsenosides belong to the group triterpenes and arebecoming a popular topic of research in pharmacologymedicine and clinical drug development in both China andabroad However there is little information as to why ginsengsynthetizes high levels of ginsenosides (contents of morethan 3) and the significance of ginsenosides for the plantrsquosgrowth development and population dynamics Ginsengcontains a wide variety of triterpene saponins (found inmorethan 60 species) which suggests that there are a large numberof metabolic pathways (including highly evolved metabolicpathways) Based on the scarcity of information this studydiscusses the effects of total ginsenosides on the feedingbehavior and two enzyme activities of Mythimna separata(Walker) larvae The results of this study will enable furtherunderstanding of the effect of triterpenes on plant populationadaptation and evolution It is also useful to investigate

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 451828 6 pageshttpdxdoiorg1011552015451828

2 Evidence-Based Complementary and Alternative Medicine

the mechanisms of Chinese herbal medicines and to add tothe reference information for phytochemotaxonomy

2 Materials and Methods

21 Insects Mythimna separata (Walker) adults were col-lected at the test base of Jilin Agricultural University inthe early summer of 2011 The offspring of these adultswere reared on grain sorghum leaves under an LD 16 8 hphotoperiod at 22 plusmn 1∘C with 70ndash80 relative humidity andnever had contact with insecticides [21]

22 Chemicals The total ginsenosides (purity ge 80 UVmethod) were purchased from Jilin Hongjiu Biotech CoLtd AChE and GST reagent kits were purchased from theNanjing Jiancheng Bioengineering Institute A Lowry ProteinAssay Kit was purchased from Beijing Dingguo ChangshengBiotech Co Ltd

23 Bioassay for Feeding Behavior A leaf disc bioassay wasused to testM separata larvae [22 23] The ginsenoside con-centrations were 20 10 and 05 (mass fraction (MF))These concentrations are within the range of ginsenosidelevels normally found in P ginseng One day prior to the assaynewly molted 4th-instar larvae were placed individually intoPetri dishes (9 cm diameter) After starvation for 48 h 6excised grain sorghum leaf discs (Φ = 15mm) soaked insolutions with different concentrations of ginsenosides for30min (control discs received distilled water only) weresupplied to all larvae The area of feeding on the leaveswas measured every 8 h and the corresponding antifeedingactivity was calculated (nonselective and selective antifeedingratios) Each test was repeated three times

24 Enzymes Activity Tests The effects of ginsenosides onlarvae of M separata larvae were tested at concentrations of20 10 and 05 (MF) The ginsenosides were dissolvedwith distilled water Fresh clean grain sorghum leaves werepunched into leaf discs with a puncher and the leaf disc wassoaked in different concentrations of ginsenoside solutionsfor 30min and air-dried prior to use The same volumeof distilled water without ginsenosides was used for thecontrol group Fourth-instar larvae were maintained for 48 hwithout access to food Larvae with the same weights werethen selected and either were allowed to feed on the leafdiscs containing ginsenosides or were placed in the controlgroup The leaves were replaced with the fresh ones every 2days because of the possible degradation of the ginsenosidesHomogenates for the two enzyme activities (glutathione s-transferase (GST) and acetylcholinesterase (AChE) activity)of M separata larvae were collected every 24 h Each test wasrepeated three times

Homogenates of the digestive tract of the larvae wereprepared as follows The digestive tract was dissected fromindividuals of each group and washed with phosphate-buffered saline (pH 70) to remove the gut contents Tothis 5mL of cold PBS was added and the solution wascentrifuged for 20min (4∘C 10000 rmin)The supernatant is

the homogenateThe protein content of the supernatant fluidwas measured using the Lowry Protein Assay Kit stainingmethod [24 25] with bovine serum albumin as the standard

GST activity was assessed according to the methodof Jaclyn and Min Lu [26 27] by measuring GSH con-jugation with 1-chloro-24-dinitrobenzene (CDNB) CDNBactivity changes with the substrate concentration in a linearrelationship A visible-ultraviolet spectrophotometer mea-sured absorption at 412 nm to detect product formation Allreactions took place in 01M potassium phosphate-buffercontaining 1mM GSH and 001ndash3mM CDNB GST activitywas determined according to the following equation

119884 =

OD120572minusOD

120573

OD120575minusOD

120576

times 119862120590times 119873 divide 119879 lowast (119860 times 119862prot) (1)

where 119884 represents GST activity (120583molminmg prot) OD120572

represents the absorbance of the control group OD120573repre-

sents the absorbance of the experiment group OD120575repre-

sents the absorbance of the reference group OD120576represents

the absorbance of the blank group119862120590represents the standard

concentration (20mM) 119873 is the dilution factor of the reac-tion system (6) 119879 is reaction time (10min) 119860 is the samplevolume (mL) and 119862prot represents the protein concentrationof the sample

AChE activity was determined using the method ofEllman et al [28] The enzyme activity was measured byassessing the increase of yellow color produced from thio-choline when it reacts with the dithiobisnitrobenzoate ion Itis based on the coupling of the following reactions

acetylthiochlineenzyme997888997888997888997888997888rarr thiocholine + acetate

thiocholine + dithiobisnitrobenzoate 997888rarr yellow color(2)

The AChE activity was spectrophotometrically measuredat 412 nm (UV-754 Shandonggaomi UV visible-ultravioletspectrophotometer) The enzyme activities were expressedas 120583molminmg protein AChE activity was determinedaccording to the following equation

1198840=OD1minusOD

2

OD3minusOD

4

times 1198620divide 119862prot (3)

In this equation 1198840

represents AChE activity(120583molminmg prot) OD

1represents the absorbance of

the experiment group OD2represents the absorbance of

the control group OD3represents the absorbance of the

reference group OD4represents the absorbance of the blank

group 1198620represents the standard concentration (1mM) and

119862prot represents the protein concentration of the sample

25 Statistical Analyses Nonselective and selective antifeed-ing ratios were calculated according to the following equa-tions

1198841=119878ck minus S119878cktimes 100

1198842=119878ck minus S119878ck + 119878times 100

(4)

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Nonselective antifeeding ratio of different total ginsenoside concentrations on 4th-instarM separata larvae

Concentration oftotal ginsenosides()

8 h 16 h 24 h

Average feedingarea (mm2)

Nonselectiveantifeeding ratio

()

Average feedingarea (mm2)

Nonselectiveantifeeding ratio

()

Average feedingarea (mm2)

Nonselectiveantifeeding ratio

()Control 166467 plusmn 10704 a mdash 177000 plusmn 000 a mdash 177000 plusmn 000 a mdash05 87633 plusmn 33310 b 4736 152967 plusmn 2802 a 1358 144067 plusmn 8980 a 186110 59267 plusmn 22413 b 6440 152400 plusmn 7529 a 1390 140800 plusmn 20127 a 204520 18867 plusmn 8159 c 8867 75767 plusmn 45324 b 5719 48333 plusmn 35860 b 7269Data are presented as the means plusmn SE Means in the same column followed by different letters are significantly different at the level 119875 lt 005 Prior to analysisof variance (SPSS 180) the homogeneity of variance was tested in each statistic test

Table 2 Selective antifeeding ratio of different total ginsenoside concentrations on 4th-instarM separata larvae

Concentration oftotal ginsenosides()

8 h 16 h 24 h

Average feedingarea (mm2)

Selectiveantifeeding ratio

()

Average feedingarea (mm2)

Selectiveantifeeding ratio

()

Average feedingarea (mm2)

Selectiveantifeeding ratio

()05 63833 plusmn 3453 3419 45033 plusmn 4045 3662 51133 plusmn 961 2328Control 130167 plusmn 7702 mdash 97067 plusmn 5278 mdash 82167 plusmn 3900 mdash10 51467 plusmn 8075 4429 47067 plusmn 2450 3200 52733 plusmn 2574 2923Control 133233 plusmn 5150 mdash 91367 plusmn 3347 mdash 96300 plusmn 3610 mdash20 26700 plusmn 9579 6249 28533 plusmn 2346 4618 39100 plusmn 2237 4246Control 115667 plusmn 12738 mdash 77500 plusmn 5048 mdash 96800 plusmn 2406 mdashData are presented as the means plusmn SE Homogeneity of variance was tested in each statistic test

In these equations 1198841

represents the nonselectiveantifeeding ratio () 119884

2represents the selective antifeeding

ratio () 119878ck represents the average feeding area of thecontrol group (mm) and 119878 represents the average feedingarea of the treated group (mm)

The nonselective and selective antifeeding ratios of larvaefed on diets containing different concentrations of ginseno-sides were compared using SPSS Statistical 180 The effectsof ginsenosides on the detoxification enzyme activity of Mseparata larvae were also analyzed using SPSS Statistical180 In all statistical tests 119875 values lt 005 were consideredstatistically significant All data are presented as the means plusmnSEM

3 Results

31 Bioassay for Feeding Behavior The nonselective antifeed-ing ratios of 4th-instar M separata larvae at 8 16 and24 h are shown in Table 1 The total ginsenosides appearedto have significant antifeeding activity against M separatalarvae The nonselective antifeeding ratios at different totalginsenoside concentrations were significantly different fromthose of the controls at 4 8 and 12 h As the total ginsenosideconcentration increased the nonselective antifeeding activitywas enhanced At 8 h the antifeeding ratios of 20 10 and05 (MF) were 8867 6440 and 4736 respectively

The selective antifeeding ratios of the 4th-instarM sepa-rata larvae are shown in Table 2 The 4th-instar M separatalarvae preferred to eat the leaves of the control however they

did consume a small amount of the treated leaves The totalginsenosides had significant inhibitory effect on M separatalarvae and this effect increased with an increase in totalginsenoside concentration

32 Enzymes Activity Tests As the total ginsenoside con-centration increased the activity of GST also increasedGST activities first decreased and then increased with thepassage of time The inhibition ratio was the highest at 48 hwith 7515 46898 and 3186 at 20 10 and 05concentrations respectively (see Figure 2)

The AChE activity of 4th-instar M separata larvae was790546 120583molminmgprot before feeding on 20 (MF) totalginsenosides (see Figure 3) however the AChE activitieswere depressed to 618998 (inhibition ratio 2170) 315114(inhibition ratio 6014) and 350698 (inhibition ratio5564) 120583molminmgprot at 24 h 48 h and 72 h respec-tively (see Figure 4) The ginsenosides inhibited the AChEactivity of 4th-instar M separata larvae and the inhibitioneffect increased with the increase in ginsenoside concentra-tion but did not decrease over time

4 Discussion

Plant secondary metabolic substances are formed via sec-ondary metabolic pathways and play indispensable roles inthe evolution of plants They have repellency antifeedingand toxic effects on phytophagous insects For exampleMomordicin I and II have significant antifeedant activity

4 Evidence-Based Complementary and Alternative Medicine

on second-instar larvae of P xylostella and the antifeedingrates were 8039 and 7409 respectively [8] Plant lectinsalso have repellency antifeeding and toxic effects on phy-tophagous insects such as caterpillars tobacco hornwormcotton leafworm and beetles [29] Azadirachtin causes mor-tality in Nilaparvata lugens (Stal) and destroys its ovarianfollicle epithelial cells [30]

We compared the larval feeding behavior of M separataon a diet containing different concentrations of total gin-senosides (MF 20 10 and 05)The total ginsenosidesimposed adverse effects on the feeding behavior of Mseparata but to different extents (Tables 1 and 2) Comparisonwith control demonstrates that the consumption of leavesby M separata was strongly affected by the total ginseno-sides If consumption is reduced the energy required forphysiological activities would also be reduced Consequentlyresistance is then reduced and physiological metabolismwould not be adequateM separata are sometimes foundnearcultivated ginseng however they rarely feed on the leaves ofginseng which is the reason for choosingM separata for thisexperiment

Total ginsenosides inhibited GST and AChE activitiesof M separata larvae If the GST activity was inhibitedthe detoxification was reduced The M separata changedfeeding behaviors and fed on the grain sorghum leaves thatwere treated with total ginsenosides In this way they couldreduce the damage from outsiders and protect themselves Itis the same for AChE their neurotransmission slows downwhen AChE activity is inhibited and M separata did feedon a small amount The total ginsenosides had antifeedingactivity against M separata larvae and inhibitory effects onthe activities of GST and AChE Glutathione S-transferase(GST) and acetylcholinesterase (AChE) play important rolesin insect metabolism and resistance to insecticides [31]This study demonstrates that after treating M separata withtotal ginsenosides GST activities decreased This indicatesthat total ginsenosides have no toxic effects in vivo andactivates the defense system of insects but could also inhibitGST activity GSTs widely exist in insects and catalyze thenucleophilic reaction of glutathione (GSH) with a varietyof electrophilic compounds GSTs play an important role inexogenous substance biotransformation drug metabolismand protection of the organism against oxidative damage [32]GST activity was inhibited by total ginsenosides and detox-ification was reduced in 4th-instar M separata larvae (seeFigure 1) Thus they suffered damage from toxic substancesand these substances are harmful to living larvae

AChE is an important neurotransmitter enzyme mainlydistributed in the brain and central nervous system [33]AChE is localized at the surface of nerve cells and can decom-pose neurotransmitter acetylcholine which is released fromthe nerve endings and guarantees normal nerve conduction[34] If its activity is inhibited the acetylcholine released inthe synaptic gap cannot be degraded and insects will havetoxicity symptoms and possibly mortality [35] Several essen-tial oils from aromatic plants monoterpenes and naturalproducts have all been shown to be inhibitors of AChE [36]Pulegone-12-epoxide isolated fromaVerbenaceaemedicinal

GST

s act

ivity

(120583m

olm

inm

g pr

ot)

0

20

40

60

80

100

120

a a a

aa

ab

bb

b

c b

b

c

c c

Control 5mgmL 10mgmL 20mgmLConcentration of total ginsenoside

0h24h

48h72h

Figure 1 Effect of total ginsenosides on GST activity in 4th-instar M separata larvae Bars with different letters are significantlydifferent from each other at 119875 lt 005 using the one-way ANOVA(SPSS 180) Data are means + SD

Concentration of total ginsenosides (mgmL)

80

60

40

20

0

Inhi

bitio

n ra

tio (

)

24h48h

72h

5 10 20

Figure 2 Inhibition ratio of total ginsenosides on GST activity in4th-instarM separata larvae

AChE

activ

ity (120583

mol

min

mg

prot

)

0

50

100

150

200

250

300

aa

a a

a

b

b b

b b

d

d c

c

c c

Control 5mgmL 10mgmL 20mgmLConcentration of total ginsenosides

0h24h

48h72h

Figure 3 Effect of total ginsenosides on AChE activity in 4th-instarM separata larvae Bars with different letters are significantlydifferent from each other at 119875 lt 005 using a one-way ANOVA(SPSS 180) Data are means + SD

plant (Lippia stoechadifolia L (Poleo)) showed an irreversibleinhibition of the AChE in house fly and Madagascar roach[37] This experiment indicates that the total ginsenosidesinhibit AChE activity directly On the basis of the researchmentioned earlier and the comprehensive analysis of ourfindings we conclude that the decline of AChE activity in

Evidence-Based Complementary and Alternative Medicine 5

Concentration of total ginsenosides (mgmL)

100

50

0Inhi

bitio

n ra

tio (

)

24h48h

72h

5 10 20

Figure 4 Inhibition ratio of total ginsenosides on AChE activity in4th-instarM separata larvae

M separata larvae indirectly indicates damage to nerve cellswhich induce AChE photoinactivation and death caused bythe disruption of normal nerve conduction Total ginseno-sides have inhibitory allelopathic effects on M separatawhich were beneficial for researching the allelopathic poten-tial of ginsenosides

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ai Hua Zhang and Shi Qiang Tan are cofirst authors theycontributed equally to the work

Acknowledgments

This work was funded by the National Natural ScienceFoundation ofChina (nos 31070316 31100239 and 31200224)the Funded Projects for Science and Technology Devel-opment Plan of Jilin (nos 20110926 20130206030YY and20140520159JH) and the Project supported by the Ministryof Science and Technology of China (no 2011BAI03B01)

References

[1] E L Rice Allelopathy Academic Press New York NY USA1984

[2] E L Rice Biological Control of Weeds and Plant DiseasesAdvances in Applied Allelopathy University of Oklahoma PressNorman Okla USA 1995

[3] S L Peng and P Nan ldquoTerpenoids in higher plants and theirroles in ecosystemsrdquo Chinese Journal of Ecology vol 21 pp 33ndash38 2002

[4] W X Gu S S Duan and S M Luo ldquoEcological characteristicof terpenoids and their allelopathic effects to plantsrdquo Journal ofSouth China Agricultural University vol 19 pp 108ndash112 2005

[5] R Ma M Wang X Zhu X Lu and K Sun ldquoAllelopathy andchemical constituents of Ligularia virgaurea volatilerdquo ChineseJournal of Applied Ecology vol 16 no 10 pp 1826ndash1829 2005

[6] R Ma M Wang K Zhao S Guo Q Zhao and K SunldquoAllelopathy of aqueous extract from Ligularia virgaurea a

dominant weed in psychro-grassland on pasture plantsrdquo Chi-nese Journal of Applied Ecology vol 17 no 5 pp 845ndash850 2006

[7] HWei YHouG Yang andMYou ldquoRepellent and antifeedanteffect of secondary metabolites of non-host plants on Plutellaxylostellardquo Chinese Journal of Applied Ecology vol 15 no 3 pp473ndash476 2004

[8] B Ling G-C Wang J Ya M-X Zhang and G-W LiangldquoAntifeedant activity and active ingredients against plutellaxylostella from Momordica charantia leavesrdquo Agricultural Sci-ences in China vol 7 no 12 pp 1466ndash1473 2008

[9] X D Li and S H Zhao ldquoThe toxic effects and mode ofazadirachtin on insectsrdquo Journal of South China AgriculturalUniversity vol 17 pp 118ndash122 1995

[10] X Zhang X L Wang and J T Fen ldquoThe development ofbotanical insecticidesmdashtoosendaninrdquo Journal of Northwest Sci-Tech University of Agriculture and Forestry vol 21 pp 1ndash5 1993

[11] W L Wang S H Zhao J Han and Y S Xu ldquoEffects of severalInsecticidal principles from chinaberry melia azedarach onthe imported cabbage worm and Asiatic corn borer Ostriniafurnacalisrdquo Acta Phytophylacica Sinica vol 19 pp 359ndash3641992

[12] Y-L Shi and W-P Wang ldquoBiological effects of toosendaninan active ingredient of herbal vermifuge in Chinese traditionalmedicinerdquo Acta Phytophylacica Sinica vol 58 no 5 pp 397ndash406 2006

[13] L Feng-Jie Z Ai-Hua X Yong-Hua and Z Lian-Xue ldquoAllelo-pathic effects of ginsenosides on in vitro growth and antioxidantenzymes activity of ginseng callusrdquo Allelopathy Journal vol 26no 1 pp 13ndash22 2010

[14] P G Xiao ldquoPreliminary investigation of wild ginseng in theNortheastrdquoActa Pharmaceutica Sinica vol 6 pp 340ndash351 1962

[15] T S Wang Chinese Ginseng Liaoning Science and TechnologyPress Shenyang China 2001

[16] A-H Zhang F-J Lei Y-H Xu G-X Zhou and L-X ZhangldquoEffects of ginsenosides on the germinating of ginseng seeds andon the activity of antioxidant enzymes of the radicles of ginsengseedlings in vitrordquoActa Ecologica Sinica vol 29 no 9 pp 4934ndash4941 2009

[17] A H Zhang F J Lei S W Fang M H Jia and L XZhang ldquoEffects of ginsenosides on the growth and activity ofantioxidant enzymes in American ginseng seedlingsrdquo Journal ofMedicinal Plants Research vol 5 pp 3217ndash3223 2011

[18] A-H Zhang F-J Lei Z-X Guo and L-X Zhang ldquoAllelopathiceffects of ginseng root exudates on the seeds germination andgrowth of ginseng and American ginsengrdquo Allelopathy Journalvol 28 no 1 pp 13ndash20 2011

[19] Y Li S-L Liu X-F Huang and W-L Ding ldquoAllelopathy ofginseng root exudates on pathogens of ginsengrdquo Acta EcologicaSinica vol 29 no 1 pp 161ndash168 2009

[20] Q J Zhang W R Ben and L H Xu ldquoAllelopathic effects ofextract from old ginseng soil on seed germination and seedlingsgrowth in ricerdquo Journal of NorthWest A amp F University (NaturalScience Edition) vol 40 pp 49ndash54 2012

[21] H C Sharma D J Sullivan and V S Bhatnagar ldquoPopulationdynamics and natural mortality factors of the Oriental army-wormMythimna separata (Lepidoptera Noctuidae) in South-Central Indiardquo Crop Protection vol 21 no 9 pp 721ndash732 2002

[22] F M Yan Chemical Ecology Science Press Beijing China 2011[23] L Z Wen Introduction to Entomology Research Methods and

Techniques Science Press Beijing China 2010

6 Evidence-Based Complementary and Alternative Medicine

[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[25] E F Hartree ldquoDetermination of protein a modification ofthe lowry method that gives a linear photometric responserdquoAnalytical Biochemistry vol 48 no 2 pp 422ndash427 1972

[26] L Z Wen Introduction to Entomology Research Methods andTechniques Science Press Beijing China 2010

[27] J M Goodrich and N Basu ldquoVariants of glutathione s-transferase pi 1 exhibit differential enzymatic activity andinhibition by heavy metalsrdquo Toxicology in Vitro vol 26 no 4pp 630ndash635 2012

[28] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash96 1961

[29] G Vandenborre G Smagghe and E J M Van DammeldquoPlant lectins as defense proteins against phytophagous insectsrdquoPhytochemistry vol 72 no 13 pp 1538ndash1550 2011

[30] S Senthil Nathan M Young Choi H Yul Seo C HoonPaik K Kalaivani and J Duk Kim ldquoEffect of azadirachtinon acetylcholinesterase (AChE) activity and histology of thebrown planthopper Nilaparvata lugens (Stal)rdquo Ecotoxicologyand Environmental Safety vol 70 no 2 pp 244ndash250 2008

[31] R M E Vos and P J van Bladeren ldquoGlutathione S-transferasesin relation to their role in the biotransformation of xenobioticsrdquoChemico-Biological Interactions vol 75 no 3 pp 241ndash265 1990

[32] Z S Jiang Z G Yan Y Z Du and Z Z Shang ldquoEffect of 120572-terthienyl on glutathione S-transferases inHelicoverpa armigeraand Ostrinia furnacalis larvaerdquo Chinese Journal of PesticideScience vol 15 no 3 pp 76ndash79 2003

[33] J I Kim C S Jung Y H Koh and S H Lee ldquoMolecularbiochemical and histochemical characterization of two acetyl-cholinesterase cDNAs from the German cockroach Blattellagermanicardquo Insect Molecular Biology vol 15 no 4 pp 513ndash5222006

[34] K Yin E-B Ma C-R Xue H-H Wu Y-P Guo and J-Z Zhang ldquoStudy on insecticidal activities and effect on threekinds of enzymes by 5-aminolevulinic acid on oxya ChinensisrdquoAgricultural Sciences in China vol 7 no 7 pp 841ndash846 2008

[35] Z H Tang ldquoResearch status and perspectives of insect resis-tance to insecticides in Chinardquo Entomological Knowledge vol37 pp 97ndash103 2000

[36] E Shaaya and A Rafaeli ldquoEssential oils as biorationalinsecticidesmdashpotency and mode of actionrdquo in InsecticidesDesign Using Advanced Technologies pp 249ndash261 SpringerBerlin Germany 2007

[37] D L Grundy andC C Still ldquoInhibition of acetylcholinesterasesby pulegone-12-epoxiderdquo Pesticide Biochemistry and Physiol-ogy vol 23 no 3 pp 383ndash388 1985-

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

the mechanisms of Chinese herbal medicines and to add tothe reference information for phytochemotaxonomy

2 Materials and Methods

21 Insects Mythimna separata (Walker) adults were col-lected at the test base of Jilin Agricultural University inthe early summer of 2011 The offspring of these adultswere reared on grain sorghum leaves under an LD 16 8 hphotoperiod at 22 plusmn 1∘C with 70ndash80 relative humidity andnever had contact with insecticides [21]

22 Chemicals The total ginsenosides (purity ge 80 UVmethod) were purchased from Jilin Hongjiu Biotech CoLtd AChE and GST reagent kits were purchased from theNanjing Jiancheng Bioengineering Institute A Lowry ProteinAssay Kit was purchased from Beijing Dingguo ChangshengBiotech Co Ltd

23 Bioassay for Feeding Behavior A leaf disc bioassay wasused to testM separata larvae [22 23] The ginsenoside con-centrations were 20 10 and 05 (mass fraction (MF))These concentrations are within the range of ginsenosidelevels normally found in P ginseng One day prior to the assaynewly molted 4th-instar larvae were placed individually intoPetri dishes (9 cm diameter) After starvation for 48 h 6excised grain sorghum leaf discs (Φ = 15mm) soaked insolutions with different concentrations of ginsenosides for30min (control discs received distilled water only) weresupplied to all larvae The area of feeding on the leaveswas measured every 8 h and the corresponding antifeedingactivity was calculated (nonselective and selective antifeedingratios) Each test was repeated three times

24 Enzymes Activity Tests The effects of ginsenosides onlarvae of M separata larvae were tested at concentrations of20 10 and 05 (MF) The ginsenosides were dissolvedwith distilled water Fresh clean grain sorghum leaves werepunched into leaf discs with a puncher and the leaf disc wassoaked in different concentrations of ginsenoside solutionsfor 30min and air-dried prior to use The same volumeof distilled water without ginsenosides was used for thecontrol group Fourth-instar larvae were maintained for 48 hwithout access to food Larvae with the same weights werethen selected and either were allowed to feed on the leafdiscs containing ginsenosides or were placed in the controlgroup The leaves were replaced with the fresh ones every 2days because of the possible degradation of the ginsenosidesHomogenates for the two enzyme activities (glutathione s-transferase (GST) and acetylcholinesterase (AChE) activity)of M separata larvae were collected every 24 h Each test wasrepeated three times

Homogenates of the digestive tract of the larvae wereprepared as follows The digestive tract was dissected fromindividuals of each group and washed with phosphate-buffered saline (pH 70) to remove the gut contents Tothis 5mL of cold PBS was added and the solution wascentrifuged for 20min (4∘C 10000 rmin)The supernatant is

the homogenateThe protein content of the supernatant fluidwas measured using the Lowry Protein Assay Kit stainingmethod [24 25] with bovine serum albumin as the standard

GST activity was assessed according to the methodof Jaclyn and Min Lu [26 27] by measuring GSH con-jugation with 1-chloro-24-dinitrobenzene (CDNB) CDNBactivity changes with the substrate concentration in a linearrelationship A visible-ultraviolet spectrophotometer mea-sured absorption at 412 nm to detect product formation Allreactions took place in 01M potassium phosphate-buffercontaining 1mM GSH and 001ndash3mM CDNB GST activitywas determined according to the following equation

119884 =

OD120572minusOD

120573

OD120575minusOD

120576

times 119862120590times 119873 divide 119879 lowast (119860 times 119862prot) (1)

where 119884 represents GST activity (120583molminmg prot) OD120572

represents the absorbance of the control group OD120573repre-

sents the absorbance of the experiment group OD120575repre-

sents the absorbance of the reference group OD120576represents

the absorbance of the blank group119862120590represents the standard

concentration (20mM) 119873 is the dilution factor of the reac-tion system (6) 119879 is reaction time (10min) 119860 is the samplevolume (mL) and 119862prot represents the protein concentrationof the sample

AChE activity was determined using the method ofEllman et al [28] The enzyme activity was measured byassessing the increase of yellow color produced from thio-choline when it reacts with the dithiobisnitrobenzoate ion Itis based on the coupling of the following reactions

acetylthiochlineenzyme997888997888997888997888997888rarr thiocholine + acetate

thiocholine + dithiobisnitrobenzoate 997888rarr yellow color(2)

The AChE activity was spectrophotometrically measuredat 412 nm (UV-754 Shandonggaomi UV visible-ultravioletspectrophotometer) The enzyme activities were expressedas 120583molminmg protein AChE activity was determinedaccording to the following equation

1198840=OD1minusOD

2

OD3minusOD

4

times 1198620divide 119862prot (3)

In this equation 1198840

represents AChE activity(120583molminmg prot) OD

1represents the absorbance of

the experiment group OD2represents the absorbance of

the control group OD3represents the absorbance of the

reference group OD4represents the absorbance of the blank

group 1198620represents the standard concentration (1mM) and

119862prot represents the protein concentration of the sample

25 Statistical Analyses Nonselective and selective antifeed-ing ratios were calculated according to the following equa-tions

1198841=119878ck minus S119878cktimes 100

1198842=119878ck minus S119878ck + 119878times 100

(4)

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Nonselective antifeeding ratio of different total ginsenoside concentrations on 4th-instarM separata larvae

Concentration oftotal ginsenosides()

8 h 16 h 24 h

Average feedingarea (mm2)

Nonselectiveantifeeding ratio

()

Average feedingarea (mm2)

Nonselectiveantifeeding ratio

()

Average feedingarea (mm2)

Nonselectiveantifeeding ratio

()Control 166467 plusmn 10704 a mdash 177000 plusmn 000 a mdash 177000 plusmn 000 a mdash05 87633 plusmn 33310 b 4736 152967 plusmn 2802 a 1358 144067 plusmn 8980 a 186110 59267 plusmn 22413 b 6440 152400 plusmn 7529 a 1390 140800 plusmn 20127 a 204520 18867 plusmn 8159 c 8867 75767 plusmn 45324 b 5719 48333 plusmn 35860 b 7269Data are presented as the means plusmn SE Means in the same column followed by different letters are significantly different at the level 119875 lt 005 Prior to analysisof variance (SPSS 180) the homogeneity of variance was tested in each statistic test

Table 2 Selective antifeeding ratio of different total ginsenoside concentrations on 4th-instarM separata larvae

Concentration oftotal ginsenosides()

8 h 16 h 24 h

Average feedingarea (mm2)

Selectiveantifeeding ratio

()

Average feedingarea (mm2)

Selectiveantifeeding ratio

()

Average feedingarea (mm2)

Selectiveantifeeding ratio

()05 63833 plusmn 3453 3419 45033 plusmn 4045 3662 51133 plusmn 961 2328Control 130167 plusmn 7702 mdash 97067 plusmn 5278 mdash 82167 plusmn 3900 mdash10 51467 plusmn 8075 4429 47067 plusmn 2450 3200 52733 plusmn 2574 2923Control 133233 plusmn 5150 mdash 91367 plusmn 3347 mdash 96300 plusmn 3610 mdash20 26700 plusmn 9579 6249 28533 plusmn 2346 4618 39100 plusmn 2237 4246Control 115667 plusmn 12738 mdash 77500 plusmn 5048 mdash 96800 plusmn 2406 mdashData are presented as the means plusmn SE Homogeneity of variance was tested in each statistic test

In these equations 1198841

represents the nonselectiveantifeeding ratio () 119884

2represents the selective antifeeding

ratio () 119878ck represents the average feeding area of thecontrol group (mm) and 119878 represents the average feedingarea of the treated group (mm)

The nonselective and selective antifeeding ratios of larvaefed on diets containing different concentrations of ginseno-sides were compared using SPSS Statistical 180 The effectsof ginsenosides on the detoxification enzyme activity of Mseparata larvae were also analyzed using SPSS Statistical180 In all statistical tests 119875 values lt 005 were consideredstatistically significant All data are presented as the means plusmnSEM

3 Results

31 Bioassay for Feeding Behavior The nonselective antifeed-ing ratios of 4th-instar M separata larvae at 8 16 and24 h are shown in Table 1 The total ginsenosides appearedto have significant antifeeding activity against M separatalarvae The nonselective antifeeding ratios at different totalginsenoside concentrations were significantly different fromthose of the controls at 4 8 and 12 h As the total ginsenosideconcentration increased the nonselective antifeeding activitywas enhanced At 8 h the antifeeding ratios of 20 10 and05 (MF) were 8867 6440 and 4736 respectively

The selective antifeeding ratios of the 4th-instarM sepa-rata larvae are shown in Table 2 The 4th-instar M separatalarvae preferred to eat the leaves of the control however they

did consume a small amount of the treated leaves The totalginsenosides had significant inhibitory effect on M separatalarvae and this effect increased with an increase in totalginsenoside concentration

32 Enzymes Activity Tests As the total ginsenoside con-centration increased the activity of GST also increasedGST activities first decreased and then increased with thepassage of time The inhibition ratio was the highest at 48 hwith 7515 46898 and 3186 at 20 10 and 05concentrations respectively (see Figure 2)

The AChE activity of 4th-instar M separata larvae was790546 120583molminmgprot before feeding on 20 (MF) totalginsenosides (see Figure 3) however the AChE activitieswere depressed to 618998 (inhibition ratio 2170) 315114(inhibition ratio 6014) and 350698 (inhibition ratio5564) 120583molminmgprot at 24 h 48 h and 72 h respec-tively (see Figure 4) The ginsenosides inhibited the AChEactivity of 4th-instar M separata larvae and the inhibitioneffect increased with the increase in ginsenoside concentra-tion but did not decrease over time

4 Discussion

Plant secondary metabolic substances are formed via sec-ondary metabolic pathways and play indispensable roles inthe evolution of plants They have repellency antifeedingand toxic effects on phytophagous insects For exampleMomordicin I and II have significant antifeedant activity

4 Evidence-Based Complementary and Alternative Medicine

on second-instar larvae of P xylostella and the antifeedingrates were 8039 and 7409 respectively [8] Plant lectinsalso have repellency antifeeding and toxic effects on phy-tophagous insects such as caterpillars tobacco hornwormcotton leafworm and beetles [29] Azadirachtin causes mor-tality in Nilaparvata lugens (Stal) and destroys its ovarianfollicle epithelial cells [30]

We compared the larval feeding behavior of M separataon a diet containing different concentrations of total gin-senosides (MF 20 10 and 05)The total ginsenosidesimposed adverse effects on the feeding behavior of Mseparata but to different extents (Tables 1 and 2) Comparisonwith control demonstrates that the consumption of leavesby M separata was strongly affected by the total ginseno-sides If consumption is reduced the energy required forphysiological activities would also be reduced Consequentlyresistance is then reduced and physiological metabolismwould not be adequateM separata are sometimes foundnearcultivated ginseng however they rarely feed on the leaves ofginseng which is the reason for choosingM separata for thisexperiment

Total ginsenosides inhibited GST and AChE activitiesof M separata larvae If the GST activity was inhibitedthe detoxification was reduced The M separata changedfeeding behaviors and fed on the grain sorghum leaves thatwere treated with total ginsenosides In this way they couldreduce the damage from outsiders and protect themselves Itis the same for AChE their neurotransmission slows downwhen AChE activity is inhibited and M separata did feedon a small amount The total ginsenosides had antifeedingactivity against M separata larvae and inhibitory effects onthe activities of GST and AChE Glutathione S-transferase(GST) and acetylcholinesterase (AChE) play important rolesin insect metabolism and resistance to insecticides [31]This study demonstrates that after treating M separata withtotal ginsenosides GST activities decreased This indicatesthat total ginsenosides have no toxic effects in vivo andactivates the defense system of insects but could also inhibitGST activity GSTs widely exist in insects and catalyze thenucleophilic reaction of glutathione (GSH) with a varietyof electrophilic compounds GSTs play an important role inexogenous substance biotransformation drug metabolismand protection of the organism against oxidative damage [32]GST activity was inhibited by total ginsenosides and detox-ification was reduced in 4th-instar M separata larvae (seeFigure 1) Thus they suffered damage from toxic substancesand these substances are harmful to living larvae

AChE is an important neurotransmitter enzyme mainlydistributed in the brain and central nervous system [33]AChE is localized at the surface of nerve cells and can decom-pose neurotransmitter acetylcholine which is released fromthe nerve endings and guarantees normal nerve conduction[34] If its activity is inhibited the acetylcholine released inthe synaptic gap cannot be degraded and insects will havetoxicity symptoms and possibly mortality [35] Several essen-tial oils from aromatic plants monoterpenes and naturalproducts have all been shown to be inhibitors of AChE [36]Pulegone-12-epoxide isolated fromaVerbenaceaemedicinal

GST

s act

ivity

(120583m

olm

inm

g pr

ot)

0

20

40

60

80

100

120

a a a

aa

ab

bb

b

c b

b

c

c c

Control 5mgmL 10mgmL 20mgmLConcentration of total ginsenoside

0h24h

48h72h

Figure 1 Effect of total ginsenosides on GST activity in 4th-instar M separata larvae Bars with different letters are significantlydifferent from each other at 119875 lt 005 using the one-way ANOVA(SPSS 180) Data are means + SD

Concentration of total ginsenosides (mgmL)

80

60

40

20

0

Inhi

bitio

n ra

tio (

)

24h48h

72h

5 10 20

Figure 2 Inhibition ratio of total ginsenosides on GST activity in4th-instarM separata larvae

AChE

activ

ity (120583

mol

min

mg

prot

)

0

50

100

150

200

250

300

aa

a a

a

b

b b

b b

d

d c

c

c c

Control 5mgmL 10mgmL 20mgmLConcentration of total ginsenosides

0h24h

48h72h

Figure 3 Effect of total ginsenosides on AChE activity in 4th-instarM separata larvae Bars with different letters are significantlydifferent from each other at 119875 lt 005 using a one-way ANOVA(SPSS 180) Data are means + SD

plant (Lippia stoechadifolia L (Poleo)) showed an irreversibleinhibition of the AChE in house fly and Madagascar roach[37] This experiment indicates that the total ginsenosidesinhibit AChE activity directly On the basis of the researchmentioned earlier and the comprehensive analysis of ourfindings we conclude that the decline of AChE activity in

Evidence-Based Complementary and Alternative Medicine 5

Concentration of total ginsenosides (mgmL)

100

50

0Inhi

bitio

n ra

tio (

)

24h48h

72h

5 10 20

Figure 4 Inhibition ratio of total ginsenosides on AChE activity in4th-instarM separata larvae

M separata larvae indirectly indicates damage to nerve cellswhich induce AChE photoinactivation and death caused bythe disruption of normal nerve conduction Total ginseno-sides have inhibitory allelopathic effects on M separatawhich were beneficial for researching the allelopathic poten-tial of ginsenosides

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ai Hua Zhang and Shi Qiang Tan are cofirst authors theycontributed equally to the work

Acknowledgments

This work was funded by the National Natural ScienceFoundation ofChina (nos 31070316 31100239 and 31200224)the Funded Projects for Science and Technology Devel-opment Plan of Jilin (nos 20110926 20130206030YY and20140520159JH) and the Project supported by the Ministryof Science and Technology of China (no 2011BAI03B01)

References

[1] E L Rice Allelopathy Academic Press New York NY USA1984

[2] E L Rice Biological Control of Weeds and Plant DiseasesAdvances in Applied Allelopathy University of Oklahoma PressNorman Okla USA 1995

[3] S L Peng and P Nan ldquoTerpenoids in higher plants and theirroles in ecosystemsrdquo Chinese Journal of Ecology vol 21 pp 33ndash38 2002

[4] W X Gu S S Duan and S M Luo ldquoEcological characteristicof terpenoids and their allelopathic effects to plantsrdquo Journal ofSouth China Agricultural University vol 19 pp 108ndash112 2005

[5] R Ma M Wang X Zhu X Lu and K Sun ldquoAllelopathy andchemical constituents of Ligularia virgaurea volatilerdquo ChineseJournal of Applied Ecology vol 16 no 10 pp 1826ndash1829 2005

[6] R Ma M Wang K Zhao S Guo Q Zhao and K SunldquoAllelopathy of aqueous extract from Ligularia virgaurea a

dominant weed in psychro-grassland on pasture plantsrdquo Chi-nese Journal of Applied Ecology vol 17 no 5 pp 845ndash850 2006

[7] HWei YHouG Yang andMYou ldquoRepellent and antifeedanteffect of secondary metabolites of non-host plants on Plutellaxylostellardquo Chinese Journal of Applied Ecology vol 15 no 3 pp473ndash476 2004

[8] B Ling G-C Wang J Ya M-X Zhang and G-W LiangldquoAntifeedant activity and active ingredients against plutellaxylostella from Momordica charantia leavesrdquo Agricultural Sci-ences in China vol 7 no 12 pp 1466ndash1473 2008

[9] X D Li and S H Zhao ldquoThe toxic effects and mode ofazadirachtin on insectsrdquo Journal of South China AgriculturalUniversity vol 17 pp 118ndash122 1995

[10] X Zhang X L Wang and J T Fen ldquoThe development ofbotanical insecticidesmdashtoosendaninrdquo Journal of Northwest Sci-Tech University of Agriculture and Forestry vol 21 pp 1ndash5 1993

[11] W L Wang S H Zhao J Han and Y S Xu ldquoEffects of severalInsecticidal principles from chinaberry melia azedarach onthe imported cabbage worm and Asiatic corn borer Ostriniafurnacalisrdquo Acta Phytophylacica Sinica vol 19 pp 359ndash3641992

[12] Y-L Shi and W-P Wang ldquoBiological effects of toosendaninan active ingredient of herbal vermifuge in Chinese traditionalmedicinerdquo Acta Phytophylacica Sinica vol 58 no 5 pp 397ndash406 2006

[13] L Feng-Jie Z Ai-Hua X Yong-Hua and Z Lian-Xue ldquoAllelo-pathic effects of ginsenosides on in vitro growth and antioxidantenzymes activity of ginseng callusrdquo Allelopathy Journal vol 26no 1 pp 13ndash22 2010

[14] P G Xiao ldquoPreliminary investigation of wild ginseng in theNortheastrdquoActa Pharmaceutica Sinica vol 6 pp 340ndash351 1962

[15] T S Wang Chinese Ginseng Liaoning Science and TechnologyPress Shenyang China 2001

[16] A-H Zhang F-J Lei Y-H Xu G-X Zhou and L-X ZhangldquoEffects of ginsenosides on the germinating of ginseng seeds andon the activity of antioxidant enzymes of the radicles of ginsengseedlings in vitrordquoActa Ecologica Sinica vol 29 no 9 pp 4934ndash4941 2009

[17] A H Zhang F J Lei S W Fang M H Jia and L XZhang ldquoEffects of ginsenosides on the growth and activity ofantioxidant enzymes in American ginseng seedlingsrdquo Journal ofMedicinal Plants Research vol 5 pp 3217ndash3223 2011

[18] A-H Zhang F-J Lei Z-X Guo and L-X Zhang ldquoAllelopathiceffects of ginseng root exudates on the seeds germination andgrowth of ginseng and American ginsengrdquo Allelopathy Journalvol 28 no 1 pp 13ndash20 2011

[19] Y Li S-L Liu X-F Huang and W-L Ding ldquoAllelopathy ofginseng root exudates on pathogens of ginsengrdquo Acta EcologicaSinica vol 29 no 1 pp 161ndash168 2009

[20] Q J Zhang W R Ben and L H Xu ldquoAllelopathic effects ofextract from old ginseng soil on seed germination and seedlingsgrowth in ricerdquo Journal of NorthWest A amp F University (NaturalScience Edition) vol 40 pp 49ndash54 2012

[21] H C Sharma D J Sullivan and V S Bhatnagar ldquoPopulationdynamics and natural mortality factors of the Oriental army-wormMythimna separata (Lepidoptera Noctuidae) in South-Central Indiardquo Crop Protection vol 21 no 9 pp 721ndash732 2002

[22] F M Yan Chemical Ecology Science Press Beijing China 2011[23] L Z Wen Introduction to Entomology Research Methods and

Techniques Science Press Beijing China 2010

6 Evidence-Based Complementary and Alternative Medicine

[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[25] E F Hartree ldquoDetermination of protein a modification ofthe lowry method that gives a linear photometric responserdquoAnalytical Biochemistry vol 48 no 2 pp 422ndash427 1972

[26] L Z Wen Introduction to Entomology Research Methods andTechniques Science Press Beijing China 2010

[27] J M Goodrich and N Basu ldquoVariants of glutathione s-transferase pi 1 exhibit differential enzymatic activity andinhibition by heavy metalsrdquo Toxicology in Vitro vol 26 no 4pp 630ndash635 2012

[28] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash96 1961

[29] G Vandenborre G Smagghe and E J M Van DammeldquoPlant lectins as defense proteins against phytophagous insectsrdquoPhytochemistry vol 72 no 13 pp 1538ndash1550 2011

[30] S Senthil Nathan M Young Choi H Yul Seo C HoonPaik K Kalaivani and J Duk Kim ldquoEffect of azadirachtinon acetylcholinesterase (AChE) activity and histology of thebrown planthopper Nilaparvata lugens (Stal)rdquo Ecotoxicologyand Environmental Safety vol 70 no 2 pp 244ndash250 2008

[31] R M E Vos and P J van Bladeren ldquoGlutathione S-transferasesin relation to their role in the biotransformation of xenobioticsrdquoChemico-Biological Interactions vol 75 no 3 pp 241ndash265 1990

[32] Z S Jiang Z G Yan Y Z Du and Z Z Shang ldquoEffect of 120572-terthienyl on glutathione S-transferases inHelicoverpa armigeraand Ostrinia furnacalis larvaerdquo Chinese Journal of PesticideScience vol 15 no 3 pp 76ndash79 2003

[33] J I Kim C S Jung Y H Koh and S H Lee ldquoMolecularbiochemical and histochemical characterization of two acetyl-cholinesterase cDNAs from the German cockroach Blattellagermanicardquo Insect Molecular Biology vol 15 no 4 pp 513ndash5222006

[34] K Yin E-B Ma C-R Xue H-H Wu Y-P Guo and J-Z Zhang ldquoStudy on insecticidal activities and effect on threekinds of enzymes by 5-aminolevulinic acid on oxya ChinensisrdquoAgricultural Sciences in China vol 7 no 7 pp 841ndash846 2008

[35] Z H Tang ldquoResearch status and perspectives of insect resis-tance to insecticides in Chinardquo Entomological Knowledge vol37 pp 97ndash103 2000

[36] E Shaaya and A Rafaeli ldquoEssential oils as biorationalinsecticidesmdashpotency and mode of actionrdquo in InsecticidesDesign Using Advanced Technologies pp 249ndash261 SpringerBerlin Germany 2007

[37] D L Grundy andC C Still ldquoInhibition of acetylcholinesterasesby pulegone-12-epoxiderdquo Pesticide Biochemistry and Physiol-ogy vol 23 no 3 pp 383ndash388 1985-

Submit your manuscripts athttpwwwhindawicom

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Behavioural Neurology

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OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Nonselective antifeeding ratio of different total ginsenoside concentrations on 4th-instarM separata larvae

Concentration oftotal ginsenosides()

8 h 16 h 24 h

Average feedingarea (mm2)

Nonselectiveantifeeding ratio

()

Average feedingarea (mm2)

Nonselectiveantifeeding ratio

()

Average feedingarea (mm2)

Nonselectiveantifeeding ratio

()Control 166467 plusmn 10704 a mdash 177000 plusmn 000 a mdash 177000 plusmn 000 a mdash05 87633 plusmn 33310 b 4736 152967 plusmn 2802 a 1358 144067 plusmn 8980 a 186110 59267 plusmn 22413 b 6440 152400 plusmn 7529 a 1390 140800 plusmn 20127 a 204520 18867 plusmn 8159 c 8867 75767 plusmn 45324 b 5719 48333 plusmn 35860 b 7269Data are presented as the means plusmn SE Means in the same column followed by different letters are significantly different at the level 119875 lt 005 Prior to analysisof variance (SPSS 180) the homogeneity of variance was tested in each statistic test

Table 2 Selective antifeeding ratio of different total ginsenoside concentrations on 4th-instarM separata larvae

Concentration oftotal ginsenosides()

8 h 16 h 24 h

Average feedingarea (mm2)

Selectiveantifeeding ratio

()

Average feedingarea (mm2)

Selectiveantifeeding ratio

()

Average feedingarea (mm2)

Selectiveantifeeding ratio

()05 63833 plusmn 3453 3419 45033 plusmn 4045 3662 51133 plusmn 961 2328Control 130167 plusmn 7702 mdash 97067 plusmn 5278 mdash 82167 plusmn 3900 mdash10 51467 plusmn 8075 4429 47067 plusmn 2450 3200 52733 plusmn 2574 2923Control 133233 plusmn 5150 mdash 91367 plusmn 3347 mdash 96300 plusmn 3610 mdash20 26700 plusmn 9579 6249 28533 plusmn 2346 4618 39100 plusmn 2237 4246Control 115667 plusmn 12738 mdash 77500 plusmn 5048 mdash 96800 plusmn 2406 mdashData are presented as the means plusmn SE Homogeneity of variance was tested in each statistic test

In these equations 1198841

represents the nonselectiveantifeeding ratio () 119884

2represents the selective antifeeding

ratio () 119878ck represents the average feeding area of thecontrol group (mm) and 119878 represents the average feedingarea of the treated group (mm)

The nonselective and selective antifeeding ratios of larvaefed on diets containing different concentrations of ginseno-sides were compared using SPSS Statistical 180 The effectsof ginsenosides on the detoxification enzyme activity of Mseparata larvae were also analyzed using SPSS Statistical180 In all statistical tests 119875 values lt 005 were consideredstatistically significant All data are presented as the means plusmnSEM

3 Results

31 Bioassay for Feeding Behavior The nonselective antifeed-ing ratios of 4th-instar M separata larvae at 8 16 and24 h are shown in Table 1 The total ginsenosides appearedto have significant antifeeding activity against M separatalarvae The nonselective antifeeding ratios at different totalginsenoside concentrations were significantly different fromthose of the controls at 4 8 and 12 h As the total ginsenosideconcentration increased the nonselective antifeeding activitywas enhanced At 8 h the antifeeding ratios of 20 10 and05 (MF) were 8867 6440 and 4736 respectively

The selective antifeeding ratios of the 4th-instarM sepa-rata larvae are shown in Table 2 The 4th-instar M separatalarvae preferred to eat the leaves of the control however they

did consume a small amount of the treated leaves The totalginsenosides had significant inhibitory effect on M separatalarvae and this effect increased with an increase in totalginsenoside concentration

32 Enzymes Activity Tests As the total ginsenoside con-centration increased the activity of GST also increasedGST activities first decreased and then increased with thepassage of time The inhibition ratio was the highest at 48 hwith 7515 46898 and 3186 at 20 10 and 05concentrations respectively (see Figure 2)

The AChE activity of 4th-instar M separata larvae was790546 120583molminmgprot before feeding on 20 (MF) totalginsenosides (see Figure 3) however the AChE activitieswere depressed to 618998 (inhibition ratio 2170) 315114(inhibition ratio 6014) and 350698 (inhibition ratio5564) 120583molminmgprot at 24 h 48 h and 72 h respec-tively (see Figure 4) The ginsenosides inhibited the AChEactivity of 4th-instar M separata larvae and the inhibitioneffect increased with the increase in ginsenoside concentra-tion but did not decrease over time

4 Discussion

Plant secondary metabolic substances are formed via sec-ondary metabolic pathways and play indispensable roles inthe evolution of plants They have repellency antifeedingand toxic effects on phytophagous insects For exampleMomordicin I and II have significant antifeedant activity

4 Evidence-Based Complementary and Alternative Medicine

on second-instar larvae of P xylostella and the antifeedingrates were 8039 and 7409 respectively [8] Plant lectinsalso have repellency antifeeding and toxic effects on phy-tophagous insects such as caterpillars tobacco hornwormcotton leafworm and beetles [29] Azadirachtin causes mor-tality in Nilaparvata lugens (Stal) and destroys its ovarianfollicle epithelial cells [30]

We compared the larval feeding behavior of M separataon a diet containing different concentrations of total gin-senosides (MF 20 10 and 05)The total ginsenosidesimposed adverse effects on the feeding behavior of Mseparata but to different extents (Tables 1 and 2) Comparisonwith control demonstrates that the consumption of leavesby M separata was strongly affected by the total ginseno-sides If consumption is reduced the energy required forphysiological activities would also be reduced Consequentlyresistance is then reduced and physiological metabolismwould not be adequateM separata are sometimes foundnearcultivated ginseng however they rarely feed on the leaves ofginseng which is the reason for choosingM separata for thisexperiment

Total ginsenosides inhibited GST and AChE activitiesof M separata larvae If the GST activity was inhibitedthe detoxification was reduced The M separata changedfeeding behaviors and fed on the grain sorghum leaves thatwere treated with total ginsenosides In this way they couldreduce the damage from outsiders and protect themselves Itis the same for AChE their neurotransmission slows downwhen AChE activity is inhibited and M separata did feedon a small amount The total ginsenosides had antifeedingactivity against M separata larvae and inhibitory effects onthe activities of GST and AChE Glutathione S-transferase(GST) and acetylcholinesterase (AChE) play important rolesin insect metabolism and resistance to insecticides [31]This study demonstrates that after treating M separata withtotal ginsenosides GST activities decreased This indicatesthat total ginsenosides have no toxic effects in vivo andactivates the defense system of insects but could also inhibitGST activity GSTs widely exist in insects and catalyze thenucleophilic reaction of glutathione (GSH) with a varietyof electrophilic compounds GSTs play an important role inexogenous substance biotransformation drug metabolismand protection of the organism against oxidative damage [32]GST activity was inhibited by total ginsenosides and detox-ification was reduced in 4th-instar M separata larvae (seeFigure 1) Thus they suffered damage from toxic substancesand these substances are harmful to living larvae

AChE is an important neurotransmitter enzyme mainlydistributed in the brain and central nervous system [33]AChE is localized at the surface of nerve cells and can decom-pose neurotransmitter acetylcholine which is released fromthe nerve endings and guarantees normal nerve conduction[34] If its activity is inhibited the acetylcholine released inthe synaptic gap cannot be degraded and insects will havetoxicity symptoms and possibly mortality [35] Several essen-tial oils from aromatic plants monoterpenes and naturalproducts have all been shown to be inhibitors of AChE [36]Pulegone-12-epoxide isolated fromaVerbenaceaemedicinal

GST

s act

ivity

(120583m

olm

inm

g pr

ot)

0

20

40

60

80

100

120

a a a

aa

ab

bb

b

c b

b

c

c c

Control 5mgmL 10mgmL 20mgmLConcentration of total ginsenoside

0h24h

48h72h

Figure 1 Effect of total ginsenosides on GST activity in 4th-instar M separata larvae Bars with different letters are significantlydifferent from each other at 119875 lt 005 using the one-way ANOVA(SPSS 180) Data are means + SD

Concentration of total ginsenosides (mgmL)

80

60

40

20

0

Inhi

bitio

n ra

tio (

)

24h48h

72h

5 10 20

Figure 2 Inhibition ratio of total ginsenosides on GST activity in4th-instarM separata larvae

AChE

activ

ity (120583

mol

min

mg

prot

)

0

50

100

150

200

250

300

aa

a a

a

b

b b

b b

d

d c

c

c c

Control 5mgmL 10mgmL 20mgmLConcentration of total ginsenosides

0h24h

48h72h

Figure 3 Effect of total ginsenosides on AChE activity in 4th-instarM separata larvae Bars with different letters are significantlydifferent from each other at 119875 lt 005 using a one-way ANOVA(SPSS 180) Data are means + SD

plant (Lippia stoechadifolia L (Poleo)) showed an irreversibleinhibition of the AChE in house fly and Madagascar roach[37] This experiment indicates that the total ginsenosidesinhibit AChE activity directly On the basis of the researchmentioned earlier and the comprehensive analysis of ourfindings we conclude that the decline of AChE activity in

Evidence-Based Complementary and Alternative Medicine 5

Concentration of total ginsenosides (mgmL)

100

50

0Inhi

bitio

n ra

tio (

)

24h48h

72h

5 10 20

Figure 4 Inhibition ratio of total ginsenosides on AChE activity in4th-instarM separata larvae

M separata larvae indirectly indicates damage to nerve cellswhich induce AChE photoinactivation and death caused bythe disruption of normal nerve conduction Total ginseno-sides have inhibitory allelopathic effects on M separatawhich were beneficial for researching the allelopathic poten-tial of ginsenosides

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ai Hua Zhang and Shi Qiang Tan are cofirst authors theycontributed equally to the work

Acknowledgments

This work was funded by the National Natural ScienceFoundation ofChina (nos 31070316 31100239 and 31200224)the Funded Projects for Science and Technology Devel-opment Plan of Jilin (nos 20110926 20130206030YY and20140520159JH) and the Project supported by the Ministryof Science and Technology of China (no 2011BAI03B01)

References

[1] E L Rice Allelopathy Academic Press New York NY USA1984

[2] E L Rice Biological Control of Weeds and Plant DiseasesAdvances in Applied Allelopathy University of Oklahoma PressNorman Okla USA 1995

[3] S L Peng and P Nan ldquoTerpenoids in higher plants and theirroles in ecosystemsrdquo Chinese Journal of Ecology vol 21 pp 33ndash38 2002

[4] W X Gu S S Duan and S M Luo ldquoEcological characteristicof terpenoids and their allelopathic effects to plantsrdquo Journal ofSouth China Agricultural University vol 19 pp 108ndash112 2005

[5] R Ma M Wang X Zhu X Lu and K Sun ldquoAllelopathy andchemical constituents of Ligularia virgaurea volatilerdquo ChineseJournal of Applied Ecology vol 16 no 10 pp 1826ndash1829 2005

[6] R Ma M Wang K Zhao S Guo Q Zhao and K SunldquoAllelopathy of aqueous extract from Ligularia virgaurea a

dominant weed in psychro-grassland on pasture plantsrdquo Chi-nese Journal of Applied Ecology vol 17 no 5 pp 845ndash850 2006

[7] HWei YHouG Yang andMYou ldquoRepellent and antifeedanteffect of secondary metabolites of non-host plants on Plutellaxylostellardquo Chinese Journal of Applied Ecology vol 15 no 3 pp473ndash476 2004

[8] B Ling G-C Wang J Ya M-X Zhang and G-W LiangldquoAntifeedant activity and active ingredients against plutellaxylostella from Momordica charantia leavesrdquo Agricultural Sci-ences in China vol 7 no 12 pp 1466ndash1473 2008

[9] X D Li and S H Zhao ldquoThe toxic effects and mode ofazadirachtin on insectsrdquo Journal of South China AgriculturalUniversity vol 17 pp 118ndash122 1995

[10] X Zhang X L Wang and J T Fen ldquoThe development ofbotanical insecticidesmdashtoosendaninrdquo Journal of Northwest Sci-Tech University of Agriculture and Forestry vol 21 pp 1ndash5 1993

[11] W L Wang S H Zhao J Han and Y S Xu ldquoEffects of severalInsecticidal principles from chinaberry melia azedarach onthe imported cabbage worm and Asiatic corn borer Ostriniafurnacalisrdquo Acta Phytophylacica Sinica vol 19 pp 359ndash3641992

[12] Y-L Shi and W-P Wang ldquoBiological effects of toosendaninan active ingredient of herbal vermifuge in Chinese traditionalmedicinerdquo Acta Phytophylacica Sinica vol 58 no 5 pp 397ndash406 2006

[13] L Feng-Jie Z Ai-Hua X Yong-Hua and Z Lian-Xue ldquoAllelo-pathic effects of ginsenosides on in vitro growth and antioxidantenzymes activity of ginseng callusrdquo Allelopathy Journal vol 26no 1 pp 13ndash22 2010

[14] P G Xiao ldquoPreliminary investigation of wild ginseng in theNortheastrdquoActa Pharmaceutica Sinica vol 6 pp 340ndash351 1962

[15] T S Wang Chinese Ginseng Liaoning Science and TechnologyPress Shenyang China 2001

[16] A-H Zhang F-J Lei Y-H Xu G-X Zhou and L-X ZhangldquoEffects of ginsenosides on the germinating of ginseng seeds andon the activity of antioxidant enzymes of the radicles of ginsengseedlings in vitrordquoActa Ecologica Sinica vol 29 no 9 pp 4934ndash4941 2009

[17] A H Zhang F J Lei S W Fang M H Jia and L XZhang ldquoEffects of ginsenosides on the growth and activity ofantioxidant enzymes in American ginseng seedlingsrdquo Journal ofMedicinal Plants Research vol 5 pp 3217ndash3223 2011

[18] A-H Zhang F-J Lei Z-X Guo and L-X Zhang ldquoAllelopathiceffects of ginseng root exudates on the seeds germination andgrowth of ginseng and American ginsengrdquo Allelopathy Journalvol 28 no 1 pp 13ndash20 2011

[19] Y Li S-L Liu X-F Huang and W-L Ding ldquoAllelopathy ofginseng root exudates on pathogens of ginsengrdquo Acta EcologicaSinica vol 29 no 1 pp 161ndash168 2009

[20] Q J Zhang W R Ben and L H Xu ldquoAllelopathic effects ofextract from old ginseng soil on seed germination and seedlingsgrowth in ricerdquo Journal of NorthWest A amp F University (NaturalScience Edition) vol 40 pp 49ndash54 2012

[21] H C Sharma D J Sullivan and V S Bhatnagar ldquoPopulationdynamics and natural mortality factors of the Oriental army-wormMythimna separata (Lepidoptera Noctuidae) in South-Central Indiardquo Crop Protection vol 21 no 9 pp 721ndash732 2002

[22] F M Yan Chemical Ecology Science Press Beijing China 2011[23] L Z Wen Introduction to Entomology Research Methods and

Techniques Science Press Beijing China 2010

6 Evidence-Based Complementary and Alternative Medicine

[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[25] E F Hartree ldquoDetermination of protein a modification ofthe lowry method that gives a linear photometric responserdquoAnalytical Biochemistry vol 48 no 2 pp 422ndash427 1972

[26] L Z Wen Introduction to Entomology Research Methods andTechniques Science Press Beijing China 2010

[27] J M Goodrich and N Basu ldquoVariants of glutathione s-transferase pi 1 exhibit differential enzymatic activity andinhibition by heavy metalsrdquo Toxicology in Vitro vol 26 no 4pp 630ndash635 2012

[28] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash96 1961

[29] G Vandenborre G Smagghe and E J M Van DammeldquoPlant lectins as defense proteins against phytophagous insectsrdquoPhytochemistry vol 72 no 13 pp 1538ndash1550 2011

[30] S Senthil Nathan M Young Choi H Yul Seo C HoonPaik K Kalaivani and J Duk Kim ldquoEffect of azadirachtinon acetylcholinesterase (AChE) activity and histology of thebrown planthopper Nilaparvata lugens (Stal)rdquo Ecotoxicologyand Environmental Safety vol 70 no 2 pp 244ndash250 2008

[31] R M E Vos and P J van Bladeren ldquoGlutathione S-transferasesin relation to their role in the biotransformation of xenobioticsrdquoChemico-Biological Interactions vol 75 no 3 pp 241ndash265 1990

[32] Z S Jiang Z G Yan Y Z Du and Z Z Shang ldquoEffect of 120572-terthienyl on glutathione S-transferases inHelicoverpa armigeraand Ostrinia furnacalis larvaerdquo Chinese Journal of PesticideScience vol 15 no 3 pp 76ndash79 2003

[33] J I Kim C S Jung Y H Koh and S H Lee ldquoMolecularbiochemical and histochemical characterization of two acetyl-cholinesterase cDNAs from the German cockroach Blattellagermanicardquo Insect Molecular Biology vol 15 no 4 pp 513ndash5222006

[34] K Yin E-B Ma C-R Xue H-H Wu Y-P Guo and J-Z Zhang ldquoStudy on insecticidal activities and effect on threekinds of enzymes by 5-aminolevulinic acid on oxya ChinensisrdquoAgricultural Sciences in China vol 7 no 7 pp 841ndash846 2008

[35] Z H Tang ldquoResearch status and perspectives of insect resis-tance to insecticides in Chinardquo Entomological Knowledge vol37 pp 97ndash103 2000

[36] E Shaaya and A Rafaeli ldquoEssential oils as biorationalinsecticidesmdashpotency and mode of actionrdquo in InsecticidesDesign Using Advanced Technologies pp 249ndash261 SpringerBerlin Germany 2007

[37] D L Grundy andC C Still ldquoInhibition of acetylcholinesterasesby pulegone-12-epoxiderdquo Pesticide Biochemistry and Physiol-ogy vol 23 no 3 pp 383ndash388 1985-

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

on second-instar larvae of P xylostella and the antifeedingrates were 8039 and 7409 respectively [8] Plant lectinsalso have repellency antifeeding and toxic effects on phy-tophagous insects such as caterpillars tobacco hornwormcotton leafworm and beetles [29] Azadirachtin causes mor-tality in Nilaparvata lugens (Stal) and destroys its ovarianfollicle epithelial cells [30]

We compared the larval feeding behavior of M separataon a diet containing different concentrations of total gin-senosides (MF 20 10 and 05)The total ginsenosidesimposed adverse effects on the feeding behavior of Mseparata but to different extents (Tables 1 and 2) Comparisonwith control demonstrates that the consumption of leavesby M separata was strongly affected by the total ginseno-sides If consumption is reduced the energy required forphysiological activities would also be reduced Consequentlyresistance is then reduced and physiological metabolismwould not be adequateM separata are sometimes foundnearcultivated ginseng however they rarely feed on the leaves ofginseng which is the reason for choosingM separata for thisexperiment

Total ginsenosides inhibited GST and AChE activitiesof M separata larvae If the GST activity was inhibitedthe detoxification was reduced The M separata changedfeeding behaviors and fed on the grain sorghum leaves thatwere treated with total ginsenosides In this way they couldreduce the damage from outsiders and protect themselves Itis the same for AChE their neurotransmission slows downwhen AChE activity is inhibited and M separata did feedon a small amount The total ginsenosides had antifeedingactivity against M separata larvae and inhibitory effects onthe activities of GST and AChE Glutathione S-transferase(GST) and acetylcholinesterase (AChE) play important rolesin insect metabolism and resistance to insecticides [31]This study demonstrates that after treating M separata withtotal ginsenosides GST activities decreased This indicatesthat total ginsenosides have no toxic effects in vivo andactivates the defense system of insects but could also inhibitGST activity GSTs widely exist in insects and catalyze thenucleophilic reaction of glutathione (GSH) with a varietyof electrophilic compounds GSTs play an important role inexogenous substance biotransformation drug metabolismand protection of the organism against oxidative damage [32]GST activity was inhibited by total ginsenosides and detox-ification was reduced in 4th-instar M separata larvae (seeFigure 1) Thus they suffered damage from toxic substancesand these substances are harmful to living larvae

AChE is an important neurotransmitter enzyme mainlydistributed in the brain and central nervous system [33]AChE is localized at the surface of nerve cells and can decom-pose neurotransmitter acetylcholine which is released fromthe nerve endings and guarantees normal nerve conduction[34] If its activity is inhibited the acetylcholine released inthe synaptic gap cannot be degraded and insects will havetoxicity symptoms and possibly mortality [35] Several essen-tial oils from aromatic plants monoterpenes and naturalproducts have all been shown to be inhibitors of AChE [36]Pulegone-12-epoxide isolated fromaVerbenaceaemedicinal

GST

s act

ivity

(120583m

olm

inm

g pr

ot)

0

20

40

60

80

100

120

a a a

aa

ab

bb

b

c b

b

c

c c

Control 5mgmL 10mgmL 20mgmLConcentration of total ginsenoside

0h24h

48h72h

Figure 1 Effect of total ginsenosides on GST activity in 4th-instar M separata larvae Bars with different letters are significantlydifferent from each other at 119875 lt 005 using the one-way ANOVA(SPSS 180) Data are means + SD

Concentration of total ginsenosides (mgmL)

80

60

40

20

0

Inhi

bitio

n ra

tio (

)

24h48h

72h

5 10 20

Figure 2 Inhibition ratio of total ginsenosides on GST activity in4th-instarM separata larvae

AChE

activ

ity (120583

mol

min

mg

prot

)

0

50

100

150

200

250

300

aa

a a

a

b

b b

b b

d

d c

c

c c

Control 5mgmL 10mgmL 20mgmLConcentration of total ginsenosides

0h24h

48h72h

Figure 3 Effect of total ginsenosides on AChE activity in 4th-instarM separata larvae Bars with different letters are significantlydifferent from each other at 119875 lt 005 using a one-way ANOVA(SPSS 180) Data are means + SD

plant (Lippia stoechadifolia L (Poleo)) showed an irreversibleinhibition of the AChE in house fly and Madagascar roach[37] This experiment indicates that the total ginsenosidesinhibit AChE activity directly On the basis of the researchmentioned earlier and the comprehensive analysis of ourfindings we conclude that the decline of AChE activity in

Evidence-Based Complementary and Alternative Medicine 5

Concentration of total ginsenosides (mgmL)

100

50

0Inhi

bitio

n ra

tio (

)

24h48h

72h

5 10 20

Figure 4 Inhibition ratio of total ginsenosides on AChE activity in4th-instarM separata larvae

M separata larvae indirectly indicates damage to nerve cellswhich induce AChE photoinactivation and death caused bythe disruption of normal nerve conduction Total ginseno-sides have inhibitory allelopathic effects on M separatawhich were beneficial for researching the allelopathic poten-tial of ginsenosides

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ai Hua Zhang and Shi Qiang Tan are cofirst authors theycontributed equally to the work

Acknowledgments

This work was funded by the National Natural ScienceFoundation ofChina (nos 31070316 31100239 and 31200224)the Funded Projects for Science and Technology Devel-opment Plan of Jilin (nos 20110926 20130206030YY and20140520159JH) and the Project supported by the Ministryof Science and Technology of China (no 2011BAI03B01)

References

[1] E L Rice Allelopathy Academic Press New York NY USA1984

[2] E L Rice Biological Control of Weeds and Plant DiseasesAdvances in Applied Allelopathy University of Oklahoma PressNorman Okla USA 1995

[3] S L Peng and P Nan ldquoTerpenoids in higher plants and theirroles in ecosystemsrdquo Chinese Journal of Ecology vol 21 pp 33ndash38 2002

[4] W X Gu S S Duan and S M Luo ldquoEcological characteristicof terpenoids and their allelopathic effects to plantsrdquo Journal ofSouth China Agricultural University vol 19 pp 108ndash112 2005

[5] R Ma M Wang X Zhu X Lu and K Sun ldquoAllelopathy andchemical constituents of Ligularia virgaurea volatilerdquo ChineseJournal of Applied Ecology vol 16 no 10 pp 1826ndash1829 2005

[6] R Ma M Wang K Zhao S Guo Q Zhao and K SunldquoAllelopathy of aqueous extract from Ligularia virgaurea a

dominant weed in psychro-grassland on pasture plantsrdquo Chi-nese Journal of Applied Ecology vol 17 no 5 pp 845ndash850 2006

[7] HWei YHouG Yang andMYou ldquoRepellent and antifeedanteffect of secondary metabolites of non-host plants on Plutellaxylostellardquo Chinese Journal of Applied Ecology vol 15 no 3 pp473ndash476 2004

[8] B Ling G-C Wang J Ya M-X Zhang and G-W LiangldquoAntifeedant activity and active ingredients against plutellaxylostella from Momordica charantia leavesrdquo Agricultural Sci-ences in China vol 7 no 12 pp 1466ndash1473 2008

[9] X D Li and S H Zhao ldquoThe toxic effects and mode ofazadirachtin on insectsrdquo Journal of South China AgriculturalUniversity vol 17 pp 118ndash122 1995

[10] X Zhang X L Wang and J T Fen ldquoThe development ofbotanical insecticidesmdashtoosendaninrdquo Journal of Northwest Sci-Tech University of Agriculture and Forestry vol 21 pp 1ndash5 1993

[11] W L Wang S H Zhao J Han and Y S Xu ldquoEffects of severalInsecticidal principles from chinaberry melia azedarach onthe imported cabbage worm and Asiatic corn borer Ostriniafurnacalisrdquo Acta Phytophylacica Sinica vol 19 pp 359ndash3641992

[12] Y-L Shi and W-P Wang ldquoBiological effects of toosendaninan active ingredient of herbal vermifuge in Chinese traditionalmedicinerdquo Acta Phytophylacica Sinica vol 58 no 5 pp 397ndash406 2006

[13] L Feng-Jie Z Ai-Hua X Yong-Hua and Z Lian-Xue ldquoAllelo-pathic effects of ginsenosides on in vitro growth and antioxidantenzymes activity of ginseng callusrdquo Allelopathy Journal vol 26no 1 pp 13ndash22 2010

[14] P G Xiao ldquoPreliminary investigation of wild ginseng in theNortheastrdquoActa Pharmaceutica Sinica vol 6 pp 340ndash351 1962

[15] T S Wang Chinese Ginseng Liaoning Science and TechnologyPress Shenyang China 2001

[16] A-H Zhang F-J Lei Y-H Xu G-X Zhou and L-X ZhangldquoEffects of ginsenosides on the germinating of ginseng seeds andon the activity of antioxidant enzymes of the radicles of ginsengseedlings in vitrordquoActa Ecologica Sinica vol 29 no 9 pp 4934ndash4941 2009

[17] A H Zhang F J Lei S W Fang M H Jia and L XZhang ldquoEffects of ginsenosides on the growth and activity ofantioxidant enzymes in American ginseng seedlingsrdquo Journal ofMedicinal Plants Research vol 5 pp 3217ndash3223 2011

[18] A-H Zhang F-J Lei Z-X Guo and L-X Zhang ldquoAllelopathiceffects of ginseng root exudates on the seeds germination andgrowth of ginseng and American ginsengrdquo Allelopathy Journalvol 28 no 1 pp 13ndash20 2011

[19] Y Li S-L Liu X-F Huang and W-L Ding ldquoAllelopathy ofginseng root exudates on pathogens of ginsengrdquo Acta EcologicaSinica vol 29 no 1 pp 161ndash168 2009

[20] Q J Zhang W R Ben and L H Xu ldquoAllelopathic effects ofextract from old ginseng soil on seed germination and seedlingsgrowth in ricerdquo Journal of NorthWest A amp F University (NaturalScience Edition) vol 40 pp 49ndash54 2012

[21] H C Sharma D J Sullivan and V S Bhatnagar ldquoPopulationdynamics and natural mortality factors of the Oriental army-wormMythimna separata (Lepidoptera Noctuidae) in South-Central Indiardquo Crop Protection vol 21 no 9 pp 721ndash732 2002

[22] F M Yan Chemical Ecology Science Press Beijing China 2011[23] L Z Wen Introduction to Entomology Research Methods and

Techniques Science Press Beijing China 2010

6 Evidence-Based Complementary and Alternative Medicine

[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[25] E F Hartree ldquoDetermination of protein a modification ofthe lowry method that gives a linear photometric responserdquoAnalytical Biochemistry vol 48 no 2 pp 422ndash427 1972

[26] L Z Wen Introduction to Entomology Research Methods andTechniques Science Press Beijing China 2010

[27] J M Goodrich and N Basu ldquoVariants of glutathione s-transferase pi 1 exhibit differential enzymatic activity andinhibition by heavy metalsrdquo Toxicology in Vitro vol 26 no 4pp 630ndash635 2012

[28] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash96 1961

[29] G Vandenborre G Smagghe and E J M Van DammeldquoPlant lectins as defense proteins against phytophagous insectsrdquoPhytochemistry vol 72 no 13 pp 1538ndash1550 2011

[30] S Senthil Nathan M Young Choi H Yul Seo C HoonPaik K Kalaivani and J Duk Kim ldquoEffect of azadirachtinon acetylcholinesterase (AChE) activity and histology of thebrown planthopper Nilaparvata lugens (Stal)rdquo Ecotoxicologyand Environmental Safety vol 70 no 2 pp 244ndash250 2008

[31] R M E Vos and P J van Bladeren ldquoGlutathione S-transferasesin relation to their role in the biotransformation of xenobioticsrdquoChemico-Biological Interactions vol 75 no 3 pp 241ndash265 1990

[32] Z S Jiang Z G Yan Y Z Du and Z Z Shang ldquoEffect of 120572-terthienyl on glutathione S-transferases inHelicoverpa armigeraand Ostrinia furnacalis larvaerdquo Chinese Journal of PesticideScience vol 15 no 3 pp 76ndash79 2003

[33] J I Kim C S Jung Y H Koh and S H Lee ldquoMolecularbiochemical and histochemical characterization of two acetyl-cholinesterase cDNAs from the German cockroach Blattellagermanicardquo Insect Molecular Biology vol 15 no 4 pp 513ndash5222006

[34] K Yin E-B Ma C-R Xue H-H Wu Y-P Guo and J-Z Zhang ldquoStudy on insecticidal activities and effect on threekinds of enzymes by 5-aminolevulinic acid on oxya ChinensisrdquoAgricultural Sciences in China vol 7 no 7 pp 841ndash846 2008

[35] Z H Tang ldquoResearch status and perspectives of insect resis-tance to insecticides in Chinardquo Entomological Knowledge vol37 pp 97ndash103 2000

[36] E Shaaya and A Rafaeli ldquoEssential oils as biorationalinsecticidesmdashpotency and mode of actionrdquo in InsecticidesDesign Using Advanced Technologies pp 249ndash261 SpringerBerlin Germany 2007

[37] D L Grundy andC C Still ldquoInhibition of acetylcholinesterasesby pulegone-12-epoxiderdquo Pesticide Biochemistry and Physiol-ogy vol 23 no 3 pp 383ndash388 1985-

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

Concentration of total ginsenosides (mgmL)

100

50

0Inhi

bitio

n ra

tio (

)

24h48h

72h

5 10 20

Figure 4 Inhibition ratio of total ginsenosides on AChE activity in4th-instarM separata larvae

M separata larvae indirectly indicates damage to nerve cellswhich induce AChE photoinactivation and death caused bythe disruption of normal nerve conduction Total ginseno-sides have inhibitory allelopathic effects on M separatawhich were beneficial for researching the allelopathic poten-tial of ginsenosides

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ai Hua Zhang and Shi Qiang Tan are cofirst authors theycontributed equally to the work

Acknowledgments

This work was funded by the National Natural ScienceFoundation ofChina (nos 31070316 31100239 and 31200224)the Funded Projects for Science and Technology Devel-opment Plan of Jilin (nos 20110926 20130206030YY and20140520159JH) and the Project supported by the Ministryof Science and Technology of China (no 2011BAI03B01)

References

[1] E L Rice Allelopathy Academic Press New York NY USA1984

[2] E L Rice Biological Control of Weeds and Plant DiseasesAdvances in Applied Allelopathy University of Oklahoma PressNorman Okla USA 1995

[3] S L Peng and P Nan ldquoTerpenoids in higher plants and theirroles in ecosystemsrdquo Chinese Journal of Ecology vol 21 pp 33ndash38 2002

[4] W X Gu S S Duan and S M Luo ldquoEcological characteristicof terpenoids and their allelopathic effects to plantsrdquo Journal ofSouth China Agricultural University vol 19 pp 108ndash112 2005

[5] R Ma M Wang X Zhu X Lu and K Sun ldquoAllelopathy andchemical constituents of Ligularia virgaurea volatilerdquo ChineseJournal of Applied Ecology vol 16 no 10 pp 1826ndash1829 2005

[6] R Ma M Wang K Zhao S Guo Q Zhao and K SunldquoAllelopathy of aqueous extract from Ligularia virgaurea a

dominant weed in psychro-grassland on pasture plantsrdquo Chi-nese Journal of Applied Ecology vol 17 no 5 pp 845ndash850 2006

[7] HWei YHouG Yang andMYou ldquoRepellent and antifeedanteffect of secondary metabolites of non-host plants on Plutellaxylostellardquo Chinese Journal of Applied Ecology vol 15 no 3 pp473ndash476 2004

[8] B Ling G-C Wang J Ya M-X Zhang and G-W LiangldquoAntifeedant activity and active ingredients against plutellaxylostella from Momordica charantia leavesrdquo Agricultural Sci-ences in China vol 7 no 12 pp 1466ndash1473 2008

[9] X D Li and S H Zhao ldquoThe toxic effects and mode ofazadirachtin on insectsrdquo Journal of South China AgriculturalUniversity vol 17 pp 118ndash122 1995

[10] X Zhang X L Wang and J T Fen ldquoThe development ofbotanical insecticidesmdashtoosendaninrdquo Journal of Northwest Sci-Tech University of Agriculture and Forestry vol 21 pp 1ndash5 1993

[11] W L Wang S H Zhao J Han and Y S Xu ldquoEffects of severalInsecticidal principles from chinaberry melia azedarach onthe imported cabbage worm and Asiatic corn borer Ostriniafurnacalisrdquo Acta Phytophylacica Sinica vol 19 pp 359ndash3641992

[12] Y-L Shi and W-P Wang ldquoBiological effects of toosendaninan active ingredient of herbal vermifuge in Chinese traditionalmedicinerdquo Acta Phytophylacica Sinica vol 58 no 5 pp 397ndash406 2006

[13] L Feng-Jie Z Ai-Hua X Yong-Hua and Z Lian-Xue ldquoAllelo-pathic effects of ginsenosides on in vitro growth and antioxidantenzymes activity of ginseng callusrdquo Allelopathy Journal vol 26no 1 pp 13ndash22 2010

[14] P G Xiao ldquoPreliminary investigation of wild ginseng in theNortheastrdquoActa Pharmaceutica Sinica vol 6 pp 340ndash351 1962

[15] T S Wang Chinese Ginseng Liaoning Science and TechnologyPress Shenyang China 2001

[16] A-H Zhang F-J Lei Y-H Xu G-X Zhou and L-X ZhangldquoEffects of ginsenosides on the germinating of ginseng seeds andon the activity of antioxidant enzymes of the radicles of ginsengseedlings in vitrordquoActa Ecologica Sinica vol 29 no 9 pp 4934ndash4941 2009

[17] A H Zhang F J Lei S W Fang M H Jia and L XZhang ldquoEffects of ginsenosides on the growth and activity ofantioxidant enzymes in American ginseng seedlingsrdquo Journal ofMedicinal Plants Research vol 5 pp 3217ndash3223 2011

[18] A-H Zhang F-J Lei Z-X Guo and L-X Zhang ldquoAllelopathiceffects of ginseng root exudates on the seeds germination andgrowth of ginseng and American ginsengrdquo Allelopathy Journalvol 28 no 1 pp 13ndash20 2011

[19] Y Li S-L Liu X-F Huang and W-L Ding ldquoAllelopathy ofginseng root exudates on pathogens of ginsengrdquo Acta EcologicaSinica vol 29 no 1 pp 161ndash168 2009

[20] Q J Zhang W R Ben and L H Xu ldquoAllelopathic effects ofextract from old ginseng soil on seed germination and seedlingsgrowth in ricerdquo Journal of NorthWest A amp F University (NaturalScience Edition) vol 40 pp 49ndash54 2012

[21] H C Sharma D J Sullivan and V S Bhatnagar ldquoPopulationdynamics and natural mortality factors of the Oriental army-wormMythimna separata (Lepidoptera Noctuidae) in South-Central Indiardquo Crop Protection vol 21 no 9 pp 721ndash732 2002

[22] F M Yan Chemical Ecology Science Press Beijing China 2011[23] L Z Wen Introduction to Entomology Research Methods and

Techniques Science Press Beijing China 2010

6 Evidence-Based Complementary and Alternative Medicine

[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[25] E F Hartree ldquoDetermination of protein a modification ofthe lowry method that gives a linear photometric responserdquoAnalytical Biochemistry vol 48 no 2 pp 422ndash427 1972

[26] L Z Wen Introduction to Entomology Research Methods andTechniques Science Press Beijing China 2010

[27] J M Goodrich and N Basu ldquoVariants of glutathione s-transferase pi 1 exhibit differential enzymatic activity andinhibition by heavy metalsrdquo Toxicology in Vitro vol 26 no 4pp 630ndash635 2012

[28] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash96 1961

[29] G Vandenborre G Smagghe and E J M Van DammeldquoPlant lectins as defense proteins against phytophagous insectsrdquoPhytochemistry vol 72 no 13 pp 1538ndash1550 2011

[30] S Senthil Nathan M Young Choi H Yul Seo C HoonPaik K Kalaivani and J Duk Kim ldquoEffect of azadirachtinon acetylcholinesterase (AChE) activity and histology of thebrown planthopper Nilaparvata lugens (Stal)rdquo Ecotoxicologyand Environmental Safety vol 70 no 2 pp 244ndash250 2008

[31] R M E Vos and P J van Bladeren ldquoGlutathione S-transferasesin relation to their role in the biotransformation of xenobioticsrdquoChemico-Biological Interactions vol 75 no 3 pp 241ndash265 1990

[32] Z S Jiang Z G Yan Y Z Du and Z Z Shang ldquoEffect of 120572-terthienyl on glutathione S-transferases inHelicoverpa armigeraand Ostrinia furnacalis larvaerdquo Chinese Journal of PesticideScience vol 15 no 3 pp 76ndash79 2003

[33] J I Kim C S Jung Y H Koh and S H Lee ldquoMolecularbiochemical and histochemical characterization of two acetyl-cholinesterase cDNAs from the German cockroach Blattellagermanicardquo Insect Molecular Biology vol 15 no 4 pp 513ndash5222006

[34] K Yin E-B Ma C-R Xue H-H Wu Y-P Guo and J-Z Zhang ldquoStudy on insecticidal activities and effect on threekinds of enzymes by 5-aminolevulinic acid on oxya ChinensisrdquoAgricultural Sciences in China vol 7 no 7 pp 841ndash846 2008

[35] Z H Tang ldquoResearch status and perspectives of insect resis-tance to insecticides in Chinardquo Entomological Knowledge vol37 pp 97ndash103 2000

[36] E Shaaya and A Rafaeli ldquoEssential oils as biorationalinsecticidesmdashpotency and mode of actionrdquo in InsecticidesDesign Using Advanced Technologies pp 249ndash261 SpringerBerlin Germany 2007

[37] D L Grundy andC C Still ldquoInhibition of acetylcholinesterasesby pulegone-12-epoxiderdquo Pesticide Biochemistry and Physiol-ogy vol 23 no 3 pp 383ndash388 1985-

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

[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[25] E F Hartree ldquoDetermination of protein a modification ofthe lowry method that gives a linear photometric responserdquoAnalytical Biochemistry vol 48 no 2 pp 422ndash427 1972

[26] L Z Wen Introduction to Entomology Research Methods andTechniques Science Press Beijing China 2010

[27] J M Goodrich and N Basu ldquoVariants of glutathione s-transferase pi 1 exhibit differential enzymatic activity andinhibition by heavy metalsrdquo Toxicology in Vitro vol 26 no 4pp 630ndash635 2012

[28] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash96 1961

[29] G Vandenborre G Smagghe and E J M Van DammeldquoPlant lectins as defense proteins against phytophagous insectsrdquoPhytochemistry vol 72 no 13 pp 1538ndash1550 2011

[30] S Senthil Nathan M Young Choi H Yul Seo C HoonPaik K Kalaivani and J Duk Kim ldquoEffect of azadirachtinon acetylcholinesterase (AChE) activity and histology of thebrown planthopper Nilaparvata lugens (Stal)rdquo Ecotoxicologyand Environmental Safety vol 70 no 2 pp 244ndash250 2008

[31] R M E Vos and P J van Bladeren ldquoGlutathione S-transferasesin relation to their role in the biotransformation of xenobioticsrdquoChemico-Biological Interactions vol 75 no 3 pp 241ndash265 1990

[32] Z S Jiang Z G Yan Y Z Du and Z Z Shang ldquoEffect of 120572-terthienyl on glutathione S-transferases inHelicoverpa armigeraand Ostrinia furnacalis larvaerdquo Chinese Journal of PesticideScience vol 15 no 3 pp 76ndash79 2003

[33] J I Kim C S Jung Y H Koh and S H Lee ldquoMolecularbiochemical and histochemical characterization of two acetyl-cholinesterase cDNAs from the German cockroach Blattellagermanicardquo Insect Molecular Biology vol 15 no 4 pp 513ndash5222006

[34] K Yin E-B Ma C-R Xue H-H Wu Y-P Guo and J-Z Zhang ldquoStudy on insecticidal activities and effect on threekinds of enzymes by 5-aminolevulinic acid on oxya ChinensisrdquoAgricultural Sciences in China vol 7 no 7 pp 841ndash846 2008

[35] Z H Tang ldquoResearch status and perspectives of insect resis-tance to insecticides in Chinardquo Entomological Knowledge vol37 pp 97ndash103 2000

[36] E Shaaya and A Rafaeli ldquoEssential oils as biorationalinsecticidesmdashpotency and mode of actionrdquo in InsecticidesDesign Using Advanced Technologies pp 249ndash261 SpringerBerlin Germany 2007

[37] D L Grundy andC C Still ldquoInhibition of acetylcholinesterasesby pulegone-12-epoxiderdquo Pesticide Biochemistry and Physiol-ogy vol 23 no 3 pp 383ndash388 1985-

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