research article the gastrointestinal irritation of polygala...

Research ArticleThe Gastrointestinal Irritation of Polygala Saponins andIts Potential Mechanism In Vitro and In Vivo

Li Wen1 Nan Xia2 PeiPei Tang1 Yi Hong1 ZiZhen Wang1 YaJie Liu1 YanJu Liu1

JianJun Liu1 and XiangQiong Li1

1Ministry of Education Key Laboratory of Traditional Chinese Medicine Resources and CompoundsHubei College of Traditional Chinese Medicine Wuhan Hubei 430061 China2Wuhan Institute of Biological Products Co Ltd Wuhan Hubei 430207 China

Correspondence should be addressed to Li Wen wenlihu123123163com

Received 26 August 2014 Revised 31 October 2014 Accepted 27 November 2014

Academic Editor Dimitrios P Bogdanos

Copyright copy 2015 Li Wen et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Processing alters the pharmacological activity and reduces the gastrointestinal toxicity of the polygalae To investigate the effect ofprocessing different glycosyl substituent productswere testedHypnotic and subhypnotic doses of pentobarbital-induced sleep testson mice were used to evaluate the sedative activity of polygala saponins with different glycosyl substituents isolated gut motilityexperiment was employed to study excitatory effects of different polygala saponins the gastrointestinal irritation effects of differentpolygala saponins were compared by measuring the levels of gastric PGE2 and intestinal TNF-120572 on mice When compared withcontrol Onjisaponin B (OJB) and tenuifolin (TEN) but not senegenin (SNG) significantly increased the number of sleeping miceand prolonged the sleeping time (119875 lt 005) 80 40 and 20mgL of OJB and 80mgL of TEN but not SNG obviously changed theamplitude and frequency of isolated jejunum (119875 lt 005) all the three compounds significantly decreased the level of gastric PGE2but had no obvious influences on the reduction of intestinal TNF-120572 level For sedative and hypnotic effects OJBgtTENgt SNG forthe protection form gastrointestinal irritation and damages OJBgtTENgt SNGTherefore in processing Polygala glycosyl breakingmay be related to the decline of pharmacological activity and gastrointestinal toxicity of polygala saponins

1 Introduction

Radix Polygalae comes from the dried roots of Polygalatenuifolia willd or Polygala sibirica L [1] which is often usedclinically for insomnia forgetfulness phlegm palpitationand so forth [2] However research shows that long-termand large doses of Radix Polygalae can cause gastric mucosalinjury [3ndash5] Nonprocessed Radix Polygalae caused obviousinhibition of gastrointestinal motility and led to flatulencethinner or necrosis intestinal [6ndash8] Pharmacy workersalways process Radix Polygalae to reduce gastrointestinalirritation such as honey-stir-baked [9 10] The major com-ponent of total saponins in Radix Polygalae is triterpenoidsaponin [11ndash18] whose nuclear parent is senegenin (SNG) Inthe process of extraction the glycosyl link at C28 breaks easilyand then produces more TEN [5] and the glycosyl link at C3breaks and becomes SNG (Figure 1)

So we selected three compounds namely OnjisaponinB (OJB) TEN and SNG to evaluate the sedative activityof the three compounds using hypnotic and subhypnoticdoses of pentobarbital-induced sleep test on mice to studyexcitatory effects of them by employing isolated gut motilityexperiment and to compare the toxic effects of them bymeasuring the levels of gastric PGE2 and intestinal TNF-120572in mice

2 Materials and Methods

21 Materials HPLC with UVD170U detector quaternarylow pressure gradient pump (Dionex-P680) purchased fromDionex Company (USA)

Radix Polygalae was purchased from Wuhan MedicinalMaterials Company (Wuhan China) and identified by DrWenjie Tang (Department of PharmacologyHubei College of

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 918048 8 pageshttpdxdoiorg1011552015918048

2 BioMed Research International

Traditional Chinese Medicine Wuhan China) The voucherspecimen was deposited at the Herbarium of Hubei college ofTraditional Chinese Medicine under the acquisition numberof WWZ0406

SNG and TEN (gt98 purity) were purchased fromChengdu Pusi Co Ltd (Chengdu China) OJB was isolatedand purified by Ministry of Education Key Laboratory ofTraditional Chinese Medicine Resources and CompoundsHubei College of Traditional Chinese Medicine

Pentobarbital sodium (Lot number F20030816) wasprovided by Sinopharm Shanghai Chemical Reagents CoLtd (Shanghai China) estazolam (Lot number 20111029)was from Anhui Bangning Pharmaceuticals (Anhui China)ELISA assay kits for PGE2 and TNF-120572 were purchasedfrom Sigma Chemicals Co (St Louis MO USA) Acetyl-choline (Lot number 091201) was purchased from ShanghaiInstitute of Biochemistry CAS (Shanghai China) BaCl2(Lot number 20110221) was purchased from Tianjin DeenChemical Reagent Co Ltd (Anhui China) Phentolamine(Lot number 101104) was purchased from Shanghai FudanForward Science and Technology CO Ltd (ShanghaiChina) Atropine (Lot number 1005051) was purchased fromZhengzhou Lingrui Pharmaceutical Co Ltd (ZhengzhouChina) Neostigmine (BCBD8573V) was purchased fromSigma-Aldrich Co (St Louis MO USA)

22 Preparation of Test Samples Preparation of PTS [1]1 kg of pieces of Polygala tenuifolia Wild were extractedthree times (2 htime) by 3-fold volumes of 85 EtOH andthe extracted solution was pooled Ethanol was recycled byevaporating until there was no alcohol smell and then wasdissolved in 1000mL of water Acetic ether saturated bywater was extracted 3 times (300mLtime) and acetic etherwas discarded and combined with aqueous phase Waterwas added to 1000mL then eluted on a D101 macroporousresin column with H

2O 30 EtOH and 70 EtOH using

3 times the column volume 1 BVh The fraction eluted by70EtOHwas condensed until there was no alcohol smell toobtain powderA (the yieldwas 532 g100 g of driedmedicinalherbs) The content of total saponins was identified to reachto 52 (calculate by the content of tenuifolin (TEN)) [13]powder A was eluted by HPLC with preparative column andthe specific parameters were as follows [19ndash22] the mobilephase of water containing 05 acetic acid and acetonitrile(38 62) C18 column (250 lowast 46mm) and evaporative light-scattering detector After the above steps compound B wasobtained andmeasured byHPLC (C18 column (250lowast46mm)area normalization method was above 95 the mobile phaseof methanol and water containing 005 phosphoric acidsolution (65 35)) As shown in Figures 1 and 2 compoundB was further identified to be polygala saponins B (OJBC75H112O35) by 13C and 1H NMR as well as infraredspectrometry (Figure 1) [23]

Estazolam (EST) was dissolved by normal saline to pre-pare the solution with concentration of 05

Preparation of OJB TEN and SNG solution was asfollows for investigating pharmacological effects on mice

all three compounds were dissolved by normal saline (con-taining 05 Tween 80) to prepare the solution with a con-centration of 20mgmL respectively the high concentrationdiluted to 20mg powdered material per mL as the lowconcentration for mice the dosing volume for mice was10mLkg For determining gastrointestinal irritation pow-dered OJB TEN or SNG dissolved in water (containing 05Tween 80) and diluted to a concentration of 20mg powderedmaterial per mL as the high concentration for mice Thesolution was prepared and diluted to different concentrations(8 4 and 2mgmL) for isolated gut motility experiment200 uL was added into per 20mL Tyrodersquos solution

Preparation of polygala total saponins (PTS) was asfollows powdered PTS dissolved in water (containing 05Tween 80) and diluted to a concentration of 20mg powderedmaterial per mL as the high concentration for mice thesolution was prepared and diluted to different concentrations(8 4 and 2mgmL) for isolated gut motility experiment200 uL was added into per 20mL Tyrodersquos solution

23 Animals Male Kunming mice weighing 18ndash22 g andmale rabbit weighing 2ndash25 kgwere procured from theCenterfor Disease Prevention andControl inHubei Province China(reg number SCXK (Hubei) 2008-0005) The animals werehoused at 22 plusmn 2∘C under a 12 h light12 h dark cycle andwith access to food and water ad libitum The mice wereacclimatized and habituated to the laboratory for at least aweek before being subjected to tests and were used only oncethroughout the experimentsThe study was carried out alongwith the ldquoPrinciples of Laboratory Animal Carerdquo [24] Theexperiment was performed according to the guidelines of theCommittee for the Purpose of Control and Supervision ofExperiments on Animals and approved by the Animal Exper-iment Ethical Committee of Hubei University of Chinesemedicine

24 Potentiation of SPB TEN and SNG on Pentobarbital-Induced Sleep in Mice Male Kunming mice were randomlydivided into 8 groups (8 mice per group) In the controlgroup mice received equal volume of normal saline whilein the seven other groups mice were intragastrically treatedwith 200mgkg of OJB 400mgkg of OJB 200 of mgkgTEN 400 ofmgkg TEN 200 ofmgkg SNG 400mgkg ofSNG and 400mgkg of PTS for one time 30min beforepentobarbital treatment 40mgkg pentobarbital was usedas hypnotic dose to treat half of the animals and theothers were administered with 25mgkg which was used assubhypnotic dose [25ndash29]The subhypnotic dosed mice wereobserved for sleep onset within 15min mouse losing rightingreflex over 1min was considered to be asleep The sleepingtime of animals receiving hypnotic dose was also observedThe sleeping latency was recorded from the injection ofpentobarbital to the sleep onset and the sleeping time wasrecorded from the loss of righting reflex to recovery Theresults were shown in Tables 1 and 2

25 Influence of Different Polygala Saponins on Isolated GutMotility of Rabbit The rabbit ileumwas dissected out and cut

BioMed Research International 3

OHO

HO

H

OH

OH

O O

O

OH

HO

H

H

OH

O

O

O

O

O

O

O

OOH

HO

O

OH

O

OH

OH

OH

O O

HO O O

OH

OH

OH

HO

OH

HO

H

HO

HO

HO

O

O

HOH

O

OH

O

OHH

HO

HO

H

H

HOH

OH COOH

HOOC

Onjisaponin B (OJB)

Tenuifolin (TEN) Senegenin (SNG)

Figure 1 Molecular structure of Onjisaponin B (OJB)

into 2 cm segment for preparing isolated intestinal smoothmuscle sample according to previous description [30] Thenormal motion curve of intestinal segment was recordedwhen spontaneous rhythmic contraction reached to equi-librium Different solutions with a volume of 02mL wereadded into the bath in order acetylcholine (1 10000) BaCl

2

(20) dopamine (10mgmL) phentolamine (10mgmL)atropine (05mgmL) and neostigmine (10mgmL) Thesegments with normal contraction after drug stimulationwere subjected to the following experiment The changes ofcurve before and after compound treatment were recorded

Specifically the mean value of tension within 3min intervalbefore and after treatment was obtained to calculate thechanging ratio as follows

Increasing of intestinal tension (IIT) = Mean oftensionafter treatment minusMean of tensionbefore treatmentChanging ratio of intestinal tension (CRIT) = (Meanof tension after treatment minus Mean of tension beforetreatment)Mean of tension before treatment times 100

The results were shown in Table 3 and Figure 3


5 10 15 20 25 30

OJB

Figure 2 Measurement of Onjisaponin B (OJB) content by HPLCthe content of OJB is 95

Table 1 Effects of polygala saponins with different glycosyl onpentobarbital-induced sleeping duration in mice

Groups Dose(mgkg)

Percentage of fallingasleep ()

Control mdash 10

OJB 200 70a

100 70a

TEN 200 60a

100 70a

SNG 200 30100 20

EST 1 802a

Mark mice were respectively administrated by intragastrical administration(IG) with high and low doses (200 and 100mgkg) of the Onjisaponin B(OJB) tenuifolin (TEN) senegenin (SNG) and 1mgkg of estazolam (EST)for one time 30min before pentobarbital treatment Mice were administeredwith 25mgkg which was used as subhypnotic dose Negative control groupswere treated with equal volume of vehicle 119899 = 10 a119875 lt 005 2a119875 lt 001versus control

Table 2 Effects of polygala saponins with different glycosyls onpentobarbital-induced sleeping animal amount in mice

Groups Dose (mgkg) Sleep latency Sleeping time (min)Control mdash 82 plusmn 20 182 plusmn 90

OJB 200 78 plusmn 26 356 plusmn 113a

100 70 plusmn 17 397 plusmn 142a

TEN 200 84 plusmn 34 312 plusmn 74a


SNG 200 87 plusmn 29 271 plusmn 111a

100 79 plusmn 26 188 plusmn 110EST 1 94 plusmn 41 268 plusmn 46a

Markmicewere respectively administrated by intragastrical administration(IG) with high and low doses (200 and 100mgkg) of the Onjisaponin B(OJB) tenuifolin (TEN) senegenin (SNG) and 1mgkg of estazolam (EST)for one time 30min before pentobarbital treatment Mice were administeredwith 40mgkg Negative control groups were treated with equal volume ofvehicle 119899 = 10 a119875 lt 005 versus control

26 Influence of Different Polygala Saponins on Gastric PGE2and Intestinal TNF-120572 Levels Male Kunming mice were ran-domly divided into 5 groups (10 mice per group) controlOJB TEN SNG and polygala total saponins Animals were

intragastrically treated with 200mgkg of OJB TEN SNGand total polygala saponins for one time while the controlmice received equal volume of normal saline (except polygalatotal saponins group animals are the ones given 25mgkgpentobarbital in 24) After 30min the animals were sacri-ficed and the stomachs and intestines were dissected out andstored at minus80∘C 1mg of tissues were homogenized in ice-cold buffer and then subjected to the ELISA measurement ofgastric PGE-2 and intestinal TNF-120572 levels The results wereshown in Figures 4 and 5

27 Statistical Analysis All data are presented as means plusmnSEM One-way ANOVA was first used to assess the dif-ferences among multiple groups followed by the Dunnettpost hoc test The significance of normal and treated groupswas using the studentrsquos 119905-test Only for subhypnotic dosageof pentobarbital-treated test chi- square test was used tocompare the number of falling asleep between control groupand each of other groups The data was assessed by thestatistical package from the Social Science Software (SPSS)program The levels of significance are indicated by differentletters

3 Results

31 Effects of Different Polygala Saponins on Pentobarbital-Induced Sleep in Mice As shown in Table 1 both OJB andTEN but not SNG significantly prolonged pentobarbital-induced sleeping duration with hypnotic dose when com-pared with control (119875 lt 005) Therefore OJB and TENcould potentiate pentobarbital-induced sleep by prolongingsleeping duration

As shown in Table 2 both OJB and TEN significantlyincreased the number of sleeping mice when compared withcontrol (119875 lt 001) indicating a potentiated effect of OJBand TEN on pentobarbital-induced sleep However SNG hadno obvious effect on the number of sleeping mice whencompared with control (119875 gt 005)

32 Influence of Different Polygala Saponins on Isolated GutMotility of Rabbit As shown in Table 3 when comparedwith control 80 40 and 20mgL of OJB increased theintestinal tension by 4813 2049 and 1284 within 30 srespectively (119875 lt 005 119875 lt 001 or 119875 lt 0001) 80mgLof OJB elevated the intestinal tension by 104 g within 30 scomparing to the control (119875 lt 005) The contractive ampli-tude presented irregular results as shown in Figure 3 SNGexhibited no significant influences on tension amplitudeand frequency Therefore only OJB had the most profoundimpact on gastrointestinal motility

33 Influence on Gastrointestinal PGE2 and TNF-120572 Levels Asshown in Figure 4 when compared to control 200mgkgof OJB TEN or SNG could significantly reduce the gastricPGE2 level (119875 lt 005) and the effects of OJB were greaterthan that of TEN (119875 lt 005)


Acetylcholine(20)

Dopamine Phentolamine Atropine Neostigmine Control

20 40 8020 40 80

OJB (mgL) TEN (mgL)

SNG (mgL) Polygala total saponins (mgL)

20 40 8020 40 80

(1 10000) (10mgmL) (10mgmL) (05mgmL) (10mgmL)200120596 were added into per 20mL Tyrodersquos solution

BaCl2

Figure 3 Effects of Onjisaponin B (OJB) tenuifolin (TEN) and senegenin (SNG) on isolated gut motility in rabbit Mark the solution wasprepared and diluted to different concentrations (8 4 and 2mgmL) for isolated gut motility experiment 200 uL were added into per 20mLTyrodersquos solution measuring the average tension 3min before and after the administration by the method of interval measurement

Table 3 Influence of polygala saponins with different glycosyls Onjisaponin B (OJB) tenuifolin (TEN) and senegenin (SNG) on isolatedintestinal tension in rabbit in vitro

Groups Concentration (mgL) IIT (g) CRIT () Amplitude (g)Control mdash 004 plusmn 029 minus053 plusmn 821 196 plusmn 055

SNG20 minus012 plusmn 026 minus014 plusmn 1298 152 plusmn 03140 minus002 plusmn 037 160 plusmn 1375 211 plusmn 04380 027 plusmn 049 831 plusmn 1614 202 plusmn 093

TEN20 009 plusmn 080 minus306 plusmn 3022 203 plusmn 08440 015 plusmn 030 minus374 plusmn 1136 187 plusmn 05080 057 plusmn 053a 1828 plusmn 2644 211 plusmn 044

OJB20 025 plusmn 042 1284 plusmn 1534a 238 plusmn 05640 049 plusmn 0282a 2049 plusmn 16612a 225 plusmn 03180 104 plusmn 0623a 4813 plusmn 34393a 236 plusmn 071

PTS20 minus003 plusmn 052 minus218 plusmn 1807 182 plusmn 02740 026 plusmn 051 896 plusmn 1654 233 plusmn 05480 058 plusmn 0472a 192 plusmn 8473a 190 plusmn 043

Mark the solution was prepared and diluted to different concentrations (8 4 and 2mgmL) for isolated gut motility experiment 200 uL was added into per20mL Tyrodersquos solution measuring the average tension 3min before and after the administration by the method of interval measurement (119899 = 8 mean plusmnSEM) a119875 lt 005 2a119875 lt 001 3a119875 lt 001 versus control Increasing of intestinal tension (IIT) = Mean of tensionafter treatment minus Mean of tensionbefore treatmentChanging ratio of intestinal tension (CRIT) = (Mean of tensionafter treatment minus Mean of tensionbefore treatment)Mean of tensionafter treatment times 100


45004000350030002500200015001000

5000

PGE2

(pg

tissu

e)

Control OJB(200) OJB(200) SNG(200) PTS(200)(mgkg)

lowast

lowast

lowast

Figure 4 Influence of polygala saponins with different glycosylon gastric PGE2 levels in mice Mark mice were respectivelyadministrated by intragastrical administration (IG) with the doses(200mgkg) of Onjisaponin B (OJB) tenuifolin (TEN) and sene-genin (SNG) negative control groups were treated with equalvolume of vehicle After the gastrointestinal tissues in micewere homogenized we determine PGE2 concentration of gastrichomogenate Each column is mean plusmn standard deviation for 10 micein each group lowast119875 lt 005 versus control

100

80

60

40

20

0Control OJB(200) TEN(200) SNG(200) PTS(200)

(mgkg)

TNF-120572

(pg

g tis

sue)

Figure 5 Influence of polygala saponins with different glycosylson gastric TNF-120572 levels in mice Mark mice were respectivelyadministrated by intragastrical administration (IG) with the doses(200mgkg) of Onjisaponin B (OJB) tenuifolin (TEN) and sene-genin (SNG) Negative control groups were treated with equalvolume of vehicle After the gastrointestinal tissues in mice werehomogenized we determine TNF-120572 levels of gastric homogenateEach column ismeanplusmn standard deviation for 10mice in each group

As shown in Figure 5 when compared to control200mgkg of OJB TEN and SNG had no significant effecton TNF-120572 level (119875 gt 005)

4 Discussion

In routine clinical usage Radix Polygalae can cause mildnausea If it is taken in larger doses it can cause nausea vom-iting abdominal distension diarrhea and adverse reaction ofhemolysis In China pharmacyworkers always process RadixPolygalae to reduce gastrointestinal irritation such as honey-stir-baked [9] because in the course of processing drugs theglycosyl link at C28 of PTS breaks easily and then producesmore tenuifolin (TEN) that is glycosyl at C3 which becomes

senegenin (SNG) on hydrolysis under alkaline condition(Figure 1) We selected these three compounds to identifythe effects of glycosyls on pharmacological and toxic effectsof polygala saponins We take the following experiments(a) hypnotic and subhypnotic doses of pentobarbital-inducedsleep tests were used to evaluate the sedative activities ofthese three compounds (b) isolated gut motility experimentwas employed to compare the gastrointestinal irritation ofthese three compounds and (c) the toxic effects of differentpolygala saponinswere determined bymeasuring the levels ofgastrointestinal mucosa protecting factor PGE2 and inflam-matory cytokine TNF-120572

This study compared the activities of them in the pen-tobarbital-induced sleeping experiment The results showedboth OJB and TEN potentiated pentobarbital-induced sleep-ing effect and also prolonged the sleeping duration butthe activities of SNG were limited It is suggested thatthe presence of glycosyl may be critical for the activity ofsaponins This phenomenon is very common in traditionalmedicine For example rhubarb dianthrone glycosides theactive component of rheum is hydrolyzed to aglycone in thelarge intestine then leading to purgationwhile the aglycone inrheum has no such purgative effect and the major reason isdue to the stereospecific blockade of glycosyls in dianthroneglycosides which protects it from gastric acid destructionTherefore it could be deduced that the glycosyls in polygalasaponins may have the function that protects it from beingdestroyed by gastric acid

The results of isolated gut motility test revealed thatOJB had much more profound impact on gastrointestinaltension than TEN whereas SNG had almost no effect onisolated gut and OJB of 80mgL can cause irregular andintense systole of isolated gut All the above indicate thatthe amount of glycosyl might highly correlate with increasedgastrointestinal irritation [31]

PGE2 is a protector of gastric mucosa However it isalso an inflammatory factor OJB and TEN could signifi-cantly reduce the level of gastric PGE2 which results inthe deprivation of protection to gastric mucosa and finallycauses gastric injury SNG without glycosyl has a weakereffect on the reduction of gastric PGE2 level than OJB andTEN dose We speculate that the presence of glycosyl couldirritate gastrointestinal itself which may be associated withthe gastric mucosa injury caused by saponins

TNF-120572 accelerates chemotaxis and necrosis of macro-phage and also promotes inflammation In the present studyOJB TEN and SNG could not significantly reduce the TNF-120572 level suggesting that polygala saponins with glycosylscaused gastrointestinal irritation via reducing PGE2 levelas well as its gastrointestinal protection but not generatinginflammatory mediator (TNF-120572)

Combined with many researchersrsquo study we infer thatpolygala saponins cannot cause gastrointestinal ulcer byproducing inflammatory irritation The use of a high doseof Polygala saponins leads to gastrointestinal congestionswelling which is mainly produced by decreasing the gastricmucosal protective factor PGE2 and has the characteristicof nonsteroidal anti-inflammatory drugs Thus we infer thatit may have anti-inflammatory effect making it possible


to be against Alzheimerrsquos hypertension and so forth Thestrength of three compoundrsquos Sedative activities is OJB gtTEN gt SNG and the strength of gastric ulcers irritationcaused by reducing the content of gastrointestinal PGE2 isalso OJB gt TEN gt SNG In summary just as our anticipationwhen Radix Polygalae was fried with honey it should havesomething to do with the fact that Radix Polygalae saponinhydrolyzes to TEN and SNG So Radix Polygalae saponinrsquosirritation to gastric mucosa decreased and its sedative effectsdecreased tooThepresent study also demonstratedTENhadfavorable sedative andhypnotic activities andpossessed lowergastrointestinal irritation than OJB which made TEN to be apotential substitute for polygala

Conflict of Interests

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

Acknowledgments

This study is supported by National Natural Science Foun-dation of China (no 81102888) The authors also appreciatethe help from colleagues in Ministry of Education KeyLaboratory of Traditional Chinese Medicine Resources andCompounds

References

[1] Y P Liu D G Wan D Yan and C Peng ldquoStudies on purifica-tion of saponins from radix polygalae with macroporous resinrdquoChinese Journal of Experimental Traditional Medical Formulaevol 10 pp 3ndash6 2004

[2] P Liu Y Hu D H Guo et al ldquoPotential antidepressant prop-erties of Radix Polygalae (Yuan Zhi)rdquo Phytomedicine vol 17 no10 pp 794ndash799 2010

[3] F Huang Y Xiong L Xu S Ma and C Dou ldquoSedative andhypnotic activities of the ethanol fraction fromFructus Schisan-drae in mice and ratsrdquo Journal of Ethnopharmacology vol 110no 3 pp 471ndash475 2007

[4] S-J Guan X-P Yan and J-K Lin ldquoStudy on acute toxicity testof different processed products of Radix polygalaerdquo ZhongguoZhong Xi Yi Jie He Za Zhi vol 32 no 3 pp 398ndash401 2012

[5] J Wang X M Liu Q Chang L W Wang and R L PanldquoChemical constituents from alkaline hydrolysis products oftotal saponins in Radix Palygalaerdquo Central South Pharmacy no11 pp 811ndash814 2010

[6] H P Zhao J Wang and H HWu ldquoEffects of crude radix poly-galae saponins and honey-stir-baked radix polygalae on PGENO andCa2+-ATPase in gaster-tissue of ratrdquo LishizhenMedicineand Materia Medica Research vol 18 pp 260ndash262 2007

[7] L Wen and C R Shu ldquoSedative and hypnotic activities of theethyl acetate fraction radix polygalae in micerdquo Herald of Med-icine vol 25 pp 998ndash999 2006

[8] L Wen ldquoScreening the anxiolytic fraction from radix poly-galaerdquo Chinese Journal of Traditional Medical Science andTechnology vol 13 pp 401ndash402 2006

[9] H H Wu J Wang and X W Liu ldquoEffects on acute toxicityand gastrointestinalmotility between variousHoney-stir-baked

and Crude Radix Polygalaerdquo Journal of Sichuan of TraditionalChinese Medicine vol 24 pp 16ndash18 2006

[10] W-F Yang JWang L-N Liu andWXiao ldquoStudy of cajal inter-stitial cells in stomach and small intestine of rats with crudehoney-stir-baked radix polygalae and its saponinsrdquo Journal ofChinese medicinal materials vol 34 no 1 pp 33ndash36 2011

[11] M Kuroda T Shizume and Y Mimaki ldquoNew acylated triter-pene glycosides from the roots of Polygala tenuifoliardquo NaturalProducts Communications vol 9 no 3 pp 379ndash382 2014

[12] C-I Lee J-Y Han J T Hong and K-W Oh ldquo345-Trimethox-ycinnamic acid (TMCA) one of the constituents of Radix Poly-galae enhances pentobarbital-induced sleeping behaviors viaGABAAergic systems inmicerdquoArchives of Pharmacal Researchvol 36 no 10 pp 1244ndash1251 2013

[13] C-J Li J-Z Yang S-S Yu et al ldquoTriterpenoid saponins andoligosaccharides from the roots of Polygala tenuifolia WilldrdquoChinese Journal of Natural Medicines vol 9 no 5 pp 321ndash3282011

[14] Y Jiang and P-F Tu ldquoXanthone O-glycosides from Polygalatenuifoliardquo Phytochemistry vol 60 no 8 pp 813ndash816 2002

[15] Y Jiang L Liu and P F Tu ldquoChemical constituents of Polygalatenuifolia IIIrdquo Chinese Journal of Natural Medicines vol 1 pp142ndash145 2003

[16] X-L Sun H Ito T Masuoka C Kamei and T Hatano ldquoEffectof Polygala tenuifolia root extract on scopolamine-inducedimpairment of rat spatial cognition in an eight-arm radial mazetaskrdquo Biological and Pharmaceutical Bulletin vol 30 no 9 pp1727ndash1731 2007

[17] K Y Zhu S L Xu R C Choi A L Yan T T-X Dong andK W-K Tsim ldquoKai-xin-san a Chinese herbal decoction con-taining Ginseng Radix et Rhizoma Radix Polygalae AcoriTatarinowii Rhizoma and Poria stimulates the expressionand secretion of neurotrophic factors in cultured astrocytesrdquoEvidence-based Complementary and Alternative Medicine vol2013 Article ID 731385 9 pages 2013

[18] H Zhang THan L Zhang et al ldquoEffects of tenuifolin extractedfrom radix polygalae on learning andmemory a behavioral andbiochemical study on aged and amnesic micerdquo Phytomedicinevol 15 no 8 pp 587ndash594 2008

[19] L Lin L Yan H Zhang et al ldquoSimultaneous analysis of poly-gala acid senegenin and 361015840-disinapoylsucrose in rat plasma byliquid chromatography-tandemmass spectrometry applicationto a pharmacokinetic study after oral administrationrdquo Biomed-ical Chromatography vol 28 no 5 pp 594ndash600 2014

[20] J Li X Dong Y Jiang et al ldquoSimultaneous determination ofphenols in Radix Polygalae by high performance liquid chro-matography quality assurance of herbs from different regionsand seasonsrdquo Journal of Separation Science vol 30 no 16 pp2583ndash2589 2007

[21] Y Ling Z Li M Chen Z Sun M Fan and C Huang ldquoAnal-ysis and detection of the chemical constituents of Radix Poly-galae and their metabolites in rats after oral administrationby ultra high-performance liquid chromatography coupledwith electrospray ionization quadrupole time-of-flight tandemmass spectrometryrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 85 pp 1ndash13 2013

[22] C Sun X Peng N Zhu et al ldquoQuantitative determination ofsibricose A

5and sibricose A

6in Radix Polygalaerdquo Zhongguo

Zhongyao Zazhi vol 37 no 11 pp 1607ndash1609 2012[23] J Liu X Yang J HeM Xia L Xu and S Yang ldquoStructure anal-

ysis of triterpene saponins in Polygala tenuifolia by electrospray


ionization ion trap multiple-stage mass spectrometryrdquo Journalof Mass Spectrometry vol 42 no 7 pp 861ndash873 2007

[24] S Y Xu L Bian and X Chen Experimental Methodology ofPharmacology Peoplersquo Medical Publishment House BeijingChina 2002

[25] A E Consolini M I Ragone G N Migliori P Conforti andM G Volonte ldquoCardiotonic and sedative effects of Cecropiapachystachya Mart (ambay) on isolated rat hearts and con-scious micerdquo Journal of Ethnopharmacology vol 106 no 1 pp90ndash96 2006

[26] A H Gilani Q Jabeen A-U Khan and A J Shah ldquoGutmodu-latory blood pressure lowering diuretic and sedative activitiesof cardamomrdquo Journal of Ethnopharmacology vol 115 no 3 pp463ndash472 2007

[27] G Perez-Ortega P Guevara-Fefer M Chavez et al ldquoSedativeand anxiolytic efficacy of Tilia americana var mexicana inflo-rescences used traditionally by communities of State ofMichoa-can Mexicordquo Journal of Ethnopharmacology vol 116 no 3 pp461ndash468 2008

[28] MRabbani S E Sajjadi A Jafarian andGVaseghi ldquoAnxiolyticeffects of Salvia reuterana Boiss on the elevated plus-mazemodel of anxiety in micerdquo Journal of Ethnopharmacology vol101 no 1ndash3 pp 100ndash103 2005

[29] A Wesołowska A Nikiforuk K Michalska W Kisiel and EChojnacka-Wojcik ldquoAnalgesic and sedative activities of lactucinand some lactucin-like guaianolides in micerdquo Journal of Ethno-pharmacology vol 107 no 2 pp 254ndash258 2006

[30] J-F Wu S-B Chen S-L Chen and P-F Tu ldquoThe chemicalconstituents of Polygala hongkongensis Hemslrdquo Acta Pharma-ceutica Sinica vol 42 no 7 pp 757ndash761 2007

[31] L Wen W Liu Y X Li et al ldquoThe effects of three saponincompounds derived from Radix Polygalae on gastric mucosalinjuries amp indexes gastrointestinal oxidation in micerdquo JokullJournal vol 7 pp 145ndash157 2014

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

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Autoimmune Diseases


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MEDIATORSINFLAMMATION

of


Traditional Chinese Medicine Wuhan China) The voucherspecimen was deposited at the Herbarium of Hubei college ofTraditional Chinese Medicine under the acquisition numberof WWZ0406

SNG and TEN (gt98 purity) were purchased fromChengdu Pusi Co Ltd (Chengdu China) OJB was isolatedand purified by Ministry of Education Key Laboratory ofTraditional Chinese Medicine Resources and CompoundsHubei College of Traditional Chinese Medicine

Pentobarbital sodium (Lot number F20030816) wasprovided by Sinopharm Shanghai Chemical Reagents CoLtd (Shanghai China) estazolam (Lot number 20111029)was from Anhui Bangning Pharmaceuticals (Anhui China)ELISA assay kits for PGE2 and TNF-120572 were purchasedfrom Sigma Chemicals Co (St Louis MO USA) Acetyl-choline (Lot number 091201) was purchased from ShanghaiInstitute of Biochemistry CAS (Shanghai China) BaCl2(Lot number 20110221) was purchased from Tianjin DeenChemical Reagent Co Ltd (Anhui China) Phentolamine(Lot number 101104) was purchased from Shanghai FudanForward Science and Technology CO Ltd (ShanghaiChina) Atropine (Lot number 1005051) was purchased fromZhengzhou Lingrui Pharmaceutical Co Ltd (ZhengzhouChina) Neostigmine (BCBD8573V) was purchased fromSigma-Aldrich Co (St Louis MO USA)

22 Preparation of Test Samples Preparation of PTS [1]1 kg of pieces of Polygala tenuifolia Wild were extractedthree times (2 htime) by 3-fold volumes of 85 EtOH andthe extracted solution was pooled Ethanol was recycled byevaporating until there was no alcohol smell and then wasdissolved in 1000mL of water Acetic ether saturated bywater was extracted 3 times (300mLtime) and acetic etherwas discarded and combined with aqueous phase Waterwas added to 1000mL then eluted on a D101 macroporousresin column with H

2O 30 EtOH and 70 EtOH using

3 times the column volume 1 BVh The fraction eluted by70EtOHwas condensed until there was no alcohol smell toobtain powderA (the yieldwas 532 g100 g of driedmedicinalherbs) The content of total saponins was identified to reachto 52 (calculate by the content of tenuifolin (TEN)) [13]powder A was eluted by HPLC with preparative column andthe specific parameters were as follows [19ndash22] the mobilephase of water containing 05 acetic acid and acetonitrile(38 62) C18 column (250 lowast 46mm) and evaporative light-scattering detector After the above steps compound B wasobtained andmeasured byHPLC (C18 column (250lowast46mm)area normalization method was above 95 the mobile phaseof methanol and water containing 005 phosphoric acidsolution (65 35)) As shown in Figures 1 and 2 compoundB was further identified to be polygala saponins B (OJBC75H112O35) by 13C and 1H NMR as well as infraredspectrometry (Figure 1) [23]

Estazolam (EST) was dissolved by normal saline to pre-pare the solution with concentration of 05

Preparation of OJB TEN and SNG solution was asfollows for investigating pharmacological effects on mice

all three compounds were dissolved by normal saline (con-taining 05 Tween 80) to prepare the solution with a con-centration of 20mgmL respectively the high concentrationdiluted to 20mg powdered material per mL as the lowconcentration for mice the dosing volume for mice was10mLkg For determining gastrointestinal irritation pow-dered OJB TEN or SNG dissolved in water (containing 05Tween 80) and diluted to a concentration of 20mg powderedmaterial per mL as the high concentration for mice Thesolution was prepared and diluted to different concentrations(8 4 and 2mgmL) for isolated gut motility experiment200 uL was added into per 20mL Tyrodersquos solution

Preparation of polygala total saponins (PTS) was asfollows powdered PTS dissolved in water (containing 05Tween 80) and diluted to a concentration of 20mg powderedmaterial per mL as the high concentration for mice thesolution was prepared and diluted to different concentrations(8 4 and 2mgmL) for isolated gut motility experiment200 uL was added into per 20mL Tyrodersquos solution

23 Animals Male Kunming mice weighing 18ndash22 g andmale rabbit weighing 2ndash25 kgwere procured from theCenterfor Disease Prevention andControl inHubei Province China(reg number SCXK (Hubei) 2008-0005) The animals werehoused at 22 plusmn 2∘C under a 12 h light12 h dark cycle andwith access to food and water ad libitum The mice wereacclimatized and habituated to the laboratory for at least aweek before being subjected to tests and were used only oncethroughout the experimentsThe study was carried out alongwith the ldquoPrinciples of Laboratory Animal Carerdquo [24] Theexperiment was performed according to the guidelines of theCommittee for the Purpose of Control and Supervision ofExperiments on Animals and approved by the Animal Exper-iment Ethical Committee of Hubei University of Chinesemedicine

24 Potentiation of SPB TEN and SNG on Pentobarbital-Induced Sleep in Mice Male Kunming mice were randomlydivided into 8 groups (8 mice per group) In the controlgroup mice received equal volume of normal saline whilein the seven other groups mice were intragastrically treatedwith 200mgkg of OJB 400mgkg of OJB 200 of mgkgTEN 400 ofmgkg TEN 200 ofmgkg SNG 400mgkg ofSNG and 400mgkg of PTS for one time 30min beforepentobarbital treatment 40mgkg pentobarbital was usedas hypnotic dose to treat half of the animals and theothers were administered with 25mgkg which was used assubhypnotic dose [25ndash29]The subhypnotic dosed mice wereobserved for sleep onset within 15min mouse losing rightingreflex over 1min was considered to be asleep The sleepingtime of animals receiving hypnotic dose was also observedThe sleeping latency was recorded from the injection ofpentobarbital to the sleep onset and the sleeping time wasrecorded from the loss of righting reflex to recovery Theresults were shown in Tables 1 and 2

25 Influence of Different Polygala Saponins on Isolated GutMotility of Rabbit The rabbit ileumwas dissected out and cut


OHO

HO

H

OH

OH

O O

O

OH

HO

H

H

OH

O

O

O

O

O

O

O

OOH

HO

O

OH

O

OH

OH

OH

O O

HO O O

OH

OH

OH

HO

OH

HO

H

HO

HO

HO

O

O

HOH

O

OH

O

OHH

HO

HO

H

H

HOH

OH COOH

HOOC

Onjisaponin B (OJB)




2






5 10 15 20 25 30

OJB



Groups Dose(mgkg)


Control mdash 10

OJB 200 70a

100 70a

TEN 200 60a

100 70a

SNG 200 30100 20

EST 1 802a














3 Results






Acetylcholine(20)


20 40 8020 40 80

OJB (mgL) TEN (mgL)


20 40 8020 40 80


BaCl2










45004000350030002500200015001000

5000

PGE2

(pg

tissu

e)


lowast

lowast

lowast


100

80

60

40

20


(mgkg)

TNF-120572

(pg

g tis

sue)



4 Discussion












Acknowledgments


References
























5and sibricose A


















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


OHO

HO

H

OH

OH

O O

O

OH

HO

H

H

OH

O

O

O

O

O

O

O

OOH

HO

O

OH

O

OH

OH

OH

O O

HO O O

OH

OH

OH

HO

OH

HO

H

HO

HO

HO

O

O

HOH

O

OH

O

OHH

HO

HO

H

H

HOH

OH COOH

HOOC

Onjisaponin B (OJB)




2






5 10 15 20 25 30

OJB



Groups Dose(mgkg)


Control mdash 10

OJB 200 70a

100 70a

TEN 200 60a

100 70a

SNG 200 30100 20

EST 1 802a














3 Results






Acetylcholine(20)


20 40 8020 40 80

OJB (mgL) TEN (mgL)


20 40 8020 40 80


BaCl2










45004000350030002500200015001000

5000

PGE2

(pg

tissu

e)


lowast

lowast

lowast


100

80

60

40

20


(mgkg)

TNF-120572

(pg

g tis

sue)



4 Discussion












Acknowledgments


References
























5and sibricose A


















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


5 10 15 20 25 30

OJB



Groups Dose(mgkg)


Control mdash 10

OJB 200 70a

100 70a

TEN 200 60a

100 70a

SNG 200 30100 20

EST 1 802a














3 Results






Acetylcholine(20)


20 40 8020 40 80

OJB (mgL) TEN (mgL)


20 40 8020 40 80


BaCl2










45004000350030002500200015001000

5000

PGE2

(pg

tissu

e)


lowast

lowast

lowast


100

80

60

40

20


(mgkg)

TNF-120572

(pg

g tis

sue)



4 Discussion












Acknowledgments


References
























5and sibricose A


















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Acetylcholine(20)


20 40 8020 40 80

OJB (mgL) TEN (mgL)


20 40 8020 40 80


BaCl2










45004000350030002500200015001000

5000

PGE2

(pg

tissu

e)


lowast

lowast

lowast


100

80

60

40

20


(mgkg)

TNF-120572

(pg

g tis

sue)



4 Discussion












Acknowledgments


References
























5and sibricose A


















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


45004000350030002500200015001000

5000

PGE2

(pg

tissu

e)


lowast

lowast

lowast


100

80

60

40

20


(mgkg)

TNF-120572

(pg

g tis

sue)



4 Discussion












Acknowledgments


References
























5and sibricose A


















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Acknowledgments


References
























5and sibricose A


















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

research article the gastrointestinal irritation of polygala...

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