Diuretic and anti-diuretic activities of fractions of Alismatis rhizome

Introduction
Natural medicine is a precious resource of the rapeutically active components,and many
studies have reported that herbal diuretics might be a useful toolin thetreatment of
hypertension and chronic kidney diseases. Alismatis Rhizoma (Zexiein Chinese),the
dried rhizome of Alisma orientale Juzepzuk(Alismata- ceae), is widely cultivated in
China and Japan.AR is known traditionally to have diuretic cand damp-heat clearing
actions,and hasbeen used for thetreatment of dysuria,edema,urinary tract infections,the
retention of fluid and phlegm,and vertigo for more than1000years A series of in
vestigations on the crude drug have revealed that protostane triterpenes,guaianeses
quiterpenes and kaurane diterpenes are its principal constituents. Modern
pharmacological investigations have shown that many terpenes have various bioactiv-
ities, suchasanti-hepatitis B virus properties,anti-tumourproperties, anti-complementary
activities,anti-typeIIV allergy activity,the ability to inhibi tosteoclast formation and
prevent bone loss,the ability to regulate the 5-HT3 receptor channel,the ability to treat
acute lung injury and the ability to reduce acute lung inflammation.
Recent toxicological studies have shown that high-doses or the long-term usage of AR
can damage the renal proximal convoluted tubules and the liver (Yuen et al., 2006; Zhu et
al., 2007; Zhao et al., 2011), while the bioactive fractions or components respon- sible for
the diuretic activity and the diuretic mechanism of AR remain unclear. It was previously
reported that the ethanol extract (EE) of AR presented a dual effect on renal function,
including promoting diuretic activity and inhibiting diuretic activity (Feng et al., 2014).
So, the primary aim of this study was to evaluate the diuretic effectiveness of the four
fractions, namely petroleum ether (Fr. PE), ethyl acetate (Fr. EA), n-butanol (Fr. NB) and
the remaining fractions (Fr. RE) obtained from the ethanol extract of AR. These fractions
were orally administrated to rats. Urinary excretion rate, pH and electrolyte excretion
were measured in normal rats.
2.2. Extractspreparation
AR (2.5kg)wasrepeatedlyextractedthreetimeswith95%
ethanolatroomtemperature.Theextractwasthenconcentrated
under pressuretoyieldareddishbrownextract(176.7g).Thenthe
EE obtainedwaspartitionedbetweenwaterandorganicsolventsof
increasingpolarities,toyieldfournewfractionsincluding:Fr.PE
(11.8g),Fr.EA(134.4g),Fr.NB(15.0g)andFr.RE(15.5g).
2.3. Experimentalanimals
The studywasconductedinaccordancewiththeExperimental
Animal AdministrationregulationsissuedbytheStateCommitteeof
ScienceandTechnologyofthePeople'sRepublicofChina.Theethical
approvalreferencenumberofthestudyisSYXK2010-004.Allthe
proceduresforthecareoftheratswereinaccordancewiththe
institutionalguidelinesforanimaluseinresearch.MaleSprague-
Dawleyrats(180220 g)wereobtainedfromthecentralanimal
breedingfacilityoftheFourthMilitaryMedicalUniversity.Allthe
animalswerehousedunderstandard environmentalconditions
(2272 1C) witha12hlightdarkcycle,withfreeaccesstotapwater
andstandardlaboratoryratfood.Theexperimentswereconducted
in accordancewithinternationallyacceptedstandardproceduresfor
animaluse.
2.4. Referencedrug
Furosemide(JiangsuHailinPharmaceuticalCorporation,Jiangsu,
China), aloopdiuretic,wasusedas a referencedrug.Itwasdissolved
inwaterpriortoadministration.
2.5. Diureticactivity
Diuretic activitywasdeterminedfollowingthereportedmeth-
ods (Zhao etal.,2009;Zhaoetal.,2012;Fengetal.,2013). Therats
weredeprivedoffoodbutnotwaterfor12hbeforetesting.Their
urinary bladderswereemptiedbygentlecompressionofthe
pelvic areaandbypulloftheirtails.Eachoftheseratswasthen
orallyadministeredwith5ml/100gbodyweight(BW)ofisotonic
saline (NaCl,0.9%w/v)toimposeauniformwaterload.30min
later,theseratswererandomlyassignedinto18groups(N10per
group) andtreatedorallyinthefollowingmanner:Group1:1ml/
100gBWofwater;Groups24: 12.5,25and50mg/kgBWofFr.
PE respectively;Groups59: 100,200,400,600and800mg/kg
BWofFr.EArespectively;Groups1014:12.5,25,50,75and
100mg/kgBWofFr.NBrespectively;Groups1517:12.5,25and
50 mg/kgBWofFr.RErespectively;Group18:20mg/kgBWof
furosemide.Eachratwasindividuallyplacedinametaboliccage,
and thecumulativeurineoutputwasdeterminedathourly
intervalsfor6h.Theelectrolyte(ofNa, K, Cl) concentrations
and pHweredeterminedinthe6hurinesamplesfromtherats.
2.6. Statisticalanalysis
Resultswereexpressedasthemean7S.E. (standarderrorof
mean). Statisticaldifferencesbetweennegativecontrolandthetest
fractionswereassessedbyanalysisofvariance(ANOVA)followedby
the Student's t-testformultiplecomparisons.Whencomparingwith
negativecontrolgroups, P-valueslessthan0.05wereconsidered
significant.
3. Results
3.1.Urinaryexcretionvolume
The dataobtainedafterorallyadministeringthefourfractions
of ARareshownin Fig. 1.
The 12.5,25and50mg/kgdosesofFr.PEcontributedslightlyto
the urineoutput(5%,2%and5%respectively),andtheincreasedidnot
arriveatstatisticalsignificance (Fig. 1A). AfteradministratedFr.EAand
Fr.NB,thedateobtainedshowedthatthe100and400mg/kgdosesof
Fr.EAandthe12.5,25and50mg/kgdosesofFr.NBsignificantly
increasedtheurinaryoutputin6h(Fig. 1B andCrespectively)(21%,
31%,19%,23%and25%respectively),whilethe800mg/kgdoseofFr.
EAandthe75and100mg/kgdosesofFr.NBproducedaremarkable
decreaseintheurinaryvolumeexcretion(21%, 38%and 44%
respectively).Itisworthnotingthatthethreedoses(12.5,25and
50 mg/kg)ofFr.REsignificantlydecreasedtheurineoutputin6h
(61%, 58% and 68%respectively)(Fig.1D).
3.2. Urineelectrolyteexcretion
Afteroraladministrationofthefourfractions,agreatvariationwas
observed inurineelectrolyteexcretion(Table1). Fr.PEata12.5mg/kg
doseproducedasignificant increaseinurineNa, K and Cl
excretion(15%,20%and15%respectively)whiletheincreaseofother
doses (25and50mg/kg)ofFr.PEdidnotreachstatisticalsignificance.
Afterthe100,200and400mg/kgdosesofFr.EAandthe12.5,25and
50 mg/kgdosesofFr.NB,thedateobtainedshowedaremarkable
parallelismamongtheurineoutputandelectrolyteexcretion(Na
and Cl). Inthisrespect,the100and400mg/kgdosesofFr.EAand
the threedosesofFr.NB(12.5,25and50mg/kg)producedanincrease
in Na (31%,61%,34%,35%and56%respectively)andClexcretion
(31%,61%,14%,16%and31%respectively).AllthedosesenhancedK
excretioninthisstudy,andthe400mg/kgdoseofFr.EAandthethree
doses(12.5,25and50mg/kg)ofFr.NBreachedstatisticalsignificance
(68%,32%,31%and38%respectively).However,the600and800mg/
kg dosesofFr.EAslightlyincreasedthe urineelectrolyteexcretion,but
the increasedidnotachievestatisticalsignificance.The75and
100mg/kgdosesofFr.NBandthe12.5,25and50mg/kgdosesof
Fr.REmarkedlydecreasedtheurineNa and Cl excretionbut
slightlyeffectedK excretion,whichmayhavecausedtheNa/K
valuetodecreasebelowthanthatofcontrolrats.
Finally,thepHvaluesofurinetreatedwiththe12.5and50mg/kg
dosesofFr.PE,the100and600mg/kgdosesofFr.EA,the50and
100mg/kgdosesofFr.NB,ofARwerelowerthanthatofthecontrol group(Po0.05, Po0.01, Po0.05, Po0.05, Po0.05and Po0.05 respectively).