Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 763720 9 pageshttpdxdoiorg1011552013763720

Research ArticleExploring Antiurolithic Effects ofGokshuradi Polyherbal Ayurvedic Formulation inEthylene-Glycol-Induced Urolithic Rats

Amol L Shirfule1 Venkatesh Racharla2 S S Y H Qadri3 and Arjun L Khandare1

1 Food and Drug Toxicology Research Centre National Institute of Nutrition Hyderabad 500007 India2Department of Pharmacology CMR College of Pharmacy Hyderabad 500007 India3 Pathology Division National Institute of Nutrition Hyderabad 500007 India

Correspondence should be addressed to Arjun L Khandare alkhandareyahoocom

Received 29 September 2012 Revised 20 December 2012 Accepted 27 December 2012

Academic Editor Raffaele Capasso

Copyright copy 2013 Amol L Shirfule et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Gokshuradi Yog (GY) is a polyherbal ayurvedic formulation used traditionally for several decades in India for the treatment ofurolithiasis The aim of the present study was to determine the underlying mechanism of GY action in the management ofcalcium oxalate urolithiasis The effect of Gokshuradi polyherbal aqueous extracts (GPAEs) was studied on various biochemicalparameters involved in calciumoxalate formation by employing in vitro and in vivomethodsGPAE exhibited significant antioxidantactivity against 1 1-diphenyl-2-picrylhydrazyl free radical and inhibited lipid peroxidation in the in vitro experiments The ratmodel of urolithiasis induced by 075 ethylene glycol (EG) and 1 ammonium chloride (AC) in water caused polyuria weightloss impairment of renal function and oxidative stress and decreased antioxidant enzyme activities in untreated control groupsHowever GPAE- (25 50 and 100mgkg) treated groups caused diuresis accompanied by a saluretic effect and revealed significantincrease in antioxidant enzyme activities along with decreased oxalate synthesizing biochemical parameters at higher doses Thisstudy revealed the antiurolithic effect of GPAE mediated possibly through inhibiting biochemical parameters involved in calciumoxalate formation along with its diuretic and antioxidant effects hence supporting its use in the treatment of calcium oxalateurolithiasis

1 Introduction

Calcium oxalate urolithiasis has become a rising problemin many countries due to geographicalgenetic variationsand modern lifestyles People living in drought and aridarea suffer more from intestinal hyper absorption of calciumoxalate leading to kidney stones It is estimated that 12of the world population is affected by kidney stones witha recurrence rate from 70 to 80 in males and from 47 to60 in females [1 2] Calcium containing stones are the mostcommon comprising about 75 of all urinary calculi whichare observed in the form of pure calcium oxalate (50) orcalcium phosphate (5) and a mixture of both (45) Manyfactors affect the growth of urinary calculi in which mineralmetabolism plays an important role [3]

The treatment modalities like surgery and drug therapyare practiced in the management of kidney stones but havesome limitations Surgical procedures like (extracorporealshock wave lithotripsy) ESWL have increased risk of stonerecurrence and are also not affordable to poor sufferersThe armamentarium of therapeutic agents also does nothave any effective drugs except for some diuretics likethiazides and furosemide This warranted a search for newdrug therapy which will be cost effective and can targetmultiple etiological risk factors in urolithiasis Ayurveda aclassical Indian authentic literature reported the traditionaluse of Gokshuradi polyherbal formulation (GPF) and is beingpracticed since many years in the management of kidneystones In our preliminary clinical studies GPF has revealedexpulsion of kidney stones in naturally occurred urolithic

2 Evidence-Based Complementary and Alternative Medicine

patients which was confirmed by X-ray analysis conductedbefore and after four weeks of treatment [4] GPF consists ofparts of five different medicinal plants that is dry fruits ofTribulus terrestris L seeds of Hygrophila spinosa T Andersand roots of Ricinnus communis L Solanum anguivi Lamkand Solanum surattense Burm GPF decoction was preparedby mixing the fine powder of these individual parts at equalproportion as mentioned in the ayurvedic literature and thenit was administered to the patients

The individual component plants of GPF are knownto possess various pharmacological activities In Ayurvedicmedicine the fruits of T terrestris L are recommended forthe treatment of urinary disorders erectile dysfunction andin traditional Chinese medicine it has been used as an anti-hypertensive in coronary heart disease [5] It also stimulatesmelanocyte proliferation and therefore is a putative treatmentfor Vitiligo along with its known antibacterial and cytotoxicactivities [6 7] H spinosa T Anders another componentof this formulation has been used in Ayurveda for variousailments like jaundice hepatic obstruction rheumatisminflammation pain diuretic and aphrodisiac and in thetreatment of urinary disorders [8 9] This plant is known topossess hypoglycemic activity in human subjects [10] antitu-mor [11] haematinic [12] antinociceptive [13] hepatoprotec-tive [14 15] free radical scavenging and lipid peroxidationactivities [16] R communis stems have anticancer antidia-betic and antiprotozoal activity [17] Solanumanguivi (Lam)also known as Solanum indicum sp distichum (SchumachandThonn) is cultivated for culinary purposes inmany partsof Africa India and the Arab Peninsula The fruits of thisplant are used as nutritious vegetables on account of theirhigh content of starch calcium vitamin A ascorbic acid andphosphate Solanum xanthocarpum (SX) Schrad amp Wendl(Family Solanaceae) is commonly known as the Indian nightshade or Yellow berried night shade found throughout Indiamostly in dry places as a weed along roadsides and wastelands [18] It has held a place of some importance in the IndianMateria Medica primarily as an expectorant and antipyreticVarious medicinal properties are attributed to it particularlyin the treatment of asthma chronic cough and catarrhal fever[19] It is one of the members of the dashamula (ten roots)of the Ayurveda [20] In Ayurveda this plant is described aspungent bitter digestive and alternative astringent Its stemsflowers and fruits are bitter carminative Root decoction isused as febrifuge effective diuretic and expectorant [21]

Although GPF is well recognized in Indian traditionalmedicine as having antiurolithic effect but no scientific datahave been published supporting its underlying mechanismof action on calcium oxalate stones Therefore this study wasproposed to determine the therapeutic potential and mecha-nism of action of aqueous extracts from this formulation inthe management of urolithiasis by employing in vitro and invivomethods

2 Material and Methods

21 Chemicals and Reagents All the chemicals usedwere of analytical grade Ethylene glycol thymol

reduced glutathione 5-51015840-dithiobis 2-nitrobenzoic acidthiobarbituric acid furosemide H

2O2 trichloroacetic acid

1133-tetraethoxypropane and guanidine hydrochloridewere obtained from Sigma Chemical Company St LouisMO USA Reagents used for histological preparationswere eosin spirit soluble hematoxylin and xylene fromHimedia Chemical Limited Bombay India Kits used in thisstudy for the determination of calcium magnesium bloodurea nitrogen (BUN) creatinine superoxide dismutaseglutathione peroxidase oxalate and citrate were purchasedfrom Spinreact Germany and Ambika diagnostics India

22 Plant Materials Plants used for the formulation werecollected from local forests of Nanded District MaharashtraIndia and authenticated by experts Voucher specimens weredeposited in the herbarium of the School of Life SciencesSRTM University Maharashtra India Respective parts ofeach plant as mentioned previously were collected and thenshade dried Dried parts were cleansed of extraneous matterand then grounded to fine powder in a grinder

23 Standard Drug Cystone polyherbal formulation fromthe Himalaya Drug Co Bangalore India was used as astandard drug in this study The formulation containedDidymocarpus pedicellata Saxifraga ligulataRubia cordifoliaCyperus scariosus Achyranthes aspera Onosma bracteatumVernonia cinerea purified Shilajeet and Hajrul YahoodBhasma

24 In Vitro Antioxidant Effect Antioxidant effect of GPAEwas determined by free radical scavenging activity and lipidperoxidation inhibitory effects 01mM solution of DPPHradical in methanol was prepared and 1mL of this solutionwas added to 3mL of GPAE at different concentrations todetermine the free radical scavenging activity [22] Solutionswere incubated for 30min at room temperature and thenabsorbance was measured at 517 nm Decrease in DPPHsolution absorbance was considered as an increase of theDPPH radical-scavenging activity This activity is given as DPPH radical scavenging which was calculated in theequation using DPPH solution as control

Lipid peroxidation inhibitory activity was determined inthe isolated rat kidneys which were electrically homogenizedin ice cold 50mMphosphate buffer saline (PBS) and adjustedto pH 74 The homogenate was processed further andlipid peroxidation inhibitory activity was determined by areported method [23] The inhibition ratio was calculatedusing the formula given for free radical scavenging activity

25 In Vivo Studies

251 Animals The animal care and handling was in accor-dance with the internationally accepted standard guidelinesfor use of animals and the protocol was approved byinstitutional animal ethical committee (IAEC number P257-2011GBR) at the National Institute of Nutrition HyderabadIndia Wistar rats (180ndash220 g) of either sex used for thisstudy were sourced and housed at the National Centre for

Evidence-Based Complementary and Alternative Medicine 3

Laboratory Animal Sciences Hyderabad India and kept inplastic cages (47 cm times 34 cm times 18 cm) with saw dust (renewedafter every 48 h) under a controlled temperature of 23ndash25∘Cand 12 h light-dark cycle Animals were given standard ratchow diet available at the centre Animals had free access tofood and water ad libitum throughout the study except 24 hbefore and during 6 h of diuretic study and while collecting24 h urine samples food was withdrawn

252 Preparation of GPAE and Cystone for Gastric FeedingInitially GPF was prepared by mixing equally the respectiveparts of each plant at a ratio of 1 1 1 1 1 respectively Inorder to prepare the decoction for gastric gavages 100 g ofGPF and 500mL of doubly distilled (dd) water were heatedin an autoclave for 15min at 121∘C After the boiling andsterilization procedure the powder turned into a jelly-likepaste which was dissolved in dd water to reach a final volumeof 750mL Then the solution was stored at 4∘C for 7 daysSubsequently the solution was poured out and centrifugedat a rate of 1500 rpm for 10min Finally the GPAE feedingsolution containing 50mg of formulation per milliliter wasprepared The same procedure was performed for Cystone tomake 100mg per milliliter of solution

253 Diuretic Activity of GPAE The diuretic activity ofGPAE was studied on Wistar rats of either sex (180ndash220 g)as described previously [24] Animals were divided withmatched body weight and sex into groups of 6 animalseach Negative and standard drug control groups were givensaline by gavage (20mLkg) and Furosemide (10mgkg ofbody weight) respectively The rest of the groups were givendifferent doses of theGPAE (25 50 and 100mgkg) dissolvedin saline Subsequently the animals were placed individuallyin metabolic and diuretic cages The urine was collectedin graduated cylinders for 6 h at 1 h intervals Total urineexcreted out was collected and the volume was determinedThe pHof the pooled urine from each animal was determinedby using pH meter Na+ and K+ concentrations on flamephotometer and Ca2+ concentration by using commerciallyavailable kits

254 Effect of GPAE and Cystone on Animal Model ofUrolithiasis Antiurolithic activity of GPAE and Cystone wasdetermined in an animal model of calcium oxalate urolithi-asis as described by Atmani et al [25] The dose which hadcaused the significant increase in urine output in diuresisstudy was selected for this study Twenty-four male Wistarrats (weighing 180ndash220 g) were divided with matched bodyweights into 5 groups of 6 animals each which were thenrandomly selected to receive various treatments as follows

Group I rats served as vehicle-treated control receivedintraperitoneal (ip) injections of normal saline (25mL)once in 24 h and water ad libitum daily for 21 days (normalcontrol)

Group II rats received stone-inducing treatment for 21days comprised of 075 (wv) EGwith 1 (wv) ammoniumchloride for 5 days following this the water supply was

switched to 075 EG alone in water along with salinetreatment (positive control urolithic group)

Group III rats received GPAE (50mgkg) through gas-tric gavages and simultaneously received stone-inducingtreatment similar to the positive control daily for 21 days(treatment group low dose)

Group IV rats received GPAE (100mgkg) through gas-tric gavages and simultaneously received stone-inducingtreatment similar to the positive control daily for 21 days(treatment group high dose)

Group V rats received standard drug-Cystone(100mgkg) through gastric gavages and simultaneouslyreceived stone-inducing treatment similar to the positivecontrol daily for 21 days (treatment group standard drug)

255 Biochemical Examination Immediately before and atthe end of a total 21 days of treatment 24 h urine samples werecollected in the presence of a few thymol crystals by housinganimals individually in the metabolic and diuretic cagesWater intake was also determined simultaneously Followingvolume and pHdetermination part of each 24 h urine samplewas acidified to pH 2 with 5MHCl Both acidified andnonacidified urine samples were then centrifuged at 1500timesgfor 10min to remove debris and supernatants were storedat minus20∘C until analyzed In acidified urine samples oxalatecalcium (Ca2+) and magnesium (Mg2+) contents were deter-mined by using commercially available kits while inorganicphosphate excretion was determined by reported method[26] In nonacidified urine samples citrate creatinine anduric acid while in serum creatinine and BUNwere estimatedwith the help of kit-based methods

Blood was collected through cardiac puncture fromanimals under ether anesthesia and consequently both thekidneys and liver were excised Animals were then sacrificedby CO

2inhalation

256 Histopathological Examination Organs were rinsed inice cold physiological saline and weighed The right kidneywas fixed in 10neutral buffered formalin processed in seriesof graded alcohol and xylene embedded in paraffin waxsectioned at 5 120583m and stained with Haematoxylin and Eosinfor examination under polarized light microscope To countthe number of crystalline deposits a field of 100x was thenrandomly selected from each region and calcium oxalatedeposits were counted The total number of deposits in eachspecimenwas reported and calcium content in kidney tissueswas determined by atomic absorption spectrophotometer

257 Lipid Peroxidation Inhibitory Effect The left kidney wasworked into 10 homogenate in PBS (50mM pH 74) cen-trifuged at 1500timesg and the supernatants were used to assessvarious antioxidant and oxalate forming enzymes activitiesand biochemical parameters like reduced glutathione (GSH)malondialdehyde (MDA) and protein carbonyl content

Kidney homogenates were estimated for MDA contentby thiobarbituric acid reactive method [27] The proteincarbonyl content was estimated by the protein derivatizationwith dinitrophenyl hydrazine (DNPH) into chromophoric

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Diuretic effect of Gokshuradi polyherbal aqueous extracts (GPAEs)

GroupsUrine vol

(mL)100 g bodyweight6 h

Na+(mmol)100 g

body weight6 h

K+

(mmol)100 gbody weight6 h

Ca2+(mmol)100 g

body weight6 hpH

Control 059 plusmn 014 009 plusmn 003 010 plusmn 002 782 plusmn 162 587 plusmn 006GPAE 25mgkg 123 plusmn 021 013 plusmn 001 015 plusmn 002 622 plusmn 229 685 plusmn 005GPAE 50mgkg 151lowast plusmn 018 019 plusmn 001 019 plusmn 005 369 plusmn 176 693 plusmn 018GPAE 100mgkg 172lowastlowast plusmn 011 026lowast plusmn 002 022lowast plusmn 003 237lowast plusmn 133 732lowastlowast plusmn 024Furosemide 10mgkg 210lowastlowast plusmn 031 038lowastlowast plusmn 007 021lowastlowast plusmn 002 126lowast plusmn 049 739lowastlowast plusmn 027Values given are mean plusmn SEM (n = 6)lowastP lt 005lowastlowastP lt 001 versus control

Table 2 Effect of GPAE and standard drug on 24 h urinary biochemical parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgChange in body weight () 1188 plusmn 153 minus478lowastlowast plusmn 108 232lowastlowast plusmn 145 886 plusmn 206 543lowastlowast plusmn 123Water intake mL24 h 622 plusmn 039 2435lowastlowast plusmn 723 143 plusmn 232 845 plusmn 117 112 plusmn 221Urine vol (mL) 582 plusmn 045 2625lowastlowast plusmn 676 1643lowast plusmn 324 1046lowast plusmn 126 1365lowast plusmn 284pH 638 plusmn 009 617 plusmn 016 627 plusmn 011 645 plusmn 014 632 plusmn 015Oxalate (mg) 038 plusmn 010 212lowastlowast plusmn 021 135lowastlowast plusmn 025 068 plusmn 011 189lowastlowast plusmn 018Ca2+ (mg) 367 plusmn 024 184lowastlowast plusmn 016 263lowast plusmn 029 316 plusmn 064 289lowast plusmn 041Mg2+ (mg) 328 plusmn 032 267 plusmn 011 342 plusmn 024 376 plusmn 031 287 plusmn 032Citrate (mg) 2310 plusmn 121 185 plusmn 190 1912 plusmn 263 1931 plusmn 122 1834 plusmn 254IPa (mg) 626 plusmn 082 789 plusmn 125 757 plusmn 055 755 plusmn 075 734 plusmn 032UAb (mg) 058 plusmn 017 123 plusmn 040 105 plusmn 025 083 plusmn 022 098 plusmn 022Values given are mean plusmn SEM (n = 6)lowastP lt 005lowastlowastP lt 001 versus control groupP lt 005P lt 001 versus urolithic groupaInorganic phosphate bUric acid

dinitrophenyl hydrazones by the method of Levine et al [28]and the carbonyl content was calculated using the DNPHmolar extinction coefficient of 22000Mminus1 cmminus1 GSH wasestimated as total nonprotein thiol (SH) group following themethod described byMoron et al [29] Superoxide dismutase(SOD) and glutathione peroxidase (GPx) were determinedby using commercially available kits Catalase activity wasdetermined by monitoring the decomposition of H

2O2at

240 nm with a spectrophotometer [30] Catalase activitywas calculated by using 120576

240(molar extinction coefficient)

= 00394mmolminus1minminus1 for H2O2and expressed as 120583moles

of H2O2decomposed per min under standard conditions at

25∘C Glycolate oxidase (GOx) activity in liver and lactatedehydrogenase (LDH) in both liver and kidney were deter-mined by reported method [31 32]

258 Statistical Analysis The data expressed are mean plusmnstandard error of mean (SEM) and the median inhibitoryconcentration (IC

50value) with 95 confidence intervals

All statistical comparisons between the groups are madeby means of one way analysis of variance with post hocStudentrsquos t-test The 119875 values less than 005 are regarded assignificantThe concentration-response curveswere analyzed

by nonlinear regression using Graph Pad Prism (Graph PadSoftware San Diego CA USA)

3 Result

31 In Vitro Studies

311 Antioxidant Effect GPAE revealed DPPH radical scav-enging activity with IC

50value of 25 (105ndash290) 120583gmL

(Figure 1(a)) and inhibited lipid peroxidation of the ratkidney homogenate in vitro by 3637 plusmn 186 and 6872 plusmn025 at 50 and 150120583gmL respectively (Figure 1(b)) Thestandard chemical BHT similarly showed DPPH radicalscavenging activity with IC

50value of 256 (102ndash285)120583gmL

and inhibited in vitro lipid peroxidation by 384 plusmn 122 and945 plusmn 46 at 50 and 150 120583gmL respectively

32 In Vivo Studies

321 Diuretic Effect The effect of various doses of GPAE onrat urine volume pH and Na+ K+ and Ca2+ excretion isgiven in Table 1 GPAE increased urine volume at 50mgkg(119875 lt 005) and 100mgkg (119875 lt 001) indicating diuretic

Evidence-Based Complementary and Alternative Medicine 5

Table 3 Effect of GPAE and standard drug on serum parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgSCc (mgdL) 086 plusmn 005 143lowastlowast plusmn 012 126 plusmn 016 089 plusmn 010 108 plusmn 012CCd (mgmin) 086 plusmn 006 050lowastlowast plusmn 006 064 plusmn 010 085 plusmn 007 072 plusmn 012BUNe (mgdL) 1989 plusmn 140 5704lowastlowast plusmn 1246 463 plusmn 1140 205 plusmn 108 362 plusmn 1020Values given are mean plusmn SEM (n = 6)lowastP lt 005lowastlowastP lt 001 versus control groupP lt 005P lt 001 versus urolithic groupcSerum creatinine dCreatinine clearance eBlood urea nitrogen

Table 4 Effect of GPAE and standard drug on kidney and liver parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgKidney weight (g) 078 plusmn 005 121lowastlowast plusmn 012 109lowast plusmn 014 082 plusmn 007 098lowast plusmn 012CDf Absent Significant Moderate Not significant ModerateMDAg (nmol) 064 plusmn 014 652lowastlowast plusmn 085 306 plusmn 110 109 plusmn 042 285 plusmn 108PCCh (nmol) 508 plusmn 068 1304lowastlowast plusmn 136 1012 plusmn 188 805 plusmn 114 980 plusmn 123GSHi (nmol) 1789 plusmn 098 1310lowast plusmn 133 1503 plusmn 182 1698 plusmn 157 1402 plusmn 131SODj (U) 622 plusmn 084 301lowastlowast plusmn 029 458 plusmn 032 548 plusmn 056 421 plusmn 021GPXk (U) 058 plusmn 005 032lowast plusmn 004 047 plusmn 008 053 plusmn 007 048 plusmn 005Catl (120583MH2O2min) 3289 plusmn 255 1689lowastlowast plusmn 198 2092 plusmn 511 2490 plusmn 232 2108 plusmn 432GOxm (nmolmg) 161 plusmn 007 235 plusmn 006lowastlowast 177 plusmn 017 150 plusmn 010 165 plusmn 017LDHn (Umg) (Liver) 154 plusmn 006 403 plusmn 009lowastlowast 149 plusmn 108 151 plusmn 006 146 plusmn 104LDHo (Umg) (Kidney) 282 plusmn 016 468 plusmn 019lowastlowast 319 plusmn 108 307 plusmn 108 332 plusmn 104Calciump (mgg) (Kidney) 0292 plusmn 016 0498 plusmn 0082 0375 plusmn 0078 0316 plusmn 0021 0369 plusmn 0038

Values given are mean plusmn SEM (n = 6)lowastP lt 005 lowastlowastP lt 001 versus control group P lt 005 P lt 001 versus urolithic groupfCrystal deposits100x field gmalondialdehyde hprotein carbonyl content ireduced glutathione jsuperoxide dismutase kglutathione peroxidase lcatalasemglycolate oxidase (nmol of glyoxylate formedmg protein) nliver lactate dehydrogenase (Umg protein) okidney lactate dehydrogenase (Umg protein) andpkidney calcium content

effect At the next higher dose (200mgkg) urine volumedid not increase above that of saline-treated group (119875 gt005) The reference diuretic drug Furosemide at 10mgkgshowed statistically significant increase in the urine output(119875 lt 001) In addition to an increase in the urine outputGPAE and similarly Furosemide increased urine excretionofNa+ andK+ compared to the saline-treated control animalsat higher dose GPAE caused significant increase in the urinepH and decrease in Ca2+ excretion (119875 lt 001) at 100mgkgcomparable to the standard drug Furosemide (10mgkg)

322 Effect Observed in the Animal Model The baselineparameters recorded before the start of treatment like bodyweights water intake along with urine volume pH andcomposition were not significantly different among all thegroupsTheparameters recorded after the treatment are givenin Table 2 As compared to normal control animals waterintake and 24 h urine volume were high in the urolithicgroup (119875 lt 001) Stone-inducing treatment also reducedpH of urine in the urolithic group as compared to thatof the normal control group although not to a significantextent Simultaneous treatment with GPAE at 100mgkg intreatment groups reduced the increase in urine volume andwater intake (119875 lt 005) as compared with the control animals(119875 lt 005) There was an increased oxalate and decreased

Ca2+ concentration (119875 lt 001) of the urine collected from theurolithic animals as compared to normal control GPAE andCystone groups exactly reversed this effect that is decreasedthe urinary oxalate (119875 lt 001) and increased Ca2+ (119875 lt 005)excretion at all the treated doses and remained significantat 100mgkg in GPAE-treated group Other changes in theurine composition like decreased levels of citrate and Mg2+and increased excretion of uric acid and inorganic phosphatein urolithic group were not statistically significant GPAE-andCystone-treated groups also did not show any statisticallysignificant change in these parameters and remained com-parable to that of control animals The urolithic treatmentrevealed loss in body weight of the urolithic animals (119875 lt001) whereas GPAE- and Cystone-treated animals showednet gain in the body weights at all the tested doses Kidneys ofurolithic group animals were heavier than those the controlanimals (119875 lt 001) whereas GPAE- (100mgkg) treated ratkidneys did not show any significant increase in weight ascompared with control however the GPAE- (50mgkg) andCystone- (100mgkg) treated animalrsquos kidneys were slightlyheavier than those the control animals (Table 4)

Urolithic treatment was found to disturb renal functionin urolithic animals as evident from the increased BUN andserum creatinine and reduced creatinine clearance GPAE-treated groups decreased the serum creatinine and BUN

6 Evidence-Based Complementary and Alternative Medicine

DPPH radical scavenging activity

0

20

40

60

80

100

120

01 02 03 04 05 1 5 10 50 100

GPAE BHT

Inhi

bitio

n (

)

(120583gml)

(a)

0

20

40

60

80

100

50 150

Lipid peroxidation inhibitory effect

BHT GPAE

Inhi

bitio

n (

)

(120583gml)

(b)

Figure 1 (a) DPPH radical scavenging activity (b) Lipid peroxi-dation inhibitory effect of GPAE and the reference drug butylatedhydroxytoluene (BHT) Symbols shown are mean plusmn SEM (119899 = 3)

levels at 100mgkg and similar observations were made inthe Cystone-treated groups at 100mgkg as compared to con-trol The creatinine clearance was significantly increased at100mgkg in GPAE-treated groups as compared to urolithicanimals but was not significant at 50mgkg (Table 3)

Histology studies of the kidney regions like cortexmedulla and papilla in the urolithic animals showed manybirefringent crystalline deposits under polarized light micro-scope (Figure 2(b)) Such deposits were found in 3 outof 6 rats from GPAE low dose (Figure 2(c)) 1 out of 6rats from GPAE high dose (Figure 2(d)) and 3 out of 6rats (Figure 2(e)) from Cystone group but visibly smallerand less abundant compared to urolithic control animalsThe tissue morphology and intactness of the kidneys ofGPAE- and Cystone-treated groups were comparable to thenormal control animals (Figure 2(a)) The atomic absorptionspectrophotometer analysis revealed decreased Ca contentin the GPAE- and Cystone- (119875 lt 005) treated groupsas compared with the urolithic control groups wherein the

GPAE at 100mgkg shown significant decrease of Ca (119875 lt001) (Table 4)

323 Lipid Peroxidation Inhibitory Effect Stone-inducingtreatment increasedMDA and carbonyl protein content (119875 lt001) and decreased GSH level (119875 lt 005) and activities ofthe antioxidant enzymes including SOD (119875 lt 001) GPX(119875 lt 005) and catalase (119875 lt 001) in kidneys of theurolithic rats as compared to control animals Activities ofoxalate forming enzymes including GOx (119875 lt 001) in liverand LDH (119875 lt 001) in both liver and kidneys were found tobe enhanced in urolithic group compared to control animalswhereas treatment with GPAE was found to be protectiveagainst this effect and oxidative changes induced by urolithictreatment at higher doses (Table 4 Figure 1(b))

4 Discussion

Phytotherapy approach is attempted in this study as analternative to primary healthcare The Gokshuradi PolyherbalFormulation (GPF) which is traditionally being utilized inIndian folk medicine since several decades to treat kidneystones was evaluated for its antiurolithic effects In our earlierstudies we have clinically evaluated the efficacy of this prepa-ration and found its potential in naturally induced urolithicpatients [4] The present study is a reverse pharmacologyapproach wherein the scientific studies were undertakenunder controlled conditions to validate the traditional use ofGPF and determine its underlying mechanism of action oncalcium oxalate urolithiasis

Calcium oxalate stone development is a multifactorialprocess involving various etiological factors Studies haveshown that calcium oxalate crystal deposition leads to thecellular injury mediated by lipid peroxidation through freeoxygen radical generation Studies revealed that these cellularinjuries favor the events of calcium oxalate retention in renaltubules which is significant for further stone development[33ndash35] Recent clinical data are also supporting this findingthat formation of urinary stones leads to the oxidative stressin patients [36] Antioxidants such as vitamin C catechinand selenium have proved to be protective against suchoxidative injury due to calcium oxalate crystal depositionas evidenced from various experimental studies [37ndash39] Inthis regard antioxidant potential of GPAE was determinedby free radical scavenging and lipid peroxidation inhibitoryactivity to study its mechanism of action InterestinglyGPAErevealed DPPH free radical scavenging and inhibited lipidperoxidation of rat kidney homogenate similar to a standardantioxidant chemical BHT

In the diuretic activity study GPAE altered urine outputin a dose dependantmanner andmaximumdiuresis observedat 100mgkg At next higher dose that is 200mgkg it did notlead to any further increase in the urine volume and remainedcomparable to the normal control group The disappearanceof the diuretic effect of these polyherbal extracts at this higherdose is not completely understood Increase in urine volumebyGPAEwas also associated with the increase in Na+ and K+electrolyte loss similar to the standard diuretic furosemide

Evidence-Based Complementary and Alternative Medicine 7

(a) (b) (c)

(d) (e)

Figure 2 (a) Normal cellular structure of control rats kidney (Gr I) (b) Ethylene-glycol-induced urolithic rats kidney showing irregularcrystals dilation and inflammation under polarized microscope (Gr II) (c) Kidneys of urolithic rats treated with GPAE (50mgkg) showingnear normal cellular structure under polarized microscope (Gr III) (d) Kidneys of urolithic rats treated with GPAE (100mgkg) showingnormal cellular Structure (Gr IV) (e) Kidneys of urolithic rats treated with Cystone (100mgkg) showing normal cellular Structure (Gr V)Arrow indicates CaOx crystals

Increase in urine volume thus suggestive of possible loss ofthese electrolytes leading to this effectGPAE-treated group isalso revealed increase in urine pH and decrease in urine Ca2+at 100mgkg like that of furosemide-treated groups Thesehypocalciuric and diuretic effects ofGPAEmay help to reduceurinary super saturation of calcium salts and thus can preventfurther stone formation

Further antiurolithic effect of GPAE was evaluated inan animal model of urolithiasis induced by ethylene glycoland ammonium chloride in combination This model isfrequently used in calcium oxalate stone formation andtherefore was selected for this study [25 40 41] After 21 daysadministration of EG +AC showed larger and aggregatedcrystals in urolithic control animals compared to the GPAE-and Cystone-treated groups These calcium oxalate crystalagglomerates get deposited in the renal tubules and furtherdevelop into renal stones [25] GPAE reduced the polyuriaassociated with lithogenic treatment as compared to urolithiccontrol groups As per the earlier reports stone inductiontreatment leads an increase in oxalate and decrease in Ca2+

excretion This is confirmed in the present study in whichurolithic control animals showed the similar effect andGPAE- and Cystone-treated groups reversed this effect [4243]

Lithogenic treatment resulted in increased calciumoxalate deposition in the urolithic control groups along withoxidative stress determined from increased levels of markersof oxidative damage that is MDA and protein carbonylcontent alongwith decreased activity of antioxidant enzymesand GSH levels in kidneys accompanied with abnormal renalfunction The renal tubules were dilated in entire kidneysof all untreated rats leading to disturbed renal flow bylarge crystal aggregates However GPAE revealed significantdecrease in all these lithogenic markers at higher doses alongwith the antioxidant activity

The present investigation thus reports the significantantiurolithic potential ofGPAE at higher doses by the combi-nation of antioxidant diuretic and hypocalciuric effects Theresults of the formulation were found significant as comparedwith Cystone which is a standard drug available in themarket

8 Evidence-Based Complementary and Alternative Medicine

used in the treatment of urolithiasis and diuretic activity wascomparable with the standard diuretic drug Furosemide

5 Conclusions

The inhibitory effect of GPAE on calcium oxalate crystalretention in renal tubules thus can be attributed to itsdiuretic hypocalciuric antioxidant effect along with itspotential to inhibit biochemical parameters involved inoxalate metabolism which rationalize its medicinal use forurinary stone diseaseThe present study will certainly benefitthe scientific community worldwide in providing a source forisolation and designing a molecule in this research pipeline

Acknowledgments

The authors are thankful to The Indian Council of MedicalResearch (ICMR) New Delhi for financial assistance to thispostdoctoral project and Director the National Institute ofNutrition Hyderabad for providing necessary laboratoryfacilities

References

[1] G C Curhan W C Willett E B Rimm and M J Stampfer ldquoAprospective study of the intake of vitamins C and B6 and therisk of kidney stones in menrdquo Journal of Urology vol 155 no 6pp 1847ndash1851 1996

[2] C L Smith and D R P Guay ldquoNephrolithiasisrdquo in Pharma-cotherapy and Pathophysiologic Approach J T Di Piro R LTalbert P E Hayes G C Yee G R Matzke and L M PoseyEds pp 720ndash736 Elsevier New York NY USA 2nd edition1992

[3] F L Coe and M J Favus Disorders of Bone and MineralMetabolism Raven press New York NY USA 1992

[4] A L Shirfule C N Khobragade P Badrinarayan Y SBorse and R H Amilkanthwar ldquoPhytochemical analysis andantiurolithiatic activity of a polyherbal formulationrdquo Journal ofHerbs Spices andMedicinal Plants vol 15 no 1 pp 66ndash72 2009

[5] A M Sharifi R Darabi and N Akbarloo ldquoStudy of antihyper-tensive mechanism of Tribulus terrestris in 2K1C hypertensiverats role of tissue ACE activityrdquo Life Sciences vol 73 no 23 pp2963ndash2971 2003

[6] R C Singh and C S Sisodia ldquoEffect of Tribulus terrestris fruitextracts on chloride and creatinine renal clearances in dogsrdquoIndian Journal of Physiology and Pharmacology vol 15 no 3pp 93ndash96 1971

[7] M Al-Ali S Wahbi H Twaij and A Al-Badr ldquoTribulusterrestris preliminary study of its diuretic and contractile effectsand comparisonwith Zeamaysrdquo Journal of Ethnopharmacologyvol 85 no 2-3 pp 257ndash260 2003

[8] A K Nadkarni Indian Materia Medica vol 1 Popular BookDepot Bombay India 3rd edition 1976

[9] R N Chopra S L Nayar and I C Chopra Glossary of IndianMedicinal Plants CSIR New Delhi India 1956

[10] M R Fernando S M D N Wickramasinghe M I Thabrewand E H Karunanayaka ldquoA preliminary investigation of thepossible hypoglycaemic activity of Asteracanthus longifoliardquoJournal of Ethnopharmacology vol 27 no 1-2 pp 7ndash14 1989

[11] U K Mazumdar M Gupta S Maiti and D MukherjeeldquoAntitumor activity of Hygrophila spinosa on Ehrlich ascitescarcinoma and sarcoma-180 induced micerdquo Indian Journal ofExperimental Biology vol 35 no 5 pp 473ndash477 1997

[12] A Gomes M Das and S C Dasgupta ldquoHaematinic effectof Hygrophila spinosa T Anderson on experimental rodentsrdquoIndian Journal of Experimental Biology vol 39 no 4 pp 381ndash382 2001

[13] P Shanmugasundaram and S Venkataraman ldquoAnti-nociceptiveactivity of Hygrophila auriculata (Schum) Heinerdquo The AfricanJournal of Traditional Complementary and AlternativeMedicines vol 2 p 62 2005

[14] A Singh and S S Handa ldquoHepatoprotective activity of Apiumgraveolens and Hygrophila auriculata against paracetamol andthioacetamide intoxication in ratsrdquo Journal of Ethnopharmacol-ogy vol 49 no 3 pp 119ndash126 1995

[15] P Shanmugasundaram and S Venkataraman ldquoHepatoprotec-tive and antioxidant effects ofHygrophila auriculata (K Schum)HeineAcanthaceae root extractrdquo Journal of Ethnopharmacologyvol 104 no 1-2 pp 124ndash128 2006

[16] M Vijayakumar R Govindarajan G M M Rao et al ldquoActionof Hygrophila auriculata against streptozotocin-induced oxida-tive stressrdquo Journal of Ethnopharmacology vol 104 no 3 pp356ndash361 2006

[17] Z A Ali andV K Singh ldquoHerbal drugs of Himalaya (medicinalplants of Garhwal and Kumaon regions of India)rdquo in Aspect ofPlant Sciences vol 15 pp 110ndash111 Today ampTomorrowrsquos PrintersNew Delhi India 1998

[18] Anonymous inTheWealth of India-RawMaterials pp 393ndash394Council of Scientific and Industrial Research NewDelhi India1989

[19] S Siddiqui S Faizi and B Siddiqui ldquoStudies in the chemicalconstituents of the fresh berries of Solanum xanthocarpumrdquoJournal of the Chemical Society of Pakistan vol 5 no 2 pp 14ndash21 1983

[20] L Mohan P Sharma and C N Srivastava ldquoComparativeefficacy of Solanum xanthocarpum extracts alone and in com-bination with a synthetic pyrethroid cypermethrin againstmalaria vector Anopheles Stephensirdquo Southeast Asian Journalof Tropical Medicine and Public Health vol 38 no 2 pp 256ndash260 2007

[21] G P Vadnere R S Gaud andA K Singhai ldquoEvaluation of anti-asthmatic property of SolanumXanthocarpum flower extractsrdquoPharmacologyonline vol 1 pp 513ndash522 2008

[22] D J Huang C D Lin H J Chen and Y H Lin ldquoAntioxidantand antiproliferative activities of sweet potato (Ipomea batata[L] Lam lsquoTaniong 57rsquo) constituentsrdquo Botanical Bulletin ofAcademia Sinica vol 45 pp 179ndash186 2004

[23] D G Kang C K Yun and H S Lee ldquoScreening and com-parison of antioxidant activity of solvent extracts of herbalmedicines used in Koreardquo Journal of Ethnopharmacology vol87 no 2-3 pp 231ndash236 2003

[24] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[25] F Atmani Y Slimani M Mimouni and B Hacht ldquoProphylaxisof calcium oxalate stones by Herniaria hirsuta on experimen-tally induced nephrolithiasis in ratsrdquo British Journal of UrologyInternational vol 92 no 1 pp 137ndash140 2003

Evidence-Based Complementary and Alternative Medicine 9

[26] J A Daly and G Ertingshausen ldquoDirect method for deter-mining inorganic phosphate in serum with the ldquocentrifichemrdquoClinical Chemistry vol 18 pp 263ndash265 1972

[27] S H Y Wong J A Knight S M Hopfer O Zaharia CN Leach Jr and F W Sunderman Jr ldquoLipoperoxides inplasma as measured by liquid-chromatographic separation ofmalondialdehyde-thiobarbituric acid adductrdquo Clinical Chem-istry vol 33 no 2 pp 214ndash220 1987

[28] R L Levine D Garland C N Oliver et al ldquoDetermination ofcarbonyl content in oxidatively modified proteinsrdquo Methods inEnzymology vol 186 pp 464ndash478 1990

[29] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[30] H E Aebi ldquoEnzymes I oxidoreductases transferasesrdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed pp 273ndash285 VCH Weinheim Germany 1987

[31] A L Baker and N E Tolbert ldquoGlycolate oxidase (ferredoxin-containing form)rdquoMethods in Enzymology vol 9 pp 338ndash3421966

[32] J King in Practical Clinical Enzymology vol 87 van Nostrand1965

[33] S R Khan ldquoCalcium oxalate crystal interaction with renaltubular epithelium mechanism of crystal adhesion and itsimpact on stone developmentrdquo Urological Research vol 23 no2 pp 71ndash79 1995

[34] S R Khan and R L Hackett ldquoRetention of calcium oxalatecrystals in renal tubulesrdquo Scanning Microscopy vol 5 pp 707ndash712 1991

[35] F Dal Moro M Mancini I M Tavolini V de Marco and PBassi ldquoCellular and molecular gateways to urolithiasis a newinsightrdquoUrologia Internationalis vol 74 no 3 pp 193ndash197 2005

[36] H S Huang M C Ma C F Chen and J Chen ldquoLipidperoxidation and its correlations with urinary levels of oxalatecitric acid and osteopontin in patients with renal calciumoxalate stonesrdquo Urology vol 62 no 6 pp 1123ndash1128 2003

[37] M Santhosh Kumar and R Selvam ldquoSupplementation ofvitamin E and seleniumprevents hyperoxaluria in experimentalurolithic ratsrdquo Journal of Nutritional Biochemistry vol 14 no 6pp 306ndash313 2003

[38] S Thamilselvan and M Menon ldquoVitamin E therapy preventshyperoxaluria-induced calciumoxalate crystal deposition in thekidney by improving renal tissue antioxidant statusrdquo BritishJournal of Urology International vol 96 no 1 pp 117ndash126 2005

[39] Y Itoh T Yasui A Okada K Tozawa Y Hayashi and K KohrildquoPreventive effects of green tea on renal stone formation and therole of oxidative stress in nephrolithiasisrdquo Journal of Urologyvol 173 no 1 pp 271ndash275 2005

[40] S Thamilselvan R L Hackett and S R Khan ldquoLipid perox-idation in ethylene glycol induced hyperoxaluria and calciumoxalate nephrolithiasisrdquo Journal of Urology vol 157 no 3 pp1059ndash1063 1997

[41] C H Tsai Y C Chen L D Chen et al ldquoA traditional Chineseherbal antilithic formula Wulingsan effectively prevents therenal deposition of calcium oxalate crystal in ethylene glycol-fed ratsrdquo Urological Research vol 36 no 1 pp 17ndash24 2008

[42] J FanM A Glass and P S Chandhoke ldquoImpact of ammoniumchloride administration on rat ethylene glycol urolithiasismodelrdquo Scanning Microscopy vol 13 pp 299ndash306 1999

[43] HK Park B C JeongMK Sung et al ldquoReduction of oxidativestress in cultured renal tubular cells and preventive effects onrenal stone formation by the bioflavonoid Quercetinrdquo Journalof Urology vol 179 no 4 pp 1620ndash1626 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

patients which was confirmed by X-ray analysis conductedbefore and after four weeks of treatment [4] GPF consists ofparts of five different medicinal plants that is dry fruits ofTribulus terrestris L seeds of Hygrophila spinosa T Andersand roots of Ricinnus communis L Solanum anguivi Lamkand Solanum surattense Burm GPF decoction was preparedby mixing the fine powder of these individual parts at equalproportion as mentioned in the ayurvedic literature and thenit was administered to the patients

The individual component plants of GPF are knownto possess various pharmacological activities In Ayurvedicmedicine the fruits of T terrestris L are recommended forthe treatment of urinary disorders erectile dysfunction andin traditional Chinese medicine it has been used as an anti-hypertensive in coronary heart disease [5] It also stimulatesmelanocyte proliferation and therefore is a putative treatmentfor Vitiligo along with its known antibacterial and cytotoxicactivities [6 7] H spinosa T Anders another componentof this formulation has been used in Ayurveda for variousailments like jaundice hepatic obstruction rheumatisminflammation pain diuretic and aphrodisiac and in thetreatment of urinary disorders [8 9] This plant is known topossess hypoglycemic activity in human subjects [10] antitu-mor [11] haematinic [12] antinociceptive [13] hepatoprotec-tive [14 15] free radical scavenging and lipid peroxidationactivities [16] R communis stems have anticancer antidia-betic and antiprotozoal activity [17] Solanumanguivi (Lam)also known as Solanum indicum sp distichum (SchumachandThonn) is cultivated for culinary purposes inmany partsof Africa India and the Arab Peninsula The fruits of thisplant are used as nutritious vegetables on account of theirhigh content of starch calcium vitamin A ascorbic acid andphosphate Solanum xanthocarpum (SX) Schrad amp Wendl(Family Solanaceae) is commonly known as the Indian nightshade or Yellow berried night shade found throughout Indiamostly in dry places as a weed along roadsides and wastelands [18] It has held a place of some importance in the IndianMateria Medica primarily as an expectorant and antipyreticVarious medicinal properties are attributed to it particularlyin the treatment of asthma chronic cough and catarrhal fever[19] It is one of the members of the dashamula (ten roots)of the Ayurveda [20] In Ayurveda this plant is described aspungent bitter digestive and alternative astringent Its stemsflowers and fruits are bitter carminative Root decoction isused as febrifuge effective diuretic and expectorant [21]

Although GPF is well recognized in Indian traditionalmedicine as having antiurolithic effect but no scientific datahave been published supporting its underlying mechanismof action on calcium oxalate stones Therefore this study wasproposed to determine the therapeutic potential and mecha-nism of action of aqueous extracts from this formulation inthe management of urolithiasis by employing in vitro and invivomethods

2 Material and Methods

21 Chemicals and Reagents All the chemicals usedwere of analytical grade Ethylene glycol thymol

reduced glutathione 5-51015840-dithiobis 2-nitrobenzoic acidthiobarbituric acid furosemide H

2O2 trichloroacetic acid

1133-tetraethoxypropane and guanidine hydrochloridewere obtained from Sigma Chemical Company St LouisMO USA Reagents used for histological preparationswere eosin spirit soluble hematoxylin and xylene fromHimedia Chemical Limited Bombay India Kits used in thisstudy for the determination of calcium magnesium bloodurea nitrogen (BUN) creatinine superoxide dismutaseglutathione peroxidase oxalate and citrate were purchasedfrom Spinreact Germany and Ambika diagnostics India

22 Plant Materials Plants used for the formulation werecollected from local forests of Nanded District MaharashtraIndia and authenticated by experts Voucher specimens weredeposited in the herbarium of the School of Life SciencesSRTM University Maharashtra India Respective parts ofeach plant as mentioned previously were collected and thenshade dried Dried parts were cleansed of extraneous matterand then grounded to fine powder in a grinder

23 Standard Drug Cystone polyherbal formulation fromthe Himalaya Drug Co Bangalore India was used as astandard drug in this study The formulation containedDidymocarpus pedicellata Saxifraga ligulataRubia cordifoliaCyperus scariosus Achyranthes aspera Onosma bracteatumVernonia cinerea purified Shilajeet and Hajrul YahoodBhasma

24 In Vitro Antioxidant Effect Antioxidant effect of GPAEwas determined by free radical scavenging activity and lipidperoxidation inhibitory effects 01mM solution of DPPHradical in methanol was prepared and 1mL of this solutionwas added to 3mL of GPAE at different concentrations todetermine the free radical scavenging activity [22] Solutionswere incubated for 30min at room temperature and thenabsorbance was measured at 517 nm Decrease in DPPHsolution absorbance was considered as an increase of theDPPH radical-scavenging activity This activity is given as DPPH radical scavenging which was calculated in theequation using DPPH solution as control

Lipid peroxidation inhibitory activity was determined inthe isolated rat kidneys which were electrically homogenizedin ice cold 50mMphosphate buffer saline (PBS) and adjustedto pH 74 The homogenate was processed further andlipid peroxidation inhibitory activity was determined by areported method [23] The inhibition ratio was calculatedusing the formula given for free radical scavenging activity

25 In Vivo Studies

251 Animals The animal care and handling was in accor-dance with the internationally accepted standard guidelinesfor use of animals and the protocol was approved byinstitutional animal ethical committee (IAEC number P257-2011GBR) at the National Institute of Nutrition HyderabadIndia Wistar rats (180ndash220 g) of either sex used for thisstudy were sourced and housed at the National Centre for

Evidence-Based Complementary and Alternative Medicine 3

Laboratory Animal Sciences Hyderabad India and kept inplastic cages (47 cm times 34 cm times 18 cm) with saw dust (renewedafter every 48 h) under a controlled temperature of 23ndash25∘Cand 12 h light-dark cycle Animals were given standard ratchow diet available at the centre Animals had free access tofood and water ad libitum throughout the study except 24 hbefore and during 6 h of diuretic study and while collecting24 h urine samples food was withdrawn

252 Preparation of GPAE and Cystone for Gastric FeedingInitially GPF was prepared by mixing equally the respectiveparts of each plant at a ratio of 1 1 1 1 1 respectively Inorder to prepare the decoction for gastric gavages 100 g ofGPF and 500mL of doubly distilled (dd) water were heatedin an autoclave for 15min at 121∘C After the boiling andsterilization procedure the powder turned into a jelly-likepaste which was dissolved in dd water to reach a final volumeof 750mL Then the solution was stored at 4∘C for 7 daysSubsequently the solution was poured out and centrifugedat a rate of 1500 rpm for 10min Finally the GPAE feedingsolution containing 50mg of formulation per milliliter wasprepared The same procedure was performed for Cystone tomake 100mg per milliliter of solution

253 Diuretic Activity of GPAE The diuretic activity ofGPAE was studied on Wistar rats of either sex (180ndash220 g)as described previously [24] Animals were divided withmatched body weight and sex into groups of 6 animalseach Negative and standard drug control groups were givensaline by gavage (20mLkg) and Furosemide (10mgkg ofbody weight) respectively The rest of the groups were givendifferent doses of theGPAE (25 50 and 100mgkg) dissolvedin saline Subsequently the animals were placed individuallyin metabolic and diuretic cages The urine was collectedin graduated cylinders for 6 h at 1 h intervals Total urineexcreted out was collected and the volume was determinedThe pHof the pooled urine from each animal was determinedby using pH meter Na+ and K+ concentrations on flamephotometer and Ca2+ concentration by using commerciallyavailable kits

254 Effect of GPAE and Cystone on Animal Model ofUrolithiasis Antiurolithic activity of GPAE and Cystone wasdetermined in an animal model of calcium oxalate urolithi-asis as described by Atmani et al [25] The dose which hadcaused the significant increase in urine output in diuresisstudy was selected for this study Twenty-four male Wistarrats (weighing 180ndash220 g) were divided with matched bodyweights into 5 groups of 6 animals each which were thenrandomly selected to receive various treatments as follows

Group I rats served as vehicle-treated control receivedintraperitoneal (ip) injections of normal saline (25mL)once in 24 h and water ad libitum daily for 21 days (normalcontrol)

Group II rats received stone-inducing treatment for 21days comprised of 075 (wv) EGwith 1 (wv) ammoniumchloride for 5 days following this the water supply was

switched to 075 EG alone in water along with salinetreatment (positive control urolithic group)

Group III rats received GPAE (50mgkg) through gas-tric gavages and simultaneously received stone-inducingtreatment similar to the positive control daily for 21 days(treatment group low dose)

Group IV rats received GPAE (100mgkg) through gas-tric gavages and simultaneously received stone-inducingtreatment similar to the positive control daily for 21 days(treatment group high dose)

Group V rats received standard drug-Cystone(100mgkg) through gastric gavages and simultaneouslyreceived stone-inducing treatment similar to the positivecontrol daily for 21 days (treatment group standard drug)

255 Biochemical Examination Immediately before and atthe end of a total 21 days of treatment 24 h urine samples werecollected in the presence of a few thymol crystals by housinganimals individually in the metabolic and diuretic cagesWater intake was also determined simultaneously Followingvolume and pHdetermination part of each 24 h urine samplewas acidified to pH 2 with 5MHCl Both acidified andnonacidified urine samples were then centrifuged at 1500timesgfor 10min to remove debris and supernatants were storedat minus20∘C until analyzed In acidified urine samples oxalatecalcium (Ca2+) and magnesium (Mg2+) contents were deter-mined by using commercially available kits while inorganicphosphate excretion was determined by reported method[26] In nonacidified urine samples citrate creatinine anduric acid while in serum creatinine and BUNwere estimatedwith the help of kit-based methods

Blood was collected through cardiac puncture fromanimals under ether anesthesia and consequently both thekidneys and liver were excised Animals were then sacrificedby CO

2inhalation

256 Histopathological Examination Organs were rinsed inice cold physiological saline and weighed The right kidneywas fixed in 10neutral buffered formalin processed in seriesof graded alcohol and xylene embedded in paraffin waxsectioned at 5 120583m and stained with Haematoxylin and Eosinfor examination under polarized light microscope To countthe number of crystalline deposits a field of 100x was thenrandomly selected from each region and calcium oxalatedeposits were counted The total number of deposits in eachspecimenwas reported and calcium content in kidney tissueswas determined by atomic absorption spectrophotometer

257 Lipid Peroxidation Inhibitory Effect The left kidney wasworked into 10 homogenate in PBS (50mM pH 74) cen-trifuged at 1500timesg and the supernatants were used to assessvarious antioxidant and oxalate forming enzymes activitiesand biochemical parameters like reduced glutathione (GSH)malondialdehyde (MDA) and protein carbonyl content

Kidney homogenates were estimated for MDA contentby thiobarbituric acid reactive method [27] The proteincarbonyl content was estimated by the protein derivatizationwith dinitrophenyl hydrazine (DNPH) into chromophoric

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Diuretic effect of Gokshuradi polyherbal aqueous extracts (GPAEs)

GroupsUrine vol

(mL)100 g bodyweight6 h

Na+(mmol)100 g

body weight6 h

K+

(mmol)100 gbody weight6 h

Ca2+(mmol)100 g

body weight6 hpH

Control 059 plusmn 014 009 plusmn 003 010 plusmn 002 782 plusmn 162 587 plusmn 006GPAE 25mgkg 123 plusmn 021 013 plusmn 001 015 plusmn 002 622 plusmn 229 685 plusmn 005GPAE 50mgkg 151lowast plusmn 018 019 plusmn 001 019 plusmn 005 369 plusmn 176 693 plusmn 018GPAE 100mgkg 172lowastlowast plusmn 011 026lowast plusmn 002 022lowast plusmn 003 237lowast plusmn 133 732lowastlowast plusmn 024Furosemide 10mgkg 210lowastlowast plusmn 031 038lowastlowast plusmn 007 021lowastlowast plusmn 002 126lowast plusmn 049 739lowastlowast plusmn 027Values given are mean plusmn SEM (n = 6)lowastP lt 005lowastlowastP lt 001 versus control

Table 2 Effect of GPAE and standard drug on 24 h urinary biochemical parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgChange in body weight () 1188 plusmn 153 minus478lowastlowast plusmn 108 232lowastlowast plusmn 145 886 plusmn 206 543lowastlowast plusmn 123Water intake mL24 h 622 plusmn 039 2435lowastlowast plusmn 723 143 plusmn 232 845 plusmn 117 112 plusmn 221Urine vol (mL) 582 plusmn 045 2625lowastlowast plusmn 676 1643lowast plusmn 324 1046lowast plusmn 126 1365lowast plusmn 284pH 638 plusmn 009 617 plusmn 016 627 plusmn 011 645 plusmn 014 632 plusmn 015Oxalate (mg) 038 plusmn 010 212lowastlowast plusmn 021 135lowastlowast plusmn 025 068 plusmn 011 189lowastlowast plusmn 018Ca2+ (mg) 367 plusmn 024 184lowastlowast plusmn 016 263lowast plusmn 029 316 plusmn 064 289lowast plusmn 041Mg2+ (mg) 328 plusmn 032 267 plusmn 011 342 plusmn 024 376 plusmn 031 287 plusmn 032Citrate (mg) 2310 plusmn 121 185 plusmn 190 1912 plusmn 263 1931 plusmn 122 1834 plusmn 254IPa (mg) 626 plusmn 082 789 plusmn 125 757 plusmn 055 755 plusmn 075 734 plusmn 032UAb (mg) 058 plusmn 017 123 plusmn 040 105 plusmn 025 083 plusmn 022 098 plusmn 022Values given are mean plusmn SEM (n = 6)lowastP lt 005lowastlowastP lt 001 versus control groupP lt 005P lt 001 versus urolithic groupaInorganic phosphate bUric acid

dinitrophenyl hydrazones by the method of Levine et al [28]and the carbonyl content was calculated using the DNPHmolar extinction coefficient of 22000Mminus1 cmminus1 GSH wasestimated as total nonprotein thiol (SH) group following themethod described byMoron et al [29] Superoxide dismutase(SOD) and glutathione peroxidase (GPx) were determinedby using commercially available kits Catalase activity wasdetermined by monitoring the decomposition of H

2O2at

240 nm with a spectrophotometer [30] Catalase activitywas calculated by using 120576

240(molar extinction coefficient)

= 00394mmolminus1minminus1 for H2O2and expressed as 120583moles

of H2O2decomposed per min under standard conditions at

25∘C Glycolate oxidase (GOx) activity in liver and lactatedehydrogenase (LDH) in both liver and kidney were deter-mined by reported method [31 32]

258 Statistical Analysis The data expressed are mean plusmnstandard error of mean (SEM) and the median inhibitoryconcentration (IC

50value) with 95 confidence intervals

All statistical comparisons between the groups are madeby means of one way analysis of variance with post hocStudentrsquos t-test The 119875 values less than 005 are regarded assignificantThe concentration-response curveswere analyzed

by nonlinear regression using Graph Pad Prism (Graph PadSoftware San Diego CA USA)

3 Result

31 In Vitro Studies

311 Antioxidant Effect GPAE revealed DPPH radical scav-enging activity with IC

50value of 25 (105ndash290) 120583gmL

(Figure 1(a)) and inhibited lipid peroxidation of the ratkidney homogenate in vitro by 3637 plusmn 186 and 6872 plusmn025 at 50 and 150120583gmL respectively (Figure 1(b)) Thestandard chemical BHT similarly showed DPPH radicalscavenging activity with IC

50value of 256 (102ndash285)120583gmL

and inhibited in vitro lipid peroxidation by 384 plusmn 122 and945 plusmn 46 at 50 and 150 120583gmL respectively

32 In Vivo Studies

321 Diuretic Effect The effect of various doses of GPAE onrat urine volume pH and Na+ K+ and Ca2+ excretion isgiven in Table 1 GPAE increased urine volume at 50mgkg(119875 lt 005) and 100mgkg (119875 lt 001) indicating diuretic

Evidence-Based Complementary and Alternative Medicine 5

Table 3 Effect of GPAE and standard drug on serum parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgSCc (mgdL) 086 plusmn 005 143lowastlowast plusmn 012 126 plusmn 016 089 plusmn 010 108 plusmn 012CCd (mgmin) 086 plusmn 006 050lowastlowast plusmn 006 064 plusmn 010 085 plusmn 007 072 plusmn 012BUNe (mgdL) 1989 plusmn 140 5704lowastlowast plusmn 1246 463 plusmn 1140 205 plusmn 108 362 plusmn 1020Values given are mean plusmn SEM (n = 6)lowastP lt 005lowastlowastP lt 001 versus control groupP lt 005P lt 001 versus urolithic groupcSerum creatinine dCreatinine clearance eBlood urea nitrogen

Table 4 Effect of GPAE and standard drug on kidney and liver parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgKidney weight (g) 078 plusmn 005 121lowastlowast plusmn 012 109lowast plusmn 014 082 plusmn 007 098lowast plusmn 012CDf Absent Significant Moderate Not significant ModerateMDAg (nmol) 064 plusmn 014 652lowastlowast plusmn 085 306 plusmn 110 109 plusmn 042 285 plusmn 108PCCh (nmol) 508 plusmn 068 1304lowastlowast plusmn 136 1012 plusmn 188 805 plusmn 114 980 plusmn 123GSHi (nmol) 1789 plusmn 098 1310lowast plusmn 133 1503 plusmn 182 1698 plusmn 157 1402 plusmn 131SODj (U) 622 plusmn 084 301lowastlowast plusmn 029 458 plusmn 032 548 plusmn 056 421 plusmn 021GPXk (U) 058 plusmn 005 032lowast plusmn 004 047 plusmn 008 053 plusmn 007 048 plusmn 005Catl (120583MH2O2min) 3289 plusmn 255 1689lowastlowast plusmn 198 2092 plusmn 511 2490 plusmn 232 2108 plusmn 432GOxm (nmolmg) 161 plusmn 007 235 plusmn 006lowastlowast 177 plusmn 017 150 plusmn 010 165 plusmn 017LDHn (Umg) (Liver) 154 plusmn 006 403 plusmn 009lowastlowast 149 plusmn 108 151 plusmn 006 146 plusmn 104LDHo (Umg) (Kidney) 282 plusmn 016 468 plusmn 019lowastlowast 319 plusmn 108 307 plusmn 108 332 plusmn 104Calciump (mgg) (Kidney) 0292 plusmn 016 0498 plusmn 0082 0375 plusmn 0078 0316 plusmn 0021 0369 plusmn 0038

Values given are mean plusmn SEM (n = 6)lowastP lt 005 lowastlowastP lt 001 versus control group P lt 005 P lt 001 versus urolithic groupfCrystal deposits100x field gmalondialdehyde hprotein carbonyl content ireduced glutathione jsuperoxide dismutase kglutathione peroxidase lcatalasemglycolate oxidase (nmol of glyoxylate formedmg protein) nliver lactate dehydrogenase (Umg protein) okidney lactate dehydrogenase (Umg protein) andpkidney calcium content

effect At the next higher dose (200mgkg) urine volumedid not increase above that of saline-treated group (119875 gt005) The reference diuretic drug Furosemide at 10mgkgshowed statistically significant increase in the urine output(119875 lt 001) In addition to an increase in the urine outputGPAE and similarly Furosemide increased urine excretionofNa+ andK+ compared to the saline-treated control animalsat higher dose GPAE caused significant increase in the urinepH and decrease in Ca2+ excretion (119875 lt 001) at 100mgkgcomparable to the standard drug Furosemide (10mgkg)

322 Effect Observed in the Animal Model The baselineparameters recorded before the start of treatment like bodyweights water intake along with urine volume pH andcomposition were not significantly different among all thegroupsTheparameters recorded after the treatment are givenin Table 2 As compared to normal control animals waterintake and 24 h urine volume were high in the urolithicgroup (119875 lt 001) Stone-inducing treatment also reducedpH of urine in the urolithic group as compared to thatof the normal control group although not to a significantextent Simultaneous treatment with GPAE at 100mgkg intreatment groups reduced the increase in urine volume andwater intake (119875 lt 005) as compared with the control animals(119875 lt 005) There was an increased oxalate and decreased

Ca2+ concentration (119875 lt 001) of the urine collected from theurolithic animals as compared to normal control GPAE andCystone groups exactly reversed this effect that is decreasedthe urinary oxalate (119875 lt 001) and increased Ca2+ (119875 lt 005)excretion at all the treated doses and remained significantat 100mgkg in GPAE-treated group Other changes in theurine composition like decreased levels of citrate and Mg2+and increased excretion of uric acid and inorganic phosphatein urolithic group were not statistically significant GPAE-andCystone-treated groups also did not show any statisticallysignificant change in these parameters and remained com-parable to that of control animals The urolithic treatmentrevealed loss in body weight of the urolithic animals (119875 lt001) whereas GPAE- and Cystone-treated animals showednet gain in the body weights at all the tested doses Kidneys ofurolithic group animals were heavier than those the controlanimals (119875 lt 001) whereas GPAE- (100mgkg) treated ratkidneys did not show any significant increase in weight ascompared with control however the GPAE- (50mgkg) andCystone- (100mgkg) treated animalrsquos kidneys were slightlyheavier than those the control animals (Table 4)

Urolithic treatment was found to disturb renal functionin urolithic animals as evident from the increased BUN andserum creatinine and reduced creatinine clearance GPAE-treated groups decreased the serum creatinine and BUN

6 Evidence-Based Complementary and Alternative Medicine

DPPH radical scavenging activity

0

20

40

60

80

100

120

01 02 03 04 05 1 5 10 50 100

GPAE BHT

Inhi

bitio

n (

)

(120583gml)

(a)

0

20

40

60

80

100

50 150

Lipid peroxidation inhibitory effect

BHT GPAE

Inhi

bitio

n (

)

(120583gml)

(b)

Figure 1 (a) DPPH radical scavenging activity (b) Lipid peroxi-dation inhibitory effect of GPAE and the reference drug butylatedhydroxytoluene (BHT) Symbols shown are mean plusmn SEM (119899 = 3)

levels at 100mgkg and similar observations were made inthe Cystone-treated groups at 100mgkg as compared to con-trol The creatinine clearance was significantly increased at100mgkg in GPAE-treated groups as compared to urolithicanimals but was not significant at 50mgkg (Table 3)

Histology studies of the kidney regions like cortexmedulla and papilla in the urolithic animals showed manybirefringent crystalline deposits under polarized light micro-scope (Figure 2(b)) Such deposits were found in 3 outof 6 rats from GPAE low dose (Figure 2(c)) 1 out of 6rats from GPAE high dose (Figure 2(d)) and 3 out of 6rats (Figure 2(e)) from Cystone group but visibly smallerand less abundant compared to urolithic control animalsThe tissue morphology and intactness of the kidneys ofGPAE- and Cystone-treated groups were comparable to thenormal control animals (Figure 2(a)) The atomic absorptionspectrophotometer analysis revealed decreased Ca contentin the GPAE- and Cystone- (119875 lt 005) treated groupsas compared with the urolithic control groups wherein the

GPAE at 100mgkg shown significant decrease of Ca (119875 lt001) (Table 4)

323 Lipid Peroxidation Inhibitory Effect Stone-inducingtreatment increasedMDA and carbonyl protein content (119875 lt001) and decreased GSH level (119875 lt 005) and activities ofthe antioxidant enzymes including SOD (119875 lt 001) GPX(119875 lt 005) and catalase (119875 lt 001) in kidneys of theurolithic rats as compared to control animals Activities ofoxalate forming enzymes including GOx (119875 lt 001) in liverand LDH (119875 lt 001) in both liver and kidneys were found tobe enhanced in urolithic group compared to control animalswhereas treatment with GPAE was found to be protectiveagainst this effect and oxidative changes induced by urolithictreatment at higher doses (Table 4 Figure 1(b))

4 Discussion

Phytotherapy approach is attempted in this study as analternative to primary healthcare The Gokshuradi PolyherbalFormulation (GPF) which is traditionally being utilized inIndian folk medicine since several decades to treat kidneystones was evaluated for its antiurolithic effects In our earlierstudies we have clinically evaluated the efficacy of this prepa-ration and found its potential in naturally induced urolithicpatients [4] The present study is a reverse pharmacologyapproach wherein the scientific studies were undertakenunder controlled conditions to validate the traditional use ofGPF and determine its underlying mechanism of action oncalcium oxalate urolithiasis

Calcium oxalate stone development is a multifactorialprocess involving various etiological factors Studies haveshown that calcium oxalate crystal deposition leads to thecellular injury mediated by lipid peroxidation through freeoxygen radical generation Studies revealed that these cellularinjuries favor the events of calcium oxalate retention in renaltubules which is significant for further stone development[33ndash35] Recent clinical data are also supporting this findingthat formation of urinary stones leads to the oxidative stressin patients [36] Antioxidants such as vitamin C catechinand selenium have proved to be protective against suchoxidative injury due to calcium oxalate crystal depositionas evidenced from various experimental studies [37ndash39] Inthis regard antioxidant potential of GPAE was determinedby free radical scavenging and lipid peroxidation inhibitoryactivity to study its mechanism of action InterestinglyGPAErevealed DPPH free radical scavenging and inhibited lipidperoxidation of rat kidney homogenate similar to a standardantioxidant chemical BHT

In the diuretic activity study GPAE altered urine outputin a dose dependantmanner andmaximumdiuresis observedat 100mgkg At next higher dose that is 200mgkg it did notlead to any further increase in the urine volume and remainedcomparable to the normal control group The disappearanceof the diuretic effect of these polyherbal extracts at this higherdose is not completely understood Increase in urine volumebyGPAEwas also associated with the increase in Na+ and K+electrolyte loss similar to the standard diuretic furosemide

Evidence-Based Complementary and Alternative Medicine 7

(a) (b) (c)

(d) (e)

Figure 2 (a) Normal cellular structure of control rats kidney (Gr I) (b) Ethylene-glycol-induced urolithic rats kidney showing irregularcrystals dilation and inflammation under polarized microscope (Gr II) (c) Kidneys of urolithic rats treated with GPAE (50mgkg) showingnear normal cellular structure under polarized microscope (Gr III) (d) Kidneys of urolithic rats treated with GPAE (100mgkg) showingnormal cellular Structure (Gr IV) (e) Kidneys of urolithic rats treated with Cystone (100mgkg) showing normal cellular Structure (Gr V)Arrow indicates CaOx crystals

Increase in urine volume thus suggestive of possible loss ofthese electrolytes leading to this effectGPAE-treated group isalso revealed increase in urine pH and decrease in urine Ca2+at 100mgkg like that of furosemide-treated groups Thesehypocalciuric and diuretic effects ofGPAEmay help to reduceurinary super saturation of calcium salts and thus can preventfurther stone formation

Further antiurolithic effect of GPAE was evaluated inan animal model of urolithiasis induced by ethylene glycoland ammonium chloride in combination This model isfrequently used in calcium oxalate stone formation andtherefore was selected for this study [25 40 41] After 21 daysadministration of EG +AC showed larger and aggregatedcrystals in urolithic control animals compared to the GPAE-and Cystone-treated groups These calcium oxalate crystalagglomerates get deposited in the renal tubules and furtherdevelop into renal stones [25] GPAE reduced the polyuriaassociated with lithogenic treatment as compared to urolithiccontrol groups As per the earlier reports stone inductiontreatment leads an increase in oxalate and decrease in Ca2+

excretion This is confirmed in the present study in whichurolithic control animals showed the similar effect andGPAE- and Cystone-treated groups reversed this effect [4243]

Lithogenic treatment resulted in increased calciumoxalate deposition in the urolithic control groups along withoxidative stress determined from increased levels of markersof oxidative damage that is MDA and protein carbonylcontent alongwith decreased activity of antioxidant enzymesand GSH levels in kidneys accompanied with abnormal renalfunction The renal tubules were dilated in entire kidneysof all untreated rats leading to disturbed renal flow bylarge crystal aggregates However GPAE revealed significantdecrease in all these lithogenic markers at higher doses alongwith the antioxidant activity

The present investigation thus reports the significantantiurolithic potential ofGPAE at higher doses by the combi-nation of antioxidant diuretic and hypocalciuric effects Theresults of the formulation were found significant as comparedwith Cystone which is a standard drug available in themarket

8 Evidence-Based Complementary and Alternative Medicine

used in the treatment of urolithiasis and diuretic activity wascomparable with the standard diuretic drug Furosemide

5 Conclusions

The inhibitory effect of GPAE on calcium oxalate crystalretention in renal tubules thus can be attributed to itsdiuretic hypocalciuric antioxidant effect along with itspotential to inhibit biochemical parameters involved inoxalate metabolism which rationalize its medicinal use forurinary stone diseaseThe present study will certainly benefitthe scientific community worldwide in providing a source forisolation and designing a molecule in this research pipeline

Acknowledgments

The authors are thankful to The Indian Council of MedicalResearch (ICMR) New Delhi for financial assistance to thispostdoctoral project and Director the National Institute ofNutrition Hyderabad for providing necessary laboratoryfacilities

References

[1] G C Curhan W C Willett E B Rimm and M J Stampfer ldquoAprospective study of the intake of vitamins C and B6 and therisk of kidney stones in menrdquo Journal of Urology vol 155 no 6pp 1847ndash1851 1996

[2] C L Smith and D R P Guay ldquoNephrolithiasisrdquo in Pharma-cotherapy and Pathophysiologic Approach J T Di Piro R LTalbert P E Hayes G C Yee G R Matzke and L M PoseyEds pp 720ndash736 Elsevier New York NY USA 2nd edition1992

[3] F L Coe and M J Favus Disorders of Bone and MineralMetabolism Raven press New York NY USA 1992

[4] A L Shirfule C N Khobragade P Badrinarayan Y SBorse and R H Amilkanthwar ldquoPhytochemical analysis andantiurolithiatic activity of a polyherbal formulationrdquo Journal ofHerbs Spices andMedicinal Plants vol 15 no 1 pp 66ndash72 2009

[5] A M Sharifi R Darabi and N Akbarloo ldquoStudy of antihyper-tensive mechanism of Tribulus terrestris in 2K1C hypertensiverats role of tissue ACE activityrdquo Life Sciences vol 73 no 23 pp2963ndash2971 2003

[6] R C Singh and C S Sisodia ldquoEffect of Tribulus terrestris fruitextracts on chloride and creatinine renal clearances in dogsrdquoIndian Journal of Physiology and Pharmacology vol 15 no 3pp 93ndash96 1971

[7] M Al-Ali S Wahbi H Twaij and A Al-Badr ldquoTribulusterrestris preliminary study of its diuretic and contractile effectsand comparisonwith Zeamaysrdquo Journal of Ethnopharmacologyvol 85 no 2-3 pp 257ndash260 2003

[8] A K Nadkarni Indian Materia Medica vol 1 Popular BookDepot Bombay India 3rd edition 1976

[9] R N Chopra S L Nayar and I C Chopra Glossary of IndianMedicinal Plants CSIR New Delhi India 1956

[10] M R Fernando S M D N Wickramasinghe M I Thabrewand E H Karunanayaka ldquoA preliminary investigation of thepossible hypoglycaemic activity of Asteracanthus longifoliardquoJournal of Ethnopharmacology vol 27 no 1-2 pp 7ndash14 1989

[11] U K Mazumdar M Gupta S Maiti and D MukherjeeldquoAntitumor activity of Hygrophila spinosa on Ehrlich ascitescarcinoma and sarcoma-180 induced micerdquo Indian Journal ofExperimental Biology vol 35 no 5 pp 473ndash477 1997

[12] A Gomes M Das and S C Dasgupta ldquoHaematinic effectof Hygrophila spinosa T Anderson on experimental rodentsrdquoIndian Journal of Experimental Biology vol 39 no 4 pp 381ndash382 2001

[13] P Shanmugasundaram and S Venkataraman ldquoAnti-nociceptiveactivity of Hygrophila auriculata (Schum) Heinerdquo The AfricanJournal of Traditional Complementary and AlternativeMedicines vol 2 p 62 2005

[14] A Singh and S S Handa ldquoHepatoprotective activity of Apiumgraveolens and Hygrophila auriculata against paracetamol andthioacetamide intoxication in ratsrdquo Journal of Ethnopharmacol-ogy vol 49 no 3 pp 119ndash126 1995

[15] P Shanmugasundaram and S Venkataraman ldquoHepatoprotec-tive and antioxidant effects ofHygrophila auriculata (K Schum)HeineAcanthaceae root extractrdquo Journal of Ethnopharmacologyvol 104 no 1-2 pp 124ndash128 2006

[16] M Vijayakumar R Govindarajan G M M Rao et al ldquoActionof Hygrophila auriculata against streptozotocin-induced oxida-tive stressrdquo Journal of Ethnopharmacology vol 104 no 3 pp356ndash361 2006

[17] Z A Ali andV K Singh ldquoHerbal drugs of Himalaya (medicinalplants of Garhwal and Kumaon regions of India)rdquo in Aspect ofPlant Sciences vol 15 pp 110ndash111 Today ampTomorrowrsquos PrintersNew Delhi India 1998

[18] Anonymous inTheWealth of India-RawMaterials pp 393ndash394Council of Scientific and Industrial Research NewDelhi India1989

[19] S Siddiqui S Faizi and B Siddiqui ldquoStudies in the chemicalconstituents of the fresh berries of Solanum xanthocarpumrdquoJournal of the Chemical Society of Pakistan vol 5 no 2 pp 14ndash21 1983

[20] L Mohan P Sharma and C N Srivastava ldquoComparativeefficacy of Solanum xanthocarpum extracts alone and in com-bination with a synthetic pyrethroid cypermethrin againstmalaria vector Anopheles Stephensirdquo Southeast Asian Journalof Tropical Medicine and Public Health vol 38 no 2 pp 256ndash260 2007

[21] G P Vadnere R S Gaud andA K Singhai ldquoEvaluation of anti-asthmatic property of SolanumXanthocarpum flower extractsrdquoPharmacologyonline vol 1 pp 513ndash522 2008

[22] D J Huang C D Lin H J Chen and Y H Lin ldquoAntioxidantand antiproliferative activities of sweet potato (Ipomea batata[L] Lam lsquoTaniong 57rsquo) constituentsrdquo Botanical Bulletin ofAcademia Sinica vol 45 pp 179ndash186 2004

[23] D G Kang C K Yun and H S Lee ldquoScreening and com-parison of antioxidant activity of solvent extracts of herbalmedicines used in Koreardquo Journal of Ethnopharmacology vol87 no 2-3 pp 231ndash236 2003

[24] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[25] F Atmani Y Slimani M Mimouni and B Hacht ldquoProphylaxisof calcium oxalate stones by Herniaria hirsuta on experimen-tally induced nephrolithiasis in ratsrdquo British Journal of UrologyInternational vol 92 no 1 pp 137ndash140 2003

Evidence-Based Complementary and Alternative Medicine 9

[26] J A Daly and G Ertingshausen ldquoDirect method for deter-mining inorganic phosphate in serum with the ldquocentrifichemrdquoClinical Chemistry vol 18 pp 263ndash265 1972

[27] S H Y Wong J A Knight S M Hopfer O Zaharia CN Leach Jr and F W Sunderman Jr ldquoLipoperoxides inplasma as measured by liquid-chromatographic separation ofmalondialdehyde-thiobarbituric acid adductrdquo Clinical Chem-istry vol 33 no 2 pp 214ndash220 1987

[28] R L Levine D Garland C N Oliver et al ldquoDetermination ofcarbonyl content in oxidatively modified proteinsrdquo Methods inEnzymology vol 186 pp 464ndash478 1990

[29] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[30] H E Aebi ldquoEnzymes I oxidoreductases transferasesrdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed pp 273ndash285 VCH Weinheim Germany 1987

[31] A L Baker and N E Tolbert ldquoGlycolate oxidase (ferredoxin-containing form)rdquoMethods in Enzymology vol 9 pp 338ndash3421966

[32] J King in Practical Clinical Enzymology vol 87 van Nostrand1965

[33] S R Khan ldquoCalcium oxalate crystal interaction with renaltubular epithelium mechanism of crystal adhesion and itsimpact on stone developmentrdquo Urological Research vol 23 no2 pp 71ndash79 1995

[34] S R Khan and R L Hackett ldquoRetention of calcium oxalatecrystals in renal tubulesrdquo Scanning Microscopy vol 5 pp 707ndash712 1991

[35] F Dal Moro M Mancini I M Tavolini V de Marco and PBassi ldquoCellular and molecular gateways to urolithiasis a newinsightrdquoUrologia Internationalis vol 74 no 3 pp 193ndash197 2005

[36] H S Huang M C Ma C F Chen and J Chen ldquoLipidperoxidation and its correlations with urinary levels of oxalatecitric acid and osteopontin in patients with renal calciumoxalate stonesrdquo Urology vol 62 no 6 pp 1123ndash1128 2003

[37] M Santhosh Kumar and R Selvam ldquoSupplementation ofvitamin E and seleniumprevents hyperoxaluria in experimentalurolithic ratsrdquo Journal of Nutritional Biochemistry vol 14 no 6pp 306ndash313 2003

[38] S Thamilselvan and M Menon ldquoVitamin E therapy preventshyperoxaluria-induced calciumoxalate crystal deposition in thekidney by improving renal tissue antioxidant statusrdquo BritishJournal of Urology International vol 96 no 1 pp 117ndash126 2005

[39] Y Itoh T Yasui A Okada K Tozawa Y Hayashi and K KohrildquoPreventive effects of green tea on renal stone formation and therole of oxidative stress in nephrolithiasisrdquo Journal of Urologyvol 173 no 1 pp 271ndash275 2005

[40] S Thamilselvan R L Hackett and S R Khan ldquoLipid perox-idation in ethylene glycol induced hyperoxaluria and calciumoxalate nephrolithiasisrdquo Journal of Urology vol 157 no 3 pp1059ndash1063 1997

[41] C H Tsai Y C Chen L D Chen et al ldquoA traditional Chineseherbal antilithic formula Wulingsan effectively prevents therenal deposition of calcium oxalate crystal in ethylene glycol-fed ratsrdquo Urological Research vol 36 no 1 pp 17ndash24 2008

[42] J FanM A Glass and P S Chandhoke ldquoImpact of ammoniumchloride administration on rat ethylene glycol urolithiasismodelrdquo Scanning Microscopy vol 13 pp 299ndash306 1999

[43] HK Park B C JeongMK Sung et al ldquoReduction of oxidativestress in cultured renal tubular cells and preventive effects onrenal stone formation by the bioflavonoid Quercetinrdquo Journalof Urology vol 179 no 4 pp 1620ndash1626 2008

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Behavioural Neurology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

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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

Laboratory Animal Sciences Hyderabad India and kept inplastic cages (47 cm times 34 cm times 18 cm) with saw dust (renewedafter every 48 h) under a controlled temperature of 23ndash25∘Cand 12 h light-dark cycle Animals were given standard ratchow diet available at the centre Animals had free access tofood and water ad libitum throughout the study except 24 hbefore and during 6 h of diuretic study and while collecting24 h urine samples food was withdrawn

252 Preparation of GPAE and Cystone for Gastric FeedingInitially GPF was prepared by mixing equally the respectiveparts of each plant at a ratio of 1 1 1 1 1 respectively Inorder to prepare the decoction for gastric gavages 100 g ofGPF and 500mL of doubly distilled (dd) water were heatedin an autoclave for 15min at 121∘C After the boiling andsterilization procedure the powder turned into a jelly-likepaste which was dissolved in dd water to reach a final volumeof 750mL Then the solution was stored at 4∘C for 7 daysSubsequently the solution was poured out and centrifugedat a rate of 1500 rpm for 10min Finally the GPAE feedingsolution containing 50mg of formulation per milliliter wasprepared The same procedure was performed for Cystone tomake 100mg per milliliter of solution

253 Diuretic Activity of GPAE The diuretic activity ofGPAE was studied on Wistar rats of either sex (180ndash220 g)as described previously [24] Animals were divided withmatched body weight and sex into groups of 6 animalseach Negative and standard drug control groups were givensaline by gavage (20mLkg) and Furosemide (10mgkg ofbody weight) respectively The rest of the groups were givendifferent doses of theGPAE (25 50 and 100mgkg) dissolvedin saline Subsequently the animals were placed individuallyin metabolic and diuretic cages The urine was collectedin graduated cylinders for 6 h at 1 h intervals Total urineexcreted out was collected and the volume was determinedThe pHof the pooled urine from each animal was determinedby using pH meter Na+ and K+ concentrations on flamephotometer and Ca2+ concentration by using commerciallyavailable kits

254 Effect of GPAE and Cystone on Animal Model ofUrolithiasis Antiurolithic activity of GPAE and Cystone wasdetermined in an animal model of calcium oxalate urolithi-asis as described by Atmani et al [25] The dose which hadcaused the significant increase in urine output in diuresisstudy was selected for this study Twenty-four male Wistarrats (weighing 180ndash220 g) were divided with matched bodyweights into 5 groups of 6 animals each which were thenrandomly selected to receive various treatments as follows

Group I rats served as vehicle-treated control receivedintraperitoneal (ip) injections of normal saline (25mL)once in 24 h and water ad libitum daily for 21 days (normalcontrol)

Group II rats received stone-inducing treatment for 21days comprised of 075 (wv) EGwith 1 (wv) ammoniumchloride for 5 days following this the water supply was

switched to 075 EG alone in water along with salinetreatment (positive control urolithic group)

Group III rats received GPAE (50mgkg) through gas-tric gavages and simultaneously received stone-inducingtreatment similar to the positive control daily for 21 days(treatment group low dose)

Group IV rats received GPAE (100mgkg) through gas-tric gavages and simultaneously received stone-inducingtreatment similar to the positive control daily for 21 days(treatment group high dose)

Group V rats received standard drug-Cystone(100mgkg) through gastric gavages and simultaneouslyreceived stone-inducing treatment similar to the positivecontrol daily for 21 days (treatment group standard drug)

255 Biochemical Examination Immediately before and atthe end of a total 21 days of treatment 24 h urine samples werecollected in the presence of a few thymol crystals by housinganimals individually in the metabolic and diuretic cagesWater intake was also determined simultaneously Followingvolume and pHdetermination part of each 24 h urine samplewas acidified to pH 2 with 5MHCl Both acidified andnonacidified urine samples were then centrifuged at 1500timesgfor 10min to remove debris and supernatants were storedat minus20∘C until analyzed In acidified urine samples oxalatecalcium (Ca2+) and magnesium (Mg2+) contents were deter-mined by using commercially available kits while inorganicphosphate excretion was determined by reported method[26] In nonacidified urine samples citrate creatinine anduric acid while in serum creatinine and BUNwere estimatedwith the help of kit-based methods

Blood was collected through cardiac puncture fromanimals under ether anesthesia and consequently both thekidneys and liver were excised Animals were then sacrificedby CO

2inhalation

256 Histopathological Examination Organs were rinsed inice cold physiological saline and weighed The right kidneywas fixed in 10neutral buffered formalin processed in seriesof graded alcohol and xylene embedded in paraffin waxsectioned at 5 120583m and stained with Haematoxylin and Eosinfor examination under polarized light microscope To countthe number of crystalline deposits a field of 100x was thenrandomly selected from each region and calcium oxalatedeposits were counted The total number of deposits in eachspecimenwas reported and calcium content in kidney tissueswas determined by atomic absorption spectrophotometer

257 Lipid Peroxidation Inhibitory Effect The left kidney wasworked into 10 homogenate in PBS (50mM pH 74) cen-trifuged at 1500timesg and the supernatants were used to assessvarious antioxidant and oxalate forming enzymes activitiesand biochemical parameters like reduced glutathione (GSH)malondialdehyde (MDA) and protein carbonyl content

Kidney homogenates were estimated for MDA contentby thiobarbituric acid reactive method [27] The proteincarbonyl content was estimated by the protein derivatizationwith dinitrophenyl hydrazine (DNPH) into chromophoric

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Diuretic effect of Gokshuradi polyherbal aqueous extracts (GPAEs)

GroupsUrine vol

(mL)100 g bodyweight6 h

Na+(mmol)100 g

body weight6 h

K+

(mmol)100 gbody weight6 h

Ca2+(mmol)100 g

body weight6 hpH

Control 059 plusmn 014 009 plusmn 003 010 plusmn 002 782 plusmn 162 587 plusmn 006GPAE 25mgkg 123 plusmn 021 013 plusmn 001 015 plusmn 002 622 plusmn 229 685 plusmn 005GPAE 50mgkg 151lowast plusmn 018 019 plusmn 001 019 plusmn 005 369 plusmn 176 693 plusmn 018GPAE 100mgkg 172lowastlowast plusmn 011 026lowast plusmn 002 022lowast plusmn 003 237lowast plusmn 133 732lowastlowast plusmn 024Furosemide 10mgkg 210lowastlowast plusmn 031 038lowastlowast plusmn 007 021lowastlowast plusmn 002 126lowast plusmn 049 739lowastlowast plusmn 027Values given are mean plusmn SEM (n = 6)lowastP lt 005lowastlowastP lt 001 versus control

Table 2 Effect of GPAE and standard drug on 24 h urinary biochemical parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgChange in body weight () 1188 plusmn 153 minus478lowastlowast plusmn 108 232lowastlowast plusmn 145 886 plusmn 206 543lowastlowast plusmn 123Water intake mL24 h 622 plusmn 039 2435lowastlowast plusmn 723 143 plusmn 232 845 plusmn 117 112 plusmn 221Urine vol (mL) 582 plusmn 045 2625lowastlowast plusmn 676 1643lowast plusmn 324 1046lowast plusmn 126 1365lowast plusmn 284pH 638 plusmn 009 617 plusmn 016 627 plusmn 011 645 plusmn 014 632 plusmn 015Oxalate (mg) 038 plusmn 010 212lowastlowast plusmn 021 135lowastlowast plusmn 025 068 plusmn 011 189lowastlowast plusmn 018Ca2+ (mg) 367 plusmn 024 184lowastlowast plusmn 016 263lowast plusmn 029 316 plusmn 064 289lowast plusmn 041Mg2+ (mg) 328 plusmn 032 267 plusmn 011 342 plusmn 024 376 plusmn 031 287 plusmn 032Citrate (mg) 2310 plusmn 121 185 plusmn 190 1912 plusmn 263 1931 plusmn 122 1834 plusmn 254IPa (mg) 626 plusmn 082 789 plusmn 125 757 plusmn 055 755 plusmn 075 734 plusmn 032UAb (mg) 058 plusmn 017 123 plusmn 040 105 plusmn 025 083 plusmn 022 098 plusmn 022Values given are mean plusmn SEM (n = 6)lowastP lt 005lowastlowastP lt 001 versus control groupP lt 005P lt 001 versus urolithic groupaInorganic phosphate bUric acid

dinitrophenyl hydrazones by the method of Levine et al [28]and the carbonyl content was calculated using the DNPHmolar extinction coefficient of 22000Mminus1 cmminus1 GSH wasestimated as total nonprotein thiol (SH) group following themethod described byMoron et al [29] Superoxide dismutase(SOD) and glutathione peroxidase (GPx) were determinedby using commercially available kits Catalase activity wasdetermined by monitoring the decomposition of H

2O2at

240 nm with a spectrophotometer [30] Catalase activitywas calculated by using 120576

240(molar extinction coefficient)

= 00394mmolminus1minminus1 for H2O2and expressed as 120583moles

of H2O2decomposed per min under standard conditions at

25∘C Glycolate oxidase (GOx) activity in liver and lactatedehydrogenase (LDH) in both liver and kidney were deter-mined by reported method [31 32]

258 Statistical Analysis The data expressed are mean plusmnstandard error of mean (SEM) and the median inhibitoryconcentration (IC

50value) with 95 confidence intervals

All statistical comparisons between the groups are madeby means of one way analysis of variance with post hocStudentrsquos t-test The 119875 values less than 005 are regarded assignificantThe concentration-response curveswere analyzed

by nonlinear regression using Graph Pad Prism (Graph PadSoftware San Diego CA USA)

3 Result

31 In Vitro Studies

311 Antioxidant Effect GPAE revealed DPPH radical scav-enging activity with IC

50value of 25 (105ndash290) 120583gmL

(Figure 1(a)) and inhibited lipid peroxidation of the ratkidney homogenate in vitro by 3637 plusmn 186 and 6872 plusmn025 at 50 and 150120583gmL respectively (Figure 1(b)) Thestandard chemical BHT similarly showed DPPH radicalscavenging activity with IC

50value of 256 (102ndash285)120583gmL

and inhibited in vitro lipid peroxidation by 384 plusmn 122 and945 plusmn 46 at 50 and 150 120583gmL respectively

32 In Vivo Studies

321 Diuretic Effect The effect of various doses of GPAE onrat urine volume pH and Na+ K+ and Ca2+ excretion isgiven in Table 1 GPAE increased urine volume at 50mgkg(119875 lt 005) and 100mgkg (119875 lt 001) indicating diuretic

Evidence-Based Complementary and Alternative Medicine 5

Table 3 Effect of GPAE and standard drug on serum parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgSCc (mgdL) 086 plusmn 005 143lowastlowast plusmn 012 126 plusmn 016 089 plusmn 010 108 plusmn 012CCd (mgmin) 086 plusmn 006 050lowastlowast plusmn 006 064 plusmn 010 085 plusmn 007 072 plusmn 012BUNe (mgdL) 1989 plusmn 140 5704lowastlowast plusmn 1246 463 plusmn 1140 205 plusmn 108 362 plusmn 1020Values given are mean plusmn SEM (n = 6)lowastP lt 005lowastlowastP lt 001 versus control groupP lt 005P lt 001 versus urolithic groupcSerum creatinine dCreatinine clearance eBlood urea nitrogen

Table 4 Effect of GPAE and standard drug on kidney and liver parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgKidney weight (g) 078 plusmn 005 121lowastlowast plusmn 012 109lowast plusmn 014 082 plusmn 007 098lowast plusmn 012CDf Absent Significant Moderate Not significant ModerateMDAg (nmol) 064 plusmn 014 652lowastlowast plusmn 085 306 plusmn 110 109 plusmn 042 285 plusmn 108PCCh (nmol) 508 plusmn 068 1304lowastlowast plusmn 136 1012 plusmn 188 805 plusmn 114 980 plusmn 123GSHi (nmol) 1789 plusmn 098 1310lowast plusmn 133 1503 plusmn 182 1698 plusmn 157 1402 plusmn 131SODj (U) 622 plusmn 084 301lowastlowast plusmn 029 458 plusmn 032 548 plusmn 056 421 plusmn 021GPXk (U) 058 plusmn 005 032lowast plusmn 004 047 plusmn 008 053 plusmn 007 048 plusmn 005Catl (120583MH2O2min) 3289 plusmn 255 1689lowastlowast plusmn 198 2092 plusmn 511 2490 plusmn 232 2108 plusmn 432GOxm (nmolmg) 161 plusmn 007 235 plusmn 006lowastlowast 177 plusmn 017 150 plusmn 010 165 plusmn 017LDHn (Umg) (Liver) 154 plusmn 006 403 plusmn 009lowastlowast 149 plusmn 108 151 plusmn 006 146 plusmn 104LDHo (Umg) (Kidney) 282 plusmn 016 468 plusmn 019lowastlowast 319 plusmn 108 307 plusmn 108 332 plusmn 104Calciump (mgg) (Kidney) 0292 plusmn 016 0498 plusmn 0082 0375 plusmn 0078 0316 plusmn 0021 0369 plusmn 0038

Values given are mean plusmn SEM (n = 6)lowastP lt 005 lowastlowastP lt 001 versus control group P lt 005 P lt 001 versus urolithic groupfCrystal deposits100x field gmalondialdehyde hprotein carbonyl content ireduced glutathione jsuperoxide dismutase kglutathione peroxidase lcatalasemglycolate oxidase (nmol of glyoxylate formedmg protein) nliver lactate dehydrogenase (Umg protein) okidney lactate dehydrogenase (Umg protein) andpkidney calcium content

effect At the next higher dose (200mgkg) urine volumedid not increase above that of saline-treated group (119875 gt005) The reference diuretic drug Furosemide at 10mgkgshowed statistically significant increase in the urine output(119875 lt 001) In addition to an increase in the urine outputGPAE and similarly Furosemide increased urine excretionofNa+ andK+ compared to the saline-treated control animalsat higher dose GPAE caused significant increase in the urinepH and decrease in Ca2+ excretion (119875 lt 001) at 100mgkgcomparable to the standard drug Furosemide (10mgkg)

322 Effect Observed in the Animal Model The baselineparameters recorded before the start of treatment like bodyweights water intake along with urine volume pH andcomposition were not significantly different among all thegroupsTheparameters recorded after the treatment are givenin Table 2 As compared to normal control animals waterintake and 24 h urine volume were high in the urolithicgroup (119875 lt 001) Stone-inducing treatment also reducedpH of urine in the urolithic group as compared to thatof the normal control group although not to a significantextent Simultaneous treatment with GPAE at 100mgkg intreatment groups reduced the increase in urine volume andwater intake (119875 lt 005) as compared with the control animals(119875 lt 005) There was an increased oxalate and decreased

Ca2+ concentration (119875 lt 001) of the urine collected from theurolithic animals as compared to normal control GPAE andCystone groups exactly reversed this effect that is decreasedthe urinary oxalate (119875 lt 001) and increased Ca2+ (119875 lt 005)excretion at all the treated doses and remained significantat 100mgkg in GPAE-treated group Other changes in theurine composition like decreased levels of citrate and Mg2+and increased excretion of uric acid and inorganic phosphatein urolithic group were not statistically significant GPAE-andCystone-treated groups also did not show any statisticallysignificant change in these parameters and remained com-parable to that of control animals The urolithic treatmentrevealed loss in body weight of the urolithic animals (119875 lt001) whereas GPAE- and Cystone-treated animals showednet gain in the body weights at all the tested doses Kidneys ofurolithic group animals were heavier than those the controlanimals (119875 lt 001) whereas GPAE- (100mgkg) treated ratkidneys did not show any significant increase in weight ascompared with control however the GPAE- (50mgkg) andCystone- (100mgkg) treated animalrsquos kidneys were slightlyheavier than those the control animals (Table 4)

Urolithic treatment was found to disturb renal functionin urolithic animals as evident from the increased BUN andserum creatinine and reduced creatinine clearance GPAE-treated groups decreased the serum creatinine and BUN

6 Evidence-Based Complementary and Alternative Medicine

DPPH radical scavenging activity

0

20

40

60

80

100

120

01 02 03 04 05 1 5 10 50 100

GPAE BHT

Inhi

bitio

n (

)

(120583gml)

(a)

0

20

40

60

80

100

50 150

Lipid peroxidation inhibitory effect

BHT GPAE

Inhi

bitio

n (

)

(120583gml)

(b)

Figure 1 (a) DPPH radical scavenging activity (b) Lipid peroxi-dation inhibitory effect of GPAE and the reference drug butylatedhydroxytoluene (BHT) Symbols shown are mean plusmn SEM (119899 = 3)

levels at 100mgkg and similar observations were made inthe Cystone-treated groups at 100mgkg as compared to con-trol The creatinine clearance was significantly increased at100mgkg in GPAE-treated groups as compared to urolithicanimals but was not significant at 50mgkg (Table 3)

Histology studies of the kidney regions like cortexmedulla and papilla in the urolithic animals showed manybirefringent crystalline deposits under polarized light micro-scope (Figure 2(b)) Such deposits were found in 3 outof 6 rats from GPAE low dose (Figure 2(c)) 1 out of 6rats from GPAE high dose (Figure 2(d)) and 3 out of 6rats (Figure 2(e)) from Cystone group but visibly smallerand less abundant compared to urolithic control animalsThe tissue morphology and intactness of the kidneys ofGPAE- and Cystone-treated groups were comparable to thenormal control animals (Figure 2(a)) The atomic absorptionspectrophotometer analysis revealed decreased Ca contentin the GPAE- and Cystone- (119875 lt 005) treated groupsas compared with the urolithic control groups wherein the

GPAE at 100mgkg shown significant decrease of Ca (119875 lt001) (Table 4)

323 Lipid Peroxidation Inhibitory Effect Stone-inducingtreatment increasedMDA and carbonyl protein content (119875 lt001) and decreased GSH level (119875 lt 005) and activities ofthe antioxidant enzymes including SOD (119875 lt 001) GPX(119875 lt 005) and catalase (119875 lt 001) in kidneys of theurolithic rats as compared to control animals Activities ofoxalate forming enzymes including GOx (119875 lt 001) in liverand LDH (119875 lt 001) in both liver and kidneys were found tobe enhanced in urolithic group compared to control animalswhereas treatment with GPAE was found to be protectiveagainst this effect and oxidative changes induced by urolithictreatment at higher doses (Table 4 Figure 1(b))

4 Discussion

Phytotherapy approach is attempted in this study as analternative to primary healthcare The Gokshuradi PolyherbalFormulation (GPF) which is traditionally being utilized inIndian folk medicine since several decades to treat kidneystones was evaluated for its antiurolithic effects In our earlierstudies we have clinically evaluated the efficacy of this prepa-ration and found its potential in naturally induced urolithicpatients [4] The present study is a reverse pharmacologyapproach wherein the scientific studies were undertakenunder controlled conditions to validate the traditional use ofGPF and determine its underlying mechanism of action oncalcium oxalate urolithiasis

Calcium oxalate stone development is a multifactorialprocess involving various etiological factors Studies haveshown that calcium oxalate crystal deposition leads to thecellular injury mediated by lipid peroxidation through freeoxygen radical generation Studies revealed that these cellularinjuries favor the events of calcium oxalate retention in renaltubules which is significant for further stone development[33ndash35] Recent clinical data are also supporting this findingthat formation of urinary stones leads to the oxidative stressin patients [36] Antioxidants such as vitamin C catechinand selenium have proved to be protective against suchoxidative injury due to calcium oxalate crystal depositionas evidenced from various experimental studies [37ndash39] Inthis regard antioxidant potential of GPAE was determinedby free radical scavenging and lipid peroxidation inhibitoryactivity to study its mechanism of action InterestinglyGPAErevealed DPPH free radical scavenging and inhibited lipidperoxidation of rat kidney homogenate similar to a standardantioxidant chemical BHT

In the diuretic activity study GPAE altered urine outputin a dose dependantmanner andmaximumdiuresis observedat 100mgkg At next higher dose that is 200mgkg it did notlead to any further increase in the urine volume and remainedcomparable to the normal control group The disappearanceof the diuretic effect of these polyherbal extracts at this higherdose is not completely understood Increase in urine volumebyGPAEwas also associated with the increase in Na+ and K+electrolyte loss similar to the standard diuretic furosemide

Evidence-Based Complementary and Alternative Medicine 7

(a) (b) (c)

(d) (e)

Figure 2 (a) Normal cellular structure of control rats kidney (Gr I) (b) Ethylene-glycol-induced urolithic rats kidney showing irregularcrystals dilation and inflammation under polarized microscope (Gr II) (c) Kidneys of urolithic rats treated with GPAE (50mgkg) showingnear normal cellular structure under polarized microscope (Gr III) (d) Kidneys of urolithic rats treated with GPAE (100mgkg) showingnormal cellular Structure (Gr IV) (e) Kidneys of urolithic rats treated with Cystone (100mgkg) showing normal cellular Structure (Gr V)Arrow indicates CaOx crystals

Increase in urine volume thus suggestive of possible loss ofthese electrolytes leading to this effectGPAE-treated group isalso revealed increase in urine pH and decrease in urine Ca2+at 100mgkg like that of furosemide-treated groups Thesehypocalciuric and diuretic effects ofGPAEmay help to reduceurinary super saturation of calcium salts and thus can preventfurther stone formation

Further antiurolithic effect of GPAE was evaluated inan animal model of urolithiasis induced by ethylene glycoland ammonium chloride in combination This model isfrequently used in calcium oxalate stone formation andtherefore was selected for this study [25 40 41] After 21 daysadministration of EG +AC showed larger and aggregatedcrystals in urolithic control animals compared to the GPAE-and Cystone-treated groups These calcium oxalate crystalagglomerates get deposited in the renal tubules and furtherdevelop into renal stones [25] GPAE reduced the polyuriaassociated with lithogenic treatment as compared to urolithiccontrol groups As per the earlier reports stone inductiontreatment leads an increase in oxalate and decrease in Ca2+

excretion This is confirmed in the present study in whichurolithic control animals showed the similar effect andGPAE- and Cystone-treated groups reversed this effect [4243]

Lithogenic treatment resulted in increased calciumoxalate deposition in the urolithic control groups along withoxidative stress determined from increased levels of markersof oxidative damage that is MDA and protein carbonylcontent alongwith decreased activity of antioxidant enzymesand GSH levels in kidneys accompanied with abnormal renalfunction The renal tubules were dilated in entire kidneysof all untreated rats leading to disturbed renal flow bylarge crystal aggregates However GPAE revealed significantdecrease in all these lithogenic markers at higher doses alongwith the antioxidant activity

The present investigation thus reports the significantantiurolithic potential ofGPAE at higher doses by the combi-nation of antioxidant diuretic and hypocalciuric effects Theresults of the formulation were found significant as comparedwith Cystone which is a standard drug available in themarket

8 Evidence-Based Complementary and Alternative Medicine

used in the treatment of urolithiasis and diuretic activity wascomparable with the standard diuretic drug Furosemide

5 Conclusions

The inhibitory effect of GPAE on calcium oxalate crystalretention in renal tubules thus can be attributed to itsdiuretic hypocalciuric antioxidant effect along with itspotential to inhibit biochemical parameters involved inoxalate metabolism which rationalize its medicinal use forurinary stone diseaseThe present study will certainly benefitthe scientific community worldwide in providing a source forisolation and designing a molecule in this research pipeline

Acknowledgments

The authors are thankful to The Indian Council of MedicalResearch (ICMR) New Delhi for financial assistance to thispostdoctoral project and Director the National Institute ofNutrition Hyderabad for providing necessary laboratoryfacilities

References

[1] G C Curhan W C Willett E B Rimm and M J Stampfer ldquoAprospective study of the intake of vitamins C and B6 and therisk of kidney stones in menrdquo Journal of Urology vol 155 no 6pp 1847ndash1851 1996

[2] C L Smith and D R P Guay ldquoNephrolithiasisrdquo in Pharma-cotherapy and Pathophysiologic Approach J T Di Piro R LTalbert P E Hayes G C Yee G R Matzke and L M PoseyEds pp 720ndash736 Elsevier New York NY USA 2nd edition1992

[3] F L Coe and M J Favus Disorders of Bone and MineralMetabolism Raven press New York NY USA 1992

[4] A L Shirfule C N Khobragade P Badrinarayan Y SBorse and R H Amilkanthwar ldquoPhytochemical analysis andantiurolithiatic activity of a polyherbal formulationrdquo Journal ofHerbs Spices andMedicinal Plants vol 15 no 1 pp 66ndash72 2009

[5] A M Sharifi R Darabi and N Akbarloo ldquoStudy of antihyper-tensive mechanism of Tribulus terrestris in 2K1C hypertensiverats role of tissue ACE activityrdquo Life Sciences vol 73 no 23 pp2963ndash2971 2003

[6] R C Singh and C S Sisodia ldquoEffect of Tribulus terrestris fruitextracts on chloride and creatinine renal clearances in dogsrdquoIndian Journal of Physiology and Pharmacology vol 15 no 3pp 93ndash96 1971

[7] M Al-Ali S Wahbi H Twaij and A Al-Badr ldquoTribulusterrestris preliminary study of its diuretic and contractile effectsand comparisonwith Zeamaysrdquo Journal of Ethnopharmacologyvol 85 no 2-3 pp 257ndash260 2003

[8] A K Nadkarni Indian Materia Medica vol 1 Popular BookDepot Bombay India 3rd edition 1976

[9] R N Chopra S L Nayar and I C Chopra Glossary of IndianMedicinal Plants CSIR New Delhi India 1956

[10] M R Fernando S M D N Wickramasinghe M I Thabrewand E H Karunanayaka ldquoA preliminary investigation of thepossible hypoglycaemic activity of Asteracanthus longifoliardquoJournal of Ethnopharmacology vol 27 no 1-2 pp 7ndash14 1989

[11] U K Mazumdar M Gupta S Maiti and D MukherjeeldquoAntitumor activity of Hygrophila spinosa on Ehrlich ascitescarcinoma and sarcoma-180 induced micerdquo Indian Journal ofExperimental Biology vol 35 no 5 pp 473ndash477 1997

[12] A Gomes M Das and S C Dasgupta ldquoHaematinic effectof Hygrophila spinosa T Anderson on experimental rodentsrdquoIndian Journal of Experimental Biology vol 39 no 4 pp 381ndash382 2001

[13] P Shanmugasundaram and S Venkataraman ldquoAnti-nociceptiveactivity of Hygrophila auriculata (Schum) Heinerdquo The AfricanJournal of Traditional Complementary and AlternativeMedicines vol 2 p 62 2005

[14] A Singh and S S Handa ldquoHepatoprotective activity of Apiumgraveolens and Hygrophila auriculata against paracetamol andthioacetamide intoxication in ratsrdquo Journal of Ethnopharmacol-ogy vol 49 no 3 pp 119ndash126 1995

[15] P Shanmugasundaram and S Venkataraman ldquoHepatoprotec-tive and antioxidant effects ofHygrophila auriculata (K Schum)HeineAcanthaceae root extractrdquo Journal of Ethnopharmacologyvol 104 no 1-2 pp 124ndash128 2006

[16] M Vijayakumar R Govindarajan G M M Rao et al ldquoActionof Hygrophila auriculata against streptozotocin-induced oxida-tive stressrdquo Journal of Ethnopharmacology vol 104 no 3 pp356ndash361 2006

[17] Z A Ali andV K Singh ldquoHerbal drugs of Himalaya (medicinalplants of Garhwal and Kumaon regions of India)rdquo in Aspect ofPlant Sciences vol 15 pp 110ndash111 Today ampTomorrowrsquos PrintersNew Delhi India 1998

[18] Anonymous inTheWealth of India-RawMaterials pp 393ndash394Council of Scientific and Industrial Research NewDelhi India1989

[19] S Siddiqui S Faizi and B Siddiqui ldquoStudies in the chemicalconstituents of the fresh berries of Solanum xanthocarpumrdquoJournal of the Chemical Society of Pakistan vol 5 no 2 pp 14ndash21 1983

[20] L Mohan P Sharma and C N Srivastava ldquoComparativeefficacy of Solanum xanthocarpum extracts alone and in com-bination with a synthetic pyrethroid cypermethrin againstmalaria vector Anopheles Stephensirdquo Southeast Asian Journalof Tropical Medicine and Public Health vol 38 no 2 pp 256ndash260 2007

[21] G P Vadnere R S Gaud andA K Singhai ldquoEvaluation of anti-asthmatic property of SolanumXanthocarpum flower extractsrdquoPharmacologyonline vol 1 pp 513ndash522 2008

[22] D J Huang C D Lin H J Chen and Y H Lin ldquoAntioxidantand antiproliferative activities of sweet potato (Ipomea batata[L] Lam lsquoTaniong 57rsquo) constituentsrdquo Botanical Bulletin ofAcademia Sinica vol 45 pp 179ndash186 2004

[23] D G Kang C K Yun and H S Lee ldquoScreening and com-parison of antioxidant activity of solvent extracts of herbalmedicines used in Koreardquo Journal of Ethnopharmacology vol87 no 2-3 pp 231ndash236 2003

[24] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[25] F Atmani Y Slimani M Mimouni and B Hacht ldquoProphylaxisof calcium oxalate stones by Herniaria hirsuta on experimen-tally induced nephrolithiasis in ratsrdquo British Journal of UrologyInternational vol 92 no 1 pp 137ndash140 2003

Evidence-Based Complementary and Alternative Medicine 9

[26] J A Daly and G Ertingshausen ldquoDirect method for deter-mining inorganic phosphate in serum with the ldquocentrifichemrdquoClinical Chemistry vol 18 pp 263ndash265 1972

[27] S H Y Wong J A Knight S M Hopfer O Zaharia CN Leach Jr and F W Sunderman Jr ldquoLipoperoxides inplasma as measured by liquid-chromatographic separation ofmalondialdehyde-thiobarbituric acid adductrdquo Clinical Chem-istry vol 33 no 2 pp 214ndash220 1987

[28] R L Levine D Garland C N Oliver et al ldquoDetermination ofcarbonyl content in oxidatively modified proteinsrdquo Methods inEnzymology vol 186 pp 464ndash478 1990

[29] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[30] H E Aebi ldquoEnzymes I oxidoreductases transferasesrdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed pp 273ndash285 VCH Weinheim Germany 1987

[31] A L Baker and N E Tolbert ldquoGlycolate oxidase (ferredoxin-containing form)rdquoMethods in Enzymology vol 9 pp 338ndash3421966

[32] J King in Practical Clinical Enzymology vol 87 van Nostrand1965

[33] S R Khan ldquoCalcium oxalate crystal interaction with renaltubular epithelium mechanism of crystal adhesion and itsimpact on stone developmentrdquo Urological Research vol 23 no2 pp 71ndash79 1995

[34] S R Khan and R L Hackett ldquoRetention of calcium oxalatecrystals in renal tubulesrdquo Scanning Microscopy vol 5 pp 707ndash712 1991

[35] F Dal Moro M Mancini I M Tavolini V de Marco and PBassi ldquoCellular and molecular gateways to urolithiasis a newinsightrdquoUrologia Internationalis vol 74 no 3 pp 193ndash197 2005

[36] H S Huang M C Ma C F Chen and J Chen ldquoLipidperoxidation and its correlations with urinary levels of oxalatecitric acid and osteopontin in patients with renal calciumoxalate stonesrdquo Urology vol 62 no 6 pp 1123ndash1128 2003

[37] M Santhosh Kumar and R Selvam ldquoSupplementation ofvitamin E and seleniumprevents hyperoxaluria in experimentalurolithic ratsrdquo Journal of Nutritional Biochemistry vol 14 no 6pp 306ndash313 2003

[38] S Thamilselvan and M Menon ldquoVitamin E therapy preventshyperoxaluria-induced calciumoxalate crystal deposition in thekidney by improving renal tissue antioxidant statusrdquo BritishJournal of Urology International vol 96 no 1 pp 117ndash126 2005

[39] Y Itoh T Yasui A Okada K Tozawa Y Hayashi and K KohrildquoPreventive effects of green tea on renal stone formation and therole of oxidative stress in nephrolithiasisrdquo Journal of Urologyvol 173 no 1 pp 271ndash275 2005

[40] S Thamilselvan R L Hackett and S R Khan ldquoLipid perox-idation in ethylene glycol induced hyperoxaluria and calciumoxalate nephrolithiasisrdquo Journal of Urology vol 157 no 3 pp1059ndash1063 1997

[41] C H Tsai Y C Chen L D Chen et al ldquoA traditional Chineseherbal antilithic formula Wulingsan effectively prevents therenal deposition of calcium oxalate crystal in ethylene glycol-fed ratsrdquo Urological Research vol 36 no 1 pp 17ndash24 2008

[42] J FanM A Glass and P S Chandhoke ldquoImpact of ammoniumchloride administration on rat ethylene glycol urolithiasismodelrdquo Scanning Microscopy vol 13 pp 299ndash306 1999

[43] HK Park B C JeongMK Sung et al ldquoReduction of oxidativestress in cultured renal tubular cells and preventive effects onrenal stone formation by the bioflavonoid Quercetinrdquo Journalof Urology vol 179 no 4 pp 1620ndash1626 2008

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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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

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OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Diuretic effect of Gokshuradi polyherbal aqueous extracts (GPAEs)

GroupsUrine vol

(mL)100 g bodyweight6 h

Na+(mmol)100 g

body weight6 h

K+

(mmol)100 gbody weight6 h

Ca2+(mmol)100 g

body weight6 hpH

Control 059 plusmn 014 009 plusmn 003 010 plusmn 002 782 plusmn 162 587 plusmn 006GPAE 25mgkg 123 plusmn 021 013 plusmn 001 015 plusmn 002 622 plusmn 229 685 plusmn 005GPAE 50mgkg 151lowast plusmn 018 019 plusmn 001 019 plusmn 005 369 plusmn 176 693 plusmn 018GPAE 100mgkg 172lowastlowast plusmn 011 026lowast plusmn 002 022lowast plusmn 003 237lowast plusmn 133 732lowastlowast plusmn 024Furosemide 10mgkg 210lowastlowast plusmn 031 038lowastlowast plusmn 007 021lowastlowast plusmn 002 126lowast plusmn 049 739lowastlowast plusmn 027Values given are mean plusmn SEM (n = 6)lowastP lt 005lowastlowastP lt 001 versus control

Table 2 Effect of GPAE and standard drug on 24 h urinary biochemical parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgChange in body weight () 1188 plusmn 153 minus478lowastlowast plusmn 108 232lowastlowast plusmn 145 886 plusmn 206 543lowastlowast plusmn 123Water intake mL24 h 622 plusmn 039 2435lowastlowast plusmn 723 143 plusmn 232 845 plusmn 117 112 plusmn 221Urine vol (mL) 582 plusmn 045 2625lowastlowast plusmn 676 1643lowast plusmn 324 1046lowast plusmn 126 1365lowast plusmn 284pH 638 plusmn 009 617 plusmn 016 627 plusmn 011 645 plusmn 014 632 plusmn 015Oxalate (mg) 038 plusmn 010 212lowastlowast plusmn 021 135lowastlowast plusmn 025 068 plusmn 011 189lowastlowast plusmn 018Ca2+ (mg) 367 plusmn 024 184lowastlowast plusmn 016 263lowast plusmn 029 316 plusmn 064 289lowast plusmn 041Mg2+ (mg) 328 plusmn 032 267 plusmn 011 342 plusmn 024 376 plusmn 031 287 plusmn 032Citrate (mg) 2310 plusmn 121 185 plusmn 190 1912 plusmn 263 1931 plusmn 122 1834 plusmn 254IPa (mg) 626 plusmn 082 789 plusmn 125 757 plusmn 055 755 plusmn 075 734 plusmn 032UAb (mg) 058 plusmn 017 123 plusmn 040 105 plusmn 025 083 plusmn 022 098 plusmn 022Values given are mean plusmn SEM (n = 6)lowastP lt 005lowastlowastP lt 001 versus control groupP lt 005P lt 001 versus urolithic groupaInorganic phosphate bUric acid

dinitrophenyl hydrazones by the method of Levine et al [28]and the carbonyl content was calculated using the DNPHmolar extinction coefficient of 22000Mminus1 cmminus1 GSH wasestimated as total nonprotein thiol (SH) group following themethod described byMoron et al [29] Superoxide dismutase(SOD) and glutathione peroxidase (GPx) were determinedby using commercially available kits Catalase activity wasdetermined by monitoring the decomposition of H

2O2at

240 nm with a spectrophotometer [30] Catalase activitywas calculated by using 120576

240(molar extinction coefficient)

= 00394mmolminus1minminus1 for H2O2and expressed as 120583moles

of H2O2decomposed per min under standard conditions at

25∘C Glycolate oxidase (GOx) activity in liver and lactatedehydrogenase (LDH) in both liver and kidney were deter-mined by reported method [31 32]

258 Statistical Analysis The data expressed are mean plusmnstandard error of mean (SEM) and the median inhibitoryconcentration (IC

50value) with 95 confidence intervals

All statistical comparisons between the groups are madeby means of one way analysis of variance with post hocStudentrsquos t-test The 119875 values less than 005 are regarded assignificantThe concentration-response curveswere analyzed

by nonlinear regression using Graph Pad Prism (Graph PadSoftware San Diego CA USA)

3 Result

31 In Vitro Studies

311 Antioxidant Effect GPAE revealed DPPH radical scav-enging activity with IC

50value of 25 (105ndash290) 120583gmL

(Figure 1(a)) and inhibited lipid peroxidation of the ratkidney homogenate in vitro by 3637 plusmn 186 and 6872 plusmn025 at 50 and 150120583gmL respectively (Figure 1(b)) Thestandard chemical BHT similarly showed DPPH radicalscavenging activity with IC

50value of 256 (102ndash285)120583gmL

and inhibited in vitro lipid peroxidation by 384 plusmn 122 and945 plusmn 46 at 50 and 150 120583gmL respectively

32 In Vivo Studies

321 Diuretic Effect The effect of various doses of GPAE onrat urine volume pH and Na+ K+ and Ca2+ excretion isgiven in Table 1 GPAE increased urine volume at 50mgkg(119875 lt 005) and 100mgkg (119875 lt 001) indicating diuretic

Evidence-Based Complementary and Alternative Medicine 5

Table 3 Effect of GPAE and standard drug on serum parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgSCc (mgdL) 086 plusmn 005 143lowastlowast plusmn 012 126 plusmn 016 089 plusmn 010 108 plusmn 012CCd (mgmin) 086 plusmn 006 050lowastlowast plusmn 006 064 plusmn 010 085 plusmn 007 072 plusmn 012BUNe (mgdL) 1989 plusmn 140 5704lowastlowast plusmn 1246 463 plusmn 1140 205 plusmn 108 362 plusmn 1020Values given are mean plusmn SEM (n = 6)lowastP lt 005lowastlowastP lt 001 versus control groupP lt 005P lt 001 versus urolithic groupcSerum creatinine dCreatinine clearance eBlood urea nitrogen

Table 4 Effect of GPAE and standard drug on kidney and liver parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgKidney weight (g) 078 plusmn 005 121lowastlowast plusmn 012 109lowast plusmn 014 082 plusmn 007 098lowast plusmn 012CDf Absent Significant Moderate Not significant ModerateMDAg (nmol) 064 plusmn 014 652lowastlowast plusmn 085 306 plusmn 110 109 plusmn 042 285 plusmn 108PCCh (nmol) 508 plusmn 068 1304lowastlowast plusmn 136 1012 plusmn 188 805 plusmn 114 980 plusmn 123GSHi (nmol) 1789 plusmn 098 1310lowast plusmn 133 1503 plusmn 182 1698 plusmn 157 1402 plusmn 131SODj (U) 622 plusmn 084 301lowastlowast plusmn 029 458 plusmn 032 548 plusmn 056 421 plusmn 021GPXk (U) 058 plusmn 005 032lowast plusmn 004 047 plusmn 008 053 plusmn 007 048 plusmn 005Catl (120583MH2O2min) 3289 plusmn 255 1689lowastlowast plusmn 198 2092 plusmn 511 2490 plusmn 232 2108 plusmn 432GOxm (nmolmg) 161 plusmn 007 235 plusmn 006lowastlowast 177 plusmn 017 150 plusmn 010 165 plusmn 017LDHn (Umg) (Liver) 154 plusmn 006 403 plusmn 009lowastlowast 149 plusmn 108 151 plusmn 006 146 plusmn 104LDHo (Umg) (Kidney) 282 plusmn 016 468 plusmn 019lowastlowast 319 plusmn 108 307 plusmn 108 332 plusmn 104Calciump (mgg) (Kidney) 0292 plusmn 016 0498 plusmn 0082 0375 plusmn 0078 0316 plusmn 0021 0369 plusmn 0038

Values given are mean plusmn SEM (n = 6)lowastP lt 005 lowastlowastP lt 001 versus control group P lt 005 P lt 001 versus urolithic groupfCrystal deposits100x field gmalondialdehyde hprotein carbonyl content ireduced glutathione jsuperoxide dismutase kglutathione peroxidase lcatalasemglycolate oxidase (nmol of glyoxylate formedmg protein) nliver lactate dehydrogenase (Umg protein) okidney lactate dehydrogenase (Umg protein) andpkidney calcium content

effect At the next higher dose (200mgkg) urine volumedid not increase above that of saline-treated group (119875 gt005) The reference diuretic drug Furosemide at 10mgkgshowed statistically significant increase in the urine output(119875 lt 001) In addition to an increase in the urine outputGPAE and similarly Furosemide increased urine excretionofNa+ andK+ compared to the saline-treated control animalsat higher dose GPAE caused significant increase in the urinepH and decrease in Ca2+ excretion (119875 lt 001) at 100mgkgcomparable to the standard drug Furosemide (10mgkg)

322 Effect Observed in the Animal Model The baselineparameters recorded before the start of treatment like bodyweights water intake along with urine volume pH andcomposition were not significantly different among all thegroupsTheparameters recorded after the treatment are givenin Table 2 As compared to normal control animals waterintake and 24 h urine volume were high in the urolithicgroup (119875 lt 001) Stone-inducing treatment also reducedpH of urine in the urolithic group as compared to thatof the normal control group although not to a significantextent Simultaneous treatment with GPAE at 100mgkg intreatment groups reduced the increase in urine volume andwater intake (119875 lt 005) as compared with the control animals(119875 lt 005) There was an increased oxalate and decreased

Ca2+ concentration (119875 lt 001) of the urine collected from theurolithic animals as compared to normal control GPAE andCystone groups exactly reversed this effect that is decreasedthe urinary oxalate (119875 lt 001) and increased Ca2+ (119875 lt 005)excretion at all the treated doses and remained significantat 100mgkg in GPAE-treated group Other changes in theurine composition like decreased levels of citrate and Mg2+and increased excretion of uric acid and inorganic phosphatein urolithic group were not statistically significant GPAE-andCystone-treated groups also did not show any statisticallysignificant change in these parameters and remained com-parable to that of control animals The urolithic treatmentrevealed loss in body weight of the urolithic animals (119875 lt001) whereas GPAE- and Cystone-treated animals showednet gain in the body weights at all the tested doses Kidneys ofurolithic group animals were heavier than those the controlanimals (119875 lt 001) whereas GPAE- (100mgkg) treated ratkidneys did not show any significant increase in weight ascompared with control however the GPAE- (50mgkg) andCystone- (100mgkg) treated animalrsquos kidneys were slightlyheavier than those the control animals (Table 4)

Urolithic treatment was found to disturb renal functionin urolithic animals as evident from the increased BUN andserum creatinine and reduced creatinine clearance GPAE-treated groups decreased the serum creatinine and BUN

6 Evidence-Based Complementary and Alternative Medicine

DPPH radical scavenging activity

0

20

40

60

80

100

120

01 02 03 04 05 1 5 10 50 100

GPAE BHT

Inhi

bitio

n (

)

(120583gml)

(a)

0

20

40

60

80

100

50 150

Lipid peroxidation inhibitory effect

BHT GPAE

Inhi

bitio

n (

)

(120583gml)

(b)

Figure 1 (a) DPPH radical scavenging activity (b) Lipid peroxi-dation inhibitory effect of GPAE and the reference drug butylatedhydroxytoluene (BHT) Symbols shown are mean plusmn SEM (119899 = 3)

levels at 100mgkg and similar observations were made inthe Cystone-treated groups at 100mgkg as compared to con-trol The creatinine clearance was significantly increased at100mgkg in GPAE-treated groups as compared to urolithicanimals but was not significant at 50mgkg (Table 3)

Histology studies of the kidney regions like cortexmedulla and papilla in the urolithic animals showed manybirefringent crystalline deposits under polarized light micro-scope (Figure 2(b)) Such deposits were found in 3 outof 6 rats from GPAE low dose (Figure 2(c)) 1 out of 6rats from GPAE high dose (Figure 2(d)) and 3 out of 6rats (Figure 2(e)) from Cystone group but visibly smallerand less abundant compared to urolithic control animalsThe tissue morphology and intactness of the kidneys ofGPAE- and Cystone-treated groups were comparable to thenormal control animals (Figure 2(a)) The atomic absorptionspectrophotometer analysis revealed decreased Ca contentin the GPAE- and Cystone- (119875 lt 005) treated groupsas compared with the urolithic control groups wherein the

GPAE at 100mgkg shown significant decrease of Ca (119875 lt001) (Table 4)

323 Lipid Peroxidation Inhibitory Effect Stone-inducingtreatment increasedMDA and carbonyl protein content (119875 lt001) and decreased GSH level (119875 lt 005) and activities ofthe antioxidant enzymes including SOD (119875 lt 001) GPX(119875 lt 005) and catalase (119875 lt 001) in kidneys of theurolithic rats as compared to control animals Activities ofoxalate forming enzymes including GOx (119875 lt 001) in liverand LDH (119875 lt 001) in both liver and kidneys were found tobe enhanced in urolithic group compared to control animalswhereas treatment with GPAE was found to be protectiveagainst this effect and oxidative changes induced by urolithictreatment at higher doses (Table 4 Figure 1(b))

4 Discussion

Phytotherapy approach is attempted in this study as analternative to primary healthcare The Gokshuradi PolyherbalFormulation (GPF) which is traditionally being utilized inIndian folk medicine since several decades to treat kidneystones was evaluated for its antiurolithic effects In our earlierstudies we have clinically evaluated the efficacy of this prepa-ration and found its potential in naturally induced urolithicpatients [4] The present study is a reverse pharmacologyapproach wherein the scientific studies were undertakenunder controlled conditions to validate the traditional use ofGPF and determine its underlying mechanism of action oncalcium oxalate urolithiasis

Calcium oxalate stone development is a multifactorialprocess involving various etiological factors Studies haveshown that calcium oxalate crystal deposition leads to thecellular injury mediated by lipid peroxidation through freeoxygen radical generation Studies revealed that these cellularinjuries favor the events of calcium oxalate retention in renaltubules which is significant for further stone development[33ndash35] Recent clinical data are also supporting this findingthat formation of urinary stones leads to the oxidative stressin patients [36] Antioxidants such as vitamin C catechinand selenium have proved to be protective against suchoxidative injury due to calcium oxalate crystal depositionas evidenced from various experimental studies [37ndash39] Inthis regard antioxidant potential of GPAE was determinedby free radical scavenging and lipid peroxidation inhibitoryactivity to study its mechanism of action InterestinglyGPAErevealed DPPH free radical scavenging and inhibited lipidperoxidation of rat kidney homogenate similar to a standardantioxidant chemical BHT

In the diuretic activity study GPAE altered urine outputin a dose dependantmanner andmaximumdiuresis observedat 100mgkg At next higher dose that is 200mgkg it did notlead to any further increase in the urine volume and remainedcomparable to the normal control group The disappearanceof the diuretic effect of these polyherbal extracts at this higherdose is not completely understood Increase in urine volumebyGPAEwas also associated with the increase in Na+ and K+electrolyte loss similar to the standard diuretic furosemide

Evidence-Based Complementary and Alternative Medicine 7

(a) (b) (c)

(d) (e)

Figure 2 (a) Normal cellular structure of control rats kidney (Gr I) (b) Ethylene-glycol-induced urolithic rats kidney showing irregularcrystals dilation and inflammation under polarized microscope (Gr II) (c) Kidneys of urolithic rats treated with GPAE (50mgkg) showingnear normal cellular structure under polarized microscope (Gr III) (d) Kidneys of urolithic rats treated with GPAE (100mgkg) showingnormal cellular Structure (Gr IV) (e) Kidneys of urolithic rats treated with Cystone (100mgkg) showing normal cellular Structure (Gr V)Arrow indicates CaOx crystals

Increase in urine volume thus suggestive of possible loss ofthese electrolytes leading to this effectGPAE-treated group isalso revealed increase in urine pH and decrease in urine Ca2+at 100mgkg like that of furosemide-treated groups Thesehypocalciuric and diuretic effects ofGPAEmay help to reduceurinary super saturation of calcium salts and thus can preventfurther stone formation

Further antiurolithic effect of GPAE was evaluated inan animal model of urolithiasis induced by ethylene glycoland ammonium chloride in combination This model isfrequently used in calcium oxalate stone formation andtherefore was selected for this study [25 40 41] After 21 daysadministration of EG +AC showed larger and aggregatedcrystals in urolithic control animals compared to the GPAE-and Cystone-treated groups These calcium oxalate crystalagglomerates get deposited in the renal tubules and furtherdevelop into renal stones [25] GPAE reduced the polyuriaassociated with lithogenic treatment as compared to urolithiccontrol groups As per the earlier reports stone inductiontreatment leads an increase in oxalate and decrease in Ca2+

excretion This is confirmed in the present study in whichurolithic control animals showed the similar effect andGPAE- and Cystone-treated groups reversed this effect [4243]

Lithogenic treatment resulted in increased calciumoxalate deposition in the urolithic control groups along withoxidative stress determined from increased levels of markersof oxidative damage that is MDA and protein carbonylcontent alongwith decreased activity of antioxidant enzymesand GSH levels in kidneys accompanied with abnormal renalfunction The renal tubules were dilated in entire kidneysof all untreated rats leading to disturbed renal flow bylarge crystal aggregates However GPAE revealed significantdecrease in all these lithogenic markers at higher doses alongwith the antioxidant activity

The present investigation thus reports the significantantiurolithic potential ofGPAE at higher doses by the combi-nation of antioxidant diuretic and hypocalciuric effects Theresults of the formulation were found significant as comparedwith Cystone which is a standard drug available in themarket

8 Evidence-Based Complementary and Alternative Medicine

used in the treatment of urolithiasis and diuretic activity wascomparable with the standard diuretic drug Furosemide

5 Conclusions

The inhibitory effect of GPAE on calcium oxalate crystalretention in renal tubules thus can be attributed to itsdiuretic hypocalciuric antioxidant effect along with itspotential to inhibit biochemical parameters involved inoxalate metabolism which rationalize its medicinal use forurinary stone diseaseThe present study will certainly benefitthe scientific community worldwide in providing a source forisolation and designing a molecule in this research pipeline

Acknowledgments

The authors are thankful to The Indian Council of MedicalResearch (ICMR) New Delhi for financial assistance to thispostdoctoral project and Director the National Institute ofNutrition Hyderabad for providing necessary laboratoryfacilities

References

[1] G C Curhan W C Willett E B Rimm and M J Stampfer ldquoAprospective study of the intake of vitamins C and B6 and therisk of kidney stones in menrdquo Journal of Urology vol 155 no 6pp 1847ndash1851 1996

[2] C L Smith and D R P Guay ldquoNephrolithiasisrdquo in Pharma-cotherapy and Pathophysiologic Approach J T Di Piro R LTalbert P E Hayes G C Yee G R Matzke and L M PoseyEds pp 720ndash736 Elsevier New York NY USA 2nd edition1992

[3] F L Coe and M J Favus Disorders of Bone and MineralMetabolism Raven press New York NY USA 1992

[4] A L Shirfule C N Khobragade P Badrinarayan Y SBorse and R H Amilkanthwar ldquoPhytochemical analysis andantiurolithiatic activity of a polyherbal formulationrdquo Journal ofHerbs Spices andMedicinal Plants vol 15 no 1 pp 66ndash72 2009

[5] A M Sharifi R Darabi and N Akbarloo ldquoStudy of antihyper-tensive mechanism of Tribulus terrestris in 2K1C hypertensiverats role of tissue ACE activityrdquo Life Sciences vol 73 no 23 pp2963ndash2971 2003

[6] R C Singh and C S Sisodia ldquoEffect of Tribulus terrestris fruitextracts on chloride and creatinine renal clearances in dogsrdquoIndian Journal of Physiology and Pharmacology vol 15 no 3pp 93ndash96 1971

[7] M Al-Ali S Wahbi H Twaij and A Al-Badr ldquoTribulusterrestris preliminary study of its diuretic and contractile effectsand comparisonwith Zeamaysrdquo Journal of Ethnopharmacologyvol 85 no 2-3 pp 257ndash260 2003

[8] A K Nadkarni Indian Materia Medica vol 1 Popular BookDepot Bombay India 3rd edition 1976

[9] R N Chopra S L Nayar and I C Chopra Glossary of IndianMedicinal Plants CSIR New Delhi India 1956

[10] M R Fernando S M D N Wickramasinghe M I Thabrewand E H Karunanayaka ldquoA preliminary investigation of thepossible hypoglycaemic activity of Asteracanthus longifoliardquoJournal of Ethnopharmacology vol 27 no 1-2 pp 7ndash14 1989

[11] U K Mazumdar M Gupta S Maiti and D MukherjeeldquoAntitumor activity of Hygrophila spinosa on Ehrlich ascitescarcinoma and sarcoma-180 induced micerdquo Indian Journal ofExperimental Biology vol 35 no 5 pp 473ndash477 1997

[12] A Gomes M Das and S C Dasgupta ldquoHaematinic effectof Hygrophila spinosa T Anderson on experimental rodentsrdquoIndian Journal of Experimental Biology vol 39 no 4 pp 381ndash382 2001

[13] P Shanmugasundaram and S Venkataraman ldquoAnti-nociceptiveactivity of Hygrophila auriculata (Schum) Heinerdquo The AfricanJournal of Traditional Complementary and AlternativeMedicines vol 2 p 62 2005

[14] A Singh and S S Handa ldquoHepatoprotective activity of Apiumgraveolens and Hygrophila auriculata against paracetamol andthioacetamide intoxication in ratsrdquo Journal of Ethnopharmacol-ogy vol 49 no 3 pp 119ndash126 1995

[15] P Shanmugasundaram and S Venkataraman ldquoHepatoprotec-tive and antioxidant effects ofHygrophila auriculata (K Schum)HeineAcanthaceae root extractrdquo Journal of Ethnopharmacologyvol 104 no 1-2 pp 124ndash128 2006

[16] M Vijayakumar R Govindarajan G M M Rao et al ldquoActionof Hygrophila auriculata against streptozotocin-induced oxida-tive stressrdquo Journal of Ethnopharmacology vol 104 no 3 pp356ndash361 2006

[17] Z A Ali andV K Singh ldquoHerbal drugs of Himalaya (medicinalplants of Garhwal and Kumaon regions of India)rdquo in Aspect ofPlant Sciences vol 15 pp 110ndash111 Today ampTomorrowrsquos PrintersNew Delhi India 1998

[18] Anonymous inTheWealth of India-RawMaterials pp 393ndash394Council of Scientific and Industrial Research NewDelhi India1989

[19] S Siddiqui S Faizi and B Siddiqui ldquoStudies in the chemicalconstituents of the fresh berries of Solanum xanthocarpumrdquoJournal of the Chemical Society of Pakistan vol 5 no 2 pp 14ndash21 1983

[20] L Mohan P Sharma and C N Srivastava ldquoComparativeefficacy of Solanum xanthocarpum extracts alone and in com-bination with a synthetic pyrethroid cypermethrin againstmalaria vector Anopheles Stephensirdquo Southeast Asian Journalof Tropical Medicine and Public Health vol 38 no 2 pp 256ndash260 2007

[21] G P Vadnere R S Gaud andA K Singhai ldquoEvaluation of anti-asthmatic property of SolanumXanthocarpum flower extractsrdquoPharmacologyonline vol 1 pp 513ndash522 2008

[22] D J Huang C D Lin H J Chen and Y H Lin ldquoAntioxidantand antiproliferative activities of sweet potato (Ipomea batata[L] Lam lsquoTaniong 57rsquo) constituentsrdquo Botanical Bulletin ofAcademia Sinica vol 45 pp 179ndash186 2004

[23] D G Kang C K Yun and H S Lee ldquoScreening and com-parison of antioxidant activity of solvent extracts of herbalmedicines used in Koreardquo Journal of Ethnopharmacology vol87 no 2-3 pp 231ndash236 2003

[24] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[25] F Atmani Y Slimani M Mimouni and B Hacht ldquoProphylaxisof calcium oxalate stones by Herniaria hirsuta on experimen-tally induced nephrolithiasis in ratsrdquo British Journal of UrologyInternational vol 92 no 1 pp 137ndash140 2003

Evidence-Based Complementary and Alternative Medicine 9

[26] J A Daly and G Ertingshausen ldquoDirect method for deter-mining inorganic phosphate in serum with the ldquocentrifichemrdquoClinical Chemistry vol 18 pp 263ndash265 1972

[27] S H Y Wong J A Knight S M Hopfer O Zaharia CN Leach Jr and F W Sunderman Jr ldquoLipoperoxides inplasma as measured by liquid-chromatographic separation ofmalondialdehyde-thiobarbituric acid adductrdquo Clinical Chem-istry vol 33 no 2 pp 214ndash220 1987

[28] R L Levine D Garland C N Oliver et al ldquoDetermination ofcarbonyl content in oxidatively modified proteinsrdquo Methods inEnzymology vol 186 pp 464ndash478 1990

[29] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[30] H E Aebi ldquoEnzymes I oxidoreductases transferasesrdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed pp 273ndash285 VCH Weinheim Germany 1987

[31] A L Baker and N E Tolbert ldquoGlycolate oxidase (ferredoxin-containing form)rdquoMethods in Enzymology vol 9 pp 338ndash3421966

[32] J King in Practical Clinical Enzymology vol 87 van Nostrand1965

[33] S R Khan ldquoCalcium oxalate crystal interaction with renaltubular epithelium mechanism of crystal adhesion and itsimpact on stone developmentrdquo Urological Research vol 23 no2 pp 71ndash79 1995

[34] S R Khan and R L Hackett ldquoRetention of calcium oxalatecrystals in renal tubulesrdquo Scanning Microscopy vol 5 pp 707ndash712 1991

[35] F Dal Moro M Mancini I M Tavolini V de Marco and PBassi ldquoCellular and molecular gateways to urolithiasis a newinsightrdquoUrologia Internationalis vol 74 no 3 pp 193ndash197 2005

[36] H S Huang M C Ma C F Chen and J Chen ldquoLipidperoxidation and its correlations with urinary levels of oxalatecitric acid and osteopontin in patients with renal calciumoxalate stonesrdquo Urology vol 62 no 6 pp 1123ndash1128 2003

[37] M Santhosh Kumar and R Selvam ldquoSupplementation ofvitamin E and seleniumprevents hyperoxaluria in experimentalurolithic ratsrdquo Journal of Nutritional Biochemistry vol 14 no 6pp 306ndash313 2003

[38] S Thamilselvan and M Menon ldquoVitamin E therapy preventshyperoxaluria-induced calciumoxalate crystal deposition in thekidney by improving renal tissue antioxidant statusrdquo BritishJournal of Urology International vol 96 no 1 pp 117ndash126 2005

[39] Y Itoh T Yasui A Okada K Tozawa Y Hayashi and K KohrildquoPreventive effects of green tea on renal stone formation and therole of oxidative stress in nephrolithiasisrdquo Journal of Urologyvol 173 no 1 pp 271ndash275 2005

[40] S Thamilselvan R L Hackett and S R Khan ldquoLipid perox-idation in ethylene glycol induced hyperoxaluria and calciumoxalate nephrolithiasisrdquo Journal of Urology vol 157 no 3 pp1059ndash1063 1997

[41] C H Tsai Y C Chen L D Chen et al ldquoA traditional Chineseherbal antilithic formula Wulingsan effectively prevents therenal deposition of calcium oxalate crystal in ethylene glycol-fed ratsrdquo Urological Research vol 36 no 1 pp 17ndash24 2008

[42] J FanM A Glass and P S Chandhoke ldquoImpact of ammoniumchloride administration on rat ethylene glycol urolithiasismodelrdquo Scanning Microscopy vol 13 pp 299ndash306 1999

[43] HK Park B C JeongMK Sung et al ldquoReduction of oxidativestress in cultured renal tubular cells and preventive effects onrenal stone formation by the bioflavonoid Quercetinrdquo Journalof Urology vol 179 no 4 pp 1620ndash1626 2008

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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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

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OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

Table 3 Effect of GPAE and standard drug on serum parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgSCc (mgdL) 086 plusmn 005 143lowastlowast plusmn 012 126 plusmn 016 089 plusmn 010 108 plusmn 012CCd (mgmin) 086 plusmn 006 050lowastlowast plusmn 006 064 plusmn 010 085 plusmn 007 072 plusmn 012BUNe (mgdL) 1989 plusmn 140 5704lowastlowast plusmn 1246 463 plusmn 1140 205 plusmn 108 362 plusmn 1020Values given are mean plusmn SEM (n = 6)lowastP lt 005lowastlowastP lt 001 versus control groupP lt 005P lt 001 versus urolithic groupcSerum creatinine dCreatinine clearance eBlood urea nitrogen

Table 4 Effect of GPAE and standard drug on kidney and liver parameters

Parameters Control Urolithic GPAE 50mgkg GPAE 100mg Standard drug 100mgkgKidney weight (g) 078 plusmn 005 121lowastlowast plusmn 012 109lowast plusmn 014 082 plusmn 007 098lowast plusmn 012CDf Absent Significant Moderate Not significant ModerateMDAg (nmol) 064 plusmn 014 652lowastlowast plusmn 085 306 plusmn 110 109 plusmn 042 285 plusmn 108PCCh (nmol) 508 plusmn 068 1304lowastlowast plusmn 136 1012 plusmn 188 805 plusmn 114 980 plusmn 123GSHi (nmol) 1789 plusmn 098 1310lowast plusmn 133 1503 plusmn 182 1698 plusmn 157 1402 plusmn 131SODj (U) 622 plusmn 084 301lowastlowast plusmn 029 458 plusmn 032 548 plusmn 056 421 plusmn 021GPXk (U) 058 plusmn 005 032lowast plusmn 004 047 plusmn 008 053 plusmn 007 048 plusmn 005Catl (120583MH2O2min) 3289 plusmn 255 1689lowastlowast plusmn 198 2092 plusmn 511 2490 plusmn 232 2108 plusmn 432GOxm (nmolmg) 161 plusmn 007 235 plusmn 006lowastlowast 177 plusmn 017 150 plusmn 010 165 plusmn 017LDHn (Umg) (Liver) 154 plusmn 006 403 plusmn 009lowastlowast 149 plusmn 108 151 plusmn 006 146 plusmn 104LDHo (Umg) (Kidney) 282 plusmn 016 468 plusmn 019lowastlowast 319 plusmn 108 307 plusmn 108 332 plusmn 104Calciump (mgg) (Kidney) 0292 plusmn 016 0498 plusmn 0082 0375 plusmn 0078 0316 plusmn 0021 0369 plusmn 0038

Values given are mean plusmn SEM (n = 6)lowastP lt 005 lowastlowastP lt 001 versus control group P lt 005 P lt 001 versus urolithic groupfCrystal deposits100x field gmalondialdehyde hprotein carbonyl content ireduced glutathione jsuperoxide dismutase kglutathione peroxidase lcatalasemglycolate oxidase (nmol of glyoxylate formedmg protein) nliver lactate dehydrogenase (Umg protein) okidney lactate dehydrogenase (Umg protein) andpkidney calcium content

effect At the next higher dose (200mgkg) urine volumedid not increase above that of saline-treated group (119875 gt005) The reference diuretic drug Furosemide at 10mgkgshowed statistically significant increase in the urine output(119875 lt 001) In addition to an increase in the urine outputGPAE and similarly Furosemide increased urine excretionofNa+ andK+ compared to the saline-treated control animalsat higher dose GPAE caused significant increase in the urinepH and decrease in Ca2+ excretion (119875 lt 001) at 100mgkgcomparable to the standard drug Furosemide (10mgkg)

322 Effect Observed in the Animal Model The baselineparameters recorded before the start of treatment like bodyweights water intake along with urine volume pH andcomposition were not significantly different among all thegroupsTheparameters recorded after the treatment are givenin Table 2 As compared to normal control animals waterintake and 24 h urine volume were high in the urolithicgroup (119875 lt 001) Stone-inducing treatment also reducedpH of urine in the urolithic group as compared to thatof the normal control group although not to a significantextent Simultaneous treatment with GPAE at 100mgkg intreatment groups reduced the increase in urine volume andwater intake (119875 lt 005) as compared with the control animals(119875 lt 005) There was an increased oxalate and decreased

Ca2+ concentration (119875 lt 001) of the urine collected from theurolithic animals as compared to normal control GPAE andCystone groups exactly reversed this effect that is decreasedthe urinary oxalate (119875 lt 001) and increased Ca2+ (119875 lt 005)excretion at all the treated doses and remained significantat 100mgkg in GPAE-treated group Other changes in theurine composition like decreased levels of citrate and Mg2+and increased excretion of uric acid and inorganic phosphatein urolithic group were not statistically significant GPAE-andCystone-treated groups also did not show any statisticallysignificant change in these parameters and remained com-parable to that of control animals The urolithic treatmentrevealed loss in body weight of the urolithic animals (119875 lt001) whereas GPAE- and Cystone-treated animals showednet gain in the body weights at all the tested doses Kidneys ofurolithic group animals were heavier than those the controlanimals (119875 lt 001) whereas GPAE- (100mgkg) treated ratkidneys did not show any significant increase in weight ascompared with control however the GPAE- (50mgkg) andCystone- (100mgkg) treated animalrsquos kidneys were slightlyheavier than those the control animals (Table 4)

Urolithic treatment was found to disturb renal functionin urolithic animals as evident from the increased BUN andserum creatinine and reduced creatinine clearance GPAE-treated groups decreased the serum creatinine and BUN

6 Evidence-Based Complementary and Alternative Medicine

DPPH radical scavenging activity

0

20

40

60

80

100

120

01 02 03 04 05 1 5 10 50 100

GPAE BHT

Inhi

bitio

n (

)

(120583gml)

(a)

0

20

40

60

80

100

50 150

Lipid peroxidation inhibitory effect

BHT GPAE

Inhi

bitio

n (

)

(120583gml)

(b)

Figure 1 (a) DPPH radical scavenging activity (b) Lipid peroxi-dation inhibitory effect of GPAE and the reference drug butylatedhydroxytoluene (BHT) Symbols shown are mean plusmn SEM (119899 = 3)

levels at 100mgkg and similar observations were made inthe Cystone-treated groups at 100mgkg as compared to con-trol The creatinine clearance was significantly increased at100mgkg in GPAE-treated groups as compared to urolithicanimals but was not significant at 50mgkg (Table 3)

Histology studies of the kidney regions like cortexmedulla and papilla in the urolithic animals showed manybirefringent crystalline deposits under polarized light micro-scope (Figure 2(b)) Such deposits were found in 3 outof 6 rats from GPAE low dose (Figure 2(c)) 1 out of 6rats from GPAE high dose (Figure 2(d)) and 3 out of 6rats (Figure 2(e)) from Cystone group but visibly smallerand less abundant compared to urolithic control animalsThe tissue morphology and intactness of the kidneys ofGPAE- and Cystone-treated groups were comparable to thenormal control animals (Figure 2(a)) The atomic absorptionspectrophotometer analysis revealed decreased Ca contentin the GPAE- and Cystone- (119875 lt 005) treated groupsas compared with the urolithic control groups wherein the

GPAE at 100mgkg shown significant decrease of Ca (119875 lt001) (Table 4)

323 Lipid Peroxidation Inhibitory Effect Stone-inducingtreatment increasedMDA and carbonyl protein content (119875 lt001) and decreased GSH level (119875 lt 005) and activities ofthe antioxidant enzymes including SOD (119875 lt 001) GPX(119875 lt 005) and catalase (119875 lt 001) in kidneys of theurolithic rats as compared to control animals Activities ofoxalate forming enzymes including GOx (119875 lt 001) in liverand LDH (119875 lt 001) in both liver and kidneys were found tobe enhanced in urolithic group compared to control animalswhereas treatment with GPAE was found to be protectiveagainst this effect and oxidative changes induced by urolithictreatment at higher doses (Table 4 Figure 1(b))

4 Discussion

Phytotherapy approach is attempted in this study as analternative to primary healthcare The Gokshuradi PolyherbalFormulation (GPF) which is traditionally being utilized inIndian folk medicine since several decades to treat kidneystones was evaluated for its antiurolithic effects In our earlierstudies we have clinically evaluated the efficacy of this prepa-ration and found its potential in naturally induced urolithicpatients [4] The present study is a reverse pharmacologyapproach wherein the scientific studies were undertakenunder controlled conditions to validate the traditional use ofGPF and determine its underlying mechanism of action oncalcium oxalate urolithiasis

Calcium oxalate stone development is a multifactorialprocess involving various etiological factors Studies haveshown that calcium oxalate crystal deposition leads to thecellular injury mediated by lipid peroxidation through freeoxygen radical generation Studies revealed that these cellularinjuries favor the events of calcium oxalate retention in renaltubules which is significant for further stone development[33ndash35] Recent clinical data are also supporting this findingthat formation of urinary stones leads to the oxidative stressin patients [36] Antioxidants such as vitamin C catechinand selenium have proved to be protective against suchoxidative injury due to calcium oxalate crystal depositionas evidenced from various experimental studies [37ndash39] Inthis regard antioxidant potential of GPAE was determinedby free radical scavenging and lipid peroxidation inhibitoryactivity to study its mechanism of action InterestinglyGPAErevealed DPPH free radical scavenging and inhibited lipidperoxidation of rat kidney homogenate similar to a standardantioxidant chemical BHT

In the diuretic activity study GPAE altered urine outputin a dose dependantmanner andmaximumdiuresis observedat 100mgkg At next higher dose that is 200mgkg it did notlead to any further increase in the urine volume and remainedcomparable to the normal control group The disappearanceof the diuretic effect of these polyherbal extracts at this higherdose is not completely understood Increase in urine volumebyGPAEwas also associated with the increase in Na+ and K+electrolyte loss similar to the standard diuretic furosemide

Evidence-Based Complementary and Alternative Medicine 7

(a) (b) (c)

(d) (e)

Figure 2 (a) Normal cellular structure of control rats kidney (Gr I) (b) Ethylene-glycol-induced urolithic rats kidney showing irregularcrystals dilation and inflammation under polarized microscope (Gr II) (c) Kidneys of urolithic rats treated with GPAE (50mgkg) showingnear normal cellular structure under polarized microscope (Gr III) (d) Kidneys of urolithic rats treated with GPAE (100mgkg) showingnormal cellular Structure (Gr IV) (e) Kidneys of urolithic rats treated with Cystone (100mgkg) showing normal cellular Structure (Gr V)Arrow indicates CaOx crystals

Increase in urine volume thus suggestive of possible loss ofthese electrolytes leading to this effectGPAE-treated group isalso revealed increase in urine pH and decrease in urine Ca2+at 100mgkg like that of furosemide-treated groups Thesehypocalciuric and diuretic effects ofGPAEmay help to reduceurinary super saturation of calcium salts and thus can preventfurther stone formation

Further antiurolithic effect of GPAE was evaluated inan animal model of urolithiasis induced by ethylene glycoland ammonium chloride in combination This model isfrequently used in calcium oxalate stone formation andtherefore was selected for this study [25 40 41] After 21 daysadministration of EG +AC showed larger and aggregatedcrystals in urolithic control animals compared to the GPAE-and Cystone-treated groups These calcium oxalate crystalagglomerates get deposited in the renal tubules and furtherdevelop into renal stones [25] GPAE reduced the polyuriaassociated with lithogenic treatment as compared to urolithiccontrol groups As per the earlier reports stone inductiontreatment leads an increase in oxalate and decrease in Ca2+

excretion This is confirmed in the present study in whichurolithic control animals showed the similar effect andGPAE- and Cystone-treated groups reversed this effect [4243]

Lithogenic treatment resulted in increased calciumoxalate deposition in the urolithic control groups along withoxidative stress determined from increased levels of markersof oxidative damage that is MDA and protein carbonylcontent alongwith decreased activity of antioxidant enzymesand GSH levels in kidneys accompanied with abnormal renalfunction The renal tubules were dilated in entire kidneysof all untreated rats leading to disturbed renal flow bylarge crystal aggregates However GPAE revealed significantdecrease in all these lithogenic markers at higher doses alongwith the antioxidant activity

The present investigation thus reports the significantantiurolithic potential ofGPAE at higher doses by the combi-nation of antioxidant diuretic and hypocalciuric effects Theresults of the formulation were found significant as comparedwith Cystone which is a standard drug available in themarket

8 Evidence-Based Complementary and Alternative Medicine

used in the treatment of urolithiasis and diuretic activity wascomparable with the standard diuretic drug Furosemide

5 Conclusions

The inhibitory effect of GPAE on calcium oxalate crystalretention in renal tubules thus can be attributed to itsdiuretic hypocalciuric antioxidant effect along with itspotential to inhibit biochemical parameters involved inoxalate metabolism which rationalize its medicinal use forurinary stone diseaseThe present study will certainly benefitthe scientific community worldwide in providing a source forisolation and designing a molecule in this research pipeline

Acknowledgments

The authors are thankful to The Indian Council of MedicalResearch (ICMR) New Delhi for financial assistance to thispostdoctoral project and Director the National Institute ofNutrition Hyderabad for providing necessary laboratoryfacilities

References

[1] G C Curhan W C Willett E B Rimm and M J Stampfer ldquoAprospective study of the intake of vitamins C and B6 and therisk of kidney stones in menrdquo Journal of Urology vol 155 no 6pp 1847ndash1851 1996

[2] C L Smith and D R P Guay ldquoNephrolithiasisrdquo in Pharma-cotherapy and Pathophysiologic Approach J T Di Piro R LTalbert P E Hayes G C Yee G R Matzke and L M PoseyEds pp 720ndash736 Elsevier New York NY USA 2nd edition1992

[3] F L Coe and M J Favus Disorders of Bone and MineralMetabolism Raven press New York NY USA 1992

[4] A L Shirfule C N Khobragade P Badrinarayan Y SBorse and R H Amilkanthwar ldquoPhytochemical analysis andantiurolithiatic activity of a polyherbal formulationrdquo Journal ofHerbs Spices andMedicinal Plants vol 15 no 1 pp 66ndash72 2009

[5] A M Sharifi R Darabi and N Akbarloo ldquoStudy of antihyper-tensive mechanism of Tribulus terrestris in 2K1C hypertensiverats role of tissue ACE activityrdquo Life Sciences vol 73 no 23 pp2963ndash2971 2003

[6] R C Singh and C S Sisodia ldquoEffect of Tribulus terrestris fruitextracts on chloride and creatinine renal clearances in dogsrdquoIndian Journal of Physiology and Pharmacology vol 15 no 3pp 93ndash96 1971

[7] M Al-Ali S Wahbi H Twaij and A Al-Badr ldquoTribulusterrestris preliminary study of its diuretic and contractile effectsand comparisonwith Zeamaysrdquo Journal of Ethnopharmacologyvol 85 no 2-3 pp 257ndash260 2003

[8] A K Nadkarni Indian Materia Medica vol 1 Popular BookDepot Bombay India 3rd edition 1976

[9] R N Chopra S L Nayar and I C Chopra Glossary of IndianMedicinal Plants CSIR New Delhi India 1956

[10] M R Fernando S M D N Wickramasinghe M I Thabrewand E H Karunanayaka ldquoA preliminary investigation of thepossible hypoglycaemic activity of Asteracanthus longifoliardquoJournal of Ethnopharmacology vol 27 no 1-2 pp 7ndash14 1989

[11] U K Mazumdar M Gupta S Maiti and D MukherjeeldquoAntitumor activity of Hygrophila spinosa on Ehrlich ascitescarcinoma and sarcoma-180 induced micerdquo Indian Journal ofExperimental Biology vol 35 no 5 pp 473ndash477 1997

[12] A Gomes M Das and S C Dasgupta ldquoHaematinic effectof Hygrophila spinosa T Anderson on experimental rodentsrdquoIndian Journal of Experimental Biology vol 39 no 4 pp 381ndash382 2001

[13] P Shanmugasundaram and S Venkataraman ldquoAnti-nociceptiveactivity of Hygrophila auriculata (Schum) Heinerdquo The AfricanJournal of Traditional Complementary and AlternativeMedicines vol 2 p 62 2005

[14] A Singh and S S Handa ldquoHepatoprotective activity of Apiumgraveolens and Hygrophila auriculata against paracetamol andthioacetamide intoxication in ratsrdquo Journal of Ethnopharmacol-ogy vol 49 no 3 pp 119ndash126 1995

[15] P Shanmugasundaram and S Venkataraman ldquoHepatoprotec-tive and antioxidant effects ofHygrophila auriculata (K Schum)HeineAcanthaceae root extractrdquo Journal of Ethnopharmacologyvol 104 no 1-2 pp 124ndash128 2006

[16] M Vijayakumar R Govindarajan G M M Rao et al ldquoActionof Hygrophila auriculata against streptozotocin-induced oxida-tive stressrdquo Journal of Ethnopharmacology vol 104 no 3 pp356ndash361 2006

[17] Z A Ali andV K Singh ldquoHerbal drugs of Himalaya (medicinalplants of Garhwal and Kumaon regions of India)rdquo in Aspect ofPlant Sciences vol 15 pp 110ndash111 Today ampTomorrowrsquos PrintersNew Delhi India 1998

[18] Anonymous inTheWealth of India-RawMaterials pp 393ndash394Council of Scientific and Industrial Research NewDelhi India1989

[19] S Siddiqui S Faizi and B Siddiqui ldquoStudies in the chemicalconstituents of the fresh berries of Solanum xanthocarpumrdquoJournal of the Chemical Society of Pakistan vol 5 no 2 pp 14ndash21 1983

[20] L Mohan P Sharma and C N Srivastava ldquoComparativeefficacy of Solanum xanthocarpum extracts alone and in com-bination with a synthetic pyrethroid cypermethrin againstmalaria vector Anopheles Stephensirdquo Southeast Asian Journalof Tropical Medicine and Public Health vol 38 no 2 pp 256ndash260 2007

[21] G P Vadnere R S Gaud andA K Singhai ldquoEvaluation of anti-asthmatic property of SolanumXanthocarpum flower extractsrdquoPharmacologyonline vol 1 pp 513ndash522 2008

[22] D J Huang C D Lin H J Chen and Y H Lin ldquoAntioxidantand antiproliferative activities of sweet potato (Ipomea batata[L] Lam lsquoTaniong 57rsquo) constituentsrdquo Botanical Bulletin ofAcademia Sinica vol 45 pp 179ndash186 2004

[23] D G Kang C K Yun and H S Lee ldquoScreening and com-parison of antioxidant activity of solvent extracts of herbalmedicines used in Koreardquo Journal of Ethnopharmacology vol87 no 2-3 pp 231ndash236 2003

[24] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[25] F Atmani Y Slimani M Mimouni and B Hacht ldquoProphylaxisof calcium oxalate stones by Herniaria hirsuta on experimen-tally induced nephrolithiasis in ratsrdquo British Journal of UrologyInternational vol 92 no 1 pp 137ndash140 2003

Evidence-Based Complementary and Alternative Medicine 9

[26] J A Daly and G Ertingshausen ldquoDirect method for deter-mining inorganic phosphate in serum with the ldquocentrifichemrdquoClinical Chemistry vol 18 pp 263ndash265 1972

[27] S H Y Wong J A Knight S M Hopfer O Zaharia CN Leach Jr and F W Sunderman Jr ldquoLipoperoxides inplasma as measured by liquid-chromatographic separation ofmalondialdehyde-thiobarbituric acid adductrdquo Clinical Chem-istry vol 33 no 2 pp 214ndash220 1987

[28] R L Levine D Garland C N Oliver et al ldquoDetermination ofcarbonyl content in oxidatively modified proteinsrdquo Methods inEnzymology vol 186 pp 464ndash478 1990

[29] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[30] H E Aebi ldquoEnzymes I oxidoreductases transferasesrdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed pp 273ndash285 VCH Weinheim Germany 1987

[31] A L Baker and N E Tolbert ldquoGlycolate oxidase (ferredoxin-containing form)rdquoMethods in Enzymology vol 9 pp 338ndash3421966

[32] J King in Practical Clinical Enzymology vol 87 van Nostrand1965

[33] S R Khan ldquoCalcium oxalate crystal interaction with renaltubular epithelium mechanism of crystal adhesion and itsimpact on stone developmentrdquo Urological Research vol 23 no2 pp 71ndash79 1995

[34] S R Khan and R L Hackett ldquoRetention of calcium oxalatecrystals in renal tubulesrdquo Scanning Microscopy vol 5 pp 707ndash712 1991

[35] F Dal Moro M Mancini I M Tavolini V de Marco and PBassi ldquoCellular and molecular gateways to urolithiasis a newinsightrdquoUrologia Internationalis vol 74 no 3 pp 193ndash197 2005

[36] H S Huang M C Ma C F Chen and J Chen ldquoLipidperoxidation and its correlations with urinary levels of oxalatecitric acid and osteopontin in patients with renal calciumoxalate stonesrdquo Urology vol 62 no 6 pp 1123ndash1128 2003

[37] M Santhosh Kumar and R Selvam ldquoSupplementation ofvitamin E and seleniumprevents hyperoxaluria in experimentalurolithic ratsrdquo Journal of Nutritional Biochemistry vol 14 no 6pp 306ndash313 2003

[38] S Thamilselvan and M Menon ldquoVitamin E therapy preventshyperoxaluria-induced calciumoxalate crystal deposition in thekidney by improving renal tissue antioxidant statusrdquo BritishJournal of Urology International vol 96 no 1 pp 117ndash126 2005

[39] Y Itoh T Yasui A Okada K Tozawa Y Hayashi and K KohrildquoPreventive effects of green tea on renal stone formation and therole of oxidative stress in nephrolithiasisrdquo Journal of Urologyvol 173 no 1 pp 271ndash275 2005

[40] S Thamilselvan R L Hackett and S R Khan ldquoLipid perox-idation in ethylene glycol induced hyperoxaluria and calciumoxalate nephrolithiasisrdquo Journal of Urology vol 157 no 3 pp1059ndash1063 1997

[41] C H Tsai Y C Chen L D Chen et al ldquoA traditional Chineseherbal antilithic formula Wulingsan effectively prevents therenal deposition of calcium oxalate crystal in ethylene glycol-fed ratsrdquo Urological Research vol 36 no 1 pp 17ndash24 2008

[42] J FanM A Glass and P S Chandhoke ldquoImpact of ammoniumchloride administration on rat ethylene glycol urolithiasismodelrdquo Scanning Microscopy vol 13 pp 299ndash306 1999

[43] HK Park B C JeongMK Sung et al ldquoReduction of oxidativestress in cultured renal tubular cells and preventive effects onrenal stone formation by the bioflavonoid Quercetinrdquo Journalof Urology vol 179 no 4 pp 1620ndash1626 2008

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

DPPH radical scavenging activity

0

20

40

60

80

100

120

01 02 03 04 05 1 5 10 50 100

GPAE BHT

Inhi

bitio

n (

)

(120583gml)

(a)

0

20

40

60

80

100

50 150

Lipid peroxidation inhibitory effect

BHT GPAE

Inhi

bitio

n (

)

(120583gml)

(b)

Figure 1 (a) DPPH radical scavenging activity (b) Lipid peroxi-dation inhibitory effect of GPAE and the reference drug butylatedhydroxytoluene (BHT) Symbols shown are mean plusmn SEM (119899 = 3)

levels at 100mgkg and similar observations were made inthe Cystone-treated groups at 100mgkg as compared to con-trol The creatinine clearance was significantly increased at100mgkg in GPAE-treated groups as compared to urolithicanimals but was not significant at 50mgkg (Table 3)

Histology studies of the kidney regions like cortexmedulla and papilla in the urolithic animals showed manybirefringent crystalline deposits under polarized light micro-scope (Figure 2(b)) Such deposits were found in 3 outof 6 rats from GPAE low dose (Figure 2(c)) 1 out of 6rats from GPAE high dose (Figure 2(d)) and 3 out of 6rats (Figure 2(e)) from Cystone group but visibly smallerand less abundant compared to urolithic control animalsThe tissue morphology and intactness of the kidneys ofGPAE- and Cystone-treated groups were comparable to thenormal control animals (Figure 2(a)) The atomic absorptionspectrophotometer analysis revealed decreased Ca contentin the GPAE- and Cystone- (119875 lt 005) treated groupsas compared with the urolithic control groups wherein the

GPAE at 100mgkg shown significant decrease of Ca (119875 lt001) (Table 4)

323 Lipid Peroxidation Inhibitory Effect Stone-inducingtreatment increasedMDA and carbonyl protein content (119875 lt001) and decreased GSH level (119875 lt 005) and activities ofthe antioxidant enzymes including SOD (119875 lt 001) GPX(119875 lt 005) and catalase (119875 lt 001) in kidneys of theurolithic rats as compared to control animals Activities ofoxalate forming enzymes including GOx (119875 lt 001) in liverand LDH (119875 lt 001) in both liver and kidneys were found tobe enhanced in urolithic group compared to control animalswhereas treatment with GPAE was found to be protectiveagainst this effect and oxidative changes induced by urolithictreatment at higher doses (Table 4 Figure 1(b))

4 Discussion

Phytotherapy approach is attempted in this study as analternative to primary healthcare The Gokshuradi PolyherbalFormulation (GPF) which is traditionally being utilized inIndian folk medicine since several decades to treat kidneystones was evaluated for its antiurolithic effects In our earlierstudies we have clinically evaluated the efficacy of this prepa-ration and found its potential in naturally induced urolithicpatients [4] The present study is a reverse pharmacologyapproach wherein the scientific studies were undertakenunder controlled conditions to validate the traditional use ofGPF and determine its underlying mechanism of action oncalcium oxalate urolithiasis

Calcium oxalate stone development is a multifactorialprocess involving various etiological factors Studies haveshown that calcium oxalate crystal deposition leads to thecellular injury mediated by lipid peroxidation through freeoxygen radical generation Studies revealed that these cellularinjuries favor the events of calcium oxalate retention in renaltubules which is significant for further stone development[33ndash35] Recent clinical data are also supporting this findingthat formation of urinary stones leads to the oxidative stressin patients [36] Antioxidants such as vitamin C catechinand selenium have proved to be protective against suchoxidative injury due to calcium oxalate crystal depositionas evidenced from various experimental studies [37ndash39] Inthis regard antioxidant potential of GPAE was determinedby free radical scavenging and lipid peroxidation inhibitoryactivity to study its mechanism of action InterestinglyGPAErevealed DPPH free radical scavenging and inhibited lipidperoxidation of rat kidney homogenate similar to a standardantioxidant chemical BHT

In the diuretic activity study GPAE altered urine outputin a dose dependantmanner andmaximumdiuresis observedat 100mgkg At next higher dose that is 200mgkg it did notlead to any further increase in the urine volume and remainedcomparable to the normal control group The disappearanceof the diuretic effect of these polyherbal extracts at this higherdose is not completely understood Increase in urine volumebyGPAEwas also associated with the increase in Na+ and K+electrolyte loss similar to the standard diuretic furosemide

Evidence-Based Complementary and Alternative Medicine 7

(a) (b) (c)

(d) (e)

Figure 2 (a) Normal cellular structure of control rats kidney (Gr I) (b) Ethylene-glycol-induced urolithic rats kidney showing irregularcrystals dilation and inflammation under polarized microscope (Gr II) (c) Kidneys of urolithic rats treated with GPAE (50mgkg) showingnear normal cellular structure under polarized microscope (Gr III) (d) Kidneys of urolithic rats treated with GPAE (100mgkg) showingnormal cellular Structure (Gr IV) (e) Kidneys of urolithic rats treated with Cystone (100mgkg) showing normal cellular Structure (Gr V)Arrow indicates CaOx crystals

Increase in urine volume thus suggestive of possible loss ofthese electrolytes leading to this effectGPAE-treated group isalso revealed increase in urine pH and decrease in urine Ca2+at 100mgkg like that of furosemide-treated groups Thesehypocalciuric and diuretic effects ofGPAEmay help to reduceurinary super saturation of calcium salts and thus can preventfurther stone formation

Further antiurolithic effect of GPAE was evaluated inan animal model of urolithiasis induced by ethylene glycoland ammonium chloride in combination This model isfrequently used in calcium oxalate stone formation andtherefore was selected for this study [25 40 41] After 21 daysadministration of EG +AC showed larger and aggregatedcrystals in urolithic control animals compared to the GPAE-and Cystone-treated groups These calcium oxalate crystalagglomerates get deposited in the renal tubules and furtherdevelop into renal stones [25] GPAE reduced the polyuriaassociated with lithogenic treatment as compared to urolithiccontrol groups As per the earlier reports stone inductiontreatment leads an increase in oxalate and decrease in Ca2+

excretion This is confirmed in the present study in whichurolithic control animals showed the similar effect andGPAE- and Cystone-treated groups reversed this effect [4243]

Lithogenic treatment resulted in increased calciumoxalate deposition in the urolithic control groups along withoxidative stress determined from increased levels of markersof oxidative damage that is MDA and protein carbonylcontent alongwith decreased activity of antioxidant enzymesand GSH levels in kidneys accompanied with abnormal renalfunction The renal tubules were dilated in entire kidneysof all untreated rats leading to disturbed renal flow bylarge crystal aggregates However GPAE revealed significantdecrease in all these lithogenic markers at higher doses alongwith the antioxidant activity

The present investigation thus reports the significantantiurolithic potential ofGPAE at higher doses by the combi-nation of antioxidant diuretic and hypocalciuric effects Theresults of the formulation were found significant as comparedwith Cystone which is a standard drug available in themarket

8 Evidence-Based Complementary and Alternative Medicine

used in the treatment of urolithiasis and diuretic activity wascomparable with the standard diuretic drug Furosemide

5 Conclusions

The inhibitory effect of GPAE on calcium oxalate crystalretention in renal tubules thus can be attributed to itsdiuretic hypocalciuric antioxidant effect along with itspotential to inhibit biochemical parameters involved inoxalate metabolism which rationalize its medicinal use forurinary stone diseaseThe present study will certainly benefitthe scientific community worldwide in providing a source forisolation and designing a molecule in this research pipeline

Acknowledgments

The authors are thankful to The Indian Council of MedicalResearch (ICMR) New Delhi for financial assistance to thispostdoctoral project and Director the National Institute ofNutrition Hyderabad for providing necessary laboratoryfacilities

References

[1] G C Curhan W C Willett E B Rimm and M J Stampfer ldquoAprospective study of the intake of vitamins C and B6 and therisk of kidney stones in menrdquo Journal of Urology vol 155 no 6pp 1847ndash1851 1996

[2] C L Smith and D R P Guay ldquoNephrolithiasisrdquo in Pharma-cotherapy and Pathophysiologic Approach J T Di Piro R LTalbert P E Hayes G C Yee G R Matzke and L M PoseyEds pp 720ndash736 Elsevier New York NY USA 2nd edition1992

[3] F L Coe and M J Favus Disorders of Bone and MineralMetabolism Raven press New York NY USA 1992

[4] A L Shirfule C N Khobragade P Badrinarayan Y SBorse and R H Amilkanthwar ldquoPhytochemical analysis andantiurolithiatic activity of a polyherbal formulationrdquo Journal ofHerbs Spices andMedicinal Plants vol 15 no 1 pp 66ndash72 2009

[5] A M Sharifi R Darabi and N Akbarloo ldquoStudy of antihyper-tensive mechanism of Tribulus terrestris in 2K1C hypertensiverats role of tissue ACE activityrdquo Life Sciences vol 73 no 23 pp2963ndash2971 2003

[6] R C Singh and C S Sisodia ldquoEffect of Tribulus terrestris fruitextracts on chloride and creatinine renal clearances in dogsrdquoIndian Journal of Physiology and Pharmacology vol 15 no 3pp 93ndash96 1971

[7] M Al-Ali S Wahbi H Twaij and A Al-Badr ldquoTribulusterrestris preliminary study of its diuretic and contractile effectsand comparisonwith Zeamaysrdquo Journal of Ethnopharmacologyvol 85 no 2-3 pp 257ndash260 2003

[8] A K Nadkarni Indian Materia Medica vol 1 Popular BookDepot Bombay India 3rd edition 1976

[9] R N Chopra S L Nayar and I C Chopra Glossary of IndianMedicinal Plants CSIR New Delhi India 1956

[10] M R Fernando S M D N Wickramasinghe M I Thabrewand E H Karunanayaka ldquoA preliminary investigation of thepossible hypoglycaemic activity of Asteracanthus longifoliardquoJournal of Ethnopharmacology vol 27 no 1-2 pp 7ndash14 1989

[11] U K Mazumdar M Gupta S Maiti and D MukherjeeldquoAntitumor activity of Hygrophila spinosa on Ehrlich ascitescarcinoma and sarcoma-180 induced micerdquo Indian Journal ofExperimental Biology vol 35 no 5 pp 473ndash477 1997

[12] A Gomes M Das and S C Dasgupta ldquoHaematinic effectof Hygrophila spinosa T Anderson on experimental rodentsrdquoIndian Journal of Experimental Biology vol 39 no 4 pp 381ndash382 2001

[13] P Shanmugasundaram and S Venkataraman ldquoAnti-nociceptiveactivity of Hygrophila auriculata (Schum) Heinerdquo The AfricanJournal of Traditional Complementary and AlternativeMedicines vol 2 p 62 2005

[14] A Singh and S S Handa ldquoHepatoprotective activity of Apiumgraveolens and Hygrophila auriculata against paracetamol andthioacetamide intoxication in ratsrdquo Journal of Ethnopharmacol-ogy vol 49 no 3 pp 119ndash126 1995

[15] P Shanmugasundaram and S Venkataraman ldquoHepatoprotec-tive and antioxidant effects ofHygrophila auriculata (K Schum)HeineAcanthaceae root extractrdquo Journal of Ethnopharmacologyvol 104 no 1-2 pp 124ndash128 2006

[16] M Vijayakumar R Govindarajan G M M Rao et al ldquoActionof Hygrophila auriculata against streptozotocin-induced oxida-tive stressrdquo Journal of Ethnopharmacology vol 104 no 3 pp356ndash361 2006

[17] Z A Ali andV K Singh ldquoHerbal drugs of Himalaya (medicinalplants of Garhwal and Kumaon regions of India)rdquo in Aspect ofPlant Sciences vol 15 pp 110ndash111 Today ampTomorrowrsquos PrintersNew Delhi India 1998

[18] Anonymous inTheWealth of India-RawMaterials pp 393ndash394Council of Scientific and Industrial Research NewDelhi India1989

[19] S Siddiqui S Faizi and B Siddiqui ldquoStudies in the chemicalconstituents of the fresh berries of Solanum xanthocarpumrdquoJournal of the Chemical Society of Pakistan vol 5 no 2 pp 14ndash21 1983

[20] L Mohan P Sharma and C N Srivastava ldquoComparativeefficacy of Solanum xanthocarpum extracts alone and in com-bination with a synthetic pyrethroid cypermethrin againstmalaria vector Anopheles Stephensirdquo Southeast Asian Journalof Tropical Medicine and Public Health vol 38 no 2 pp 256ndash260 2007

[21] G P Vadnere R S Gaud andA K Singhai ldquoEvaluation of anti-asthmatic property of SolanumXanthocarpum flower extractsrdquoPharmacologyonline vol 1 pp 513ndash522 2008

[22] D J Huang C D Lin H J Chen and Y H Lin ldquoAntioxidantand antiproliferative activities of sweet potato (Ipomea batata[L] Lam lsquoTaniong 57rsquo) constituentsrdquo Botanical Bulletin ofAcademia Sinica vol 45 pp 179ndash186 2004

[23] D G Kang C K Yun and H S Lee ldquoScreening and com-parison of antioxidant activity of solvent extracts of herbalmedicines used in Koreardquo Journal of Ethnopharmacology vol87 no 2-3 pp 231ndash236 2003

[24] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[25] F Atmani Y Slimani M Mimouni and B Hacht ldquoProphylaxisof calcium oxalate stones by Herniaria hirsuta on experimen-tally induced nephrolithiasis in ratsrdquo British Journal of UrologyInternational vol 92 no 1 pp 137ndash140 2003

Evidence-Based Complementary and Alternative Medicine 9

[26] J A Daly and G Ertingshausen ldquoDirect method for deter-mining inorganic phosphate in serum with the ldquocentrifichemrdquoClinical Chemistry vol 18 pp 263ndash265 1972

[27] S H Y Wong J A Knight S M Hopfer O Zaharia CN Leach Jr and F W Sunderman Jr ldquoLipoperoxides inplasma as measured by liquid-chromatographic separation ofmalondialdehyde-thiobarbituric acid adductrdquo Clinical Chem-istry vol 33 no 2 pp 214ndash220 1987

[28] R L Levine D Garland C N Oliver et al ldquoDetermination ofcarbonyl content in oxidatively modified proteinsrdquo Methods inEnzymology vol 186 pp 464ndash478 1990

[29] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[30] H E Aebi ldquoEnzymes I oxidoreductases transferasesrdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed pp 273ndash285 VCH Weinheim Germany 1987

[31] A L Baker and N E Tolbert ldquoGlycolate oxidase (ferredoxin-containing form)rdquoMethods in Enzymology vol 9 pp 338ndash3421966

[32] J King in Practical Clinical Enzymology vol 87 van Nostrand1965

[33] S R Khan ldquoCalcium oxalate crystal interaction with renaltubular epithelium mechanism of crystal adhesion and itsimpact on stone developmentrdquo Urological Research vol 23 no2 pp 71ndash79 1995

[34] S R Khan and R L Hackett ldquoRetention of calcium oxalatecrystals in renal tubulesrdquo Scanning Microscopy vol 5 pp 707ndash712 1991

[35] F Dal Moro M Mancini I M Tavolini V de Marco and PBassi ldquoCellular and molecular gateways to urolithiasis a newinsightrdquoUrologia Internationalis vol 74 no 3 pp 193ndash197 2005

[36] H S Huang M C Ma C F Chen and J Chen ldquoLipidperoxidation and its correlations with urinary levels of oxalatecitric acid and osteopontin in patients with renal calciumoxalate stonesrdquo Urology vol 62 no 6 pp 1123ndash1128 2003

[37] M Santhosh Kumar and R Selvam ldquoSupplementation ofvitamin E and seleniumprevents hyperoxaluria in experimentalurolithic ratsrdquo Journal of Nutritional Biochemistry vol 14 no 6pp 306ndash313 2003

[38] S Thamilselvan and M Menon ldquoVitamin E therapy preventshyperoxaluria-induced calciumoxalate crystal deposition in thekidney by improving renal tissue antioxidant statusrdquo BritishJournal of Urology International vol 96 no 1 pp 117ndash126 2005

[39] Y Itoh T Yasui A Okada K Tozawa Y Hayashi and K KohrildquoPreventive effects of green tea on renal stone formation and therole of oxidative stress in nephrolithiasisrdquo Journal of Urologyvol 173 no 1 pp 271ndash275 2005

[40] S Thamilselvan R L Hackett and S R Khan ldquoLipid perox-idation in ethylene glycol induced hyperoxaluria and calciumoxalate nephrolithiasisrdquo Journal of Urology vol 157 no 3 pp1059ndash1063 1997

[41] C H Tsai Y C Chen L D Chen et al ldquoA traditional Chineseherbal antilithic formula Wulingsan effectively prevents therenal deposition of calcium oxalate crystal in ethylene glycol-fed ratsrdquo Urological Research vol 36 no 1 pp 17ndash24 2008

[42] J FanM A Glass and P S Chandhoke ldquoImpact of ammoniumchloride administration on rat ethylene glycol urolithiasismodelrdquo Scanning Microscopy vol 13 pp 299ndash306 1999

[43] HK Park B C JeongMK Sung et al ldquoReduction of oxidativestress in cultured renal tubular cells and preventive effects onrenal stone formation by the bioflavonoid Quercetinrdquo Journalof Urology vol 179 no 4 pp 1620ndash1626 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

(a) (b) (c)

(d) (e)

Figure 2 (a) Normal cellular structure of control rats kidney (Gr I) (b) Ethylene-glycol-induced urolithic rats kidney showing irregularcrystals dilation and inflammation under polarized microscope (Gr II) (c) Kidneys of urolithic rats treated with GPAE (50mgkg) showingnear normal cellular structure under polarized microscope (Gr III) (d) Kidneys of urolithic rats treated with GPAE (100mgkg) showingnormal cellular Structure (Gr IV) (e) Kidneys of urolithic rats treated with Cystone (100mgkg) showing normal cellular Structure (Gr V)Arrow indicates CaOx crystals

Increase in urine volume thus suggestive of possible loss ofthese electrolytes leading to this effectGPAE-treated group isalso revealed increase in urine pH and decrease in urine Ca2+at 100mgkg like that of furosemide-treated groups Thesehypocalciuric and diuretic effects ofGPAEmay help to reduceurinary super saturation of calcium salts and thus can preventfurther stone formation

Further antiurolithic effect of GPAE was evaluated inan animal model of urolithiasis induced by ethylene glycoland ammonium chloride in combination This model isfrequently used in calcium oxalate stone formation andtherefore was selected for this study [25 40 41] After 21 daysadministration of EG +AC showed larger and aggregatedcrystals in urolithic control animals compared to the GPAE-and Cystone-treated groups These calcium oxalate crystalagglomerates get deposited in the renal tubules and furtherdevelop into renal stones [25] GPAE reduced the polyuriaassociated with lithogenic treatment as compared to urolithiccontrol groups As per the earlier reports stone inductiontreatment leads an increase in oxalate and decrease in Ca2+

excretion This is confirmed in the present study in whichurolithic control animals showed the similar effect andGPAE- and Cystone-treated groups reversed this effect [4243]

Lithogenic treatment resulted in increased calciumoxalate deposition in the urolithic control groups along withoxidative stress determined from increased levels of markersof oxidative damage that is MDA and protein carbonylcontent alongwith decreased activity of antioxidant enzymesand GSH levels in kidneys accompanied with abnormal renalfunction The renal tubules were dilated in entire kidneysof all untreated rats leading to disturbed renal flow bylarge crystal aggregates However GPAE revealed significantdecrease in all these lithogenic markers at higher doses alongwith the antioxidant activity

The present investigation thus reports the significantantiurolithic potential ofGPAE at higher doses by the combi-nation of antioxidant diuretic and hypocalciuric effects Theresults of the formulation were found significant as comparedwith Cystone which is a standard drug available in themarket

8 Evidence-Based Complementary and Alternative Medicine

used in the treatment of urolithiasis and diuretic activity wascomparable with the standard diuretic drug Furosemide

5 Conclusions

The inhibitory effect of GPAE on calcium oxalate crystalretention in renal tubules thus can be attributed to itsdiuretic hypocalciuric antioxidant effect along with itspotential to inhibit biochemical parameters involved inoxalate metabolism which rationalize its medicinal use forurinary stone diseaseThe present study will certainly benefitthe scientific community worldwide in providing a source forisolation and designing a molecule in this research pipeline

Acknowledgments

The authors are thankful to The Indian Council of MedicalResearch (ICMR) New Delhi for financial assistance to thispostdoctoral project and Director the National Institute ofNutrition Hyderabad for providing necessary laboratoryfacilities

References

[1] G C Curhan W C Willett E B Rimm and M J Stampfer ldquoAprospective study of the intake of vitamins C and B6 and therisk of kidney stones in menrdquo Journal of Urology vol 155 no 6pp 1847ndash1851 1996

[2] C L Smith and D R P Guay ldquoNephrolithiasisrdquo in Pharma-cotherapy and Pathophysiologic Approach J T Di Piro R LTalbert P E Hayes G C Yee G R Matzke and L M PoseyEds pp 720ndash736 Elsevier New York NY USA 2nd edition1992

[3] F L Coe and M J Favus Disorders of Bone and MineralMetabolism Raven press New York NY USA 1992

[4] A L Shirfule C N Khobragade P Badrinarayan Y SBorse and R H Amilkanthwar ldquoPhytochemical analysis andantiurolithiatic activity of a polyherbal formulationrdquo Journal ofHerbs Spices andMedicinal Plants vol 15 no 1 pp 66ndash72 2009

[5] A M Sharifi R Darabi and N Akbarloo ldquoStudy of antihyper-tensive mechanism of Tribulus terrestris in 2K1C hypertensiverats role of tissue ACE activityrdquo Life Sciences vol 73 no 23 pp2963ndash2971 2003

[6] R C Singh and C S Sisodia ldquoEffect of Tribulus terrestris fruitextracts on chloride and creatinine renal clearances in dogsrdquoIndian Journal of Physiology and Pharmacology vol 15 no 3pp 93ndash96 1971

[7] M Al-Ali S Wahbi H Twaij and A Al-Badr ldquoTribulusterrestris preliminary study of its diuretic and contractile effectsand comparisonwith Zeamaysrdquo Journal of Ethnopharmacologyvol 85 no 2-3 pp 257ndash260 2003

[8] A K Nadkarni Indian Materia Medica vol 1 Popular BookDepot Bombay India 3rd edition 1976

[9] R N Chopra S L Nayar and I C Chopra Glossary of IndianMedicinal Plants CSIR New Delhi India 1956

[10] M R Fernando S M D N Wickramasinghe M I Thabrewand E H Karunanayaka ldquoA preliminary investigation of thepossible hypoglycaemic activity of Asteracanthus longifoliardquoJournal of Ethnopharmacology vol 27 no 1-2 pp 7ndash14 1989

[11] U K Mazumdar M Gupta S Maiti and D MukherjeeldquoAntitumor activity of Hygrophila spinosa on Ehrlich ascitescarcinoma and sarcoma-180 induced micerdquo Indian Journal ofExperimental Biology vol 35 no 5 pp 473ndash477 1997

[12] A Gomes M Das and S C Dasgupta ldquoHaematinic effectof Hygrophila spinosa T Anderson on experimental rodentsrdquoIndian Journal of Experimental Biology vol 39 no 4 pp 381ndash382 2001

[13] P Shanmugasundaram and S Venkataraman ldquoAnti-nociceptiveactivity of Hygrophila auriculata (Schum) Heinerdquo The AfricanJournal of Traditional Complementary and AlternativeMedicines vol 2 p 62 2005

[14] A Singh and S S Handa ldquoHepatoprotective activity of Apiumgraveolens and Hygrophila auriculata against paracetamol andthioacetamide intoxication in ratsrdquo Journal of Ethnopharmacol-ogy vol 49 no 3 pp 119ndash126 1995

[15] P Shanmugasundaram and S Venkataraman ldquoHepatoprotec-tive and antioxidant effects ofHygrophila auriculata (K Schum)HeineAcanthaceae root extractrdquo Journal of Ethnopharmacologyvol 104 no 1-2 pp 124ndash128 2006

[16] M Vijayakumar R Govindarajan G M M Rao et al ldquoActionof Hygrophila auriculata against streptozotocin-induced oxida-tive stressrdquo Journal of Ethnopharmacology vol 104 no 3 pp356ndash361 2006

[17] Z A Ali andV K Singh ldquoHerbal drugs of Himalaya (medicinalplants of Garhwal and Kumaon regions of India)rdquo in Aspect ofPlant Sciences vol 15 pp 110ndash111 Today ampTomorrowrsquos PrintersNew Delhi India 1998

[18] Anonymous inTheWealth of India-RawMaterials pp 393ndash394Council of Scientific and Industrial Research NewDelhi India1989

[19] S Siddiqui S Faizi and B Siddiqui ldquoStudies in the chemicalconstituents of the fresh berries of Solanum xanthocarpumrdquoJournal of the Chemical Society of Pakistan vol 5 no 2 pp 14ndash21 1983

[20] L Mohan P Sharma and C N Srivastava ldquoComparativeefficacy of Solanum xanthocarpum extracts alone and in com-bination with a synthetic pyrethroid cypermethrin againstmalaria vector Anopheles Stephensirdquo Southeast Asian Journalof Tropical Medicine and Public Health vol 38 no 2 pp 256ndash260 2007

[21] G P Vadnere R S Gaud andA K Singhai ldquoEvaluation of anti-asthmatic property of SolanumXanthocarpum flower extractsrdquoPharmacologyonline vol 1 pp 513ndash522 2008

[22] D J Huang C D Lin H J Chen and Y H Lin ldquoAntioxidantand antiproliferative activities of sweet potato (Ipomea batata[L] Lam lsquoTaniong 57rsquo) constituentsrdquo Botanical Bulletin ofAcademia Sinica vol 45 pp 179ndash186 2004

[23] D G Kang C K Yun and H S Lee ldquoScreening and com-parison of antioxidant activity of solvent extracts of herbalmedicines used in Koreardquo Journal of Ethnopharmacology vol87 no 2-3 pp 231ndash236 2003

[24] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[25] F Atmani Y Slimani M Mimouni and B Hacht ldquoProphylaxisof calcium oxalate stones by Herniaria hirsuta on experimen-tally induced nephrolithiasis in ratsrdquo British Journal of UrologyInternational vol 92 no 1 pp 137ndash140 2003

Evidence-Based Complementary and Alternative Medicine 9

[26] J A Daly and G Ertingshausen ldquoDirect method for deter-mining inorganic phosphate in serum with the ldquocentrifichemrdquoClinical Chemistry vol 18 pp 263ndash265 1972

[27] S H Y Wong J A Knight S M Hopfer O Zaharia CN Leach Jr and F W Sunderman Jr ldquoLipoperoxides inplasma as measured by liquid-chromatographic separation ofmalondialdehyde-thiobarbituric acid adductrdquo Clinical Chem-istry vol 33 no 2 pp 214ndash220 1987

[28] R L Levine D Garland C N Oliver et al ldquoDetermination ofcarbonyl content in oxidatively modified proteinsrdquo Methods inEnzymology vol 186 pp 464ndash478 1990

[29] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[30] H E Aebi ldquoEnzymes I oxidoreductases transferasesrdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed pp 273ndash285 VCH Weinheim Germany 1987

[31] A L Baker and N E Tolbert ldquoGlycolate oxidase (ferredoxin-containing form)rdquoMethods in Enzymology vol 9 pp 338ndash3421966

[32] J King in Practical Clinical Enzymology vol 87 van Nostrand1965

[33] S R Khan ldquoCalcium oxalate crystal interaction with renaltubular epithelium mechanism of crystal adhesion and itsimpact on stone developmentrdquo Urological Research vol 23 no2 pp 71ndash79 1995

[34] S R Khan and R L Hackett ldquoRetention of calcium oxalatecrystals in renal tubulesrdquo Scanning Microscopy vol 5 pp 707ndash712 1991

[35] F Dal Moro M Mancini I M Tavolini V de Marco and PBassi ldquoCellular and molecular gateways to urolithiasis a newinsightrdquoUrologia Internationalis vol 74 no 3 pp 193ndash197 2005

[36] H S Huang M C Ma C F Chen and J Chen ldquoLipidperoxidation and its correlations with urinary levels of oxalatecitric acid and osteopontin in patients with renal calciumoxalate stonesrdquo Urology vol 62 no 6 pp 1123ndash1128 2003

[37] M Santhosh Kumar and R Selvam ldquoSupplementation ofvitamin E and seleniumprevents hyperoxaluria in experimentalurolithic ratsrdquo Journal of Nutritional Biochemistry vol 14 no 6pp 306ndash313 2003

[38] S Thamilselvan and M Menon ldquoVitamin E therapy preventshyperoxaluria-induced calciumoxalate crystal deposition in thekidney by improving renal tissue antioxidant statusrdquo BritishJournal of Urology International vol 96 no 1 pp 117ndash126 2005

[39] Y Itoh T Yasui A Okada K Tozawa Y Hayashi and K KohrildquoPreventive effects of green tea on renal stone formation and therole of oxidative stress in nephrolithiasisrdquo Journal of Urologyvol 173 no 1 pp 271ndash275 2005

[40] S Thamilselvan R L Hackett and S R Khan ldquoLipid perox-idation in ethylene glycol induced hyperoxaluria and calciumoxalate nephrolithiasisrdquo Journal of Urology vol 157 no 3 pp1059ndash1063 1997

[41] C H Tsai Y C Chen L D Chen et al ldquoA traditional Chineseherbal antilithic formula Wulingsan effectively prevents therenal deposition of calcium oxalate crystal in ethylene glycol-fed ratsrdquo Urological Research vol 36 no 1 pp 17ndash24 2008

[42] J FanM A Glass and P S Chandhoke ldquoImpact of ammoniumchloride administration on rat ethylene glycol urolithiasismodelrdquo Scanning Microscopy vol 13 pp 299ndash306 1999

[43] HK Park B C JeongMK Sung et al ldquoReduction of oxidativestress in cultured renal tubular cells and preventive effects onrenal stone formation by the bioflavonoid Quercetinrdquo Journalof Urology vol 179 no 4 pp 1620ndash1626 2008

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

8 Evidence-Based Complementary and Alternative Medicine

used in the treatment of urolithiasis and diuretic activity wascomparable with the standard diuretic drug Furosemide

5 Conclusions

The inhibitory effect of GPAE on calcium oxalate crystalretention in renal tubules thus can be attributed to itsdiuretic hypocalciuric antioxidant effect along with itspotential to inhibit biochemical parameters involved inoxalate metabolism which rationalize its medicinal use forurinary stone diseaseThe present study will certainly benefitthe scientific community worldwide in providing a source forisolation and designing a molecule in this research pipeline

Acknowledgments

The authors are thankful to The Indian Council of MedicalResearch (ICMR) New Delhi for financial assistance to thispostdoctoral project and Director the National Institute ofNutrition Hyderabad for providing necessary laboratoryfacilities

References

[1] G C Curhan W C Willett E B Rimm and M J Stampfer ldquoAprospective study of the intake of vitamins C and B6 and therisk of kidney stones in menrdquo Journal of Urology vol 155 no 6pp 1847ndash1851 1996

[2] C L Smith and D R P Guay ldquoNephrolithiasisrdquo in Pharma-cotherapy and Pathophysiologic Approach J T Di Piro R LTalbert P E Hayes G C Yee G R Matzke and L M PoseyEds pp 720ndash736 Elsevier New York NY USA 2nd edition1992

[3] F L Coe and M J Favus Disorders of Bone and MineralMetabolism Raven press New York NY USA 1992

[4] A L Shirfule C N Khobragade P Badrinarayan Y SBorse and R H Amilkanthwar ldquoPhytochemical analysis andantiurolithiatic activity of a polyherbal formulationrdquo Journal ofHerbs Spices andMedicinal Plants vol 15 no 1 pp 66ndash72 2009

[5] A M Sharifi R Darabi and N Akbarloo ldquoStudy of antihyper-tensive mechanism of Tribulus terrestris in 2K1C hypertensiverats role of tissue ACE activityrdquo Life Sciences vol 73 no 23 pp2963ndash2971 2003

[6] R C Singh and C S Sisodia ldquoEffect of Tribulus terrestris fruitextracts on chloride and creatinine renal clearances in dogsrdquoIndian Journal of Physiology and Pharmacology vol 15 no 3pp 93ndash96 1971

[7] M Al-Ali S Wahbi H Twaij and A Al-Badr ldquoTribulusterrestris preliminary study of its diuretic and contractile effectsand comparisonwith Zeamaysrdquo Journal of Ethnopharmacologyvol 85 no 2-3 pp 257ndash260 2003

[8] A K Nadkarni Indian Materia Medica vol 1 Popular BookDepot Bombay India 3rd edition 1976

[9] R N Chopra S L Nayar and I C Chopra Glossary of IndianMedicinal Plants CSIR New Delhi India 1956

[10] M R Fernando S M D N Wickramasinghe M I Thabrewand E H Karunanayaka ldquoA preliminary investigation of thepossible hypoglycaemic activity of Asteracanthus longifoliardquoJournal of Ethnopharmacology vol 27 no 1-2 pp 7ndash14 1989

[11] U K Mazumdar M Gupta S Maiti and D MukherjeeldquoAntitumor activity of Hygrophila spinosa on Ehrlich ascitescarcinoma and sarcoma-180 induced micerdquo Indian Journal ofExperimental Biology vol 35 no 5 pp 473ndash477 1997

[12] A Gomes M Das and S C Dasgupta ldquoHaematinic effectof Hygrophila spinosa T Anderson on experimental rodentsrdquoIndian Journal of Experimental Biology vol 39 no 4 pp 381ndash382 2001

[13] P Shanmugasundaram and S Venkataraman ldquoAnti-nociceptiveactivity of Hygrophila auriculata (Schum) Heinerdquo The AfricanJournal of Traditional Complementary and AlternativeMedicines vol 2 p 62 2005

[14] A Singh and S S Handa ldquoHepatoprotective activity of Apiumgraveolens and Hygrophila auriculata against paracetamol andthioacetamide intoxication in ratsrdquo Journal of Ethnopharmacol-ogy vol 49 no 3 pp 119ndash126 1995

[15] P Shanmugasundaram and S Venkataraman ldquoHepatoprotec-tive and antioxidant effects ofHygrophila auriculata (K Schum)HeineAcanthaceae root extractrdquo Journal of Ethnopharmacologyvol 104 no 1-2 pp 124ndash128 2006

[16] M Vijayakumar R Govindarajan G M M Rao et al ldquoActionof Hygrophila auriculata against streptozotocin-induced oxida-tive stressrdquo Journal of Ethnopharmacology vol 104 no 3 pp356ndash361 2006

[17] Z A Ali andV K Singh ldquoHerbal drugs of Himalaya (medicinalplants of Garhwal and Kumaon regions of India)rdquo in Aspect ofPlant Sciences vol 15 pp 110ndash111 Today ampTomorrowrsquos PrintersNew Delhi India 1998

[18] Anonymous inTheWealth of India-RawMaterials pp 393ndash394Council of Scientific and Industrial Research NewDelhi India1989

[19] S Siddiqui S Faizi and B Siddiqui ldquoStudies in the chemicalconstituents of the fresh berries of Solanum xanthocarpumrdquoJournal of the Chemical Society of Pakistan vol 5 no 2 pp 14ndash21 1983

[20] L Mohan P Sharma and C N Srivastava ldquoComparativeefficacy of Solanum xanthocarpum extracts alone and in com-bination with a synthetic pyrethroid cypermethrin againstmalaria vector Anopheles Stephensirdquo Southeast Asian Journalof Tropical Medicine and Public Health vol 38 no 2 pp 256ndash260 2007

[21] G P Vadnere R S Gaud andA K Singhai ldquoEvaluation of anti-asthmatic property of SolanumXanthocarpum flower extractsrdquoPharmacologyonline vol 1 pp 513ndash522 2008

[22] D J Huang C D Lin H J Chen and Y H Lin ldquoAntioxidantand antiproliferative activities of sweet potato (Ipomea batata[L] Lam lsquoTaniong 57rsquo) constituentsrdquo Botanical Bulletin ofAcademia Sinica vol 45 pp 179ndash186 2004

[23] D G Kang C K Yun and H S Lee ldquoScreening and com-parison of antioxidant activity of solvent extracts of herbalmedicines used in Koreardquo Journal of Ethnopharmacology vol87 no 2-3 pp 231ndash236 2003

[24] A E Consolini O A N Baldini and A G Amat ldquoPharmaco-logical basis for the empirical use of Eugenia uniflora L (Myr-taceae) as antihypertensiverdquo Journal of Ethnopharmacology vol66 no 1 pp 33ndash39 1999

[25] F Atmani Y Slimani M Mimouni and B Hacht ldquoProphylaxisof calcium oxalate stones by Herniaria hirsuta on experimen-tally induced nephrolithiasis in ratsrdquo British Journal of UrologyInternational vol 92 no 1 pp 137ndash140 2003

Evidence-Based Complementary and Alternative Medicine 9

[26] J A Daly and G Ertingshausen ldquoDirect method for deter-mining inorganic phosphate in serum with the ldquocentrifichemrdquoClinical Chemistry vol 18 pp 263ndash265 1972

[27] S H Y Wong J A Knight S M Hopfer O Zaharia CN Leach Jr and F W Sunderman Jr ldquoLipoperoxides inplasma as measured by liquid-chromatographic separation ofmalondialdehyde-thiobarbituric acid adductrdquo Clinical Chem-istry vol 33 no 2 pp 214ndash220 1987

[28] R L Levine D Garland C N Oliver et al ldquoDetermination ofcarbonyl content in oxidatively modified proteinsrdquo Methods inEnzymology vol 186 pp 464ndash478 1990

[29] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[30] H E Aebi ldquoEnzymes I oxidoreductases transferasesrdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed pp 273ndash285 VCH Weinheim Germany 1987

[31] A L Baker and N E Tolbert ldquoGlycolate oxidase (ferredoxin-containing form)rdquoMethods in Enzymology vol 9 pp 338ndash3421966

[32] J King in Practical Clinical Enzymology vol 87 van Nostrand1965

[33] S R Khan ldquoCalcium oxalate crystal interaction with renaltubular epithelium mechanism of crystal adhesion and itsimpact on stone developmentrdquo Urological Research vol 23 no2 pp 71ndash79 1995

[34] S R Khan and R L Hackett ldquoRetention of calcium oxalatecrystals in renal tubulesrdquo Scanning Microscopy vol 5 pp 707ndash712 1991

[35] F Dal Moro M Mancini I M Tavolini V de Marco and PBassi ldquoCellular and molecular gateways to urolithiasis a newinsightrdquoUrologia Internationalis vol 74 no 3 pp 193ndash197 2005

[36] H S Huang M C Ma C F Chen and J Chen ldquoLipidperoxidation and its correlations with urinary levels of oxalatecitric acid and osteopontin in patients with renal calciumoxalate stonesrdquo Urology vol 62 no 6 pp 1123ndash1128 2003

[37] M Santhosh Kumar and R Selvam ldquoSupplementation ofvitamin E and seleniumprevents hyperoxaluria in experimentalurolithic ratsrdquo Journal of Nutritional Biochemistry vol 14 no 6pp 306ndash313 2003

[38] S Thamilselvan and M Menon ldquoVitamin E therapy preventshyperoxaluria-induced calciumoxalate crystal deposition in thekidney by improving renal tissue antioxidant statusrdquo BritishJournal of Urology International vol 96 no 1 pp 117ndash126 2005

[39] Y Itoh T Yasui A Okada K Tozawa Y Hayashi and K KohrildquoPreventive effects of green tea on renal stone formation and therole of oxidative stress in nephrolithiasisrdquo Journal of Urologyvol 173 no 1 pp 271ndash275 2005

[40] S Thamilselvan R L Hackett and S R Khan ldquoLipid perox-idation in ethylene glycol induced hyperoxaluria and calciumoxalate nephrolithiasisrdquo Journal of Urology vol 157 no 3 pp1059ndash1063 1997

[41] C H Tsai Y C Chen L D Chen et al ldquoA traditional Chineseherbal antilithic formula Wulingsan effectively prevents therenal deposition of calcium oxalate crystal in ethylene glycol-fed ratsrdquo Urological Research vol 36 no 1 pp 17ndash24 2008

[42] J FanM A Glass and P S Chandhoke ldquoImpact of ammoniumchloride administration on rat ethylene glycol urolithiasismodelrdquo Scanning Microscopy vol 13 pp 299ndash306 1999

[43] HK Park B C JeongMK Sung et al ldquoReduction of oxidativestress in cultured renal tubular cells and preventive effects onrenal stone formation by the bioflavonoid Quercetinrdquo Journalof Urology vol 179 no 4 pp 1620ndash1626 2008

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 9

[26] J A Daly and G Ertingshausen ldquoDirect method for deter-mining inorganic phosphate in serum with the ldquocentrifichemrdquoClinical Chemistry vol 18 pp 263ndash265 1972

[27] S H Y Wong J A Knight S M Hopfer O Zaharia CN Leach Jr and F W Sunderman Jr ldquoLipoperoxides inplasma as measured by liquid-chromatographic separation ofmalondialdehyde-thiobarbituric acid adductrdquo Clinical Chem-istry vol 33 no 2 pp 214ndash220 1987

[28] R L Levine D Garland C N Oliver et al ldquoDetermination ofcarbonyl content in oxidatively modified proteinsrdquo Methods inEnzymology vol 186 pp 464ndash478 1990

[29] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[30] H E Aebi ldquoEnzymes I oxidoreductases transferasesrdquo inMethods of Enzymatic Analysis H U Bergmeyer Ed pp 273ndash285 VCH Weinheim Germany 1987

[31] A L Baker and N E Tolbert ldquoGlycolate oxidase (ferredoxin-containing form)rdquoMethods in Enzymology vol 9 pp 338ndash3421966

[32] J King in Practical Clinical Enzymology vol 87 van Nostrand1965

[33] S R Khan ldquoCalcium oxalate crystal interaction with renaltubular epithelium mechanism of crystal adhesion and itsimpact on stone developmentrdquo Urological Research vol 23 no2 pp 71ndash79 1995

[34] S R Khan and R L Hackett ldquoRetention of calcium oxalatecrystals in renal tubulesrdquo Scanning Microscopy vol 5 pp 707ndash712 1991

[35] F Dal Moro M Mancini I M Tavolini V de Marco and PBassi ldquoCellular and molecular gateways to urolithiasis a newinsightrdquoUrologia Internationalis vol 74 no 3 pp 193ndash197 2005

[36] H S Huang M C Ma C F Chen and J Chen ldquoLipidperoxidation and its correlations with urinary levels of oxalatecitric acid and osteopontin in patients with renal calciumoxalate stonesrdquo Urology vol 62 no 6 pp 1123ndash1128 2003

[37] M Santhosh Kumar and R Selvam ldquoSupplementation ofvitamin E and seleniumprevents hyperoxaluria in experimentalurolithic ratsrdquo Journal of Nutritional Biochemistry vol 14 no 6pp 306ndash313 2003

[38] S Thamilselvan and M Menon ldquoVitamin E therapy preventshyperoxaluria-induced calciumoxalate crystal deposition in thekidney by improving renal tissue antioxidant statusrdquo BritishJournal of Urology International vol 96 no 1 pp 117ndash126 2005

[39] Y Itoh T Yasui A Okada K Tozawa Y Hayashi and K KohrildquoPreventive effects of green tea on renal stone formation and therole of oxidative stress in nephrolithiasisrdquo Journal of Urologyvol 173 no 1 pp 271ndash275 2005

[40] S Thamilselvan R L Hackett and S R Khan ldquoLipid perox-idation in ethylene glycol induced hyperoxaluria and calciumoxalate nephrolithiasisrdquo Journal of Urology vol 157 no 3 pp1059ndash1063 1997

[41] C H Tsai Y C Chen L D Chen et al ldquoA traditional Chineseherbal antilithic formula Wulingsan effectively prevents therenal deposition of calcium oxalate crystal in ethylene glycol-fed ratsrdquo Urological Research vol 36 no 1 pp 17ndash24 2008

[42] J FanM A Glass and P S Chandhoke ldquoImpact of ammoniumchloride administration on rat ethylene glycol urolithiasismodelrdquo Scanning Microscopy vol 13 pp 299ndash306 1999

[43] HK Park B C JeongMK Sung et al ldquoReduction of oxidativestress in cultured renal tubular cells and preventive effects onrenal stone formation by the bioflavonoid Quercetinrdquo Journalof Urology vol 179 no 4 pp 1620ndash1626 2008

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