)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jtm/2015/961205.pdf · (Azadirachta...

Research ArticleProtective Effect of Aqueous Crude Extract of Neem(Azadirachta indica) Leaves on Plasmodium berghei-InducedRenal Damage in Mice

Voravuth Somsak Sukanya Chachiyo Ubonwan Jaihan and Somrudee Nakinchat

Department of Clinical Chemistry Faculty of Medical Technology Western University Kanchanaburi 71170 Thailand

Correspondence should be addressed to Voravuth Somsak voravuthsomsakgmailcom

Received 14 June 2015 Revised 14 August 2015 Accepted 18 August 2015

Academic Editor Shyam Sundar

Copyright copy 2015 Voravuth Somsak 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

Malaria is a major public health problem in the world because it can cause of death in patients Malaria-associated renal injuryis associated with 45 of mortality in adult patients hospitalized with severe form of the disease Therefore new plant extracts toprotect against renal injury induced by malaria infection are urgently needed In this study we investigated the protective effectof aqueous crude extract of Azadirachta indica (neem) leaves on renal injury induced by Plasmodium berghei ANKA infectionin mice ICR mice were injected intraperitoneally with 1 times 107 parasitized erythrocytes of PbANKA and neem extracts (5001000 and 2000mgkg) were given orally for 4 consecutive days Plasma blood urea nitrogen (BUN) and creatinine levels weresubsequently measured Malaria-induced renal injury was evidenced as marked increases of BUN and creatinine levels Howeverthe oral administration of neem leaf extract to PbANKA infected mice for 4 days brought back BUN and creatinine levels to nearnormalcy and the highest activity was observed at doses of 1000 and 2000mgkg Additionally no toxic effects were found innormal mice treated with this extract Hence neem leaf extract can be considered a potential candidate for protection against renalinjury induced by malaria

1 Introduction

Malaria caused by Plasmodium parasites is a major publichealth problem in tropical zones including Africa South andCentral America and Asia where it is most prevalent withestimates of 216 million cases and 655000 deaths More than85 of malaria cases and 90 of malaria deaths occur insub-Saharan Africa mainly in young children [1] Causesof death in malaria have been reported including cerebralmalaria acute hemolysis and severe anemia and vital organdamage and failure [2] For organ damagemalaria-associatedrenal injury occurs in between 1 and 4 of hospitalizedpatients with a mortality that can reach up to 45 [3] Thepathogenesis of malaria-associated renal injury is multifac-torial and not well characterized but several hypothesessuggest involvement of cytoadherence of parasitized erythro-cytes Recent studies suggest that proinflammatory responseoxidative injury and apoptosis probably explain part of thisinjury [4 5] During malaria infection it causes generation

of reactive oxygen species (ROS) such as superoxide anionand hydroxyl radical which deplete glutathione levels andinhibit the activity of antioxidant enzymes especially inrenal tissues [6 7] Therefore oxidative stress induced bymalaria infection may produce cellular injury and necrosisvia several mechanisms including peroxidation of membranelipids protein denaturation and DNA damage [8]

In this respect medicinal plants are potential targets forresearch and development of alternative drugs In particu-lar Azadirachta indica (neem) is one of the most promis-ing medicinal plants having several biological activitiesespecially as antioxidant anti-inflammatory antibacterialantifungal and antiulcer ones [9ndash12] Several studies havebeen undertaken on the protective effects of neem [13ndash15]It has been described that aqueous crude extract of neemleaves showed significant hypoglycemic hypolipemic hep-atoprotective and hypertensive activities [16ndash19] Moreoverprotective effect on diabetic nephropathy in rats of neem leafextract has also been reported [20] However the protective

Hindawi Publishing CorporationJournal of Tropical MedicineVolume 2015 Article ID 961205 5 pageshttpdxdoiorg1011552015961205

2 Journal of Tropical Medicine

effect of neem extract on renal damage induced by malariainfection has not yet been reported Hence the aim of thepresent study was to evaluate the possible protective effect ofaqueous crude extract of neem leaves on P berghei-inducedrenal damage in mice

2 Materials and Methods

21 Crude Extract Preparation The neem leaves were col-lected from Kanchanaburi province Thailand The plantswere identified in the Faculty of Pharmacy Payap UniversityThe leaves were cleaned and dried using hot air oven at 55∘Cfor 6 h and then they were powdered The powder was usedfor the preparation of aqueous crude extract according tothe procedure previously described with some modification[10] The dried powders of neem leaves were boiled withdistilled water (plant water = 1 20 wv) for 6 h and thenfiltered The filtrate was evaporated to dryness in a vacuumevaporator to yield the aqueous crude extract of neem leafBefore experiment the neem leaf extract was dissolved in20 Tween-80 at the doses of 500 1000 and 2000mgkg

22 Mice Female ICR mice obtained from the NationalLaboratory Animal Center Mahidol University Thailand 4ndash6 weeks old weighing 25 to 30 g were used for the studyTheywere given clean tap water and pelleted diet ad libitum Allmouse procedures were approved by the Ethical Committeeon Animal Experimentation Faculty of Medical TechnologyWestern University Thailand

23 Parasite Strain and Infection of Animals PlasmodiumbergheiANKA(PbANKA)obtained fromDrChairatUthaip-ibull from BIOTEC NSTDA Thailand was used Naıve ICRmice were inoculated with 1 times 107 parasitized erythrocytesof PbANKA by intraperitoneal (IP) injection Parasitemiawas daily monitored by microscopy of Giemsa stained thinblood smear Additionally renal markers including BUN andcreatinine levels were also measured

24 Measurements of Renal Markers For determination ofrenal damage BUN and creatinine levels were used asmarkers in this study Blood was collected from tail vein ofmice into heparinized hematocrit tubes Centrifugation wasthen performed with 10000 g for 10min Next plasma wascollected into a new 15-mL tube for measurement of BUNand creatinine using commercially available diagnostic kits(BioSystems S A Costa Brava Barcelona Spain) accordingto the manufacturerrsquos instructions

25 Efficacy Test In Vivo The standard 4-day suppressivetest against PbANKA infection in mice was employed [21]Groups of naıve ICR mice (5 mice of each) were inoculatedwith 1 times 107 parasitized erythrocytes of PbANKA by IPinjection They were subsequently treated with 500 1000and 2000mgkg of neem leaf extract orally by gavage twicea day for 4 consecutive days Normal mice treated with orwithout 2000mgkg of extract were used as healthy controlswhile PbANKA infected mice treated with 20 Tween-80

were used as disease controls On day 5 of experiment bloodwas collected for measurement of BUN and creatinine levels

26 Statistical Analysis The one-way ANOVA was used toanalyze and compare the results at a 95 confidence levelValues of 119875 lt 005 were considered significant Results wereexpressed as mean plusmn standard error of mean (SEM)

3 Results

31 Renal Damage Induced by Plasmodium berghei Infectionin Mice ICR mice were infected with 1 times 107 parasitizederythrocytes of PbANKA by IP injection parasitemia andrenal markers were then daily monitored It was found thatPbANKA was firstly detectable on day 2 after infection witha parasitemia less than 1 and reached gt60 on day 12(Figure 1(a)) Moreover during blood stage propagation ofPbANKA in mice BUN and creatinine levels were markedlyincreased in the response to the presence of the parasitewhich reached significant values firstly on day 4 after infec-tion (Figures 1(b) and 1(c)) The infected mice died within 2weeks (Figure 1(d))

32 Protective Effect of Neem Leaf Extract on Renal Dam-age Induced by Plasmodium berghei Infection in Mice ICRmice were infected with 1 times 107 parasitized erythrocytesof PbANKA by IP injection and given 500 1000 and2000mgkg of the neem leaf extract for 4 consecutive dayssubsequently withmeasurement of renal markers As showedin Figures 2(a) and 2(b) during PbANKA infection renaldamage was developed as indicated by increasing of BUNand creatinine levels significantly (119875 lt 001) Interestinglyneem leas extract exerted a dose-dependent protection ofrenal damage induced by PbANKA infection as indicated byreduction of BUN and creatinine levels in the extract treatedgroups Significant (119875 lt 005) protections were observed atdoses of 1000 and 2000mgkg of neem leaf extracts com-paredwith untreated groups and no significances were foundwhen compared to normal groups Moreover prolongedsurvival time was also observed in infected mice treated with1000 and 2000mgkg of the extracts (25 days for 1000mgkgand 30 days for 2000mgkg) Additionally no toxic effectswere found in normal mice treated with 2000mgkg of thisextract

4 Discussion

In the current study we provide evidence that malaria-associated renal injury could be protected against by usingthe aqueous crude extract of neem leaves in PbANKAinfected mouse model Impairment of renal function duringmalaria infection has been reported by clinical reports andit is an important life-threatening complication of malariainfection that goes beyond the classical clinical symptomsof malaria [22 23] Moreover the adversities to access ofmedical services or delay in diagnosis in their place of originis implicated in the severity of disease The onset of renalinjury in PbANKA infected mice came out from day 4

Journal of Tropical Medicine 3

144 6 8 10 1220Day after infection

0

20

40

60

80Pa

rasit

emia

()

(a)

0

20

40

60

80

BUN

(mg

dL)

2 4 6 8 10 12 140Day after infection

(b)

0

1

2

3

4

Crea

tinin

e (m

gdL

)


(c)

0

20

40

60

80

100

Cum

ulat

ive s

urvi

val


(d)

Figure 1Malaria-associated renal injury induced byPlasmodiumberghei infection inmice Groups of ICRmice (5mice of each)were infectedwith 1 times 107 parasitized erythrocytes of PbANKA by IP injection (a) Parasitemia (b) BUN and (c) creatinine levels were daily measured (d)Cumulative survival of infected mice was also observed Results were expressed as mean plusmn SEM

after infection and the incidence of renal injury was con-firmed through manifestation including marked increases ofplasma BUN and creatinine levels It can be suggested thatpathophysiology of malaria infection was usually associatedwith an activation of immune system and comprehends acomplex network with production of ROS oxidative stressand inflammation [24ndash28] It has also been described thatthe erythrocyte destruction during blood stage of infectionaccumulated high levels of toxic free heme in circulation thathad the ability to induce oxidative stress from productionof hydroxyl radicals via the FentonHaber-Weiss reactionand resulting renal injury [29ndash31] Apoptosis of renal tissuesduring malaria infection has also been suggested in renalinjury [30] Additionally pathogenesis of malaria-associatedrenal injury is most likely to be due to immune-complex-mediated glomerulonephritis caused by immune-complexdeposition and endothelial damage which may lead to fatalforms of malarial nephropathies [32]

The present study here has demonstrated that aqueouscrude extract of neem leaves significantly reduced plasmaBUN and creatinine levels as indicators for renal injuryinduced by PbANKA infection This protective effect of

neem leaf extract on renal injury may be due to its uniquecomposition where the leaf extract neem is rich in flavonoids(rutin and quercetin flavonoglycosides polyphenols andtannins) [13] Flavonoids in neem have been described topossess both antioxidant and anti-inflammatory activities viascavenging free radicals and inhibit lipid peroxidation [33]Furthermore neem leaves are rich in polyphenols whichare known for their potent antioxidant and free radicalscavenging properties [9 11] Therefore antioxidant and freeradical scavenging activities of neem leaf extract might playa critical role to protect against renal injury induced byPbANKA infection in mice In addition it has been reportedthat neem leaf extract in both crude extract and activeingredient azadirachtin showed the antimalarial propertyagainst PbANKA infectedmice andmight be due to the effectof this plant to protect against renal injury induced bymalaria[34]

5 Conclusion

The results obtained in this study showed the aqueous crudeextract of neem (A indica) leaves exerted a dose-dependent


N NE UN 500 1000 20000

10

20

30

40

50

Extract (mgkg)

BUN

(mg

dL)

lowastlowast

1 times 107 PbANKA

(a)

N NE UN 500 1000 200000

05

10

15

20

25

Extract (mgkg)

Crea

tinin

e (m

gdL

)

1 times 107 PbANKA

lowastlowast

(b)

Figure 2 Protective effect of aqueous crude extract of neem leaves on renal injury induced by Plasmodium berghei infection in mice Groupsof ICR mice (5 mice of each) were infected with 1 times 107 parasitized erythrocytes of PbANKA by IP injection and given 500 1000 and2000mgkg of neem leaf extracts orally for 4 consecutive days On day 5 of experiment (a) BUN and (b) creatinine levels were measuredand compared to normal and untreated groups Results were expressed as mean plusmn SEM lowastlowast119875 lt 001 compared to normal group 119875 lt 005and 119875 lt 001 compared to untreated group N normal mice NE normal mice treated with 2000mgkg of neem leaf extract and UN

untreated mice

protective activity of renal damage induced by P berghei Itwasmost effective at the dose levels of 1000 and 2000mgkgThis plant can be recommended for use since it possessed ahigh protective effect against malaria and can be obtained atrelatively no cost from nature

Conflict of Interests

The authors have declared no conflict of interests regardingthe publication of this paper

Acknowledgments

The authors thank Dr Sakaewan Ounjaijean for helpfuldiscussion on plant extraction The authors also want toacknowledge Dr Chairat Uthaipibull and Associate ProfessorDr Somdet Srichairatanakool for their contributions anddiscussions on P berghei infectionmodel and the efficacy testin vivo

References

[1] WHOWorld Malaria Report 2011 World Health Organization2011

[2] N J White S Pukrittayakamee T T Hien M A Faiz O AMokuolu and A M Dondorp ldquoMalariardquo The Lancet vol 383no 9918 pp 723ndash735 2014

[3] S Eiam-Ong ldquoMalarial nephropathyrdquo Seminars in Nephrologyvol 23 no 1 pp 21ndash33 2003

[4] R Sinniah L Rui-Mei and A Kara ldquoUp-regulation of cy-tokines in glomerulonephritis associated with murine malariainfectionrdquo International Journal of Experimental Pathology vol80 no 2 pp 87ndash95 1999

[5] R S Barsoum ldquoMalarial acute renal failurerdquo Journal of theAmerican Society of Nephrology vol 11 no 11 pp 2147ndash21542000

[6] V Jeney J Balla A Yachie et al ldquoPro-oxidant and cytotoxiceffects of circulating hemerdquo Blood vol 100 no 3 pp 879ndash8872002

[7] N Sibmooh P Yamanont S Krudsood et al ldquoIncreased fluidityand oxidation of malarial lipoproteins relation with severityand induction of endothelial expression of adhesionmoleculesrdquoLipids in Health and Disease vol 3 article 15 2004

[8] LDOMora LMGAntunesHD C Francescato andMDL P Bianchi ldquoThe effects of oral glutamine on cisplatin-inducednephrotoxicity in ratsrdquo Pharmacological Research vol 47 no 6pp 517ndash522 2003

[9] W Chaisawangwong and W Gritsanapan ldquoQuality assessmentand scavenging activity of Siamese neem flower extractyrdquoNatural Product Research vol 27 no 4-5 pp 394ndash401 2013

[10] P Sithisarn R Supabphol and W Gritsanapan ldquoCompari-son of free radical scavenging activity of Siamese neem tree(Azadirachta indica A Juss var siamensis Valeton) leaf extractsprepared by different methods of extractionrdquoMedical Principlesand Practice vol 15 no 3 pp 219ndash222 2006

[11] P Sithisarn R Supabphol and W Gritsanapan ldquoAntioxidantactivity of Siamese neem tree (VP1209)rdquo Journal of Ethnophar-macology vol 99 no 1 pp 109ndash112 2005

[12] R Paul M Prasad and N K Sah ldquoAnticancer biology ofAzadirachta indica L (neem) a mini reviewrdquo Cancer Biology ampTherapy vol 12 no 6 pp 467ndash476 2011

[13] M A Dkhil S Al-Quraishy A E Abdel Moneim and D DelicldquoProtective effect of Azadirachta indica extract against Eimeriapapillata-induced coccidiosisrdquo Parasitology Research vol 112no 1 pp 101ndash106 2013

[14] R Subapriya R Kumaraguruparan S K Abraham and SNagini ldquoProtective effects of ethanolic neem leaf extract on


DMBA-induced genotoxicity and oxidative stress in micerdquoJournal of Herbal Pharmacotherapy vol 5 no 4 pp 39ndash502006

[15] R Subapriya R Kumaraguruparan S K Abraham and SNagini ldquoProtective effects of ethanolic neem leaf extract onN-methyl-N1015840-nitro-N-nitrosoguanidine-induced genotoxicityand oxidative stress inmicerdquoDrug andChemical Toxicology vol27 no 1 pp 15ndash26 2004

[16] A L Sunarwidhi S Sudarsono and A E Nugroho ldquoHypo-glycemic effect of combination of Azadirachta indica A Jussand Gynura procumbens (Lour) Merr ethanolic extracts stan-dardized by rutin and quercetin in alloxan-induced hyper-glycemic ratsrdquo Advanced Pharmaceutical Bulletin vol 4 pp613ndash618 2014

[17] V S Kumar and V Navaratnam ldquoNeem (Azadirachta indica)prehistory to contemporary medicinal uses to humankindrdquoAsian Pacific Journal of Tropical Biomedicine vol 3 no 7 pp505ndash514 2013

[18] A Koul VMohan and S Bharati ldquoAzadirachta indicamitigatesDMBA-induced hepatotoxicity a biochemical and radiometricstudyrdquo Indian Journal of Biochemistry amp Biophysics vol 51 no1 pp 37ndash45 2014

[19] T A Babu and S Ananthakrishnan ldquoIdiopathic intracranialhypertension secondary to ingestion of Morinda coreia andAzadirachta indica leaves extract in infantrdquo Journal of Pharma-cology amp Pharmacotherapeutics vol 4 no 4 pp 298ndash299 2013

[20] A Oluwole Busayo Z Laura D Olufunke Olubusola AOluwafunmike Sharon D Luciana and CM Ezekiel AdemolaldquoAmeliorative effects of ethanolic leaf extract of Azadirachtaindica on renal histologic alterations in streptozotocin-induceddiabetic ratsrdquoTheAmerican Journal of ChineseMedicine vol 39no 5 pp 903ndash916 2011

[21] W Peters J H Portus and B L Robinson ldquoThe chemotherapyof rodent malaria XXII The value of drug resistant strainsof Plasmodium berghei in screening for blood schizontocidalactivityrdquo Annals of Tropical Medicine and Parasitology vol 69no 2 pp 155ndash171 1975

[22] M Al Rohani H Aljawshaei and E Aduolimi ldquoAcute renalfailure in Yemeni patientsrdquo Saudi Journal of Kidney Diseases andTransplantation vol 22 no 4 pp 829ndash833 2011

[23] T William J Menon G Rajahram et al ldquoSevere Plasmodiumknowlesi malaria in a tertiary care hospital Sabah MalaysiardquoEmerging Infectious Diseases vol 17 no 7 pp 1248ndash1255 2011

[24] N Narsaria C Mohanty B K Das S P Mishra and R PrasadldquoOxidative stress in children with severe malariardquo Journal ofTropical Pediatrics vol 58 no 2 pp 147ndash150 2012

[25] R Bilgin M S Yalcin G Yucebilgic I S Koltas and SYazar ldquoOxidative stress in vivax malariardquo The Korean Journalof Parasitology vol 50 no 4 pp 375ndash377 2012

[26] P Sobolewski I Gramaglia J A Frangos M Intaglietta andHvan der Heyde ldquoPlasmodium berghei resists killing by reactiveoxygen speciesrdquo Infection and Immunity vol 73 no 10 pp6704ndash6710 2005

[27] K Becker L Tilley J L Vennerstrom D Roberts S Rogersonand H Ginsburg ldquoOxidative stress in malaria parasite-infectederythrocytes host-parasite interactionsrdquo International Journalfor Parasitology vol 34 no 2 pp 163ndash189 2004

[28] A Pabon J Carmona L C Burgos and S Blair ldquoOxidativestress in patients with non-complicated malariardquo Clinical Bio-chemistry vol 36 no 1 pp 71ndash78 2003

[29] F F Dutra L S Alves D Rodrigues et al ldquoHemolysis-induced lethality involves inflammasome activation by hemerdquo

Proceedings of the National Academy of Sciences of the UnitedStates of America vol 111 no 39 pp E4110ndashE4118 2014

[30] R M Elias M Correa-Costa C R Barreto et al ldquoOxidativestress and modification of renal vascular permeability areassociated with acute kidney injury during P berghei ANKAinfectionrdquo PLoS ONE vol 7 no 8 Article ID e44004 2012

[31] Z-W Zhang J Cheng F Xu et al ldquoRed blood cell extrudesnucleus andmitochondria against oxidative stressrdquo IUBMBLifevol 63 no 7 pp 560ndash565 2011

[32] H M Elsheikha and H A Sheashaa ldquoEpidemiology patho-physiology management and outcome of renal dysfunctionassociated with plasmodia infectionrdquo Parasitology Research vol101 no 5 pp 1183ndash1190 2007

[33] W Kitdamrongtham K Ishii K Ebina et al ldquoLimonoids andflavonoids from the flowers ofAzadirachta indica var siamensisand their melanogenesis-inhibitory and cytotoxic activitiesrdquoChemistry amp Biodiversity vol 11 no 1 pp 73ndash84 2014

[34] L Lucantoni R S Yerbanga G Lupidi L Pasqualini FEsposito and A Habluetzel ldquoTransmission blocking activityof a standardized neem (Azadirachta indica) seed extract onthe rodent malaria parasite Plasmodium berghei in its vectorAnopheles stephensirdquoMalaria Journal vol 9 article 66 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


effect of neem extract on renal damage induced by malariainfection has not yet been reported Hence the aim of thepresent study was to evaluate the possible protective effect ofaqueous crude extract of neem leaves on P berghei-inducedrenal damage in mice

2 Materials and Methods

21 Crude Extract Preparation The neem leaves were col-lected from Kanchanaburi province Thailand The plantswere identified in the Faculty of Pharmacy Payap UniversityThe leaves were cleaned and dried using hot air oven at 55∘Cfor 6 h and then they were powdered The powder was usedfor the preparation of aqueous crude extract according tothe procedure previously described with some modification[10] The dried powders of neem leaves were boiled withdistilled water (plant water = 1 20 wv) for 6 h and thenfiltered The filtrate was evaporated to dryness in a vacuumevaporator to yield the aqueous crude extract of neem leafBefore experiment the neem leaf extract was dissolved in20 Tween-80 at the doses of 500 1000 and 2000mgkg

22 Mice Female ICR mice obtained from the NationalLaboratory Animal Center Mahidol University Thailand 4ndash6 weeks old weighing 25 to 30 g were used for the studyTheywere given clean tap water and pelleted diet ad libitum Allmouse procedures were approved by the Ethical Committeeon Animal Experimentation Faculty of Medical TechnologyWestern University Thailand

23 Parasite Strain and Infection of Animals PlasmodiumbergheiANKA(PbANKA)obtained fromDrChairatUthaip-ibull from BIOTEC NSTDA Thailand was used Naıve ICRmice were inoculated with 1 times 107 parasitized erythrocytesof PbANKA by intraperitoneal (IP) injection Parasitemiawas daily monitored by microscopy of Giemsa stained thinblood smear Additionally renal markers including BUN andcreatinine levels were also measured

24 Measurements of Renal Markers For determination ofrenal damage BUN and creatinine levels were used asmarkers in this study Blood was collected from tail vein ofmice into heparinized hematocrit tubes Centrifugation wasthen performed with 10000 g for 10min Next plasma wascollected into a new 15-mL tube for measurement of BUNand creatinine using commercially available diagnostic kits(BioSystems S A Costa Brava Barcelona Spain) accordingto the manufacturerrsquos instructions

25 Efficacy Test In Vivo The standard 4-day suppressivetest against PbANKA infection in mice was employed [21]Groups of naıve ICR mice (5 mice of each) were inoculatedwith 1 times 107 parasitized erythrocytes of PbANKA by IPinjection They were subsequently treated with 500 1000and 2000mgkg of neem leaf extract orally by gavage twicea day for 4 consecutive days Normal mice treated with orwithout 2000mgkg of extract were used as healthy controlswhile PbANKA infected mice treated with 20 Tween-80

were used as disease controls On day 5 of experiment bloodwas collected for measurement of BUN and creatinine levels

26 Statistical Analysis The one-way ANOVA was used toanalyze and compare the results at a 95 confidence levelValues of 119875 lt 005 were considered significant Results wereexpressed as mean plusmn standard error of mean (SEM)

3 Results

31 Renal Damage Induced by Plasmodium berghei Infectionin Mice ICR mice were infected with 1 times 107 parasitizederythrocytes of PbANKA by IP injection parasitemia andrenal markers were then daily monitored It was found thatPbANKA was firstly detectable on day 2 after infection witha parasitemia less than 1 and reached gt60 on day 12(Figure 1(a)) Moreover during blood stage propagation ofPbANKA in mice BUN and creatinine levels were markedlyincreased in the response to the presence of the parasitewhich reached significant values firstly on day 4 after infec-tion (Figures 1(b) and 1(c)) The infected mice died within 2weeks (Figure 1(d))

32 Protective Effect of Neem Leaf Extract on Renal Dam-age Induced by Plasmodium berghei Infection in Mice ICRmice were infected with 1 times 107 parasitized erythrocytesof PbANKA by IP injection and given 500 1000 and2000mgkg of the neem leaf extract for 4 consecutive dayssubsequently withmeasurement of renal markers As showedin Figures 2(a) and 2(b) during PbANKA infection renaldamage was developed as indicated by increasing of BUNand creatinine levels significantly (119875 lt 001) Interestinglyneem leas extract exerted a dose-dependent protection ofrenal damage induced by PbANKA infection as indicated byreduction of BUN and creatinine levels in the extract treatedgroups Significant (119875 lt 005) protections were observed atdoses of 1000 and 2000mgkg of neem leaf extracts com-paredwith untreated groups and no significances were foundwhen compared to normal groups Moreover prolongedsurvival time was also observed in infected mice treated with1000 and 2000mgkg of the extracts (25 days for 1000mgkgand 30 days for 2000mgkg) Additionally no toxic effectswere found in normal mice treated with 2000mgkg of thisextract

4 Discussion

In the current study we provide evidence that malaria-associated renal injury could be protected against by usingthe aqueous crude extract of neem leaves in PbANKAinfected mouse model Impairment of renal function duringmalaria infection has been reported by clinical reports andit is an important life-threatening complication of malariainfection that goes beyond the classical clinical symptomsof malaria [22 23] Moreover the adversities to access ofmedical services or delay in diagnosis in their place of originis implicated in the severity of disease The onset of renalinjury in PbANKA infected mice came out from day 4



0

20

40

60

80Pa

rasit

emia

()

(a)

0

20

40

60

80

BUN

(mg

dL)


(b)

0

1

2

3

4

Crea

tinin

e (m

gdL

)


(c)

0

20

40

60

80

100

Cum

ulat

ive s

urvi

val


(d)





5 Conclusion



N NE UN 500 1000 20000

10

20

30

40

50

Extract (mgkg)

BUN

(mg

dL)

lowastlowast

1 times 107 PbANKA

(a)

N NE UN 500 1000 200000

05

10

15

20

25

Extract (mgkg)

Crea

tinin

e (m

gdL

)

1 times 107 PbANKA

lowastlowast

(b)


untreated mice




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















0

20

40

60

80Pa

rasit

emia

()

(a)

0

20

40

60

80

BUN

(mg

dL)


(b)

0

1

2

3

4

Crea

tinin

e (m

gdL

)


(c)

0

20

40

60

80

100

Cum

ulat

ive s

urvi

val


(d)





5 Conclusion



N NE UN 500 1000 20000

10

20

30

40

50

Extract (mgkg)

BUN

(mg

dL)

lowastlowast

1 times 107 PbANKA

(a)

N NE UN 500 1000 200000

05

10

15

20

25

Extract (mgkg)

Crea

tinin

e (m

gdL

)

1 times 107 PbANKA

lowastlowast

(b)


untreated mice




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















N NE UN 500 1000 20000

10

20

30

40

50

Extract (mgkg)

BUN

(mg

dL)

lowastlowast

1 times 107 PbANKA

(a)

N NE UN 500 1000 200000

05

10

15

20

25

Extract (mgkg)

Crea

tinin

e (m

gdL

)

1 times 107 PbANKA

lowastlowast

(b)


untreated mice




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jtm/2015/961205.pdf · (Azadirachta...

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Transcript of )JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jtm/2015/961205.pdf · (Azadirachta...