Protective effect of Pongamia pinnata flowers

Indian Journal of Experimental Biology Vol. 4 1, January 2003, pp. 58-62

Protective effect of Pongamia pinnata flowers against cisplatin and gentamicin induced nephrotoxicity in rats Annie Shirwaikar l *, S Malini2, S Chandrika Kumari I I

Department of Pharmacognosy, Co llege of Pharm . Sci ences; 2Department of Pharmacology, K M C, MAHE, Manipal 576 1 19, India Receil'ed ! 8 Allglls! 2002: revised 2 ! October 2002

Ethanoli c ex tract of fl owers of PUlIgolllia !Jillll{/fo was studi ed for its protective effe ct again st cisplatin and gentami cin induced renal injury in rats. When the ex tract (300 & 600 mg kg- I) was adm ini stered orall y ror 10 days rollow ing cispl atin 1 (5 mg kg.. ip) on day 5, toxicit y of cispl at in , as meas ured by loss or body weig ht , elevated blood urea and serum creatinine declined signifi cantl y. Similarly in ge ntamicin (40 mg kg· 1 s.c) induced rena l injury, th e ex tract (600 mg kg· l ) normali zed th c raised blood urea and serulll creatinine levels. Reversa l or cispl atill and ge ntami cin renal eell damage as induced by tubular necros is ie, marked congestion or th e glomeruli with glome rul ar atroph y, degenerati on or tubul ar epi theli al ce ll s wit h casts in the ILI bular lumen and infiltration of infl ammatory cell s in the interstitium was confirmed on hi sto athological examination. In th e preve nti ve regimen. co-admini stration or th e ex tract with ge ntamicin signiricantl y pre ve nt ed th e ren al injury both functionally and histologically. Ethan oli c ex tract or fl owers had a marked nitri c ox ide free radical scave nging crfect, sugges ting an anti ox idative propert y. Two fla vo noids, known ror th eir anti ox idant activ it y vi:. kaemprcrol and 3. 5. 6. 7, 8- pcnt:tmeth oxy fla vone we re iso la ted from th e ex tract. The res ults sugges ted that the flo we rs of POlIgwllia pillllo /([ had a protective effect aga in st cisplatin and gen tamici n induced ren al injury throu gh anti ox idant propert y.

Various studies have reported that th e nephrotoxicity assoc iated as a limiting side-e ffect of the antin eo plastic, cisplatin an d the am ino glycoside antibiotic, gentamicin is due to the involvement of oxidative stress via free rad ical formation 2 . Synthetic antioxidants and free radical scavengers like glycine' and se lenomethionine 4 have been found to show partial protection against damage caused by cisplatin. Similarly cyclodex trin sulphates 5, polyaspartic acid(' etc also have been found to partially red uce gentamicin induced renal damage. Search for nephroprotecti ve agents has made man turn to alternative sources viz. indigenous system of medic ine. It is a well-documented fact that most medicinal plants are enriched with bioflavonoids, which have antioxidant property. POllgalllia pinnal~1 (L.) Pierre (Fabaceac) is one such plant containing a number of bioactive compounds. Commonly known as Indian Beech Tree, it is a medium sized (up to ]8 m high) glabro us tree, , found in India, Burma, Malaya, N. Australia, Polynesia, China and Philippine Islands. Flowers of thi s plant are ri ch in bioflavonoids and ex tensively used for various sk in di seases, diabetes and in renal disorders by the tribes of ChiUoor District 7 (Andhra Pradesh, India). A survey of litera*Correspondent author : E- mail: ann ic.shirwaikar@co ps.maniral.edu ann iesh irwai kar @yahoo.co m

ture showed th e absence of any experimental data whatsoever to justi fy the nephroprotect ive rol e of the flowers of thi s pl ant. Thus, the present study was undertaken to evaluate the efficacy of' the flowers of Pongalllia pillnala against nep hrotox icity induced by commonly known nephrotoxic agents cisplatin and gentamici n.

Materials and Methods Plant lIIalerial-Flowers of Pongal11ia pinnala were collected from Kolar Di strict, Karnataka, India, in the month of April-May, 2001 and authenticated by botanist Dr. Gopalkrishna Bhat, Professor of Botany , Poorna Prajna College, Udupi, India. A herba rium spec imen bearing voucher No. PP. 508 has been deposited in the Department of Pharmacog nosy, College of Pharmaceutical Sciences, Manipal , India. Drugs and chemicals-Ci splatin and gentami cin were obtained from Biochem Pharmaceutical Industri es, Mumbai ; Urea estimation kit and Creatinine estimation kit were procured from Agappe Diagnostics, Maharash tra and Dr. Reddy' s Laboratories, Hyderabad respecti vel y. Preparation of alcoholic exlracl - The shade dri ed coarsely powdered flowers (I kg) were subjected to Soxhlet ex traction using ethanol(9SC7o) for 6 hr. The solvent was removed in vacuo and the extract was used for chemical and pharmacological st udi es.

SHIRWAIKAR el (/1.: NEPHROPROTECTI VE EFFECT OF PONGA MIA FLOWERS

Phytochemical screening - Preliminary phytochemi cal sc reenin g revea led th e presence of stero ids, carbohydrates, phenoli c co mpounds, fl avonoids, fixed oiIs and fat ss. CheJllical studies - Alcoholic ex tract was fracti onated into petrol eum ether, ether, eth yl acetate fracti on. Ether fraction ( 13 g) was di ssol ved in a small qu antity of methan ol and mi xed with silica gel-G (200 g) co lumn prepared in chl orofo rm . The column was eluted with chl oroform fo ll owed by varying percentages of meth anol in chl oroform. The elu ates with I % methanol in chl oroform deposited a li ght brown compound whi ch had ultra violet spectral data matchin g with 3, 5, 6, 7, 8-pentameth oxy ll avo ne9 . The elu ates (4 and 5% meth anol) in chl oroform precipitated a ye ll ow co loured co mpound whi ch showed UV spectral charac teri sti cs matchin g th at of kae mpferol9 .

PharmacoLogicaL stlldy AniJllals - The study was perfo rmed on male Wi sta r albino rats (60-90 days old) weighin g 150-200 g. They were maintained on stand ard co nditions (temperature and humidity co ntroll ed), di et (Hindustan Leve r Ltd ) and water ad libituJII . The stud y was co nducted after obtaining loca l animal ethi cal co mmittee clearan ce. Acute tuxicity studies -Animals were fed with increas ing doses of (30, 100, 300, 600, 1000 and 3000 mg/kg) of alcoholic ex trac t of fl owe rs of P. pinl1ata suspend ed in 2% w/v gum acac ia. The anim als were observed co ntinu ously fo r 2 hI' fo r th e gross behav ioral changes and then intermittently once every 2 hI' and fin all y at the end of 24 and 72 hI' to note an y oth er tox ic signs inclLiding death . Cisplatin -indllced rellol illjllrv - Seven gro ups (II = 8) were used to stu dy the effec t of P. pillllow on cisplatin induced renal tox ici ty in rats. Group I admini stered with equiv alent volumes of ve hicl e for 10 days , se rved as norm al contro l. Group 2 was trea ted orall y with P. pill nata ex tract (600 mg kg- I) for 10 days. The foll ow ing day renal fun ction was assessed. Groups 3, 4, 5 and 6 were ad mini stered with eispl atin (5 mg kg- I body we ight;, single dose, ip) lo. The blood was withdraw n on da y 5 in Group 3 and day 15 in Group 4 to chec k th e pe rsistence of renal injury. Groups 5 and 6, serving as curati ve reg imen, were treated with P. pinnuw ex tract (300 an d 600 mg kg- I po respect ive ly) fro m day 6 onwa rds fo r 10 days. The fo llow ing days renal fun cti on was assessed. Group 7, which served as preve ntive reg imen, was treated with 600 mg kg- I of ext ract fo r I hr prior to the admini stra-

59

tion of cisplatin . Treatment was co ntinu ed fo r next 4 days. Gentalllicin-indll ced renal illjury - Five gro ups of rats were used. Group I served as normal control. Remainin g groups were admini stered with gen t'l micin (40mg kg- I sc) fo r 13 days ll . Bl ood was withdra wn on day 14 in Group 2 and on day 24 in Group 3 to check the persistence of renal injury. Group 4 anima ls were treated orally with 600 mg kg- I of ex tract from day 14 onward s for 10 days, after whi ch bl ood was assessed for renal clea rance. In Group 5 ge ntam icin and the fl owe r ex tract was admini stered simultaneously for 13 days follow ing whi ch renal func ti on tests 12 were perfo rmed. Assesslll ent of renal ./ill/ ction - (a) Perce ntage chan ge in the body we ight was meas ured for eac h rat before treatment and at the end of th e trea tmcnt; (b) bl ood urea leve l was es timated by urease enzy ma ti c meth od using UV spec tro mete r Shimadzu UV-240; and (c) serum creatinin e level was meas ured by alka line picrate meth od using UV spectrometer Shi madzu UV-240. Histopath ologica l stlldies- Two anim als from eac h group were sac ri ficed on th e day of blood wi th drawa l and kidneys were iso lated. The kidney secti ons were stain ed with hematoxy lin and eos in and observed under light mi crosco pe. St atistical {fnalysis - The data we re analysed using one way analys is of va ri ance fo ll owed by post hoc Student-New man-Keuls Test using SPSS comp uter softwa re. Stati stical sign ificance was set at P < 0.05. In vitro antiox idant study Nitric oxide scaveng ill g aCl/Flty itropruss ide (5 111M) was mi xed with diffe ren t co ncentrati ons of tes t ex trac t and incubated at 25 °C fo r 5 hr. After 5 hr Greiss reagent was added and abso rba nce of chrolllophore formed was read at 546 nm . Co ntrol experimcnt was also ca rri ed out in simil ar mann er. The expe riment was repeated in tripli ca te. Perce ntage scav enging clr ec t was ca lculated and th e res ults are prese nted in Fig. 3.

Results and Discussion PharmacologicaL studies Toxicity-O ral administrati on o r th c alcohol ic extract of PongO/llia pinll ara prod uced no tox ic erfec ts up to 3 g/k g in rats even after 24 and 72 hr. Cisplatin-illduced renat da /1/ age - Res ults on effects of oral admini strati on of P. p inl10ta on ci splati n induced elevation of biood urca, serulll creatinine and

60

INDIAN J EXP BIOL, JANUARY 2003

on reduction of body weight in rats are given in Table I. The control animals had an increase in body weight gain of 13%. The body weight of animals, which received cisplatin (ip), was reduced by 30% at the end of two weeks. However, when P. pinnata extract of 300 and 600 mg kg- I was given in curative regimen the extent of reduction in body weight showed significant decline at both the doses. Co-administration of cisplatin and flower extract, failed to prevent the reduction in body weight. The levels of blood urea and serum creatinine increased significantly in cisplatin treated animals and this elevation persisted for 2 weeks. The elevations of serum markers were significantly reduced following 300 and 600 mg kg-I of flower extract. However, the extent of elevation in the serum markers was not affected wh en the flower extract was administered simultaneously. The sect ions of the kidneys isolated from rats treated with cisplatin exhibited marked congestion of the g lomeruli with glomerular atrophy, desquam ation of tubular epithelial cells with casts in the tubular lumen, infiltration of inflammatory cells in the interstitium indicating cisplatin induced acute renal necrosis (Fig. I). Following the treatment with extract (600 mg kg- I) in the curative regimen congestion of the g lomeruli were reduced, degeneration of tubular cells were not observed and a very few tubular casts were observed in the interstitium indicating marked protection against the injury caused by cisplatin. However, in preventive regimen of 600 mg kg-I features of tubular necrosis persisted.

Gentamicin- induced renal damage- The results are depicted in Table 2. The daily subcutaneous administration of gentamicin (40 mg kg- I) for 13 days caused renal impairement. This was evidenced by significant decrease in body weight after 13 days of injection . However, the body weight rose gradually in the next 10 days. This may be due to sodium water retention . The extract at 600 mg kg- I normalized the change in body weight both in curative and preventive regimen. Renal dysfunction was observed further as significant increase in blood urea and serum creatinine levels after 13 days of gentamicin administration, which persisted even after 10 days of cessation of therapy. The treatment of the rats with flower extract (600 mg kg- I) after gentamicin induced damage caused a marked decrease in the blood urea and serum creatinine levels . The co-administration of the extract also showed the same effect indicating its protective

Fig. I - Pho tomicrographs of (a) normal control, (b) cisplatin intoxicated and (c) Pongalllia pinnala extract trea ted rat kidn ey. /Inllammatory cells, casts in the tubular lume n (arrow) and tubular dege neration (arrow head) in b which was normalized in c. G-Glomerulus, T-Renaltubules, H x E stain ed, Scale bar 40IL.]

=

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SI-IIRW A IKA R el af.: NEPI-IROPROTECTIV E EFFECT OF PONGAMfA FLOWERS

activity. The histopa thol og ic examin ati on reported features of acute renal nec ros is like tubular desqu amati on along with number of casts in th e tubular lumen, after 10 days of cessation of th erapy (Fig. 2), whi ch was not seen on day 13. In fac t, th e relationship among tubular cell dysfun cti on, morphological damage, impairement of glomerular filtration and renal bl ood tl ow in acute renal failure are poorl y understood l '. Hence, it could be reasoned that functional damage or renal dysfuncti on occ ured befo re stru ctural damage. The flow er ex tract norm ali zed th e hi stopathol ogical features of acute tubular nec rosis by ge ntamicin in the curati ve regimen. The present results of our study co nfirmed that cisplatin at 5 mg kg- I ip and gentamicin 40 mg kg- I sc produces significant nephrotox icity as characteri zed by increase in blood urea, serum creatinine and renal tubul ar necrosis. Induction of nephrotoxicity by cisplatin is ass umed to be a rapid process invol ving reacTab lc 1 -

ti on with proteins in renal tubul es. As renal damage occurs within an hour after ad mini strati on of cisplatin , it is important that th e protecti ve agent be prese nt in sufficient conce ntrati on in the renal tubul es before injury occ urs. Thi s mi ght ex plain why even oral ael ·· ministration of the fl ower ex tract in multipl e doses fail ed to protec t the rats of group 7 after cisplatin admini stration . A relationship between oxidative stress and nephrotoxicity has been we ll demonstrated in man y ex perimental animal models2. J. Evidence points out th at cisplatin and ge ntamicin induce nephrotoxicity partl y via ox idati ve stress. One of th e mechanisms proposed, by which cisplat in induces free rad ical damage, is by increasing th e activity of ca1ciulllindependent nitric oxide sy nthase. Flavono ids arc potent anti ox idants and are known to modu late the activities of various enzy me systems due to their interac ti on with va ri ous biomo lecul es 2 . The flowers o/"

Effcct of alcoholic cx tract of P. pilllla/a in cisplatin induccd rcnal damagc I Valucs arc mcan ± SE of R rcplication sl

Groups (II = R) I 2 3 4 5

6 7

Trcatmcnl rcg imcn

Vchiclc Alc. cx trac t Cispl at in 5 Ih day Cispl atin IS Ih day Cispl atin + alc. cx tract 300 mg/k g (cu rati vc rcgimcn) Cisplatin + alc. cx trac t 000 mg/kg (curati vc regimcn) Cisplatin + alc. cx tract 600 mg/kg (prevcnti ve rcgimcn)

Changc in body wc ighl (%) 13.20 ± 2.24 13.20 ± 2. 15 - 13.39 ± l AY - 30.68 ± 3AO" -5.93 ± 2.86h

Bloodurca (mg/d I)

Sc rum crcatininc (mg/d l)

32.60 ± 3.6 1 3 1A2 ± 1.7 90.96 ± 7.1 4" 7 1.53 ± 4.5 I " 50.27 ± I A6"

0.96 ± 0.05 0.90 ± 0.00 1.8 1 ± 0.08" 1.6 1 ± 0.04" l AO ± 0.08

- 3.30 ± l.73 h.,

28.2 1 ± 2A3 b -,

1.29 ± 0.50

109.0 ± R. ll

1.65 ± 0. 11

- 1 .· J . ~

± 1.28

h

"I' < 0.05 vs group I ; h< 0.05 vs group 4; and '< 0.05 vs group 2 One way ANOVA followcd by post hoc Studcnt-Ncwman-Kcul s Tcst Table 2 -

Effcc t of alcoholi c cx trac t of P. pin ll a/({ in gcntamici n induccd rc.nal damagc IValucs arc mcan ± SE of 8 rcp li cati onsl

Groups

Trca tm cn t rcg imcn

Changc in body wc ight

Blood urca (mg/dl )

Scrum crca tin ine (mg/d l)

13. 19 ± 2.24 13.20 ± 2.1 5

32.60 ± 3.6 1 3 1A2 ± 1.7

0.96 ± 0.057 0.90 ± 0.00

-9.79 ± 1.7" 13.6 1 ± 3.70< -6.73 ± 0. 831>

55.34 ± 7. 14" 68.92 ± 4.5 1" 39 .34 ± 1.09"

1.72 ± 0.064" 1.29 ± 0.029" 1.07 ± 0.04h

1.54 ± 1.62