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International Journal of Pharmaceutical Science Invention ISSN (Online): 2319 – 6718, ISSN (Print): 2319 – 670X www.ijpsi.org Volume 2 Issue 5 ‖ May 2013 ‖ PP.55-65

Trypsin inhibitors from Lavatera cashmeriana Camb. seeds: isolation, characterization and in-vitro cytoxicity activity Syed Rakashanda 1 , Syed Mubashir 2 , Yasrib Qurishi 3 , Abid Hamid 3 , Akbar Masood 1 and Shajrul Amin 1 * 1

Depart ment of Biochemistry, The University of Kashmir, Srinagar (J&K), 1900 06, India. 2 Depart ment of Botany, The University of Kashmir, Srinagar (J&K), 190006, India 3 Cancer Pharmacology Division, CSIR Indian Institute Of Integrative Medicine, Jammu, I ndia 180001

ABSTRACT: P ro t e a se in h ib ito r s (PI s ) a re s ma l l p ro te in s th a t a r e q u ite co m mo n in n a tu r e. PIs a re p r es en t in mu lti p le fo r ms in n u me ro u s t i ssu e s o f a n i ma l s a n d p la n t s a s we ll a s in mic ro o rg a n i sm s. PI s a re e me rg in g with p ro m i sin g th e ra p eu ti c u se s in th e tr ea t men t o f d is ea s e s lik e ca n c er s. Wo rk in g u n d er th e sa me t r en d we i so la ted a n d ch a ra c te ri z ed p ro tea s e in h ib i to r s f ro m La va t er a ca sh me ri a n a Ca mb . t o ch ec k th ei r in vit ro cyto xi cit y a c ti vi ty. Fo u r s er in e p ro tea se in h ib ito r s n a med L C - p i I, L C- p i II , L C- p i III a n d L C - p i IV w er e p u ri fi ed to el ec tro p h o r eti c h o mo g en ei ty f ro m seed s o f La va te ra ca sh me ria n a Ca mb . b y p ro ced u r e th a t en ta iled a mmo n iu m su lp h a te p r ecip ita tio n , a n io n e xch a n g e ch ro ma to g ra p h y a n d g e l fi lt ra tio n o n S ep h a d e x G - 1 0 0 . Th e c ru d e p ro tein re so lved in to fo u r ma jo r p ea k s o n io n exch a n g e co lu mn . Th e i so la ted p ro te in s e xe rt ed a n in h ib ito ry a ctio n a g a in s t th e a c ti v ity o f tr yp sin , ch ymo tr y p sin a n d ela s ta se. Th e a cti vi ty o f t ryp s in a n d ch ymo t ryp s in wa s in h ib i ted a lmo s t co m p let ely in a mo la r ra t io o f 1 :1 wh e rea s ela sta se wa s in h ib it ed o n ly in a mo la r ra t io o f 0 .2 :1 . Th e in h ib ito r s a p p ea red a s 2 0 .9 , 1 4 .1 , 1 6 .8 a n d 7 .9 kDa p ea ks in g el fi lt ra t io n a n d 1 0 , 1 4 , 1 6 a n d 7 kDa b a n d s b y S DS PAGE es ta b l i sh in g th a t L C - p i I i s co n st itu ted o f t wo si mi la r su b u n i t s o f 1 0 kDa . Th e in h ib i to r s we re fo u n d to b e g lyco p ro te in s. Th e i n h ib it io n o f t ryp s in wa s ma xi mu m a t p H 7 , 8 , 7 .6 a n d 8 .5 fo r L C - p i I , L C - p i II, L C - p i II I a n d L C - p i I V r e sp ec ti vely . Th e o p t imu m temp era tu re wa s fo u n d to b e 3 0 ˚ C fo r a l l th e fo u r in h ib i to r s. We a lso exa min ed th e su p p re ss iv e a ct iv ity o f L C - p i I a g a in st P ro sta te (P C - 3 ) a n d Br ea s t (M C F - 7 ), Co lo n (H C T 1 5 ) a n d Neu ro b la sto ma (I MR - 3 2 ). Th e i n h ib it o r wa s fo u n d to su p p r es s in - v it ro c el l g ro wth o f P ro s ta t e ( PC - 3 ) a n d B rea s t (M CF - 7 ) cel l lin e s, b u t co u ld n o t in h ib it th e g ro wth o f Co lo n (H CT - 1 5 ) a n d Ne u ro b la s to ma (IM R - 3 2 ) cel l lin e s. K ey w o rd s - Ch ro ma to g r a p h y, ch ymo tr yp s in , cy to xi ci ty, ela s ta s e, La va te ra ca sh me ria n a Ca mb ., p ro t ea s e in h ib i t o r s, t ryp s i n

I.

INTRODUCTION

P la nt s p r o d uc e a var ie t y o f p ro te i ns t ha t ser ve i n t h e d e fe n ce a g ai n st p at ho ge n s and i n v ad i n g o r ga ni s ms , i nc l ud i n g r ib o so me i n a cti v at i n g p ro tei n s [1 ] , le cti n s [2 ] , p ro tea s e in h ib i to r s [3 ] a nd a nt i f u n ga l p r o te i n s [4 -6 ]. Am o n g t h es e p ro te as e i n hib ito r s are b e li e ved to p la y a n i mp o r ta nt r o l e i n t he d e fe n se a g ai n st att ac k o f b o t h p h yt o p h ago u s i n s ect s a nd mi cro o r ga n i s ms [ 7 , 8 ] . P r o tea se i n hib ito r s a re o f, v er y co m mo n o cc u rr en ce a nd ha ve b ee n iso la ted fro m a lar ge n u mb e r o f o r ga n i s ms i n c ud in g p la n t s, a n i ma l s a nd mi cro o r ga n i s ms

*C o rre sp o nd i n g a u t ho r : S haj r ul A mi n , D ep ar t m en t o f B io c he mi s tr y, U n iv er si t y o f Ka s h mir ,Sr i na ga r (J &K) -1 9 0 0 0 6 , I nd ia e ma i l: s haj r ul @ red i f fma i l.co m , mo b i le : +9 1 - 9 4 1 9 0 1 8 1 7 4 www.ijpsi.org

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Trypsin inhibitors from Lavatera cashmeriana Camb. Seeds: isolation, characterization and in… [ 9 -1 9 ]. Na t ur a ll y o cc ur r i n g p r o tea se i n h ib i to rs ar e e s se n t ia l fo r re g u la ti n g t h e ac ti v it y o f t he ir co r r esp o nd i n g p r o tea se s a nd p l a y k e y re g ul ato r y ro le s i n ma n y b io lo gi cal p ro ce ss es e. g. b lo o d c lo tti n g , ap o p to s is , s i g nal l in g r ecep to r i n ter act io n s i n a n i ma l s [2 0 ] . Re ce nt st ud ie s i n vo l vi n g i n vi tr o a nd i n vi v o mo d e l s ys t e ms a nd h u ma n tri al s h a ve ev id e nc ed t h at t he se in h ib i to r s ha v e a nt i– c arci no g e nic p ro p ert ie s [2 1 -2 5 ] . T hi s is s up p o rt ed b y t he o b ser va tio n t ha t p o p ul at io n s co n s u mi n g re la ti v el y hi g h a mo u n ts o f le g u me s eed s s ho w r e la ti ve l y lo w i nc id e nc e s o f p ro s ta te, co lo n, b re as t a nd s ki n ca n cer s [2 6 ] . La va te ra ca sh me r ia n a Ca mb . (Ma l vac eae ), wh ic h i s e nd e mi c to K a s h mi r val le y, ha s grea t med i ci na l i mp o r ta nc e. I t s p art s are b e in g u sed to tr eat so re t hro a ta nd co m mo n co ld . I n t hi s co m mu n i cat io n we ha ve rep o r t ed th e i so la tio n a nd c har ac teri za tio n o f p ro tea se i n h ib i to r s fr o m t he s eed s o f t h is p l a nt wh i c h a re cap ab le o f i n hi b it i n g tr yp si n, ch y mo tr yp si n a nd e la st a se.

II.

MATERIALS AND METHODS

a . See d Co l lect io n Mat ur e se ed s o f La va t e ra ca sh me r ia n a Ca mb . we re p ro c ur ed fro m De p art me n t o f B o ta n y, U ni v er si t y o f Kas h mi r . T r yp si n, c h y mo tr yp si n , e la sta s e, N - α -b e nzo yl - D Larg i ni n e -p - ni tr o a n il id e ( B AP N A) , ace t yl - L- t yr o si n e et h yl e s ter ( AT E E), N -s u cc i n yl - L(ala) 3 – p - ni tr o a n al id e, D E AE - ce ll u lo se a nd Sep had e x G -1 0 0 we re p urc ha sed fro m ( S i g ma Ald r ic h Co mp a n y, U S A ) . b. I so la t io n a n d p ur if i ca t io n o f pro tea se i n hi bit o rs f ro m La va te ra ca s h m eria na Ca mb . see d s T we n t y f i ve gr a m d r i e d seed s o f La ve s te ra c a sh m er ia n a C a mb . we r e so a ked i n 0 .1 5 N Na Cl fo r 1 6 ho ur s. T he s wo l le n se ed s were ho mo g e niz ed wi t h 3 0 0 ml o f i ce co ld sa li ne T ri s b u f f er ( 2 0 m M, p H 8 .0 ) co nt ai n i n g 1 mM so d i u m me tab i s ul fite i n a R e mi a uto mi x b le nd e r at 4 ° C fo r 1 0 mi n. T h e ho mo g e na t e wa s fil tered t hro u g h 4 la yer s o f c h ee se clo t h. T h e fi ltr a te wa s c en tr i f u g ed at 1 2 ,0 0 0 g fo r 2 0 mi n, t he p el le t wa s d is card ed a nd t he s up er n ata n t wa s r e ta i ne d and sto r ed at 4 ° C. Fro m t h e s up er n at a nt, th e p ro t ei n s wer e p recip it ated b y a m mo n i u m s u lp hat e a t 9 0 % sa t ura tio n . C r ud e e x tra ct was fur t her p uri fied b y io n e x c ha n ge c hr o ma to g r ap h y o n DE AE -c el l ulo s e co l u mn u si n g 2 0 m M T ris b u ffer, p H 8 . T he p ro te i n wa s el u t ed u si n g li n ear so d i u m ch lo rid e gr ad ie n t fro m 0 .0 5 to 0 .6 M. T he fra ct io n s e l ut ed fr o m io n -e x c ha n ge co l u mn wer e c hro ma to gr ap hed sep a rate l y o n S ep had e x G -1 0 0 co l u mn i n 0 .1 M T r is b u f fer , p H 8 a nd 0 . 4 M Na C l. T he c har g e ho mo ge ne it y o f t he fra ct io n s el u ted fr o m ge l f il tr a tio n co l u m n wa s c h ec ked b y p o l yacr yl a mi d e ge l elec tro p ho r e s is . c. Pro tei n est i ma t io n P ro tei n co nc e ntr atio n was d et er mi n ed b y t h e met ho d o f Lo wr y et a l . [2 7 ] us i n g B S A as t he s ta nd a r d p r o tei n. d. Po ly a cry la mi de g el ele ct ro p ho r e si s P o l yacr yl a mid e gel ele c tr o p ho re si s i n p r es e nce and ab s e nce o f SD S wa s p erfo r med at p H 8 .3 o n 1 0 % ge l wi t h a d is co nt i n uo u s b u ffer s ys te m. T he g el s we re s tai n ed wit h Co o ma s si e B r i ll ia n t B l u e R - 2 5 0 . e. M o lecula r w eig ht det e r mi n a t io n T he s ub u n it mo l ec ul a r ma s s o f t he p ro t ea se i n h ib i to r s wa s e st i mat ed b y d is co nt i n uo us S D S -P AG E ac co r d i n g to met ho d o f La m me l i [2 8 ] . N at i v e mo l ec u lar ma s s wa s d e ter mi n ed b y g e l f il tr at io n o n S ep had e x G -1 0 0 co l u mn u s i n g t h e fo llo wi n g p ro t ei n ma r ker s: B S A ( 6 6 k D a ) , O va lb u mi n (4 5 kD a) , P ep s i n (3 4 .7 k D A) a nd c yto c hro me c (1 2 .4 k Da). T he g el fi ltr atio n d at a wa s tr ea ted a cco rd i n g to t he r el at io n g i ve n b y An d r e w [2 9 ] . f . M ea sure me nt o f a nt it r y pt i c a ct iv ity T he a nt itr yp ti c ac ti v it y o f i n hib ito r s wa s e st i ma ted b y me as ur i n g t h e % in h ib i tio n o f tr yp si n, u s i n g s yn t h eti c s ub s tr a te b e n zo yl - D L-ar g i ni n e -p - ni tro a n il i d e [3 0 ] . V ari ab le vo l u me s o f p ur i f ied i n h i b ito r s we r e i n c ub at ed wit h 1 0 mg o f tr yp s i n (d i l ut ed i n 1 mM H Cl ) and 0 .1 M T r is - Hc l b u f f er ; p H 8 .0 , co nta i ni n g 0 .0 1 M C a Cl 2 , ( fi n al vo l u me 5 0 0 µl) fo r 1 0 mi n at 3 7 0 C. T h e n 5 0 0 µl o f B AP N A (1 mM i n DM SO) wa s ad d ed , i n c u b ated fo r 4 0 mi n a t 3 7 0 C a nd rea ct io n wa s sto p p ed b y t he ad d it io n o f 5 0 0 µ l o f a cet ic ac i d (3 0 % v/ v) . T h e co n tro l , wh i c h d id no t c o nt ai n i n hib ito r, wa s p r ep ared u nd e r str ic tl y id en ti cal co nd i tio n s. T he b la n k fo r eac h vo l u me o f t he i n hib ito r te s t ed was o b tai n ed b y t h e ad d it io n o f a cet ic www.ijpsi.org

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Trypsin inhibitors from Lavatera cashmeriana Camb. Seeds: isolation, characterization and in… acid j u s t b e fo r e t h e i n cub at io n wi t h t he s ub st rate. T h e i nt e n si t y o f t he ye llo w co lo u r p ro d uc ed wa s me as ur ed b y r eco r d i n g t he ab so rb an ce a t 4 0 5 n m. g . M ea sure me nt o f a nt ic h y mo t ry pt i c a ct iv ity Di ff er e nt vo l u me s o f eac h i n hib ito r wa s p re - i nc ub a ted wi t h 1 0 µ g o f e nz y me so l u tio n fo r 5 mi n a t r o o m te mp e r at ur e i n 1 ml o f 4 6 mM -T r is , 0 .5 mM Ca C l 2 b u ffer, p H 8 .1 . T he reac tio n wa s st ar ted b y t he ad d it io n o f 3 ml o f s ub s tra te (a ce t yl - L- t yro s i ne e t h yl es ter) so l u tio n to p r e -i nc ub ated e nz y me p l u s i n hi b ito r so l ut io n. B la n k was p r ep ared fo r eac h vo l u me o f e xtr ac t te s ted b y o mi tt i n g t he e nz y me fro m t h e r eac tio n mi x t ure. T he in te n s it y o f t he ye l lo w co lo r p r o d u ced wa s me a s ured b y r eco rd i n g t h e ab so rb a nce at 4 1 0 n m a ga i ns t a n ap p r o p r iat e b la n k [ 3 1 ] . A co nt ro l, wh e re c h y mo tr y p si n wa s no t p re in c ub a ted wi t h i n h ib i to r wa s a l so p r ep a red . h. M ea sure me nt o f a nt ie l a st a se a c t iv ity Di ff er e nt vo l u me s o f t he p ur i fi ed i n hib ito r s we re i n c ub a ted wi t h t he re ac tio n mi x t ur e (6 5 0 µl ) co nt ai n in g 1 0 0 mM T ri s -H C l, 1 0 mM C a Cl 2 , p H 7 .5 , 0 . 1 3 5 mM s ub s trat e ( N -s u cc i n yl - L- ( al a) 3 -p - n itr o a n al id e) a nd e la sta s e (1 .1 3 U/ ml ). T he co n tr o l, wh i c h d id no t co n ta i n i n hib ito r , wa s p r ep ar ed u nd er s tri ct l y id e nt ic al co nd i tio n s [3 2 ] . B lan k wa s p rep ared fo r ea c h vo l u me o f e x tr ac t t es ted b y o mi t ti n g t he e nz y me fro m t he rea ctio n mi x t ur e. T he r ea ct io n wa s car r ied o ut at 3 6 0 C, a nd t h e ab so rb a nce wa s me as u red at 4 1 0 n m. i. Effe ct o f pH o n t he st a bi lit y o f t h e In h ib ito r Di ff er e nt vo l u me s o f t he p ur i fi ed i n hib ito r s we re i n c ub a ted wi t h t he re ac tio n mi x t ur e (6 5 0 µl ) co nt ai n in g 1 0 0 mM T ri s -H C l, 1 0 mM C a Cl 2 , p H 7 .5 , 0 . 1 3 5 mM s ub s trat e and e la st a se ( 1 .1 3 U / ml ) . T he co n tr o l , wh i c h d id no t co nta i n i n h ib i to r, was p rep ared u nd er str ic tl y id e n ti cal co nd it io n s. B la n k s we re p r ep ared fo r ea c h vo l u me o f e xtr act t es ted b y o mit ti n g t h e e nz y me fr o m t h e r eac tio n mi x t ur e. T he reac tio n wa s carr ie d o ut a t 3 6 0 C, a nd th e ab so rb a n ce wa s me a s ur ed at 4 1 0 n m. j . Effe ct o f t e mp e ra t ure o n t he a c t iv ity o f In h ib i to rs 2 0 µl o f t he p u r i f ied sa mp le s we re i nc ub ated a t var io us te mp era t ure s fro m 1 0 to 7 0 o C, fo r 1 0 mi n u t es a n d act i vi t y wa s d e ter mi n e d . k. Ca r bo hy dra t e A na ly s i s T he p u r i f ied i n h ib i to r s we re a n al ys ed fo r carb o h yd ra te co n te n t b y p he no l s ulp h ur ic acid r ea ct io n [ 3 3 ] u s i n g D - g ala cto se a s st a nd ard . l. Cy to to xi cit y A ssa y ( S R B a s sa y ) T he c yto to xic it y o f LC - p i I wa s d et er mi n ed b y S RB as sa y [3 4 ] u si n g C o lo n (H CT 1 5 ), P ro s tat e ( P C -3 ) , B r ea st ( M CF - 7 ) a nd N e u ro b la sto ma (I M R -3 2 ). Ce ll s (3 ×1 0 3 /ce ll s) we re p l at ed i n 1 0 0 µl o f med i u m/ we ll i n 9 6 - we ll p late s. After i n c ub a tio n o ver n i g ht, LC -p i I wa s ad d ed i n var io u s co nce n tr a tio n s (5 0 0 , 3 0 0 , 1 0 0 , 5 0 µ g / ml) ; trip lic ate we l ls wer e in cl ud ed fo r e ac h co nc e nt r at io n. T he p l at es we re i nc ub a ted fo r 4 8 h rs at 3 7 0 C a nd i n a n at mo sp here o f 5 % n C O 2 . T he ce ll s we re t h e n fi xe d b y ad d i n g 5 0 µl o f ice - co ld 5 0 % T C A to eac h we ll f o r 6 0 mi n i n ca se o f ad he re nt ce l ls T he p la te s were wa s hed fi ve t i me s i n ru n n i n g t ap wa ter a nd st ai ned wi t h 1 0 0 ml p er we ll S RB rea g e nt (0 .4 % w/ v S RB i n 1 % acet ic ac id fo r 3 0 mi n. T he p lat e s were wa s h e d fi ve ti me s i n 1 % ace tic a cid to re mo v e u nb o u nd S RB a nd a llo wed to d r y o ver n i g ht. S R B wa s so l ub i li sed wi t h 1 0 0 µl p er we ll 9 6 we l l p la te 1 0 mM T r i s -b ase , s ha ke n fo r 5 mi n a nd t he O D wa s me a s ur ed a t 5 7 0 n m wi t h refere n ce wa ve le n g t h o f 6 2 0 n m.

III.

RESULTS

a . I so la t io n a n d P ur if i ca t io n T he cr ud e e xtr ac t wa s fo u nd to ha v e 8 5 % a nt it r yp t ic a ct i vi t y, 8 0 % an ti c h y mo tr yp ti c act i vi t y a nd 9 4 .6 % a n ti ela st as e ac ti v it y. O n DE AE ce ll u lo s e maj o r p o rt io n o f t he p ro t ei n in cr ud e e x tr a ct p a s sed u nb o u nd t h ro u g h t he co l u mn . T h e frac tio n d id no t s ho w a n y p ro tea se i n h ib i to r a ct i vi t y. T h e b o u nd p ro te i n wa s e l ut ed u si n g a li n e ar so d i u m c hl o r id e grad ie n t fro m 0 .0 5 to 0 .6 M o f 2 0 mM T ris b u ffer, p H 8 i n 5 ml frac tio n s at a flo w rat e o f 3 0 ml/ hr. At t h i s p H, b i n d in g o f i n h ib i to r s to t h e io n -e xc h a n ger wa s al m o st co mp l et e. T h e b o u nd p ro te i n el ut ed i nt o ma n y s ma ll mi n o r p e a k s a nd fo ur maj o r p e a ks fr o m t he co l u mn as s ho wn i n Fi g. 1 . T he fr a ct io n s u nd er mi no r p ea ks d id n ’t s ho w a n y a n tip ro tea se a ct i vi t y wh ere as fra ct io n s u nd e r p eak I , I I , III a nd I V s ho we d a nt ip ro t ea se acti v it ie s a ga i ns t tr yp s i n, c h y mo tr yp si n and ela s ta se. T he fra ct io n s u nd er ea c h p ea k we re p o o led a nd www.ijpsi.org

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Trypsin inhibitors from Lavatera cashmeriana Camb. Seeds: isolation, characterization and in… co n ce nt rat ed . T he i nd i v i d ua l p ea k s were lab e led as LC -p i I, LC -p i II, LC -p i III & LC - p i IV a nd furt h er c h r o ma to gr ap h ed o n to Sep had e x G -1 0 0 co l u mn . Al l t he fo ur p ea k s r eso l ved in to si n g le p r o t ei n p e a k s ho wi n g t ha t t he p r ep a ratio n s we re ho mo g e no u s wi t h re sp e ct to si ze. T he re s u lt s ar e g i ve n i n Fi g . 2 . T h e fr act io n s u nd er e ac h p ea k were fo u nd to ha v e an ti tr yp t ic, a n ti c h y mo tr yp t i c a nd a n tie la st a se act i vi t y. At e ac h p u ri fica tio n s tep s, t h e sp ec i fi c a ct i vi t y o f i n hi b ito r s i n cr e as ed o ver t h e h o mo ge na te (T ab l e 1 ).

Fig .1 E l utio n pro f il e o f cru de e xt ra ct o n D E AE c el lu lo se co l u mn . 3 0 0 mg o f p ro te in w a s a p pl ie d o n t h e co l u mn ( 3 5 ×2 c m) eq ui li br a ted w it h 2 0 mM Tri s buf fer, pH 8 . T he pro te i n w a s el ut e d us i ng a li nea r so d iu m c h l o ri de g ra d ie nt fro m 0 . 0 5 to 0 .6 M in 5 ml fra ctio n s a t f lo w ra t e o f 3 0 ml / hr. Ser ie s 1 re pre se nt a b so rba nce a t 2 8 0 n m a n d s erie s 2 re pr es ent % a nt itry ptic a ct iv ity .

Fig. 2 Elutio n pro f ile o f DE A E - c el lu lo se fra cti o ns o n Se p ha d e x G - 1 0 0 co l u mn . Tw e nty mi l l ig ra m o f ea c h pr o t ei n w a s a p pl ie d o n the Sa p he de x G - 1 0 0 co l u mn (4 0 ×2 c m) eq ui li bra t ed w it h 0 .1 M Tri s b uf f er pH 8 , 0 .4 M Na Cl. T he pr o tei n w a s el ute d a t a ra te o f 3 0 ml / hr in 5 ml f ra ct io n s. T he v o i d v o l u me o f co l u mn w a s 3 0 ml Se rie s 1 re pr e se nt a b so r ba n ce a t 2 8 0 n m o f LC - p i I, se rie s 2 o f LC - pi I I, s eri e s 3 o f LC - p i II I a n d ser ie s 4 o f L C - p i IV .

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Trypsin inhibitors from Lavatera cashmeriana Camb. Seeds: isolation, characterization and in…

Ta ble 1 : P ur if ica t io n o f pro t ea s e i n hi bi to r s fr o m La v a te ra ca s hm er ia na Ca mb . se ed s Steps

Total p r ot e i n ( mg )

Crude extract A mmo n i u m s ul p h at e f r ac t i o n DEAEc e l l ul os e f r ac t i o n L C - pi I L C - pi I I L C - pi I I I L C - pi I V Gel f i l t e r at i o n L C - pi I L C - pi I I L C - pi I I I L C - pi I V

A nt i t r y pt i c a c t i vi t y

160000

I n hi bi t o r U ni t s * 8000

Specific ac t i vi t y 0.05

A nt i c hy mo t r y pt i c ac t i vi t y I n hi bi t o Specific r U ni t s * ac t i vi t y 7500 0.04

A nt i e l as t a s e ac t i vi t y I n hi bi t o Specific r u ni t s * ac t i vi t y 8800 0.055

Per cent R e c ov e r y

Purifica -tion factor

100

1

24100

5000

0.20

4000

0.16

6800

0.28

75

5.09

450 500 600 500

1000 950 1000 900

2.2 1.9 1.6 1.8

900 900 945 800

2 1.8 1.57 1.6

2600 2400 2500 2250

5.7 4.8 4.1 4.5

29.5 27.2 28.4 25.6

103.6 87.2 74.5 81.8

17.5 26 47 36

1000 950 980 800

57.14 36.58 20.85 22.22

950 900 1000 800

54.28 34.61 21.27 22.2

1540 1500 1580 1480

88 57.69 33.61 41.11

17.5 17 17.9 16.8

1600 1048 611 747

* O ne i nh ib it o r u nit ( 1 IU) i s def in ed a s t he a mo u nt o f i nh i bito r w hi ch s up pr es se d li bra t io n o f o n e µ mo l s ub st ra te per mi n by a c tiv e e nzy me . b. El ectro p ho re t ic a na ly si s T he p ur i f ied LC -p i p r ep ar a tio n s we re ho mo g en eo us wi t h re sp e ct to ch ar ge a s rev ea led b y t he s i n gle b and wh e n s ub j e cted to p o l yacr yl a mi d e g el el e ctro p ho re s is (F i g. 3 ). I II I II IV V

Fig . 3 Po ly a c ry la mi de g el e lec tro pho reti c pa t tern o f pro tea se in hi b it o r s f ro m La va t er a ca s hm er ia na Ca mb . Ab o u t 5 0 µg o f e ac h p ro t ei n wa s ap p li ed o n p o l yacr yl a mi d e ge l at p H 8 .3 , u si n g 1 0 % sep a r at i n g ge l a nd 4 % s ta c ki n g ge l . C urre n t o f 8 m A p er we l l wa s ap p li ed . T h e st ai n i n g s o l ut io n u sed wa s Co o ma s sie b r il li a nt b lu e R -2 5 0 . La n e I cr u d e e xtr act ; l a ne II, LC - p i I ; l a ne I I I , LC - p i,I I ; la n e I V , LC - p i III and la n e V, LC - p i IV .

i.

c. Cha ra cte riza t io n M o lecula r w eig ht det e r mi n a t io n T he mo le c ula r we i g ht s o f La va t era ca sh me ri a n a Ca mb . p ro tea s e i n hib ito rs a s d eter mi n ed b y g el f il tr a t io n c hr o ma to grap h y wa s 2 0 .9 , 1 4 .1 , 1 6 .8 a nd 7 .9 k Da fo r LC p i I , www.ijpsi.org

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Trypsin inhibitors from Lavatera cashmeriana Camb. Seeds: isolation, characterization and in‌ LC p i I I, Lc p i I I I a nd LC p i I V re sp e ct i v el y. B as ed o n S DS p o l yacr yl a mi d e g el elec tro p ho r e s is d a ta t he mo lec u lar we i g ht wa s fo u nd to b e 1 0 , 1 4 , 1 6 a nd 7 kDa fo r LC - p i I, II, III and I V r e sp ec ti ve l y ( F i g 4 ) . T he res u lt s o f SD S -P AGE to ge t her wi t h ge l fil tra tio n d ata r e vea led t ha t LC -p i I e xi st s a s d i me r o f t wo id e nt ic al s ub u n it s o f 1 0 k Da . M .W

I

II

II I

IV

V

22 kDa 18 kDa 14 kDa 10 kDa 7 kDa Fig . 4

S DS - po ly a cry la mi d e g el el ectro p ho ret i c pa tt er n o f pro t ea s e i nh i bito r s.

E lec tr o p ho r e s is wa s car r ied o ut i n T ris - gl yc i n e b u ffer p H 8 .3 a t a c urre n t o f 8 mA p er wel l us i n g 1 0 % sep ar at i n g g el a nd 4 % s tac ki n g g el. Ab o u t 8 0 Âľ g o f ea c h p ro te i n wa s ap p li ed i n eac h we l s. La ne I , mo l ec u lar wei g h t s ta nd ard s; la n e II, LC - p i III; l a ne III , LC p i IV; la ne I V, LC - p i I I and la ne V, LC - p i I . T he g el s we re sta i ned wit h Co o ma s s ie b ril lia n t b l ue R -2 5 0 . ii.

Sp ec ific it y T he sp ec i fi ci t y o f p ur i f ied p ro tea se i n h ib i to r s wa s d e ter mi n ed a ga i n st tr yp si n, ch y mo tr yp si n a nd el as ta se. LC -p i I , I I, III a nd I V i n h ib i ted t he ac ti v it y o f tr yp si n b y 9 9 %, 9 6 %, 9 2 % a nd 8 2 % a nd o f c h y mo tr yp s i n b y 8 7 %, 8 8 %, 7 9 % a nd 7 2 % resp ect i ve l y a t t h e mo lar r at io o f 1 :1 ( Fi g. 5 A a nd 5 B ) . T he act i vi t y o f el as ta se wa s i n hib i ted b y 9 9 %, 7 7 %, 9 1 % a nd 7 9 % r e sp e ct i ve l y b y LC -p i I, II, III a nd IV o n l y at t h e mo lar r at io o f 0 .2 :1 (F i g.5 C) .

(A)

(B)

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Trypsin inhibitors from Lavatera cashmeriana Camb. Seeds: isolation, characterization and in‌

(C) Fig . 5 I nh ib it io n o f t h e t ry p si n ( A), c hy mo tr y ps i n (B ) a nd e la s ta s e (C) by La va t era ca s hm er ia na Ca mb . p r o t ea s e i n hi bito rs . B io lo g ica l a ctiv ity o f i nh i bit o rs w a s mea su re d by the deg r ee o f % i nh i bit io n o f e nzy ma ti c a ctiv ity , u s ing B A PN A, A T E E a n d N su cc iny l - L- ( a la ) 3 - p- n it r o a na l id e a s su b stra t e fo r try p s in, c hy mo try ps i n a n d ela sta s e res pe ctiv ely . Ser ie s 1 re pre se nt L C - pi I, s eri es 2 LC - pi I I, ser ie s 3 L C - pi I I I a n d s erie s 4 L C - pi I V. iii .

pH o pti mu m T he o p t i mu m p H fo r t he ma xi mu m a n ti tr y p tic ac ti v it y o f p uri fi ed p ro t ea se in h ib i to r s wa s 7 f o r LC -p i I , 8 fo r LC - p i I I, 7 .6 fo r LC - p i III a nd 8 .5 fo r LC - p i IV (Fi g.6 ) .

Fig . 6 Eff ect o f pH o n a ct iv it y o f La va te ra ca s hm eria n Ca mb. p ro tea s e i nh i bito r s. Ser ie s 1 re pre se nt L C - pi I, s eri es 2 LC - pi I I, ser ie s 3 L C - pi I I I a n d s erie s 4 L C - pi I V iv .

Effe ct o f t e mp e ra t ure o n a ct iv it y o f p ro tea se i nh i bito r s T he ma x i mu m a n ti tr yp t ic act i vi t y o f t h e i n hi b ito r s wa s o b ta i ned at 3 0 o C. T he in h ib i to r s s ho wed a s ha r p d ecr ea se i n ac ti v it y after ward a nd wer e co m p let el y i na ct i va ted at 7 0 Ëš C ( Fi g .7 ) .

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Trypsin inhibitors from Lavatera cashmeriana Camb. Seeds: isolation, characterization and in…

Fig .7 Effe ct o f t e mp era t ure o n t h e a ct iv ity o f pro tea s e i n hi bito rs Ser ie s 1 , 2 , 3 , 4 re pre se nt LC - p i I, LC - p i II, L C - p i II I a n d LC - p i IV res pe ctiv ely . v.

v i.

Ca r bo hy dra t e Co nt e nt T he i n hib ito r s wer e fo u nd to b e g l yco p ro te i ns wi t h LC -p i I co n ta i ni n g 9 .1 µ g s u gar, LC -p i II 7 .0 µ g s u gar , LC -p i I I I 3 .8 µ g a nd L C -p i IV 2 0 µ g o f s u gar p er mg o f p ro tei n (T ab le 2 ). Ta ble 2 : Ca rbo hy dra te co nt ent o f p ro tea se i n hi bito r s In h ib it o r s Ca r bo hy dra t e co nte nt (µg / mg o f pro t ei n) LC - p i I 9 .1 LC - p i II 7 .0 LC - p i II I 3 .8 LC - p i IV 20 Ana ly s i s o f ca rbo hy dra te w a s do n e by D u bo i s et a l ., 1 9 5 6 . Gro w t h i n hi bi t io n by L C - p i I i n h u ma n ca n cer cel l s Gro wt h i n h ib i tio n o f P r o st ate (P C -3 ) a nd B r ea s t (M C F -7 ) ce ll li ne b y LC -p i I at var io u s co n ce n tr a tio n s in - vi t ro wa s d eter mi ned . T he i n h ib i to r s ho we d a d o se -d ep e nd e n t in h ib i to r y e f fec t o n ce ll gr o wt h . N e gl i gib le e ffe ct wa s see n o n gro wt h o f co lo n ( H CT -1 5 ) and Ne uro b la s to ma ( I M R -3 2 ) cel l s. T h e re s ul t s are gi v e n i n T ab le 3 . Ta ble 3 : Cy to to xic ity o f p uri fie d L C - p i I

Ti s s ue Ty pe Cel l l i ne Ty pe S. No Co d e 1. 2. 3. 4. 5. 6.

Co lo n Pro sta te B rea st H C T- 1 5 PC- 3 M CF - 7 % G RO W TH I NH IB I T IO N

Ne uro bla sto ma IM R- 3 2

Co n c. ( µg / ml) 38 LC - p i I 500 89 72 52 34 32 47 LC - p i I 300 54 2 6 1 1 4 1 30 LC - p i I 100 5 5 36 23 LC - p i I 50 12 34 22 31 Pa cl it a xel 1 µM 16 26 37 M it o my ci n C 1 µM 76 The bold values are shown for those compounds which have proved to be active and those in normal font represent least significant. www.ijpsi.org

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Trypsin inhibitors from Lavatera cashmeriana Camb. Seeds: isolation, characterization and in… IV.

DISCUSSION

P ro tea se i n hib ito r s we r e i so l at ed fro m s eed s o f La va t era ca sh m e ri a n a C a mb . wh ic h i s e nd e mi c to Ka s h mi r va lle y o f I nd i a. I t wa s fo u nd to b e u sed b y ma n y K as h mir i p eo p le fo r so me med ic i n al p ur p o se s. T he cr ud e p ro tei n sep ar ated i n to fo ur maj o r p e a ks o n DE AE - ce ll u lo s e co l u mn s u g g es ti n g t h at fo ur i n hib ito rs e x is t i n La v a te ra ca sh me ria n a Ca mb . seed s ( LC -p i I , L C -p i I I , LC -p i III a nd LC -p i IV ). Si mi l ar r es u lt s were rep o r ted b y O gat a et al. [ 3 5 ] fo r o k r a ( Ab el mo sch u s e scu le n tu s L.) p ro t ea se i n h ib i to rs . T he p ur i fied in h ib i to r s wer e ho mo g e no u s wi t h r e sp ec t to s iz e and c h ar ge a s wa s e vi d en t fro m t he ir g el fil trat io n b e h a vio r a nd p o l yacr yl a mi d e ge l el ectro p ho r es i s i n p re s e n ce a nd ab se n ce o f SD S. T he mo le c ular wei g ht o f t h e i n hib ito r s d eter mi n ed b y SD S -P AGE wa s ab o u t 6 % le ss er t ha n mo le c ular we i g h t s o f i n hib ito r s d eter mi n ed b y ge l fil tra tio n d ata . T h is i s u nd er s ta nd ab le b ec a us e in h ib i to r s b ei n g gl yco p r o tei n s s ho w a no ma lo us b eh a vio r o n SD S P AGE ge l [ 3 6 ] . S D S -P AGE al so r e vea led t h at LC -p i I i s co mp o sed o f t wo s ub u n it s o f 1 0 k Da e ac h. T h e t wo -c h ai n str u ct ur e ha s b ee n rep o rted p re vio u s l y fo r 2 0 k Da K u ni tz p ro tei n as e i n hib ito r s f o u nd i n seed s o f Ad en a n th e ra p a vo n in a [3 7 ] , Arch id en d ro n ell ip t icu m [ 3 8 ] and p o ta to t ub er s [ 3 9 ] . Fo ur p ro tei n as e i n hib ito r s ha v e al so b ee n p uri fied and c har ac ter i zed f r o m Arch id en d ro n el lip ticu m o n e wi t h mo le c ul ar wei g ht o f 2 0 kDa [3 8 ] and Ph a seo lu s vu lg a ri s L. [ 4 0 ] . T he i so lat ed p r o tea se i n h ib i to r s wer e s tro n g i n hib ito r s o f ela st as e t h a n tr yp si n a n d ch y mo tr yp si n. T hi s i s co mp arab le wi t h i n hib i to rs iso la ted fro m j ack fr u it se ed s wer e al s o fr a ct io n I I a nd III i n hib it ed e la sta s e s tro n gl y t h a n tr yp s i n a nd ch y mo tr yp si n [ 4 1 ] . T he o p ti mu m p H o f fo u r i n hib ito rs LC -p i I, II, III and I V wa s fo u nd b et we e n 7 , 8 , 7 .6 and 8 .5 r esp e ct i vel y, wh ic h i s co ns i ste n t wi t h ear li er rep o rts [3 8 , 4 2 ] . Si n g h al [ 4 3 ] r ep o r ted t h at t he tr yp si n i n h ib i to r fro m mu n g b e a n seed s was ac ti ve b e t we e n p H 4 .0 a nd 1 0 .0 . An nap ur na [ 4 4 ] r ep o rt ed t ha t th e tr yp si n i n hib ito r fro m j ac k fr u it seed s wa s s tab le o ver s ti ll a wi d er r a n ge o f p H (3 .0 to 1 2 .0 ). I n co n tra st , t h e tr yp si n i n hib ito r fro m p i geo n p e a s eed s r eta i ned i t s fu ll act i v it y b e t we e n p H 7 .0 a n d 1 0 .0 , b u t wh e n exp o sed to a cid ic p H fr o m 3 .0 to 5 .0 , 2 0 % o f t he ac ti v it y wa s lo s t [4 5 ] . J o h ns to n et al . [4 6 ] o b ser v ed t h at t he mi d g ut o f lep id o p te ra n lar va e i s hi g h l y a l ka li n e a nd t h e d i ge s ti ve p ro tea se s ha v e o p t i ma l ac ti v it y b e t wee n p H 1 0 .0 a nd 1 1 .0 , wh il e t he lar va e o f p h yt o p ha go u s co leo p t er an h a ve acid ic co nd i tio n s i n t he ir mi d g ut r e gio n , wi t h p H o p t i ma fo r d i ge st i ve e n z y me s t yp i ca ll y i n t he r a n ge 4 . 0 -5 .0 . T he o p t i mu m te m p erat ur e wa s fo u nd to b e 3 0 ˚ C fo r al l i n hi b ito r s. Ho we v er t he i n hib ito rs wer e a ct i ve up to 6 0 ˚ C b ut we re co mp le te l y i nac ti v ated at 7 0 ˚ C. T r yp s i n i n h ib i t o rs fro m o kr a seed s we r e a lso rep o r ted to b e s tab le up to 6 0 ˚ C a nd r ap id l y i n act i va ted a t 8 0 ˚C . T he p r o tea se i n hib ito r s iso la ted fro m s eed s o f L a va te ra ca sh me r ia n a C a mb . W hi c h in h ib i ted tr yp s i n, c h y mo tr yp si n a nd ela s ta se ca n b e cla s si fied a s a K u ni tz t yp e o f in h ib i to r b a sed o n t heir mo lec u lar si ze. K u n it z - t yp e p ro t ea se i n h ib i to r s ha vi n g d i ffer e nt mo lec u lar ma s se s ha v e al so b e e n r ep o r ted i n s o yb e a n (1 9 k Da) , mu s t a rd s eed s (2 0 k Da) and Ca ja n u s ca ja n ( 1 4 k Da) [ 4 7 , 4 8 ] . T he i n hi b ito r s we re fo u nd to b e gl y c o p ro t ei n s. T h e p res e nce o f car b o h yd r at es i n p la nt p ro tea se i n h ib ito r o f o t her fa mi l ie s ha s b ee n r ep o rt ed earl ier al so [ 3 5 , 4 0 a nd 4 1 ] . In s u m ma r y, t he p o te n tia l a n tic a ncer act i vi t y o f LC -p i I a ga i n st t h ese h u ma n ca ncer ce ll li ne s wa s i n ve s ti ga ted . LC - p i I e x hib it ed a st ro n g i n h ib i t o r y e ffec t o n t h e p ro li fe rat io n o f P r o st at e ( P C -3 ) a nd B rea st ( MC F -7 ) ca n cer ce ll li n es i n in v it ro . T he an ti ca nc er act i vi t y o f LC -p i I co u ld b e attr ib ut ed in p ar t to i ts i n h ib i ti o n o f p ro li fera tio n d ue to i ts p r o te as e i n hi b ito r ac ti v it y a s LC -p i I i s s tro n g i n h i b ito r o f tr yp s i n, ch y mo tr yp si n a nd e la st a se.

ACKNOWLEDGEMENT T he a u t ho r e xp r es se s g r ati t ud e to U ni ver s it y o f Ka s h mi r, Sri n a gar fo r p ro v id i n g fi na nc ia l a s si st a nce a nd to t he d ep ar t me n t o f B io ch e mi s tr y fo r t he i n fra st ruc t ure.

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